Rodriguez Bacte

C& Publishing, Inc.
REVIEW HANDB0OK IN
Di:agnostic
Bacteridolog.y
THIRD EDITION
Maria Teresa T. Rodriguez
REVIEW HANDBOOK IN
Diagnostic
Bacteriology
THIRD EDITION
REVIEW HANDBO0K IN
Diagnostic
Bactericolog(y
THIRD EDITION
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c& E Publishing, Inc.
2022
CONTENTS
Dedication
Preface
Acknowledgments
Discovery of Microorganisms
Chapter 1
Assessment Quiz .
Bacterial Taxonomy
Chapter 2
Assessment Quiz . .1 4
17
Prokaryotes, Eukaryotes, and Archae
Chapter 3 25
Assessment Quiz.
Bacterial Genetics . 27
Chapter 4
Assessment Quiz
Bacterial Metabolism 33
Chapter 5 36
Assessment Quiz.
Microbial Nutrition 37
Chapter 6 Assessment Quiz. . 41
Pathogenesis of Infection. 43
Chapter 7 60
Assessment Quiz.
Biosafety and Quality Control in a Clinical Microbiology Laboratory . 63

Chapter 8 .78
Assessment Quiz.
Sterilization and Disinfection 81
Chapter 9 92
Assessment Quiz .
Specimen Collection, Transport, and Processing .

. 95
Chapter 10
113
Assessment Quiz.
Culture and Culture Media 115

Chapter 11 132
Assessment Quiz.
135
Microscopy.
Chapter 12 140
Assessment Quiz .
143
Bacterial Staining
Chapter 13 Assessment Quiz . 152
Methods for Bacterial Identification® 155

Chapter 14 168
Assessment Quiz .
Antimicrobials (Antibiotics) 171

Chapter 15 180
Assessment Quiz .
Antimicrobial Susceptibility Testing 183

Chapter 16 196
Assessment Quiz.
Gram-positive Cocci. 199

Chapter 17
Assessment Quiz. 245
Gram-negative Cocci 247

Chapter 18
Enterobacteriaceae (Gram-negative Bacilli) 263

Chapter 19
Non-enteric Gastrointestinal Pathogens 307

Chapter 20
Non-fermentative Gram-negative Bacilli 323

Chapter 21
Assessment Quiz . 335
Small, Pleomorphic Gram-negative Bacilli 339

Chapter 22
Aerobic Gram-positive Bacilli. 361

Chapter 23
Assessment Quiz.
387
Chapter 24
Mycobacteria (Acid-fast Bacilli) 391
Anaerobic Bacteria 425

Chapter 25
441
Chapter 26 Rickettsiaceae and Related Organisms
449
Chapter 27 Chlamydiaceae
455
Chapter 28 Mycoplasmataceae (Cell wall-deficient Bacteria).
461
Spirochetes and Miscellaneous Bacteria
Chapter 29 475
Assessment Quiz
. . . . . . . . . . . . . . . . . . . 479
References
The Author
PREFACE
Review Handbook in Diagnostic Bacteriology is conceptualized and designed for Medical

Technology and Allied Health Sciences students as a simplified guide for understanding the
fundamentals of diagnostic microbiology.
This guidebook provides information on every clinically significant bacterial species, their
morphological and cultural characteristics, related infections and diseases, and associated
laboratory diagnoses. Molecular techniques for rapid detection and characterization of prokaryotes
are also included in the discussion. Each chapter is presented in an outline form for easy reading
and comprehension.
This learning material is also useful to medical technologists who are reviewing for their
certification examinations.
- Maria Teresa T. Rodriguez

ACKNOWLEDGMENTS
I extend my sincerest thanks and appreciation to:

My son Gabriel, for his unwavering love, constant support, and understanding; and
Mr. Nardito D. Moraleta, academic colleagues, and students, for their encouragement,
inspiration, and valuable contributions.
All praise and highest glory to our God Almighty for the wisdom and strength.
CHAPTER 1
DISCOVERY OF
MICROORGANISMS
INTENDED LEARNING OUTCOMES

At the end of this chapter, the students should be able to:
1. describe the significant discoveries that led to the development of
microbiology;
2. explain the Koch's postulates and their relationship to the progression of
disease; and
3. discuss the contributions of the early scientists and microbiologists.
Microbiology is defined as the study of organisms that are too small

to be seen by the naked eye. In this opening chapter, the great minds and
their contributions to the discovery and evolution of microbiology; and the
relationship of this discipline to medicine and other areas of biology are listed.
The Discovery of Microorganisms
Francesco Stelluti (1577-1652)
He made the earliest observations on bees and weevils using a
microscope supposedly supplied by Galileo.
Antonie van Leeuwenhoek (1632-1723)

He was considered as the "first true microbiologist."
He was the first person to observe and accurately describe living
microorganisms, such as bacteria and protozoa, thus, he was regarded
as the "Father of Bacteriology and Protozoology."
He used the term "animalcules," or the tiny living and moving cells
seen under the microscope, to describe microorganisms.
his self-made single lens microscope with 50x to 300x
He used
magnification to study bacteria and protozoa.

REVIEW HANDBOOK IN
DIAGNOSTIC BACTERIOLOGY
Spontaneous Generation
It states that life arises from non-living matter.
Aristotle (384-322 B.C.)

He mentioned that simple invertebrates could come from spontaneous generation.
Francesco Redi (1626-1697)
from
could not arise spontaneously decaying meat
In 1668, he demonstrated that maggots
belief that life forms could
the long-held arise
The results of his investigation invalidated from
non-living things.
John Needham (1731-1781)

He observed that the sealed flask with boiled mutton broth became cloudy after standing.
"vital force" that could give rise to life.
He asserted that organic matter possessed a
Lazzaro Spallanzani (1729-1799)

improved the previous experiments of Needham by heating the broth
that
He was transferred
into a sealed jar.
remained sealed.
He observed that no growth took place as long as the flasks
He proposed that air transports microorganisms into the culture medium.
He concluded that microorganisms from the air probably had entered Needham's concoction
after they were boiled.
Biogenesis
It states that living cells could only arise from pre-existing living cells.
Rudolf Virchow (1821-1902)

He challenged the concept of spontaneous generation with biogenesis.
Theodor Schwann (1810-1882)

He observed that no growth occurred in a flask that contained a nutrient solution after allowing
the air to pass through heated tube.
Heinrich Schroder (1810-1885) and Theodore von Dusch (1824-1890)

They noticed that no growth took place after allowing the air to pass through a sterile cotton
wool placed on a flask with heat-sterilized culture medium.
Louis Pasteur (1822-1895)

He disproved the theory of spontaneous generation.
He proved that while the air does not generate microbes, microorganisms are, indeed, presen
and can contaminate a sterile solution.
VOOJOIRBTOAR DISCOVERY OF MICROORGANISMS
He proposed the use of heat in killingmicroorganisms, which is now called the aseptic technique
or a method utilized
in preventing contamination by unwanted microorganisms.
He provided evidences that microorganisms could not originate from "mystical forces" present in
non-living materials. mood hedoa
He developed the vaccine against anthrax (1881) and rabies (1885). .i worle of leth 2sw sH
He improved the wine-making processes by introducing the concept of fermentation and
"pasteurization".
developed the anthraxt
John Tyndall (1820-1893)
He showed that dust carry germs that could contaminate a sterile broth. end ads 26W ori
Tyndallization is a form of sterilization the 19th century that uses moist heat for three
in
consecutive days to eradicate vegetative cells and endospores.
Ferdinand Cohn (1828-1898) hoabsatbris8o

He discovered that there are bacteria that could withstand a series of boiling because of heat-
resistant structures known as endospores.
Antoine-Laurent Lavoisier (1743-1794)

He showed the importance of oxygen to life.
it mont hins huealoat ad laum metnegto omea odT
Fermentation and Pasteurization
Theodor Schwann explained that yeast cells are responsible for the conversion of sugars to
alcohol. glbemn ennhiun in ead onl booubommiaH
Louis Pasteur described that certain microorganisms known as yeasts convert sugar to alcohol in
the absence of air, a process known as fermentation.
Pasteur stated that the souring and spoilage of wine are caused by different bacteria. He also
proved that in the presence of air, bacteria convert the alcohol in the beverage into vinegar or
acetic acid.
To resolve the problem in the wine industry, Pasteur suggested the minimal heating of beers and
wines that is sufficient to kill most of the bacteria also known as pasteurization.
Theory of Antisepsis • lo sau orl bAR supin ibel attllbe-Inoninonne orl! bagoiavob vedT
Ignaz Semmelweis (1816-1865)

He demonstrated that routine handwashing can prevent the spread of diseases.
Joseph Lister (1827-1912)

He introduced the system of antiseptic surgery.
He pioneered in promoting among surgeons the handwashing before and after an operation, the
wearing of gloves, sterilizing of surgical instruments, and the use of phenol as an antimicrobial
agent for surgical wound dressing.
6 REVIEW HANDBOOK IN DIAGNOSTIC BACTERIOLOGY
TABLE 1-1. Important Events in the Development of Microbiology

Date Significant Event
1590-1595 Hans Jansen developed the first compound microscope.

1676 Antonie van Leeuwenhoek discovered animalcules. w ferl avog of alds sis venT
1786 Otto Friedrich Muller produced the first classification of bacteria. oslo1g enisrnoy
1798 Edward Jenner introduced the smallpox immunization through cowpox inoculation.
1838-1839 Theodor Schwann and Matthias Jakob Schleiden introduced the cell
theory. Ke5 614 2
1858 Rudolf Virchow proposed the theory of biogenesis.
1861
Louis Pasteur used the aerobic and anaerobic terminologies in describing the metabolism of
yeast.
1867 Joseph Lister published his work on antiseptic surgery.

1875 Ferdinand Cohn used the genus name Bacillus in classifying bacteria.
1880 Louis Pasteur described the weak strain of organisms as attenuated in developing vaccines.
1881
Robert Koch cultured bacteria on gelatin.
Louis Pasteur developed the anthrax vaccine.
1882 Robert Koch discovered the tubercle bacilli.
Koch's postulates were first published.
1884
Autoclave was developed..
Hans Christian Gram introduced Gram stain.
1885 Louis Pasteur produced the rabies vaccine.
1887 Julius Richard Petri introduced the use of the Petri dish.
1890 Emil von Behring prepared antitoxins for diphtheria and tetanus.
1895 Jules Bordet discovered the complement.
1896 Ronald Ross showed that malarial parasites are carried by mosquitoes 08) venold buswol
1900 Walter Reed proved that yellow fever is transmitted by mosquitoes. orli shem vorlT
1901 Karl Landsteiner discovered the blood group system.
1906 Fritz Schaudinn and Erich Hoffman showed that the Treponema pallidum causes syphilis.
1907 Recognition of the role of protozoa in the disease process
1909
Sigurd Orla-Jensen established the importance of including the physiologic characteristics of
bacteria in their classification
1910 Paul Ehrlich developed chemotherapeutic agent for syphilis.
1923 First edition of Bergey's Manual of Determinative Bacteriology was published. Subsequent
editions were titled Bergey's Manual of Systematic Bacteriology.
to sou orl al il
1933 Ernst Ruska developed the first transmission electron microscope. f
Edouard
1937
Chatton introduced living organisms as either prokaryotes or eukaryotes.
1944
Selman Waksman discovered streptomycin.
1945 Alexander Fleming discovered penicillin.
1951
Max Theiler developed the yellow fever vaccine.
1953 Francis Crick and James Watson discovered the structure of DNA.
1954
Rosalyn Yalow and Solomon Berson developed the radioimmunoassay.
pJo aieAS hDiScoVERY OF MICROORGANISMS 7
)
Cornelius van Niel and Roger Stanier described prokaryotes.
1976
Recognition of archaeobacteria as a distinct microbial group
1980
Development of scanning tunneling microscope (STM)
1 –1 4
Robert Gallo and Luc Montagnier identified and isolated HIV.tt hadaitaen odi
Kary Mullis developed polymerase chain reaction (PCR). 2o blan
1986 The fifirst hepatitis B vaccine created through genetic engineering was approved for human
1Se.
F trer
1995 ChickenpoX vaccine was approved.
Craig Venter, Hamilton Smith, and Claire Fraser released the first complete genome
pr to tan shsequence of a microorganism utilizing Haemophilus influ h no▇ or
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REVIEW HANDBOOK IN DIAGNOSTIC BACTERIOLOGY
ASSESSMENT QUIZ
Encircle the letter that corresponds to the correct answer.
1. Who established the theory that bacteria indeed cause diseases? ns olse hodoh
a. Ronald Ross
b. Charles Chamberland
C. Robert Koch
d. John Needham
Who introduced the concept of amplification of microorganisms through the use

2.
of the
polymerase chain reaction procedure?
a. Max Theiler
b. Kary Mullis
C. Selman Waksman
d.
Sigurd Orla-Jensen
3.
It states that living cells can only arise from
pre-existing living cells.
a. Biogenesis
b. Spontaneous generation
C. Germ theory of disease
d. Fermentation
4.
Who was the first to use the term
"attenuated" in developing vaccines?
a. Louis Pasteur
b. Rudolf Virchow
C. Robert Koch
d. Jules Bordet
5. He first demonstrated the

significance of hand
disease. washing in preventing the spread of infection and
a.
Theodor Schwann
b. Antoine-Laurent Lavoisier
C. Emil von Behring
d. Ignaz Semmelweis
DISCOVERY OF MICROORGANISMS 9
6.
Which antibiotic did Alexander Fleming discover?
a. Salvarsan
b. Bacitracin
c. Penicillin
d. Erythromycin
7. Based on the experiment of Cohn, which cellular structures are heat-resistant?
a. Endospores
b. Yeast
C. Maggots
d. Animalcules
8. All of the following correctly states the postulates of Robert Koch EXCEPT
a. The microorganism must be present in every case of the disease and the healthy host.
b. The suspected microorganism must be isolated from diseased host and grown in a pure
culture.
C. The same disease must be absent when the isolated microorganism is inoculated into a
healthy host.
d. The same organism must be isolated again from the diseased host.
9. Who introduced the concept of aseptic technique?

a. Edward Jenner
b. Sergei Winogradsky
c. Joseph Lister
d. Louis Pasteur
10. He was the first to use the term "prokaryotes and eukaryotes" when describing microorganisms.
a. Antonie van Leeuwenhoek
b. Hans Jansen
¢. Walter Reed
d. Édouard Chatton
CHAPTER 2
obto feltmie to beeogmo
BACTERIAL TAXONOMY Aslimic to boaogmop

At the end of this chapter, the students should be able to:™
2. explain how microorganisms are classified;

3. write correctly the names of the bacteria; and
differentiate phenotypic from genotypic characteristics.
onten auring orl pndinw n
Taxonomy
It is an area of biological science that comprises three distinct
concentrations, namely classification, nomenclature, and
bon identification.
It is a formal system of organizing, classifying, and naming living

things.
It is based on the similarities and differences in the genotype and
phenotype of organisms.
Carl von Linne, a Swedish botanist, laid down the basic rules for aib to eesbor
taxonomic categories (binomial system).
I. Classification
It is the organization of microorganisms that have similar™z8T

morphologic, physiologic, and genetic traits into specific groups ouostab
or taxa.
The classification system is hierarchic and consists of the following taxa

designations:
1. Domain - Bacteria, Archaea, Eukarya
2. Kingdom - composed of similar phyla; similarities of DNA and
RNA
3. Division/Phyla - composed of similar classes

4. Class - composed of similar orders
5. Order - composed of similar families
6. Family - composed of similar genera JOHOXAT JAIRSTOAS
7. Genus - composed of various species with common characteristics
8. Species is the basic
group or the collection of bacterial strains with common physiologic
and genetic features
Note: Tribe is included in the
hierarchy of taxonomy. It is in between family and genus but is
not commonly utilized in bacterial taxonomy.
Subspecies are species which are subdivided based on the following differences:
Biotype
similar genetic traits but different biochemical
a. is
-
having
and
physiological characteristics within the same species
b. Serotype is based on
serological (surface antigens) differences within the same
species
II. Nomenclature
It is the
naming
International Code of microorganisms according to established guidelines provided by the
of Nomenclature of Bacteria or the Bacteriological Code.
In writing the genus name,
1.
The first letter should be
capitalized and followed by
which begins
with a lower-case letter.
the species epithet (specific name),
2. Both
the genus and species should be italized in
(e.g., Staphylococcus aureus or print but underlined when written in script
Staphylococcus aureus).
3.
When bacteria are referred to as group, their names are
leg, staphylococci, streptococci, etc). neither capitalized nor underlined
IIl. Identification
It is
the process by which laced af A
the microorganisms' key features are described. "3:0 1o seryhisnt
the process of discovering and recording the traits of organisms so that
It is
in an
overall taxonomic scheme. they may be placed
Genotypic Characteristics
It refers to the organism's genetic make-up.
It involves the detection of a gene or a part thereof,
An example is the base oran RNA product of a specific organism.
sequencing of DNA or RNA to
organisms. determine the relationship of two
Molecular characterization involves the study of nucleic acid compostion and proteins.
POO JOISTDA& DITRONDAIQ BACTERIAL TAXONOMY 13
Phenotypic Characteristics
It is based on the features beyond the genetic level.
It includes readily observable characteristics, such as the morphological features, as well as those
pi19n99 traits that may require extensive analytical procedures. to nomoallop orl of maston it
Some examples are colony morphology, staining, and biochemical and susceptibility tests.
edion blachemizal and mumumolog

Classical Characteristics d
It is useful in
routine phylogenetic studies of microorganisms such as the understanding of
morphology, physiology and metabolism, ecology, and genetic analysis.
Phylogenetic or phyletic classification is based on evolutionary relationships of microorganisms
starting from its genetic characteristics instead of just studying the general biological
resemblance.
and
madel basilatigs2 i netinw ad avawls blwoda ebmon eunes)
960D alvosned adt ewollot sisloed to gnirach/

1. Biogroup is a population of species that share the same biochemical properties
bsand al gniwollol onft to flbldW
2. Epithet - is the proper word for the name of the species
3. Genotype is the collection of genes that describes the characteristics of an organism
4. Morphovar - also known as a morphotype, is a prokaryotic strain which differs morphologically
from other strains
bios
5. Phenotype - is the observable physical and biochemical properties of the organisms
Polyphasic Taxonomy - is a modern system of bacterial classification and identification combining

phylogenetic, phenotypic, and genotypic characterizations, likewise utilizing molecular sequences
and epigenetic factors
7. Serogroup - is a serovar having similar antigens nived shabed to gniquorg erls allI .5
8. Strain - is an altered or a variant microorganism within the same species evlovn # .b
estoage smee onlt midtiw mainsgroomimn tnshsv s et it

CHAPTER 3
PROKARYOTES,
EUKARYOTES, AND
ARCHAE

1. describe the cellular structures of bacteria and their functions;

2. differentiate the properties of Gram-positive bacteria and Gram-
negative bacteria;
3. discuss the specific features of the acid-fast bacilli cell wall;
4. distinguish the characteristics of eukaryotes from those of prokaryotes;

and
5. cite the traits of archae and compare with the prokaryotes and
eukaryotes.
Prokaryotes
These are organisms that do not contain a true nucleus.
These organisms also do not contain organelles such as mitochondria,

endoplasmic reticulum, and Golgi apparatus.
All the functions of this organisms take place in the cytoplasm or
cytoplasmic membrane.
The word "prokaryote" is formed by the words "pro," which means
before, and the Greek word "karyon," which means nucleus, nut, or
kernel.
A common example of these cells are the bacteria.
Bacterial Cell Structures
Cell Envelope
It is the outermost structure of the bacterial cell.
It is composed of an outer membrane (Gram-negative bacteria), cell

wall, periplasm (Gram-negative bacteria), and plasma membrane.
18 REVIEW HANDBOOK IN DIAGNOSTIC BACTERIOLOORR
Cell Wall
It is also referred to as the peptidoglycan or murein layer.
of the cell.
It is a rigid structure that maintains the shape
subunits and teichoic acid or lipoteichoic acid
It is composed of disaccharide-pentapeptide
and structure have been the primary

target of antimicrobial agents.
Its synthesis
Functions
osmotic pressure inside the cell is
a. It prevents bacterial cells from rupturing when the greater
than the pressure outside the cell.
b. It serves as a point of anchorage for flagella.
C. It determines the staining characteristics of a species.
Types of Cell Walls

1. Gram-positive cell wall
It is composed of a very thick protective peptidoglycan (murein) layer.
It consists of glycan chains of alternating N-acetyl-D-glucosamine (NAG) and N-acety-D-
muramic (NAM) acid.
It contains a negatively charged teichoic acid and contributes to the total electric charge of
the cell wall.
target of antimicrobial agents, like penicillin, which prevent the synthesis o

It is the prime
peptidoglycan.
2. Gram-negative cell wall

It has porins that
contribute to the permeability of the cell wall.
It contains a periplasmic space which is involved in
peptidoglycan synthesis.
It does not contain teichoic acid.
Layers of the Gram-negative Cell Wall

a. Outer membrane/wall
It is composed of
proteins, phospholipids, and lipopolysaccharide (LPS).
contributes
the negative charge
It to the
of
the bacterial surface, which stabilizes
membrane structure.
It allows hydrophilic compounds to enter the cell

through the porins.
It acts as a barrier to toxic
substances that prevents movement inside the cell.
It is vital in
evading the host defenses the of the
strong negative charge
membrane is an important factor in evading phagocytosis.
It is also considered as an endotoxin
and
3 regions of LPS: Lipid
A (endotoxin, major component), core polysacchari
antigenic O-specific polysaccharide
YOD/OIF PROKARYOTES, EUKARYOTES, AND ARCHAE 19
b. Inner membrane/wall
It is composed of a thin peptidoglycan layer, which is the reason for its high
lsatmers to susceptibility to mechanical breakage.
TABLE 3-1. Comparison Between Gram-positive Bacteria and Gram-negative Bacteria

Properties Gram-positive bacteria Gram-negative bacteria
Cell wall The thick cell wall is composed of The outer membrane is composed of
peptidoglycan; teichoic acids may also lipids, proteins, LPS, and a thin inner
be present.
Shape Spherical, rod-shaped, or filamentous Spherical, oval, straight or curved

rods, helical, or filamentous
Metabolism chemoorganoheterotrophic
present absent
Endospore present in some groups absent
Periplasmic space absent present
Flagellar structure 2 rings in the basal body#89112 85 Diosi: rings in the basal body
Resistance to physical high low
disruption
Resistance to lysozyme low high

disruption
Inhibition by basic dyes high low
Reproduction binary fission
3. Acid-fast cell wall
It has Gram-positive cell wall structure.

Aside from a peptidoglycan layer, it contains a waxy layer of glycolipids and fatty acids
(mycolic acid) that is bound to the exterior of the cell wall.
Mycolic acid has a strong hydrophobic structure that affects the permeability of the acid-fast
01 SONDINIRO cell wall.
Some examples of bacterial cells that have this kind of cell wall are Mycobacterium and
tack leet at - brimsslo Rame
4. Absence of cell wall
Prokaryotes that do not have a cell wall contain sterols in their cell membrane.
Examples: Mycoplasma and Ureaplasma
Plasma Membrane
It is the deepest layer of the cell envelope.
It consists of a phospholipid bilayer that surrounds the cytoplasm, and the layer is embedded
with lipoproteins.
It functions as the mitochondria, Golgi complexes, and lysosomes. printaun tannl

It acts as an osmotic barrier.
It regulates the transport of solutes across the membrane as well as the generation of chemical
energy (ATP).
It is the site of respiration and photosynthesis.
Cytoplasmic Structures in a Prokaryotic Cell

1. Ribosome (Non-membranous structure)
It is the site of protein biosynthesis and gives the cytoplasm a granular structure.
It consists of RNA and proteins.
It is 70S in size and separates into two subunits, 50S and 30S.
2. Genome
It consists of single, circular chromosome.

It appears as diffused nucleoid or chromatin body that is attached to a mesosome (sac-like
structure).
3. Plasmid
It is an extrachromosomal, double-stranded element of DNA that is associated with
virulence.
It is cytoplasm and serves

located in the as a site for the genes to encode for antibiotic
resistance and toxin production.

It is a self-replicating cellular structure that may be transferred to a daughter cell (vertical
transfer) or may be transferred asexually through conjugation (horizontal transfer).
It is not essential for bacterial growth thus, a bacterial cell may or may not contain a plasmid.
It sometimes disappears during cell division, and it can make bacteria (mostly Gram-
negative bacteria) pathogenic.
Kinds of Plasmids
ass-bit
a. Large plasmid - is responsible for the production of p-lactamases that provide resistance to
-lactam antibiotics like penicillin and oxacillin
b. Small plasmid is resistant to tetracyclines and chloramphenicol
4. Inclusion bodies
They serve as the energy source or food reserve of the bacteria.

They are composed mainly of polysaccharides, which lessen osmotic pressure.
Examples: Glycogen, cyanophycin granules, poly-B-hydroxybutyrate granules, carboxysomes
(cyanobacteria, nitrifying bacteria, and thiobacilli), gas vacuoles (cyanobacteria,
halobacterium, and Thiothrix), and polyphosphate granules (volutin or metachromatic
granules)
Examples of polyphosphate granules: Babes-Ernst bodies (Corynebacterium diphtheriae),
bipolar bodies (Yersinia pestis), and Much's granules (Mycobacterium tuberculosis)
YODJO PROKARYOTES, EUKARYOTES, AND ARCHAE 21
5.
Endospores/Asexual spores (Resistant structures)
These are small, dormant structures located inside the bacterial cell.
They aid in the survival of bacteria against external conditions.
eant d
They are produced within vegetative cells of some Gram-positive bacteria.
They are composed of dipicolinic acid and calcium ions (calcium dipicolinate).
The locations of these structures aid in the microscopic identification of bacteria.
Examples of endospore-forming bacteria: Bacillus and Clostridum
Types of Spores According to their Location and the Associated Organism and T
a. Terminal spore Clostridium tetani
b. Subterminal spore - Clostridium botulinum

C. Central spore Bacillus anthracis
Terminologies
1. Sporogenesis/Sporulation - is the process of spore formation

2. Germination - is the end of the spore's dormant stage
Cell Appendages fifin oua mue duence
1.
It is an outward complex of polysaccharides on the bacterial surface and other cells.
It helps the bacteria in attaching to the surface of tissues or solid objects. adviliadn0d
It appears as a capsule or a slime layer.
Types of Glycocalyx
a. Capsule
It is an organized material that is firmly attached to the cell wall.
It is mostly made up of polysaccharide polymers.

It protects the bacteria (virulence factor) from the attacks of human defense system cells
since it resists phagocytosis and dessication.
b. Slime layer
It is an unorganized material that is loosely attached to the cell wall.
It also consists of polysaccharides.

Itcan either inhibit phagocytosis or aid in the adherence of the bacteria to the host tissue
or synthetic implants.
2. Flagellum (Organ of Locomotion)

It is an exterior protein filament (flagellin) that rotates and causes bacteria to be motile.
It is important in the survivability and the pathogenic ability of bacteria.

Flagellar arrangement
a. Atrichous ~ without flagellum

b. Monotrichous single flagellum on one end
C.
Amphitrichous - single flagellum on both ends
d.
Lophotrichous - tuff/group of flagella on one end or both ends logranb
e. Peritrichous - spread over the whole surface
3. Pili (Fimbriae)
These are
hair-like, proteinaceous structures, about 2 um in length, that extend from the cell
membrane to the external environment.
Types of Pili
a. Common/Somatic Pili
They are the organ of attachment - aid in the
attachment of bacteria to tissues and
surfaces.
They are considered a virulence factor.

Examples of bacteria with common pili: Neisseria gonorrhoeae and Pseudomonas
b. Sex Pili
They are an essential part of the

genetic transfer/conjugation process.
Example of bacteria with sex pili: E. coli (also
with somatic pili)
Notes to Remember
Gram-positive bacteria have two basal rings

flagellar structure. while Gram-negative bacteria have four basal rings in the
Gliding motility is exhibited by

cyanobacteria, myxobacteria, and Capnocytophaga, d2226.2401
True motility and Brownian
movement are best observed through the hanging drop method.
With true motility, the bacteria seem to be
going in a definite direction.
molecules.movement,
with Brownian the bacteria bounce back and forth rapidly due to the bombardment of
The movement of bacteria toward or
away from a particular stimulus is called taxis. mule
TABLE 3-2. Presence or Absence of Cell

Appendages
Motile bacteria
Non-motile bacteria
Bacillus anthracis
Bacillus cereus
Bacillus subtilis
Clostridium perfringens
Campylobacter
Corynebacterium diphtheriae
Clostridium tetani
Erysipelothrix
YODLan PROKARYOTES, EUKARYOTES, AND ARCHAE 23
Escherichia coli
Klebsiella pneumoniae
Listeria Mycobacterium tuberculosis

Pseudomonas Neisseria
Vibrio Staphylococci and Streptococci

Bacteria with capsules Bacteria with spores
Bacillus anthracis Bacillus
Klebsiella pneumoniae Clostridium

Streptococcus pneumoniae
Bacteria with inclusion bodies Or granules
Corynebacterium Babes-Ernst bodies
Mycobacterium Much's granules
Nocardia and Actinomycetes Sulfur granules

Pasteurella and Bordetella Bipolar bodies
Eukaryotes
These are microorganisms that contain a true nucleus (with chromosome bound by a nuclear
membrane).
These are cells of higher plants, animals, fungi, protozoa, and other morphologically complex
and larger organisms than prokaryotes.
They contain many membrane-bound organelles such as nucleus, endoplasmic reticulum, Golgi
body, mitochondria, and lysosomes, in which cellular functions are performed.
26
TABLE 3-3. Comparison Between a Prokaryotic Cell and a Eukaryotic Cell
Characteristic Prokaryotes Eukaryotes
Size of the cell 0.20 um 2.0 um 10 um - 100 um
Cell wall usually present; mostly contains usually absent except in fungi
peptidoglycan (chitin); if present, it is chemically
simple
Nucleus absent; absence of nuclear present; true nucleus with nuclear

membrane or nucleoli membrane and nucleoli
Genome ___location in the nucleoid or at the mesosome in the nucleus
Chromosomal DNA single, circular chromosome; multiple linear chromosomes; with

complexed with RNA; histones
without histones
Membrane-bound absent present
organelles
Ribosomes present; smaller size (70S) present; larger size (80S)
Lysosomes and absent present in some species
peroxisomes
absent
Pili and fimbriae present
BACTERIOLOGY
24 REVIEW HANDBOOK IN DIAGNOSTIC
Prokaryotes Eukaryotes
Characteristic
simple; consists of two protein- complex (multiple microtubules)
Flagella
building blocks
Sterols absent (except in Mycoplasmataceae) present
Introns in genes absent present
Cytoskeleton and no cytoskeleton and cytoplasmic with cytoskeleton and cytoplasmic

cytoplasmic streaming streaming streaming
Gas vesicles present absent
Cell division asexual (binary fission) sexual and asexual
Terminologies
1. Introns - a sequence of base pairs in DNA that interrupts the continuity of genetic information
2. Cytoskeleton - is a cytoplasmic element, which includes the keratin and other microfibrils, that
functions as a
supportive system within a cell, especially with epithelial cells
Archaea (Archaeobacteria)
Archae is from the Greek word "archaics,' which means ancient.
The cellular structures of archaea include the cell wall, plasma membrane, ribosomes, and
flagella.
They do not contain a nucleus and membrane-bound organelles. tam vrom misinoo vent
The cell walls of archaebacteria do
contain the
typical peptidoglycan as observed
not
in
prokaryotes, instead a protein or glycoprotein wall structure known as the "S-layer"

a protective coat.
is present as
These organisms are classified as aerobes, facultative anaerobes, or

obligate anaerobes.
They may be stained either
as Gram-positive or Gram-negative organisms in various shapes such
as
spherical, rod, and spiral (pleomorphic).
They
grow and survive under extreme environ mental conditions
They reproduce
through binary fission, fragmentation, or budding.
PROKARYOTES, EUKARYOTES, AND ARCHAE 25
ASSESSMENT QUIZ
1. What is the major component of the acid-fast cell wall?
a. muramic acid
c. lipopolysaccharide
d. mycolic acid
2. It is the organ of attachment that helps bacteria during tissue invasion.

a.
b. somatic pili
c. flagella
d. teichoic acid
3. It serves as the mitochondria of the prokaryotic cell.

a. plasma membrane
b. periplasmic space
C. ribosomes
d. genome
4. It protects the bacteria against phagocytosis, hence, it is considered as a virulence factor.

a. capsule
b. cell membrane
C. plasmid
hen ir
d. spores
5. Which of the following inclusion bodies are found on Corynebacterium diphtheriae?

a. Much's granules
b. Bipolar bodies
C. Polyphosphate
d. Babes-Ernst bodies
6. It is an extrachromosomal structure that serves as a site for the development of bacterial resistance
to antimicrobial agents.
a. plasmid
c. porins
d. carboxysomes
26 KEVI W ND▇ ▇ ▇ ▇ DACT▇ RIOLO Y
7. Which of the following organisms lacks a cell wall?

a. Bacillus
b. Capnocyłophaga
Ebaam ol aEbnoeno2 1sdt eie utsb
c. Nocardia le a s Duarail serM
e D
d. re a p l a s m
Which of the following terms n ks to organisms with a "true nucleus"?ainea

asdote vicepu
hipiooyum h
a. Archaebacteria
b. Bacteria
c. Prokaryotes plaVionest pniuh cho rd eler iart tnembens io nnyo sdt etl
d. Eukaryotes hsonvly
iG S i i
9. It has a centrally-located endospore.
a. Mycobacterium tuberculosis B :a A
baioet
b. Bacillus anthracis
Ei atl tae if5
C.Halobacteriu icrwor se doupeiepln prt s 29ym8ul
d. Clostridium tetani nS dnar Mde
s pinanicnsą d
10. It is the endotoxin layer of the gram-negative cell wall.
fa
a.Antigenic-0-specific polysaccharide t i
b. Lipid A l1 EA)
haahabeniitosa
c. intro. rt saboubiancoaoror eisoy ▇ oiratoad ed abaiozq
d. periplasmic space lhnaq
sgden lse
thbinerla
td
nahhuM
slbnd loql
euo
ia eeral
plisadda ast ihunteheacurbaietn

hgeldserian
baads
eroi
at t
CHAPTER 4
BACTERIAL GENETICS

1. define terminologies;
2. explain the fundamentals of bacterial genetics;
3. discuss the replication process in prokaryotes;

4. distinguish the steps of gene expression; and
5. discuss the mechanisms of gene transfer.
A.T. Levine presented in the 1920s that DNA is composed of phosphates, five-riog sllrw
carbon sugars, and nitrogen-containing bases. Three decades later, Rosalind an09
Franklin introduced the helical structure using x-ray crystallography. Then in
the 1950s, James Watson and Francis Crick described the three-dimensional
structure of the DNA molecule.
The ability of microorganisms to change rapidly, acquire new genes, and

clinical microbiologists
undergo mutations presents continual challenges to
as they isolate and characterize the microorganisms associated with humans. bobnstt nob
Currently, the study of prokaryotes through genetics is considered as

the new nonsen
generation of microbial assay.
Deoxyribonucleic Acid (DNA)

It is a double-stranded helical chain of deoxynucleotides.
The nitrogen bases are adenine, guanine, thymine, and cytosine.

The helix formed from the twisted double-stranded structure appears
like a "spiral staircase."
The information contained in the DNA is determined primarily by

the sequence of letters along the staircase.
It is involved in the RNA synthesis.I
Ribonucleic Acid (RNA)

ribose instead of deoxyribose.
acid, and it contains the sugar
It is a single-stranded, short nucleic the thymine, which is replaced by
the DNA, except for
It has the same nitrogen bases found in
uracil.
Genetics
It is the process of heredity and variation.

cellular pathways,
functions, and structures originate.
It is the starting point from which all other
Major Aspects of Genetics

1. Structure and organization of genetic material
The nitrogenous bases (adenine,
guanine, cytosine, and thymine), which are covalently
to the genetic code.
linked to each deoxyribose sugar, are the key
(ribose or
A nucleoside consists of a nitrogenous base attached to pentose sugar
deoxyribose) while a nucleotide is composed of a nucleoside and a phosphate group.
(3' to 5' and 5' to 3).
The complementary strands are in opposite directions or antiparallel
The order of bases in DNA or RNA strand is the base sequence.
A DNA sequence that encodes for a specific product (RNA or protein) is defined as a gene.
All genes taken together within an organism comprise a genome.
Active genes are referred to as constitutive genes, such as those involved in the transcription
process, while genes that are expressed in a certain condition are the inducible genes.
The size of a gene and of an entire genome is usually expressed in the number of base pairs
(bp) present.
A genome organized in discreet elements is known as chromosomes.
A bacterium contains a single, unpaired (haploid) chromosome.
The bacterial chromosome contains a single, unpaired (haploid) chromosome, and exists as a
double-stranded, closed, circular, naked macromolecule; most eukaryotes have linear DNA.
The information for protein synthesis is encoded in the bacterial DNA and transmitted to
chromosome.
The general flow of information in a bacterial cell is from the DNA to the messengerRNA
(mRNA).
The mRNA acts as "blueprint" for protein construction.
A codon is composed of three nucleotides which "codes" or corresponds to specific amino
acid.
2. Replication and expression of genetic information

Replication in genetics is a process where there is for
duplication of chromosomal DNA
insertion into a daughter cell.
It results from a division of one
parent cell, then each of
the resulting daughter cells receives
the full and identical genetic complement contained in the original parent cell.
YOOUIOIARTOAE DITROMONG BACTERIAL GENETICS 29
The process of replication takes approximately 40 minutes with rapidly growing bacteria.
The process of encoding information in genetic elements is known as gene expression.
Transcription
It is the synthesis of a single-stranded RNA using one strand of the DNA as a template.
It converts the DNA base sequence of gene into a mRNA molecule.
RNA polymerase is the enzyme that is central to the transcription process.

In prokaryotes, only one RNA polymerase is present.
Translation
It is
a process wherein a specific protein is synthesized from the mRNA.
The genetic code with mRNA molecules is translated into a specific amino acid sequence
that is responsible for the protein structure and function.
3. Mechanisms by which genetic information is changed and exchanged among bacteria

Mutation
It is an alteration in the original nucleotide sequence of gene or genes within an organism's

genome (genotype). avioguingo lo aldegso ens abimaslg
Causes: Induced through chemical exposure, by physical factors in the environment, by
biologic factors such as the introduction of a foreign DNA into the cell, or as a result of an
error during DNA replication
Recombination (Homologous Recombination)rulent

It is a process by which genes are transferred or exchanged between homologous regions on
two DNA molecules.
It allows organisms to acquire and copy new combinations of biochemical pathways as an

outcome of the modifications in their environment.
It occurs when portion of the genetic material that originates from one bacterial cell (donor)
is transferred into a second bacterial cell (recipient).
It involves a number of binding proteins with the RecA protein, which is essential for DNA
repair and maintenance, playing a central role.
Mechanisms of Gene Transfer

1. Transformation
It involves the uptake of free DNA that is released into the environment when another
bacterial cell (donor) dies and undergoes lysis or cell disintegration caused by a rupture in
the cell wall.
2. Transduction
It is the transfer of bacterial genes by bacteriophage from one cell to another.

In this mechanism, DNA from two bacteria may come together in one cell.
Bacteriophage is a virus that infects bacteria.

Types of Transduction eatunim Ob vole

a. Generalized transduction gls 3116.18
It is the process in which the bacterial DNA is incorporated into another bacterium,
specifically during the lysis of the virulent bacteriophage.
b. Specialized transduction
It is the process in which of bacterial DNA with viral nucleic acid
part or a fragment
is transferred to another bacterium by the temperate bacteriophage during lysogenic
ong vino eato odon al
process.
3. Conjugation
It is the transfer of genetic material from a donor cell to recipient cell.
It occurs between two living cells (cell-to-cell contact).
It requires the mobilization of the donor's chromosome.
The sex pilus (donor) establishes a conjugative bridge that serves as the conduit for DNA
transfer to the recipient cell.
The plasmid could be transferred by conjugation (horizontal transfer), but not all
plasmids are capable of conjugative transfer.
YOO IONS DAB DITRONDAI BACTERIAL GENETICS 31
ASSESSMENT QUIZ
Encircle the letter that corresponds to the correct answer. sdgaodq bru evand avon
1. It is not included in the nitrogenous bases of RNA.

a. uracil
b. thymine
C. adenine
d. guanine
2. Which of the following is the correct description of the bacterial chromosomes?

a. single, unpaired
b. single, paired
double, haploid
d. double, diploid
3. Which enzyme is vital in the transcription process?

a. restriction enzyme
b. DNA polymerase
c. transposon
d.
4. It is the transfer of genetic materials through a virulent bacteriophage.

a. specialized transduction
b. replication
C. generalized transduction
d. mutation
5. It serves as the channel for the movement of the DNA during conjugation.
a. nucleotide
b. sex pili
c. plasmid
d. bacteriophage
6. It is the correct composition of a nucleoside.

a. nitrogenous bases and deoxyribose sugar
b. nitrogenous bases and phosphate group
C. pentose sugar and phosphate group
d. nitrogenous bases only
e. all of the above
7. It is the synthesis of a single-stranded RNA from a DNA template.

a. replication
b. generalized translation
C. transcription
d. transformation
8.
Which of the following is the correct description of the complementary strand
during
amplification?
a. anti-parallel
b. 3' to 5' and 5' to 3'
C. both
d. neither
9. It is described as the DNA sequence that encodes for a specific protein.

a. genome
b. base pair
C. gene
d. nucleic acid
10. It acts as
blueprint for protein synthesis.
a. DNA
b. chromosome
C. nucleotide
d. mRNA
CHAPTER 5
BACTERIAL METABOLISM

1. discuss and contrast the biochemical pathways that generate energy;
2. differentiate respiration from fermentation; and
3. explain the relationship of prokaryotic cell function and energy
utilization.
res noba acondledue auonspo
Metabolism is defined as the sum of all chemical processes that take

place in a living organism and results in its growth, energy generation, waste
disposal, and other functions in relation to cell nutrient distribution.
Bacterial metabolism is divided intotwo major parts: anabolism or the
constructive phase and catabolism or the destructive phase. In anabolism,
there is synthesis of complex molecules and energy production; whereas in
catabolism, the large complex molecules are broken down into simpler, smaller
molecules accompanied by energy utilization.
Bacterial metabolism consists of biochemical reactions that break down
organic compounds and produce new bacterial structures from the resulting
carbon skeleton. All biochemical reactions in the cell depend on the presence
and activity of specific enzymes.
Energy Production
the degradative process of
It is accomplished by the breakdown of chemical substrates through
catabolism that is coupled with oxidation-reduction reactions.
atoms, are highly reduced compounds,
Compounds, such as glucose that have many hydrogen
and thus contain large amount of potential energy.
Glucose is an in organisms.
essential nutrient for energy production
two general processes: respiration and
To produce energy from glucose, microorganisms use
fermentation.
Respiration
It is an efficient ATP-generating process in which molecules are oxidized and results in an
inorganic molecule as the final electron acceptor.
In this process, glucose is completely broken down and results in a high-energy production.
In the presence of oxygen, glucose is changed into carbon dioxide and water.
It is carried out by obligate aerobes and facultative anaerobes.
In an aerobic respiration, oxygen is the final electron acceptor, while in anaerobic respiration, one
of the exogenous substances, such as nitrate, sulfate, and fumarate, is the final electron acceptor.
Processes in Respiration
1. Glycolysis (Embden-Meyerhof-Parnas pathway)
It is the first stage in carbohydrate metabolism.
It is the oxidation of glucose to pyruvic acid.
It is the major route of glucose metabolism in most cells.
2.
It is the most important process for the complete oxidation of a substrate under aerobic
conditions.
In this process, an enzyme system converts pyruvate into carbon dioxide and
an acid.
cycle is used
generate energy in the form of adenosine triphosphate (ATP).-10
This to
The substrate for this process is the acetyl coenzyme A. notaey
Fermentation
It does not require oxygen (anaerobic process), the use of Krebs cycle, or an electron transport
chain.
It releases energy from sugars or other organic molecules, such as

amino acids and purines.
It forms a mixture of
end products (lactate, butyrate, ethanol, and acetoin) in the medium; the
analysis of these products is useful for the identification of
anaerobic bacteria.
It is carried out by both obligate and facultative anaerobes
ocuoinaro8 areo6 BACTERIAL METABOLISM 35
Types of Fermentation
1. Alcoholic Fermentation
It
turns sugar into ethanol and carbon dioxide (for fungi, algae, protozoa, and some bacteria).
2. Homolactic Fermentation
In this process, pyruvate is reduced to lactate (for Streptococcus and Lactobacillus), which is
used to make yogurt, sauerkraut, and pickles; it means that only one acid is produced after
fermentation.
3. Heterolactic Fermentation
This process produces substances other than lactate, such as alcohol, carbon dioxide, formic
acid, and acetic acid.
4. Mixed acid fermentation
It involves the production of ethanol and acids, such as lactic, acetic, succinic, and formic
acid.
It utilizes formic hydrogenlyase that converts formic acid into an equal amount of hydrogen
and carbon dioxide.
food
5. Butanediol fermentation
In this process, pyruvate is converted into acetoin then reduced to 2,3-butanediol with
NADH; small amounts of ethanol and mixed acids are also synthesized.
Some bacteria that undergo this process are Enterobacter, Serratia, Erwinia, and Bacillus.
6. Butyric acid fermentation

It involves the conversion of pyruvate into butyric acid along with acetic acid, carbon dioxide,
bios
and hydrogen.
Some bacteria exhibiting this kind of reaction are Clostridium, Fusobacterium, and Eubacterium
(obligate anaerobes).
Energy Utilization
Once energy is obtained, bacteria, as well as other organisms, utilize it in various ways:
1. for the biosynthesis of new cell components;
2. for the maintenance of the physical and chemical integrity of the cell;
3. for the activity of the locomotor organelles;
4. for the transport of solutes across membranes; and
5. for heat production.

ASSESSMENT QUIZ
of ethanol and acids?

1. What type of fermentation involves the production
a. Mixed acid fermentation
511097
b. Butanediol fermentation
c. Homolactic fermentation
d. Heterolactic fermentation
2. What substrate is used in the Krebs cycle that generates ATP?

a.
b. Carbon
C. Acetyl coenzyme A
d. Lactate
3. For which of the following mechanisms do bacteria utilize energy?

a. Locomotion
b. Binary fission
C. Both
d. None of the above
4. Which of these statements best describes the fermentation process?

a. It does not require oxygen.
b. It oxidizes glucose to pyruvic acid.
C. It is the main method of glucose metabolism in most cells.
d. It is an
enzyme-utilizing system that converts pyruvate into carbon dioxide and an acid.
5. What is the basic source of energy that is essential for
prokaryotes?
a. Fatty acid
b. Glucose
C. Amino acid
d. Purine
CHAPTER 6
MICROBIAL NUTRITION

2. discuss the requirements for bacterial growth and their role in microbial
physiology; and
3. describe the types of bacteria according to their physiologic conditions.
Microorganisms require specific nutrients that are essential for their

growth and multiplication. Elements such as oxygen,
carbon, hydrogen, a nodsD of anibi0o
nitrogen, and sulfur are some of the needed substances in energy production
and biosynthesis. Moreover, temperature, pH, and moisture are also necessary
to facilitate their growth and survival.
Physiologic Requirements of Bacteria
901008 mod
According to Oxygen Requirement
1. Aerobes
These organisms require oxygen and grow well with room air.
Air contains 15% to 21% oxygen and 1% CO,.
Examples: Bordetella, Brucella, Mycobacteria, and Pseudomonas
2. Anaerobes
They do not require oxygen to grow and survive.
Types of Anaerobes
a. Obligate anaerobes
They do not require the presence of oxygen, and they die
after prolonged exposure to air.
Examples: Clostridium and Bacteroides
b. Facultative anaerobes
bacteria.
They are the most clinically significant
absence of oxygen; hence they are
as Te osthat onm growianmetobitahly?

the presence or
These organisms grow either
consi d ered
They do not require oxygen but grow better in
its presence.
Example: Enterobacteriaceae
C. Aerotolerant anaerobes
but unable to perform metabolic
These organisms can survive in the presence of oxygen
processes unless situated in an anaerobic environment.
Example: Lactobacillus and Cutibacterium
3. Microaerophile
for growth.
It is an organism that requires only 2% to 10% oxygen
Example: Campylobacter, Helicobacter, and Treponema
Notes to Remember
Obligate aerobes and facultative anaerobes contain protective enzymes (superoxide dismutase
and
catalase) that counter the toxic effects of oxygen.
According to Carbon Dioxide Requirement

Capnophiles require an increased CO, (5% to 10% CO.).
Most aerobic and facultative aerobic bacteria need 0.03% CO,.
Example: Haemophilus influenzae, Neisseria gonorrhoeae, and Streptococcus pneumoniae
According to Nutritional Requirement

1. As to the carbon source
Autotrophs use carbon dioxide as the sole source of carbon.
Heterotrophs utilize reduced, preformed, organic molecules from other bacteria.
As to the energy source
Phototrophs are organisms that use light as their energy source.

Chemotrophs make use of the
energy produced by the
oxidation of organic or inorganic
compounds.
3. As to the electron source
Lithotrophs reduce inorganic molecules to

be used in biosynthesis or energy conservation.
Organotrophs require organic substances
multiplication. carbohydrates and lipids) for growth and
MICROBIAL NUTRITION 39
Notes to Remember
Autotrophs are also lithotrophs because they obtain energy either photosynthetically or oxidatively.
Heterotrophs are also organotrophs since they obtain energy through oxidation or fermentation of
organic substances such as glucose.
All bacteria that inhabit the human body are classified into the heterotrophic or organotrophic group.
Fastidious bacteria need additional substances such as vitamins, purines, pyrimidines, and
hemoglobin for growth and survival.
Saprophytes require dead organic substances.
Parasites necessitate organic substances from living tissues. od ainogoring feom tol Bg mumdgp afll
According to Temperature Requirement

The optimum temperature for most clinically significant bacteria is 35°C to 37°C.
1. Psychrophiles/Cryophiles
These organisms grow well at 0°C to a maximum of 20 °C.
Examples: Listeria monocytogenes and Yersinia enterocolitica
They grow between 20 °C to 45°C.

These are the most commonly encountered pathogenic bacteria in the clinical laboratory.
3.
Thermophiles/Hyperthermophileseok-lwng minobes
These organisms grow between 50°C to 60 °C, just like the archaea.
Examples: Geobacillus stearthermophilu, Therms aquaticus, Sulfolobus, Pyrococcus, and
4. Extremophiles
Theseare prokaryotes that are able to survive in unusual conditions like the absence of
oxygen, increased temperature, and living below the earth's surface.

Example: Bacillus infernus (strict anaerobe)
Terminologies
1. Thermal death time is the lowest/minimum time required to kill organisms under constant
temperature
2. Thermal death point - is the lowest temperature required to kill organisms in a constant time
IN DIAGNOSTIC BACTERIOLOGY
40 REVIEW HANDBOOK
According to pH
pH scale is a measure of the hydrogen ion concentration.
Types of Organisms According to pH Tolerance ingotonsgto

a. Acidophiles grow between pH 0 and 5.5 (Picrophilus and Sulfolobus)
b. Neutrophiles grow between pH 5.5 and 8.0 (most clinically significant bacteria)
C. Alkalophiles. grow between pH 8.5 and 11.5 (Bacillus alcalophilus, Natronobactrim)
Notes to Remember
The optimum pH for most pathogenic bacteria is from pH 6.5 to 7.5.edue pinez1o staliermoon ast
Diagnostic laboratory culture media for bacterial isolation are usually adjusted to final pH between
7.0 and 7.5.
According to Moisture
Moisture is vital for bacterial growth and susceptibility testing. Hdnt Peatalini/bret
According to Salt Concentration®

Halophiles require and grow in increased concentration of sodium chloride.
Examples: Staphylococcus aureus and Listeria monocytogenes
According to Pressure sinaihed pin poring bmelsbodhs dnommoo loom bdl ens sporT
Barophiles are organisms that grow rapidly in high-pressure environments (600 to 1100 atm
pressure).
Examples: Photobacterium, Shewanell, and Colwellia
According to Growth Factors

substances that are required by fastidious bacteria for their growth and
Growth factors are
multiplication.
vitamins.
Some examples of growth factors are amino acids, purines, pyrimidines, and
YUC IDISICAB DINORDAIO MICROBIAL NUTRITION 41
ASSESSMENT QUIZ
1. Which of the following is the most commonly isolated bacteria as to temperature requirement?
a. Thermophiles
b. Psychrophiles ve:/co to etostts lifmned onh
C.
Hyperthermophiles
d. Mesophiles
2. In which pH scale do most clinically significant bacteria grow?

a. 5.5 and 8.0
b. 8.5 to 11.5
C. 6.5 to 8.0
d. 0 to 5.5
3. These are organisms that survive in increased salt concentration.

a. Capnophiles
b. Barophiles
c. Halophiles
d. Litotrophs
iniog disob tormod !
4. Which bacteria only require 5% of oxygen?
a. Microaerophiles
b. Aerotolerant anaerobes ent nl alb iliw bns edmsnms olsgtido maal
c. Facultative anaerobes
d. Anaerobes
5. Which bacteria utilize organic substances as source of carbon?
a. Lithotrophs
b. Heterotrophs
C. Chemotrophs
d. Autotrophs
42
6. Which type of bacteria requires growth factors like hemoglobin and pyrimidines?
a.
b. Alkalophiles
c. Fastidious bacteria
d. Saprophytes
7. Which enzyme protects bacteria against the harmful effects of oxygen? ingodove
a. Catalase
b. Glucuronidase
C. Oxidase
d. Coagulase
0.81
8. Which of the following organisms is NOT a capnophile?

a. Neisseria gonorrhoeae
b. Haemophilus influenzae
C. Streptococcus pneumoniae
d. Treponema pallidum
9. It is the lowest temperature that will destroy organisms in a constant time. Slidgonad
a. Thermal death time
b. Thermal reduction time
C. Thermal death point
d. Thermal reduction point
10. This organism is an obligate anaerobe and will die in the presence of oxygen. 01019/
a. Enterobacteria
b. Clostridium
C. Pseudomonas
d. Neisseria
CHAPTER 7
PATHOGENESIS OF
INFECTION
lynes of Infections cording TO How

2. differentiate the types and routes of infections; o dis suppe af tedld nodbeini
3. discuss the classifications of diseases;
4. compare the host resistance factors with the infectious agent factors;
5. explain the phases of infectious diseases;

6. distinguish true pathogens from opportunistic pathogens;
7. relate the virulence factors with the bacteria causing diseases and
infections; and
8. cite the epidemiologic steps of an outbreak investigation.
Pathogenesis the development of an infection and disease. Certain

is
virulent agents with mechanisms of resistance against the host protective

factors are involved in the proliferation of microorganisms and the progress of nor ante
diseases.
Infection
It involves the growth and multiplication of microorganisms that

cause damage to their host.
Itthe bodily invasion of pathogenic microorganisms that
is
reproduce, multiply, and cause diseases through local cellular injury,

toxin secretion, or antigen-antibody reaction in the host.
44 REVIEW HANDBOOK
Cause
Types of Infections According to
1. Autogenous Infection
It is caused by a microorganism from the microbiota of the host.
2. Iatrogenic Infection
It is an infection that occurs as a result of a medical treatment or procedure.
3. Opportunistic Infection
It is an infection that affects immunocompromised hosts but not the healthy individuals.
The overuse of antibiotics, immunosuppressive drugs, and chemotherapeutic agents may
cause this infection.
4. Nosocomial Infection
It is also known as the hospital-acquired infection.

It is a type of infection that is acquired in a healthcare facility.
Handwashing is still the cornerstone of modern infection control programs.
Common examples: urinary tract infection (UTI), lung infection (pneumonia), surgical site
infection, and bloodstream infection
Predisposing Factors to Nosocomial Infections

a. Wide variety of microbes in the hospital environment
b. Weakened or immunocompromised patients penhe
C. Chain of transmission (direct or indirect)
from health workers to patient
from patient to patient

vector-borne transmission
airborne transmission - inhalation of droplets ≤ 5 um (tuberculosis) or inhalation of

droplets > 5 um (pertussis)
use of fomites (catheters, needles, dressings, beds, and wheelchairs)
Types of Infections According to Settings

1. Acute Care
a. Surgical-Site Infection (SSI)

infection at a site where a surgical procedure was performed; usually risk is stratified by
length of surgery, site of infection, and degree of anticipated contamination
b. Central Line-Associated Bloodstream Infection (CLABSI)
a central
bloodstream infection related to the presence of an intravascular device such as
venous catheter
C.
Catheter-Associated Urinary Tract Infection (CAUTI)
infection of the urinary tract, frequently associated with a urinary catheter
PATHOGENESIS OF INFECTION 45
2.
Community-Acquired Infection (CAI)
a. Health Care-Associated Infection (HAI) boold adf otnl nob
b. Iatrogenic Infection
HAIs and iatrogenic infections occur because of instrumentation, increased use of
antimicrobial agents, breaks in aseptic techniques, and lack of hand hygiene.
Types of Infections According to Host Distribution

1. Local Infection
It
means signs and symptoms are confined in one area.
Some examples are infected wounds, boils, and abscesses.
2. Focal Infection
It starts as a
local infection before spreading to the other parts of the body.
Some examples are tooth infection, tonsillitis, appendicitis, and wound infections caused by
Clostridium tetani.
3. Systemic Infection (Generalized Infection)

It means the microbes spread throughout the body via blood or lymph (general invasion).
Kinds of Systemic Infections
a. Bacteremia
It is the presence of bacteria in the blood.

The organisms invade the bloodstream without active multiplication.
The highest concentration of bacteria in the blood occurs before the fever spikes.
Bacteremia as to Source/Origin
Primary Bacteremia
armo?
It occurs when bacteria are present in an endovascular source, such as an infected

cardiac valve or an infected intravenous catheter.
It happens when bacteria arise from an infected extravascular source, such as the
lungs in patients with pneumonia. -oiemolqoyes e
Bacteremia as to Duration
Transient Bacteremia
It bloodstream for a brief period following a dental

occurs when bacteria enter the
or medical procedure; and subsequently the organisms are immediately cleared
by the host immune system specifically by the reticuloendothelial system without

presenting any serious symptoms.
Intermittent Bacteremia
It is characterized by the presence ofinfections, such as abscess in any part of the

body, or certain clinical manifestation, such as meningococcemia, gonococcemia, or
microorganisms from the

pneumonia, resulting in periodic released of the Primary
site of infection into the blood. QAH) colbelhI beisos
Continuous Bacteremia
in the blood, originating from
It is the consistent presence of bacteria an
b. Septicemia
blood. oiloalnl to
It isthe active multiplication of the invading bacteria in the
Examples of Biomarkers for Sepsis: CRP, D-dimer, LPS-binding protein, procalcitonin
lactate, neutrophil gelatinase-associated lipocalin, copeptin, and endocan
C.
It is a condition wherein pus-producing organisms repeatedly invade the bloodstream

and become localized at different parts of the body.
d. Toxemia
It is the presence of toxins in the blood.

Bacteria are localized in one area, but they produce a toxin that spreads and gets
absorbed by the body cells.
Extent of Infection
1. Primary Infection
It is the initial infection that causes the illness.
Dool silt ni priedond to somseong ond at
An example is the common cold.
2. Secondary Infection d11b bootd balt of cristond to nottmutneomoo feonsin snl
It is caused by opportunistic pathogens after the primary infection has weakened the host's
immune system.
Some examples are pneumonia and bronchitis that may develop from common cold.
3.
It is clinically silent inside the body and causes no noticeable illnesses in the host. Then,
An example is the asymptomatic-type polio infection. 23no116c R a9001
4. Mixed Infection
It is caused by two or more organisms.

An example is wound infection.
5. Acute Infection
It is a type of infection that develops rapidly and usually with a short duration.
An example is whooping cough.
VOO JOISTOAB SITED PATHOGENESIS OF INFECTION 47
Chronic Infection
It is an infection which progresses slowly from weeks to a period of years. polbing2

An example is tuberculosis.
Routes of Infection
1. Direct Transmission
Congenital: Streptococcus agalactiae, Neisseria gonorthoeae, and Treponema pallidum subsp.

pallidum
Sexual contact:
N. gonorrhoeae, T. pallidum subsp. pallidum, and Chlamydia trachomatis
Infectious respiratory secretions or droplets: Streptococcus pyogenes and Neisseria meningitidis
Hand-to-hand transmission: Rhinovirus
2. Indirect Transmission
Fomites: Bed and wheelchair
Water: Salmonella, Shigella, and Vibrio

Require arthropod vectors: Borrelia, Francisella, and Yersinia
Disease
specific illness or disorder that is characterized by a recognizable

It is a set of signs and
symptoms which are attributable to heredity, infection, diet, or environment.

It results when the infection produces notable changes in the human physiology, specifically
those that cause damage to the body's organ system.
Predisposing factors: Gender, genetics, climate and weather, nutrition, fatigue/stress,

environment, life style, age and occupation
ont 10 sommroggs arls at

Classification of Infectious Diseases
1. Communicable or Contagious Disease
It spreads from one host to another, either directly or indirectly.
Examples: Tuberculosis, herpes, flu, and chickenpox
not 2. Non-communicable Disease
It does not spread from one host to another.
It is caused by external microbes (true pathogens) or by opportunistic pathogens living

inside the body.
Example: Tetanus and botulism (both caused by true pathogens)
Classification of Disease According to Occurrence

1. Sporadic Disease - occurs occasionally
2. Endemic Disease - is constantly present in a particular ___location or population
3. Epidemic Disease - it affects large number of people in a given population within a short SPan
of time
4. Pandemic Disease - it affects populations across large countries and continents globally
207600010313
Effects of Infectious Diseases
1. Signs
These are objective changes that can be measured.

Examples: Fever, redness, swelling, and paralysis
2. Symptoms
These are subjective indications of a disease.
Examples: Pain and malaise
3. Syndrome
It is a group of signs and symptoms that are associated with a disease.

Examples: Acquired immune deficiency syndrome (AIDS)
Phases of Infectious Diseases
1. Incubation Period
It is the time between the exposure to a pathogenic organism and the onset of symptoms.
2. Prodromal Period
It is the appearance of the signs and symptoms.
3. Clinical or Illness Period
It is the peak of characteristic signs and symptoms of an infection or a disease.
4. Decline Period
It is the period in which the signs and symptoms begin to subside as the host's condition
improves.
5. Convalescence or Period of Recovery
It is the period in which the surviving host is recuperating towards full recovery.
Strategies for Preventing Infectious Diseases

1. Preventing Transmission
Isolate sick persons.
Avoid direct contact with infected persons.

Wear personal protective equipment, like face mask and gloves.
2. Controlling Microbial Reservoirs

Sanitation and disinfection
Food preservation
Water and sewage treatment
Vector and pest control
3. Minimizing Risk
Vaccination
Use of antiseptics
Proper use of antimicrobials
Source: Tille, Patricia M. Bailey and Scott's Diagnostic Microbiology. 14th ed. 2017.
Classifications of Pathogenic Microorganisms

1. True Pathogens
These organisms are able to invade the tissues of healthy individuals through some inherent
ability-causing various diseases.
These are normally found outside the host.
Examples: Streptococcus pyogenes, Neisseria gonorrheae, Salmonella, Shigella, and Yersinia
2. Opportunistic Pathogens
These organisms normally do not cause diseases in their natural habitat.
They cause diseases if the host is immunocompromised, if they enter a different part of the
body outside their habitats, or enter sterile sites such as the brain and the blood vessels.
Examples: Neisseria meningitidis and Escherichia coli
Neisseriameningitidis is usually harmless in the respiratory tract but may still cause
meningitis to the host.

Escherichia coli can also cause urinary tract infection (UTI).
50 REVIEW HANDBOOK IN
Host-Microbe Relationship
1. Symbiosis - is the association of two organisms living in close proximity st gpuiszow
2. Mutualism - is symbiotic relationship in which both organisms benefit from each another
a relationship in which one organism benefits while
Commensalism
there is no beneficial om
3. is
harmful effect to the other organism

4. Parasitism - is a
relationship in which one organism (parasite) benefits at the expense of its hoss
Terminologies
1. Biofilm Production
It is a complex interaction among the host, indwelling device, and bacteria. It is

a key
component in bacterial pathogenesis. And
Examples of organisms capable of biofilm formation: S. aureus, K. pneumoniae, P.

coagulase-negative staphylococci (CoNS), and Candida albicans (fungi)
2. Pathogenicity
It pertains to the ability of pathogenic agent to produce a disease in susceptible individual
3. Pathogenicity Islands
These are genomic regions found in pathogenic microorganisms where virulence factors are
encoded.
Virulence
It is the ability of the microorganisms to cause diseases. ssalb thortsy antenco-uid
It is the degree of pathogenicity.
A very pathogenic microorganism or a rapidly progressive condition is considered when there is
high virulence factor.
Organisms that can establish infection with a relatively low infective dose are considered more
virulent than those that require high numbers for infection.Istiion anieinng
141115
Factors Influencing Microbial Virulence ilenena

1. Toxic factors
and cellulat
Toxins are substances produced by pathogenic microorganisms causing tissue
damage.
and streptococca
Examples: Diphtheria toxin, tetanospasmin, botulism toxin, enterotoxin,
erythrogenic toxin
2. Enzymatic factors
disease.
These are produced by bacteria that aid in the spread of infection and
lecithind%
Examples: Hyaluronidase, coagulase, collagenase, streptokinase, hemolysin, and
YOCJOIST0ARbITED PATHOGENESIS OF INFECTION 51
brr 3. Cellular structure
It provides an additional protection to the bacteria like the capsule which resists
phagocytosis. auglongoretron
Examples of encapsulated bacteria: Bacillus anthracis, Klebsiella pneumoniae, and Streptococcus
pneumoniae
Host Resistance Factors
1. Physical Barrier
The skin serves as the
physical and chemical barrier to microorganisms., ritaini flemns enl
The acellular, outermost layer of the skin and the tightly packed cellular layers underneath
provide an impenetrable physical barrier to all microorganisms unless they are damaged.
Examples: Stricture at the urethral opening, the flushing action of urination, and the thick
mucus plug in the cervical opening
lome tic antnels from
2. Cleansing Mechanism
The nasal hairs keep out airborne particles that may contain microorganisms.tg
The "cough-sneeze" reflex contributes to the removal of potentially infectious agents.
The cells lining the trachea contain cilia (hair-like cellular projections) that move the trapped
al pogidg mucus and microorganisms upward and away from the delicate cells of the lungs.
3. Indigenous Antimicrobial
Some examples of these substances are lysozymes and bile salts.
Lysozymes destroy bacterial cell walls while bile salts disrupt bacterial membranes.
Indigenous Microbiota (Microbial Flora) mnislnit ros etasibal vamedl emia omod
These microorganisms are commonly found on or in the body sites of healthy persons.
It is more accurate to refer to the population of microorganisms as "microbiota" instead of
is aa misnomer that originated from the time when bacteria
"microbial flora" since the latter is
tholaunodo were still part of the plant kingdom.
Types of Microbiota
a. Resident Microbiota - temporarily inhabit, multiply in, and colonize an area for months or
years
b. Transient Microbiota inhabit (but not multiply) and colonize an area until they are
do
eliminated by either the host's inherent immune defense or competition with the resident
microbiota
Different Body Sites and their Microbiota
a.
b. Mouth and Oral Cavity: Viridans streptococci

C. Upper Respiratory Tract: Viridans streptococci, diphtheroids, and Staphylococcus epidermidis
d. Nasopharynx: S, aureus, S. epidermidis, and N. meningitidis (asymptomatic carriers)
Bacteroides, Bifidobacterium, Clostridium,

e. Gastrointestinal Tract: E. coli, Eubacterium, and
anaerobic gram-positive cocci
6. Urethra: S. epidermidis, diptheroids, and alpha- and non-hemolytic streptococci
Notes to Remember
H. influenzae, S. pneumoniae, and N. meningitidis are found in the nasopharynx of healthy individuals,
and stomach but are present
Microorganisms usually do not reproduce in the esophagus
as transient
microbiota in contaminated food.

from the colon.
The small intestine contains few bacteria, which usually originate
microbials although
tubes are normally sterile or free of there are
The kidneys, bladder, and fallopian
some organisms that originate from the perineum particularly in the urethra of females,
5. Phagocytosis
It is the process by which certain cells called phagocytes engulf and dispose of
microorganisms and cell debris.

and destroy bacteria
Phagocytes (polymorphonuclear leukocytes and macrophages) ingest
and other foreign particles through process known as endocytosis.
Factors involved in Phagocytosis: Chemotaxis, attachment of the particle to the phagocyte,
ingestion, and killing of the invading organisms
6. Inflammation
This condition serves as a reinforcement mechanism against microbial survival and

proliferation in tissues and organs.
Some signs that may indicate an inflammation are swelling, redness, burning sensation,
and
pain in the affected area.

The components of inflammation are phagocytes, complement system, coagulation system
and cytokines.is
Infections caused by anaerobes exhibit purulent appearance with many polymorphonucleal
leukocytes.
7. Immune response
It provides the human host with the ability to create a specific protective
response against
microorganisms.
an immune
The immune system "recalls" all of the encountered microorganisms so that
mediated defensive response is immediately available in the second or
third time that they
they come across.
within 30 minutes
The normal immune system removes the bacteria from the blood
45 minutes.
The host's immune response may be reduced or altered due to immunosuppressive

chemotherapy, or radiation.
Dad brreo: PATHOGENESIS OF INFECTION 53
Types of Specific Immunity

a. Humoral Immunity
It is also known as
the antibody-mediated immunity.
It is based on
the action of soluble proteins called antibodies that occur in the body fluids
and on the plasma membrane of B-lymphocytes
b.
Cellular Immunity
It is also known as the cell-mediated immunity.
It is based on the
of a specific T-lymphocytes that directly attack the host cells that
action
are infected with viruses, parasites, and cancer cells. The T-lymphocytes also target the
transplanted cells.
Terminologies sogel/etontanlo solvitos for senogser offloage-mon s el dl
1. Active Immunization
It is the protection of susceptible humans and domestic animals from communicable diseases
through the administration of vaccines (vaccination).
For this type of immunization, vaccines may be prepared from living, weakened (attenuated),
or killed microorganisms, inactivated bacterial toxins (toxoids), purified macromolecules,
3090208
recombinant vectors (modified polio vaccine), or DNA vaccines.
2. Acquired Active Immunity
It is the specific response of the host to an invading organism.
3. Anamnestic Response
It is the ability of the B-lymphocytes to recall pathogens during the primary encounter leading
to a higher antibody response on the second encounter. bodnns berrustantons Sat lisms
4. Antigen
It is high-molecular weight substance that illicit antibody formation.
It is made up of carbohydrates (polysaccharides) or proteins.
are said to be immunogenic,
Antigens that are recognized as foreign substances by a host
On capable of eliciting an immune response.

5. Antigenic Drift
It is a minor antigenic change as a result of a mutation in the organism strains.
It facilitates the pathogen in avoiding host-immune responses.
6. Antigenic Shift
It is major genetically determined change in the antigenic property of an organism in which
it becomes unrecognizable by the host's immune system. bns Meildate cf robro nt
7. Antibody
It is a protein secreted by the plasma cells against an invading antigen or a pathogen.
Major regions: Fab region (antigen-binding site) and Fc region (phagocyte- and complement-
binding site)
Examples of antibodies: IgG, IgA, IgM, IgD, and IgE
8. Complement-fixing Antibodies
These are antibodies that are attached to the
surface of pathogens and kill the bacteria by lysis.
9. Cytokines and other cells.
as the macrophages
These are proteins produced by phagocytes, such bodies such as an
It helps in the stimulation of

the immune system to respond against foreign
invading antigen (pathogen).
10. Heterophile Antibody
It is a type of an antibody that is secreted in
response to a molecule which also reacts against
an antigen from an unknown or unrelated source.
11. Natural (Innate) Immunity
process by which phagocytes are
It is a non-specific response that activates chemotaxis, or the
directed to the site of replication and engulf the invading organism.
12. Neutralizing Antibody
This type of antibody is atfached to the surface of microorganisms and block surface receptors.
13. Opsonizing Antibody
It is a type of antibody that is attached to the surface
of microorganisms and render pathogens
susceptible to phagocytosis.
14. Passive Immunization
individuals without fully
transient-type of immunization that is administered
to
It is a
activating the person's immune system for the mere purpose of producing the corresponding
antibodies to diseases.
Protection does not require the participation of the recipient's immune system and only lasts
until the transferred antibodies remain in the recipient's body.
Infectious Agent
1. Adherence
In order for a microorganism to cause diseases, it must penetrate the mucous layer and attach
to the epithelium.
The host cells must produce the necessary receptors for adhesion.
The main adhesins in the bacteria are the common
pili (fimbriae) and surface
polysaccharides.
2. Proliferation
order to establish and cause a disease, a pathogen must multiply, following its attachment
In
to host cells.
Secretory antibody, lactoferrin, and lysozyme inhibit proliferation, and these are produced
by the host.
of phagocytosis is essential for most pathogens to survive and reproduce.

The evasion
3. Tissue damage
It is a result of either a
preformed toxin (Clostridium botulinum, Bacillus cereus, and
Staphylococcus aureus) or the disruption of the normal functioning of the intestinal cells
(Escherichia coli, Vibrio cholerae, Shigella, and Salmonella).
disease or
A an
infection is noticeable only if tissue damage occurs.
4. Production of Toxins
a. Exotoxins
They are mostly present in Gram-positive bacteria.

They are known to be one of the most lethal substances.
These toxins do not require bacterial death to be released into the circulation.
They do not produce fever to the host.

These are either secreted or creted by living microorganisms.
Some examples are cytotoxins, neurotoxins, and enterotoxins.
Some bacteria that produce this kind of toxins are Clostridium botulinum, Corynebacterium
diphtheriae, Staphylococcus aureus, and Streptococcus pyogenes.
b. Endotoxins
They are commonly found in Gram-negative bacteria.

They are composed of the lipopolysaccharide portion of the cell wall.
They are released when the bacteria die and their cell walls undergo lysis, which
consequently liberates the endotoxin.
This kind of toxin stimulates the fever center in the hypothalamus. roe diw
The toxicity is due to the lipid A portion of the lipopolysaccharide (LPS).
The LPS may elicit fever, chills, hypotension, granulocytosis, thrombocytopenia, and
disseminated intravascular coagulation.
Endotoxic shock is result of the Gram-negative septicemia.5270. mb
The effects of this kind of toxin include changes in the following: blood pressure,
clotting, body temperature, blood cell circulation, metabolism, humoral and cellular
immunity (activation of both the classic and alternate complement pathways), and
resistance to infection.
In the development of fever, five (5) substances are essential:

a. Endotoxin
903 t0 6 b. Peptidoglycan
C. Cytokines
d. Interleukin-1 and tissue necrosis factor
e. Acute phase reactants
and Endotoxin
TABLE 7-1. Differential Characteristics of Exotoxin Endotoxin
Exotoxin
Gram-negative bacteria
mostly Gram-positive
bacteria
Parent organism
LPS - lipid A
Chemical nature simple protein
part of outer membrane
Location outside the living cell
heat-stable at
heat-labile
Heat stability
inactivated at 60 °C
low
Toxicity high
typhoid fever, UTI, and
Representative diseases/ gas gangrene, tetanus, botulism, meningococcal meningitis
infections diphtheria, and scarlet fever
produce fever by releasing
Fever usually does not produce fever interleukin-1
carried by extrachromosomal synthesized directly by

Genetics
genes such as plasmids
chromosomal genes
highly antigenic weakly antigenic
Immune response
Effects on host destroys a particular part of the disruption of clotting (DIC), fever,
host's cell hypotension, shock, and death
Chemical Treatment by susceptible/detoxified resistant/not detoxified

Formaldehyde
5. Invasion
It is the process of penetrating and growing in tissues.
With some organisms, the invasion involves only a few layers of cells while others involve
deep-tissue invasion.
brun 6. Dissemination
It is the spread of microorganisms to distant body sites. roum bote minusaenu
Certain organisms that survive phagocytosis may be disseminated rapidly to several body
Routes of Transmission
1. Airborne Transmission
Respiratory spread of infectious diseases is common.

Infections of the lower respiratory tract are less common but more serious than those of the
upper respiratory tract.
Secretions are aerosolized by
coughing, sneezing, and talking.
Infectious diseases,such astuberculosis, brucellosis, tularemia, legionellosis, and plague,
may be acquired through inhalation of infectious particles in liquid droplet.
Droplet nuclei are the small
airborne for long periods.
residues from larger droplets and arelight enough toremain
Some infectious agents
have become airborne.
may be transmitted by dust particles that
00101/A8 ord PATHOGENESIS OF INFECTION 57
Other examples of illnesses that can be transmitted in the air are streptococcal sore throat,
sinusitis, acute epiglottis, and diphtheria.
2. Transmission by Food and Water

Infections occur via the fecal-oral route.
Gastric enzymes and juices in the stomach prevent the survival of most organisms.
Examples of pathogens related to waterborne infections are Salmonella, Campylobacter, Yersinia
enterocolitica, Pseudomonas, E. coli O157:H7, Legionella, and Aeromomas. n B nea80
3. Close Contact
It refers to the passage of organism through salivary (herpes simplex), skin (warts, syphilis),
and genital contact (gonorrhea, syphilis, hepatitis).
4. Cuts and Bites

anno tnovs won seoran to sedman edd al t1
Cuts and bites cause spread of infection and may progress to debilitating diseases.
Pathogenic staphylococci gain entry through skin cuts and abrasions causing life-threatening
sickness.
5. Arthropods
The infectious agents multiply within the arthropod which transmits the microorganisms
while feeding off of a human host.
Diseases like plague and tularemia are passed by arthropod vectors to susceptible host.
Examples of bacteria transmitted by vectors: Yersinia pestis, Francisella tularensis, Borrelia,
Orientia, and Rickettsia
6. Zoonoses plmabnel golmoall to boodtladld
These are animal-related diseases that depend on the contact with animals or animal
by-product for transmission.

Diseases may be passed through contact with animal secretions (animal bites or scratches,
Pasteurella and Bartonella henselae) and animal carcasses and by-products (aerosolized,
brucellosis and Q fever) or by ingestion of unpasteurized animal products like milk and
dairy (brucellosis).
Carrier
It is a person or animal that harbors and spreads a microorganism that causes a disease or
infection but does not become ill.
Types of Carriers
a. Casual/acute/transient carrier - harbors the microorganism temporarily for a few days or weeks
b. Chronic carrier remains infected for a relatively long time, sometimes throughout its entire life
C. Convalescent carrier - is an individual who has recovered from an infection or a disease but
continue to harbor a large number of the pathogen
d. Active carrier - is an individual who has an overt clinical case of the disease and is capable to
transfer it with or without showing any signs and symptoms of the sickness
and effects of disease and injury.

It is the study of occurrence, distribution, causes,
Factors Contributing to Epidemiology
It is the pattern of susceptibility and resistance of certain bacteria to antimicrobial agents

essential in the monitoring and progress of therapy.
2. Infection Rate
It is the frequency of an infectious disease within a specified population.
3. Incidence rate
It is the number of times a new event occurs in a given period.
4. Likelihood of Becoming Endemic

The organisms or diseases are indigenous to and/or constantly present in a geographic area
or population.
When an organism or disease is constantly present in a population
5. Likelihood of Becoming Epidemic

A disease is considered as epidemic if it affects a significantly large number of people in a
short period of time in a geographic area.
The influenza is a classic cause of an epidemic.
Likelihood of Becoming Pandemic

A disease is considered pandemic if it
affects huge populations across large regions like
several countries or a continent.
bas
7. Morbidity Rate
It is the number of cases of disease in a specified population during a defined time interval.
It is a measure of the infectiousness
of an organism.
8. Mortality Rate
10 6889
It is the number of deaths due to
a disease in a population. lemina To noaren
9. Outbreak
It is the sudden increase in

the number of rare microorganisms or the occurrenceof
disease.
10. Reservoir
It
is the source of
environment.
an
infection, which may be a person, animal, or any object from the
VOO JOIRETDAMI DIT2D PATHOGENESIS OF INFECTION 59
11. Prevalence Rate

It refers to the number of
persons or cases in a population infected with particular disease.
12. Surveillance
It is a systematic collection of the data and the analysis and interpretation of the details
surrounding a disease or event.
Molecular Epidemiology
It is the study of the occurrence of genetically-related infections and diseases.
It utilizes molecular science to identify, locate, and characterize the etiology or microorganisms,
likewise to
generate specific markers to determine the outcome of the situation in any given
community.
Objectives of an Outbreak Investigation

1. To identify the causal agent
2. To determine the mode of transmission
3. To establish contact tracing specifically on the carriers

4. To account for the population at risk
5. To establish the risk factors
Source: https://www.who.int/hac/techguidance/training/outbreak
Epidemiologic Steps of an Outbreak Investigation

o don ob vismion ameinsggo dalrIW
1. Prepare for field work.
2. Establish the existence of an outbreak.
3. Verify the diagnosis.

4. Construct a working case definition.
5. Find cases systematically and record information.
6. Perform descriptive epidemiology.
7. Develop hypotheses.
8. Evaluate hypotheses epidemiologically.
9. As necessary, reconsider, refine, and re-evaluate hypotheses.
10. Compare and reconcile with laboratory and/or environmental studies.
11. Implement control and prevention measures.
12. Initiate or maintain surveillance.
13. Communicate findings.
Source: https://www.cdc.gov
ASSESSMENT QUIZ
answer.
the correct
Encircle the letter that corresponds to
the presence of a certain clinical manifestation, source
such meningococco
as
characterized by
1. It is
microorganisms from the of infection;into
or pneumonia resulting in the release of the the
bloodstream.
a. continuous bacteremia
b. intermittent bacteremia
c. transient bacteremia
10 Smootno adi eninslab of alsatsm

disease.
2. It is the sudden increase in the occurrence of the
outbreak
a. tunes a nov event
b. surveillance rate
C. pandemic Becoming Endemi
d. epidemic
3. These are substances that illicit the development of fever. 998

a. peptidoglycan and cytokines
b. capsule and exotoxin
C. somatic pili and coagulase
d. acute phase reactants only
4. Which organisms normally do not cause disease in their natural habitat unless the host is
immunocompromised?
a. resident microbiota
b. virulent pathogens
c. transient microbiota
d. opportunistic pathogens
5. It is the ability of B-lymphocytes to recall pathogens during primary infection, which could resulf
in a higher antibody response against preceding infections from the same pathogens.
a. anamnestic response
b. antigenic drift
C. antigenic shift
d. innate immunity
6. It is : type of infection that is clinically silent and without observable illness in the host.
a. chronic infection
b. primary infection
c. acute infection
d. latent infection
7. These antibodies are attached to the surface of the microorganisms and render the invading
pathogens susceptible to phagocytosis.
a. neutralizing antibodies
b. heterophile antibodies
C. opsonizing antibodies
d. natural antibodies
8. This occurs when one organism gains benefit from a relationship but not causing any harm to
other organisms.
a. parasitism
b. mutualism
c. commensalism
d. symbiotic
9. This type of immunity is based on the response of the antibodies that occur on the membrane of
the B-lymphocytes.
a. passive
b. cellular
c. acquired
d. humoral
10. It is the onset of signs and symptoms of disease.
a. incubation period
b. prodromal
C. convalescence
d. clinical phase
CHAPTER 8
BIOSAFETY AND
QUALITY CONTROL IN A
LABORATORY

1. discuss the principle of biosafety in a clinical microbiology laboratory; thich ahrie
2. differentiate the types of biological safety cabinets;
3. compare the biological safety level agents with the potential agents of
bioterrorism;
4. explain the classification of infective microorganisms by risk group;
5. distinguish the hierarchy of control and relate with the work in an
70 10 academic microbiology laboratory;

1 6. describe the parameters to ensure the reliability of procedures and-dlr
laboratory test results;
A 7. design an algorithm of biosafety protocols; and
8. apply the concept of infection control in performing laboratory
activities.
Clinical laboratory specimens are potential hazards since they may contain
infectious agents, such as bacteria, viruses, and fungi. Universal precautions
recommended by the Centers for Disease Control and Prevention (CDC)
include the use ofpersonal protective equipment (PPE), such as but not limited itama
to laboratory gowns, face mask/respirator, disposable gloves, goggles, and
face shields. This measure should be observed and strictly implemented to

avoid acquisition of infectious agents.
Blood and body fluid precautions recommended by the CDC should be
applied to all clinical specimens like blood, tissue, secretions, body fluids with
visible blood, cerebrospinal fluid, etc.
Work practice controls should be strictly implemented in the clinical and

molecular laboratories, such as the basics of surface/benchtop disinfection,
proper disposal of sharps, and absolute restrictions on eating, drinking, and
mouth pipetting.
provisions
All specimens should be treated as infectious.
Compliance to engineering controls, like
are requisites to
and closed tube sampling technology,
for biological safety cabinets, eyewash stations, available to all
eradicate potential hazards in the workplace. Therefore, safety guidelines must be readily
laboratory personnel and must be periodically revised when the necessity arises.
Principle of Biosafety
The fundamental objective of any biosafety program is the containment of potentially
hazardous
biological agents and toxins.

Source: CDC Biosafety in Microbiology and Biomedical Laboratories, 6th ed.,
2020
Biological Safety Cabinet (BSC)

It is a device or a primary containment that encloses a working area to protect workers from
aerosol exposure and or infectious disease agents.

It is also known as a ventilated cabinet.
It is an engineering control that is utilized in the clinical microbiology laboratory and other areas
that are handling biological samples.
It is considered as the standard device used in the academic and clinical laboratory to contain
hazardous biological agents and its products.
This is used when splashes and aerosol may occur while processing the samples.
In BSC, the air that contains the infectious material is sterilized,
either by heat, UV light, or by
passage through a high-efficiency particulate (HEPA) filter that removes particles larger than
0.3 um.
The air going in and exhausted by the BSC undergoes sterilization mostly by using the HEPA
filter.
Types of Biological Safety Cabinets

1. Class I Cabinet
It is an open-fronted type of cabinet that uses

an exhaust fan to allow movement of air going
through the open front.
It allows room (unsterilized) air into the cabinet, to
circulate around the workspace, and
expose
the material within, hence, samples are exposed to contamina(in
In this type of BSC, only air to be exhausted
is sterilized using HEPA filter.
It protects the laboratory worker and environment but
no provision for samples.
It is mostly used for BSL 1 agents though it
can also process BSL 2 organisms.
2. Class II Cabinet
an open-fronted cabinet
It is
same with the BSC class
worker, environment, and the samples. but provides protection for the
BIOSAFETY AND QUALITY CONTROL IN A CLINICAL MICROBIOLOGY LABORATORY 65
It sterilizes the air using HEPA filter that flows over the infectious material and air to be
exhausted.
It is mostly utilized for BSL and 2 agents, with provisions also for BSL 3 organisms.
In this type of BSC, contaminated air is
maintained by a negative pressure.
Air
is exhausted either to room/thimble connection (class II A) or outside the facility through
a hard duct (class II B).
Processing of samples from a possible bioterrorism event should occur within at least a class
a. Class II, Type A1
It is only utilized for biological samples.

Airflow: 70% recirculated and 30% exhausted
b. Class II, Type A2
It is the most commonly used type of BSC in the clinical and microbiology laboratory.
mal
It is used for biological and clinical (infectious) samples.
It is utilized for specimens treated with minimal concentration of volatile chemicals.
C. Class II, Type B1
It is used for biological samples and minimal concentration of volatile or toxic chemicals.
d. Class II, Type B2
It is appropriate for processing chemicals, radioisotopes, and carcinogens aside from

biological samples treated with toxic or hazardous chemicals.
The exhaust air is discharged completely outside of the facility or the laboratory (exhaust
to the atmosphere).
3. Class III Cabinet
It is a close-fronted BSC with an airtight system which completely separates the worker from
the sample.
It is also known as the glove-box cabinet.
It provides the highest level of safety to the laboratory worker.
The interior of the cabinet is under a negative pressure.
In this type of BSC, the infectious sample/material is handled with rubber gloves that are
attached and sealed onto the cabinet.
The air coming into and going out of the cabinet is sterilized using HEPA filter.
It is preferred for BSL 4 agents.
DIAGNOSTIC BACTERIOLOGY D YILIAUO OMA YT3ARDE
Classification of Biological Agents

1. Biosafety Level 1 Agents
These are agents that have no known potential for infecting healthy people and
immunocompetent adult humans.

are used for laboratory activities of students academic purposes/educations
(for
They
they pose
minimal threat to laboratory personnel and the environment.
training) since
exercises, the workplace
In utilizing these microbial agents for routine instructional
should install a provision for a biohazard sign entrance of
at the main the laboratory and
of PPE and adherence to work practice protocols.
infographics on proper use
Examples: Bacillus subtilis, Escherichia coli, and other non-infectious microorganisms

These are agents acquired by ingestion or exposure to percutaneous or mucous membrane
They include all the common agents of infectious diseases and pose a moderate potential
hazard for the employees and the environment.
are considered indigenous and cause

The microorganisms severe diseases.
In handling these agents, access to the laboratory is limited while it requires the personnel to
use the recommended laboratory suit before going to their specific stations.
The personnel handling these agents should also receive immunization.
Human Immunodeficiency Virus (HIV) is either a BSL 2 or BSL 3 agent.
Examples: Bacillus anthracis, Yersinia pestis, Salmonella, Shigella, Human Immunodeficiency

41021 abigh Virus (HIV), and Hepatitis B Virus (HBV) g0ie2s201g not sistgofags er
laun!3.
These are potential agents for aerosol transmission.

The microorganisms are either indigenous or exotic and transmitted through respiratory
route.
In processing these lethal pathogens, the air movement in the laboratory must be controlled
JI0TA 19810M to contain the infectious materials, that is, from the clean area to the contaminated area
(negative air pressure).

Y. pestis can also be classified as BSL-3 agent since it can be transmitted through respiratory
route.
Examples: Mycobacterium tuberculosis, Francisella tularensis, Brucella spp., Coxiella burnett,

St. Louis encephalitis virus, and systemic fungi

These are agents that cause life-threatening infections and diseases, classified as dangerole
and exotic.
have an
Aerosol transmission with pressure is very high with these microorganisms, or may
unknown risk of transmission.
BIOSAFETY AND QUALITY CONTROL IN A CLINICAL MICROBIOLOCY LABORATORY 67
In handling these organisms, maximum containment and decontamination of all personnel

and materials before leaving the area are strictly observed.
Class III BSC is used when handling these biological agents. aomor hsblmened
No exact treatment yet
or vaccine is available. pritring basin
The laboratory or facility handling these organisms has strict access control, and it should
either be separated from the main building or situated in an isolated area within the
building.
Examples: arbovirus, arenavirus, filovirus (Ebola and Marburg), smallpox virus
High Risk Microbes
91621
gov gnilodsl ineghne gnigedosd
Low Risk Microbes sanh
FIGURE 8.1. Hierarchy of Biosafety Levels

Source: https://www.cdc.gov/training/quicklearns/biosafety/
Classification of Infective Microorganisms by Risk Group inAhnehfbomed
1. Risk Group 1: Without/Low Individual and Community Risk 3slaths tns penilt nis
a microorganism that is unlikely to cause human or animal disease
2. Risk Group 2: Moderate Individual Risk, Low Community Risk ignsxo

a pathogen that can cause human or animal disease but is unlikely to be a serious hazard to
laboratory workers, the community, livestock, or the environment stsmxt
Laboratory exposures may cause serious infection, but effective treatment and preventive
measures are available, and the risk of spread of infection is limited.
3. Risk Group 3: High Individual Risk, Low Community Risk

a pathogen that usually causes serious human or animal disease but does not ordinarily
spread from one infected individual to another
Effective treatment and preventive measures are available.

REVIEW HANDBOOK IN
DIAGNOSTIC BACTERIOLOGY D YMAUCIONA YT!A20IE
68
4.
a pathogen that usually causes serious

human or disease and that can be
animal
readil,
transmitted from one individual to another, directly or indirectly
Effective treatment and preventive measures are not usually available.

Source: WHO Laboratory Biosafety Manual, 3rd ed., Geneva, Switzerland, 2004. MILLEDE 10
Molmodal edT
Classification of Infectious Substances as to Transport of Samples

1. Category A Infectious Substances
any material() known or reasonably expected to contain, biological agents capable

These are
of causing permanent disability, life- threatening or fatal disease

or
otherwise healthy
in
humans or animals.
These substances pose the greatest biosafety and biosecurity risks; and
as a result, they
are subject to the most stringent risk control procedures, such as controlled triple-layer
packaging, stringent labeling regulations, and thorough documentation.
Only those personnel with appropriate trainings and certifications are permitted to handle
this type of samples.
2. Category B infectious substances

These are defined as
any material() containing biological agents capable of causing infection
in humans or animals, but which do not meet the criteria for inclusion in Category A.
These substances are also subject to strict regulation
with a triple packaging system.
These
are generally with lower risk and severity compared to Category A infectious
substances.
3.
Exempt Human/Animal Specimens
These are substances or
materials derived from human or animal patients (that are clinical
specimens)
with a minimal likelihood that infectious biological agents present. are
Triple packaging system using redundant layers of packaging is still required.

These are exempted from stringent criteria applied to Category A and
Category B infectious
substances on labelling and documentation.
Source: WHO Laboratory Biosafety Manual, 4th ed., 2020
Categories of Potential Agents of Bioterrorism

1.
Category A agents
These are
agents that
pose the greatest public health threat.
They are
easily transmitted and highly infectious.
These
agents result
high mortality rates and might
in social
disruption. cause public panic and
Examples: Bacillusanthracis,Clostridium botulinum, Francisella

major (small pox) and tularensis, Yersinia pestis,
Ebola, Marburg, Lassa and Machupo viruses
2. Category B agents
These are
agents with moderate morbidity and low mortality. ban stnsos amnipolni,
They are not as
easily transmitted compared to category A agents. ong
These agents require surveillance and diagnostic assistance from the CDC facility.
Examples: Staphylococcus aureus (Enterotoxin B), Clostridium perfringens, Escherichia coli
0157:H7, Salmonella, Shigella, Vibrio cholerae, Burkholderia mallei, Burkholderia pseudomallei,
Brucella,
Chlamydia psittaci, Coxiella burnetti, Rickettsia prowazeki, and Cryptosporidium parvum
3. Category C agents
These are the
emerging pathogens. alb nonw bos boaroelb oldiaelmaned vldaid
These are agents easily produce and disseminate.
They have potential for high
morbidity and mortality rates. d bovomos ad taum 199
Examples: Hanta virus and Nipah virus
Laboratory Acquired-Infection (LAI)

LAIs are mostly transmitted through parenteral inoculations like needlesticks or contaminated
sharps, spills and splashes, ingestion, and inhalation.
The infectious agents that pose the greatest risk are those that are transmitted by aerosols.
The laboratory procedures that create aerosol are pipetting, flaming loops, agar plates streaking,
and centrifugation. 00 1998 vidietv ed bluorie lodnaye brexsrok ond
Microorganisms, such as M. tuberculosis, can be aerosolized while Brucella and F. tularensis can be
acquired through inhalation of an aerosol.

HIV and hepatitis viruses (HBV and HCV) are bloodborne pathogens that may be transmitted
from contaminated specimens through a needlestick injury or percutaneous route. d vs.m
The five most frequently acquired laboratory infections iare shigellosis, salmonellosis,
tuberculosis, brucellosis, and viral hepatitis.
Statistics shows that males and younger employees are involved in more LAIs than females and
older employees.
Laboratory personnel are also advised to orient their family
members on the potential
occupational risk.
Handwashing is the cornerstone for preventing the spread of infections and diseases including
Personal Protective Equipment
Complete PPE should be provided by the health care facility administrator or employer, and it
must be worn when handling potentially infectious samples. I ernon Ibnomon flori ovesl
Proper use and disposal of PPE (laboratory gowns/coats, PPE suit, face mask, respirators, safety
eye glasses/goggles, ear plugs, face shields, gloves, closed-toe laboratory footwear, and shoe
covers) is an integral component of the biosafety practices.
In the absence of a BSC or in case it is

unvallable, PPE serves as the primary protection against
infectious agents and hazards. material.
penetrate the PPE
For PPE to be protective, blood and body fluids should not
cover all potentially exposed skin, hence long
Laboratory gowns and coats should be able to
recommended.
sleeves with provisions for closed wrists are
of the personal clothing.
Laboratory coats/gown are prevent contamination
used to
laboratories as they can help
Disposable laboratory coats are
ideal for clinical microbiology
prevent the chain of infection.
tuberculosis or any
HEPA respirators are required when attending to patients confirmed with
highly transmissible diseases and when cleaning biological spills.
Fit-test is important before using N95 mask HEPA respirators.
or
area and must be disposed of in PPE-designated

PPE must be removed before leaving the work
waste bin.
Proper handwashing is a must before and after using PPE.
Infection Control
All laboratory-related accidents must be reported immediately to the head of the laboratory and
safety officer.
Benchtops and work surfaces should be decontaminated.
The biohazard symbol should be visibly seen on all equipment and instruments that process and
50 mm contain infectious and potentially infectious samples and materials.
Contact precautions designed to prevent the spread of infectious agents such as multidrug-
are
bettime resistant organisms like vancomycin-resistant enterococci, MRSA, and Clostridium difficile that
may be transmitted through direct or indirect contact with the person or the environment.
Droplet precautions aim to stop the transmission of microorganisms like the Neisseria
meningitidis, Bordetella pertussis, and even influenza virus, by close respiratory contact while
airborne precautions include control of M. tuberculosis, varicella virus, and rubeola virus.
For HIV testing, serum samples should be examined at an
interval of six weeks, three months,
and six months.
Strict handwashing must be implemented in the clinical

laboratory hands and forearms are the
most exposed
body parts to blood-containing fluids and biological fluids.
Hand washing and maintaining physical distance, aside from wearing face mask, are
the
recommended measures by
COVID-19.
the World Health Organization (WHO) to avoid contracting
A regular
orientation on biosafety and biorisk assessment should
worker with emphasis on basic protocol, such as the be provided to the laboratory
creation of a work station where staff can
leave their personal items like
pens, writing pads, journals, and even mobile gadgets since these
are potential sources of infections, thus they should not be taken hormd
AND QUALITY CONTROL IN A CLINICAL MICROBIOLOGY LABORATORY 71
Clinical Laboratory Air-Handling System

The ideal clinical microbiology laboratory should be under negative pressure; the air should not
be recirculated
after passing through the laboratory.
The air-handling system should
move air from lower-risk to higher-risk areas and never the
reverse.
Maximum precaution in handling specimens with Mycobacterium tuberculosis should be observed

to prevent accidental spills that can aerobically spread or lead to aerosol infection.
Hazardous Wastes
These are substances, which singly or in combination, have a significant threat or hazard to
human health or
to the environment and require special handling, processing, or disposal.
They could be flammable, explosive, reactive, corrosive, toxic, infectious, carcinogenic,
bioconcentrative-persistent in nature, potentially lethal, an irritant, an oxidizer, or a strong
sensitizer.
Disposal of Hazardous Wastes

All materials contaminated with potentially infectious agents should be decontaminated before
disposal using an autoclave, incinerator, or alternative waste treatment methods.
Pipettes, swabs, and other glass objects should be placed inside a firm cardboard container before
disposal.
Sharp objects (needles and scalpels) are placed in containers that are autoclaved or incinerated.
TABLE 8-1. Categories of Segregated Laboratory Waste Material and Recommended Treatment
Laboratory Waste Recommended Treatment
Can be reused or recycled or disposed of as general

Uncontaminated (Non-infectious) Material
municipal waste
Contaminated sharps (hypodermic needles, Must be collected in puncture-proof containers fitted with
scalpels, knives and broken glass) covers and treated as infectious
Must be first decontaminated (chemically or physically)

Contaminated material for reuse or recycling and then washed; thereafter it can be treated as
uncontaminated (non- infectious) material
Must be decontaminated onsite or stored safely before
Contaminated material for disposal transportation to another site for decontamination and
disposal
Must be incinerated onsite or stored safely before
Contaminated material for incineration transportation to another site for incineration
Liquid waste (including potentially Should be decontaminated before disposal in the sanitary
contaminated liquids) for disposal in the sewer
sanitary sewer system

DIAGNOSTIC BACTERIOLOGY TLIALD OA YITABD
Standard Microbiological Practices

Not eating, drinking, or applying cosmetics in the laboratory
Washing hands after working with infectious materials and before leaving the laboratory
Routinely decontaminate work surfaces
Source: (https://www.cdc.gov/training/quicklearns/biosafety/)
Most
effective
Physically remove
Elimination
the hazard
Substitution Replace the hazard
Isolate people
Engineering
Controls from the hazard
Administrative Change the way

Controls people work strrop elstranom l16
Protect the worker with

Personal Protective Equipment
Least
effective
FIGURE 8-2. Hierarchy of Controls

Source: https://www.cdc.gov/niosh/topics/hierachy/default.html
Biorisk Assessment
It is a process
where laboratory practices, control measures, biosafety standards, and the
hazardous characteristics of infectious agents are reviewed to
prevent laboratory-acquired
infections (LAIs) or exposure to high-risk pathogens.
It determines the potential risk to individuals, like
the possible outcome or consequences.
those who are working in a laboratory, and
It also provides measures to mitigate the risk.

It is conducted by the respective
checklist for systematic assessment.
institution periodically using standardized or reference-based
Steps in Performing Risk Assessment

Gather information: Identify the
1.
hazards associated with an infectious agent or material.
2. Evaluate the risk: Identify the risk or activities that
material. might cause exposures to the agent or
Develop a risk control strategy:

Consider the competencies and experience oflaboratory staff
3.
and the resources

needed includingits availability.
4.
Select and implement risk control measures: Review the national and institutional regulations
and evaluate the process
of dissemination of information. burh
5.
Review risks and risk control measures: Develop, implement, and review control measures to
reduce the risk for exposure.
Source: WHO Laboratory Biosafety Manual, 4th ed., 2020 and CDC Guidelines for Safe Work Practices in Human and Animal
Medical Diagnostic Laboratories
Notes to Remember
All
clinical laboratories must adhere to at least biosafety level 2 guidelines.
Processing and handling of specimens and cultures are the major sources of the biological hazards in
a clinical microbiology laboratory.
Samples for the isolation and detection of viral

pathogens, such as the severe acute respiratory
syndrome corona viruses and emerging and re-emerging pathogens of public health concern, should
be handled with extreme precaution and only by personnel with appropriate trainings for processing
highly infectious agents.
Laminar flow hood (LFH) is not an example of a biological safety cabinet since it does not provide any
form of protection the laboratory staff rather it ensures protection to the sample by maintaining the
to
environment free of contamination.
yon ore fori siboM
When using LFH, the potentially contaminated air arising from the environment and sampling may
be acquired by the laboratorian, hence, it should only be used when processing non-infectious and
non-toxic specimens and in media and reagent preparation.
The administrators of the clinical microbiology unit and all healthcare facilities should be regularly
informed of the standard protocols on patient and laboratory safety issued and released by the World
Health Organization, Centers for Disease Control and Prevention, as well as the advisories from the
local and regional health offices.
The classification of the infective microorganisms by risk group issued by the WHO is related to the
biosafety level (BSL) agents.
Quality Control in a Microbiology Laboratory
Instruments and Reagents Requiring Quality Control Monitoring T

1. Thermometer Calibration
Thermometers should be checked periodically against a reference thermometer from the

National Institute of Standards and Technology (NIST).
Thermometers that differ by more than 1°C from the reference thermometers should be
disposed of.
Thermometers should be checked daily for the presence of gas bubbles to ensure accuracy of
reading.
immersed in glycerol since it

easier to read if they are
permanently
Thermometers are
the door is opened. 920717
prevents temperature fluctuations when
Reference calibration point: 0°C, 37°C, and 56°C f
Other temperature used for calibration: -20°C and 2°C to 8°C
Temperature factor for 0°C, 37°C, and 56°C: +0.1
2. Carbon Dioxide Incubator
The percentage of carbon dioxide must be checked daily.
3. Centrifuge
be checked twice a year using a tachometer.
The speed or revolution per minute (rpm) must
4. Culture Media
All media should be checked based on their performance and sterility, and records should be
kept for at least two years.
the Clinical and
quality control (QC) of culture media are established by
The criteria for
Laboratory Standards Institute (CLSI).

The organisms that are selected for the QC of culture media should represent the most
fastidious organisms for which the medium was designed.
Media that are not quality-controlled by the laboratory must still undergo observation for
5. Reagents
Reagents should be tested daily with both positive and negative controls. osmeinimbsodl
Reagents that require quality control monitoring: Catalase, coagulase, gelatin, hippurate,
nitrate, oxidase, Kovac's, PYR, B-Lactamase, Voges-Proskauer, X and V strips for Haemophilus,
and antibiotic discs.
6. Antimicrobial Susceptibility
The recommended control organisms are specific strains from the American
Type Culture
Collection (ATCC).
Susceptibility testing of control organisms is usually done daily for 20
days to 30 days.
All results from the 20- to
30-day evaluation should be kept while the antimicrobial agent is
being used.
Antimicrobial solutions must not
be refrozen after thawing. tore
150 to 200 isolates with comparable growth rates and
agent are tested by both the
different levels of susceptibility to the
standard disk diffusion and
dilution MIC tests.
7. Personnel competence
All tests performed on patients must be subjected to

proficiency testing twice a year.
Participation in continuing education is another form of
8. Stock cultures
A
stock culture should be grown in large volume of broth and then divided into vials to
last for a year.
A vial of the stock culture can be taken from the batch preparation on a weekly basis.
To maintain the viability of an organism, it should be subcultured twice after thawing.
Organisms stored in a freezer should be kept at -70°C.
The media for stock
cultures are the Schaedler broth with glycerol, chopped meat
(anaerobes), skim milk, tryptic soy agar deeps, and cystine tryptic agar.
Notes to Remember
Daily monitoring of the temperature is required for incubator, anaerobic chamber, water bath, heating
block, refrigerator, and biofreezer.
For microscopes, its functionality should be checked quarterly while centrifuge and biohazard hoods
should be checked biannually and at least once a year, respectively.
TABLE 8-2. Strains Used in Quality Control of Routine Antimicrobial Susceptibility Tests
Strains Purpose
Bacteroides fragilis ATCC* 25285

Anaerobes
Eubacterium lentum ATCC 43055
Enterococcus faecalis ATCC 51299 Synergy screen test (positive) 30

Monitors thymidine and synergy test for
Enterococcus faecalis ATCC 29212
aureus (negative)
Enterococci and S.
Escherichia coli ATCC 25922 Gram-negative bacteria

Escherichia coli ATCC 35218 Gram-negative bacteria 6-lactamase positive
Hemophilus influenzae ATCC 49247 H. influenzae (non-B-lactamase)
Extended spectrum B-lactamases or ESBL
Klebsiella pneumoniae ATCC 700603
(screening and confirmatory tests)
Neisseria gonorrhoeae ATCC 49226 N. gonorrhoeae B-lactamase negative
Monitoring of the Susceptibility Media (Calcium
Pseudomonas aeruginosa ATCC 27853 and Magnesium content)
Staphylococcus aureus ATCC 25923 Gram-positive bacteria
Staphylococcus aureus ATCC 29213 Gram-positive bacteria (MIC)

Staphylococcus aureus ATCC 43300 Oxacillin-resistant
Streptococcus pneumoniae ATCC 49619 Streptococcus pneumoniae and other streptococci

*ATCC - American Type Culture Collection
4 BIOSAFETY LAB LEVELS

coC
CDC
1 controlled access
2 hand washing sink
3
sharp hazards warning
policy
personal protective
equipment
laboratory bench
6 autoclave
controlled access
(2 hand washing sink
policy
physical containment
device
personal protective
equipment
6 laboratory bench
autoclave
BSL3 (WITH RISK-BASED ENHANCEMENTS)

R TIGHT (WHEN DISINFECTING) 1 self-closing double-
door access
2 controlled access
3 personal shower out
policy
hand washing sink
6 sealed penetrations
device
& powered air purifying
IR TIGHT (WHEN DISINFECTING) respirator
@ laboratory bench
10 autoclave
11 exhaust HEPA filter
12 effluent
decontamination
system
AIR TIGHT
self-closing double-
door access
2 controlled access
policy
hand washing sink
sealed penetrations
device
positive pressure
protective suit
TIGHT
laboratory bench
autoclave
10 chemical shower out
personal shower out
12 supply exhaust HEPA
filters
13 effluent_
decontamination
system
Required safety equipment Risk-based enhancements

www.cdc.gov/24-7
FIGURE 8.3. CDC Biosafety Levels

Source. https://www.cdc.gov/cpr/infographics/biosafety.htm
BiOSAFETY AND QUALITY CONTROL IN A CLINICAL MICRO IOLOcY LABORATORY 77
e a 6,H A
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ldh ioange osasonttor nitie gps h
e1agclte Aieollenud
i omuenq inisford
ailisqpr aiy
dtiw babnndd bior 2olqmr2 orsdbeuioz 1snod ns a bexiny ▇ i maing1o erT
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ASSESSMENT QUIZ

is described as a pathogen
1. As to classification of infective microorganisms byrisk group (RG), it
to laboratory workers.
that can cause human disease but unlikely to create a serious hazard
a.
b.
C.
d. RG 4
of organisms should be utilized for checking the efficacy of

culture
2. In quality control, what type
media?
a. most fastidious organisms for which the medium was designed

b. non-fastidious organisms for verifying the efficacy of the primary plated media
C. gram-positive bacteria for using selective media
d. gram-negative bacteria for using differential media
3. Which of the following is the most commonly used engineering control in clinical microbiology
laboratory?
a. eye wash station
b. laminar flow hood
C. biological safety cabinet

d. negative pressure laboratory room
Which of the following is NOT listed in the

4.
most frequently acquired laboratory infections?
a. salmonellosis
b. brucellosis
c. bacterial pneumonia
d. viral hepatitis
5. This organism is recognized as "can be aerosolized,"

hence, samples should be handled with
utmost care and safety.
a. Bacillus anthracis, BSL 2 agent

b. Yersinia pestis, BSL 2 agent
C. Francisella tularensis, BSL 3 agent
d. Mycobacterium tuberculosis, BSL' agent
BIOSAFETY AND QUALITY CONTROL IN A 79
CLINICAL MICROBIOLOGY LABORATORY
These reagents require

daily monitoring to ensure the reliability of test results.
a. TSI agar for carbohydrate fermentation test
b. PYR and B-Lactamase
C. RPR and VDRL
d. wet mount and SIM for motility test
7. These substances pose the highest biosafety and biosecurity risks during transport of samples
between and across healthcare facilities.
a. Exempt specimens
b. Category A infectious substances
c. Category B infectious substances
d. Category C infectious substances
8. How frequently should Biohazard hoods be checked?
a. weekly
b. monthly
c. quarterly
d. once a year
9. In which type of PPE should "fit test" be performed?
a. respirators
b. face shield
C. gloves
d.
10. Which of the following is the typical mode of transmission for microorganisms that pose the
greatest risk in a microbiology laboratory?

a. vector bite
b. needle stick
C. aerosol
d. ingestion
CHAPTER 9
remains noowitn
STERILIZATION AND tood
DISINFECTION

2. discuss the various sterilization techniques and their roles in infection
control;
3. explain the factors that influence the activity of disinfectants; s

4. differentiate the chemical agents utilized in disinfection;
5. describe the importance of gas sterilization in healthcare institutions;
and
6. apply the concept of sterilization and disinfection in an academic
laboratory.
Microbial control involves physical and chemical agents that destroy

microorganisms and potential pathogens or inhibit their growth and prevent
their transmission.
Sterilization .
Sterilization refers to the removal or destruction of all forms of life,

including bacterial spores. _
.Physical Methods of Sterilization

Co
Application of Heat
Heat is the most commonly used method for the removal
of
microorganisms.
Moist Heat Procedure

structural proteins
It destroys microorganisms through the coagulation of enzymes and
and degradation of nucleic acids.
1. Boiling'
It destroys vegetative bacteria (non-sporulating)

Temperature and time of exposure: 100*C for 10 minutes to 15 minutes
2. Autoclaving
It is the fastest and simplest method of sterilization through which all organisms
spores, are killed within 15 minutes.
(except for prions), including those that contain
It is used to sterilize biohazardous trash and heat-stable objects.
Principle: Steam under pressure
indicator:
Bacillus
Biological
stearothermophilus)
hot steam under pressure.
Autoclave is a chamber which is filled with
Autoclaving Conditions and Purpose
a. 121°C, 15 psi for 15 minutes to 20 minutes: Culture media, liquids, glass pipettes,
and laboratory utensils
b. 132°C, 15 psi for 30 minutes to 60 minutes: Decontamination of medical waste
3. Fractional or Intermittent Sterilization (Tyndallization)

It destroys vegetative cells and spores after three consecutive days of sterilization.
Temperature and time of exposure: 100°C for 30 minutes
Instrument: Arnold's sterilizer (free-flowing steam)
4. Inspissation
It is utilized to sterilize protein-rich medium such as the Lowenstein-Jensen

medium.
Principle: Thickening of culture media through evaporation

Temperature and time of exposure: 70°C to 80°C
for two hours (three consecutive
days)
5. Pasteurization/Partial Sterilization
It is used
to sterilize milk, dairy products, and alcoholic
beverages.
It eliminates food-borne pathogens
and organisms responsible for food spoilage.
This method cannot eliminate
bacterial endospores.
STERILIZATION AND DISINFECTION
83
Types of Pasteurization
a. Low-Temperature Holding (LTH)/Batch Method

It
destroys milk-borne pathogens.
Treatment at this temperature reduces spoilage of food without affecting its taste.
Temperature and time of exposure: 63 'C for 30 minutes
b.
High-Temperature Short-Time (HTST)/Flash Pasteurizations lamen
Principle: Quick heating then immediate cooling

Temperature and time of exposure: 72°C for 15 seconds
C.
Ultra-High Temperature (UHT)
It is ideal for milk
products and beverage additives like coffee creamer.
Temperature and time of exposure: 140°C for 3 seconds (cooled very quickly in a
vacuum chamber)
Advantage: Milk can be stored at room temperature for two months without
affecting its flavor.
Dry Heat Procedure

This is a sterilization method that does not require water.
It kills microorganisms by denaturating proteins.

It is recommended for the sterilization of glassware, oil products, and powder.
Biological indicator: Bacillus atrophaeus (ATCC 9372)
1. Flaming or Direct Heating

It is the procedure for sterilization of inoculating loops and needles.
2. Oven-heating
It is used for glassware, oil, petrolatum or powders. penlullen anew
Temperature and time of exposure: 160°C to 170 °C for 1.5 hours to 2 hours
3. Incineration
It is the most common method of treating infectious waste and infected laboratory
animals.
300°C to 400°C
Principle: Burning of materials into ashes at
Temperature for hazardous materials: 870°C to 980°C
4. Cremation
It is utilized to control the spread of communicable diseases.

Terminologies
the lowest temperature at which a suspension of bacteria

1. Thermal Death Point (TDP) refers to
is killed in 10 minutes
Thermal Death Time (TDT) refers to

the shortest period of time needed to kill bacteria at a
one
prescribed temperature and under specific conditions prt
3. Decimal Reduction Time (DRT/D/D Value) - refers to the time in minutes to reduce the
this is widely used or observed
bacterial population or spores by 90% at a specified temperature;
in the food industry
2 Filtration
antibiotic solutions, toxic chemicals,
It is the method of choice for the sterilization of
radioisotopes, vaccines, and carbohydrates (heat-sensitive solutions).
It may be used with both liquid and air substances.
Types of Filters
1. Depth Filters
These are made up of fibrous or granular materials.
Example: Berkefield filter (diatomaceous earth), Chamberland filter (unglazed porcelain),

and asbestos
2. Membrane Filters (Circular filters)

These are porous membranes (almost 0.1 um thick).
They are composed of cellulose acetate or polycarbonate.
They are designed to sterilize pharmaceuticals, ophthalmic solutions, culture media,
antibiotics, and oil products.
a. Liquid filtration
It uses cellulose acetate or cellulose nitrate membrane with a vacuum.

b. Air filtration
It utilizes high-efficiency particulate air (HEPA) filters.
HEPA filters remove organisms larger than 0.3 um from isolation rooms, operating
C. Filtration of bacteria, yeasts, and molds

It utilizes membrane filters with
0.45-um pores.
Membrane filters with pores ranging in size from 0.2 um to 0.45 um in diameter
most bacteria as well as fungi but not viruses.
d. Critical sterilizing
It uses membrane filters
with 0.22-pm pores ideal for parenteral solutions and alcohol.
Membrane filters with 0.22 um pores are used to eradicate but
not viral agents. vegetative cells and spores
85
010lharoAe STERILIZATION AND DISINFECTION
It is considered bacteriostatic because it educes the rate of metabolism.
It is important in food microbiology.

Exposure to 2°C to 8°C for 72 hours kills
the agents of syphilis.
7) Desiccation and Lyophilization

Desiccation destroys bacteria through the disruption of metabolism that involves removing water
from microbes, and that is considered as bacteriostatic.
Lyophilization destroys bacteria through changes in proteins and decrease in chemical reactions.
Examples of bacteria which remain active in a dry environment:

a. Neisseria gonorrhoeae - viable for one hour
b. Mycobacterium tuberculosis viable for several months eri to elmenoamop ddiw undoss¾
C. Bacillus and Clostridium - viable for ten years
Osmotic Pressure
It is the use of high concentrations of salts and sugars in food to create a hypertonic environment.
Inplasmolysis, the plasma membrane shrinks away from the cell wall (cell may not die, but
usually stops growing) as water leaves the cell.
¢) Radiation
The underlying principle is that when the radiation passes through the cells, free hydrogen and
hydroxyl radicals and some peroxidases are created. These free radicals, in turn, cause different
intracellular damage.
Biological indicator: Bacillus pumilus
1. Ionizing radiation (Cold sterilization)

It causes mutation in the DNA and generates peroxides.
It destroys vegetative cells and endospores of both prokaryotes and eukaryotes.

It has a shorter wavelength.
It is usually designed to sterilize plastic syringes, sutures, catheters, gloves, hormone

solutions, and antibiotics.
It is also used to "pasteurize" meat products. 1
The types of radiation rays used are gamma rays (1500 to 2500 radiation) and x-rays.
co butiogs vilsoau al d
2. Non-ionizing radiation
It causes damage to cellular DNA by producing thymine dimers.
It has a longer wavelength (> 1 m) and low energy.
It is used to sterilize exposed surfaces, operating rooms, nursery rooms, and cafeterias.
rays (10 um to
400 um) in which 260 pmis
are ultraviolet
The types of radiation rays utilized
the most lethal.
Microorganism in water are destroyed when passed under UV hamps,

•)Chemical Methods of Sterilization
Disinfection
of microorganisms which include potential
It refers to the removal, inhibition, or killing
pathogens by utiliezing chemical agents usually on inanimate objects although it does not remove
all bacterial spores.
Destroy Microorganisms
Mechanisms by Which Chemical Disinfectants
1. Reaction with components of the cytoplasmic membrane
2. Denaturation of cellular proteins
3. Reaction with the thiol groups of enzymes

4. Damage to DNA and RNA
Factors That Influence the Activity of Disinfectants

1. Types of organism present
2. Number of organisms present (microbial load)
3. Temperature and pH level of the process
4. Concentration of disinfectants
5. Amount of organics and biofilms

6. Length of contact time between the chemical agent and the microorganism
7. Type of water used for disinfection
Terminologies
1. Antiseptic
It is applied topically on the skin.
It inhibits sepsis formation.
Examples: Hexachlorophene and

tincture of iodine/povidine alcohol (iodophor)
2. Disinfectant
It is usually applied to inanimate objects.

Sodium hypochlorite (NaOCI) in 1:10 dilution is an
effective disinfectant.
Examples: Cresols,
chlorine, and sodium hypochlorite
Yoouoigana STERILIZATION AND DISINFECTION
87
3. Bactericidal
It precipitates bacterial protein and kills all bacteria in the specimen.

Some examples are strong acids such as sulfuric acid and hydrochloric acid.
4. Bacteriostatic
It inhibits the of organisms.

growth
Common Chemical Agents Used in Microbial Control

1. Acid and Alkaline Solutions
It hydrolyzes and coagulates proteins.
2. Phenol (Tuberculocidal)
The first widely used antiseptic and disinfectant.
It destroys plasma membranes and denatures cell proteins.
It is effective even in the presence of organics.
5% phenol with 10 to 30 minutes contact time is effective against mycobacteria.
3. Alcohol (Non-sporicidal)
It causes denaturation of proteins and dissolution of lipid membranes.
It is used as both an antiseptic and a disinfectant (bactericidal and fungicidal).
It should be used in 60% to 90% concentration to achieve its antimicrobial property.
It should be allowed to evaporate from the surface to achieve complete antisepsis.
Examples: Isopropanol (rubbing alcohol) and ethanol
Disadvantage: Poor activity against non-enveloped viruses and ineffective in the presence of
organic material
4.
It destroys microorganisms through the oxidation process. antiliod

lsituo bord
Examples: Chlorine, iodine, fluorine, bromine, and astatine

10 minutes to 30 minutes of contact time
NaOCl should be freshly prepared every day with
to make it an effective tuberculocide - 1:10 dilution is an effective bleach. i
Halozone (parasulfone dichloraminobenzoic acid) contains chloride and is used to disinfect
drinking water.
Preparations of lodine: Tincture and Iodophor

Tincture of iodine contains 2% iodine in 70% alcohol and is used as antiseptic.
an
Iodophor is composed of iodine and a neutral polymer carrier and acts as either an antiseptic
or a disinfectant.
after 70% alcohol.

Before drawing blood for culture, iodophor is applied due to the lackof free
Improperly diluted iodophors may not kill microorganisms iodine in
the solution.
is the oxidative
effects of molecular iodine
The antibacterial effect of iodophors and
hypoiodic acid.
the skin prior to blood collection
Contact time for lodine: 30 to 60 seconds onto
Disadvantage: Non-sporicidal and nontuberculocidal
Chlorine
It is used in the form of hypochlorite.

the oxidative and disinfecting effects of
Its antibacterial activity is manifested by
are dissolved in water).
hypochlorous acid (formed when chloride ions
Concentrated solutions (corrosive) should not be used for disinfection.
The CDC recommendation for this chemical agent is 1:10 dilution of 5.25% solution of
sodium hypochlorite, especially for disinfecting table tops and floors with
blood spills.
Contact time: 3 minutes for organic materials and 10 minutes to 30 minutes for mycobacteria
Disadvantage: Ineffective in the presence of large amounts of proteins

bns alidgmn asnanguto dbrxed alonsly
5. Salts of Heavy Metals
It destroys microorganisms by inactivating and precipitating cell proteins.
Some examples are copper, arsenic, mercury, silver, and zinc (bacteriostatic).
AgNO, is an eye drop solution used historically against N. gonorrhoeae while mercuric
chloride is an antiseptic. D
b. Quaternary Ammonium Compounds (Non-tuberculocidal and Non-sporicidal)

It is widely used as
surface-active agents.
Its antibacterial effect is the disruption of the cell membrane that results in the leakage of cell
contents.
It is
used on laboratory bench-tops and floors, in cleaning dairy utensils, and in disinfecting
food outlet facilities.
Some examples are zephiran

(benzalkonium chloride and
chloride.
grit los
Culture of Pseudomonas aeruginosa in an ammonium acetate medium is resistant to quats.

7. Phenolic
derivative of
It is a
phenol withreduced toxicity.
ths a stabe surface disinfectant and effective in the presence of organics.
disrupts microbial cell walls and membranes and
It
precipitates proteins.
Common phenolics: Ortho-phenylphenol
and Ortho-benzyl-para-chlorophenol
VOO ONETOAG> STERILIZATION AND DISINFECTION 89
CHG
inactivates certain lipid-enveloped viruses, such as HIV, respiratory virus, and
cytomegalovirus.
CHG binds to the skin and remains active for at least 6 hours.
CHG is an effective body disinfectant used in the hospital prior to surgery.
PCMX effective for surface cleansing contaminated with body fluids, such as blood
is and
sputum; it is also used as a skin antisepsis.
Triclosan is more effective compared to CHG, and it is utilized for clothing and furniture
decontamination and, as an additive to dental care liquid.
Disadvantage: Nonsporocidal
8. Aldehyde- Cold/Chemical Sterilant

Its antibacterial effect is the inactivation of proteins and nucleic acids.
It is recommended for sterilizing medical instruments.

Example: Formaldehyde and 2% glutaraldehyde mol gniwollol adt 39 er tol anivice nl
a. Formaldehyde (HCHO)
It is generally used as formalin which consists of 37% aqueous solution or HCHO gas.
It is best for sterilizing HEPA filters in a biological safety cabinet (formaldehyde vapor).
Its usefulness, however, is limited because it has an irritability factor, and it is a known
carcinogen.
Recommended concentration: 8% HCHO
For mycobacteria, 3% to 8% HCHO is used with a contact time of at least 30 minutes.
b. Glutaraldehyde (Pseudomonacidal, Tuberculocidal, Fungicidal, and Virucidal)

It is commonly utilized on medical instruments (heat-labile) that are made of plastic and
rubber materials.
It is effective against human immunodeficiency virus (HIV) and hepatitis B (HBV) when
10 minutes.
organisms are exposed for
It has a rapid killing action but does not penetrate organic materials well when used as a
sterilant.
Recommended concentration: 2% Glutaraldehyde

2% glutaraldehyde is bactericidal (including tuberculocidal) in 10 minutes and sporicidal
in 3 hours to 10 hours.
9. Gas Sterilant
a. Ethylene oxide (EtO)

It is the most commonly used gas for sterilization.
catheters, heat-sensitive equipment
It is utilized to sterilize plastic Petri dishes, sutures,
such as the heart-lung machines.
It has an explosive property so it should be mixed with nitrogen or carbon dioxide before
use.
BACTERIOLOGY
in spores and vegetative cells.

is antiacierial effect is the allylation of nucleic acids
The concentration used is
from 450 mg/L to 700 mg/L of chamber space at 55P3*C teto 60°c
for two hours.
Biological indicator: Bacillus species

b. Periacetic acid sod more botenin
It is active against all vegetative microorganisms and fungal spores.

It is used to sterilize pieces/parts of medical equipment.
bus moltentmnsimosst
Coefficient (PC)
Disinfectant Screening Test - Phenol
10-minute exposure. 671l biA
It is the highest dilution that kills the bacteria after
The potency of a disinfectant is compared with phenol.
Test organisms: Staphylococcus aureus and
Salmonella serotype Typhi (20°C or 37°C)
In solving for the PC, the following formula is used:

time 104
PChighest dilution of disinfectant that will kill organisms at a given
=
B69 Of highest dilution of phenol that will kill organisms at a given time
Result: PC 1, which indicates that the disinfectant is more effective than phenol
Interpretation: The higher the PC value, the more effective the disinfectant.
o0 9aO45 STERI니ZATION AND DiSINFECTION 91
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ASSESSMENT QUIZ
decontaminate
1. Which of the following is the preferred sterilization technique to hospital
apparatus such as the heart-lung machine?
a. gas sterilization
b. Tyndallization
C. cold sterilization
d. chemical treatment y formaldehyde
2. Which of the following is NOT true regarding HEPA filter?

a. used for air filtration
b. removes organisms larger than 0.3 um

C. utilized in critical sterilization
d. installed in biological safety cabinet
3. What is the ideal contact time for tincture of iodine to be an effective antiseptic?
a. 45 seconds
b. 90 seconds
C. 2 minutes
d. 3 minutes
4. This chemical disinfectant is effective against bacteria with high lipid content, fungi, and even
viruses such as HIV.
a. alcohol
b. glutaraldehyde
C. quats
d. phenol
5. It is the recommended technique to sterilize gloves and plastic syringes.

a. using peracetic acid
b. treatment with ethylene oxide
c. partial sterilization
d. Ionizing radiation
STERILIZATION AND DISINFECTION 93
b. This is the biological indicator to

determine effective sterilization after autoclaving.
a. Bacillus pumilus
b. Bacillus atrophaeus
d. Pseudomonas aeruginosa
7.
Which of the following is the best sterilization technique for protein-rich culture media?
a. pasteurization
b. tyndallization
C. filtration
d. inspissation
8. What are the components of an iodine tincture?

a.
iodine and hypochlorite
b. 2% iodine in 70% alcohol
c. 5% iodine in 95% ethanol

d. pure iodine
9. What are the recommended temperature, pressure, and time of exposure when decontaminating
medical waste through autoclaving?
a. 100°C, 15 psi for 30 minutes to 45 minutes
b. 121°C, 15 psi for 15 minutes to 20 minutes
C. 132°C, 15 psi for 30 minutes to 60 minutes
d. 160°C, 15 psi for 10 minutes to 20 minutes
10. It is an effective skin disinfectant as it inactivates microorganisms with increased lipid content,
such as the respiratory viruses and cytomegalovirus.
a. chlorhexidine gluconate
b. 95% ethanol
C. hydrogen peroxide
d. triclosan
CHAPTER 10
SPECIMEN COLLECTION,
TRANSPORT. AND
PROCESSING

1.
discuss the significance of proper specimen collection and handling in
relation to
the reliability of diagnostic tests;
2. explain the basic
and critical protocolsin specimen collection including
labeling, transport, and storage;
3. discuss the parameters of specimen rejection;
4. cite the relevance of specimen prioritization in achieving quality uwava hoganmd bisind
laboratory data;
describe the panic results in a clinical microbiology laboratory; and
6. design a flowchart on sample management to releasing of test result. 21010
Specimen collection, handling, and transport are critical steps in diagnostic

medicine because the results that the laboratory provides to the clinicians
greatly depend on the quality of the specimen.

During the collection of specimens, indigenous microbiota are found in
clinical samples as a result of the contamination of normally sterile sites or the
involvement of the colonizers in the infection.
Proper skin cleansing prior to blood cultures and spinal taps decreases the
presence of contaminants and skin microbial flora in the specimen. In addition,
the specimen should be collected from the actual site of infection whenever
possible. The sample container should be labelled with the appropriate patient
0/18
demographic including the site of collection.
The laboratory can make accurate and useful determinations only if a
specimen has been collected and transported properly.

109712
General Guidelines for Specimen Collection

1. Specimens should be collected during the acute or early phases of an illiness.
the specimen.
2. The correct anatomic site must be identified/selected for collection of
for diagnostic testing.
should be sufficient
3. The quantity of the collected specimen
4. If possible, specimens should be collected before antibiotics are administered,
for nares and
throat specimens) compared with
5. Swabs are generally poor specimens (except
tissues or needle aspirates.
For lesions, wounds, and abscesses, the specimen is collected from
the advancing margin of the
6.
lesion, preferably by needle aspiration rather than by swabs.

a sterile tube or transport vial and not squirted onto a
7. Aspirated specimens must be placed into
swab.
8. For patient-collected specimens, clear instructions should be given by the authorized healthcare
personnel, and it should never be assumed that the patient knows how to properly collect a
specimen.
The specimen must be labeled accurately with the specific anatomic site and the patient's
demographic profile.
10. Appropriate transport system should be utilized in case the specimen will be submitted to
microbiology laboratory outside the premise of the healthcare institution.
Points to Consider in Sample Collection

For blood culture, 70% alcohol followed by 2% tincture of iodine are the recommended skin
antiseptics for cleansing the site prior to specimen collection.
Due to skin contaminants or indigenous microbial flora, cleansing the site before collection of
sample is a strict requirement as in the case of blood culture to avoid false-positive bacteremia.
For wound infections and
abscesses, these sites should not be cleansed with antiseptics prior to
collection of samples because it would reduce the desired pathogens and it may kill the organims
leading to false-negative result. mns Stoldunoimn
The antiseptic should be in contact with the skin or site of

collection for at least 30 seconds.
The common
skin contaminants are coagulase-negative staphylococci (CoNS), Micrococcus
species, and Cutibacterium (Propionibactrium) acnes.
Routine Samples for Clinical Microbiology

Ideally, 10 mL of blood should be
drawn and transferred into each blood culture bottle to
increase the chances of having : positive blood culture.
The optimal ratio of blood to culture medium is about 1:5
to 1:10.
Specimens should be submitted
on two swabs, each for direct smear and culture. However,
swabs
are never recommended for anaerobic culture
Abscess specimens collected by aspiration are highly suggested anaerobic culture compared
for
swab samples because the former are less likely to be contaminaura
to
microbiota. with skin

SPECIMEN COLLECTION, TRANSPORT, AND PROCESSING 97
It
is recommended that after
sample aspiration of the specimen, dead space in the syringe should
be
removed since it may be filled with atmospheric air which can "contaminate" the samples for
anaerobic bacteriology.
For
viral culture, samples should be collected within 2 to 3
days of the acute phase.
Specimen Containers and Other Materials for Collection

1. Specimens for microbiology cultures should be collected in sterile containers.
Stool specimens can be collected in clean, non-sterile containers.
2. Swabs are used for specimens from the upper respiratory tract, external ear, eye, and genital
tract.
Nonetheless, swabs are not recommended for routine collection because they are easily
contaminated and dried out.
If swabs are
used, Dacron or calcium alginate swabs are preferred rather than cotton-tipped
ones.
Cotton-tipped swabs may have excessive fatty acids which are inhibitory and toxic to some
bacteria.
CDC Recommendation for Virology

Calcium alginate swabs or swabs with wooden shafts are not recommended for viral collection
including samples for SARS-CoV-2 studies since they may contain substances that inactivate
some viruses and may inhibit molecular tests. Instead, use only synthetic fiber swabs with thin
plastic or wire shafts that have been designed for sampling the nasopharyngeal mucosa
Source: https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html
Specimen Labelling
The patient's information, which includes the patient's complete name and identification number
(hospital number), must be provided on the specimen label.
The age and birth date should be included in the specimen label if they are part of the
institutional protocol. 120001
The label should include the date and time of collection, source of the specimen, and the
antibiotics provided to the patient, if possible.
The verification of a mislabeled specimen or requisition should not be done, if possible, over the
telephone.
Specimen Transport
should be transported to the laboratory immediately and preferably
1. Ideally, most specimens
within 30 minutes of collection and not longer than two hours for both aerobic and anaerobic
culture.
and maintained as much as possible to their original state.
Specimens should be transported
For anaerobic bacteria, the transport should not take more than 10 minutes.
cerebrospinal
Aluid (CSF) and body fluids (amniotic, pericardial,
Fer torele sarepies, adh arnorial, they should be transported preferably within 15 minutes
after collection.
caused by prolonged transport can reduce
Changes in oxygen, pH, and temperature contaminants or undesired
the overgrowth of
the recovery of certain bacteria and allow
organisms.
2. All specimen containers should be placed in sealable, leak-proof plastic bags.
Patient specimens or culture isolates must be
triple-packaged before being shipped.
Specimen bags should be marked with a biohazard label.
collection.
3. All specimens should be transported to the laboratory immediately after
Any delay may decrease the number of pathogens and hasten the multiplication of
indigenous microbiota.
916 a06wa 916/18

Air Transport of Samples
Prior to air shipment, biological samples must meet the International Air Transport Association
(IATA) and International
Civil Aviation Organization (ICAO) packing regulations.
be deposited first
Samples containing infectious or potentially infectious agents should in
a primary receptacle (plastic biohazard bag), then in a secondary container, and finally in a
tertiary container (a receptacle that protects the sample from physical damage).
When handling biosamples for air transport, trained personnel are required.
910108
Triple Packaging of Infectious Substances

To limit the risk of exposure and/or release during transportation, redundant layers of packaging
are commonly used to control any leakage or breach of containment of an infectious sample.
A triple package consists of three layers

rodr 1. Primary receptacle
it contains the infectious substance, and the receptable should be watertight, leak-proof, and
arit do bin labelled properly including the contents of the sample.
It should be protected against spill by wrapping with absorbent material.uldent
A cushioning material should be used in case
of multiple primary receptacles in one
container, to prevent contact between them.
2. Secondary container
It is
watertight, leak-proof packaging, and is used to enclose and protect the primary
receptacle(s).
Several wrapped primary receptacles may be placed in a single secondary packaging.

3. Third receptacle
It protects
the secondary container from physical damage during transport.
TABLE 10-1. Transport Requirements of Specimens for Clinical Microbiology

Transport Condition
Specimen
Immediately at room Body fluids
temperature
Inner ear
Gastric aspirate
Suprapubic aspirate of urine
Within 15 minutes at room

Cerebrospinal fluid (CSF)
temperature
Within two hours at room Blood

or bone marrow specimens™ad bris ameniqusil
temperature
Within 24 hours at room Abscess

temperature
Outer ear
Conjunctiva specimens
Genital tractspecimens (For urethral specimens, within
2 hours using John E. Martin Biological Environment
Chamber or JEMBEC)
Respiratory tract specimens 08A Vloa fboe

Tissue specimens ind koold bihrbsbbh mneluscopins
Immediately at 4°C Gastric biopsy
Within 2 hours at 4°C Indwelling catheter (urine sample) nows ase ud bandiar
Straight catheter (urine sample)
Within 24 hours at 4°C Rectal swab
Stool culture
Clean voided midstream urine
Specimen Preservation aT
boot
If the transport of the specimen to the laboratory or its processing is delayed, the specimen can
be maintained with the use of holding media or appropriate temperature.
Ideally, samples for clinical bacteriology should not contain any preservatives since it may cause
inhibition of the microbial growth.
Preservatives should not be added on fecal specimens intended for bacteriological testing.
Stool specimens for Clostridium difficile toxin assay should be collected without a preservative and
can be refrigerated. However, if the specimen cannot be processed within 48 hours, it should be
stored at -70°C.
Boric acid orPolyvinyl alcohol (preservatives) may be added to urine samples to maintain the
appropriate colony count at room temperature for 24 hours if the specimen cannot be processed
immediately.
Transport or Holding Media

present in a specimen but
do not allow
These media sustain the viability of microorganisms
further multiplication.
medium, Cary Blair, Transgrow, and JEMBEC (John

E. Martin
Examples: Stuart medium, Amies
Environmental Chamber)
acids present in the
Charcoal is sometimes added to a transport medium to absorb fatty
specimen that could affect the pH of the medium and eventually killing fastidious organisms
like Neisseria gonorrhoeae and Bordetella pertussis.
For anaerobic culture, tissue and biopsy samples should be placed in anaerobic transport
tubes or
vials containing PRAS (pre-reduced anaerobically sterilized) medium to maintain

the moist and
prevent dryness.
bacterial (Mycoplasma,
Viral transport media (VTM) preserves the samples for viral and
Ureaplasma, and Chlamydia) isolation and maintains the viability of the organisms for
48 hours at 2°C to 8'C and even at room temperature while long storage requires -70°C.
VTM is ideal for respiratory and genital samples, eye and ear discharge, and tissue and skin
specimens intended for molecular test. It contains antimicrobials, such as gentamicin and
amphotericin B, to suppress the growth of the contaminants and indigenous flora.
Anticoagulants
These are used to prevent clotting of specimens such as blood, bone marrow, and synovial fluid.
The sodium polyanetholsulfonate (SPS) in 0.025% to 0.050% concentration is the preferred
anticoagulant added into blood culture media; 0.025% SPS is commonly used.
Certain organisms such as Neisseria species, Gardnerella vaginalis and some anaerobic bacteria are
inhibited by SPS even when the concentration is at 0.025%.
Culture tubes or
bottles containing heparin, ethylenediaminetetraacetic acid (EDTA), and
sodium citrate should be avoided because they may suppress the
growth of organisms.
Heparin may inhibit Gram-positive bacteria
positive cocci.
while citrate diminishes the activity of Gram-
Sodium amylosulfate (SAS) has structural homology with SPS but is less
effective in neutralizing
serum bactericidal activity and has inhibitory property to some
pneumoniae. Gram-negative bacterial like K.
Synovial and peritoneal fluids can also be inoculated into
samples.
broth media intended for blood
Reasons for Utilizing 0.025% to 0.050% SPS as the Preferred

Anticoagulant for Blood Culture:
a. To
inhibit phagocytosis and complement activation
To neutralize the
b.
activity of aminoglycoside antibiotics
C. To neutralize the bactericidal effect of plasma
Specimen Storage
If specimens cannot be processed immediately after transporting them to the laboratory, they
be stored under these temperature conditions: 2302
must
a.
Refrigerated Temperature (4°C)
Specimens that should be kept at this temperature sputa, viral samples, fomites such as
are
catheters and unpreserved samples like urine and stool.

Stool specimens that are not transported immediately to
the laboratory can be refrigerated;
but if the delay
is longer than two hours, the specimen can be placed in a Cary Blair
transport medium.
Specimens that are suspected of containing anaerobic bacteria should never be stored in the
refrigerator. Nonetheless, stool samples for the Clostridium difficile toxin assay can be stored
up to three days at 2°C to 8°C.
b. Incubator Temperature (35°C)

CSF should always be kept at this temperature. Lisr Tish
This is the recommended preservation temperature for CSF samples intended for Gram
staining, culture, and sensitivity tests.
C. Ambient (room) Temperature (22°C to 25°C) guorlila) hild

Specimens for anaerobic culture, sterile body fluids, genital specimens, wound and lesion
discharge, tissues, and swabs (ear and eye) are held in this temperature.
Samples from the URT (nasal, nasopharynx, throat) may also be kept at ambient temperature
prior to testing.
Urine and stool samples may be stored at this temperature provided a preservative will be
added to these specimens. Again, preservatives will only be added to these two samples if
the bacteriologic test will not be affected, otherwise, urine and stool samples should be free
from additives.
d. Freezer Temperature (-20°C or -70°C)

Sera for serological studies are frozen up to one week at -20°C.
Tissues or specimens that are to be stored for a long time should be kept at -70°C.
Fecal specimens for the study of Clostridium difficile toxin may be kept at -70°C, if the storage
will be longer than three days.
BACTERIOLOGY
TABLE 10-2. Storage Requirements Prior to Specimen Processing

Specimen
Storage conditions
Plate as soon as possible Body fluids
Bone
Gastric biopsy
Suprapubic aspirate of urine
Abscess
24 hours at room temperature
Inner ear0
For inner ear specimen
Eye and conjunctiva
Genital tract specimen (For urethral specimens, put them in JEMBEC at
35°C immediately upon receipt in the laboratory) thef
Upper respiratory tract specimens
Tissue specimens
Indefinitely at room temperature Hair, nail, or skin scrapings
Immediately at 35°C Blood
Bone marrow
Corneal scrapings
6 hours at 35°C CSF (although immediate processing is recommended)
24 hours at 4 °C Lower respiratory tract sample
Midstream urine (unpreserved)
Straight catheter urine(unpreserved)
Indwelling catheter urine (unpreserved)
Foreign devices such as catheter
72 hours at 4°C Rectal swab
Stool culture
Specimen Rejection
All rejected specimens require the acknowledgment of the person in collecting the
charge of
specimen.
The presence of squamous epithelial
cells and bacteria without the cells of inflammation
are
indicators that samples may have been altered or contaminated.

Specimens that are impossible to recollect or
invasive
that would require the patient to undergo another
procedure (specimen collection of bone marrow or CSF or may need
to be
surgery)
processed regardless of the condition of the specimen.
Reasons for Specimen Rejection
1. The information on the label does
not match
the information on the requisition slip.
2. It is
transported atan improper temperature.
3. The transported specimen has not been placed in the

proper holding medium.
4. The quantity of specimen is not
sufficient (QNS)for testing.
5.
The transport of specimen exceeds the two-hour post-collection period.
6.
It is preserved in a fixative (formalin).
7. It has been reserved
for anaerobic culture from a site known to have anaerobes as part of the
indigenous microbiota (vagina and mouth).
8. The specimen has already dried up upon
transporting to the laboratory.
9.
The specimen container is leaking.
10. It has questionable medical value.
More
11.
than one specimen from the same source (except for blood) and from the same patient is
submitted on the same day.
12. A single swab is submitted with multiple requests for various organisms.
13. Expectorated sputum in which the Gram stain reveals < 25 WBCs and > 10 epithelial cells
Specimen Prioritization
When a specimen is received with multiple requests but the amount of specimen is insufficient,
staff which of the tests should be prioritized.
the clinician should inform the laboratory
When multiple specimens arrive at the same time, priority should be given to those that are most
critical such as CSF, tissue, blood, and sterile fluids.
Urine, throat, sputa, stool, or wound drainage are specimens that can be stored and processed
later.
Acid-fast bacilli (AFB), viral, and fungal specimens can be processed by batch.
TABLE 10-3. Levels of Specimen Prioritization

Specimens
Level Description
Amniotic fluid, blood, brain, CSF, heart valves, pericardial fluid
Critical/Invasive
Bone, feces, sputum, tissue, other body fluids not listed under level
Unpreserved
Catheter tip, urine, tissue
Quantitation required
Preserved Urine, feces, and swabs in holding media
Source: Textbook of Diagnostic Microbiology. Mahon and Lehman. 5th ed, 2016,
Level 1 specimens are collected

from individuals with potentially life-threatening illnesses.
Level 2 specimens may quickly deteriorate or may have overgrowth of contaminating organisms.
need to be digested and decontaminated can be refrigerated
Specimens for AFB like sputum that
and processed once a day.
sal t neod ton asn

Specimen Processing
It involves the preparation of samples for microscopy and culture
Gross Examination
of blood, mucus, and rice-watery appearance.
Stool specimens are examined for evidence
(black discoloration) is one of the indicators of an
The presence of a necrotic tissue samples
anaerobic infection. mnodaIorl
Preparation of Smears
submitted to the laboratory can be smeared directly
1. Specimens, such as secretions and discharge
on slides.
be submitted to the
2. For samples collected on swabs, one swab each for smear and culture should
laboratory.
Smears from swabs are made by gently rolling over back and forth across the slide.
Swabs should not be rubbed over the slide as it may cause distortion of the bacterial
morphology.
Smears should not be prepared from a swab that has already been used for inoculation.
If only a single swab is submitted to the laboratory, the culture should be prioritized while
the smear can be prepared from the plates or broths after incubation. eferitlism ned
016 60610 steen
3. Opaque materials must be thinly spread on a slide.
4. Granules within the material should be crushed using two glass slides to assess their contents.
5. For thin fluid specimens such as urine, CSF, and transudates, place a drop on the slide while
avoiding to spread it all over.
Thin fluids should not be spread over a larger area unless it is turbid.
Cytocentrifugation is preferred for thin fluid specimens as well as bronchoalveolar lavage.

Cytocentrifugation is a process in which cells and microorganisms from the specimens are
bruh deposited onto a glass slide as a monolayer. DonoinmA
Purpose of Direct Microscopic Examination

1. The quality of the specimen can be determined.
Sputum is rejected if it represents the saliva and not the lower respiratory tract secretions.
For sputum samples to be accepted for cultivation, there should be less than 10 epithelial
cells and more than 25 pus cells per microscopic field (Bartlett's criteria).
2. The clinician can be given an early indication of illnesses of the
3. The result of the microscopy provides information on what to expect from the culture. g
If
three different morphotypes are seen on the direct Gram stain but only two organisms
grow on the culture, the third organism may be an anaerobic bacterium
O Score
TABLE 10-4. Grading the Quality of the
Clinical Sample Using the
(Bartlett's Q Scoring of Sputum Samples) Average Number of
Average Number of Score Squamous Epithelial Cells/LPF
Score
Neutrophils/LPF 0 (none)
0 (none)
-1
1-9 (few)
+1 1-9 (few)
-2
10-24 (moderate numbers)
+2 10-24 (moderate numbers)
+3 ≥ 25 (many, numerous)
-3 antineeon. 2 25 (many, numerous)
Interpretation:
Points for average number of squamous cells
O score: Points for average number of neutrophils
Minimum score: -3
Maximum score: +3 anD oferi
The higher the score, the better the specimen.

A specimen with a composite score 2 +1 should be cultured.
cultured.
A sputum specimen from a leukopenic patient with ciliated respiratory epithelial cells should be
Note: This is adopted from Mahon and Lehman: Textbook of Diagnostic Microbiology, 6th ed, 2019, Elsevier.
TABLE 10-5. Criteria to Determine Suitability of Sputum Sample for Culture

(Murray-Washington Method for Contamination Assessment)
Group Epithelial Cells/LPF Leukocytes/LPF
1 >25 <10
>25 10-25
>25
10-25
>25
<10
>25
Interpretation:
Ideally, only samples that fall under groups 4 and 5 are suitable for culture. However, immune
suppressed individuals will have reduced numbers of leukocytes in their secretions as well when
their cell
counts diminish. Therefore, the criteria have been revised so that the number of epithelial
cells (>25 per low-power field) is a better indicator
ofmucosal or saliva contamination.
Note: The is adopted from Mahon
and Lehman: Textbook of Diagnostic Microbiology. Gth cd., 2019. Elsevier
Specimens For Clinical Bacteriology

1. Abscess (Lesion, wound, pustule, ulcer)
Needle aspiration enhances the
recovery of anaerobic bacteria.
Types of Abscesses
a. Superficial abscess
aerobic swab that is moistened with Stuart or Amial medium

An
Swab along the leading edge of the wound may be used.

b. Deep abscess
The specimen is aspirated from the wall of the abscess or the advancing margin of the
lesion.
Aspirated specimen should be transferred into sterile tube or transport vial.

Since this specimen is recommended for anaerobic culture, include Brucella Blood Agar
(BBA), Laked Kanamycin Vancomycin Blood Agar (LKVBA), and Bacteroides Bile Esculin
Agar (BBEA) in the list of culture media.
2. Blood
Ideally, blood should be drawn during the time of febrile (fever) episode.
The venipuncture site should be cleansed with 70% alcohol followed by 2% tincture of iodine.
If there is a difficulty in the collection of samples, the aerobic bottle should be inoculated first
with the appropriate volume and the remaining sample will be for the anaerobic bottle; at
least one
anaerobic blood culture bottle should be utilized in difficult situations.
Studies reveal that three sets of two bottles each collection will satisfy the required volume
of the sample to detect bacteremia.
Sample container: A set of blood culture bottle is composed of one each of aerobic and
anaerobic culture bottle.
No more than three sets of samples should be drawn in a 24-hour period.

Sample preparation: To ensure complete antisepsis, the iodine should be in contact with the
skin for 30 seconds to 60 seconds.
Sample collection: Two sets of culture bottles should be drawn preferable from two different
sites, with 10 mL of blood every collection for adults and 5 mL to 10 mL for children.
CLSI: Chlorhexidine gluconate is the recommended skin disinfectant for blood culture, for
infants 2 months and older, and for patients with iodine sensitivity.
3. Body Fluids (amniotic, abdominal, ascites/peritoneal, bile, joint/synovial, pericardial, and
pleural)
Sample preparation: The skin should be disinfected prior to sample collection (needle
aspiration), same as the procedure for blood culture.
Sample concentration through centrifugation or filtration may be required prior to smear
preparation and cultivation.
The sample should be inoculated as soon as it is received in the microbiology laboratory.
4. Bone
The skin should be disinfected before the surgical procedure. noluticed ngqh
The specimen may require homogenization.

Prior to anaerobic culture of bone fragments or
tissue samples, 1 mL of sterile thioglycollate
broth is added to a grinder to achieve homogenization. This procedure
sterile tissue
is performed in an
anaerobic chamber or the grinding must be done quickly to reduce
aerobiosis in the absence of the chamber.
108 REVIEW HANDBOOK
5. Cerebrospinal Fluid
The skin should be disinfected before aspiration.
mL (microbiology and chemical tests)
Required volume: 10
Storage: Up to 6 hours at 35°C (if CSF cannot be processed immediately)
cryptococcal antigen test
Rapid diagnostic testing like Gram staining and is the
recommended method for this specimen.
Bacteria associated with meningitis (Neisseria meningitidis and Haemophilus influenzae) are
fastidious and susceptible to cold temperature or drying.
Smear and staining can be performed after cytocentrifugation.
Ear
a. Inner ear
Sample Preparation: The ear canal should be cleansed with a mild soap solution before
performing myringotomy or the puncturing of the eardrum.
the material should be aspirated behind it
Sample collection: If the eardrum is intact,
with a sterile syringe. A swab should be used to collect materials from the ruptured
eardrum.
b. Outer ear
The crust should be wiped off with a sterile saline.

An aerobic swab moistened with Stuart or Amies medium may be used; the swab should
be firmly rotated in the outer canal to collect the sample.
7. Eye
a. Conjunctiva
Sample collection: Samples should be obtained from both eyes using two separate swabs.
An aerobic swab that is moistened with Stuart or Amies medium or pre-moistened with
sterile saline may be utilized.

b. Corneal scrapings
Sample preparation: The clinician should administer local anesthetic before collecting
the specimen.
Sample collection: Using a spatula, scrape the discharge and inoculate directly onto/ into
medium.
Bedside inoculation should be done utilizing the following media: BAP, CAP Sabouraud
dextrose agar (SDA), Middlebrook 7H10, and thioglycollate.
8. Respiratory Tract
a. Upper Respiratory Tract
For nasal specimens
The premoistened swab should be inserted at least one inch into nares.
for nasopharyngeal specimens (Nas

(Nasopharynx)
the specimen of choice for the recovery of Bordetella pertussis. elushsal
It is
that is moistened with Stuart or Amies medium is used for the collection of the
A swab
specimen.
Sample Collection: A flexible swab is inserted through the nose into the posterior
nasopharynx and rotated for five seconds.
ad bluore For pharyngeal (throat) specimens
It is the recommended specimen for the routine culture of group A streptococci.
A
swab that is moistened with Stuart or Amies medium is used for the collection of
specimen.
Sample Collection: Posterior pharynx and tonsils should be swabbed without touching
the palate and sides of the mouth and tongue.
b.
Lower Respiratory Tract (Bronchoalveolar lavage, bronchial brush, bronchial wash)
An anaerobic culture is appropriate only if a sheathed (protected) catheter is used.
Sputum
Prior to sample collection, the patient should rinse his/her mouth with sterile water.
A first-morning specimen is preferred for an acid-fast bacilli (AFB) microscopy.
For mycobacterial infections, two to three consecutive early-morning sputum specimens
should be submitted.
The methods of collecting sputum can be done through deep cough (expectorated sputum)
or can also be aerosol-induced (induced sputum) for pediatric and "uncooperative" patients.
9. Gastrointestinal Tract
a. Gastric aspirate
The specimen should be collected early in the morning before the patient rises from the
bed or takes his/her first meal.

Gastric aspirates are the best specimens for infants. bled ad bluods sidal
It is used for the examination of AFB.
hour of collection.
The sample must be neutralized within one
b. Gastric biopsy
This specimen is recommended for the detection and isolation of Helicobacter pylori.
C. Rectal swab
Formicroscopy, methylene blue is utilized to observe fecal leukocytes.

1 cm to 1.5 cm through the anal sphincter, and feces
Sample collection: Insert the swab
collection. Ideally, two rectal swabs should be submitted to the
should be visible after
laboratory.
be utilized for rectal swabs.
Cary-Blair transport medium can
BACTERIOLOGY
10. Genital Tract
Hsinfected before collectingspecimen.

a. Bartholin cyst
The skin should be

The specimen should be aspirated.
b.
prior to sample collection.
The mucus should be removed the cervical canal should be
be used on
the speculum, and
A lubricant should not
thoroughly swabbed. medium.

The swab must be
moistened with Stuart or Amies
C. Cul-de-sac specimen (Sample aspiration)

d. Endometrium
Surgical biopsy or transcervical aspirate is performed using a sheathed catheter,
e. Urethra
Amies medium.
The swab must be moistened with Stuart or
2 cm to 4 cm into the urethra and
Sample collection: A flexible swab should be inserted
rotated for two seconds or alternatively; some discharge can be collected on a JEMBEC
transport system.
Vagina
Exudates should be removed before specimen collection.
The swab must be moistened with Stuart or Amies medium.
The secretions from the mucous membrane of the vagina are swabbed.
11. Urine
..5
a. Clean-catch voided midstream (clean-voided specimen or CVS) of urination
The first-morning urine is preferred because it provides a more concentrated specimen.
Females: The external genitalia should be cleansed first with soap and water, and the
labia should be held apart to begin voiding.
Males: The external genitalia should be cleansed with soap and water; the foreskin
should be retracted if necessary.
A pure culture of E. coli that is
greater than 105 CFU/mL represents a true infection.
b. Straight catheter
The urethral area
should be cleansed with soap and water.
Sample collection: After inserting the
urine should be
catheter into the bladder, the first 15 mL of the
voided or expelled by the bladder before collecting the ensuing urinary
stream as specimen.
C.
Indwelling catheter (Foley catheter)
The catheter collection port should be
sterile before starting the collection procedure.
Sample volume: 5 mL to 10 mLof urine should be aspirated with needle and syringe.
d. Suprapubic aspirate
Sample preparation: The skin should be disinfected prior to specimen collection.
Sample collection: The needle is inserted above the symphysis pubis through the
and then into the full urinary bladder where the urine sample is
aspirated.
Only authorized healthcare personnel should perform the sample collection.
12. Feces
It is the specimen of choice for detecting gastrointestinal pathogens.

Sample collection: A freshly-collected stool specimen without a preservative should be
submitted to the laboratory. The sample should not be contaminated with water or urine.
If a patient has received antiparasitic drugs, the specimen collection should be done after 7 to
10 days.
In case microscopy will be considered, methylene blue is utilized to observe fecal leukocytes.
13. Tissue Samples
The skin should be disinfected prior to the collection of specimens.

The specimen cannot be dried out so it should be moistened with sterile, distilled water if not
bloody.
Homogenization may be needed.
For anaerobic culture, tissue samples placed in a sterile container may be transported with
moistened gauze to prevent dryness and ideally in oxygen-free pouches.
14. Catheter and IV Tube Tips
Sample preparation: Skin disinfection prior to removal of the sample from the site, same as
the procedure for blood culture
Specimen "tips" should be aseptically placed into a sterile container with screw-cap.
It should be transported immediately to the microbiology laboratory.
Foley catheter is not accepted for culture.
15. Foreign bodies

a. Intrauterine device (IUD)
usually cultured for the detection of Actinomyces species.
It is
the removal of the IUD.
Sample collection: The skin is disinfected before
b. Catheters, pins, and prosthetic valves
A segment of the catheter (5 cm to 7 cm) is rolled four times across the agar using sterile
forceps (Maki roll technique).
When using catheters and valves, more than 15 colonies are required to perform
identification and susceptibility tests.
Critical (Panic) Results in a Clinical Microbiology Laboratory cut ezeBe
1. Positive blood culture
2. Positive peptide nucleic acid fluorescence in situ hybridization results for Staphylococcus aureus
and Enterococcus
3. Presence of Streptococcus pyogenes in a surgical wound
4. Positive capsular swelling test for Streptococcus pneumoniae

5. Presence of Streptococcus agalactiae in vaginal/rectal swabs among pregnant women
Positive AFB smear and Mycobacterium tuberculosis culture
7. Positive antibiotic-resistant bacteria
8. Positive CSF Gram stain, antigen test, and culture
9. Gram stain suggestive of Clostridium perfringens (gas gangrene)

10. Positive identification: Bacillus anthracis, Legionella, Francisella, and Brucella
ASSESSMENT QUIZ
Encircle the
letter that corresponds to the correct answer.
1. It is utilized as a transport medium for both viruses and bacteria such as Mycoplasma and
Chlamydia.
a.
b. Cary-blair
c. Amies
d. Stuart
2. Which of the following is NOT true regarding SPS?

a. inhibits phagocytosis
b. used in 0.25% concentration
C. suppresses the growth of Neisseria
d. neutralizes the bactericidal effect of plasma
3. It should be maintained at 37°C if there is delay in the processing of this sample.

a. nasopharyngeal swab
b. tissue samples
C. sputum
d.
4. Which substance that may be present in cotton-tipped swabs is inhibitory and toxic to some
bacteria?
a. Fatty acid
b. Charcoal
c. Carbon
d. Formalin
5. Which of the statements is correct regarding sample collection for microbiological studies?
a. Nasopharyngeal swab should be rotated at least 10 minutes to achieve enough sample.
b. 10 mL of blood is a requirement for pediatric blood culture.
C. A single random sputum sample is enough for AFB microscopy.
d. Calcium alginate swab is preferred in bacteriology but not in virology.
114
that are suspected of

6. Which procedure is required for processing samples containing
mycobacteria?
a. Cytocentrifugation
b. Homogenization
C. Decontamination
d. Concentration
7. All are considered "panic laboratory results" EXCEPT:

a. presence of antibiotic resistant bacteria
b. positive coagulase
c. microscopy suggestive of Clostridium perfringens oun TOvat snlwollot edi to moidw
d. positive capsular swelling test for pnetimococcus
in a refrigerated temperature while waiting
8. Which of the following samples should not be stored
for processing?
a. specimens for anaerobic bacteriology necle
b. sputum
c. viral samples
d. fomites
culture media to inhibit the accumulation of fatty acid in

9. Which of the following is added to
cotton-tipped swab?
a. heparin
b. bile salt
c. alcohol
d. charcoal
10. Which of the following is the best skin disinfectant prior to blood culture?
a. isopropanol then tincture of iodine
b. chlorhexidine gluconate only
c. tincture of iodine only
d. 95% alcohol
CHAPTER 11
CULTURE AND
CULTURE MEDIA

1. discuss the types of culture media and their purpose;

2. cite the specific components of selective and special media;
3. explain the requirements for incubation of various organisms including
anaerobic culture;
4. distinguish the types of inoculation techniques;

5. describe the colonial growth of bacteria and generation time; and
6. differentiate the stages of bacterial growth.
Cultures are the growth of microorganisms in a culture medium. Since the

study of microorganisms relies on their ability to replicate
the host, the appropriateness of culture media is certainly important.
A culture medium is composed of a mixture of nutrients such as carbon, ado
nitrogen, sulfur, phosphorus, hydrogen, oxygen, and buffers. A liquid, semi-
solid, or solid medium are essential to observe patterns of microbial growth

apart from their purpose for transport and storage.
Inhibitory agents like dyes, salts, and antimicrobials are added into the
medium to facilitate the isolation of the desired bacteria while suppressing the
other organisms present in the sample.
The selection of the medium for inoculation is based on the type of
specimen submitted for culture and the characteristics of organisms that

are involved in the infection process. For instance, if several plates will be
inoculated for specimen, the
media should be arranged starting with
a given
the most enriched to selective. In this manner, inhibitors added to the selective
media will not be carried over from one medium to another.
isolation and identification of microorganisms in the areas
of
The
industrial and food microbiology require a specialized culture media. thritat
Types of Culture
1. Pure culture
It contains a single species.
2. Mixed culture
It contains more than one species.
3. Stock culture
medium (one species per
t is composed of several species contained in a separate culture
culture medium).
then divided into small vials; this
It should be cultivated in a large volume of broth and
year.
lengthens the shelf life the sample culture to at least a
It is used for academic and industrial purposes.
Classification of Culture Media
According to Consistency
1. Liquid medium
It does not contain any amount of agar.
It allows the growth of aerobes, anaerobes, and facultative anaerobes.
Examples: Brain heart infusion (BHI), trypticase soy broth (TSB), and thioglycollate
It contains 0.5% to 1% agar.
It is used to observe bacterial motility and detect indole and sulfide production.
Example: Sulfide indole motility (SIM) medium 110V20
loa
3. Solid medium
It contains 2% to 3% agar
Examples: Triple sugar iron (TSI) agar, MacConkey (MAC)

agar, blood agar plate (BAP), and
chocolate agar plate (CAP)
Notes to Remember
Agar is a sulfated polymer made up of

D-galactose, 3,6-anhydro-L-galactose, and D-glucuronic acid
and is usually derived from red algae.
Agar will melt if the temperature reaches 80°C to 90°C and

solidify at 40 °C to 50°C.
The cooling temperature for distribution of culture medium into
Petri plates is 55°C to 60°C.
Molten agar in the amount of 20 mL to 25 mL should be transferred to
sterile plates.
YOQJOIRETDAS D1T2 CULTURE AND CULTURE MEDIA 117
According to Composition
1. Synthetic or defined medium
It is a medium in which
all the components are known.
designed for research purposes as either a liquid or solid medium.
It is
It is preferred for the isolation of cyanobacteria and chemoorganotrophs."

edf not ai 4 stinole?
An example is the BG-11
2.
Non-synthetic or complex medium
to nonibh
It is a medium in which some of the substances are unknown (peptones, meat, and yeast
extracts).
useful for
It is
the isolation of medically significant bacteria."onarlan dhond svits.om-nre1D
Some examples are nutrient broth (NB) medium, TSB, and MAC agar.
Pliod el stond Vo
3. Tissue culture medium

It is utilized for
Chlamydia).2n3 its el tond mint
obligate intracellular bacteria (Rickettsia and
Some
examples are W138 cells, HeLa 229 cells, and McCoy cells.
HeLa 229 cells are human cervical tissue cells while McCoy cells and W 138 cells are
fibroblasts. All of these are used for the isolation of Chlamydia. Gibomn gis 99901
Embryonated eggs are utilized for the propagation of Rickettsia.

sibam sgvi-biloe gun geodT
According to Dispensing or Distribution of Culture Media lamnke smod

Plated media are distributed into the dish or plate. il! siaimmallib of basiliar ai GAa
Tube media are prepared as either liquid, slant, butt and slant, or butt. nitrot : 9D
Examples: SIM, Triple sugar iron (TSI) agar, Simmons' citrate agar (SCA), and lysine iron agar
(LIA)
These are routine in the laboratory and without additional supplements. ortite D
These are media that support the growth of most non-fastidious bacteria. DAM
of meat and soybean extracts.
They are usually composed
Examples: Nutrient agar (NA), nutrient broth (NB), and
2. Enrichment media (Liquid-type media)
They propagate certain group of bacteria from a mixture of organisms. aMa

These contain specific nutrients and without additional supplements. b) 10106
These media are incubated for a certain period and then subculture to isolate the desired
organism.
These can also be used as a supplement to agar plates to detect aerobes, anaerobes, and
microaerophiles.
Examples: Alkaline peptone water, Selenite F, thioglycolate, tetrathionate, Lim broth, and
Gram-negative (GN) broth
BACTERIOLOGY
Notes to Remember
8.4 is best for

Vibrio species before inoculation into a
pH
Alkaline peptone water adjusted to
thiosulfate-citrate-bile salt-sucrose (TCBS) agar.

and water samples.
Selenite F is for the isolation of Salmonella from feces, urine,
that promotes the growth of
almost all non-
enrichment medium
Thioglycolate is a general support
fastidious bacteria.
is a selective enrichment broth for the isolation

of Salmonella and Proteus; the addition of
Tetrathionate
bile salt and thiosulfate into the medium suppresses other coliform bacilli.
Gram-negative broth enhances the growth Salmonella and Shigella.
GN broth is both an enrichment and selective medium; it contains sodium citrate and sodium
which inhibit Gram-positive bacteria.
Lim broth is an enrichment medium for group B streptococci.
3. Enriched media and non-selective media
These are media with additional supplements such as blood, vitamins, and yeast extract,
which are necessary for the growth of fastidious organisms.
These are solid-type media.
Some examples are BAP and CAP. to noilsmaig 10
BAP is utilized to
differentiate the hemolytic patterns of bacteria.
CAP is routinely used for the recovery of Haemophilus species.
1628 noa
P BAP and CAP promote the isolation of fastidious organisms.
4. Differential media
These media allow

the visualization of metabolic differences between groups of bacteria.
Some examples are MAC, BAP, eosin methylene blue (EMB), Hektoen enteric agar (HEA), and
Cystine-Lactose-Electrolyte-Deficient(CLED) agar.
MAC separates lactose fermenters (pink
colonies).
colonies) from non-lactose fermenters (colorless
distinguishes hemolytic patterns of streptococci.

BAP
Neutral red is the

the production of acid. of sugar and eventually
EMB differentiates
E. coli(greenish metallic sheen
bacteria
(dark purple colonies). colonies) from other lactose fermenting
HEA identifies theSalmonella(bluish green colonies with black centers)

colonies) while the other coliforms Shigella (green
CLED agar
appear orange or salmon pink colonies.from
also characterizes those bacterial that utilize
medium), and it is recommended for urine lactose
(both yellow colonies and
culture.
YOGICIPTIDAS DIT CULTURE AND CULTURE MEDIA 719
5.
These media are incorporated with antibiotics, dyes, or chemicals to inhibit

other organisms the growth of
while promoting the growth of the desired bacteria.
Some examples are HEA, MAC, xylose
(BSA), mannitol salt lysine deoxycholate (XLD) agar, bismuth sulfite agar
agar (MSA), and Thayer-Martin agar (TMA).
Other selective media and their purpose:
Gentamicin blood
agar for Streptococcus
b.
Bacitracin chocolate agar for
C. Blood agar plate with ampicillin for Aeromonas rmtnos eslhtod muthin sidetes edT
d. Phenylethyl alcohol agar (PEA) for Gram-positive bacteria
e.
Columbia colistin-nalidixic acid (CNA) agar for Gram-positive bacteria
f. Bacteroides Bile Esculin Agar (BBEA) for
anaerobes such as Bacteroides fragilis
To inhibit
a.
Gram-positive bacteria: Crystal/Gentian violet, basic/carbol fuchsin, and bile salt
long b. To inhibit Gram-negative bacteria: Potassium tellurite and sodium azide cunaly
C. To
inhibit swarming of swarming bacteria: Alcohol and chloral hydrate
Notes to Remember
HEA contains bile salt, acid fuchsin (dye), and pH indicator bromothymol blue. The bile salt and dye
inhibit the indigenous microbiota of the lower GIT (non-enteric pathogens).
MAC contains bile salt and crystal violet that are against Gram-positive bacteria.
XLD agar contains xylose, lysine, sucrose, 0.25% sodium desoxycholate salt, and sodium thiosulfate.
agar differentiates Salmonella (red/colorless colonies with black centers) from Shigella (red/
colorless colonies without black centers).
HEA, XLD, and MAC promote the recovery of fecal bacteria.

bna onits sunk biull shod shote
MSA supports the growth of Staphylococcus aureus.
TMA is primarily selective for Neisseria species.

BAP, CAP and MAC are generally used for routine isolation of aerobic bacteria, while BBE, PEA (also
used for aerobic bacteriology), and *CDC-BAP are for anaerobic cultures.
*CDC - Centers for Disease Control and Prevention
6. Special media
It is used to solate bacteria with specific growth requirements.
Some examples are Lowenstein-Jensen (L) medium and thiosulfate-citrate-bile salt-sucrose
(TCBS) agar.
LJ medium is protein-rich medium that is composed of whole eggs and malachite green.
and sterilization is through inspissation and not by
It supports the growth of mycobacteria
autoclaving.
BACTERIOLOGY
TCBS is selective for the isolation

of Vibrio in which sterilization is
through boiling, and
never by autoclaving.
media is not by routine autoclaving, they are
Since the sterilization of both LJ and TCBS
169 guilde classified as "special culture media"
Blood Culture System

The blood culture bottle is utilized for the isolation of aerobic and anaerobic bacteria,
microaerophiles and fungi.
The aerobic culture bottles contain combinations of trypticase soy broth, brain-heart infusion
broth, Brucella broth, or Columbia broth with soybean casein digest and peptones; while for the
anaerobic culture bottle, it has the same basic components as the aerobic culture system and 0.5%
cysteine with decrease exposure to oxygen to support the growth of anaerobes.
An antimicrobial removal device (ARD) or resins is added into the medium to absorb any
antibiotics that have been taken by the individual and are present in the blood at the time of
fica • sample collection.

Manual Blood Culture System: Septi-Chek (biphasic-CAP and MAC) medium, Oxoid Signal
(also used formicroaerophiles), and Isostat/Lysis Centrifugation (for fastidious bacteria,
mycobacteria and fungi)
Automated Blood Culture System: BACTEC system (the detection of carbon dioxide produced
after the consumption of the substrate by the bacteria, indicates microbial growth)
After plating or transferring the media to tubes, a representative uninoculated sample of each
ir batch of preparation should be incubated overnight at 35°C to observe for the presence of
contaminants (bacterial growth).
Inoculation of Specimen
Sterile body fluids, pus, urine, and sputum are inoculated directly into the culture media.
Specimens received on swabs can be inoculated directly into the desired culture media even in
the absence of a transport medium.
Some examples specimens that require a direct or "bedside" inoculation are blood,
of
genital specimens, corneal scrapings, sterile fluids like synovial

and peritoneal fluids, and
nasopharyngeal swabs for isolation of Bordetella pertussis.
Inoculation Techniques
Streaking is the most common manner of inoculation.
A specimen that is collected through a swab is inoculated by gently "rolling the tip of the
swab" onto the upper edge of the plate, then
the inoculated area should be streaked (quadrant
streaking technique) by a sterile loop to distribute the specimen.
Liquid samples, such as urine, blood, and synovial fluid, are directly inoculated into appropriate
culture medium.
CULTURE AND CULTURE MEDIA 121
Purulent samples like aspirates in transport containers should

be vortexed, and a drop should be
placed onto agar plates and streaked into liquid medium.
vol winlt belsse
The "stabbing" of the medium is usually
performed with group A streptococci to create
anaerobiosis and promote sub-surface hemolysis.
In caseof an
catheter, it is transferred into a thioglycolate tube
IV
tip may be rolled over onto

or by using sterile forceps, the
solid culture media four times, then streak out.
Overlapping inoculation is used for antimicrobial susceptibility test (disk diffusion method).
Manner of Inoculation
For culture
plates, the inoculating loop is sterilized and allowed to coolthoroughly before use.
For urine culture,
it suggested that the inoculating loop should be flamed in between agar plates
to prevent carry over contamination.
Urine specimens are inoculated using a quantitative isolation technique with a calibrated loop
91 25 (0.01 mL or 0.001 mL) to deliver a specified volume. ko
inoculation is used for the isolation of the pathogens that are sensitive to drying
Bedside or direct
or extreme temperature. However, specimens collected at the bedside are more susceptible to
contamination.
When more than one agar plate is used, inoculation should start from the nonselective to the
selective agar plates.
The selection of culture media for inoculation considers the source of the specimen or the
anatomical site for which the Organism is possibly located. Algptionpttoa.snica oel esdl fl
Anaerobic Cultivation
Ways to Facilitate Anaerobic Cultivation

Pi 1. The culture media should be protected from oxygen exposure, freshly prepared, and should be
held in an anaerobic condition until needed.
2. The use of special culture medium incorporated with thioglycolate and cysteine (reducing
agents).
3. The boiling of culture medium to remove (drive off) oxygen.
4. The use of an anaerobic chamber with a vacuum pump for processing of samples and storage of
culture media prior to inoculation.
gloveless chamber are the two (2) types of the
anaerobic
the
The sealed glove box and
chamber system.
hood and incubator.
The anaerobic chamber serves as the inoculation
The chamber system also allows isolation, susceptibility testing, and reading of plates.
an anaerobic chamber, a gas-pak jar and plastic pouch (anaerobic biobags)
In the absence of
containing a palladium catalyst can be utilized.

both anaerobic chamber and holding
5. Increase concentration of nitrogen
and hydrogen gases in
system (gas-pak jar and anaerobic biobags).

Components of an Anaerobic Chamber:

Sealed Glove Box and Gloveless
anaerobic atmosphere
Nitrogen gas - acts as a filler for the remaining percentage of the
9060.
1.
and isolation of anaerobes
facilitate the growth
2. Hydrogen and carbon dioxide gases
remove residual oxygen
from the chamber by combining with
3. Palladium catalyst pellets
hydrogen to form water
hydrogen combines with the free
4. Silica gel (dessicant) - absorbs the water that is formed when
oxygen
5., Methylene blue/reazurin an oxygen-reduction indicator that becomes colorless in the absence
of oxygen
The ideal anaerobic incubation system is the anaerobic chamber using neoprene gloves
attached and sealed to the incubator ('gloveless chamber").
When the CO, content of an anaerobic incubator is increased to 10%, the oxygen is lowered to
approximately 18%.
The College of American Pathologist (CAP) requires laboratories to conduct daily monitoring
of the anaerobic chamber using either of the oxygen reduction indicators to determine if the
incubator achieves the desired purpose.
Gas-pak Jar
It has the same components of the anaerobic chamber, except that the gases envelope
are in the
pouch.
To generate gas, the envelope pouch can be treated with or without water (waterless gas
generator).
When water added to the
envelope pouch, carbon dioxide and hydrogen
is
anaerobiosis takes 30 minutes to almost an hour to achieve

are produced, and
the desired condition.
For waterless gas
generator, it should be utilized immediately once opened; and carbon dioxide is
produced without hydrogen and water vapor
inside the jar while oxygen is rapidly absorbed.
Failure to achieve an anaerobic condition
could be the result of a "poisoned" catalyst crack in
or a
the O-ring, jar, or lid (for water-treated gas generator).
The disadvantage of using an anaerobic jar is that the
plates have to be removed from the jar
every time it is examined.
Incubation Conditions
1. Aerobes: 21% O, + 0.02% CO,
2. Anaerobes: 0% ©, 5%-10% CO, 5%-10%

chambers) H, + 80%-90% N, (anaerobe jars, bags/pouches, of
Capnophiles: 5%-10% CO, + 15%
O (CO,incubator or bag;3% CO in candle jars)
3.
4. Microaerophiles: 5%-10% O,
8%-10% CO, 80% N,
rod lomanbas 0112 CULTURE AND CULTURE MEDIA 123
Grading of Growth on Plate

a.
4t: Signifies many, heavy growth, if growth is up to the 4th quadrant
b. 3+: Signifies a
moderate growth; if growth is up to the 3rd quadrant
C.
2+: Signifies a few or light growth; if growth is in the 2nd quadrant
d.
1t: Signifies a rare growth; if growth is in the 1st quadrant only
Notes to Remember
For most bacteria, the ideal incubation temperature is 35°C to 37•

For initial reading of plates and tubes, aerobic bacterial cultures should be examined at least after 18
hours of incubation while anaerobic cultures require 24 hours to 48 hours of incubation.
Most routine bacterial cultures whether in broth or solid media are held for 48 hours to 72 hours.
Cultures for anaerobes may be held for 5 days to 7 days.

Blood culture bottles (broth and biphasic medium) are incubated up to 7 days for manual method and
up to 5 days for automated procedures.
For the automated systems, blood culture bottles are exposed
to mechanical agitation to increase
oxygenation of the broth and ulteriorly enhance the faster detection of organisms.
Slow growing bacteria, such as mycobacteria, require 14 days to more than a month of continuous
incubation under the desired conditions before visible colonies appear.

Atypical bacteria may require special media or atmosphere beyond the routine setup.
TABLE 11-1. Culture Media and Their Purposes

Culture medium Purpose
Vibrio
Alkaline peptone broth
Anaerobic blood agar (CDC) Obligate
Bacteriodes bile esculin agar Gram-negative anaerobes
Bile esculin agar Enterococci and group D streptococci PDt o ebeatop

Bismuth sulfite agar
Selective media for Salmonella (stool)
Blood agar Primary plated medium; Differentiation of hemolytic
patterns
Bordet-Gengou agar Bordetella pertussis and Bordetella parapertussis

Selective medium for Salmonella
Brilliant green agar
Francisella tularensis
Blood cystine dextrose agar
Brain heart infusion agar and broth Fastidious organisms
Buffered charcoal yeast extract (BCYE) Legionella spp. and Nocardia

Campylobacter spp.
Campy-blood agar
BACTERIOLOGY
Purpose
Culture medium
Selective holding medium for Campylobacter; used for
Campy-thioglycollate broth cold enrichment technique (4°C)
Obligate, slow-growing anaerobe
CDC anaerobe 5% sheep blood agar
Campylobacter spp.
Cefoperazone vancomycin amphotericin (CVA) agar Yersinia enterocolitica and Aeromonas
Cefsulodin-irgasan-novobiocin (CIN) agar
Pseudomonas aeruginosa
Cetrimide agar
Chlamydia and Rickettsia
Chick embryo
Fastidious bacteria such as Haemophilus and
Chocolate agar
pathogenic Neisseria
Haemophilus spp.
Chocolate agar with horse blood
Chromogenic media
Methicillin-resistant Staphylococcus aureus (MRSA)
Columbia colistin-nalidixic acid agar (Columbia Gram-positive cocci
Note: Anaerobic CNA for anaerobes
Cystine tellurite blood agar (CTBA)
Cycloserine cefoxitin fructose agar (CCFA) Clostridium difficile
Nocardia asteroides
Czapek agar
Dorset egg medium Mycobacterium tuberculosis and
Nocardia asteroides
Deoxycholate citrate agar 6114

Salmonella and Shigella (stool specimen)
Dieudonne's medium Vibrio cholerae
Dubos oleic agar Mycobacterium tuberculosis
Edward-Hayflick agar Mycoplasma

Eosin methylene blue or Levine's medium Differential medium for lactose fermenters (LFs) and
non-lactose fermenters (NLFs)
Ellinghausen, McCullough, Johnson, and Harris Leptospira interrogans

(EMJH) medium
Fletcher semi-solid medium
Leptospira
Fetal bovine serum agar with vancomycin Haemophilus ducreyi
Footpads of mice (living tissue)
Mycobacterium leprae
Gram-negative broth Selective and enrichment medium for enteric
pathogens like Salmonella and Shigella
Human blood
Tween bilayer medium Gardnerella vaginalis
Hektoen enteric (HE) agar
Differentiation of Salmonella and Shigella
Kelly medium
Borrelia burgdorferi
Laked Kanamycin-Vancomycin with Blood (LKVB)
agar
Bacteroides and Prevotella
Loeffler blood serum medium
Lowenstein-Jensen (LJ) agar
Mycobacterium
Lysine iron agar
Litmus milk media
Clostridium
YOOJOIRTBAE.511EM CULTURE AND CULTURE MEDIA 125
Culture medium
Purpose
MacConkey (MAC) agar
Differential medium for LF and NLF; selective for
Gram-negative bacteria
MacConkey (MAC) agar with sorbitol
E. coli 0157:H7 in fecal specimen
Mannitol salt agar
Staphylococcus aureus
Martin-Lewis agar
Neisseria gonorrhoeae
McBride medium
Listeria monocytogenes
Methylene blue milk
Enterococcus
Middlebrook 7H10 agar
Mycobacteria
Modified Lombard-Dowell medium Aerobes and anaerobes
Modified Thayer-Martin Agar Neisseria gonorrheae and Neisseria meningitidis
Mueller-Hinton agar Susceptibility test (antimicrobial testing)
New York City (NYC) agar Neisseria gonorrhoeae, Ureaplasma urealyticum and
Mycoplasma spp.
Oxidative-fermentative (OF) medium Differential media for non-fermentative bacilli®
Petragnani medium Mycobacteria

Phenylethyl alcohol (PEA) medium Selective isolation of aerobic and anaerobic Gram-
positive bacteria
Rabbit blood agar with yeast extract Haemophilus influenzae #6A0 8312010
Regan-Lowe agar Bordetella pertussis
Salmonella-Shigella agar (SSA) ang iridi Salmonella and Shigella netbed anod
Schaedler agar mort bNon-selective medium for aerobes and anaerobes
Seller agar
Selenite broth (enrichment medium) Salmonella SPP. Ssgge win anol ware les Iutal.
agar
Campylobacter spp.
Tetrathionate broth Salmonella and Shigella spp.
Neisseria gonorrhoeae and
Thayer-Martin agar Neisseria meningitidis
Aerobes, anaerobes, microaerophiles, and
Thioglycollate broth aerotolerant anaerobes
Vibrio
Thiosulfate-citrate-bile salts-sucrose (TCBS) agar
08001 Corynebacterium diphtheriae-naianop
agar
Enrichment and selective medium for Streptococcus
Todd-Hewitt broth with nalidixic acid and
gentamicin or colistin
Blood culture bottle base and enrichment broth
Tryticase soy broth (TSB)
Aerobic actinomycetes
Tyrosine agar
Salmonella and Shigella
Xylose lysine deoxycholate (XLD) agar
BACTERIOLOGY
Cultural Characteristics
One of the major features of bacteria is their appearance followingtheir growth on
various
media.
The color of the colonies and the

culture medium, the abundance of growth, and odor of the
culture make each genus or species unique.
Manner of Reporting
1. Agar Plate Colonies
a. Size
while others appear mucoid or slimy.
Coloniesrange from small (pinpoint) to large
Pseudomonas and Proteus spread across the entire agar surface (swarming).
b. Margin and Elevation
or entire.
Margin of colonies is mostly smooth
Filiform, lobate, and undulate margins are also observed.
sometimes umbonate.
As to elevation, colonies may appear raised, flat, convex or
C. Chromogenesis
Colonies may be pigmented or colored, but not all bacterial species have this distinctive
feature.
Some bacteria retain the pigment within the cell while other bacteria color the medium.
Pigment production is best observed from a growth on solid media.
d. Optical features DR1gr
. Colonies may appear opaque, translucent, shiny, or opalescent.
e. Odor
Growth of bacteria may produce putrid, fruity, ammoniacal, or aromatic odor.
2. Growth on Agar Slant

Amount growth may be scanty, moderate, or abundant
Margin or edge - same with the description on agar plate

Consistency - colonies may appear butyrous or butter-like, viscous or stingy, dry and brittle
Chromogenesis - colonies may be pigmented
Odor - same with the description on agar plate
3. Growth in Nutrient Brotha
It may appear turbid (10° CFU/mL of broth is needed to create turbidity).
It is confined on
the surface of the broth as a film (pellicle) or accumulated as sediment.
It may also be reported
as viscous in appearance.
r CULTURE AND CULTURE MEDIA 127
4. Growth in Gelatin Slant
It is confined within the zone of inoculation also known as a "thread-like" or "beaded-like/

string of pearl" pattern.
015 21115
There are microorganisms that demonstrate varying degrees
inoculation and the
of of spreading outside the line
("spiny"), growth appears like rhizoid, arborescent ("tree-like), echinulate
or effuse ("common spreading growth"),
Sometimes the bacterial growth exhibits liquefaction of gelatin that starts evenly from the
top of the agar slant and occurs as a funnel-like pattern.
5. Growth in Blood Culture Bottles
For manual procedure, blood culture bottles are examined
macroscopically for turbidity and
hemolysis using transmitted light.
For biphasic medium
(Septi-Chek), bacterial colonies are observed in or on the agar, while
the presence of fluids
are noted on other culture bottles (Oxoid bottle).
In the automated system, microbial growth
is evident through production of gas such as
nobel
carbon dioxide (BACTEC system) or multiple gases such as carbon dioxide, hydrogen, and
oxygen (Versa TREK).
Culture bottles from manual or automated system with evidence of growth and "flagged"
should be removed from the incubator and equipment. A portion of the fluid should be
processed for Gram staining and subculture (agar plates) to confirm whether a pathogen is
present in the samples.
Bacterial Growth Curve
The generation time of bacteria in a culture varies according to their cellular properties. It takes
20 minutes for a fast-growing bacterium such as Escherichia coli or as long as 24 hours for a slow-
growing bacterium such as Mycobacterium tuberculosis.
Stages of Bacterial Growth

1. Lag Phase or Period of Rejuvenescence
the cell number.
It is the period when there is no cell division or an abrupt increase in
It is the start of biosynthesis although there is no increase in cell mass.
It is the adjustment phase to a new environment.
ow! ofni aabivib Moolighia 6
2. Log or Exponential Phase
It is the period when microorganisms are actively growing and dividing.
It is the stage in which the bacteria increase logarithmically since cellular production is most
active during this period.

It is the phase in which
microorganisms are utilized in physiological, biochemical. and
antimicrobial testing.
BACTERIOLOGY
3. Stationary/Plateau phase division and dying organisms although

balance between cell
is
It is the period when there
remains constant.
the number of viable microorganisms
of surviving cells slow down andnutrientsare
It is the phase in
which metabolic activities
becoming limited. GR
accumulate.
It is the phase in which dead debris starts to
4. Death or decline phase
growth as the number of dead cells
is a cessation of bacterial
It is the period when there
exceeds the living microorganisms.
increase in the amount of toxic waste.
It is the stage in which there is a loss of nutrients and
Terminologies
1. Generation - is the doubling of the cell number

2. Generation time/Doubling time is the time required for bacteria to double their population
Stationary Phase
Decline Phase
Log Phase
Lag Phase
FIGURE 11-1. Bacterial Growth
Reproduction
Transverse Binary Fission

It is the most common asexual
reproductive process in which a single cell divides into two
daughter cells after developing a transverse cell wall.
Growth Measurement
1. By cell count .
microscopy, use of electronic particle counter, or colony count
2. By cell mass
-weighing, measuring the nitrogen content, and turbidimetry
3. By cell activity - observation of
the biochemical activity
VOG IDIGATOND SIT3 CULTURE AND CULTURE MEDIA 129
TABLE 11-2. Methods for

Measuring Bacterial Growth
Methods
Application
1. Microscopic count
Enumeration of bacteria in milk and vaccine
A measured volume
of a bacterial suspension Breed count method (milk)
is placed
on a microscope slide or on a
counting chamber. Petroff-Hausser counter - for prokaryotes
It
does not distinguish between living cells Hemocytometer for prokaryotes and eukaryotes
and dead cells.
2. Plate count
Enumeration of bacteria in milk, food, water, and soil
It is the most commonly used method.
It measures the number of: viable cells.
It determines the CFU/mL of bacteria.

30 to
300 colonies (non-aggregate cells)
should be counted.
3. Membrane or molecular filter Enumeration of bacteria in food and water (including
It utilizes a polycarbonate membrane filter or lakes and streams)

millipore filter.
4. Turbidimetric method (Cell mass) It is used to prepare the standard inocula for
It requires 10 to 100 million cells/mL. antimicrobial testing.
5. Dry weight determination (Cell mass) For filamentous organisms (fungi)
6. Biochemical activity Enumeration of bacteria in milk

130 REVIEW HANDB0OK IN DIAGNOSTIC BACTERIOLOGY
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nm t0e eu
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mu BAPrp CAP
MAC
FIGURE 11-3. Blood agar plate [BAPJ, chocolate agar plate [CAPJ,
and MacConkey agar (MACJ
FIGURE 11-4, Colonies on blood agar plate

oldEe 3piCULTURE AND CULTURE MEDIA 131
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ASSESSMENT QUIZ

bottle?
medium in blood culture
1. Which of the following is the commonly used
a. thioglycollate
b. trypticase soy broth
¢. nutrient broth
d. tetrathionate broth
which bacteria does "stabbing of the agar

medium" serve as the recommended inoculation
2. For
technique?
a. obligate anaerobic Bacteroides
b. microaerophilic Campylobacter®
C. facultative anaerobic Klebsiella
d. group A streptococci
3. It is classified as a special culture medium since sterilization

is not done by the usual autoclaving
but by inspissation.
a. L-J medium
b.
C. BBE
d. CLED
4. It removes residual oxygen from the gas pack jar to promote anaerobiosis.
a. Palladium pellets
b. reazurin
c. silica gel
d. methylene blue
5. Which does not represent the typical growth patterns of microorganisms in liquid medium?
a.
b. scanty
C. sediment
d. turbid
CULTURE AND CULTURE MEDIA 133
6. Which of the following is added to

agar medium
a.
chloral hydrate to inhibit the Gram-negative bacteria?
b. sodium azide
C. bile salt
d. basic fuchsin
What term refers to

7.
the time required for the bacteria to double its
population?
a. lag phase
b. doubling time
c. exponential
d. proliferation time
8. It is both differential and

selective medium.
a. CAP
b. BAP
d. TMA
9. It is a phase of bacterial growth where isolates are utilized for phenotyping testing.
a. stationary phase
b. rejuvenescence
c. plateau
d. log phase
10. Which of the following methods is used for measuring bacterial growth?
a. Microscopic count
b. Turbidimetric method
C. Biochemical activity
d. All of the above

CHAPTER 12
MICROSCOPY

2. differentiate the various types of microscopes; and
3. explain the parts of a bright-field microscope.
Microscopy is a fundamental method used for both the detection and

characterization of bacteria. A microscope is vital in magnifying these
microorganisms to make them visible for identification purposes. Ideally, a loo boeit
microscope should be parfocal, that is, the image should remain in focus even
when the objectives are changed.
Types of Microscopes
1./ Light Microscope disk
In thismicroscope, the visible light passes through the specimen
and then through a series of lenses that reflect the light, resulting potent blus
in the magnification of the organisms that are present in the
specimen.
It has glass lenses to bend and focus light rays, thus creating
magnified images of small objects.
To effectively visualize cells through light microscopy, at least 105
cells per milliliter of specimen are required.
136 BEVIEW HANDBOOK IN DIAGNOSTIC BACTERIOLOGY
Notes to Remember
size of the bacteria is micrometer (um).

The unit of measurement for determining the
lens of a light microscope.
An ocular micrometer is located within the eyepiece
is a measure of the relative velocity at which light passes through a material Or
Refractive index
specimen.
The total magnification is the product of the lens and the objectives that are used.
a. Brightfield Microscope
clinical laboratory.
It is the most commonly used light microscope in
It forms a dark image against a "brighter" background.
It can distinguish between two dots or cells that are 0.2 um apart.
Magnification of the Lens
a. Ocular (Eyepiece) lens - 10x

b. Objective lenses
Low power - 10x
High power - 40x

Oil immersion - 100x
Purpose of the Cedar Wood Oil
a. It enhances the resolution of the microscope.

b. It is used to fill the space between the objective lens and the glass slide.
C. It
prevents light rays from dispersing and changing their wavelength after passing
through the samples.
Microscopic Resolution or Resolving Power

Resolving power refers to the ability of the lenses to
separate closely distant objects.
Resolution is determined
by numerical aperture and wavelength of light. 15M
The
resolution is increased by decreasing the wavelength of coming from the
light
MINOOR MICROSCOPY 137
TABLE 12-1.
Parts of a Brightfield Microscopy
Parts of the Microscope
Function
Ocular (Eyepiece) Lens It is
through this part of the microscope that the object is observed.
To produce high magnification with good resolution, the lens
must be small.
Diopter Adjustment Ring It is
used to compensate for the difference in the observer's sight or
vision between the left eye and the right eye.
Interpupillary Distance Scale It determines the distance between the left eye and the right eye.
Interpupillary Distance Adjustment Seat It is used to adjust the interpupillary distance scale.
Observation Tube
amelitimnogmim It is a monocular or binocular tube that transmits the image from
the objective lens to the ocular.
Arm
It is used for holding the microscope.
Revolving Nosepiece It holds the objectives.

Rotating this part easily changes the magnification of the objectives.
Objective Lens It is the most important part of the microscope producing a clear
image of the specimen.
It is the primary lens that magnifies the specimen.
The higher the magnification. of an objective, the greater is the
radio Ils enduin doldw agne 9 feature or detail seen in a specimen. 2128110
Coarse Focus Adjustment Knob It is rotated to locate into focus the specimen® by moving the stage
up and down.
Fine Focus Adjustment Knob frocked It is rotated to obtain a clearer view of the image.
Coarse Focus Adjustment Knob and It rotates the ring to adjust the tension of the coarse focus
adjustment knob.
Tension Adjustment Ring
Vertical Feed Knob It is rotated to move the specimen glass plate in a vertical direction.
Horizontal Feed Knob It is rotated to move the specimen glass plate in horizontal
direction.
Observation Tube Securing It is used to secure the observation tube.

Knob
It should be loosened when rotating the observation tube.
MENING It is the part where the specimen is placed.
Mechanical Stage
Specimen Holder bus-beld nt alien It holds the glass slide in position.
It focuses the light from the illumination source to the specimen and
Condenser
is magnified by the objective lens.
It controls the amount of light entering the condenser.
Condenser Iris Diaphragm Dial
It is a filter with a diameter of 45 mm.
Window Lens
It can be rotated to adjust the illumination or brightness of the light.
Brightness Adjustment Knob
Base
It supports the entire microscope.
b. Phase-contrast Microscope
It permits a detailed examination of internal structures in living organisms.
in a culture.
It is used to identify medically significant fungi grown
be used to examine
Staining is not included in this type of microscopy, hence, it can
unstained living cells.
It has the same magnification as the dark-field microscope.
C. Fluorescence Microscope
It involves the excitation of fluorochromes using light. inemuts
It uses fluorochromes (dyes) to stain or attach to microorganisms.

It is designed to observe chlamydia, legionella, mycobacteria, and fungi.
The magnification of this microscope is 10x to 400x.

Examples of fluorochrome dyes: Acridine orange, auramine and rhodamine, calcofluor
white, and fluorescein isothiocyanate (FITC)
d. Dark field Microscope
It uses a dark field condenser that blocks light that will enter the objective directly.
light to hit the specimen at an oblique angle which makes all other light
It directs the
that passes through the specimen miss the objective, thus, creating a "dark field" while
the organisms appear extremely bright. 10t c
It is utilized to observe spirochetes (Treponema pallidum).
It examines unstained microorganisms suspended in liquid against a dark background.
It
detects living microorganisms that are invisible under ordinary light.
The magnification of this microscope is 10x to 400x.
2. Electron Microscope
It utilizes electrons instead of light to visualize small objects.

It has electromagnetic fields instead of lenses to form an image on a fluorescent screen. thald
It has a built-in camera to capture images of the cells in black-and-white transmission
electron micrographs.
It is ideal for studying the morphology of bacteria
The advantage in using this microscope is that objects smaller than 0.2 um can be visualized.
The fixative used is glutaraldehyde or osmium tetroxide while the dehydrating agent IS
alcohol or acetone.
The stains are

lead citrate and uranyl acetate.
a
Transmission electron microscope (TEM)
It allows the visualization of the internal structures of cells.
It has the greatest resolution that is approximately 1,000 times the light
higher than
microscope.
used to examine very thin specimens and microorganisms since it can magnify'
It is
specimen a million times.

139
In this
type of microscope, specimens are placed into the path of the electron beam.
Resolving power: 0.2 nm
b. Scanning electron microscope (SEM)
It scans the surface of the cells or specimens.

In this microscope, the specimen is positioned at the bottom of the column.
Resolving power: 200 nm
Magnification: 20x to 10,000x
3.
Digital Microscopy
It is
technology that utilizes the science of staining like the Gram reaction and capture
the cellular
images through web-based interface.
It uses an
automated microscope and the interface to present images on screen or
computer monitor.
Advantage: Standardization in microcopy and cost-effective mi leom rll. al b6dW

Example: Leica and PathXL
b
ASSESSMENT QUIZ
1. It compensates for the difference in

the observer's eyesight.
a. fine coarse adjustment

b. interpupillary distance scale
c. window lens
d. diopter adjustment ring 100.01
2. Which of the following terms refers to the ability of the lenses to separate closely distant objects?
a. magnification
b. resolving power
c. refractive index
d. phase-contrast
3. What is the most important characteristic of the bright-field microscope?

a. parfocal
b. high resolution
C. reduce ergonomics
d. reasonable cost
4. Which of the following dyes is the most recommended for fluorescent microscopy?
a. acridine orange
b. calcaflour white
C. both
d. neither
5. Which type of microscope allows the detailed examination of internal structures in living
organisms?
a. Dark field microscope
b. Phase-contrast microscope
C. Electron microscope
d. Brightfield microscope
MICROSCOPY 141
6. It is used to examine
verythin organisms and their
a. Scanning electronmicroscope cellular structures.
b.
Dark field microscope
C.
Fluorescent microscope
d.
Transmission electronmicroscope
7.
What is the magnification ofthe eyepiece?
a.
b. 10x
C.
d.
8. What is the amount of the bacterial cells needed to

effectively perform microscopy?
a. 105 cells/mL
b. 108 cells/mL
C. 1.5 x108 CFU/mL
d. Any concentration is acceptable.
9. Which of the following is NOT a function of cedar wood oil?

a.
It enhances the resolution of the microscope.
b. It stains the specimen to improve visualization.
C. It prevents light rays from dispersing and changing wavelength after passing through the
samples.
d. It is used to fill the space between the objective lens and the glass slide.
10. Which of the following is the most commonly used microscope in the clinical microbiology unit?
a. Electron microscope
b. Fluorescent microscope
C. Bright field microscope
d. Binocular microscope
CHAPTER 13
BACTERIAL STAINING

1.
discuss the types of bacterial staining and their significance;
2.
bacteria;
3. explain the significance of Gram sure technique;

4. discuss the principle of acid fast staining and negative staining; and
5. design a flow chart on differential staining.
Stains chromogenic solutions that are essential in the microscopic

are
identification and characterization of organisms. The differences in the staining to
properties of the bacterial cell wall is the first step in microbial identification.
Objectives of Staining
1. To determine the morphology of bacteria mste Isinswitib beau vInommroo
2. To differentiate groups of bacteria

3. To identify organisms with special structures
Kinds of Ionizable Dyes Used in Staining Bacteria

1. Basic Dyes
These are commonly used in the clinical bacteriology.

These are cationic dyes with positively charged groups
to negatively charged
(pentavalent nitrogen) that adhere
molecules like nucleic acids and proteins.
violet,
Examples: Methylene blue, basic/carbol fuchsin, crystal
safranin, and malachite green
2. Acidic Dyes
These are anionic dyes with negatively charged groups (carboxyls and phenolic) that bind to
positively charged cell structures.
Examples: Eosin, rose bengal, and acid fuchsin
Staining Techniques
1. Simple Staining
In this procedure, a single stain is used.
It is directed towards coloring the forms and shapes of the cells.
The inoculum size is 105 CFU/mL.
Example: Methylene blue staining
2. Differential Staining
It divides bacteria into separate groups. v11200-
It is directed towards coloring the components of the cells.
The inoculum size is 105 CFU/mL
Examples: Gram staining and Acid-fast bacilli (AFB) staining
Steps in Differential Staining:
a. Application of the primary stain

b. Application of the mordant (it increases the binding of primary stain to the cell wall)
C. Application of the decolorizing agent istlib orl1 amainemto lo nouaxholonds brs nodsoin
d. Application of the secondary stain/counterstain Jarll orl af low Ioo Iomotbed orft to eathnlg
Gram Staining
It is the most commonly used differential staining technique in the clinical microbiology
laboratory.
It utilizes
crystal violet as the primary stain while safranin is the secondary stain or counterstain.
In thisstaining procedure, iodine acts as the mordant and acetone-alcohol mixture is the
decolorizing agent.
The "differential" process in Gram staining is the decolorization step because of the distinct
appearance of bacteria after the application of the acetone-alcohol mixture.
TABLE 13-1. Gram Staining Reaction Theories

Theory Gram-positive Bacteria Gram-negative Bacteria
Less permeable to decolorizing agent L Permeable to decolorizing agent
Stearn and Stearn Low isoelectric point
U OITROMDAID BACTERIAL STAINING 145
Principle of Gram Staining emione

Bacteria
with thick cell walls containing teichoic acid retain the crystal violet-iodine complex
after decolorization and appear purple, hence, they are Gram-positive.
Those
bacteria with thinner cell walls that contain
lipolysaccharide do not retain the primary
dye-iodine complex and appear deep pink or red after treatment with the counterstain safranin;
and they are
called Gram-negative.
TABLE 13-2. Differential Characteristics of Gram-positive and Gram-negative Bacteria

Gram-positive bacteria Gram-negative bacteria
Primary stain (crystal violet)
Mordant (iodine)
Decolorization (acetone alcohol)
Secondary stain (safranin)
Precautions in Gram Staining d evilog

1. If the crystal violet is rinsed too vigorously prior to the application of iodine, the organisms may
appear unstained.
2. If the decolorization is prolonged, the Gram-positive complex will be removed and the bacteria
will not be stained.
3. If the decolorization is insufficient, the organism may falsely appear as Gram-positive cells.
4. If the safranin dye is applied for more than minute, the Gram-positive complex will be washed
brt. a off from the previously stained cells.
5. Failure to leave the safranin for a sufficient time will result in unstained Gram-negative bacteria
and background materials.
Quality Control
25923
Gram-positive bacteria: Staphylococcus aureus ATCC
ATCC 25922
Gram-negative bacteria: Escherichia coli
General Rule in Gram Staining

NVM (B)
All cocci are Gram-positive except Neisseria, Veillonella, and Branhamella (Moraxella).
All bacilli are Gram-negative except Actinomadura, Arcanobacterium,
Bacillus, Clostridium,
2.
Kuthria, Listeria, Mycobacterium, Nocardia, Rhodococcus,
Corynebacterium, Erysipelothrix, Gordonia,
and Tsukamurella.
Streptomyces, Tropheryma whipplei,
Reasons Why Gram-positive Bacteria Become Gram-negative Bacteria o slqonin

1. Removal of MgRNA
2. Aged, dying, and autolyzing cells
Old cells may lose their ability to retain stains.
Antibiotic-treated bacterial cells have atypical staining reaction.
3. By using acidic iodine during staining
4. Due to a technical error or the wrong use of stains
Exceptions in Gram Staining

1. Organisms that exist almost exclusively within host cells (Chlamydia)
2. Organisms that lack cell walls (Mycoplasma and Ureaplasma)
3. Organisms with insufficient dimension to be resolved by light microscopy (Spirochetes)
Gram Sure
It differentiates aerobic Gram-positive bacilli or coccobacilli from Gram-negative rods or Gram-

variable.
It detects aminopeptidase secreted by Gram-negative bacteria which in turn hydrolyzes the

substrate.
It is used as an adjunct staining procedure.

Reagent Disk: L-Alanine-7-amido-4-methylcoumarin (substrate) t rits
Procedure: Isolate an 18-hour old
pure colony and transfer into a tube with 0.25 mL
demineralized water, then add the reagent disk. Incubate for 5 minutes to 10 minutes at room
temperature.
(+) result: Blue fluorescence (under long wave UV light) of aerobic Gram-negative rods and
Quality Control
Positive: Escherichia coli ATCC 25922 = Blue fluorescence
Negative: Staphylococcus aureus ATCC 25923 = Absence of fluorescence (colorless)
Acid-Fast Staining
It is the technique applied to bacteria with high lipid content in their cell walls.
It utilizes carbol fuchsin as the primary stain and methylene blue or malachite green as the
secondary stain.
In this procedure, the cell wall of acid-fast bacteria resists the acid-
acid-alcohol (hydrochloric
ethanol mixture) decolorization step.
Heat is applied a mordant in
as
the Ziehl-Neelsen method while tergitol is for the Kinyoul
method.
BACTERIAL STAINING 147
Principles of Acid-fast Staining

The primary stain binds to
the mycolic acid in the cell walls of the acid-fast bacteria, like in
mycobacteria, and is retained after
decolorizing with acid alcohol.
Acid-fast bacilli
(AFB) retain the primary stain creating a deep pink or red color on bacterial cells
while non-AFB are either blue- or
counterstain.
green-colored due to the methylene blue or malachite green
TABLE 13-3. Differential Characteristics of Acid-fast and Non-acid-fast Bacteriatono-bisA

Acid-fast Non-acid-fast
Primary stain (carbol fuchsin)
Mordant (heat/tergitol)
Decolorization (3% acid-alcohol)
Secondary stain (methylene blue)

6110 g16.bs
green
Acid-fast Staining Methods
bd 1. Ziehl-Neelsen/Hot Staining Method
2. Kinyoun/Cold Staining Method
3. Pappenheim Method - differentiates Mycobacterium smegmatis from Mycobacterium tuberculosis
4. Baumgarten Method - differentiates Mycobacterium leprae from Mycobacterium tuberculosis
cell wall of AFB
5. Auramine-Rhodamine Method- selective for the
Quality Control (Ziehl.

(H37Ra)s wbasompieongntb\xbqb\v
Positive-AFB: Mycobacterium tuberculosis ATCC 25177
3308
Positive-Partially AFB: Nocardia asteroides ATCC
Negative-NonAFB: Escherichia coli ATCC 25922
Ways to Facilitate Acid-fast Staining

for five to seven minutes to temporarily remove the mycolic acid
1. Heating or steaming process
while the smear is flooded with stain
2. By increasing the concentration
of dye and phenol in the staining reagent ammete aviless!
3. Prolonged contact of the specimen with the primary stain

4. Addition of a wetting agent like tergitol
BACTERIOLOGY
Notes to Remember
Mycolic acid renders the cells resistant to decolorization, thus the term "acid-fast."
Acid-fastness is influenced by colonial age, medium for
growth, and ultraviolet light.
contact with the bacterial smear for 5 minutes to 7 minutes to ensure
The carbolfuchsin should be in
staining of the organisms while the methylene blue is 1 minute.

Acid-alcohol decolorizing agent is composed of hydrochloric acid and ethanol.
The Ziehl-Neelsen method is ideal for concentrated smears and staining of partially acid-fast bacill:
like the Nocardia species.
Modified Acid-Fast Staining Method (Modified Kinyoun)

It is useful for the identification of intestinal coccidian oocysts.
It is ideal for the detection of cryptosporidia and cyclospora parasites in stool specimens.
The reagents used are the same with those of the conventional acid-fast reagents except for the
concentration of the acid alcohol (1% H,SO).
For the counterstain, either methylene blue or malachite green can be utilized.
Aside from fecal specimens (fresh or formalin-preserved), tissue biopsy, duodenal aspirate, and
respiratory samples are acceptable.
The results show oocysts appearing as magenta-stained organisms against a blue/green
background.
Quality Control TA 1o low foole
Cryptosporidium spp. from a formalin preserved-sample should be included every testing

10%
pinkish-red against a uniformly green background (https://www.cdc.

specimen, and it stains
gov/dpdx/diagnosticprocedures/stool/staining.html).
DNA-Probe Mediated Staining

It specifically identifies selected pathogens, such as Chlamydia trachomatis, Bordetella pertussis,
Legionella pneumophila, herpes simplex virus, varicella-zoster virus, cytomegalovirus, adenovirus,
Negative Staining
It demonstrates the presence of a diffuse capsule surrounding some bacteria.
It is an excellent
technique for studying bacterial gas vacuoles and viral morphology.
In this staining procedure, the bacteria appear as
light-colored bodies against a dark background
since the cell surface repels the acidic stain as a result of the cell wall having a negative charge.
Example: India ink or Nigrosin staining
DOJOISATDAS SITEONDAIG H BACTERIAL STAINING 149
It utilizes special dyes (fluorophores) to

microscope. visualize bacterial cells through a
720trirni in
fluorescent
It supports the microscopy

of acid-fast bacilli smears, hence, it is preferred for mycobacteria.
It has the ability to stain the nucleic acids
aside from the protein content of the organisms.
Types of Fluorescent Staining

1. Fluorochrome Staining
This technique used a single fluorescent
dye (fluorophore) and is directed towards staining
the bacterial cell.
It is more
sensitive than the routine differential staining methods in determining bacterial
microscopy.
It enhances the microscopic property of the organisms and provides greater contrasting
factor.
Sample size: 104/mL of specimen

Examples of dyes: Acridine orange, auramine, rhodamine, and calcofluor white (for fungi)
Acridine Orange
It is a nonspecific dye which binds to the nucleic acid component of every host cell and
microorganism. leg6
It can be used to detect the presence of bacteria in blood cultures and sterile samples like
cerebrospinal fluid (CSF).
It confirms the presence of bacteria in a sample that has not been detected by Gram staining.
cocci and bacilli)
Positive staining reaction: Bright orange fluorescence (both
with the mycoplasma grown in culture.
Advantage: It has affinity
the category of "Gram-
Disadvantage: It has no ability to distinguish whether it belongs to
bacteria" since all microorganisms have the same staining
positive or Gram-negative
reaction.
Auramine-Rhodamine
the mycolic
mycobacteria across species, specifically
acid.
the
It stains the cell wall of
green background
Positive reaction: Bright yellow or orange bacterial cells against i
It increases the identification of mycobacteria directly in patient specimen.
Major advantage:
2. Immunofluorescence
fluorescent dye and a specific antibody reagent to identify
It involves the use of a
microorganisms. conjugated antibody

microbial antigen (bacteria) and
the
the
forms "tagging" between this tagging renders the organism
and antibody specific for the antigen)
It
(fluorescent dye
easily detectable by
fluorescent microscopy.
and small pleomorphic bacilli such as Bordetellaand

Advantage: It detects Chlamydia spp.,
and virologic staining.
Legionella; it is also utilized in parasitic
(FITC)
Commonly used dye: Fluorescein isothiocyanate
Positive staining reaction: Apple-green fluorescence
TABLE 13-4. Special Staining Methods

Stain Cellular structure/Bacteria
Staining technique
Albert Malachite green and toluidine blue Metachromatic granules
Crystal violet Capsule
Anthony, Hiss, and Gin
Cell wall
Congo red
Carbol fuchsin and nigrosin dye Endospore
Carbol fuchsin DNA
Fontana-Tribondeau Ammoniacal silver nitrate and

tannic acid
Carbol fuchsin and tannic acid

Gray Flagella
(mordant)
Carbol fuchsin, tannic acid, and
Flagella ambx3
methylene blue
Silver nitrate
Neisser Methylene blue and crystal violet Metachromatic granules

Nigrosin dye (black dye)
Schaeffer-Fulton m Malachite green and safranin red
151
ceLo A9 aeo BACTERIAL STAINING
11
o tnt ntelot oaionE
dlo eitLa dat

uspt
▇
D ht
si
uno
SeE
FIGURE 13-1. Gram stain reaction of Staphylococcus aureus epaLadt

(Photo by R. Facklam)
e a pGroh S veut
Cocói in clushe
tnaai
aie oghny
teiena p sie tinotah
.lnatu
i bea ei incbomarl
tEan
fboi
eateei
be1 ninche b
p rl to 20qu▇ ▇ esd
tEet
tei m 1o -f r ab sisir9relib ols
Sple at uta a d
ns FIGURE 13-2. Photomicrograph of Gram stain of Escherichia coli
(Photo from USCDCP) stb
titats iaot illodo00h Aron Neyative
Ba |i
a an
lon sbivo1g
st
trt 1 E
ie
du uouho led
t
FIGURE 13-3. Leifson staining of Vibrio cholerae

(Photo by W. A. Clark)
ASSESSMENT QUIZ
the correct answer.
Encircle the letter that corresponds to
for Gram staining?
1. Which of the following are the qualitycontrol organisms
coli
a. Staphylococcus aureus and Escherichia
b. Staphylococcus epidermidis and Enterobacter
c. Bacillussubtilis and Neisseria nonpathogenic species
d. Bacillus spp. and Moraxella spp.
2. What is the most commonly used stains in clinical bacteriology?

a. Acidic dyes
b. Basic dyes
C. Negative stain
d. Anionic stain
3. What mordant is used in Ziehl-Neelsen method?

a. heat
b. iodine
c. tergitol
d. safranin red
technigue?
* What is the purpose of the Gram sure staining
bacteria from Gram ghosk
to strerentiate the true Cram positive
b. to study gas vacuoles of prokaryotes
to detect intracellular
C.
bacteria like Chlamydia
d. to distinguish coccobacilli from Gram-variable
5. It provides color to the nucleic acid of the prokaryotes.
a. auramine
b.
calcofluour white
C.
d. acridineorange
BACTERIAL STAINING 153
Which of the
following statements is NOT attributed to the change
6.
Gram-negative? of Gram-positive bacteria to

a. removal of mRNA
b. aged, dying, and autolyzing cells
C. use of acidic iodine
d.
incorrect application of stains
7.
Which of the following statements is NOT true about modified Kinyoun method?
a. It is
used for the identification of Cryptosporidium.
b.
Stool is the preferred specimen.
C. Acetic acid is used in
combination with ethanol as a decolorizer.
d. Acid-fast organisms exhibit a magenta color after staining.
8. In Gram staining, what happens to the organism if the decolorizing agent is insufficiently applied?
a. The organism may appear as falsely Gram-positive cells.
b. The organism will be unstained.
C. The Gram-positive complex will be removed.
d. The organism will exhibit a red color.
9. It colors the background but not the bacterial cell wall or membrane inclusions.
a. Negative staining
b. Baumgarten method
C. DNA probe-mediated
d. Simple staining
e. Fluorescent staining.
10. Which of the following methods is used for DNA staining?

a. Dyar stain
b. Feulgen stain
c. India ink method
d. Fontana-Tribondeau stain
CHAPTER 14
METHODS FOR
BACTERIAL
IDENTIFICATION

At the end
of this chapter, the students should be able to:
1. explain the procedure for automated bacterial
identification;
2. differentiate the types of
immunodiagnostic methods;
3.
discuss the principle and significance of MALDI-TOF in microbial
identification;
4.
contrast the types of polymerase chain reaction;
5. explain the basic steps of polymerase chain reaction; and
6. distinguish the fluorescence in
situ hybridization with the emerging Mais bik ubiusrooid
molecular diagnostic tests,
of To1
The identification of microorganisms over the last decades has been

revolutionized to meet the ever growing and changing needs of medical
diagnosis. Irans
From the staining techniques in 1884 that was introduced by Hans eavlertn
Christian Gram to the utilization of amplification procedures by polymerase <in ons
chain reaction (PCR) in 1985 by Kary Mullis, the advancement in microbiology 10012
is continuously evolving, reshaping the morphology of bacterial detection and

characterization.
characterization of microorganisms serves as the

The biochemical
for the identification of most
fundamental source of information
bacterial species after microscopy. ord bits secs
1. Motility test - Wet mount and

hanging drop preparation
2. Staining Gram-staining, acid-fast staining, and structural staining
carbohydrate fermentation (LIA);
3. Manual biochemical test
catalase and coagulase tests
4. Use of routine and selective media
Analytical Profile Index (API)

It consists of plastic strips and microtubes that contain dehydrated biochemical substrates.
It is commonly used for identification of Gram-negative enteric bacteria.
It has the same principle with the biochemical manual tubed method.
Procedure: The biochemical substrates (pH-based substrates) are inoculated with pure culture
suspended in sterile physiologic saline and incubated for 18 hours to 24 hours at 35°C; some
reagents may be added to the cupules after incubation.
Examples: API 20E (Enterobacteriaceae), Rapid 20E (rapid identification of the Enterobacteriaceae
in 4 hours), API 20NE (non-enteric), API 20S (streptococci), API 20C (yeast), and API 20A
(anaerobes)
Automated Method for Identification of Bacteria
In this method, known patterns of microbial growth are compared with the test organism using
a computer software.
The growth of microorganisms is detected through colorimetric, flourometric, or turbidimetric

analysis.
used to various microorganisms such as Gram-positive and Gram-negative cocci, fermenters

It is
and nonfermenters, corynebacteria, and even yeast, both combining the phenotyping assays like
biochemical and antimicrobial susceptibility tests.
Lyophilized reagents are utilized as chromogenic substrates, reaching more than 30 reagent
substrates for biochemical tests.
The underlying principle is that the pure bacterial isolate during incubation at 35° C for 16
hours to 24 hours utilized the carbohydrate in the reagents, thus producing acid or alkaline end
products as early as 2 hours. bris
The advantages for using this method are reduced turnaround time, minimal analytical errors,
simultaneous analysis of multiple isolates, and automated recording of bacterial identification.
Examples: BD Phoenix System, MicroScan System (Autoscan and WalkAway), Sensititre (TREK)
and Vitek System
Serological Test/Immunodiagnosis
It is a basic detection of antibody response from an antigenic stimulation, either through a single
identification or serial dilution.
With the exception of specific illnesses like Legionnaire's disease, where titers may not grow
until months after infection, a serum sample for antibody detection should be collected during
the acute phase of the disease and the period of convalescence.
The presence of antibodies implies either
a pathogen exposure or a subclinical infection; a
positive IgM antibody indicates a current or recent infection whereas
a positive IgG antibody
denotes a prior infection or exposure to an organism.
It is
one of the diagnostic tests for organisms that are difficult to culture like the
atypical bacteria
(Rickettsia and spirochetes).
Vodlola
METHODS FOR BACTERIAL IDENTIFICATION 157
It is also utilized to assist in the

detection of
caused by the *TORCH agents. congenital infections in newborn, such as those
CSF and urine samples are also accepted
for serological test aside from serum. 01100
*TORCH -
Toxoplasma gondi, Other
B19, Varicella-Zoster, organisms (Treponema pallidum subsp. pallidum, Parvovirus
sift ni ete) Rubella virus, Cytomegalovirus, and Herpes simplex virus
Bacterial Agglutination
It is performed by adding
antibodies (agglutinins) to the bacterial suspension that will bind to
the surface of the organisms.
It is used to identify bacteria that are difficult to cultivate in a culture medium.
(+) Result: Presence of visible clumps or agglutination
Interpretation: Specific antibodies bind to the bacterial surface antigens, causing the organism to
clump.
Examples: Widal test (Typhidot), Weil-Felix test, and febrile agglutinin test for the diagnosis of
tularemia, leptospirosis, and brucellosis
Particle Agglutination
It uses artificial carriers, such as latex particles or treated red blood cells, or biological carriers
like bacterial cells as reagents, which can adsorb test antigen and react with the specific antibody
present in the patient's serum.
(+) Result: Presence of visible clumps or agglutination ni to eleo0ps

Examples: Microhemagglutination test for antibody to Treponema pallidum (MHA-TP),
hemagglutination treponemal test for syphilis (HATTS), and passive hemagglutination test for
streptococci
Latex Agglutination (Antigen Test)

antibody that
in the patient's specimen binds
to the is present on the
In this test, the antigen
B streptococci; serotypes of N. meningitidis;

used for direct identification of group
It is
urine; S. pneumoniae; and E. coli
a bori H. influenzae type in CSF, serum, and
molecular assays
limits the utilization of this method for bacterial
The advent of the
identification.
bound to a particle to increase the visibility of the agglutination

It uses the antibody that is
reaction between an antigen and an antibody.
It assists in the detection of N. meningitidis, Hemophilus, and streptococci. 1120-lite.)
binds to the test-strain (Cowan strain) while
1 the test-
In this test, the antibody (Fc portion) site in the antibody (Fab portion).
the specific target
antigen (suspected pathogen) binds to contains protein A in its cell walls is utilized in this
S. aureus organism (Cowan
strain) that
procedure as part of the reagent.
BACTERIOLOGY
that causes the disease

which includes the antigen
It is composed of two parts: a test system the sheep's red blood cells,
in the patient serum and an indicator system which consists of
an exogenous complement.
Euthvioy
complement-fixing antibody like the immunoglobulin G (IgG), and
blood cells which means presence of antibody in the
(+) Result: Absence of lysis with the red
patient serum
(-) Result: Occurrence of lysis with the red blood cells and the absence of antibodies in the test
serum
of baid
Flocculation Test nd vidnabt of boen a
It uses a soluble antigen that reacts with the antibody in a serum sample.
(+) Result: Occurrence of macroscopic or microscopic flocculation (precipitation)

Some examples of this test are venereal disease research laboratory (VDRL) test and rapid plasma
reagin (RPR) test.
Enzyme-linked Immunosorbent Assay (ELISA)

It is a sensitive and specific method for the detection of antibodies against certain pathogens.
This method consists of enzyme-bound antibodies with the antibody-binding sites free to react
with their specific
antigen.
It is utilized for the diagnosis of infectious diseases such as those caused by Legionella. 4)
Alkaline phosphatase or horseradish peroxidase is used as an enzyme-conjugate antibody

101
fest reagent. 6inon syidenq bas (ell
(+) Result: Colored end product
Immunofluorescent Assays
It is commonly used for the rapid identification of bacterial and viral antigens in body fluids,
In this method, monoclonal or

polyclonal antibodies (conjugates), which are attached to
lenow fluorescent dyes, are applied to an antigen (patient's specimen) that is previously treated with
either formalin, acetone, or alcohol, then the reaction is visualized under the fluorescent
microscope where final results can be released within an hour.
It is the preferred method for the detection of
Borrelia, Legionella, Mycoplasma pneumoniae,
Rickettsia, and TORCH.
Examples of fluorescent dyes: Auramine, rhodamine, and

fluorescein isothiocyanate (FITC)
(+) Result: Occurrence of fluorescence as seen under the
microscope
Advantage: It can determine the source of the specimen.
allow 'os ati at

YOOIO1 METHODS FOR BACTERIAL IDENTIFICATION 159
1. Direct Fluorescent
Antibody (DFA) Test
utilizes a fluorescent dye and a specific labeled-antibody or conjugate that binds to the
It
ant to alevlo antigen (bacterial isolate) in the sample.

A positive
result is achieved if fluorescence (illumination) is seen under the microscope.
elevlens
2.
Indirect Fluorescent Antibody (IFA) Test lostab of beau at ydqmgotsmonb bippil-as2
It is a two-step or a sandwich technique that is more sensitive than DEAD a aRS0
It determines the
presence of pathogens by using the patient's serum that is placed directly
on the
microscopic slide which contains the specific antigen or the target organism. XTIEM
Western Blot
It is
based on the electrophoretic separation of bacterial proteins in supporting media.
mind
It detects several antibody reactions from single infectious agent.

It is used to confirm the presence of antibodies to human immunodeficiency virus type 1 (HIV-1)
in patients whose serahave been repeatedly reactive in EIA tests.
Supporting media: Agarose gel and polyacrylamide gel

(+) Result: Bands on the strips (pattern of antibodies)
dsiom nistotd supinu orld alosloh il
Pulsed-field Gel Electrophoresis

It is a strain-typing technique that can be a useful addition to epidemiologic research.
Enzyme-digested chromosomal fragments of bacteria are isolated electrophoretically in this
approach.
The probable relatedness is another epidemiologic tool that can be used in an outbreak
investigation.
The fragment patterns are examined among microbe strains retrieved after a probable outbreak:
a. The strains can be identified as possibly related if the patterns are comparable.'1 1
b. Unrelated strains would be identified by their different patterns.
Bacteriophage Typing
This method is based on the specificity of phage surface receptors for cell surface receptors.
that can attach to surface receptors can infect bacteria and cause lysis.
Only bacteriophages
A bacteriophage is a virus that attacks bacteria.
doua inutaon olnogomouda alidw anistmo Bas

bior
Chromatographic Method (Gas Chromatography and

High Performance Liquid Chromatography)
cellular fatty acids,
and products of pyrolysis of the
It involves analysis of microbial metabolites,
whole bacterial cells.
Gas-liquid chromatography is used to detect the cellular fatty acids

HIPLC is utilized for the fatty acid analysis of the Nocardia species.
Matrix-Assisted Laser Desorption Ionization-Time-of-Flight povnint orlt ito

Mass Spectrometry (MALDI-TOF-MS)
the rapid identification of a wide range of pathogenic species,
It is an excellent tool for
Enterobacteriaceae, non-enterics, anaerobes, and even mycoses.

It is utilized for species, subspecies, and strain identification of bacteria.
It can be used for blood culture samples provided a portion of the fluid is subcultured in pure
plate.
It provides faster result in terms of bacterial identification (in minutes) compared to the current
semiautomated biochemical methods.
It detects the unique protein moieties of every organism, which is then important for bacterial
detection.
It has 2 components: Ionization Phase and the TOF Phase

For the procedure, the bacterial isolate is ionized by transferring it from the culture plate
to a metal plate, where it is treated with matrix solution (hydroxycinnamic acid, water, and
acetonitrile) until it forms a crystallized microbial protein or matrix lattice which is then
analyzed using the MALDI-TOF instrument.
The protein signal that is generated at the end of the process is specific for each bacterial species.
This method creates unique mass spectral fingerprints that are compared to a massive database
of mass spectra.
Bioinformatics profiling is used in this technology to accurately identify microorganisms at the
genus and species levels since spectral fingerprints are unique signals for each microorganism.
Limitation: Antimicrobial susceptibility test
Molecular Diagnosis
It is currently considered the most important method for microbial identification.
confirming the taxonomy of the emerging and re-emerging pathogens.
It is very useful in
In this method, agarose gel

electrophoresis is one of the basic adjuncts to separate nucleic acids
and proteins while chromogenic reagent such as SYBR Green is for
staining the nucleic acid.
BACTERIAL IDENTIFICATION 161
Polymerase Chain Reaction (Nucleic Acid

Amplification Assay)
the most commonly used amplification technique in molecular
It is
It is the first nucleic acid

amplification method
It is
technique that increases the nucleic acid of the test sample or target microorganism from a
very small amount to a
million copies.
It is utilized for rapid
detection of nucleic acid in biological samples, a very significant tool in the
diagnosis of the etiologies of diseases.
Types of PCR
1. Conventional PCR
Principle: The DNA sequence is amplified utilizing a Taq polymerase. &.n niache seeniomt
3
Basic Steps: Denaturation, Annealing, and Elongation
Main Reagents: Taq DNA polymerase and Primers
Other components: DNA template, buffer, cation, and deoxyribonucleotide triphosphates
Instrument: Thermal Cycler
Major Drawback: Environmental and Carry-Over Contamination
Clinical/Diagnostic Application: Microbial detection, phylogenetic study, gene analysis,

therapeutic cancer detection, therapeutic management, and DNA profiling and cloning
The result of the PCR amplification is visualized through gel electrophoresis.
TABLE 14-1. PCR Components

PCR Components Purpose
Taq DNA Polymerase (E.C. 2.7.7.7) ersatioNn Amplification of the target DNA or DNA template
Source: Thermus aquaticus (Taq), a heat-tolerant bacterium
Primers - DNA oligonucleotides (single 01 Provide the starting point for DNA synthesis
stranded); 2 primers every PCR cycle Bind to the opposite strands of the DNA template
Taq polymerase can only make copies of DNA in the
presence of i 1 primer.
DNA
Pattern for amplification
template
Either a complementary DNA (cDNA) or genomic DNA
Building blocks of the new DNA strand

Consist of dATP (deoxyadensine triphosphate), dCP
BACTERIOLOGY
DIAGNOSTIC
Purpose
PCR Components
Cofactor for Taq DNA polymerase
relationship of the primer (3-OH')
and
Cation (Magnesium) Strengthens the
Vital
of the completion of the amplification process
the form of MgC2
to the function of the Taq polymerase
and Potassium
Provides stability between 8.0 to 9.5
Buffer (Tris-Hydrochloric acid pH environment
Creates
chloride) increasing the specificity
Promotes a reliable PCR process by
of the reaction
Polymerase chain reaction - PCR
original DNA to
be replicated
5'
5' 3'
5' 3'
5'
3' 3'
3'
5' 5'
DNA primer
nucleotide
_ Denaturation (Strand separation): The separation of the two hydrogen-bonded complementary chains of
DNA into a pair of single-stranded polynucleotide molecules by
process of heating (94°C to 96°C).
2 - Annealing (Primer binding): The temperature is lowered (45 °C to 60°C) so the primers can attach
themselves to the single-stranded DNA strands.
3 - Elongation/Extension (Synthesis of New DNA): It starts at the annealed primer and works its way along the
DNA strand (72°C).
FIGURE 14-1. Steps of Polymerase Chain Reaction And
Ad sine Source: https://microbeonline.com/polymerase-chain-reaction-pcr-steps-types-applications/
Notes to Remember
Magnesium helps maintain the electric charge (negative charge) of the phosphate group and in the
annealing step.
The concentration of the magnesium is essential in the reliabilty of the amplification process if high
concentration, thiswill lead to erroneous copies of DNA by the Taq polymerase while low concentration
may result in insufficient activity or inactivation of the DNA polymerase and eventually decrease in
the level of PCR products.
Increased level of
dNTP's can slow down or inhibit the polymerase chain reaction.
METHODS
FOR BACTERIAL IDENTIFICATION 163
2. Real-time PCR (qPCR)

It is also known as
It has all the
the quantitative PCK, a variation of the classic polymerase chain reaction.
three steps of the conventional PCR with detection of the amplicons.
It qmantfies the target nticleic acid after cach replication cycl tin the same PCR equipment
utilizing commercially available fluorescence deterting ehermioya the
It measures gene expression and viral load asselent avromt or basilian eill
It detects single
nucleotide polymorphism (SNP) and base pair differences.
It is ideal for monitoring the progress of treatment
It
uses fluorescent dyes to mark specific DNA, and the amount of
fluorescence produced is
proportional fo the amount of DNA present.
In comparison to traditional PCR tests,
qPCR devices measure the amplified product
(amplicon). In
addition, it determines the number of copies of target substance present in the
original specimen.
It does not utilize an
agarose gel since it has a built-in imaging device.
Principle: The accumulation of amplicon is monitored by labeling primers with fluorescent
dyes, and these labels create a change in fluorescence signal that is proportional to the
quantity of amplified product present in each cycle, and it grows as the number of species
increases.
Examples of qPCR techniques: SYBR green and 5' Nuclease Test TaqMan PCR
(1909
Advantages
1. It combines amplification and product detection at one time following the closed system.
2. It is used for multiple sample analyses.
3. It reduces cross-contamination with the amplified production. proensiT-ersvadl
4. It lowers turn-around time (TAT).
SYBR green
but not to single-stranded DNA
dye that binds to double-stranded DNA (dsDNA)
It is a
(sSDNA) and fluoresces when thus bound. viles bite

During the PCR cycle, the amount of fluorescent signal produced increases as more
double stranded products are generated to which SYBR green dye attaches and fluoresces.
The quantity of
double-stranded DNA molecules present in the reaction at any one time
the reaction. Yale 61
determines the amount of fluorescence in
has tendency to bind and fluoresce all double-stranded products
Disadvantage: SYBR green
in the reaction. sUp
5' Nuclease Test TagMan PCR

nucleic-acid probe complementary to an internal region of
In this procedure, a dye-labeled
used.
the target DNA is
one of the template strands - the fluorophore is linked to
The dye-labeled probe anneals to to the probe's 3' end, and the probe is tagged with
the probe's 5' end, the quencher is coupled
two fluorescent moieties.
DIAGNOSTIC BACTERIOLOOY
PCR or STNPCK)
a.
Nested PCR (Single-tube Nested that improves
the sensitivity and specificity
conventional PCR
Tt is a modification of the
of the assay.
acid samples such those isolated
from
formalin-fixed
nucleic
It is effective for divergent
paraffin-embedded tissue. of the assay, especially when detecting low count
Th is utilized to improve the sensitivity
and mycobacteria and viruses such as the herpes virus.
Rickettsia
of prokaryotes such reduced by this type of PCR.
the target sequence is
The nonspecific amplification of nucleic acid target, as well as two
the same
of primers directed at
It requires two sets
PCR reactions in quick succession. and an extended
put to
the initial reaction vessel
In this method, both sets of primers
are
PCR is performed. of the second

is intended to anneal to sequences upstream
The first set of primers is nested within the first set of
set of primers, whereas the second set of primers
primers.
also known as "outer primers," amplify a large fragment of
The first set of primers,
thegene, which is used as template in the second round of PCR.
or nested primers," targets
The second set of primers, also known as "inner primers
a smaller section of the amplicon.
Amplicons are seen by electrophoresis with a molecular weight marker in a 2% ethidium
bromide-stained agarose gel.
Advantage: It will only prime a specific product created in the original PCR, ensuring
that PCR specificity is maintained.
b. Reverse-Transcription PCR (RT-PCR)
of the DNA.
In this type of PCR, the RNA template is the starting sample instead
This method transcribes the RNA "reversely" into complementary DNA (cDNA) by way
the differential phase
of the reverse transcriptase enzyme, hence, reverse transcription is
compared to the conventional PCR.

It detects prokaryotes and eukaryotes.
It is very significant for the determination of the RNA viruses such as dengue and SARS.
It is vastly used in biomedical research like in cancer institute.
Advantage: To study unstable nucleic acid such as the RNA and determine the gene
expression
Points to consider: The success of RT-PCR depends on the quality of the RNA template.
First Procedure in RT-PCR: Synthesis of DNA/RNA Hybrid
Characteristic of the Reverse Transcriptase: RNA Dependent-DNA Polymerase
00:01 METHODS FOR BACTERIAL IDENTIFICATION 165
C.
Real-time RT-PCR (RT-PCR/Reverse Transcription
T
Quantitative PCR
It is useful for
measuring the abundance of
expression. certain RNAs to determine the gene
It quantifies viral load such
Dhole nd Zines oih bes hnern immunodefie no pins (in)

those of the human
hepatitis C virus, and immunodeficiency virus (HIV),
It is
the gold standard methodfor
the detection of SARS-CoV-2 (agent of COVID-19). di
Methods: One-step RT-qPCR and Two-step RT-PCR
Points to consider: Oligo(GT) and random hexamer primers are only included in two-step
Advantage: In one-step RT-qPCR, minimal cross contamination (closed-tube system),
low volume sampling, reduced TAT (8 hours to 8 hours), and ideal for high-throughput
applications are the add-on features; while for two-step is the flexibility in enzyme
selection and reaction optimization. 129vni
Disadvantage: It may not detect previous infection/disease. boeu ei 11
(OMD) emeinsgro bailihom vilapitensg

a tud
mRNA
Sequence-specific Primers clania

DNA Pol
Buffer +
Sequence-
specific
Primers
901 10 521 Sequence-

bal anlonsupse oriog AM181 specific
Primers
Two-Step
One-Step
both circumstances, which is then utilized as template for

RNA is reverse-transcribed into cDNA in
in a single reaction vessel
GPCR amplification. One-step RT-qPCR combines cDNA synthesis and aPck in one tube. In two-step RT-
using the same reaction buffer with the primers and all the PCR components
@PCR, cDNA is made in one reaction (RT) with the same PCR components but different set of primers and
then an aliquot from the first tube in a subsequent qPCR step utilizing another portion of the
standard
sequence-specific primers.
FIGURE 14-2. RT-4PCR Step Methods
wweentermolecutar-biology-resource-library/spotli
166 REVIEW HANDBOOK
Notes to Remember
the cDNA synthesis and amplification
of
specific target.
Sequence specific primers jump start
anneal at higher temperatures than random primers, one-step
Because specific primers typically employ novel RT.
it reaction
becauses. bitem ise higherreaction temperatures than two-step workfows
and
that can tolerate higher

temperatures.
d. Multiplex PCR
finvolves amplifyingnumerous target sequences using multiple sets of primersin the
same PCR mixture.
rather than separate
t allows simultaneous amplification of multiple gene segments test
runs for each. 29107051
It is recommended for genetic investigations that must be done several times.

It is used in genotyping, mutation and polymorphism analyses, and detection of
genetically modified organisms (GMOs).
but causing the same disease.
It is applicable to studies of various organisms
DNA quality.
It is best for amplification of samples with poor
Advantage: Low sample volume requirement and different organisms can be detected in
a single reaction
Disadvantage: Cross-reaction between primers and no single ideal melting temperature
DNA (Sanger) Sequencing

It determines the order of nucleotides in a DNA fragment.
most
It is useful for phylogenic recognition of certain bacterial species through the use of the
common 16S rRNA gene sequencing technique.
Fluorescence In Situ Hybridization (FISH)

31-6019v9?
It is a molecular approach that does not necessitate nucleic acid amplification.
It
uses an oligonucleotide or peptide nucleic acid (PNA) probe with a fluorescent label to target
ribosomal ribonucleic acid (rRNA) in an organism.
It has a high specificity and a high sensitivity of species-specific probes. " ma

Procedure: An assay can take anywhere between 1.5 hours and 3 hours to complete.
blood culture samples using various

It is used to detect S. aureus and Enterococcus
species in
probes.
FOR BACTERIAL IDENTIFICATION 167
Microarrays and Nanoarrays

It refers to
the grouping of DNAmolecules at the micro ornano level.
It detects the
gene expression
of the target microorganism.
It identifies bacteria, new genes, and mutations and analyzes the success of treatment.
It recognizes transcriptional levels of
genes in a disease process.
It is the study of microbial genes collected from the environment.

It utilizes the
combination of the science of sequencing and gene expression in determining
specific genomes.
It is useful in the study of the indigenous flora in the human body and gut.
It identifies pathogens
that are difficult to cultivate in primary and special media.
Proteomics
It is the study of proteins (proteome) on cellular level.
In clinical microbiology, it identifies protein expression in biosamples suspected with pathogens.

It also determines the effectiveness of 910050010
drug therapy across diseases.
adl ni neglinn adt nsowted nollenitulggs

ASSESSMENT QUIZ
Encircle the letter that corresponds to the correct answer. 9003 911 210915b
like the confirmation of immune

1. It detects antibody reactions in single infectious agent just
reactions in individuals with HIV.
a. Western blot
b. Indirect Fluorescent Antibody Test

c. RT-PCR
d.
moieties with
2. It is a rapid assay for identification of pathogenic bacteria utilizing the protein
a. MALDI-TOF-MS
b. Pulsed-field Gel Electrophoresis
C. Proteomics 2010031019
d. Microarrays
the completion of the

3. It is a cofactor in the polymerase chain reaction, and it is important in
procedure.
a. Buffer Tris-Hydrochloric acid
b. Primer oligonucleotides
C.
d. Magnesium
4. This serological assay uses the protein A in Staphylococcus aureus as a reagent to enhance the
agglutination between the antigen in the sample and the antibody.
a. Particle agglutination
b. Coagglutination
c. Flocculation
d. Complement Fixation
5. This type of PCR is recommended for samples that have been preserved in formalin and paraffin
wax and improves the recovery of mycobacteria and
atypical organisms like Rickettsia.
a. 5' Nuclease Test TaqMan PCR
C. Multiplex PCR
d.
METHODS FOR BACTERIAL IDENTIFICATION 169
6.
Immunofluorescent methods determino the TORCH antigens polyclonal
antibodies attached to fluorescent using monoclonal or
dyes.
Whatis the meaningofthe acronym TORCH?
Tetanus, Oligella, Rickettsia, Campylobacter, and Helicolne h
a.
Tetanus, Oligella, Rickettsia, Cytomegalovirus, and Herpes Simplex

b.
C. Toxoplasmosis, Other
d. Toxoplasmosis,
agents, Rubella, Cytomegalovirus, and Herpes Simplex
Other agents, Rubella, Chlamydia, and Helicobacker
7. What is the required temperature for the denaturation step in PCK?
a.
b.
C. 60 °C
d.
technique does not require amplification, and it utilizes oligonucleotides with fluorescence
8. This
labels to detect
the rRNAin microorganisms.
a. Metagenomics
b. MALDI-TOF-MS
c. FISH
d.
Bacteriophage typing
9. It is an
automated procedure for the identification of bacteria coupled with antimicrobial
susceptibility testing.
a. API
b.
C. ELISA
d. Sanger
10. What is the source of the DNA polymerase that is used in PCR?
a. Thermus aquaticus
b. Thermophilus aquaticus
C. Bacillus infernus
d.
CHAPTER 15
ANTIMICROBIALS
(ANTIBIOTICS)

2. discuss the sources of antibiotics;
3.
distinguish between the bacteriostatic agents and the bactericidal
agents;
4. explain the mechanism of antibacterial action and the target cellular
structure;
5. differentiate intrinsic resistance from acquired resistance; and

6. discuss the beta-lactamases and their contributions to antimicrobial
resistance.
Antibiotics
These chemical substances are produced by microorganisms with airbed af
the capacity to inhibit (bacteriostatic) or kill (bactericidal) other
microorganisms.
They can also be synthesized via chemical procedures that are
independent from microbial activity.
Some antimicrobial drugs are narrow-spectrum such as
bacitracin,
clindamycin, dapsone, erythromycin, gentamicin, isoniazid, penicillin,

against a limited number of milnA lo nolisaliel.
polymyxin B, and vancomycin. They are effective
pathogens.
Other drugs are broad-spectrum such as ampicillin, cephalosporins,
trimethoprim,
and
chloramphenicol, ciprofloxacin, rifampicin, sulfonamides,
tetracycline. They destroy different kinds of organisms.
of Antibiotics
TABLE 15-1. Sources Produced
Antibiotics
Source (Microorganism] Bacitracin
Bacillus subtilis
Bacillus polymyxa
Cephalosporins
Cephalosporium Gentamicin
Micromonospora purpurea Penicillin
Penicillium notatum
Erythromycin
Streptomyces erythraeus
Neomycin
Streptomyces fradiae
Amphotericin B
Streptomyces
Nystatin 0110
Streptomyces nourse
Chloramphenicol
Streptomyces venezuelae
Classification of Antibacterial Drugs

1. Natural Drugs
bacteria or fungi.
These drugs are produced by
nystatin, rifampicin,
Examples: Amphotericin B, erythromycin, kanamycin, neomycin,
streptomycin, tetracycline, vancomycin, bacitracin, gentamicin, polymyxin, griseofulvin,
penicillin, and cephalosporin
2. Semi-synthetic Drugs
These are modified natural drugs with added chemical groups.
Examples: Ampicillin, carbenicillin, and methicillin
3. Synthetic Drugs
These are chemically-produced drugs.
and
Examples: Sulfonamides, trimethoprim, chloramphenicol, ciprfloxacin, isoniazid,
dapsone
Antimicrobial Agents
Classification of Antimicrobial Agents

1 Bacteriostatic Agents
These are
antimicrobial agents that inhibit bacterial growth; but generally, they do not kill
the microorganisms.
Examples: Chloramphenicol,
and tetracyclines
dapsone, erythromycin, clindamycin, isoniazid, sulfonamides
2. Bactericidal Agents
These are antimicrobial
agents that usually kill or destroy organisms.
They are used for
the treatment of life-threatening infections.
STUDINSTONEOT ANTIMICROBIALS (ANTIBIOTICS) 173
Examples:
Aminoglycosides (gentamicin, amikacin, and streptomycin), p-lactams
ceftriazone, imepenem, penicillin, and ceotaxim. daptomycin, glycopeptides
vancomycin), isoniazid, rifampicin, quinolones, bacitracine and nelonycaole
Groupings of Antimicrobial Agents
1.
Group A Primary Test
Examples: Ampicillin, cefazolin, gentamicin, and
2.
Group B Primary Test Selectively nonsuls.
3.
Group C Supplemental Report Collectively
Examples: Aztreonam, ceftazidime, chloramphenicol, and tetracycline
Terminologies
1. Integrons - are genetic elements that are capable of integrating genes (cassettes) by an integron
encoded site-specific recombinase
2. Minimal-inhibitory concentration (MIC) is the lowest concentration of a drug that inhibits
bacterial growth
3. Penicillin-binding proteins (PBPs) - are enzymes (transpeptidase or transglycolase) that mediate
peptidoglycan cross-linking with reduced affinity for B-lactam antibiotics
4. Therapeutic index - is the ratio of the toxic dose to the therapeutic dose and as such, the higher the
therapeutic index, the more effective the chemotherapeutic agent

5. Transposons - are DNA elements that encode transposition and excision functions and which are
also able to carry antibiotic resistance genes among plasmids and the chromosome
Characteristics of Antimicrobial Agents enLid

1. The antimicrobial agent must be in an active form. lindia
2. The antibiotic must be able to achieve sufficient concentration at the site of infection that is higher
than the MIC to be considered effective.
3. The antimicrobials must have selective toxicity.
Types of Antimicrobial Agents According to the Mechanism of Action

I 1. Cell Wall Inhibitors nder
with high therapeutic index.
These are the most selective antibiotics
They inhibit the activity of the transpeptidase enzymes in which cell growth stops and the
death of the cells often follows.
These drugs are

effective against Gram-positive bacteria.
174 REVIEW HANDBOOK
IN DIAGNOSTIC BACTERIOLOGV
S-lactams, carbenicillin, isoniazid, methicillin, and vancomycin

Examples: Bacitracin, precursors
Bacitracin inhibits the
synthesis of peptidoglycan
of antibacterial agents
transpeptidation; largest group
B-lactams - inhibit can either be bactericidal or bacteriostatic
Isoniazid - acts only on growing celisand inhibits the translocationand
is a glycopeptide antibiotic that elongation
Vancomycin
of peptidoglycan
Cell
Specific Examples of the Cell
Wall Inhibitors
a. B-actam antibiotics: Pentellin, cephalosporins, monobactamns, and carbapenes

cefoxitin, ceftriaxone, and
b. Cephalosporins: Cephalothin,
C. Penicillinase-resistant penicillin drugs: Methicillin,
Notes to Remember
B-lactam antibiotics differ from each other in their basic ring structure and moieties attached to the
rings.
The $-lactam ring is the core of the antibacterial property of the 6-lactam drugs.
The structure of the penicillin "ring" resembles the terminal D-alanyl-D-alanine of the peptidoglycan
subunit.
Penicillins and cephalosporins remain as the recommended drugs for treating pneumococcal
infections. 77701716 0201091 7010
Vancomycin is a cup-shaped glycopeptide which binds to the terminal D-alanyl-D-alanine of the

pentapeptidyl-glycosyl peptidoglycan intermediates.
2. Protein Synthesis Inhibitors

These antibiotics bind to the 30S subunit that results in the misreading of mRNA, and thus
interfere with aminoacyl-tRNA binding.
It also binds with a 50S subunit
that results in the inhibition of peptidyl transferase and
peptide chain elongation.
These drugs target
the aerobic and anaerobic Gram-positive and Gram-negative species,
including M., tuberculosis, B. fragilis, N. meningitidis, H. influenzae, and S. pneumoniae.
a. Drugs which bind to the and
30S ribosomal unit
spectinomycin
-
aminoglycosides, tetracyclines,
b.
Drugs which bind to the 50S ribosomal unit
and streptogramins Chloramphenicol, erythromycin, lincomycin
Blocks
the initial step in protein synthesis - linezolid
C.
YOOJOIRETDNE DITH ANTIMICROBIALS (ANTIBIOTICS) 175
Notes to Remember
Protein biosynthesis requires the

sequential binding of the 30s and 50s ribosomal subunits to mRNA;
thisleads to the translocation ofthe genetic message id ad
Aminoglycosides such as gentamicin,
kanamycin, neomycin, streptomycin, and tobramycin have a
cyclohexane ring and may cause deafness and loss of balance! consielegt bmetbom-oipoloia
Chloramphenicol may cause a temporary or permanent depression of the bone marrow that leads to
aplastic anemia and leukopenia which are
the toxic effects of the drug.
Tetracyclines bind to 16S rRNA
near the aminoacyl-tRNA acceptor site, reversibly inhibit protein
synthesis, hence disrupting the rotation of bound
tRNA into the A site during translation.ite
Chloramphenicol and erythromycin bind to 23S rRNA on the 50S ribosomal unit.
The increased dosage of tetracycline may lead to hepatic and kidney damage and yellowing of the
teeth of children. 316
Macrolide antibiotics such as erythromycin, clindamycin, and azithromycin inhibit the assembly of
lo 3. Nucleic Acid Synthesis Inhibitors

a. Rifampicin - inhibits RNA polymerase; inhibits RNA synthesis
Quinolones - interfere with DNA gyrase and topoisomerase IV and highly effective for
addqu sl enteric bacteria like E. coli
Metronidazole - disrupts DNA and is effective against anaerobic bacteria

Topoisomerase IV is found in Gram-positive, and DNA gyrase is present in Gram-
negative bacteria
4. Cell Membrane Inhibitors
Polymyxin B and E are effective against Gram-negative bacteria, like Pseudomonas aeruginosa,
and also as a topical antibiotic.
(.8.$.8.5:32) agasmslosl-8
Some examples are polymyxin and daptomycin.
5. Essential Metabolite Inhibitors
a.
b.
C. Cord factor inhibitor: Isoniazid

molecules that are needed in
The folic acid pathway provides the essential precursor
DNA biosynthesis.
in nature. S1 12760
SMZ and TMP are synthetic drugs that do not exist
Antibiotic Resistance against the target pathogens.

antimicrobialsclinical,
T involves alleration of the supposed action of theenvironmental, or due to theorganism
resistance may be biologic,

The antimicrobial
itself.
in the organism's
physiology while
involves disturbance the
Biologic-mediated resistance to changes in the pH of environment, oxygen level, or cation
environmental-mediated is due
concentrations of the antibiotics.
various bacterial strains
create different
is observed when to inhibit or kill the
Clinical-mediated resistance of the drugs
to antibiotics resulting to inability
susceptibility patterns ran either be intrinsic or extrinsic resistance.
pathogens whereas microorganism-mediated
Intrinsic Resistance result of the normal physiologic and biochemical
resistance that develops as
a
It is a form of
makeup of the bacteria.

cell.
It is passed vertically into a new
of the antibiotic and on the impermeability
It depends on the hydrophobic or hydrophilic nature
of the cell wall to the antimicrobials.
of resistance through the enzymatic inactivation of
All Gram-negative bacteria mediate this type
anand/ternaryye
penicillin.
the secretion of by bacteria, presence
Some examples
of aminoglycosides (enterococci and anaerobes), and resistance

to glycopeptide antibiotic
(Gram-positive bacteria).
Acquired Resistance
It is caused by the changes in the genetic structure ofthemicroorganisms.
It is usually acquired by a certain species or bacterial strain. AnotididatbosidmnoM1
Some examples are through mutations and gene transfer.
p-lactamases (EC 3.5.2.6.)

Other name: Penicillinase or cephalosporinase
Substrate: -lactam ring
Location of the $-lactamase gene: Plasmids, transposons, integron, or chromosome
These are enzymes that chemically inactivate [-lactam drugs by disrupting and consequently
opening the p-lactam ring component molecule to form the inactive penicilloic acid.
of the
The production of theseenzymes by somebacteriasuch as staphylococci,Haemophilus ingluenzne,
species
Neisseria gonorrhoeae,
enterobacteriaceae, Pseudomonas, Moraxella catarrhalis, and
/ Bacteroides
is a significant mechanism
contributing the resistance some P-lactam drugs.
Eatery
In Gram-positive
to as to to the
these are secreted
bacteria, less protection
offer
ANTIMICROBIALS (ANTIBIOTICS) 177
The synthesis
of these bacterial enzymes may be inducible of constitutive; that is, inducing
like oxacillin (another g-lactam drug) is
agent an
essential to achieve sufficient amount of the

enzyme, or
same concentration of $-lactamase produced regardless of exposure to an inducing
agent.
Major Classifications of G-Lactamase

Class
1. A and
D (Serine Peptidases)
2. Class B (Metalloenzymes)
3. Class C (Cephalosporinase)
Class
the most clinically important B-lactamases.
A and C are
Class A p-lactamase is found on

plasmids and mostly in Gram-negative bacteria, such as
TEM, CTX-M, SHV, SME, PSE, IMI and KPC.
Class B g-lactamases are VIM, IMP, and NDM.
Class C f-lactamases such as AmpC and CMY are chromosomally located.
Class D .-lactamase that is commonly identified is the OXA.
Discussion Points on B-lactamases

TEM-1 is the most commonly isolated p-lactamase in Gram-negative bacteria.
TEM-1 is found in plasmids and detected in E. coli, K. pneumoniae, E. aerogenes, and H. influenzae.
SHV-1 has been identified in K.
pneumoniae isolates and considered as the most common of the
chromosomal -lactamases found in this species.

CTX-M has been found in strains of Enterobacteriaceae. af
Metallo-B-lactam resistance is caused by the VIM, IMP, and New Delhi metallo-B-lactamase-1
(NDM-1).
NDM-1 hydrolyzes penicillins, cephalosporins, and carbapenems, with the exception of
Oxacillinase-48 (OxA-48), which belongs to class D of the Ambler classification, inactivates
oxacillin and methicillin.
Specific examples of OXA are OXA-1, OXA-23, OXA-33, and OXA-48.
blaCMY genes code for CMY.

Zinc is a requirement in the
class B B-lactamase, to execute its function.
Patterns of Resistance to B-lactam Antibiotics

the antibiotic
1. Production of -lactamase that hydrolyses the p-lactam ring of
the peptidoglycan structure in bacterial cell wall
2. Penicillin binding proteins (PBPs) that maintain
3. Alteration of porin channels
4. Initiation of efflux exporter proteins
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331424/
BACTERIOLOGY
B-lactamase Inhibitors function as substrates for

(-lactam antibiotics and
They have structural similarity with the
B-lactamase, trus reducing their harmful effects on the S-lactam antibiotics.core)
(they have -lactam
Examples: Clavulanic acid, sulbactam, and tazobactam
possess a P-lactam core.
Avibactam and relebactam are also G-lactamase inhibitors but do not
Extended Spectrum -lactamases (ESBLS) antimicrobials such as
are (-lactamases that

can inactivate extended-spectrum
These
and aztreonam.
cephalosporin, penicillin,
to the properties of the parent enzymes.
These enzymes are differentiated with regard
meropenem, and ertapenem) are actively against ESBL-producing
strains.
ESBL activity is inhibited by clavulanic acid.
Notes to Remember (012203210
PBPs and B-lactamases can serve as receptors for B-lactam antibiotics that enter the cell.
the
of altered penicillin-binding proteins which
are
The penicillin resistance is due to the presence
drug targets in the cell wall.
and Yersinia
The IncA/C multidrug-resistant plasmid has been detected in E. coli, Salmonella serovars
chloramphenicol,
pestis, and it carries resistance to B-lactams, aminoglycosides, tetracyclines,
quaternary ammonium compounds, sulfonamides, and trimethoprim.
mcr-1 gene has been identified in Enterobacteriaceae, and it creates plasmid-encoded polymyxin
resistance.
It is important to test penicillin-resistant isolates with other antimicrobial agents such as cefotaxime,
erythromycin, vancomycin, and tetracycline through MIC method and not disk diffusion.
Ceftriaxone is the drug of choice for penicillin-resistant H. influenzae and N. gonorrhoeae. 1i socre
Examples of How Antibiotic Resistance Spreads

5 1601
Animals get
antibiotics and George get
develop resistant antibiotics and
bacteria in their develops resistant
guts. bacteria in his guts.
Drug-resistant
bacteria on meat George stays at home
from animals. and in the general
When not handles community. Spreads
or cooked properly, resistant bacteria.
the bacteria can consistsfovi George gets care at
spread to humans. hospital, nursing
home or other
inpatient care
aten facility.
Fertilizer or water containing

animal feces and drug. Resistant germs spread
resistant bacteria is used on directly to other patients
food crops. or indirectly on unclean ant to noul
hands of healthcare
providers.
Healthcare
facility
Resistant bacteria
spread directly to other
patients from surfaces
within the healthcare
facility.
Patients go
Vegetable farm home.
DIM d
FIGURE 15-1. Development of antibiotic resistance

(Photo from Public Health Image Library)
BACTERIOLOGY
ASSESSMENT QUIZ
anemia as one of its toxic

1. This antimicrobial is a protein synthesis inhibitor that can cause aplastic
effects.
a. Gentamicin
b.
C. Chloramphenicol
d.
the normal metabolism of the bacteria and can be passed

2. This type of resistance is an outcome of
vertically to an offspring.
a. Passive resistance
b. Acquired resistance
C. Extrinsic resistance
d. Intrinsic resistance
3. Which of the following is the correct enzyme commission (EC) numbers of p-lactamase? n
a. E.C 3.5.2.6
b. E.C 3.1.3.1
C. E.C 1.1.1.27
d. E.C 2.7.7.7
4. It is the lowest concentration of an antimicrobial agent that can inhibit the growth of bacteria.
a.
b. MIC
C.
d. Therapeutic index
5. This drug inhibits the RNA polymerase, and it is classified as a nucleic acid inhibitor.
a. Metronidazaole
b.
c. Lincomycin
d. Rifampicin
What is the most

commonly encountered [-lactamase in Gram-negative bacteria?
6.
a. OXA
b. TEM-1
c. SHV-1
d. blaCMY
7. These are mobile DNA elements that are able to carry antibiotic resistant genes among plasmids.
a. Transposons
b. p-lactamase
C. Integrons
d. Transglycolase
8. In which class do the clinically significant -lactamase enzymes are found?

a. A and C
b. A and D
C. B and C
d. B and D
9. These enzymes are involved in the synthesis of the peptidoglycan and alterations in their
biochemical make-up can lead to antibiotic resistance.
a.
b. B-lactamase
C.
d. Gyrase
10. It is the source of the amphotericin B, an antifungal drug added into gonococcal media.
a. Streptomyces venezuelae
b. Micromonospora purpurea
c. Streptomyces
d. Bacillus subtilis
CHAPTER 16
SUSCEPTIBILITY TESTING

At the end of this chapter, the students should be able to;
1. define related terms;
2. discuss and contrast the antimicrobial susceptibility methods;
3. explain the preparation of the turbidity standard;

4. cite the factors that influence the result of the disk diffusion method;
5. differentiate the causes of false susceptibility and resistance;
b. discuss the principle of Etest: and
7. explain the principle of the automated antimicrobial sensitivity test.
Clinically, the antimicrobial susceptibility testing is significant because

it determines whether the bacterial isolate will be inhibited by the desired
antibiotics or killed, or has a pattern of resistance.
Antimicrobial susceptibility test supports reliable therapeutic management
since it identifies those drugs that can be effective against certain bacteria.
Purpose of the Antimicrobial Susceptibility Test (AST)

disease
the bacterial isolate to cause
1. To determine the capacity of
2. To identify the susceptibility patterns of the bacterial isolate
3. To ascertain the availability of
the standard susceptibility methods
184 REVIEW HANDBOOK
Terminologies
of an antimicrobial agent that coincides
1. Breakpoint (cutof) - refers to the concentration
particular drug.
for
with a
susceptible or intermediate MIC range
value (ECV)
is the minimum inhibitory concentration or
minimal
2. Epidemiologic cut-off
epaemetonrcentration (MEC) that separates a population into isolates with
phenotypic MIC value
and those
without
acquired or mutational resistance based on their
the lowest concentration of the antimicrobial
3. Minimum bactericidal concentration (MBC) -
is
agent that is needed to kill the bacteria

4. Minimum lethal concentration (MLC) - is the lowest concentration of the antimicrobial agent that
bacterial isolates when subcultured in a fresh medium
kills the
5. Paradoxic (eagle) effect is defined as the decreased bactericidal activity at a higher drug
concentration
the bacterial growth at higher drug concentrations and
6. Skipped wells - involves the presence of
no growth at one or more of the lower drug concentrations; this result may indicate contamination,
the antimicrobials, and the presence of an unusual
improper incubation, improper concentration of
resistant isolate
7. Trailing growth - involves heavy bacterial growth lower concentrations of drugs that is
at
followed by one or more wells that show a greatly reduced growth in the form of a small button or
a light haze
Standard Components of the Antimicrobial Susceptibility Test

Inoculum size, susceptibility medium, incubation condition, and antibacterial agents
Use of Control Tubes/Plates

It is a matter of requirement that each AST should include control tubes/plates - a growth control
tube/plate (broth/agar with the test inoculum) and uninoculated control tube/agar (culture
medium only).
shotued disnoo lenises avity ad
They should be incubated same with the test broths and plates.
Controltubes/plates should be read prior to the results of the patients to ensure that the AST
procedure is reliable.
Mueller Hinton Agar/Broth (MHA/MHB) 2sel orts to ento BAP

It is the
standard susceptibility
It is composed of
fatty acid), and agar.beef infusion, nucleic acids, vitamins, casein hydrolysate, (casein neutraties
It is the recommended medium
for susceptibility testing due to reduced concentration of
sulfonamide, trimethoprim, and tetracycline inhibitors
An MH broth with
2% NaCI is used to improve the detection of oxacillin-resistant staphylococcl
An MH broth with 5% lysed
horse blood
testing the susceptibility
or sheep blood is utilized for
for streptococci Neisseria meningitidis,and other
fastidious organisms.
ANTIMICROBIAL SUSCEPTIBILITY TESTING 185
Quality Control
Gram-positive
bacteria: Staphylococcus aureus ATCC 25923
Gram-negative bacteria: Escherichia coli ATCC 25922
Types of Antimicrobial Susceptibility Tests

1.
It involves challenging the organism of interest with antimicrobial agents in a broth

environment, preferably an MH broth.
Principle: The lowest concentration of the
antimicrobial drug or the minimum inhibitory
concentration(MIC) that will inhibit bacterial growth as shown by the absence of turbidity.
Preferred method: Serial twofold-dilution concentrations (1g/mL)
Standard inoculum size: 5 x 105 U/mL
Procedure: A specific amount of antibiotic is prepared in a decreasing concentration in
broth by a serial dilution technique in which a standard amount of the test organism is then
inoculated.
Interpretation: The absence of turbidity signifies the inhibition of bacterial growth by the
antibiotics being tested while persistent turbidity of the broth indicates resistance.
Mueller-Hinton broth with 2% to 5% lysed horse blood is utilized for fastidious bacteria like
streptococci.
a. Broth Macrodilution
MH broth volume: ≥1 mL
Incubation condition: 16 hours to 24 hours at 35°C
Advantage: For fastidious bacteria and identifying MBC endpoints

Disadvantage: Impracticability when used to test several antimicrobial agents or isolates
b. Broth Microdilution
It is the commonly used broth susceptibility test.
It is recommended for testing different antimicrobials (10 to 15 antimicrobials) and can

be used against a single isolate or
for susceptibility testing of fastidious bacteria.
It is also suggested for challenging the anaerobes with several antibacterial agents.
It is utilized for susceptibility testing of antibiotic-resistant bacteria such as the MRSA
(high-level producers of beta-lactamase).

MH broth volume: 0.05 mL to 1 mL
bacteria: 1 x 10t CFU/mL
Standard inoculum size for anaerobic
hours at 35°C; but for anaerobes, it takes up
Incubation condition: It takes 16 hours to 24
to 48 hours.
produce a penicillin MIC that is consistently inside the

Disadvantage: Inability to
resistant range for staphylococci (low-level p-lactamase producers)
BACTERIOLOGY
Notes to Remember
in the accuracy of the results of the antimicrobial testing.

Properly prepared inocula are vital
the broth dilution method.
Usually, 1 mL of the inoculum is used for
be carefully transferred into
the test inocula should the
During inoculation, all bacteria in
antimicrobial solution, otherwise, the
bacteria may adhere to the wall of the tube or well above
the
meniscus of the antimicrobial solution and

may remain viable
terms of the pH, cation concentrations (magnesium
The susceptibility medium should be checked in
and calcium), thymidine content, and amounts of supplements (blood).
2. Agar Dilution Method

It is the reference method for testing anaerobes and N. gonorrhoeae.
reproducibility.
It is frequently used in research studies because of its accuracy and
It determines both the MIC and MBC.
agar medium with
Principle: Different bacterial isolates are spot-inoculated onto an pre-
defined concentration of an antibiotic.

Standard inoculum size: 1 x 104 CFU/mL
Procedure: A standard inoculum of bacteria is spot-inoculated onto a 100-mm plate, then

incubate at 35 °C for 16 hours to 24 hours.
Shelf life of an agar dilution plate: 7 days

The susceptibility medium is MHA while Brucella-laked sheep blood (Wadsworth method)
is for the anaerobes.
For fastidious and microaerophilic bacteria, 5% defibrinated sheep blood is added into the
Advantage: For testing one or more bacterial isolates (up to 32 different isolates) utilizing ?
90- or 100-mm Petri dish
3. Disk Diffusion Method (Kirby-Bauer Test)

It is limited to aerobic and facultatively anaerobic bacteria.
It is a qualitative method that provides the greatest flexibility and cost-effectiveness.
It allows the laboratory to test
any 12 antimicrobial agents on a 150-mm MHA plate.
Principle: The larger the zone of inhibition, the lower the MIC since the zone of inhibition is
inversely related to MIC.
Standard inoculum size is 1.5 x 10s CFU/mL
Procedure: A filter paper
disk impregnated with an antimicrobial agent of specific
concentration is carefully placed on an agar plate that is inoculated with the test
previously
organism.
Disadvantage: It is not recommended for testing slow-growing organisms like mycobacteria

and anaerobes.
YOOJON ANTIMICROBIAL SUSCEPTIBILITY TESTING 187
Turbidity Standard (0.5% McFarland Solution/Barium Sulfate Suspension)qphrl

It is composed of 99.5 mL of 1% H,SO, and 0.5 mL of 1.175% of barium chloride.
It is
equivalent to 1.5 x 108
Pure cultures are
grown or are directly prepared on agar plates to match the turbidity of the 0.5%
McFarland standard.
Preparation of Pure Inocula

1.
Using a sterile inoculating loop or needle, isolate four or five colonies of the same morphology
from a non-selective culture medium.
2. Transfer the isolated colonies into 2 ml of sterile saline.roileplal af esanemont antlaob
3. The bacterial suspension and McFarland solution are compared by matching the turbidity of the
tubes against a dark background.
bacterial suspension initially does not match the turbidity of the McFarland standard, the
If the
suspension may be diluted or supplemented with additional organisms until it achieves the same
transparency.
5. The inoculum should be used within 15 minutes. Ad nouidinnllo anos s dmanq
Note: To ensure accurate results, isolates must be in the log phase of growth.
BBL Prompt Inoculation System

It can be used to achieve a standard bacterial inoculum usually for disk diffusion method.
Components: Inoculum rod (wand) that can hold 1.5 x 108 CFU/mL and saline tube
Procedure: Using the sterile loop, five to ten colonies from a pure plate are "touched" by the rod,
then transfer the isolated colonies by placing the wand into the small plastic vial with saline. Mix
the suspension with a vortex and use the inoculum within 6 hours.
During Streaking and Placement of Disks

Ene 1.0 The MHA plate must be turned 60 degrees between each streaking (overlapping streaking),
ideally three times without resuspending the swab into the inoculum. MHA supplemented with
5% sheep's blood is recommended for fastidious bacteria like streptococcci.
surface of the agar to dry for three to five minutes but not longer than 15 minutes.
2. Allow the
3. The antimicrobial agents are applied onto MHA with a sterile forceps or disk dispenser.
mm from disk center to disk center and no closer
4. The disks must be positioned no closer than 24
than 10 mm to 15 mm from the edge of the plate. The disks are pressed firmly to ensure contact
with the agar.
5. Within 15 minutes of inoculation, plates are inverted and incubated at 35 °C for 16 to 18 hours.
Plates are inverted to
avoid the moisture contamination on the agar surface that can interfere
with the interpretation of test results.
Susceptibility plates with suspected methicillin-resistant and vancomycin-resistant strains

are incubated for 24 hours.
For fastidious bacteria such as S.

pneumoniae and N.gonorrheae, susceptibility plates may be
incubated in an environment
with an increased CO,.
Interpretation of Kirby-Bauer Test

1. Susceptible (S)
the antibiotic employed in the study
could be a good therapy option.
It suggests that
resistance or a clinically negligible level of resistance.

It is represented by the lack of bacterial
It shows a well-defined zone of inhibition aroundthe antibiotic disk.
2. Susceptible-Dose Dependent (SDD)
It specifies that the isolate's susceptibility is dependent on the dosage protocol. rtieian
It implies that changing the antimicrobial dose, such as by increasing or more frequent
dosing, increases the likelihood of growth inhibition. ne6r2 contuar
3. Intermediate (I)
It shows possible effectiveness of the antimicrobial agent against the isolate, but it is less
potent than the drug interpreted as susceptible.
There will be less clinical response compared to a susceptible strain. VOLG InGeN5y)
It presents a zone of inhibition but smaller compared to susceptible zones. boont erft
4. Resistant (R)
It means that the antimicrobial agent might not be the best option for therapy.
There is no zone of inhibition, or the inhibition does not meet the criteria for
susceptible or
intermediate.
boy 5. Nonsusceptible (NS) inolos hol of

It describes antimicrobials with MICs above or zone diameters below the susceptible
breakpoint.
Reading of Plates
The growth must be confluent to be
accepted for plate reading, hence Petri plate with poor and
non-confluent growth should be
2. Plates are examined two to
three inches above a black, non-reflective surface and the zones are
measured from the back side of the plate. o951ft 1o1
3. The diameter of each zone of
inhibition is measured using a caliper or ruler.
4.
Transmitted light (plate held up to the light source), rather than
124370 accuracy of tests with a reflected light, will improve the
penicillinase-resistant penicillin.
5. For media that contain
blood, the plate is examined with the lid removed.
6.
Zone measurement is compared with
the interpretive tables of CLSI, and the results are usually
reported as susceptible, intermediate, or resistant
7.
Control plates should be
read prior to the interpretation of results to determine whether the test
was performed correctly.
VOCION ANTIMICROBIAL SUSCEPTIBILITY TESTING 189
Causes of False Resistance shorticidto

1.
Use of heavy inocula or undiluted specimen®
2.
Late examination of plates (prolonged incubation)
3. Use
of disk with inadequate drug concentration (prolonged drug storage)
4.
Failure to immediately refrigerate the disk after use
5. Use
of wrong bacterial isolate
Notes to Remember
The
drug concentration decreases at increasing distances from the disk.
A
wide zone surrounding a disk signifies a greater susceptibility of the organism to the antibiotic.
Zone width is related to
antibiotic concentration, diffusion rate, and solubility.
Colonies appearing within a clear zone as a result of contamination or presence of mixed culture
should not be ignored; the original colony should be retested.
The zone size of a swarming organism such as Proteus should be ignored when reading the area of
inhibition zone sizes are measured at the point where bacterial growth is inhibited.
The presence "haze" is a sign of bacterial growth especially when testing sulfonamides and
trimethoprim, and this should be ignored only when the two antimicrobials are used.
artisibl
Routine susceptibility testing on p-hemolytic streptococci is generally unnecessary.
Mueller- Hinton base supplemented with growth factors (X and V) also known as the Haemophilus test
medium (HTM) is preferred for H. influenzae.
HTM broth and agar are utilized for susceptibility testing. operfe svitmells nE 26 been af fl
Factors Affecting the Zone of Inhibition

1. Amount of inoculum or test organism
Only pure cultures can be tested.
If the inoculum is too light, it would result in a false susceptibility; if the inoculum is too
heavy, it would result in a false resistance.
2. Thickness of the susceptibility agar plate 4 mm)
If the agar is too thick, the zone sizes will be smaller; if the agar is too thin, the zone sizes
will be larger.
Growth rate of the test organism

best for most bacteria with 16 hours to 18 hours of incubation.
A temperature of 35 C is
to the false detection of MRSA.
Temperatures higher than 35*C may lead
zones of inhibition.
Lower temperature may lead to larger
is not recommended except
Incubation with increased 5% to 10% carbon dioxide for
meningitidis and S. pneumoniae.

as N.
capnophilic bacteria such
The pH of the medium (pH 7.2 to pH 7.4)

in decreased pH.
Incubation of plates in CO2 could result
Increased pH results in the decreased activity of tetracycline
Low pH decreases the activity of aminoglycosides and erythromycin.
5. Number of disk per plate
A 150-mm plate can have a maximum of 12 disks.
Placement of more than 12 disks may result in overlapping zones (erroneous results).
6. Concentration of divalent cations (calcium and magnesium) in susceptibility media bn

it can affect the testing of aminoglycosides and tetracyclines against P. aeruginosa. sinolo
Elevated concentrations of the divalent cations may result in the diminished activity of the
10 6915 ar aminoglycosides against P. aeruginosa and the decreased activity of tetracyclines against all
bacteria.
It is dilution test that is based on the diffusion of a continuous varying concentrations of an

antimicrobial agent.
It uses thin plastic strips that is impregnated with a single antibiotic of decreasing concentrations
(MIC concentration scale).
It is used as an alternative
susceptibility test for fastidious bacteria like the S. pneumoniae,
H. influenzae, and anaerobic bacteria.
Procedure: A strip is placed on the surface of the culture medium that is inoculated with the
desired organism, and the antibiotic is diffused into the surrounding agar.
Result: Ellipse of growth inhibition
Interpretation: The MIC is read at the point on the scale where the ellipse intersects the strip.
Minimum Bactericidal Concentration Test

It determines the lowest level of the antimicrobial agent that kills bacterium.
Standard inoculum size: 5 x 105
Procedure: 0.01-mL aliquot of each clear MIC tube or well is subcultured to an agar medium,
usually BAP, and incubated overnight.
Volume spread per plate: 0.1 mL
YOCJOISD ANTIMICROBIAL SUSCEPTIBILITY TESTING 191
(+) Result: A 99.9% reduction in

the CFU/mL ascompared to the concentration in the original
inocula which is recorded asthe MBC
Interpretation: The subculture plate observed for bactericidal effect or almost complete
is
absence of bacterial growth, that is 99.99% reduction in the CFU/mL. MiVn 12 ahholpb
an
Time-Kill Assay
It involves the addition of a
bacterial isolate to a broth medium with an antimicrobial agent.
The rate of
the killing of bacteria is measured over a specified period.allay
Procedure: The test bacteria are inoculated into several broth tubes that contain various
concentrations of antibiotics. After incubation at 35 °C, small aliquots are diluted and plated for
colony count.
Result and
Interpretation: Three or more log"0 reduction in bacterial count in the test plate as
compared with the control plate, which indicates an
adequate bactericidal response oriet s
Synergy Test
It detects theeffectiveness of combined antimicrobials against a single bacterial isolate.D
It is
performed using a broth dilution checkerboard method or time-kill assay.
Procedure: Two antimicrobial agents are tested in each well or tube.
bits Result: Microorganisms are killed by a hundred-fold or more as compared to the most active
antibiotic that is tested
singly after 24 hours of incubation.
Interpretation: Antagonism is seen when the two antibiotics appear less potent than the most
active single antimicrobial.
Similar to the MIC/MBC test, its inhibitory and bactericidal parameters are evaluated.
It measures the activity of one or mor antimicrobial agents in the patient's serum against an
organism that is obtained from the patient.
The specimens are trough and peak
sera sample for trough level is obtained 0-30 minutes
can be 30 minutes to 60 minutes after IV infusion,
before the next dose while the peak sample
dose. tur B.emabsd t0woto
60 minutes after IM injection, or 90 minutes after oral
The culture medium is the patient's serum with the antibiotics given to the patient.
Final inoculum size: 5 x 105 CFU/mL
is plated on MHA supplemented with blood or BAP and
Procedure: An aliquot of each dilution,
then incubated.
that inhibits visible growth is the serum-static titer.
Result: The highest dilution
in a 99% reduction in CFU/mL as compared
Interpretation: The serum dilution which results
with the original inoculum,
is the serum-static
Carba NP (CNP) Test
t is a confirmatory test for carbapenemase production of some Gram-negative bacili,

It detects KPC, VIM, NDM, and IMP.
Test antimicrobial: Imipenem in a broth solution
pH indicator: Phenol red
Incubation condition: 35 •C for 2 hours
Positive result: Yellow color
Negative result: Red-orange color (both tubes)
Automated Antimicrobial Susceptibility Test
It follows the principle of turbidimetric detection of bacterial growth in a broth medium by using
a photometer to examine the test wells.
lack of turbidity, whereas an
In this method, susceptibility to antimicrobial agents is based on the
indication of resistance is based on the increase in turbidity.
For the interpretation of results, growth endpoints of broth microdilution panels are detected
using an automated reader device.
of using this method is that it has a shortened analysis period of
5 hours to 15
The advantage
hours.
Some examples are the Phoenix system, MicroScan WalkAway system, TREK Sensititre, and
Vitek 2 and Vitek Legacy systems.
Growth detection can also be performed through the hydrolysis of a fluorogenic growth
substrate that is added into a special test medium.
Fluorometer is also used for growth detection in which microbial growth is detected as an
emission of a fluorescent signal when microorganism consumes a fluorophore-labeled substrate
in the test medium during growth.
Examples of Automated Susceptibility Testing Methods

1. Phoenix system
It uses a manual gravity-based inoculation technique.
Growth patterns are automatically read following the redox indicator system. gin on
It has mechanism to confirm ESBL of Gram-negative bacteria.

Test results are given after 8 hours to 12 hours and resistant bacteria are also routinely
identified.
2.
MicroScan WalkAway system
In this method, the inocula are manually introduced into the broth microdilution tray.
Growth patterns are automatically read and interpreted after incubation.
Bacterial growth could be detected using or
spectrophotometry (overnight incubation)
fluorometry (provides results after three to five hours).
YOOUDI ANTIMICROBIAL SUSCEPTIBILITY TESTING 193
3. VITEK system
It is a fully automated
equipment that is designed for the identification of bacteria and
antimicrobial susceptibility test.
Inocula are mechanically added to
specified concentrations
of antibiotics placed in
miniaturized plastic card.
a
In VITEK 2 analyzer, optical readings are made

every 15 minutes to measure transmitted
light through each well; total of 64
wells are used with up to 22 different antimicrobials in
various concentrations
Components: Sample processor and reader/incubator, computer work station, Smart Carrier
Station, and barcode scanner
Final reading: 6 hours to hours
MIC and antimicrobial testing results can be identified using the Advanced Expert System
(AES).
BACTERIOLOGY
TE SITTIV
Disk Diffusion Method

FIGURE 16-1. Measurement of the zone of inhibition in
(Photo from USCDCP)
weve hogxa boonsybA adiigatad bellion
FIGURE 16-2. Disk diffusion method using Mueller-Hinton agar

(Photo from USCDCP)
pH 8.0 pH 7.2
TE = 18mm
TE - 22mm
$ - 20mm
E - 27mm
FIGURE 16-3. Kirby-Bauer method, different measurements of zone of inhibition

(Photo from Public Health Image Library)
p0uoneANTIMICRO IAL SuSCEPTIBILITY TESTING 195
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FIGURE 16-4. McFarland Standard against a Wickerham card n noiauiib odt
tnsSource: Textbook of Diagnostic Microbiology, Mahon and Lehman, 6th ed, 2019i mn
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FIGURE 16-5. Etest gentamicin strip for Escherichia coli susceptibility testnsnnon
Source: Textbook of Diagnostic Microbiology, Mahon and Lehman, 6th ed, 2019
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ASSESSMENT QUIZ
answer.
Encircle the letter
that corresponds to the correct
1. It measures
the effectiveness of two antimicrobials against a single bacterial isolate.
a. Time-kill asay
b. Disk Diffusion
c. Schlichter test
d. Synergy test
2. Which of the following is

an automated system for routine susceptibility testing?
a.
b. MicroScan WalkAway
c. both
d. neither of the choices
3. This is the distance of the antibiotic disc from the edge of the plate to avoid erroneously reading
the diffusion method.
a. 5 mm to 10 mm
b. 10 mm to 15 mm
c. 15mm to 20mm
d. 20 mm to 25 mm
4. Which cations are added to the Mueller-Hinton medium?
a. phosphorus and magnesium®

b. magnesium and ionized calcium
C. magnesium and total calcium
d. ionized calcium and phosphorus
5. It is described as having decreased bactericidal activity in the presence of a high drug

concentration.
a. eagle effect
b. trailing growth
C. breakpoint
d. MBC
ANTIMICROBIAL SUSCEPTIBILITY TESTING 197
6. What is the next step that should be done if

haziness
appears on the MH medium after
challenging a bacterial growth with sulfonamides and trimethoprim?
a. change the disk, expired
b.
prepare another inoculum, sign of contamination
c. change MHA plate, improper storage
d. ignore, acceptable result
7. It determines carbapenemases such as KPC and VIM.

a. Etest
b.
C. Kirby-Bauer
d. Broth microdilution
8. Which of the following can cause false resistance?

a.
inoculum is too light compared with the Mc Farland standard
b. use of carbon dioxide incubator
c. improper storage of antibiotic disc

d. thickness of the agar is < 4 mm
9. Which of the following may lead to false detection of the resistant organisms such as the MRSA?
a.
incubation temperature higher than 35°C
b. use of methicillin disc
c. increased divalent cations in the medium
d. low pH of MHA
10. What is the equivalent inoculum size of the 0.5% BaSO,?

a. 1.5 x 108 CFU/mL
b.
c. 5 x 105 CFU/mL
d. 5 > 108 CFU/mL
CHAPTER 17
GRAM-POSITIVE COCCI

1. differentiate the general characteristics of staphylococci, streptococci,

and enterococci;
2. virulence factors in Gram-positive cocci and their relation to the

cite the
progression of disease;
3. compare the characteristics of Staphylococcus aureus with those
species in the group;
4. explain the pattern of antimicrobial resistance of staphylococci;
5. describe the hemolytic reactions of streptococci and the Lancefield
classification;
6. differentiate the pathogenecity of the medically significant streptococci;

7. distinguish between the properties of Enterococcus and Streptococcus
bovis; and
8. design an algorithm of diagnostic tests to determine the clinically

significant species in the group of Gram-positive cocci.
Staphylococci
The genus name is derived from the Greek words staphule, which
which means "berries."
means "bunches of grapes," and kokkos,
These are Gram-positive cocci that belong to the family
Staphylococcaceae.
and facultatively anaerobic bacteria
They are catalase-producing
except S. saccharolyticus, which is an
obligate anaerobe.
They are non-motile, non-spore-forming glucose fermenters.

the skin, mucous membranes, and
They are normal inhabitants of
intestines.
with human infections are
The species which are associated
surfaces.
colonizers of various skin and mucosal
BACTERIOLOGY
clusters or sometimes singly

Microscopy: Gram-positive spherical cells that appear in
Culture: BAP Colonies are creamy,
white light gold, with butyrous appearance while other
or
B-hemolytic like S. aureus.

species may have gray colonies; some species may be
Common species: 5. aureus, 5. epidermidis, S. saprophyticus, S. hemolyticus, S. lugdunensis, S. hyicus,
S. intermedius, S. delphini, S. lutrae, and S.
TABLE 17-1. Differential Tests Between Staphylococci and Micrococci

Result
Quality Control
Test
Susceptible:
Bacitracin/Taxo A (0.04 units) Susceptible - Micrococci
(2 10 mm zone of inhibition)
Streptococcus pyogenes ATCC 19615
Medium: BAP or MHA
Micrococcus luteus ATCC 10240
Resistant - Staphylococci
(≤ 6 mm or no zone of inhibition) Resistant:
Streptococcus agalactiae ATCC 27956
Furazolidone Susceptibility Susceptible - StaphylococciSusceptible:
(100-ug) (≥ 15 mm zone of inhibition) Staphylococcus aureus ATCC 25923
Medium: BAP Micrococci Resistant Resistant:
(≤ 9 mm or no zone of inhibition) Micrococcus luteus ATCC 10240
Lysostaphin Susceptibility Staphylococci - Susceptible Susceptible:

(10-ug or 200 g/L) (10 mm to 16 mm zone of Staphylococcus aureus ATCC 25923
Medium: MHA or broth inhibition) Resistant:
Micrococci - Resistant Micrococcus luteus ATCC 4698
Modified Oxidase/Microdase Positive - Blue color within

minutes Micrococcus luteus ATCC 10240
Reagent: Tetramethyl-p-
phenylenediamine in dimethyl Negative - No color change Negative: inc ont easons
Staphylococcus aureus ATCC 25923
Growth on Furoxone-Tween-80-Oil Positive - Micrococci Positive:
Red O Agar Micrococcus luteus ATCC 10240

Negative - Staphylococci

Anaerobic Acid Production from Positive - Staphylococci Positive:
Carbohydrates Open and Closed Tubes: Yellow Staphylococcus aureus ATCC 25923
Medium: Baird Parker's Negative Micrococci Staphylococcus aureus ATCC 9144
Modification OF
Closed/Anaerobic Tube: Purple/ Negative:
Glucose: 1% No color change (but in open/ Micrococcus luteus ATCC 10240
aerobic tube it is yellow color)
pH indicator: Bromcresol purple
dimet enlt of gnolid ted 1bm0s Note: Staphylococci are both
positive with OF (open and closed
Note: Sometimes it is called the
tubes) test while Micrococci only in
b1 oxidation test (open tube).
Anaerobic Acid Production from
Positive/Yellow color
Glycerol-Erythromycin Medium Staphylococci
Erythromycin: 4-ug Bits Negative - Micrococci
PH indicator: Bromcresol purple
YE0 I0 BTDAS OIROMONId GRAM-POSITIVE COCCI 201
Staphylococcus aureus
It is the
true coagulase-positive and
the most virulent species of staphylococci.
It is chiefly responsible for the various skin,
wound, and deep tissue infections.
This organism causes infection when it enters a normally sterile site (trauma or abrasion of the
skin or mucosal surfaces).
It is acquired through direct exposure with unwashed hands, as well as contact with inanimate
objects.
baailq mon sirafond arb pntbelong
It is also one
of the common causes of food poisoning.
It can
be cultivated by adding 7.5% to 10% NaCl (halophilic microorganism).
Principal virulence factor: Coagulase
Culture: BAP Colonies have a golden yellow color and narrow zone of B-hemolytic pattern
(sometimes no hemolysis).
Related Infections and Diseases

Staphylococci are inhabitants of the skin surfaces and nares, hence, transmission from person to
person IS common through contaminated hands and fomites.
Aside from cutaneous and toxin-induced infections and diseases, S. aureus has been implicated
in several conditions such as septicemia, acute bacterial endocarditis, spinal epidural abscess,
osteomyelitis (musculoskeletal infection), septic arthritis in children, cystic fibrosis, suppurative

intracranial phlebitis, urinary tract infection, and nosocomial pneumonia.
Patients in nurseries and burn units, as well as those who have had surgery or other invasive
treatments, are susceptible to hospital and hospice outbreaks caused by this organism.
Methicillin-resistant Staphylococcus aureus (MRSA) infections, both community acquired and
nosocomial, are a key health alert.
she ent ap abled asturnsh il
1. Cutaneous infections (Skin and soft tissue infections)
a. Folliculitis - is a mild inflammation of the hair follicle or sebaceous gland
b. are large, raised, superficial abscesses which can be an extension of folliculitis
C. Carbuncles develop from multiple furuncles which may advance into the deeper tissues
and cause fever and chills leading to systemic infection
d. Staphylococcal impetigo contagious superficial infection that is commonly seen

is a
vesicles surrounded by a red border;

in children and characterized by crusty lesions and
S. aureus causes both
the bullous and non-bullous impetigo.
2. Toxin-induced cases
one or more preformed
a. Staphylococcal food poisoning - it comes from ingesting
enterotoxins found on S. aureus-contaminated
food or beverages; self-limiting; typically
onset
resolves within 24 to 48 hours of
is an extensive exfoliative dermatitis that occurs primarily
b. Scalded skin syndrome (SSS)
af in newborns and previously healthy children
Toxic shock syndrome (TSS)

is a rare butpotentially fatal, multi-organ system illness that
of fever, chills, vomiting, diarrhea, muscle aches, and
C.
is characterized by a sudden onset

shock
rashes, desquamation; and it rapidly progresses to hypotension and
Virulence Factors: Enzymes and Toxins wine it noriv n0. ali eaun
1. Coagulase
It promotes the formation of a fibrin layer around
the staphylococcal abscess, thereby
protecting the bacteria from phagocytosis.
It coagulates the fibrinogen in the plasma.
Types of Coagulase
a. Cell-bound coagulase or clumping factor - is bound to the cell wall and clots human, rabbit,
maling
or pig plasma by directly converting fibrinogen into fibrin
b. Unbound or free coagulase - is extracellular enzyme that is not bound
an to the cell wall and
promotes clot formation when bacterial cells are incubated with plasma
2. Hyaluronidase (Spreading-factor enzyme)

It enhances invasion and survival in the tissue.
It breaks down the hyaluronic acid that is present in the intracellular ground substance of
connective tissues, resulting in the spread of bacteria.
3. Staphylokinase (Fibrinolysin)
It causes fibrinolytic activities by dissolving fibrin clots.
Lipase (Fat-splitting enzyme)

It is produced by both coagulase-positive and coagulase-negative staphylococci.
It degrades lipids on the skin surface allowing bacterial entry into epidermal layers.
It is essential for bacterial survival in sebaceous areas
of the body. -0
It is vital in the formation of furuncles, carbuncles, and boils.
5.
Deoxyribonuclease (DNAse)
It lowers the viscosity of exudates giving the pathogen more mobility.

It destroys DNA.
6. p-lactamase
It breaks down penicillin and other beta-lactam drugs.

More than 90% of clinical
staphylococci isolates are penicillin-resistant as a result of enzyme
production
or the synthesis of the inducible plasmid-encoded -lactamase
7.
Enterotoxin (Heat-stable toxin)
It is produced by the majority
of S. aureus isolates causing staphylococcal food poisoning
It is stable to heating
eliminate the
at
100°C for 30 minutes; reheating contaminated food does
not
organisms.
203
It appears to act as
neurotoxins that stimulate vomiting through the vagus nerve. oT feat
It is resistant to hydrolysis by gastric and jejunal enzymes:.n- ONES R!
Examples: A, B, C1, C2, D, E, and G to ]
Enterotoxins A, B, and D
infected food handler
are heat-stable and mostly responsible for food poisoning (the
is the source of contamination) with enterotoxin A being the most
common etiology.
Enterotoxins B, C, G, and I are associated with enterocolitis.

Enterotoxin B is related to
pseudomembranous enterocolitis and is often found in
contaminated milk products.
8.
Panton-Valentine Leukocidin (PVL-Cytolytic toxin)
It attacks and kills white blood
cells (PMN, macrophage, and monocytes). ns Pf sf
It is a pore-forming exotoxin that suppresses phagocytosis.
It is responsible for necrotizing skin and soft tissue infections.
9. Hemolysin (Cytotoxin)
It
causes anemia and makes iron available for microbial growth.
ofgnia lagd ent elfl
Types of hemolysins
a. Alpha-hemolysin
It is the predominant hemolysin that is produced by S. aureus.
It destroys red blood cells, platelets, and macrophages; and it causes severe tissue
damage.
b. Beta-hemolysin
It degrades spingomyelin and RBC around nerves.
It enhances hemolytic activity on incubation at
35°C (heat-labile) and subsequent
exposure to 4°C.
C. Gamma-hemolysin
It is less toxic than alpha- and beta-lysin. 40201s7d nouron of bods st of
It is produced by all S. aureus strains that cause RBC injury in culture and produces
edematous lesions.
d. Delta-hemolysin
it is less potent compared to other hemolysins.
It destroys RBC, but
It is associated with the Panton-Valentine leukocidin.
and Bho tol) Hrmd (s
10. Exfoliatin Serotypes A and B /Epidermolytic Toxins A
toxin is a serine protease that divides the intracellular bridges of
Exfoliatin or epidermolytic
the epidermis and causes extensive sloughing of the epidermis to produce a burn-ike effect
on the patient.
It degrades the stratum granulosum (surface layers of the skin).
It causes scalded-skin syndrome (SSS) or Ritter disease.
11. Toxic Shock Syndrome Toxin 1 (TSST-1/Superantigens)/Enterotoxin F/Pyrogenic Exotoxin C

It is a chromosomal-mediated toxin. o bib P.1u63
It causes almost all conditions
of menstruation-associated TSS.
of a large amount of cytokines
that are responsible for the
It stimulates the production
It is absorbed through the vaginal mucosa,

thus allowing the systemic effects seen in TSS.
It has a tendency to cross imucosal surfaces and can reactivate bacterial cell wall-induced
arthritis.
It enhances the lethal dose of endotoxin on renal tubular cells.
12. Protein A
It isan immunologically active substance that is found in the cell wall.

It is antiphagocytic since it competes with neutrophils for the Fc portion of specific opsonins.
Differential Tests for Staphylococcus aureus

1. Coagulase Test
It is the best single criterion of pathogenicity of Staphylococcus aureus.
Reagent: Rabbit plasma
Anticoagulant: Ethylene diaminetetra-acetic acid (EDTA)
Citrate-utilizing organisms may vield false-positive results, so EDTA-containing plasma
QUEal BTw should be used instead of citrate.
Quality Control
Positive: Staphylococcus aureus ATCC 25923
Negative. Staphylococcus epidermidis ATCC 12228
Types of Coagulase Tests

a. Slide method
It is used to screen catalase-positive colonies.

It detects cell-bound coagulase or clumping factor on the surface of the bacterial cell
wall.
In this test, the clumping factor reacts directly with the fibrinogen in the plasma without
incubation.
Procedure: Using a sterile wire loop, an isolated colony is emulsified in a drop of rabbit
plasma. Then rotate the slide for 10 seconds.
(+) Result: Clot or coagulum formation within 30 seconds
Other slide coagulase-positive staphylococci: S. lugdunensis and S. schleiferi

b. Tube method one
It is considered as a sensitive but definitive method.
It detects extracellular or free coagulase.

X3QJOIREIDAR SITRONDAIC GRAM-POSITIVE COCCI 205
Procedure: A
tube containing 0.5 mL of
35°C initially for hour to 4 hours. A
the reagent is inoculated and then incubated at
positive and negative control should be included.
(+) Result:
Clot or coagulum formation after one to four hours of incubation px
If no clot appears after 4 hours of incubation, the tube should be left at room temperature
for an
additional 20 hours of
incubation (overnight incubation). di-Buhih s wrie
Other tube coagulase-positive staphylococci: S. hnyicus, S. intermedius, S. lutrae, S delphini,
and S.
schleiferi subsp. coagulans
2. Mannitol Fermentation
It is used to differentiate the pathogenic staphylococci from non-pathogenic species.

Culture medium: Mannitol salt agar (MSA)Rom Brus hebtuoa lo tnondeed anoigyd
(t) Result: Yellow-colored colonies of S. aureus (colonies are surrounded by a yellow halo)
For
the interpretation, the pathogenic staphylococci ferment mannitol and produce acid.
The fermentation of mannitol gives the colonies and the medium a yellow color.
Quality Control omsiilesrabs
Positive: Staphylococcus aureus ATCC 6538 1 1D a0s MagS MO6D pinsgomonrD (+)
Negative: Escherichia coli ATCC 25922
shemped tod0o 1o A2AM-non
3. Deoxyribonuclease (DNase) Test

It identifies pathogenic species of staphylococci that produce DNAse.
It detects organisms that can hydrolyze DNA just like S. aureus and use it as a carbon source
for metabolism.
Culture Medium: DNase agar with methyl green dye and sodium chloride (light green color)
pH of the medium: 7.5
incubated at 35°C to 37°C for
Procedure: Test organism is streaked onto the medium and
18 hours to 24 hours.
(+) Result: Clear/colorless zone around the test organism mnithobie bistol ef
green in color.
(-) Result: DNAse agar remains

25922
Negative: Escherichia coli ATCC
4. Tellurite Glycine Agar Test

This results in jet black colonies of S. aureus.
5. Polymyxin Sensitivity Test

The S. aureus is resistant to this test.
Methicillin-resistant Staphylococcus aureus (MRSA)

as methicillin, nafcillin, and
It is type of S. aureus strain that is resistant to antibiotics such
401652
It is resistant to all A-lactam drugs including cephalosporins and carbapenems, though they may
show a susceptibility pattern to ceftaroline (Source: https://www.cdc.gov/mrsa/lab/index.html)
It has a weakly positive or sometimes negative
slide coagulase test.
It can be acquired after a prolonged stay in

the hospital (ICU and burn patients) close contact
with individuals who are carriers of this organism, after using broad spectrum antibiotics, and
exposure to nasal secretions.
It is controlled by the proper isolation of the organism, rapid identification of the bacteria, hand
to infection control
hygiene, treatment of sources, and most importantly, a strict compliance
programs.
It may also be resistant to other semi-synthetic penicillins.

It is probable cause of necrotizing/cavitary pneumonia and influenza-like illness.
MRSA infection is predicted by Gram-positive cocci on sputum samples. 1rts) edlT
Nasal swab is the common specimen used for detection of this resistant strain. vileuQ
(+) Chromogenic/CHROMagar test: Changes in the color of MRSA colonies from pink to mauve
color within 24 hours to 48 hours of incubation at 35 C against colorless or light blue colonies of
non-MRSA or other bacteria
Types of MRSA
1. Hospital-acquired (HA) MRSA

of hospitalization, use of intravenous catheters, past MRSA infections,
It is related to history
roles no living in a long-term-care facility or had a surgical procedure, which are all risk factors in
acquiring this strain.
It is resistant to antibiotics except glycopeptides (vancomycin).
2. Community-acquired (CA) MRSA

It is found predominantly in
younger individuals, generally linked to skin and soft-tissue
infections.
It has been isolated from correctional facilities of inmates, military

camps, crowded housing
conditions, and even among athletes causing outbreaks.
It is susceptible to non-f-lactam drugs compared to other types of MRSA, exhibiting less
resistance.
The use of injection drugs and MSM (men having sex with men) are risk factors. list
It shows a significant pattern of resistance to erythromycin and seldom to fluroquinolones.
It is reported
to be associated with a high rate of mortality and morbidity, and it frequently
carries the Panton-Valentine leukocidin (PVL) genes.
207
Health care-associated community-onset (HACO) MRSA

3.
It comprises the majority of MRSA infection

It manifests after months or
a year of hospitalization.
It is resistant to some antibiotics such as
clindamycin, erythromycin,and fluroquinolones.
Resistant Genes Associated with Staphylococcus aureus inrne astonlylopte
mecA gene is
responsible for the
which is present in the cell
production of a novel penicillin-binding protein (PBP2a or PBP2)
wall of the bacteria notably in S. aureus.
Because of the presence of mecA gene, S. aureus
becomes resistant to all -lactam antibiotics
and penicillinase-resistant penicillin drugs (methicillin, oxacillin, nacillin, cloxacillin, and
dicloxacillin).
The PBP2a has
low affinity for beta-lactams and it does not bind to penicillinase-resistant
penicillin drugs, thereby rendering any of those drugs ineffective for treatment.
A variant mecA
gene known as the mecC gene (mecC MRSA) is resistant to cefoxitin but with
differentiated susceptibility to oxacillin.
mecC gene produces PBP2c, which also renders the organism resistant to all beta-lactams.
2 stou
The mecA and mecC genes are not found in methicillin-susceptible strains.
Research shows that mecC gene has zoonotic
origin and has been isolated from Staphylococcus
stepanovicii.
Strict infection control measures should be designed to reduce the emergence of highly resistant
strains such as the vancomycin- resistant Staphylococcus aureus (VRSA).
The members are Staphylococcus aureus, Staphylococcus argenteus, and Staphylococcus schweitzeri.
Continuous research is on-going to completely characterize the members of this group.
Characteristics of the Staphylococcus aureus complex

a. S. argenteus is tube coagulase-positive and PYR negative, same with S. aureus. 2120Oyq2
as blaZ (most
b. S. argenteus was found to be carrying resistance genes such predominant), erm(C),
isolated from bats. 00

Staphylococcus argenteus and Staphylococcus schweitzeri are
said to be
C.
Differential Biochemical Test for Staphylococcus aureus complex pyel

1. D-Ribose Fermentation: (+) S. schweitzeri brn
2. N-acetyl-D-glucosamine: (+) S. aureus
S. argenteus
3. D-Galactose Fermentation: (+) S. aureus and
U KEVIEW HANDB0OK IN DIAGNOSTIC BACTERIOLO Y
Groups of Coagulase-Negative Staphylococci (CoNS)is Isolated

itnst Humans
from ued
1.Staphylococcus epidermidis Group
S. epidermidis, S. haemolyticus, S. capitis, S. hominis, S, pasteuri, S. auricularis, S. saccharolyticus,
moloS, warneri, and S. capraee pbvno araulaihbanenhor2 nsiy idrtni e
2. Staphylococcus saprophyticus Group th 4 cana0 ndan
(EE ro saprophyticus, S, equ S. nepalensis, and S. cohmnii5bo ailtia
aon bntie lelieetime bias
Staphylococcus intermedius Group
S. schleiferi subp, schieiferimuoed anntgy An lo soeaengodli to paael
3.
nitetdi
bas rilie iaan unauiueaeahianoeun
pannintenE
4.Staphylococcus simulans Group Wol ed ord
o ataS1t
S. simulans
the ualonieten tordyaisbre vderoll egub nilibine
t5.Staphylococcus sciuri Groupnnn entat en nvorme Auhtindiev A
S. sciuri and S. lentus n,iturzilliozntncitkiiga rua balatineeitib
Note: S. lugdunensis belongs to the CoNS Unspecified Group.abiiniietha pneg D
WiE ALti i1 i
StaphylococCus épidermidisbns hdo ehonoas Ca edan sdtewionedbiern

liniooaga
It is an indigenous microbiota of the skin.
in It is a contaminant of medical instruments, catheters (indwelling and IV), CSF shunts, and
prosthetic heart valve implants (implanted medical devices).n0ons7 dtas dbue antsa
It secretes an exopolysaccharide and a delta exotoxin that seems to contribute to its virulence.
Culture: BAP - Colonies appear white, opaque, small to medium-sized pin-heads, and are non-
hemolytic.ni Ln6 ritnyn anto nip prti Enoiylon e E usdem el
Biochemical test: Coagulase-negative; (-) MSApos 0 gninsan ridhrRe 2aoDaano
Antimicrobial test: Susceptible with 5- g novobiocin (16 mm to 27 mm zone of inhibition)
spae sOs wEbcan uleat untn 2aipnaoarD
Staphylococcus saprophyticustnen ndvitpalpeoeiehntieprtr
) .It is associated with community-acquired UTI in young, sexually active females.p 2 i
adheres more effectively to the epithelial cells that line the urogenital tract than other
coagulase-negative staphylococci (CoNS).rlinhp bat anhnuamopip
Culture: BAP - Colonies appear white although some strains produce yellow pigments, opaque,
slightly larger than pin-heads, and are non-hemolytic1e ahkain e
Urine culture: 10,000 CFU/mL (significant findings)tztEn e
Biochemical test: Coagulase-negative; (-) MSA
Antimicrobial test: Resistant to 5- g novobiocin (6 mm to 12 mm zone of inhibition)h
Staphylococcus lugdunensis
It can be confused with S, aureus if the slide coagulase method is performed.
YOG IDRITDAL SITE CVDAIC GRAM-POSITIVE COCCI 209
It is more
aggressive than the other CoNS in terms of infectivity. ibuilsr-nibadloo
contains the
It
mecA gene that codes for oxacillin resistance.
It is a CoNS by tube method.
Some related infections include infective endocarditis, meningitis, septicernia, UTL, and skin and
soft tissue infections. loal orli a
Staphylococcus haemolyticus
It
is becoming clinically significant due to its isolation from blood culture.in niliyols>
Notes to Remember
The novobiocin sensitivity test differentiates S. epidermidis from S. epidermidis.

The novobiocin-susceptible CoNS species are S. epidermidis, S. capitis, S. haemolyticus, S. hominis subsp.
hominis, S. lugdunensis, S. saccharolyticus, and S. warneri.
The novobiocin-resistant CoNS species are S. saprophyticus, S. cohnii, S. kloosii, and S. xylosus.
Other Resistant Genes Produced by Staphylococci Per bidene i 1e 2proeor

1. Erythromycin ribosomal methylase (erm) gene
It is class of enzyme-inactivating genes.
It codes for methylation of the 23S rRNA, which results in the resistance to erythromycin and
either inducible or constitutive resistance to clindamycin.
2.
It codes for an efflux mechanism, which results in the resistance to erythromycin but
susceptibility to clindamycin.
Specimens: Aspirated secretions (best sample), purulent exudates, joint fluids, blood, and tissue
1. Microscopy
Gram-positive spherical cells that appear singly, in pairs, or in clusters
2. Culture
Culture media: BAP, MSA, PEA, CNA, CAP, BHI, Thioglycolate, and CHROM agar
routine culture media.
Staphylococci grow easily in
A low colony count for S. saprophyticus (urine culture) is still considered significant.
sterile sites
and from the sites associated with indwelling devices
CoNS recovered from
should be considered potential pathogens.
BACTERIOLOGY
for purulent
Colistin-nalidixic agar (CNA) is used and is selective for Gram.
a enriched with 5% sheep blood
Phenylethyl alcohol (PEA) agar
positive bacteria.
MSA and PEA are used for heavily contaminated specimens, antdlioolni bulsloromo?
the isolation of MRSA.2it Doa
CHROM agar is
selective and differential medium for
MRSA produced colored colonies in CHROM agar. isolates and enhance the
the growth of nonstaphylococci and non-MRSA
to suppressihehe strea organisims, high salt (NaCI) concentration and antimicrobials such as
cefoxitin are added to culture media.
colored colony compared
After 24 hours or 48 hours of incubation, MRSA isolates produce
to colorless isolates of other bacteria.
3. Biochemical Test
Catalase Test
It differentiates staphylococci (catalase 4) from streptococci (catalase -)

Catalase is a heme enzyme that lyses hydrogen peroxide to water and oxygen. orr
be taken from BAP because of the
The colonies that will be used for this test should not
presence of peroxidase, which creates a false effervescence.

Reagent: 3% H,O,
(+) Result: Presence of bubble formation or effervescence.
Types of catalase test: Aerobic catalase test (3% H,O) and Anaerobic catalase test (15%
H,O,)
Quality Control
Negative: Streptococcus pyogenes ATCC 19615
b. Coagulase Test
S. aureus is frequently separated from less pathogenic species by being coagulase-
positive in both slide and tube methods.
Isolates that do not produce either a clumping factor or staphylocoagulase are reported
as coagulase-negative staphylococci.
Types of coagulase tests: Slide coagulase test and Tube coagulase test
Note: For complete assay procedure, refer to the differential tests for S. aureus.
C. Mannitol Fermentation Test
The fermentation of mannitol provides the colonies and the medium a yellow color.
Note: For complete assay procedure, refer to the differential tests for S. aureus.
d.
Pyrrolidonyl Arylamidase (PYR) Test
It differentiates coagulase-positive staphylococci (CoPS) through slide method.
WODIRITDAS BITEOMBAId GRAM-POSITIVE CoCCI 211
t detects staphylococci-producing arylamidase enzyme.

Final reagent: p-dimethylaminoc innamaldehyde
Procedure: Rub the isolated colonies onto the substrate disk. Then, add a drop of the
final reagent.
(+) Result: Cherry red
(-) Result: No color change on the paper disk sirlon

End
products: L-pyrrolidone and [-naphthylamine reoke mol brt T
(+) PYR: S. lugdunensis and S. schleiferi tiny mot egnatb toloD dnasl ()
(-) PYR: S. aureus
Other PYR-positive staphylocoeci: S. intermedius p o o1 8:916 A

Quality Control
Positive: Enterococcus faecalis (ATCC 29212) loa bprolop-biqtu Taluesh (-)

Negative: Streptococcus agalactiae (ATCC 10386)
e. Voges-Proskauer (VP)/Barritt's Method
It differentiates coagulase-positive staphylococci via tube method. millision

It detects glucose fermentation in the medium through the formation of acetoin which is
the intermediate product of 2,3 butanediol.
Culture medium: VP broth with 0.5% glucose s oflired rustlesras binsbianios
Reagents: 5% alpha-naphthol and 40% potassium hydroxide .xo stive ALM
Procedure: The reagents are added to the broth with the test organism after overnight
incubation at 35°C.
(+) Result: Deep pink or red (acetoin or acetylmethylcarbinol) 1r1t SolomothA
(+) VP: S, aureus

(-) VP: S. intermedius and S. hyicus
Other VP-positive staphylococci: S. lugdunensis, S. haemolyticus, and S. schleiferi
Quality Control
Positive: Enterobacter aerogenes ATCC 13048
f. 6-lactamase Test
Cephalosporin/Nitrocefin Test
It is the most widely used -lactamase test.
The substrate is nitrocefin incorporated in the cephalosporin or cefinase disk/strip.

Procedure: A loopful of organism is applied directly onto a pre-moistened disk/strip.
red color within 60 minutes (5 minutes to 10 minutes for other
(+) Result: Deep pink or
bacteria) grom
Quality Control
Positive: Hemophilus influenzae ATCC 33533

25240
Negative: Branhamella catarrhalis ATCC
Acidometric method
The reagent
is etrate-buffered penicillin (benzylpenicillin) impregnated in strips, and
the loopful of the colonies is smeared onto
pH indicator: Bromcresol purple or phenol red 1 1g1 ouboy
to 10 minutes
(+) Result: Color change from purple to yellow within 5 minutes
Iodometric method
The reagents are a phosphate-buffered penicillin and starch-iodine complex.

(+) Result: Colorless solution
(-) Result: Purple-colored solution
4. Antimicrobial Susceptibility Test

The traditional treatment drugs for staphylococcal infections include methicillin, oxacillin,
commonly used class representative.30

Oxacillin is the most
90100
When an isolate shows resistance to one of the penicillinase-resistant penicillins, it must be

considered as resistant to the entire group.
MHA with oxacillin and 2% NaCl is used for microdilution testing.

Disk diffusion and automated susceptibility testing have limitations in detecting resistant
isolates.
Automated antimicrobial susceptibility systems using cefoxitin can detect MRSA.

Vancomycin is one of the antibiotics that are still used in the therapy although resistance to
this antibiotic has also been reported.
MRSA isolates are susceptible to
ceftaroline and ceftobiprole, the new subclass of
cephalosporins with anti-MRSA action.
Vancomycin Agar Screen Plate

It identifies resistance of
enterococci and staphylococci to vancomycin.
CDC recommendation: S. aureus isolates should be
screened with 6-ug/mL vancomycin
added into : brain-heart infusion agar.
Procedure: Spot
inoculation onto BHI with vancomycin; then, incubate at 35°C for 24
hours.
(+) result: Any growth or colony is considered as a significant result.

Quality Control
Enterococcus fnccalis ATCC 29212 - Susceptible orcompletely no growth

Enterococcus faecalis ATCC 51299 - Resistant or
with more than one colony
YOQ JOIFETDAB SITZOMDAIG GRAM-POSITIVE COCCI 213
b. Oxacillin-screen Plate
It is used to detect MRSA in clinical samples but not as reliable as the cefoxitin disk test.
Susceptibility plate: MHA with 4% Naci and 6-ug/mL oxacillin irri
Inoculum size: 1.5
* 10% CFU/mL 3 oi 5ob af nohididhi to bnos bodedacf
Procedure: The test organism is spot-inoculated or introduced by overlapping streaking
using a sterile swab; then, plates are incubated for 24 hours at 35 °C. s10
Interpretation: The
growth of more than one colony is an indication of oxacillin
resistance while absence of growth on the agar plate indicates that an isolate is sensitive
to oxacillin.
Advantage: It detects the presence of

borderline oxacillin-resistant S. aureus (BORSA) in
clinical specimens.
All oxacillin-resistant staphylococci must be reported as resistant to all B-lactam drugs.
Quality Control
586061
notbubong edl en nowe eniude fitsiales
Staphylococcus aureus 25923 (Susceptible or no growth)

Staphylococcus aureus ATCC 43300 (Resistant or with growth)
It is the preferred method for detecting oxacillin resistance in both S. aureus and
S. lugdunensis as recommended by the Clinical and Laboratory Standards Institute
It is a specific marker for mecA-mediated methicillin/oxacillin resistance.
It improves the detection of MRSA.

Cefoxitin is used to induce expression of PBP2a in mecA-containing strains of
staphylococci, and it is considered the best antimicrobial for disk diffusion test.
Broth dilution method using 4 "g cefoxitin is acceptable, and the presence of turbidity in
this antibiotic concentration indicates oxacillin resistance.
Results: Susceptible - 22mm zone of inhibition (mecA negative)

Resistant - 21mm zone of inhibitino (mecA positive)
CoNS other than S. lugdenensis: Susceptible - ≥25 mm me leamasr-aillibexo
Resistant - $ 24 mm edsefegelnstbe
Quality Control
Staphylococcus aureus ATCC 25923 - Susceptible or no growth (possibly mecA-negative)

or with growth
Staphylococcus aureus 33591 - Resistant
d. Double-Disk Diffusion Test (D-test)

ulus lols
It is used to detect inducible clindamycin resistance in staphylococci.
Procedure: A 15-1g erythromycin disk (E) and a 2-1g clindamycin disk (C) are placed 15
mm to 26 mm apart on
an MHA or BAP with the isolate to be tested and then incubated
overnight at 35°C.
Result: Positive "D shaped" zone of inhibition (Inducible resistance)
Negative - Absence of blunting
Interpretations of the D-test result

There is a flattening on one side (near the erythromycin disk) of the clindamycin zone of
inhibition, which gives the appearance of a "D zone.'
due to ermA gene or emC gene. mplupont
D-shaped zone of inhibition is
The absence of blunting indicates erythromycin resistance. Toben
Quality Control
Zone of inhibition around the
Staphylococcus aureus subsp. aureus ATCC BAA-977 -
of inhibition
Staphylococcus aureus subsp. aureus ATCC BAA-976 - No zone
e. Penicillin Disk Diffusion Zone Edge Test
detection of antibiotic
It is considered more sensitive than the nitrocefin test in the
resistant strains such as the production of the -lactamase.
Antibiotic disc: Penicillin G
Results: Positive - Edge of the zone appears sharp like a "cliff"

Negative - Edge of the zone appears fuzzy like "beach"
Interpretation of Result: Positive or sharp edge result is defined as resistant to penicillin
while a fuzzy edge indicates absence of B-lactamase.
Broth MIC to Penicillin: 2 0.12
Quality Control
B-lactamase positive: Staphylococcus aureus ATCC 29213

B-lactamase negative: Staphylococcus aureus ATCC 25923
5. Immunodiagnosis
Latex Agglutination Test

It identifies both the clumping factor and protein A.
It also useful for detecting MRSA by using anti-PBP2a monoclonal antibodies - identification
of MRSA denotes detection of altered penicillin-binding proteins (PBP), and eventually
oxacillin-resistant strains. mond todo 2/oD
Its advantage is that it can be utilized for both S. aureus and CoNS.
6. MALDI-TOF Mass Spectrometry

This method is used to determine
the different species of the Staphylococcus.
It is a rapid and an accurate
detection parameter for the members of the staphylococci.
7. Molecular Test - Nucleic
Acid Probes or PCR amplification
It is the
gold standard for the direct of mecA gene in MRSA.
It assists specifically in the identification of CA-MRSA strains. As
The nucA gene is
amplified by PCR and is used as a standard confirmatory marker for
S.
aureus, especially for patients under antimicrobial treatment.
GRAM-POSITIVE COCCI 215
The 16S ribosomal RNA

level.
(rRNA) analysis is also used to identify Staphylococcus at the species
Specimen:
nasal swab (preferred for MRSA), blood culture, urine, etc mno drorn sood
Molecular Assays for MRSA: Gene-OHM, Xpert MRSA and Light Cycler MRSA Advanced
Gram-positive Cocci
Staphylococci
Coagulase Test
Positive Negative
S. aureus Bacitracin/Oxidase
Susceptible (+) Resistant (-)

Of ts wora monsien Iliw 41
Micrococci Coagulase-negative Staphylococci (CONS)
02071
Novobiocin Susceptibility
Susceptible Resistant
S. epidermidis S, saprophyticus
FIGURE 17-1. Schematic Diagram for Identification of Staphylococci
Streptococci
They belong to the family Streptococcaceae.

They are facultative anaerobes except peptostreptococci, which are obligate anaerobes, while
unable to use it for respiration like the
but are
other species grow in the presence
of oxygen
aerotolerant anaerobes.
thus require an increased consumption of CO, for growth.
Some species are capnophilic and
Their growth is enhanced by
blood, serum, or glucose that is incorporated into the agar plate.
viridans group and S. pneumoniae are included in the

All streptococci except the
classification.
Some members are part of the indigenous human microbiota, but they can cause life-threatening
sites is gained.
boor infections if access to normally sterile
lancet-shaped cells, that are arranged in
Microscopy: Gram-positive spherical cells some exhibit
chains or pairs
Culture: BAP - Colonies appear grayish, pinpoint, and translucent to slightly opaque while some
species have mucoid colonies.

Biochemical tests: (-) Catalase, oxidase, and gas production; non-motile; ferments carbohydrate
Species: Streptococcus pyogenes, Streptococcus agalactiae, and S. pneumoniae
Notorious pathogens: S. pyogenes and S. pneumoniae
Groups of streptococci: Viridans streptococci and nutritionally variant streptococci (NVS)
Classification of Streptococci
A. Academic/Bergey's Classification
2051b 2
It is based on temperature requirement.
1. Pyogenic group
It will neither grow at 10 °C nor at 45°C, but only at 37°C.
It mostly has beta-hemolytic reactions.

Some examples are S. pyogenes, agalactiae, and groups C and G streptococci.
Pyogenic streptococci: Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus

dysgalactiae subsp. equisimilis, and Streptococcus anginosus group
2. Viridans group
It will grow at both 45°C and 37 °C.

It is not part of the Lancefield group and may be a-hemolytic or non-hemolytic.
It is an indigenous microbiota in the upper respiratory tract (URT) in humans.

Some may have A, C, G, or N Lancefield antigen.
Some examples are Streptococcus mutans, Streptococcus mitis, and Streptococcus salivarius.
3. Lactic group
It will grow at 10'C and 37°C.
It is non-hemolytic and has a Lancefield N antigen.
It is often found in dairy products.
of
An example is the Streptococcus lactis, which causes normal coagulation or souring
milk.
LOTROMDAIC GRAM-POSITIVE COCCI 217
4. Enterococcus
group (formerly streptococci)
It will grow at 10°C, 45°C, and 37°C.
It is
part of the indigenous microbiota of the human intestine.
Some examples are Enterococcus faecalis
and Enterococcus faecium.
B. Smith and Brown Classification
It is based on hemolytic patterns. BAG

1. Alpha-hemolytic (a) Streptococci
These organisms create a partial/incomplete hemolysis of red blood cells around the
colonies.
The partial hemolysis is seen as a greenish discoloration on the culture medium.

An example is the Streptococcus pneumoniae (green streptococci).
2. Beta-hemolytic (p) Streptococci

They exhibit a complete lysis of red blood cells around the colony.
For the culture, there is a clear zone around the colony. hmnud of plaspoding al it
Some examples are Streptococcus pyogenes and Streptococcus agalactiae. upnsat 1
3. Non-hemolytic Streptococci
They do not exhibit lysis of red blood cells around the colony. For culture, the red blood
cells that immediately surround the colony are unaffected (no change).
It is sometimes called the gamma hemolytic (y) streptococci.

An example is Streptococcus bovis.
C. Lancefield Classification (Antigen Serogrouping)

It is based on the extraction of C carbohydrate from the streptococcal cell wall. Air!
Rebecca Lancefield, the microbiologist who developed the serogrouping of streptococci,

found out that the C carbohydrate could be extracted from the streptococcal cell wall by
for 10 minutes.r
placing the organisms in dilute acid and heating the suspension
It is mostly significant in classifying and identifying the beta-hemolytic streptococci.
TABLE 17-1, Lancefield Classification of Streptococci Species

Species
Hemolytic reaction Lancefield group
S. agaclatiae*
B
S. dysgalactiae subsp. equisimilis,

C, G S. equi
D
S. bovis group
a- or non-hemolytic
Enterococci
D
a-, B-, or non-hemolytic
None but some strains may Viridans streptococci
a-, 3-, or non-hemolytic have A, C, F, G, or N or none
None S. pneumoniae
*Occasional isolates that are non-hemolytic
Group A Streptococci (GAS)
It is not considered as part of the indigenous microbiota.

It is pathogenic to humans.
It is acquired through contaminated droplets that are released during coughing or sneezing.
It is resistant to drying and can be recovered from swab specimens several hours after the
collection.
The representative species for this group is Streptococcus pyogenes.
Streptococcus pyogenes
It is a "fever-producing and flesh-eating bacterium" that affects the deeper tissues and organs.
Primary reservoir: Humans
Principal virulence factor: M protein (attached to the peptidoglycan; antiphagocytic; for
adherence to mucosal cells).
Culture: BAP Colonies are grayish-white, small, translucent, and smooth with a well-defined
B-hemolysis.
Other virulence factors of GAS

a. Protein F - mediates epithelial cell attachment
b. Lipoteichoic acid - strengthens the adherence of the organism to the respiratory mucosal cells
c. Hyaluronic acid capsule - prevents phagocytosis; weakly immunogenic; and masks its antigens
d. Hemolysins - Streptolysin O and S (inhibit chemotaxis)
e. Toxins and
Enzymes - Erythrogenic toxin, DNAse, hyaluronidase, and streptokinase
Group A Streptococci Hemolysins
a. Streptolysin O
It is an oxygen-labile hemolysin.
SMRONDAId GRAM-POSITIVE COCCI 219
It is responsible for
subsurface hemolysis on BAP that is incubated anaerobically. 6D22
this highly antigenic and induces an antibody response. i -sdihit horsisining A
It isdetected a serological test known as
by
the anti-streptolysin O (ASO), and a positive
elolgo result indicates a recent infection.
Inoculation technique: To create anaerobiosis, it is best to "stab the agar" using an
inoculating loop.
incubation.
It is also important to observe the subsurface lysis after overnight
It is an oxygen-stable hemolysin.
tinal lesi nollisnD-xilnded deaf phaompsid
It is non-antigenic (nonimmunogenic).
It is responsible for surface
hemolysis on BAP that is incubated aerobically.
Enzymes and Toxins Produced by Streptococcus pyogenes ur srisli s aist
1. Pyrogenic toxins - Serotypes A, B, C, and F (Streptococcal Superantigens) u Asth1
These are formerly known as erythrogenic toxins.
Exotoxin B (cysteine protease) degrades proteins and mediates the rashes that are caused by
hollowe scarlet fever.
2. Deoxyribonuclease (DNase)
It lowers the viscosity of exudates giving the pathogens more mobility. bolbelni
Types of DNase/antigenic enzymes: A, B, C, and D
Streptokinase
It causes fibrin clot lysis. elacesy boold asbeum arfi antogqua
It is a protein that binds to plasminogen and activates the production of plasmin.

It activates a host blood factor that dissolves fibrin clots. I br9snd yd bsanns ar i
4. Hyaluronidase (Spreading-factor enzyme)

It solubilizes the ground substance of mammalian tissues. ramolqrave vITET

Development of resistance against S. pyogenes may be attributed to the type-specific antibodies to
the M protein.
1.
Pharyngitis or tonsilitis (Strep throat)
It is spread by air droplets and close contact (aerosolized droplet).
cause outbreaks of sore throat and scarlet fever in schools and
Highly virulent strains can
camps.
Its diagnosis relies on
the culture of specimen (throat swab) or direct antigen detection.
associated with pharyngitis is Ml. ian mours
The most common serotype
2. Scarlet fever (Scarlatina)

initially on the
neck and upper
A punctate exanthem overlying a diffuse erythema appears
chest, one to two days following strep throat.
the inhalation of infectious respiratory droplets.
It is communicable and spread through
It is caused by the release of pyrogenic exotoxins.

Some cardinal signs for this illness
include diffused red rash on the upper chest, trunk, and
extremities and a "strawberry-colored" tongue.
Susceptibility test: Dick's test (erythrogenic toxin test) - positive result shows erythema or
redness of the skin
Diagnostic test: Schultz-Charlton test (anti-erythrogenic toxin test) - positive result exhibits a
site of inoculation
"blanching phenomenon" or rash fade at the
101
3. Streptococcal skin infection

It is a cutaneous infection caused predominantly by group A streptococci strains. coVEn
If left untreated, it may progress to necrotizing fasciitis. "e? anixot aimgoryt
characterized by
Cellulitis - is a contagious infection of subcutaneous skin tissue that is
redness (erythema) and an accumulation of fluid (edema)1012073) 8 mixoloxd
b. Erysipelas - is an acute infection of the dermal layer of the skin with painful, swollen,
reddish spots
C. Strep impetigo - is a contagious skin infection that spreads through close contact with an
infected person 91010
4. Necrotizing fasciitis (NF/Galloping Gangrene/Flesh Eating Bacteria Syndrome)

It is a rare life-threatening infection affecting the skin and the tissues under the skin that
supports the muscles, blood vessels, and nerves.

It can be acquired even in the absence of skin trauma or break in the skin.
It is caused by bacteria ranging from the aerobic to anaerobic category. tswitos it
Types of NF: Type NF (aerobic and anaerobic bacteria), Type 2 NF (S. pyogenes) and Type 3
NF (Clostridium perfringens)
Early symptoms: Fever with erythematous, painful, swollen area of the skin
Late symptoms: Black spots on the skin or ulcers, pus dripping from the infected area, and
5. Rheumatic fever
It is characterized by fever, inflammation of the heart, joints, and blood vessels.

It is a complication of pharyngitis.
6.
Acute glomerulonephritis or Bright's disease
It is an inflammatory disease of the renal
glomeruli.
It results from the deposition of antigen-antibody complexes to the
kidneys.
It is a complication of pharyngitis that develops 2 weeks after infection.
221
7.
Streptococcal toxicshock syndrome (Strep
Itis a condition in which the whole
organ system shuts down and leads to death.
The initial
strep infections
leading to this condition include
pharyngitis, cellulitis, and
wound infections.
Spec A or
pyrogenic exotoxin is vital in the pathogenesis of this disease.
Notes to Remember
Group
A streptococci (GAS) also have superantigens that are associated with necrotizing fasciitis or
toxic shock syndrome (TSS).
The resistance to infection with S. pyogenes may be due to type-specific antibodies to M protein.
Streptolysin O and
DNase B antibodies can be detected serologically and results are utilized in the
diagnosis of
acute rheumatic fever and acute glomerulonephritis. n
Laboratory Tests for Group A Streptococci autel ort no roitoini 910 Seus> vadI
(Streptococcus pyogenes)
Specimens: Pharyngeal and tonsillar swabs (throat swabs) and tissue biopsy (necrotizing
fasciitis).
1. Bacitracin Disk Test/Taxo A (0.04 units)

It differentiates S. pyogenes from other -hemolytic groups.
It is helpful in the detection of group A streptococci in throat cultures.

Groups C and G are also susceptible to bacitracin.
(+) Result: Susceptible or any zone of inhibition 94.10 9258 nomertion leom sul at it
Quality Control
Susceptible: Streptococcus pyogenes ATCC 19615981,192717MAR knotbel Inail

Micrococcus luteus ATCC 10240
Resistant: Streptococcus agalactiae ATCC 27956
2. Sulfamethoxazole and Trimethoprim (SXT) Test brus SAJ 8MAD (o eteat Irolmedacid
the identification of Group A streptococci, same with Taxo A.

It is a presumptive test for
Group B streptococci are also resistant to SXT.

Group C streptococci are sensitive (negative result) to
absence of a zone of inhibition
(+) Result: Exhibits resistance or
Quality Control
ATCC 19615
Susceptible: Streptococcus pyogenes
ATCC 6249
Resistant: Streptococcus mitis
Test
3. Pyrrolidonyl arylamidase (PYR)
It is more specific than bacitracin test.
It detects the presence of PYR
S. pyogenes is the only B-hemolytic Strepiococeus species that is positive to PYR test
(+) Result: Cherry red color fronds
Other PYR test-positive organism: Enterococci to A buge

Quality Control
Positive: Streptococcus pyogenes ATCC 19615
Negative: Streptococcus agalactiae ATCC 12386
(22T) amotbnive dborle dixor

Group B Streptococci (GBS)
and lower gastrointestinal
These are part of the indigenous microbiota of the female genital tract
These are nosocomially transmitted by unwashed hands of mother and healthcare personnel to
the newborn or infant.
They cause the infection on the fetus during passage through the colonized birth canal and
premature rupture of mother's membranes.
serotypes identified in this group, and Serotype III clone ST-17 is cited the major
as
There are ten
cause of Group B streptococci (GBS) neonatal infections.
As the recommendation of CDC, all pregnant women should be screened for group
streptococci at 35 to 37 weeks of gestation.
It is the representative species of GBS. crined of aldifcooaue beln 9u D bns D equond
It is the most common cause of neonatal meningitis.nox yr TO aldiigesaue aluesA (t)

Virulence factor: Capsule (with sialic acid as the significant component)
and protease
Culture: BAP - Colonies appear glossy grayish-white and mucoid with a small zone of beta-
Biochemical tests: (t) CAMP, LAP, and Hippurate hydrolysis mift bns slosexorionsi

As stated by CDC, pregnant
women with history of child with GBS disease or positive urine
culture for GBS should receive antimicrobial treatment to
prevent the risk of early-onset
infection.
1. Neonatal meningitis and sepsis

2. Pneumonia
3. Postpartum infection
4. Osteomyelitis
YOOJDIST MAE DITROMONO GRAM-POSITIVE COCCI 223
5. Urinary tract infection

6. Endocarditis
Laboratory Tests for Group B Streptococci

(Streptococcus agalactiae)
brid
bite Tags boold nohsfsetedt sunffuodoa

1. CAMP (Christie, Atkins, Munch-Peterson) Test
It is used to
differentiate S. agalactiae from other [-hemolytic streptococci.
It is performed in an aerobic environment and the plate is incubated at 35 °C.
Culture medium: BAP
Reagent: B-lysin strip or B-lysin-producing

strain of S. aureus. M aepeepo oels vorlT
Procedure: A well-isolated colony of S. aureus
from a pure plate is streak down the center
of the BAP while the test organism is streak perpendicular to the reagent organism. Plate is
incubated overnight at 35°C to 37°C. mish brits
(+) Result: Arrow-head or bowtie-shaped beta-hemolysis near S. aureus growth aboM
Positive: Streptococcus agalactiae ATCC 13813 (Arrowhead hemolysis)

Negative: Streptococcus pyogenes ATCC 19615 (With beta hemolysis but not arrowhead
shaped)
2. Hippurate Hydrolysis Test

It is used to differentiate S. agalactiae from other (-hemolytic streptococci. ol600 adgyT
S. agalactiae possesses the enzyme hippuricase or hippurate hydrolase. vrolea 9319.1
The reagents are sodium hippurate and ninhydrin. islcar antmmol-tnolos sgidt
Procedure: Place the hippurate disk into a bacterial suspension; then, incubate the tube at
35°C for 2 hours. Ninhydrin reagent is added about 0.2 mL and reincubate for 30 minutes.
(t) Result: Exhibits a purple color after adding ninhydrin reagent
(-) Result: No color change
Quality Controlbnn o eqpong nitw astaloat ailvlotod-& animmot-ynolop llame
Positive: Streptococcus agalactiae ATCC 12386
ATCC 19615
Negative: Streptococcus pyogenes
3.
It is performed using coagglutination or latex agglutination.

4. Molecular Assay
can be identified with
Isolates of GES from Lim broth or other selective culture media
chemiluminescent DNA probe.
It can also detect GBS directly in vaginal and rectal swabs (Cepheid GeneXpert GBS Assay).
BACTERIOLOGY
Notes to Remember
is the recommended drug for treatment.

B-hemolytic streptococci are susceptible to penicillin which
S. agalactiae is resistant to both bacitracin and SXT.o0lqpite
the Centers for Disease Control and Prevention (CDC) for the
identification
The recommendation of
of Group B streptococci is to inoculate swabs (vaginal and rectal swabs) in a selective broth and
subculture thereafter on blood agar and selective culture medium.
mon snitonlsgn 2 edcimewollib of boas ef ft

Group C G and F Streptococci -135110
These organisms are recovered from the upper respiratory tract, female genital tract (vagina),
and skin as indigenous flora.
They also possess M protein similar to the group A streptococci.

with latex agglutination
Isolates of this group with beta hemolysis are detected by serotyping
ex gia! reagents.
They are associated with pharyngitis and skin infections such as impetigo. sdiani Cantal
Mode of acquisition: Person-to-person contact jsrle sftwod 10
Culture: Colonies are small flat and grayish white with regular to narrow zone of alpha or beta
Group C streptococci (S. equi subsp. zooepidemicus) are animal pathogens and serve as the main
source of streptokinase.
Species: S. dysgalactiae subsp. equisimilis, and S. equi subsp. zooepidemicus
1. Large colony-forming isolates

Large colony-forming isolates with groups A, C, and G antigens belong to the pyogenic
streptococci.
Large, colony-forming, B-hemolytic isolates with groups A, C, G, and L antigens belong to
the S. dysgalactiae subsp. equisimilis.
2. Small colony-forming isolates
Small, colony-forming, -hemolytic isolates with groups A, C and F antigens belong to the
S. anginosus group,
Viridans Streptococci
These are also known as the

alpha-prime streptococci that lack the Lancefield group antigens.
These are indigenous microbiota of the and
upper respiratory tract, female genital tract,
gastrointestinal tract.
These are the most common cause
of subacute bacterial endocarditis (SBE).
SETIDAIO GRAM-POSITIVE COCCI 225
They are
opportunistic pathogens and
may gain entry to humans through dental procedures.
Virulence factorsi: Capsule, cytolysin, extracelluh.
Culture: Colonies are
smooth small grayish-white with alpha
Related infection: SBE,
gingivitis, dental caries,
meningitis, bacteremia, osteomyelitis, and
Laboratory test: (+) LAP, (-) PYR, bile-insoluble, no
growth in 6.5% NaCI, and optochin-resistant
Groups of Viridans Streptococci
streptococcus mitis Group: S. mitis, S. oralis, $. pneumoniae, and
1.
S. sanguis pu 1A.1-)
2.
Streptococcus mutans Group: S. mutans and S. sobrinus
3. Streptococcus salivarius Group: S. salivarius and S. vestibularis
Streptococcus bovis Group: S. alactolyticus, S. equinus, S. galloyticu, and s. infantarius
4.
5.
Streptococcus anginosus Group: S. anginosus, S. intermedius, and S. constellatus
Notes to Remember
S.
mutans group is the most commonly isolated species of viridans streptococci.
S. mitis group is the common cause of SBE.
S. mutans group is frequently isolated in dental caries.

S. bovis group and the enterococci have group D antigens in their cell walls.
S. gallolyticus
subsp. gallolyticus of the S. bovis group is often isolated in blood cultures of individuals
with gastrointestinal carcinoma.
The members of the S. anginosus group have Lancefield group A, C, F, G, or N antigen or none at all.
The most common viridans streptococci responsible for liver and brain abscesses is the milleri
streptococci complex or the S. anginosus group producing a distinct "caramel" odor in culture medium,
and susceptible to penicillin.
The alpha-prime hemolytic pattern observed in viridans streptococci is characterized by a small inner
zone of intact red blood cells and a wider outer zone of beta hemolysis.
Laboratory Diagnosis for Viridans Streptococci

and pus secretions.
Specimens: Blood, gingival scrapings,
Leucine Aminopeptidase (LAP) Test

for catalase- negative, Gram-positive cocci.
It is presumptive test
LAP is a peptidase that hydrolyzes peptide bonds that are adjacent to a free amino group.
Substrate: Leucine- 6-naphthylamide
BACTERIOLOGY
onto substrate disk by a rubbing motion.

Procedure: Place loopful of the test organism
of incubating the disk at
Then, add drop of the cinnamaldehyde reagent after 5 minutes
room temperature.
End product: -naphthylamine
(+) Result: Red color
() Result: No color change
(+) LAP: Viridans streptococci, enterococci, S. pyogenes, S. agalactiae, and S. pneumoniae
(-) LAP: Aerococcus and Leuconostoc
Quality Control
Positive: Enterococcus faecalis ATCC 29212

Negative: Aerococcus viridans ATCC 11563
2. Voges-Proskauer (VP) Test

Culture medium: VP broth
Reagents: 5% a-naphthol and 40% KOH
End product: Acetoin

End color: Deep pink or red color
(+) VP: S. anginosus, S. bovis, and S. mutans groups
Quality Control
Positive: Enterobacter aerogenes ATCC 13048
3. -D-Glucuronidase (BGUR) Test

It differentiates the B-hemolytic streptococci in large colony group C and G from the small
colony group C and G of the S. anginosus group or milleri complex.
BGUR is an enzyme that is found in isolates of large, colony-forming, B-hemolytic groups
and G streptococci.
Substrate: Methylumbelliferyl-f-D-glucoronide motiag offvlomod aming-sdale of

boold bsr fakini to onos
(+) Result: Flourescence (large colony $-hemolytic Group C and G)
Quality Control
Positive: Escherichia coli ATCC 11775

Negative: Raoultella planticola NCTC* 9528
*National Collection of Type Cultures

Group C streptococci are susceptible to bacitracin and SXT.
Group G streptococci may be resistant or susceptible to bacitracin
SITEOHUAI GRAM-POSITIVE COCCI 227
Diagnostic Tests for

S. bovis Group (Group D streptococci)
5.
Growth in bile esculin medium

Reagents: Esculin and 1% to 4% bile
salt
(+) Result: Black color
complex is exhibited in the agar within 48 hours.
b. 6.5% NaCI (Nutrient Broth Base) Test
The result shows no growth (negative reaction/absence of turbidity) in this high

medium.
salt
C. PYR Test
The
result explains it is negative (no color change).. Pore
d. Penicillin Test
The result shows S.

bovis group is susceptible to this antimicrobial. lud b et it
Streptococcus pneumoniae
It is also known as diplococcus or pneumococcus.
It is an
asymptomatic member of a normal respiratory tract such as the nasopharynx.
It is the causative agent of lobar pneumonia.
It is the most common cause of bacterial pneumonia in the elderly and immunocompromised
individuals.
It has been identified as an etiology of the community acquired pneumonia (CAP) in adults and a
major cause of healthcare associated pneumonia (HCAP).
It is the most common organism associated with bacterial meningitis in adults.
Its cell wall contains an antigen known as the C substance which is not related to the C
carbohydrate of the Lancefield groups.

Principal virulence factor: Capsule (polysaccharide)
Microscopy: Gram-positive cocci in pairs that are oval or lancet-shaped

Culture: BAP Young colonies appear "dome-shaped," glistening, mucoid, and alpha hemolytic
while old colonies exhibit a "coin with a raised rim" sometimes "dimple-shaped" morphology.

Mode of acquisition: Person to person contact with infected respiratory droplets
and secretions.
should be given starting at age 2

CDC recommendation: 13-valent conjugated vaccine (PCV 13)
months as part of the pediatric immunization while the 23-valent polysaccharide vaccineon(PSV
may be given vaccination depending the
23) is for adults. Persons older than 65 years old
advice of the clinician. $
bacteremia, endocarditis, peritonitis, and typical hemolytic
S.pneumoniae is also an agent of
uremic syndrome.
1. Lobar pneumonia
It is characterized by the presence of voluminous fluid which hastens as the bacteriaspread

to the lungs.
Predisposing factors: Alcoholism, malnutrition, and viral infection of the upper respiratory
tract (URT)
cells and white blood cells
Sputum microscopy: Presenceof a large number of S. pneumoniae
(WBCs) while absence oropharyngeal microbiota is observed
of
Community-acquired pneumonia (CAP) in adults

S. pneumoniae is the most common cause of CAP in adults. nude climaaven't
It is the leading cause of mortality among geriatric patients.
It is a pulmonary parenchymal infection that occurs outside the hospital. AD
Predisposing factors: advanced age, alcoholism, impaired respiratory airway, smoking,

immunocompromised, malnutrition, substance abuse, and populous urban house settings
Comorbidities: organ dysfunctions such as the heart and lungs, respiratory viral infection,
21
diabetes mellitus, and stroke
3. Streptococcal meningitis
It is a complication of pneumonia and otitis media, and it commonly affects infants and the
elderly.
4. Otitis media auromgrang bentmpendinumtmon ando holol, me
It is an inflammation of the middle ear.
S. pneumoniae
is the most common isolate in children under 3 years old with recurrent otitis
media.
Notes to Remember
The capsule of S. pneumoniae is immunogenic and can be identified with the appropriate antiserum.
The
opsonization of the capsule renders the organism avirulent.
Laboratory Diagnosis for Streptococcus pneumoniae

Specimens: Sputum, blood, and CSF
A rust-tinged sputum may indicate S. pneumoniae.
1. Gram stain
The cells are oval or

lancet-shaped, and they appear in pairs or in short chains.
Direct smears of CSF reveal WBCs and Gram-positive cocci in pairs.
2. Culture
Culture media: CAP BHI, and TSA with

5% sheep blood.
SITCOMOAID GRAM-POSITIVE COCCI 229
that progresses to flat colonies with depressed conters beyond 24 hours of incubation.
3.
Bile Solubility Test
It evaluates
the ability of S, pneumoniac to lyse (autolysis) in the presence of bile salts, utilizing
not only
sputum but also blood and CSF cultures as specimens.
It distinguishes S. pneumoniae from
other alpha-hemolytic streptococci.
Procedure: Place 1 to 2
drops of 10% sodium deoxycholate onto 18-hour to 24-hour old
colony, or alternatively, a well-isolated colony is transferred into a test tube with 2% sodium
deoxycholate. Incubate for 10 minutes at 35•c to 37°C in increased carbon dioxide. If the
result is
negative, continue the incubation until 2 hours before releasing the final result.
(+) Result: Absence of
turbidity (lysis of the organism)
(-) Result: Turbid
Quality Control
Positive: Streptococcus pneumoniae ATCC 49619 (Bile soluble) oSocnusg ss.ahnowub it

Negative: Enterococcus faecalis ATCC 29212 (Bile insoluble) bims ho wivilis ath cbalst f
4. Neufeld-Quellung Reaction (Capsular Swelling Test) rueng ho rotenoneus wnodanaw

It allows the detection of S. pneumoniae as well as the serotyping of isolates.
The underlying principle is that the capsule swells in the presence of a specific anticapsular
serum.
(+) Result: Swelling of capsule
5. Urine Antigen Test (Point of Care Test)

It is used as a nonculture diagnostic method for severe pneumococcal infection and otitis
media.
It is not recommended for individuals who just received pneumococcal vaccinations (pcv 13
and ppsv23).
Test Method: Alere BinaxNOW (with results within 15-20 minutes) esegge ails
Antimicrobial Susceptibility Test (AST)

x11g
The AST for S. pneumoniae is known as the Optochin Disk Test/Taxo P Test.
and the viridans streptococci.
It is a differential test between the pneumococcus
(+) Taxo P Test: 2 14 mm zone of inhibition on

macrolides, doxycycline, beta-lactams
S. pneumoniae has a known pattern of resistance with
however, penicillin is still considered as one of the
like penicillin and fluoroquinolone,
infections.
antimicrobials for treating pneumococcal
Quality Control
Postive. Streptococcus pneumonine ATCC -49619 Susceptibe,zone of inhibition)

no
Negative: Streptococcus mitis

ATCC 49456 (Resistant, of
7. Skin Test (Francis Test)
This test will help detect the presence of antibodies against pneumococci.
8. Co-agglutination
It utilizes particle-bound antibodies to enhance the visibility of the agglutination reaction
between the antigen and antibody.
It assists in the detection of pneumoniae.
TABLE 17-2. Differential Tests for Streptococcus pneumoniae

Result
Test
≥ 14 mm
1. Optochin susceptibility/Taxo P disk test (Presumptive test)
Disk: 6-um or 10-um optochin /ethylhydroxycuprein hydrochloride
Susceptibility medium: BAP

(+) bile-soluble or
2. Bile solubility (Confirmatory test)
absence of turbidity
It differentiates pneumococci from viridans streptococci.
It detects the activity of amidase (autocatalytic enzyme).
The reagent is sodium deoxycholate (bile salt).

When heavy suspension of pneumococcus is added to the bile salt, the "an fi5d O-blata/f
cloudiness of the broth clears after three hours of incubation.
3. Neufeld-Quellung reaction (+) swelling of capsule
The anti-pneumococcal serum is mixed with sputum, CSF, and other

sources along with methylene blue (examined under oil immersion
objective).
4. Mouse virulence (+) death of the animal
General Laboratory Tests for Lancefield Groups of Streptococci and

Other Streptococci
1. Microscopy
Cells appear as Gram-positive but they are more elongated than spherical.
Cells are more likely to appear in chains when they are grown in broth cultures.
2. Culture fou TeA ont
Culture media: BAP, PEA, CNA, CAP, Carrot broth, Lim broth, and Granada agar
The plates should be incubated for 48 hours before the culture is reported as negative.
CAP is incubated with 5% to 10% CO for capnophilic streptococci. situsrstr
Carrot broth (selective broth) or Granada agar (selective agar) is utilized for vaginal and
rectal swabs from pregnant women.
Carrot broth a chromogenic medium in which the presence of Group B streptococci gives
is
the clear medium orange or red color after 6 hours to 24 hours of incubation.
Granada agar gives Group B streptococci yellow to orange colonies.
231
Lim broth
is a selective enrichment and transport medium that
group B streptococci. improves the recovery of
Cultivation and isolation procedures for streptococci

ban nou a.
For isolating GAS from throat swabs, BAP with SXT is used to suppress the growth of the
microbiota.
b.
To better observe the beta-hemolytic pattern of s. pyogenes, BAP should be inoculated by
sterose vedi "stabbing" the inoculating loop into the agar several times to achieve anaerobiosis.
C. For detecting GBS during pregnancy,
vaginal or anal swab is inoculated into transport
medium such as Lim broth or Trans-Vag broth with 8 ug/mL gentamicin and 15 ug/mL
nalidixic acid to inhibit vaginal microbiota. Both tubes are incubated for 18 hours to 24 hours;
then they are subcultured to BAP. 901190
brt o d." Lim broth is an improved Todd-Hewitt broth (THB). It contains all the components of THB
and with added
antimicrobial agents such as10 ug/mL of colistin and 15 ug/mL of nalidixic
acid.
3. Biochemical Test - Catalase Test

Reagent: 3% H,O,
Result: Negative (without bubble formation)
Weak or false-positive catalase reactions can be seen when colonies are taken from blood-
containing media.
OW DO BOUNU boold tanemicane

4. Antimicrobial Susceptibility Test - Bacitracin and SXT
Group A streptococci are susceptible to bacitracin and resistant to SXT. mala mend
Group B streptococci are resistant to both bacitracin and SXT.

Group C streptococci are susceptible to bacitracin while Group G streptococci have variable
resistance. d-producing carbohyd
Group A and B streptococci are susceptible to penicillin, and they are considered as the
AMID 15g6 target antimicrobials for treatment with GAS having greater sensitivity. 390 Burle
5. Molecular Diagnosis
These studies are utilized to determine the genetic relationships among different phenotypes
of the members of the family Streptococcaceae.
Real-time PCR is utilized to detect Group A and B streptococci utilizing the ptsI gene and cfb
gene, respectively.
shotoel or lo virsons oill eanimmstab i
Enterococci
KILOS They belong to the family Enterococcaceae.

because all the species produce the group D
They are formerly known as group D streptococci
antigen same with the Streptococcus bovis
microbiota of human and animal intestinal tracts and female
The species are indigenous
genitourinary tract.
pathogens and are frequent causes of nosocomial infections.

They are opportunistic
antimicrobial agents with temperature-dependent growth
They are resistant
to multiple
polite pi a to . or iuboaong norialon ons notmelhig
properties.
infections and
diseases are attributed to person-to-person transmission and
The development of
The dine of iornites and contaminated medical instruments.
beta-hemolytic. redo rotted
of
non-hemolytic or may be
alpha- or
The members are
reaction (weak bubble formation) because they secrete
The species produce "pseudocatalase"
peroxidase.
smooth with varied hemolysis.
Culture: Colonies are creamy white, glistening
gelatinase, and cytolysis r15714
Contributes to virulence: Adhesins, serine protease,
endocarditis, and wound
Related infections: nosocomial urinary tract infection, bacteremia,
infection
6.5% NaCl broth
Laboratory test: () PYR, LAP, and bile esculin; growth in
E. durans, and E. raffinosus
Species: E. faecalis, E. faecium, E. avium, E. gallinarum,
Common isolates: E. faecalis and E. faecium
Differential test for the common isolates: (+) Tellurite Test - E. faecalis
(+) Arabinose Fermentation - E. faecium
Laboratory Diagnosis for Enterococci

Specimens: Blood, urine, or wound secretions
1. Gram stain
Presence of Gram-positive cocci in pairs and long chains.
2. Culture
Culture media: Trypticase soy broth (TSB) or brain heart infusion (BHI) with 5% sheep blood
The recommended media for contaminated
specimens are bile esculin azide agar, CNA,
PEA, and cephalexin-aztreonam-arabinose agar.
Enterococci grow well at 35 'C with CO,.
E. faecalis can be identified by its ability to grow in a medium that contains tellurite.
3. Biochemical Test
a.
Bile Esculin Test (Growth in a bile esculin medium)
It determines the capacity of the bacteria to grow in the
presence of increased bile salts
and the ability to hydrolyze esculin.
a differential test between enterococei and Group D streptococci (S. bovis group
It is
from non-group D viridans streptococci.

Reagents: Esculin and 4% bile salt
WED OUSTBA8 SITZOMDAIN GRAM-POSITIVE COCCI 233
(+) Result:
A black color complex is exhibited in the agar (for both the enterococci and
non-enterococci) within 48 hours.
(-) Result: No growth
or no color change in the medium
Quality Control
Positive: Enterococcus faecalis ATCC 19433

b.
6.5% NaCl Tolerance Test (Nutrient Broth Base)
(+) Result: Turbidity (Growth of enterocococci)
(-) Result: No turbidity (Non-enterococci or S.
bovis group)
Quality Control
Positive: Enterococcus fecalis ATCC 29212

Negative: Streptococcus pyogenes ATCC 19615
C. PYR Test
(+) Result: Cherry red
(-) Result: No color change on the paper disk

All enterococci isolate from humans hydrolyze PYR.
Quality Control
Positive: Enterococcus faecalis ATCC 29212 seorll of talimia enotbetn ssto vorlT
Negative: Streptococcus agalactiae ATCC 12386

d. Acid Production
The enterococci utilize an acid-producing carbohydrates and methyl-a-glucopyranosides
The result is positive.

Enterococci are resistant both to penicillin test and efromycin acid disc (100 ug)
test.
vancomycin-resistant strains are predominantly E. faecium due to the resistant

Majority of
(vanA, vanB, and vanC).
genes known as the van genes
Enterococci are also resistant to cephalosporins and some aminoglycosides like gentamicin
and erythromycin.
vancomycin, and it is located in the plasmids and
The vanA is highly resistant to
transposons.
(NVS).
They are formerly known as nutritionally variant streptococa
They are also known as pyridoxal-dependent or vitamin B,-dependent streptococci, thiol.
dependent streptococci, and symbiotic streptococci.
They are part of the human oral and gastrointestinal microbiota.
Abiotrophia will not grow on BAP or

CAP unless pyridoxal (B.) is supplied.
They exhibit alpha or gamma hemolysis.
Microscopy: Gram-variable and pleomorphic forms
or tiny colonies around an organism that produces
Culture: Colonies appear as "satellites"
pyridoxal such as S. aureus ("staphylococcal streak test"); they resemble viridans streptococci.
Growth factors: Thiol compounds (cystein, vitamin By, and pyridoxal)
Biochemical test: (+) PYR and LAP
Species: Granulicatella adiacens and Granulicatella elegans
Related infections: Endocarditis, otitis media, breast implant-associated infection,
endophthalmitis, and septic arthritis
Streptococcus-like Organisms
The genera are Aerococcus, Gemella, Lactococcus, Leucont Pediococcus, and Stomatococcus (Rothia).
Hemolytic patterns: Alpha hemolytic or non-hemolytic

They cause infections similar to those of streptococci and enterococci.
Some are vancomycin-resistant (Leuconostoc and Pediococcus) while others are vancomycin-
susceptible (Aerococcus).
It is a common airborne bacterium.
It resembles viridans
streptococci in culture plate but is microscopically similar to staphylococci
in arrangement.
It presents similar biochemical reactions with enterococci such as being salt-tolerant.

It exhibits a pseudocatalase reaction.
Microscopy: Gram-positive cocci in tetrads or

clusters (staphylococci-like arrangement)
Common isolates: A. viridians [(+) Bile Esculin and PYR
tests] and A. urinae I(-) Bile Esculin and
PYR tests]
Urinary tract pathogens: A. urinae and A. sanguinicola

It has
similar morphology with the Neisseria species, that is, "diplococci with adjacent sides
flattened" but the colonial
appearance resembles viridans streptococci.
It is an indigenous flora of humans
particularly in the oral cavity and respiratory tract.
Microscopy: Gram-negative cocci in pairs clusters or short chains
Species: G. haemolysans, G. morbillorum,
G. bergeriae, and G. sanguinis
Common isolates: G.
haemolysans (aerobic) and G. morbillorum (anaerobic)
Biochemical Test for the
Common Isolates: (+) PYR; (-) Bile Esculin and Growth in 6% NaCl Broth
Antimicrobial Susceptibility Test: Common isolates are susceptible to penicillin. O
RUCA
It is formerly under the group N. streptococci.

It is similar to enterococci, but it
does not produce acid from carbohydrates.
It has
been isolated from dairy and plant products.
It can be
alpha or non-hemolytic and may grow at 10°C. elusinv
It has similar biochemical characteristics with enterococci and viridans streptococci.
It is frequently found on plant surfaces, on vegetables, and in milk products.

It is a gas producer by MRS (Mann, Rogosa and Sharp) method and with alpha or beta hemolytic
pattern.
Microscopy: Irregularly coccoid
Species: L. citreum, L. dextranicum, L. lactis, and L. mesenteroides
Laboratory tests: (+) Bile esculin hydrolysis and Growth in 6.5% NaCl broth; (-) PYR and LAP
It is isolated from natural environment like vegetables.

It shows resistance to vancomycin that is similar to Leuconostoc.
Microscopy: Appears in pairs, tetrads, and clusters

P. damnosus
Species: P. acidilactici and
Laboratory tests: (+) BE and LAP; (-) PYR
TABLE 17-3. Summary of the Biochemical Tests for

Streptococci and Enterococci
Group D
Streptococci Enterococc:
Tests S. agalactiae S. pneumoniae Streptococci
(S. bovis group)
R R
R
Bacitracin S* R* S
S R
R R S
R R
R
R S
R
CAMP
Bile esculin
Growth in 6.5% +
NaCl broth
PYR
LAP 10116 + +
'S - susceptible; R - resistant; V variable, positive or negative reaction flomed-hon .0 srigis ad M1501
+, most strains with positive result

-
most strains with negative result
1oL Ib1a iaD0GRAM-POSITIVE COCCI 237
A
238 REVIEW HANDBOOK
FIGURE 17-3. Photomicrograph of Staphylococcus aureus,

spherical Gram-positive cocci, magnified 250x
FIGURE 17-4. Gram stain reaction of Staphylococcus aureus

(Photo from USCDCP)
25 Spoti
FIGURE 17-5. Micrograph of methicilin-resistant Staphylococcus aureus

(Photo by 1. H. Carr 61. Biddle)
239
Coagulase tubes
Staphylococcus
epidermidis
Staphylococcus
aureus
FIGURE 17-6.
Positive coagulum formation for Staphylococcus aureus in a coagulase test
(Photo by R. Watson)
FIGURE 17-7. Coagulase-negative Staphylococci (CoNS) showing white,

creamy, and nonhemolytic colonies
Source: Textbook on Diagnostic Microbiology, Mahon and Lehman, 6th ed, 2019
FIGURE 17-8. Novobiocin Susceptibility Test

Source: Textbook on Diagnostic Microbiology. Mahon and Lehman, oth ed, 2019
uphylo
FIGURE 17-9. Mannitol Fermentation Test Result

Source: https://microbeonline.com/mannitol-salt-agar-msa-composition-uses-and-colony-characteristics/
VP +ue Vp -Ve
VP
A
TEST
FIGURE 17-10. Voges Proskaver Test

Source: https://microbiologyinfo.com/ oges-proskauer-vp-test-principle-reagents-procedure-and-result.
FIGURE 17-11. DNAse Test Result. A and B, Positive; C, Negative

pource: https://microbeonline.com/deoxyribonuclease-dnase-test-principle-procedure-results/
easoerdameuIdGRAM-POsITIVE CocCI 241
FIGURE 17-12. CHROMagar Test

Source: http://www.chromagar.com/food-water-chromagar-staphylococcus--68.html#YPZ
3 L t5 H "aenissr"Ari 3tol
FIGURE 17-13. Catalase test showing a positive result with the presence
of bubble formation/effervescence
FIGURE 17-14. PYR Testt e ia s

anaSource: http://universe84acom/pyr-test
FIGURE 17-15. Beta-lactamase (Nitrocefin) Test Result

Note: The "haziness" typical growth of the heteroresistant oxacillin- resistant Staphylococcus aureus
FIGURE 17-16. Oxacillin Screen Plate

Sharp zone edge ("cliff) _ ( inclamasu poaitive

Figure below this table).
Fuzzy zone edge ("beach") -lactamase negative

(see Figure 2 below this table)
FIGURE 17-17. Penicillin G Test. a) Sharp "cliff" zone edge. b) Fuzzy "beach" zone edge
Source: https://www.iammdelhi.org/wp-content/uploads/2017/10/2017-Difficult-or-Tricky-Antibiotic-Resistance-Phenotypes.pdf
FIGURE 17-18. D-Test

FIGURE 17-19. Streptococcus pneumoniae on chocolate agar plate

Beta Haemolysis Alpha Haemolysis®

Gamma Haemolysis
FIGURE 17-20. Hemolytic Patterns of Streptococci

iass.com/blood-agar-haemolysis-test/
Source: https://microbiologyclass.c
FIGURE 17-21. CAMP Test. Streptococcus agalactiae shows the classic arrow shape near the
streptococcal streak
Source: Textbook on Diagnostic Microbiology, Mahon and. Lehman, 6th ed, 2019
MITRCHONIC GRAM-POSITIVE COCCI 245

It is responsible for the
1.
a. mecC gene
inducible resistance of Staphylococcus aureus to clindamycin.
b. erm gene
C.
d. mec A gene
2. It has a "diplococcal morphology" similar to the genus Neisseria.

a.
b.
C.
d. Aerococcus
3. What is the negative control organism for mannitol fermentation test?

a. Escherichia coli ATCC 25922
b
b. Streptococcus pyogenes ATCC 19615
C. Enterococcus faecalis ATCC 29212
d. Streptococcus agalactiae ATCC 10386
4. It is an Staphylococcus aureus-dependent organism that requires the pyridoxal secreted by that

bacterium.
a.
b. Granulicatella
c. Staphylococcus lugdenensis
d. Group C streptococci
5. It has an autolytic property resulting in "coin with a raised rim" appearance of the colonies after
48 hours of incubation.
a. Staphylococcus saprophyticus
b. Staphylococcus agalactiae
C. Staphylococcus pneumoniae
d. Staphylococcus bovis
induce expression of PBP2a in mecA-containing strains,

and it is consideredas the
6. It is used to
best
test.
antimicrobial for staphylococcal disk diffusion
SlotonG
a. oxacillin
b. vancomycin
C. methicillin
d.
7. What is the most common cause of

subacute bacterial endocarditis among Gram-positive cocci?
a. Streptococcus mutans
b. Streptococcus bovis
c. Streptococcus mitis
d. Streptococcus simulans
8. What is the principal virulence factor of Streptococcus pyogenes?

a. polysaccharide capsule
b. sialic acid capsule
C. exotoxin
d. M protein
9. It assists in the recovery of enterococci from contaminated specimens.
a. bile esculin azide agar 8400000
b. cephalexin-aztreonam-arabinose agar
C. Granada agar
d. a and b
10. Which of the following is the best medium for early detection of group B streptococci from
prenatal vaginal samples?
a. Carrot broth
b. Lim broth
c. phenyl ethyl alcohol

d. colistin nalidixic
agar
CHAPTER 18
GRAM-NEGATIVE COCCI

1. differentiate the characteristics of the pathogenic Neisseria from other
2. explain the importance of proper specimen collection and transport in

the isolation of the pathogenic Neisseria species;
3. describe the colony appearance of Moraxella catarrhalis and related
infections and diseases;
4. cite the samples and culture media used in the identification of
5. develop an algorithm of diagnostic tests to identify the Neisseria

species and Moraxella catarrhalis.
The family Neisseriaceae includes the major genera Neisseria, Kingella,

Eikenella, Simonsiella, and Alysiella. mot
Since it has
Moraxella catarrhalis belongs to the family Moraxellaceae.
similar morphological characteristics with the genus Neisseria, it is discussed in
this 1:916
chapter.
Neisseria
The species in this genus are obligate aerobes, non-motile, and non-
The species are fastidious and capnophilic; and they grow optimally
in a moist temperature.
Their natural habitats are the mucous
membranes of the respiratory
and
urogenital tracts.
Most species are carbohydrate fermenters.
cholesterol.
They grow best in media with blood and
They have non-pigmented colonies except

N. flava, N. flavescens, and N. subflava.
The members are sensitive to heat and drying, thus requiring a direct inoculation of specimens
"at the bedside."
Microscopy: Gram-negative diplococci that are coffee- or kidney bean-shaped, except
N. bacilliformis, N. elongata, and N. weaveri which are rod-shaped

mucoid with
glistening small to large, grayish-white
convex; some are
sticky to granular appearance.

Biochemical tests: (f) Oxidase; (+) Catalase except N. elongata and N. bacilliformis
Human pathogens: N. gonorrhoeae and N. meningitidis
Neisseria gonorrhoeae (Gonococcus)

It is not part of the human microbiota although humans are the only known host.
It is the leading cause of sexually transmitted diseases.
It is found on the mucous membranes of urogenital tract, anorectal area, oropharynx, and
conjunctiva.
It can be transmitted by an infected mother to a newborn during birth.
It is a glucose fermenter.
Principal virulence factor: common pili

Culture: CAP - Colonies appear small, shiny, gray to tan-colored, translucent, and raised.
Colonial types: 11 and T2 are virulent with pili, and T3 to T5 are avirulent without pili.
Other virulence factors: Receptors for transferrin, capsule, IgA cellular membrane proteins
(Por B), and lipooligosaccharide (LOS) endotoxin

1. Gonorrhea
It came from the Greek words gonos, which means "seed," and rhoia, which means "flux."
It is an acute pyogenic infection of non-ciliated, columnar, and transitional epithelium.
The sites of infection are the
urethra, endocervix, conjunctiva, and pharyngeal surface or
anorectal area.
The
incubation period is between two to seven days.
The symptoms may include a purulent discharge, lower abdominal for men, and
pain
vaginal bleeding for women. straildo on aunsg
It may also be asymptomatic among females.
2. Purulent urethritis and cervicitis
Untreated gonococcal cervicitis may cause

sterility and perihepatitis (Fitz-Hugh-Curtis
syndrome).
3. Pharyngitis - is the chief complaint in symptomatic oropharyngeal infections

4. Anorectal infections
trectal gonorrhea) - causes rectal pain and a bloody stool
GRAM-NEGATIVE COCCI 249
5.
Ophthalmia neonatorum - is a gonococcal eye infection acquired by newborns
infected birth canal through an
Purulent arthritis,
6.
epididymnitis, and pelvic inflaminatory disease loo ST bas IT
General
Species:
Guidelines on Specimen Collection and Handling for the Identification of Nelseria
Specimen collection and transport is the single most important factor in the accurate diagnosis of
the pathogenic species such as N. gonorrhoene and N. meningitidis.
Direct inoculation
of samples onto plates is acceptable since Neisseria is susceptible to drying.
If samples cannot be processed
immediately, it should be held at room temperature and avoid
wod refrigeration since Neisseria species are sensitive to cold temperature.
Swabs should be placed in
transport system like Amies medium with charcoal if direct plating
cannot be performed.
Cotton swabs should not be used due to the
presence of toxic fatty acids in the cotton fibers since
these are inhibitory to growth of Neisseria.
The pathogenic species are temperature-dependent, and they prefer immediate incubation at
35°C - N. gonorrhoeae and N. meningitidis require prompt incubation in increased carbon dioxide
syringe after plating; both organisms are sometimes lost in subcultures due to their fastidious nature,
and both require iron as growth enhancers.
Laboratory Diagnosis AMT
Specimens: Pus secretions from the urethra, cervix, prostate, rectal mucosa, pharynx, and joint
fluid
Specimens from the urethra and endocervix are preferred.
1. Microscopy
The presence of intracellular Gram-negative diplococci, coffee- or kidney bean-shaped is
If more than five polymorphonuclear leukocytes (PMNLs) per immersion field is observed
but without any bacteria, it suggests non-gonococcal urethritis which
is commonly seen in
chlamydial infections (C. trachomatis).

Smears for the Gram staining is best prepared from urogenital specimens and ideally not
"contaminate" the
from pharyngeal specimens because commensal Neisseria species can
collection, and it can be mistaken as the pathogenic Neisseria.
2. Culture (For confirmation)

GC-Lect.
Culture media: CAP TMA, MTM, ML, NYC, and
Direct inoculation at the bedside is optimal.
will not be recovered
N. gonorrhcne is sensitive to sodium polyanethol sultonate (SPS) and
from routine blood culture due to possible inhibition.
N. gonorrhoeae does not grow on BAP. in an atmosphere of increased
72 hours
35°C to 37 °C for
of the plates at
Prompt incubation candle jar and JEMBEC (John E. Martin
carbon dioxide incubator,
CO, such as the use of
tablet is
Biological Environmental Chamber) system with citric acid-bicarbonate required to
achieve optimum growth.
T1 and 12 colonial types have smaller and raised colonies compared to 1 3 to 15 types which
are larger colonies and appear flat.
Specimen Transport
Transport Media: Cary Blair and Amies with charcoal

Transport System: Transgrow, JEMBEC, and Gono-Pak
onto an agar medium following a "Z"
For inoculation, the swab specimen should be rolled
pattern; then they should be cross-streaked with : sterile loop. od jonnso eblgmne l
composed of selective media and a provision for an increased carbon
The transport system is
dioxide that will support the growth of the capnophilic Neisseria. ed blvode adawa
Selective Culture Media for Neisseria gonorrheae

a. Thayer-Martin agar (TMA)
It is a chocolate agar base with a supplement (IsoVitaleX) and antibiotics.
The antimicrobial agents (inhibitors) are vancomycin, colistin, and nystatin. p0 3
Vancomycin inhibits gram-positive bacteria while colistin is against gram-negative
bacteria except Neisseria.
Nystatin is an anti-fungal agent.
N. meningitidis, N. lactamica, and N. flavescens grow on TMA.
b. Modified Thayer-Martin (MTM) agar
It has all TMA components and trimethoprim lactate (prevents swarming).
C. Martin-Lewis (ML) medium
It contains all the ingredients of MTM agar except nystatin which is substituted by
anisomycin.
It has an increased vancomycin concentration.
d. New York City (NYC) medium
It is a transparent medium with lysed horse blood and yeast dialysate.
The antimicrobial agents are vancomycin, colistin, trimethoprim, and amphotercin b
(anti-fungal agent).
Advantage: The growth of genital Mycoplasma (Mycoplasma hominis and Ureaplasma
urealyticum) can be observed."
e. GC-Lect medium
It contains the same NYC antibiotics and lincomycin.
3. Biochemical Test
The confirmation of Neisseria species is based on its growth and biochemical characteristics.
Carbohydrate Utilization Test (Cystine Trpticase Agar/CTA Test)
It is the standard method of identifying
N. gonorrhoeae.
It detects acid production from
glucose, maltose, fructose, lactose, and sucrose.
It requires a pure culture plate to ensure accuracy of the results.
Due to
its long incubation period and limitation, it is
Medium: no longer used.
Cystine trypticase agar (CTA) with 1% carbohydrate ...
Control: Carbohydrate-free medium

(+) Result: Exhibits yellow color within 24
to 72 hours of incubation at 35°C.
N. gonorrhoeae and N. meningitidis have
positive reactions with CTA medium.
Rapid Acid Detection Tests Plus
To make detectable acid from

carbohydrates, these assays rely on preformed enzymes
secreted by the bacteria in heavy concentrations to
lyze the substrates in the reaction.
Major components: Four carbohydrates (glucose, maltose, lactose and sucrose)

Additional tests: DNAse and
beta-lactamase (hence the term "plus", 6-in-1 assay package)
Procedure: A pure culture from MTM
agar or CAP is used to create a bacterial
suspension, then 200 ul transferred to each well, sealed, and incubated at 35°C for
is
2 hours in a non-CO2 incubator. A control well is included, hence a total of 7 wells.
Substrate: 4 sugars, DNA, and penicillin (for -lactamase test) metemOnn
(+) Result for QuadFERM+ Red to yellow color
Quality Control
Positive for Glucose and Negative for DNAse: Neisseria gonorrhoeae ATCC 31426
Positive for Glucose, Maltose, and Lactose: Neisseria lactamica ATCC 23971
Positive for Glucose, Maltose, and Sucrose: Neisseria mucosa ATCC 19695
Negative Acid Production and Positive DNAse: Moraxella catarrhalis ATCC 25240
b. Oxidase Test
It detects the presence of cytochrome oxidase that degrades the substate cytochrome c.
an isolated colony is
Procedure: A drop of the reagent is applied directly onto a plate, or
rubbed onto a filter paper disk that is impregnated with the reagent.
bA
(+) Result: Dark purple color within 10 seconds

Quality Control
Positive: Pseudomonas aeruginosa ATCC 27853
C. Superoxol Test
The reagent is 30% H,O:
slide that is treated with
placed on a clean glass
For the procedure, an isolated colony
is
the reagent.
( Result Exhibits a vigorous bubbling (N. gonorrhocae)
d. DNase Test
The culture medium is DNase agar with methyl green.

after 18 hours to 24 hours of
clear halo around the colonies
(+) Result: Exhibits a
incubation
If OUADFerm+ test is utilized, a positive result indicates a yellow color.

in this test
M. catarihalis secretes DNase which signifies a positive reaction while
Neisseria provides a negative reaction.
Quality Control (DNAse Agar/DNA Hydrolysis Test)

B-lactamase Test (Cephalosporin/Nitrocefin Test)
within 10 minutes)
(+) Result: Positive (deep pink or red
Quality Control
Negative: Branhamella catarrhalis ATCC 25240

f. Chromogenic Substrate
It detects atypical strains of Neisseria with differentiated results
in the carbohydrate
utilization test.
Colonies used for this test should only be isolated from selective media like Thayer
Martin agar (TMA).
It identifies bacterial enzymes that hydrolyze the colorless substrates to colored end
products.
Disadvantage: Misidentification of nonpathogenic species as N. gonorrhoene or

N. meningitidis.
Multitest (Conventional-Chromogenic Enzyme Test)

It utilizes enzyme substrates coupled with the principles of the biochemical tests - hence
the term "multitest".
It identifies colonies from both selective and nonselective culture media.
Advantage: It detects Moraxella catarrhalis and Haemophilus species aside from Neisseria.
Example: API NH, Microscan HNID Panel, Vitek, and BBL Crystal
4. Antimicrobial Susceptibility Test (AST)

Since N. gonorrhoeaeand also N. meningitidis are resistant to vancomycin, colistin, nystatin,
and lincomycin, these antibiotics can be added into the culture media to inhibit the growth
of normal flora and other bacteria while promoting the selective isolation of the two species.
Antibiotics such
as extended-spectrum cephalosporins and quinolones are utilized.
Etest may be used as an alternate method.
Preferred medium: Gonococcal (GC) agar

CLSI Recommendation: Disk diffusion or Agar dilution
5. Immunodiagnosis
It employs monoclonal antibodies for bacterial identification

It does not require pure selective culture plate - this can be
such as CAP. performed on primary media
a.
Fluorescent Antibody Test (FAT)
This test is highly specific and sensitive. A r1sa erthinle Saisinb st
It uses monoclonal antibodies that recognize epitopes on the principal outer membrane
protein (Por) of N. gonorrhoeae.
b.
It serves
as a confirmatory test for the biochemical characteristics of N. gonorrheae.
It employs monoclonal antibodies to identify the gonococci _ the monoclonal antibodies
aredirected against N. gonorrhoeae attached to Staphylococcus aureus cells which are
destroyed by the gonococci in the reaction.
(+) Result: Exhibits agglutination
b. Molecular Diagnosis
a.
Nucleic Acid Amplification Test (NAAT)
It is currently the recommended and reliable method for the identification of
N.
gonorrhoeae in clinical samples including urine specimens. 2 m9arf it 512
It
detects gonococcal antigen or nucleic acid directly in specimens both in symptomatic
and asymptomatic population.
Specimens: Endocervical or urethral swabs and urine
Methods: Abbott RealTime CT/NG, BD ProbeTec ET, Cobas CT/NG, and Xpert CT/NG
Advantage: High sensitivity and capability to detect Chlamydia trachomatis together with
N. gonorrheal
Disadvantage: Lower detection rate in extragenital infections among young individuals
involving the rectal and orophyrngeal areas
b. Chemiluminescent Nucleic Acid Probe
It is a rapid test for directly detecting gonococcal rRNA in genital and conjunctival
specimens.
two hours.
It provides results within one hour to
Specimen: Endocervical or urethral swabs
recommended for pharyngeal or rectal specimens.
Disadvantage: It is not
Neisseria meningitidis (Meningococci)

It is the causative agent of meningococcemia or spotted fever,
It is the leading cause of fatal bacterial meningitis.
It is only isolated from

humans specifically in the nasopharynx and oropharynx - the
ISNU
asymptomatic human carriage in

the upper respiratory tract is common.
and a true pathogen
of the upper respiratory tract.
It is both considered as commensal
contacts especially in crowded housing

It spreads by respiratory droplets easily through close
facilities.
It is both a glucose and maltose fermenter, secretes

beta-lactamase, and requires iron for growth.
bon
a carrier
Mode of Transmission: Respiratory droplet from
Principal Virulence Factor: Lipooligosaccharide (LOS)-Endotoxin Complex S8:*9167
Microscopy: Encapsulated strains can have a halo around the organism.
potentia
Culture: BAP - Colonies appear large, smooth, bluish-gray and convex with greenish dots under
the colonies.
and mucoid.::00
CAP - Colonies appear small gray to tan colored, convex,
218:171010 TMA - Colonies exbibit smooth, convex, with colorless to gray appearance.
Major Serogroups: A, B, C, Y, and.W-135 b cha fiboditgs Isnebonom exolgfie

Other Virulence factors: Pili, polysaccharide capsules, IgA1, and cellular membrane proteins
(Por A and Por B)
Related Infection and Disease

N. meningitidis has the ability to invade serous membranes and joint tissues, resulting in pleuritis,
pericarditis, and arthritis.
The LOS-endotoxin complex produces hemorrhage in the adrenals known as the Waterhouse-
Friderichsen syndrome.
Though N. meningitidis is considered pathogenic, continues study has been made to determine the
correlation of human carriage and the development of the disease.
Penicillin is the drug of choice for the treatment of meningococcal meningitis.
It refers to the presence of N. meningitidis in the blood and can occur in an acute or chronic form.
It develops with or without meningitis.

The source of epidemic is oral secretions and respiratory droplets (person-to-person spread).
The incubation that leads to the development of the disease occurs from day of the exposure to
199710 101
Signs and symptoms: Frontal headache, stiff neck, and fever (epidemic
meningitis in adults)
Petechial skin lesions may develop during bacteremic spread due to the release of endotoxin after
a bacterial cell lysis.
Laboratory Diagnosis
Specimens: CSF, blood, nasopharyngeal swabs, and petechial skin lesions
CSF
specimens should be kept at room temperature or placed at 35°C before plating.
1. Microscopy
Sediments of $F after centrifugation is best for staining and culture.

Microscopy reveals Gram-negative intracellular and extracellular diplococi.
2. Culture
Culture media: BAP CAP, and TMA
Nasopharyngeal swabs should be
transport media with charcoal. plated immediately in the JEMBEC system or placed in
Culture plates should
for 24 hours be incubated in an environment with increased catbon diovide at 35°C
to 72 hours.
N. meningitidis is sensitive to
0.025%.
SPS, so the content in blood culture broth should not exceed
TMA allows the growth of N.
gonorrheae and N. meningitidis while most Neisserisa species
have either variable or
no growth.
Both N. gonorrheae and N. meningitidis will not
grow on BAP or CAP incubated at room
temperature compared to other Neisseria species including N. bacilliformis.

3. Oxidase Test
(+) Result: Purple color
Quality Control
Positive: Pseudomonas aeruginosa ATCC 27853

4.
Antimicrobial Susceptibility Test
Though routine sensitivity test is not a requirement, CLSI recommends that either a broth
microdilution or an agar dilution MIC test with cation-supplemented MH broth and laked
horse blood or MHA with 5% sheep blood should be used.
The third-generation cephalosporins is recommended for meningococcemia.

Penicillin G remains as the primary drug for treatment of meningococcal meningitis.
5.
Immunodiagnosis
Identification can be confirmed by using slide agglutination with pooled polyvalent
grouping antisera. tulcal
a. Latex agglutination direct identification of antigens in CSF, serum, and urine;

N. meningitidis can be confirmed by slide agglutination
accelerates the antibody and antigen reaction through a
Counterimmunoelectrophoresis -
buffered diffusion medium
0. Gamma-glutamyl Aminopeptidase (GGAP) Test

N. meningitidis produces this enzyme and creates a positive reaction.
Reagent substrate: Gamima-glutamyl-p-nitroanilide (CPNA)
4) Result: Yellow color (p-nitroaniline end product)
Quality Control
13077
Positive: Neisseria meningitidis
Negative: Moraxella catarrhalis ATCC 25240
256 REVIEW HANDBOOK IN DIAGNOSTIC BACTERIOLOOY
Species
TABLE 18.1. Differential Characteristics of the Non-pathogenic Neisserta
Distinguishing Feature
Non-pathogenic Culture (CAP)
Biochemical Test
Neisseria Species Colistin susceptibility test
Neisseria cinerea Translucent, raised and CTA: Negative but differentiates N. cinerea (sensitive, ≥10
sometimes exbibit Gothel
slightly granular mm) from N. gonorrhoeae (resistant)
colonies variability in acid
production
Similar to the T3
colonies of N. Nitrate Reduction:
Positive
gonorrhoeae
CTA: Negative (+) Growth on BAP and CAP at 25°c
Neisseria elongata Colonies are large, flat
with clay-like
appearance and has SOT- Weakly positive
tendency to "corrode or negative Gram-negative "rod-shaped" coccus
the agar
CTA: Negative (+) Growth on BAP and CAP at 25°C
Neisseria flavescens Smooth colonies with
yellow color (+) Growth on TMA
Yellow pigmented colonies
Neisseria lactamica Small and translucent CTA: Positive (+) Growth on TMA bIoNI
and
colonies with a "yellow (glucose, maltose, (+) ONPG within 30 minutes
ring"
Lactose-fermenting species
Similar colonial growth Nitrate Reduction:

Isolated from meningococcal carrier
Positive
surveys, con monly from younger
children and rarely from adults
Neisseria mucosa Colonies are large, CTA: Positive (+) Growth on BAP and CAP at 25°C
smooth, with mucoid- (glucose, fructose, 0is. at 25°C
(+) Growth on NA
sticky appearance maltose, and sucrose)
Positive reaction to all biochemical

SOT: Weakly positive tests except lactose fermentation
or negative
Isolated from the nasopharynx of

Nitrate Reduction:
Positive
children or young adults
Neisseria sicca Colonies are dry and CTA: Positive n (+) Growth on BAP and CAP at 25°C
wrinkled exhibiting (glucose, fructose,
'bread crumbs"
maltose, and sucrose)'
Nitrate Reduction:
Positive
Neisseria weaveri
Small and semi-opaque CTA: Negative Gram-negative "rod-shaped" coccus
colonies with smooth
Catalase: Positive
appearance
PAD: Positive
Nitrate Reduction: Associated with dog-bite infection
Positive
NA - Nutrient
agar, SOT -Superoxol Test; PAD. Phenylalanine Deaminase
257
Moraxela catarrhalis (Branhamellacatarrhalis)

It is the most
commonly isolated member of the genus Moraxella:n
indigenous microbiota of the oropharynx, and itis considered
It is part of the
pathogen. as an opportunistic
It resembles Neisseria
species by
It is a non-motile,
exhibiting Gram-negative coccal morphology.
fastidious, B-lactamase producer, and it is encapsulated with common pili.
It causes
upper respiratory
tract infections in otherwise healthy children and the elderly.
It is the third most common cause
of otitis media and sinusitis in children. n (-)
Microscopy: Small, Gram-negative cocci that tend to
grow in pairs
from end to end with their
adjacent sides flattened
Culture: BAP - Colonies are
smooth, opaqu( grayish-white with "hockey puck" appearance; old
colonies with "wagon wheel" appearance
Biochemical test: (+) oxidase and catalase
Related Infection: otitis
media and sinusitis; bronchitis and pneumonia as co-morbidities
Laboratory Diagnosis fon3hben

Specimens: Ear discharge, nasopharyngeal swab, sputum, and bronchial and sinus aspirates
1. Microscopy
M. catarrhalis appears as Gram-negative intracellular diplococci.
2. Culture - BAP and CAP
There is growth on BAP and CAP at 25°C and on nutrient agar at 35°C.
On CAP, M. catarrhalis has pink to brown colonies and the same hockey-puck consistency
observed on BAP.
M. catarrhalis is inhibited in gonococcal media by colistin.
3. Biochemical Test
a. Carbohydrate Utilization Test

the CTA test.
M. catarrhalis does not utilize any sugar in
b. DNase Test/DNA Hydrolysis Test

It is the definitive test for
the identification of M. catarrhalis.
or colorless halo around the colonies after 18 hours to 24 hours
(+) Result: Exhibits a clear
of incubation
In OUADFerm+ test, a positive

DNAse reaction exhibits a yellow color.
Quality Control

Moraxella catarrhalis ATCC 25240
ATCC 25922
Negative: Escherichia coli
Neisseria sonorrhoene ATCC 31426
258 REVIEW HANDBOOK IN DIAGNOSTIC BACTERIO
Butyrate Esterase/Tributyrin
It is a definitive test for M. catarrhalis.
or tributyrin (hydrolyze by
Reagent substrate. Bromo-chlor-indolyl butyrate organisme
that secrete butyrate esterase)

placed onto a pre-moistened disk
Procedure: A loopful of the organism is
detection of butyrate esterase.
that
impregnated with the substrate for the

(+) Result: Blue color (Indigo)
6 Result. No color change on the disk
Quality Control
Positive: Moraxella catarrhalis ATCC 25240
43069
Negative: Neisseria gonorrheae ATCC

M. catarrhalis isolates are sensitive to cephalosporins and trimethoprim- sulfamethoxazole.
Raveendran, et al (2020) explains that M. catarrhalis is susceptible to amoxicillin/clavulanic
acid combination, in the presence of beta-lactamase production.
PCR assay reveals the production of beta-lactamases BRO-1 and BRO-2 encoded by bro-1 and
bro-2 genes.
These bro-1 and bro-2 genes are responsible for the penicillin resistance of M. catarrhalis.
TABLE 18-2. Differential Tests for Pathogenic Neisseria and Moraxella catarrhalis
Test N. gonorrhocae N. meningitidis M. catarrhalis
02000
Superoxol
Growth on MTM, ML, and +
NYC
Growth on BAP at 25°C
Growth on NA* at 35°C
Acid Production
Glucose
Fructose
Maltose
Sucrose
Lactose
Nitrate reduction
Tributyrin hydrolysis
f-galactosidase
Y-glutamyl aminopeptidase
+
*NA - Nutrient agar; - variable, v
+, most
strains with positive reactionvariable, positive or negative reaction
, most
strains with negative reaction
FIGURE 18-1.
Gram stain of Neisseria gonorrhoede using cervical smear
(Photo by J. Miller)
FIGURE 18-2. Photomicrograph of Gram stain of Neisseria gonorrhoeae

(Photo from USCDCP)
FIGURE 18-3. Gram staining reaction of Neisseria gonorthoece

(Photo from ]. Miller)
FIGURE 18-4. Oxidase test. A positive result (left) showing purple

coloration and a negative result [right) shows no coloration
(Photo by J. Reynolds)
eanal E
N. gonorrhoeae
CKKK
DNaseB-Lac
B. catarrhalis
M
DNas -Lac
FIGURE 18-5. QuadFERM+ Test Results

Source: https://www.cdc.gov/std/gonorrhea/lab/tests/dnase.ntn
QUOITIDAD DRaOnoN 261
GRAM-NEGATIVE COCCI
ASSESSMENT QUIZ Elishe t otheee
Encircle the letter that corresponds to the

correct answer
Which of the following is
the positive control organism used in Tributyrin tes1t2
1.
a. Moraxella catarrhalis ATCC 25240
b. Neisseria gonorrhoeae ATCC 31426

C. Escherichia coli ATCC 25922 Vest loxo reque rol hagporodi
d. Neisseria gonorrheae ATCC 43069
2.
It has "bread crumbs" looking colonies on CAP and positive with carbohydrate fermentation
except fructose.
a. Neisseria flavescens
b. Neisseria sicca
C. Neisseria mucosa
d. Neisseria cinerea
3. All of the following is true about TMA culture EXCEPT:

a. positive growth of both Neisseria gonorrheae and Neisseria lactamica
b. it contains neomycin as anti-fungal agent
C. CAP is the based medium
d. allows growth of Neisseria meningitidis
4. How many sugars are utilized in the QuadFERM+ test?

a. 2
b. 3
C. 4
d. 5
7112
5. This
enzyme test differentiates Neisseria gonorrheae from Neisseria meningitidis.
a. ALP
c. B-lactamase
d. SOD
BACTERIOLOGY
6. What is the distinguishing appearance

a. yellow ring around colonies
b. slimy colonies
C. hockey puck
d. clay-like colonies
7. What is the reagent for superoxol test?

a. p-dab
b. 30% hydrogen peroxide
nollstnan c. Bromo-chlor-indolyl butyrate
d.
8. Which of the following is the substrate used for -lactamase test?

a. penicillin
b. methicillin
C.
d. nitrocefin
9. It allows detection of other causes of genital diseases aside from gonorrhea.

a.
b.
C.
d. GC-Lect Medium
10. Which genes are

responsible for the antibiotic resistance of Moraxella catarrhalis?
a. bro-1 and bro-2
b.
C.
d.
CHAPTER 19
ENTEROBACTERIACEAE
(GRAM-NEGATIVE
BACILLI)

1. outline the general characteristics of the Enterobacteriaceae;
2. explain the antigenic structures and their purpose in bacterial tester omd o fusm
identification;
3. discuss the principle of the different biochemical tests;
4. explain the fermentation patterns of enterobacteria in culture media;
5. relate the result of the biochemical test with the unknown organism;
6. cite the infections and diseases associated with Enterobacteriaceae; and

7. design an algorithm of diagnostic tests to determine the clinically
significant genus and species.
General Characteristics of Enterobacteriaceae ai anomiooce yrs ni songst
The members of this family are commonly referred to as
"enterobacteria."
They species are facultatively anaerobic, non-spore-forming, Gram-

negative bacilli.
All members are motile at 35°C with peritrichous flagella except for
Klebsiella, Shigella, and Yersinia.
Enterobacter.
All members are non-encapsulated except Klebsiella and
other sugars
All species ferment glucose, with some species utilizing
like lactose and sucrose.
and
except Photorhabdus
All species reduce nitrate to nitrite
Most of them are present in

the gastrointestinal tract (GIT) of humans
except for Plesiomonas, Salmonella, Shigella,
and
as commensal flora
to 5 •C (psychrophiles) especially
Some organisms may grow at 1°C
of Serratia.
Yersinia enterocolitica and some strains
with rounded ends

Microscopy: Straight Gram-negative rods
or
gray except Klebsiella and Enterobacie

and
Culture. BAB/CAP - Colonies appear as smooth, large, some strains of E. coli, which
with mucoid colonies; and they are
non-hemolytic except are
Plesiomonas shigelloides
Biochemical tests: (+) Catalase; (-) oxidase except
Genera in the family Enterobacteriaceae

Budvicia, Buttiauxella, Cedecea, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ewingella, Hafnia,
Obesumbacterium, Pragia, Pantoea,
Tatumella,
Salmonella, Serratia, Shigella,
Photorhabdus, Proteus, Providencia, Rahnella,
Xenorhabdus, Yersinia, and Yokenella
Groups of Enterobacteria
1. Opportunistic Pathogens
and animals.
They are part of the intestinal microbiota of both humans
They generally do not initiate disease

in healthy, uncompromised human hosts.
their normal body sites (extraintestinal
They may produce serious infection outside
infection).
They produce significant virulent factors.
examples are Escherichia coli, Citrobacter, Enterobacter, Klebsiella, Proteus,
and Serratia.
Some
2. Overt/True pathogens
commensal microbiota of the human GIT.
They are not present as
They are acquired through ingestion of contaminated food or water.
Their presence in any specimens is considered very significant.

Some examples are Salmonella, Shigella, Yersinia, and Plesiomonas shigelloides oinrom onle
Notes to Remember
E. coli, K. oxytoca, K. pneumoniae, and P. mirabilis are isolates of the urinary tract infection.
E. coli, Enterobacter, K. pneumoniae, and P. mirabilis are agents of bacteremia.
Shigella, Salmonella, E. coli, and Yersinia are associated with diarrhea.slgson-nom OTB STOCHIENY
Citrobacter, Enterobacter, and Serratia are antibiotic-resistant genera. awheabbulp Inanmol olong-
E. coli, K. pneumoniae, and K. oxytoca
are ESBL producing enterobacteria including carbapenemasesand
cephalosporinases.
Antigen-determinants for Serological Identification of Enterobacteria

1.
Somatic O antigen - heat-stable; located in the cell wall; used for E.
coli and Shigella serotyping
2. Flagellar H antigen - heat-labile; found in the flagellum; used for
Salmonella serotyping
3. Capsular K antigen. heat-labile of
polysaccharide; found as K1 and Vi antigen
antigen of E. coli
S.
enterica subsp. enterica serotype Typhi.
ENTEROBACTERIACEAE (GRAM-NECATIVE BACILL) 265
TABLE 19-1. BacterialSpecies andIts AssociatedInfections

Bacteria
Infections/Diseases
Escherichia coli
Shigella
Bacteriuria, epticemtia,neonatalsepsis,meningilis,anddissionl
Diarrhea, dysentery syndrome
Edwardsiella
Diarrhea,wound infection,septicemia, meningitis, and enteric fever
Salmonella Septicemia, enteric fever. and diarrhea
Opportunistic
and nosocomial infections
bacteriuria, pneumonia, septicemia, and liver disease
Opportunistic and
Enterobacter
septicemia nosocomial infections, wound infections, bacteriuria, and
Serratia
Opportunistic
septicemia
and nosocomial infections, wound infections, Iackeruria and
Proteus
Bacteriuria, wound infections, and septicemia
Providencia Opportunistic and nosocomial infections, wound infections, bacteriuria, and
septicemia
Morganella Opportunistic and nosocomial infections
Yersinia pestis Plague
Yersinia pseudotuberculosis Mesenteric adenitis, diarrhea
Yersinia enterocolitica Mesenteric adenitis, diarrhea
Erwinia Wounds that are contaminated with soil
or vegetation
Source: Textbook of Diagnostic Microbiology, 4th ed. by C. R. Mahon, D. C. Lehman, and G. Manuselis, Jr., 2010.
Escherichia
Escherichia coli (Colon bacillus)

It is the most significant species in the genus Escherichia.
It may inhabit the female genital tract although it is a microbiota of the large intestine.
It is a primary indicator of fecal contamination in water purification.

It is the leading cause of nosocomial urinary tract infection (UTI).
It has both the adhesive fimbriae and sex pili.
serotypes causing diarrhea and UTI.

It has several
Mode of transmission: person-to-person contact, fecal-oral route, and through consumption of

contaminated food and water
intimin
Virulence factors: Endotoxin, common pili, K1 antigen, and
Antigenic determinants: O, H, and K antigens
and dry, and they exhibit pink color (lactose fermenter or
Culture MAC - Colonies appear flat
non-lactose fermenters or NLF.
some strains may be
some exhibit a B-hemolytic pattern.
BAP - Most strains are non-hemolytic though
EMB - Colonies appear as greenish metallic sheen.
and citrate) reaction: + + -
IMViC (Indole, methyl red, Voges-Proskauer,
slant/acidic butt), (+) gas, -) hydrogen sulfide

TSIA (triple sugar iron agar) reaction: A/A (acidic
or H,S
Related infection/disease: Bacteremia,
UTI, hemolytic uremic syndrome (HUS), and neonatal
meningitis
Escherichia hermanii
It is formerly called E. coli atypical or enteric group II.
It has been isolated from CSF, wound infection, and blood.
Culture: Colonies have yellow pigmentation.

Other Species of the Genus Escherichia
E. vulneris
E. albertii, E. blattae, E. fergusonii, and
TABLE 19-2. Differential Characteristics of E. coli Strains

Infection Virulence Characteristics
Types of E. coli strains
Factors/ Serotype
Enteropathogenic E. coli (EPEC) Infantile diarrhea Pathogenicity (+) H antigen and

(stool without blood) islands intimin
It causes the loss of microvilli; there is no
toxin production. Lab test:
Serotypes:
• It is attached to the brush border of the
HeLa cell assay
intestinal epithelial cells, causing cellular 055:H6
damage (with adhesive property).
O111:H2
Enterotoxigenic E. coli (ETEC) Traveler's diarrhea Heat-stable (ST) Persons with
Colonization occurs in the proximal small

and heat-labile achlorhydria are at a
intestine. (LT) enterotoxins high risk.
Infective dose: 10t to 1010
Serotypes:
Enteroinvasive E. coli (EIEC) Dysentery-like Invasin

It penetrates and multiplies within the or Shigella-like
infection
intestinal epithelial cells. Serotypes: (+) large plasmid
• Infective dose: 106 Watery diarrhea O124:H30 (+) Sereny test*
with WBCs
Enterohemorrhagic E. coli (EHEC) Hemorrhagic colitis Verotoxin I and II (+) intimin
•
The toxins produced by this strain destroy Hemolytic uremic or Shiga-like toxin (-) MUG*
vascular endothelial cells. syndrome (HUS) • Associated with
Bloody diarrhea thrombotic
without WBCs 0157:H7 thrombocytopenic
Enteroaggregative E. coli (EAEC) and • Persistent watery EAEC has:
Diffusely adherent E. coli (DAEC) diarrhea (EAEC) "stacked-brick
EAEC adheres to the surface of the
Serotype: appearance.
intestinal mucosa. and UTI (DAEC) O44:H18
EAEC adheres to HEp-2 cells which form Lab test:
clumps of bacteria. HeLa cell assay
ENTER
(GRAM-NEGATIVE BACILLI) 267
of E. coll strains
Types
Infection
Virulence Characteristics
Uropathogenic E. coli (UPEC) UTI

Factors/ Serotype
UPEC iS the most common cause of UTI Common pili,
in humans aerobactins, and adheres strongly to
the urinary tract.
• MUG - 4-methlyumbellifery-B-D glucuroide
Sereny test determines the organism's ability to produce
test for Shigella and EIEC keratoconjunctivitis in guinea pigs; is a virulence
EHEC 0157:H7
It is the
most virulent serotype causing hemolytic uremic syndrome (HUS). Up-histuol
It appears colorless on MAC and produces
heavy growth compared to other strains which are
pink colored.
It rarely produces beta-glucuronide that results in a negative MUG test result.
Notes to Remember
K1 antigen is responsible for neonatal meningitis. & InrvstawdommneD as nwonf homeot af fl
Enterotoxigenic E. coli only grows on BAP, and it is acquired from contaminated food or water.
Enteroinvasive E. coli requires higher infective dose (10°) than Shigella species to produce a disease.
E coli secretes enterotoxins such as heat-labile (LT) and heat-stable (ST), which activates the cyclic
adenosine monophosphate (cAMP) and guanylate cyclase, respectively.
The
4-methylumbelliferyl--D-glucuronide (MUG) test is a screening biochemical test which detects
Verotoxin I is similar to the Shiga toxin that is produced by S. dysenteriae type 1, and it causes damage
to the kidney (Vero) cells of the African green monkey.
Intimin which is
encoded by the eaeA gene and hemolysin from the hly gene are the other virulent
factors for EHEC.
Shiga toxin can be identified by nucleic acid test or enzyme immunoassay by isolating colonies from
nonselective media.
animals.
The species of this genus are usually found in the GIT of humans and
mucoid consistency.
Culture: MAC - Colonies exhibit a pink color (LF) with
Growth on media with potassium cyanide (KCN): Positive
IMViC reaction: - - + +
ISIA reaction: A/A, (+) gas, (-) H,S
K. oxytoca, K. ozaenae, K. rhinoscleromatis and
Species: K. pneumoniae subsp. pneumoniae,K. planticola (Raoultella planticola), and K. terrigena
K.
ornithinolytica (Raoultella ornithinolytica),
(Raoultella terrigena).
268 BACTERIOLOGY
REVIEW HANDBOOK IN DIAGNOSTIC
Klebsiella pneumoniae subsp. pneumoniae (Friedlander's bacilus)

It is the most commonly isolated species of
infections among hospitalized patients
It is the frequent cause of lower respiratory tract hosts such as newborns, elderly
(hospital-acquired pneumonia) and in immunocompromised
patients, and patients on respirators.
It is an agent of ventilator associated pneumonia due to carbapenemase-producing . prcunominc
infected individuals.
It produces a "currant jelly-like" sputum among
Virulence factor: Polysaccharide capsule
Differential Test: (+) String test
DAM IO
Growth on media with potassium cyanide (KCN);: Positive
IMViC reaction: _ ++
TSIA reaction: A/A, (+) gas, (-) H,S
Klebsiella granulomatis
It is formerly known as Calymmatobacterum granulomatis. 1s:6nogd 10
It is the etiologic agent of granuloma inguinale or donovanosis.

It is a pleomorphic Gram-negative bacillus that is encapsulated and non-motile.
It has antigenic and molecular determinants similar to the Klebsiella species.
It is best cultivated in 1 yolk sac or in a fresh egg yolk medium.
It is the only member of the Enterobacteriaceae that will not grow in the primary plated media or
Diagnostic Marker: Blue or bluish-purple rods with a "safety pin" appearance; surrounded
by a pink capsule and the presence of Donovan bodies (inclusion bodies) in mononucleated
endothelial cells
Preferred Specimen: Tissue from the Ulcer (wound)

Preferred stains for microscopy: Wright-Giemsa stain and Warthin-Starry
Donovanosis is a sexually transmitted disease that is characterized by nodules which enlarge
and evolve to form a tender, erythematous, granulomatous, and painless lesions that bleed easily.
Notes to Remember
K. pneumoniae and K. oxytoca are

agents of urinary tract infection, pneumonia, and bacteremia.
K. oxytoca is linked to antimicrobial-associated hemorrhagic colitis.
K. ozaenae is the causative agent of chronic atrophic rhinitis which is also known as the "foul-smelling
atrophic rhinitis; it is characterized by the presence of plasmid-mediated ESBLs in nosocomial
resulting
antibiotic resistance.
K. pneumoniae subsp. rhinoscleromatis causes granuloma of the nose

and oropharynx.
ENTEROBACTERIACEAE(GRAM-NEGATIVE BACILLI) 269
K. oxytoca
and K. ornithinolytica (Raoultella
ornithinolytica) are both indole-positive. BY BOSK
The genus Raoultella belongs to the
family
and biochemical characteristics as the other genera
Enterobacteriaceae havingthesame morphological, cultural,
except that studies show that it is
may grow in cold temperature(psychrophiles). encapsulated and
K. ornithinolytica, K. planticola, and K. terrigena

are now under the genus Raoultella.
TABLE 19-3. Differential Tests for

Commonly Isolated Klebsiella Species
Biochemical tests K. pneumoniae subsp.
K. oxytoca
pneumoniae
Indole
Methyl red
-/+ +
Urease
Lysine decarboxylase (LDC) +
Gelatin liquefaction (22•C)

Growth in potassium cyanide (KCN) + +
Alginate utilization
+, most strains with positive reaction
-/+, most strains are negative
-, most strains with negative reaction
Enterobacter
The members of this genus resemble those of Klebsiella when grown on a MacConkey agar.
The species can cause bacteremia, nosocomial pneumonia, and urinary tract infection.
Culture: MAC - Colonies exhibit a pink color and are sometimes mucoid (LF).
Growth on media with KCN: Positive
Ornithine decarboxylase test: Positive

Lysine decarboxylase (LDC) test:
Positive (all species except E. cloacae and E. gergoviae) .01DVEA
(E. aerogenes and E. cloacae)
Sorbitol fermentation: Positive
Urease and Malonate test: Positive (E. cloacae)
IMViC reaction:
ISIA reaction: A/A, (+) gas, (-) H,S

cloacae, E. cancerogenus, and E. hormaechei
Clinically significant species: E. aerogenes, E. cloacae subsp
cloacae
Common isolates: E. aerogenes and E.
Enterobacter cancerogenus tormerly E. luyiornay has been isolated from individuals with
osteomyelitis following traumatic wounds.
270 REVIEW HANDBOOK IN DIAGNOSTIC BACTERIOLOOV
TABLE 19-4.
Differential Tests for Commonly solated Enterobacter species os
E. cloacac
E. aerogenes
Biochemical tests
+
Arginine dihydrolase
Ornithine decarboxylase
Urease
Alginate utilization
Gelatin Hydrolysis (22°C)
+, most strains with positive reaction
, most strains with negative reaction
(w), weakly positive;
with positive reaction after > 3 days of incubation
Cronobacter sakazakii
nolbsm eviliaog ttw entmule lom
It is formerly known as Enterobacter sakazakii.
It is found as a contaminant of powdered infant formula.
It is isolated from individuals with brain abscesses and respiratory and wound infections.
Culture: MAC - Colonies exhibit pink color (LF).
BHIA - Colonies exhibit a yellow pigmentation with mucoid appearance.

IMViC reaction:
TSIA reaction: A/A, (+) gas, (-) H,S
Pantoea
Pantoea agglomerans
It is formerly known as Enterobacter agglomerans.
It causes nosocomial outbreaks of
septicemia due to contaminated IV fluids.
It
shows a triple decarboxylase negative reaction.
Culture: MAC Colonies are clear or
colorless (NLF).
BAP - Colonies are
large with yellow color.
IMViC reaction: - v + v
TSIA reaction: K/A (Alkaline slant/Acidic butt), (-) gas, (-)H,S

Serratia
The species in this group are
nosocomial outbreaks such as opportunistic pathogens that are usually associated with
pneumonia.
The species are resistant to
a wide range of antibiotics." CLAT) DaRim ad anionlclonang
Culture: MAC - Colonies are clear
fermentation. and colorless (NLF); some strains may exhibit late lactose
IMViC reaction: - - + +
TSIA reaction: K/A or A/A, (+)
Other biochemical
test: (+) DNAse, gelatinase, lipase, and ONPGe.e.
Species: S. marcescens subsp. marcescens, S. rubidaea, S.
S. fonticola and S. liquefaciens complex
plymuthica, S. ficaria, S. entomophila,
Biogroups: S. odorifera biogroups 1 and 2
Serratia marcescens
It is
the most clinically significant species of the genus.
It causes bacteremic outbreaks in nurseries and cardiac surgery and burn units.
A few strains of this species
are late lactose fermenters.
Biochemical test: (+) Urease, gelatinase, and ONPG; (-) arabinose fermentation the bearg
Notes to Remember moilosen evitspon to ovilbog aldnney
S. marcescens, S.
plymuthica produce pink to red colonies (due to
rubidaea, S. liquifaciens, and S. the
production of prodigiosin pigment) after incubation at 25 °C.

S. odorifera has a musty and pungent odor or a "rotten potato-like" odor..m9g
S. liquefaciens ferments arabinose and exhibits growth in a culture medium with KCN.doe
DNAse test distinguishes Serratia spp. (positive) from Enterobacter spp. (negative).
Proteus
infection.
The members are isolated from urine, wound, and ear
tubules and can cause acute glomerulonephritis
Thespecies can infect the proximal kidney
catheterization.
(AGN), particularly in patients with UTI or
The members of this genus are rapid urease producers - urease splits urea leading to increased
urine pH and eventually promote renal stone formation.
Species: P. mirabilis, P. vulgaris, P. penneri, and P. myxofaciens
Human mirabilis and P. vulgaris
pathogens: P.
Most common isolate: P. mirabilis

"swarming phenomenon" and
Culture: MAC
are clear and colorless (NLF) with
Colonies
"burnt-chocolate" or "burnt-gunpowder" odor.
Positive
Phenylalanine deaminase (PAD) test:
iron agar (LIA) reaction: R/A
(red slant/acid butt) are aplnolg.
9201 Lysine
IMViC reaction: P. mirabilis: - + VV
P. vulgaris: + + - v
TSIA reaction: P. mirabilis: K/A, (+) gas, (+) H,S
P. vulgaris: K/A, (+/-) gas, (+) H,S oleg
TABLE 19.5. Differential Tests for the Commonly Isolated Proteus Species
Biochemical tests P. mirabilis
Indole
Phenylalanine
deaminase (PAD)
LIA R/A
K/A, (+) gas, (+) H.S K/A, (+/-) gas, (+) H,S
T, most strains with positive reaction
-, most strains with negative reaction
V, variable, positive or negative reaction
Providencia
units. roba
The species of this genus are the cause of nosocomial outbreaks involving burn
Species: P. alcalifaciens, P. rettgeri, P. rustigianii, P. stuartii, and P. heimbachae
Culture: MAC - Colonies are clear and colorless (NLF).

PAD test: Positive
IMViC reaction: ++-+
LIA reaction: R/A
TSIA reaction: K/A, (-) gas, (-) H,S
Providencia rettgeri
It is a pathogen of the urinary tract.
It also causes diarrheal disease among travelers.
It is mostly resistant to antimicrobial agents.
Providencia stuartii
It is found in nosocomial outbreaks in burn units.
It is resistant to antimicrobial agents same with P rettgeri.
ENTEROEACTERIACEAE (GRAM- NEGATIVE BAGILLI) 273
Providencia
It is the most commonly isolates
in fecal specimen of children with diarrhea.
TABLE 19-6. Differential Tests
Biochemical test
for the Commonty Isolated Providencia Species T
P. rettgeri
P. stuartii
Urease
Phenylalanine
deaminase (PAD)
Arabitol Fermentation
t, most strains with positive reaction

most strains with negative reaction
The species of this genus has the same biochemical reaction

as those of P. vulgaris, except that the
latter is citrate-negative. RODL37 18060071/
It has been identified as a

cause of neonatal sepsis. tts loote Isodmelh mi botslod ad neo il
Species: M. morganii subsp. morganii and M. morganiisubsp. siboniing ntiw baisibozas 21 h
Culture: MAC Colonies are clear and colorless (NLF).
PAD test: Positive
LIA reaction: R/A

IMViC reaction: + + - -
TSIA reaction: K/A. (+) gas, (-) H,S

Other biochemical tests: (+) Urease, KCN, and ornithine decarboxylase
Edwardsiella
The species of this genus have been isolated from cold-blooded and warm-blooded animals.
Mode of transmission: Ingestion of food and water contaminated with infected animal products
and excreta
Species: E. tarda, E. hoshinae, and E. ictaluri

Human pathogen: E. tarda
Culture: MAC Colonies are clear and colorless (NLF).
Urease test: Negative

LDC test: Positive
IMViC reaction: + + _ -le m111510?
reaction: K/A, (+) gas, (+) H,S

E. coli on MacConkey agar

and Salmonella in biochemicaltests.
The species of this genus resemble
with Salmonella.
The species can cause false-positive
agglutination test results
All species grow in Simmons
citrate aga (SCA).
colorless (NLF) after 24 hours; may appear with a
Culture: MAC - Colonies become clear and
light pink color (LF) after 48 hours.
C. braakii, and C. farmeri
Common isolates: C. freundii, C. koseri, and
Urease test: Positive
IMViC reaction: C. freundii:

C. koseri: + + - +
TSIA reaction: C. freundii: K/A or A/A, (+) gas, (+) H,S

C. koseri: K/A, (+) gas, (-) H,S
Citrobacter freundii
It can be isolated in diarrheal stool cultures (extraintestinal pathogen).
It is associated with pneumonia, intraabdominal abscess, and endocarditis in intravenous drug
users.
It produces group 1 cephalosporinase.
Citrobacter koseri (formerly C. diversus)

It causes outbreaks of neonatal meningitis and brain abscess in nursery units.
TABLE 19-7. Differential Tests for Citrobacter Species

Biochemical tests C. koseri
Indole +
H,S production +
Growth in KCN
A/A or K/A, (+) gas, (+) H.S bas s: K/A, (+) gas, (-) H.S
Salmonella
The species of this genus are the most pathogenic enterobacteria that cause enteric fever(typhoid
fever) and acute gastroenteritis (food poisoning) to humans.
They are not part tract of
the gastrointestinal
of the
human intestinal flora but commensals of
animals.
They may be transmitted by human carriers.

Modes of acquisition: Ingestion of contaminated animal food
products or improperly
cooked
poultry, milk, eggs, and dairy;
and direct human contact
ENTE
ENTEROBACTERIACEAE 275
(GRAM-NEGATIVE BACILLI)
Species: S. enterica (type species) and S. bongori

Subspecies of S. enterica:
S. enterica subsp. enterica (I),
subsp. arizonae (IIIa), S. S. enterica subsp. salamae (II), S. enterica
enterica subsp. diarizonae (Illb), S. enterica
S. enterica subsp. indica (VI) subsp. houtenae (IV),
and
Most commnon human isolate: S, ernterica subsp. enterica ()

Virulence factor: Fimbriae and
enterotoxin (S. enterica)
Culture: MAC - Colonies are clear
and colorless (NLF) dns yd boligui ad mo behnsd sdT
black centers 10 nori pallanomles To noltsloal
Antigenic structures: Somatic O and
flagellar H - for serologic grouping
Vi antigen (Salmonella serotype Typhi) - antiphagocytic; may interfere with
intracellular killing
Salmonella bongori
It is named after the town of Bongor in Chad, Africa, where it was isolated from a host lizard in
end
It can also be isolated from other cold-blooded animals. volud funsag es rowe bool
Salmonella Serotypes and its Significance bs)srtms1nod :noisnimserib 10 boM
All former species of Salmonella have been designated as serotypes under S. enterica subsp.
enterica (e.g., S. enterica subsp. enterica serotype Typhi). plimoy sseuslamoq2
The genus Salmonella has thousands of serotypes. (Source: https://www.cdc.gov/salmonella/reportspubs/
salmonella-atlas/serotyping-importance.html) those
Examples of Salmonella serotypes: Salmonella serotype Typhi, Salmonella serotype Paratyphi A, B,
and C, and Salmonella serotype Choleraesuis, and Salmonella serotype Typhimurium
Main causative agent of enteric fever: Salmonella serotype Typhi

Etiologic agents of paratyphoid fever: Salmonella serotype Paratyphi A, B, and C, and Salmonella
big serotype Choleraesuis
Biochemical Characteristics of Salmonella Species 10

Salmonella serotype Gallinarum.
All species are motile except Salmonella serotype Pullorum and
All species produce gas except for Salmonelia serotype Gallinarum and Salmonella serotype Typhi.
All species produce H, S except for Salmonella serotype Paratyphi A.
LDC: Positive (except for Salmonella serotype Paratyphi A)
Urease: Negative
Growth on media with KCN: Negative
IMViC reaction: + -+
(Salmonella serotype Typhi)

reaction: K/A, (+) gas, (+) H,S
K/A, (-) gas, (+) H.S (Salmonella serotype Typhi)
BACTERIOLOGY
General Categories of Salmonella Infection

reservoirs and sources of infections
as
Salmonellae are found in various animals that serve
Paratyphi.
except Salmonella serotype Typhi and Salmonella serotype
The carriers of Salmonella
excrete the organisms along with their feces and thus Cause
if there are issues on water
ccidental" contamination of food if the person is unhygienic or
sanitation.
gallbladder infection is not evident.
The carriers can be treated by antimicrobial therapy if the
from culture plates is significant
and specific for diagnosis of typhoid
Isolation of salmonellae
fever.
chronic state.
Cholecystectomy can be recommended to enteric carriers in
1. Acute Gastroenteritis
It is one of the most common forms of food poisoning.

comes from animals.
commonly caused by S, enterica subsp. enterica that
It is
outbreak from contaminated
The Salmonella serotype Typhimurium has been involved
in
food such as peanut butter crackers and cereals. balaloal ed cals mes
Sources of infection: Poultry products, milk, and handling of pets

Mode of dissemination: Contaminated kitchen utensils
Infective dose: 106 bacteria
Symptoms: Nausea, vomiting, fever and chills, watery diarrhea, and abdominal pain
2. Enteric fever (Typhoid fever)

It is a febrile disease that develops from eating contaminated food prepared by infected
individuals or carriers.
Direct transmission through fomites is also possible.

The site of long-term carriage is the gallbladder.
Most common agent causing enteric fever: Salmonella enterica subsp. enterica serotype Typhi
Other agents of enteric fever (paratyphoid fever): Salmonella serotype Paratyphi A, B, and C
and Salmonella serotype Choleraesuis
Sources of infection: Human carriers (food handlers), contaminated food, and water
Causes of outbreaks: Improper sewage disposal, poor sanitation, and lack of clean water
source
Symptoms: Malaise, anorexia, myalgia, and severe frontal headache

Complications: Necrosis in the gallbladder (necrotizing cholecystitis) and Peyer's patches
Hallmark of infection: Appearance of "rose spots" during the second week of fever
3. Bacteremia
It occurs with and without

extraintestinal infection that is caused by non-typhoidal
Salmonella species.
It is
characterized by prolonged fever and intermittent bacteremia.
ENTE
Causative agents: Salmonella

Salmonella serotype Choleraesuis serotype Typhimurium, Salmonella serotype Paratyphi, and
Specimens for Salmonella Identification

1. Blood - first week of infection
2. Stool - second to third week of infection POXs amidt. oe
3. Urine third week of infection 9 Vihieng anad boosolnofutsl esthonl shel s et Beths
TABLE 19-8. Biochemical Differentiation of Salmonella Serotypes ashetnoab sllegin?

Biochemical tests S
serotype S. serotype Other
Typhi Paratyphi Cholerasuis
Citrate utilization
+
Gas production + +
H2S production
Lysine decarboxylase
Arabinose fermentation + +
variable (10% to 89% of strains are positive) humen Glf and la a nemamoble u
Shigella
The species of this genus are closely related to those of Escherichia.

The members are neither human microflora or animal commensals.
The species are non-motile, intracellular pathogens that multiply within the cells of the intestinal
epithelium.
Most of thespecies can cause bacillary dysentery.
Modes of transmission: Four F's (flies, fingers, food, fecal); food and water from infected persons,
and fecal-oral route
Reservoir: Human carriers

sonnei
Species: S. dysenteriae, S. flexneri, S. boydi, and S.
Most virulent species: S. dysenteriae
C (s. boydii), and D (S. sonnei)
Serogroups: A (S. dysenteriae), B (S. flexneri),
Antigenic structure: Somatic O
and colorless (NLF).
Culture: MAC - Colonies are clear, fragile,
SSA . Colonies are
colorless without black centers.
LDC test: Negative
IMViC reaction: v + - -
reaction: K/A, (-) gas, (-) H,S

Biochemical Characteristics of Shigella Species

not produce gas from glucose
except some strains of S. flexnert.
All species do
except S. dysenteriae.
All species are mannitol fermenters
All species do not decarboxylate lysine.
All species
S. sonnei is a late lactose fermenter and has a positive reaction with the ONPG test.
Shigella dysenteriae
It is the most virulent of the species and causes bacillary dysentery.
Virulence factor: Shiga toxin
Shigella sonnei
The infection from this organism is self-limiting, and it is usually characterized by fever and
watery diarrhea (stool without blood).
It has one serotype as opposed to the other species.
Shigella flexneri
It is one of the agents of gay bowel syndrome.
It causes gastroenteritis among males who have sexual relationship with another male.
Bacillary Dysentery
It is an enteric infection that is most commonly caused by S. dysenteriae type 1. 3h15m
It is characterized by acute
inflammatory colitis and bloody diarrhea (blood, mucus, and WBCs
in the stool) due to the attachment of the organisms to mucosal surfaces and formation of ulcers
after epithelial penetration.
It ishighly communicable because of the low infective dose that is required to produce the
disease (< 200 bacilli).
In young children, rectal prolapse occurs due
to the excessive straining.
Source of infection: Human carriers
Modes of transmission:
persons
4Fs, person-to-person contact, and contaminated water from infected
symptoms: Fever, chills, abdominal cramps, painful bowel movement, and

Complications: Ileus (obstruction of the
(HUS) intestine), seizure, and
hemolytic-uremic syndrome
ENTEROBACTERIACEAE (GRAM- NECATIVE BACILLS 279
Remember
Notes to
Poor personal hygiene is an important factor in the transmission

of
Shigella spp.
Shigella causes outbreak in areas where houses are in
centers and among travelers. close proximity, in military camps, in daycare
sensitive
Shigellae are to pH
changes (susceptible to acidic stool) so stool
specimens should
inoculated immediately after collection to increase the recovery of the ougansms'
be
Aside from stool specimens, rectal swabs can be used for the isolation of Shigella.
Versinia
The species are predominantly isolated from the environment such as soil and water.
Human pathogens: Y. pestis, Y. enterocolitica subsp. enterocolitica, and Y. pseudotuberculosis
Yersinia pestis (Plague bacillus)

It is a class A bioterrorism agent.
It is a recognized zoonotic bacterium.

It is not part of the indigenous microbiota of human GIT and is a non-motile enterobacterium.
It is the only enterobacteria that is transmitted to humans through the bite of an infected flea.
It is the causative agent of the bubonic plague. 10016700

It can be isolated on a routine culture media, and it grows best at 25 'C to 30°C. rons
Vector: Xenopsylla cheopis (Oriental rat flea) isw 10

Reservoir: Rats
Virulence factors: Endotoxin, coagulase, and fibrinolysin iris frort botale ad Bant
Microscopy: Gram staining - Short, plump rod

exhibiting "closed safety
Wayson stain or Methylene blue Appear as bipolar bodies
pin"
Culture: MAC _ Colonies are clear and colorless (NLF).
BAP . Colonies are pinpoint at 24 hours.
Broth - Colonies have a "stalactite-shaped" pattern.
IMViC reaction: - +
ISIA reaction: K/A, (-) gas, (-) H,S

Plague
Yersinia pestis, and
it is transmitted
the rodents that is caused by to humans
It is a disease of
through flea bites.

Humans may also acquire it by
ingestion of contaminated animal tissues and inhalation of
contaminated airborne droplets.
the blood and lymph.
Once inside the human body, the bacteria multiply in
Clinical Forms of Plague
a. Bubonic/Glandular Plague
of the vector.
It is the most common form and results from the bite
It is the painful inflammatory swelling of the axilla and groin

onset of high fever and
(buboes) and is usually observed less than a week after exposure.
b. Pulmonary Plague/Respiratory Transmission
acquired by close contact with infected individuals though inhalation
of
It is respiratory
droplets.
It occurs secondarily to bubonic plague.
It is the active multiplication of the bacteria in the blood and respiratory tract. oft
It is the most commonly isolated species of Yersinia, onnluo antiuors no bsteloai edinsa fl
It is the causative agent of enterocolitis or waterborne gastroenteritis.
It is motile in SIM at 25 'C by peritrichous flagella but not at 35°C.
It
has been isolated from contaminated blood bag (packed RBC) units, hence it is considered as a
blood transfusion hazard.
It has the ability to survive in cold temperature ("cold enrichment").

Modes of acquisition: Ingestion of undercooked food (pork and pork intestines, vacuum-packed
meat and
chicken) and dairy products (chocolate milk), and handling pets
Reservoir: Swine, dogs, cats, rabbits, and cows
Related
infections: Appendicitis-like syndrome, arthritis, and erythema nodosum
Selective culture medium: Cefsulodin-irgasan-novobiocin (CIN) agar
Microscopy: Coccobacilli with bipolar bodies
Culture: MAC. Colonies are clear and
colorless (NLF).
CIN - "Bull's-eye" appearance
of colonies or burgundy centers with transparentborders
after incubation at room temperature for 48 hours®
IMViC reaction: v+--
TSIA
reaction: K/A, (-) gas, (4-) H,S
ENTEROEACTERIACEAE (GRAM-NEGATIVE BACILL) 281
Versinia pseudotuberculosis
It is a
pathogen of the rodents, particularly guinea pigs.
It has been isolated
In humans, it
in avian animals such as turkey and pigeons.
causes septicemia with
in animals.
lymphadenitis while pseudotubercles infection is observed
It has similar morphological characteristics with the plague bacillus.

It is motile in SIM at 18°C to 25°C but not at 35°C.
Modes of acquisition: Direct contact with

contaminated food and water
infected animals or their feces, and ingestion of
Reservoir: Birds
Culture: MAC - Colonies are clear and colorless

CIN - Small, "red pin-like"
colonies without transparent borders
IMViC reaction:
TSIA reaction: K/A, (-) gas, (-) H.S

Other biochemical test: (+) Urease and rhamnose fermentation
TABLE 19-9. Differential Tests for Versinia Species d mesolng
Biochemical tests Y. pestis Y. enterocolitica pseudotuberculosis

Indole
Methyl red + +
Voges-Proskauer (25°C)
Motility
+
Urease
Sucrose fermentation
Beta-galactosidase
Plesiomonas
Plesiomonas shigelloides
Plesiomonas.
It is the only species in the genus
It is not part of the indigenous human microbiota and is considered a true pathogen.
from warm- and cold-blooded
it has been isolated
It is found in fresh and estuarine water, and
animals.
BACTERIOLOGY
the species name "shigelloides".

It often cross-agglutinates with Shigella, hence
member of the Enterobacteriaceae.
It is the only oxidase-positive
It is motile by monotrichous or lophotrichous flagella.
it has been isolated from HIV-positive individuals withinflammatory bowel disease.
It isconsidered occupational hazard among veterinarians, fish handlers, and those working
as an
with aquatic sports. sullend st
Mode of acquisition: Ingestion of undercooked seafood (oysters and shrimps) and contaminated
water; they gain entry thru skin cuts
Virulence factor: hemolysins, cytotoxins, production of exoenzymes

Microscopy: Straight bacilli which can occur singly, in pairs, in short chains, or filamentous
Vibriostatic Test 0/129: Sensitive
Related infection: Secretory diarrhea, neonatal meningitis, and septicemia DAM STIED
Cultural Characteristics
a. BAP: Colonies are shiny, opaque, and non-hemolytic.
b. MAC: Colonies are clear and colorless (NLF). Some strains will not grow on MAC.
C. green-bile salt agar: Colonies exhibit white or green to pink color while
Inositol-brilliant
Aeromonas species are colorless. This medium enhances the recovery of plesiomonads from
specimens.
d. HEA: Colonies exhibit growth.
e. TCBS: Colonies do not exhibit growth.
f. Media with NaCI: Colonies do not exhibit growth.
Biochemical and Serological Characteristics

Carbohydrate fermentation test: Glucose, maltose, trehalose
Oxidase test: Positive
Decarboxylase test: Positive trio decarboxylate test

Inositol fermentation: Positive
IMViC reaction: + + _ -
TSIA reaction: K/A, (-)

gas, (-) H,S
Antigenic determinant: Somatic O and flagellar H
Laboratory Diagnosis of Enterobacteriaceae

Specimens: stool, rectal swab, blood, urine,
wound discharge, and CSF
The members of Enterobacteriaceae are commonly isolafed from stool specimens hence,fecal
samples should not be
intestinal pathogens. used for routine testing unless the diagnosisinvolves possible true
Stool specimens submitted
for routine testing should also include culture or toxin detection to
identify EHEC.
ENTEROBACTERIACEAE (GRAM-NEGATIVE BACILL) 283
Enterics that are
highly significant.
isolatedfromsterile sites, such as Salmonellafrom hone marrow aspirates, are
1. Microscopy
The members of Enterobacteriaceze
with rounded ends. are seen as straight Gram-negative rods or coccobacilli
Wayson stain can be used to observe the bipolar bodies of Y. pestis. Top rtal
In the identification of enterics, direct
smears of stool specimens are not routinely performed.
2. Culture
Culture media: BAP, CAP, MAC, HEA, XLD, CIN, Salmonella-Shigella agar (SSA), Eosin
Methylene Blue (EMB), Bismuth Sulfite agar (BSA), and
CHROMagar Orientation Medium
Enrichment Media: Selenite F and GN broth
Transport media: Amies, Cary-Blair, and Stuart media
Colony morphology: BAP and CAP Colonies are large, gray, and smooth.
The optimal temperature for the growth of enterobacteria is between 35°C to 37°C, except
for Serratia and Yersinia (1°C to 5°C), and E. coli (can also grow at 45 'C to 50°C).
Selenite F and GN broths promote the multiplication of Salmonella and Shigella in stool
samples.
Fecal pathogens are generally non-lactose fermenters.
Salmonella species produce colonies with black centers in media with H,S indicators such as
HEA, BSA, and XLD agar.
CIN medium includes neutral red as the pH indicator, and it is selective for the growth of
enterocolitica and Aeromonas since it contains crystal
Gram-negative bacteria especially Y.
violet and bile salts.
Colorless colonies on media designed for enterobacteria mostly suggest that

the colonies have
same color with the culture medium. bns stalluevth mulbnd holmabnteh
Plated and tube media should be read within 18 hours to 24 hours of incubation to avoid false
results.
Differential and Selective Media for the Isolation of Enterobacteria nothe

a. MacConkey-Sorbitol agar (SMAC)
negative) from other strains of
(sorbitol fermentation
It differentiates E. coli 0157:H7
E. coli (sorbitol fermentation positive).

MAC except lactose was replaced by D-sorbitol
Main composition: Same with
pH indicator: Neutral red
colonies while the other strains have the
exhibits colorless
Result: E. coli 0157:H7
common pink colonies.

CHROM Agar 0157 increase the recovery of
and tellurite and
SMAC with cefixime
EHEC from stool specimens.

dftixime-tellurite (SMAC CT) helps in the identitieatim end
isolation of
imie non-verocytotoxigenic and non-sorbitol termenter strains

SMAC with of
E. coli 0157:H7,
E. coli are inhibited.
IN DIAGNOSTIC
BACTERIOLOGY
284 REVIEW HANDBOOK
Hektoen enteric agar (HEA)

isolation of Salmonella and Shigella from food, water, and dairy products.
b.
It supports the other Gram-negative

of Gram-positive and
The bile salts and dye inhibit the growth
bacilli in the GIT.
acid fuchsin
Major composition: Lactose, sucrose, salicin, and
Inhibitory agent: Bile salts
pH indicator: Bromothymol blue
H,S indicator: Ferric ammonium citrate
Growth on HEA
colonies (with black centers for slow lactose
LF: Yellow-orange colonies or pink to orange
fermenting C. freundii)
and Salmonella species)
NLF: *Colorless colonies (with black centers for Proteus
Other coliforms: Colonies exhibit orange color.
Uninoculated HEA: Green color
of the uninoculated medium.
*It means green or blue-green colonies, same color
C. Xylose-lysine deoxycholate (XLD) agar
It identifies the deamination of lysine by bacteria.
Both HE agar and XLD agar are useful for the

isolation of Salmonella and Shigella species
such as stool.
from heavily contaminated samples
The uninoculated XLD is red in color and though colonies appear also red or deep pink
after incubation, they are sometimes reported as "colorless"
Major composition: Lactose, sucrose, xylose, lysine, and sodium deoxycholate

Inhibitory agent: Sodium deoxycholate
H,S indicators: Sodium thiosulfate and ferric ammonium sulfate roloo omea
Growth on XLD agar
Shigella: Colonies are colorless without black centers.
Salmonella: Colonies are colorless with black centers. bals bassbinewing
Eosin methylene blue (EMB)

It is basically utilized for the differentiation and isolation of E. coli and Enterobacter.
Major composition: Eosin Y, methylene blue, lactose, and sucrose

Inhibit Gram-positive bacteria: Eosin Y and
pH indicator: Eosin and methylene blue winks Tero 110g
modification of EMB is
the Levine's medium with increased concentration of lactose
A
and contains no sucrose.
Growth on EMB agar
LF - E. coli: Greenish metallic sheen

LF - Klebsiella and
Enterohacter: Dark purple colonies with mucoid consistency
ENTEROEACTERIACEAE (GRAM-NEGATIVE BACILLI) 285
pink colonies
Other bacteria: Colorless colonies

e. Salmonella-Shigella agar (SSA)
It is both
differential and sclective for Salmonella and Shigella species.
It inhibits growth
in stool
specimens.
of Gram-positive bacteria and some lactose fermenters that are found
Carbohydrate source: Lactose

Inhibitory agents: Bile salts, brilliant
green dye, and sodium citrate
pH indicator: Neutral red
H,S indicator: Sodium thiosulfate and ferric ammonium citrate

Growth on SSA
Salmonella: Colonies are colorless with black centers.

Shigella: Colorless colonies without black centers.
Other coliforms: Colorless colonies (with mucoid for
encapsulated bacteria)
f. CHROMagar Orientation
It is utilized for isolation and differentiation of urinary pathogens such as E. coli and
enterococci.
It is a nonselective medium which contains chromopeptone and chromogens the
artificial substrates (chromogens) release differentiated colored compounds after
degradation by specific microbial enzymes.

It allows isolation of the agents of urinary infection such as S. saprophyticus and
S. agalactiae strains, and the Klebiella-Enterobacte-Serratia (KES) and Proteus-Providencia-
Morganella (PPM) groups.
CHROMagar Salmonella medium produce mauve-colored colonies of Salmonella.

Growth on CHROMagar
pink transparent colonies with or without halos
E. coli: Dark rose to
KES: Blue to dark blue colonies with or without violet halos

PPM: Pale to beige colonies surrounded by brown halos
Enterococcus: Small blue-green colonies
to blue pinpoint colonies with or without halos
S. agalactiae: Light blue-green
Other organisms (including yeasts): Colonies exhibit a cream-colored pigmentation
Plated Media, PA-257481.04, May 2019
Source: BD Instructions For Use Ready-To-Use
3.
(Slide agglutination test)
Immunodiagnosis: Serotyping or
It is a serological test that
identifies strains (serovars or serotypes) of microorganisms that
differ in their antigenic composition.
antigen) of Salmonella and Shigella species and
the
It determines the O serogroups (somatic
strains.
of enterovirulent E. coli
on the surface of bacteria.
Antibodies are utilized to detect specific antigens
Preferred medium for isolation
of colonies: Sheep blood agar (SBA)
BACTERIOLOGY
0157:H7
Commonly tested organisms®

Salmonella,Shigella,and E. coli
to G and Vi antigens
(reagents): Polyvalent
A
Antisera and G sllogine pilsnomise
E,
C1, C2, D,
Serogroups A, B. pure bacterial suspension prepared
Procedure (Reverse Passive Agelatination) A dropadded
ofa with a drop of antisera.
then
in sterile saline is placed on a clean slide and
Positive result: Agglutination
a. Salmonella and Shigella serotyping

and H antigens while Shigella serotyping is based
Salmonella serotyping identifies the O
only on the somatic O antigen.
Salmonella serotype Typhi also produces heat-labile capsular polysaccharide
Vi antigen
and carries the D serogroup.

from humans belong to serogroups A to E1 while Shigella
Most of the Salmonella isolates
has A to D serogroups. bald toorDw
b. E. coli serotyping
Sorbitol-negative E. coli can be serotyped to identify whether the somatic antigen

0157
and the flagellar H antigen are present.

when grown in SIM to enhance the
The H antigen of E. coli 0157 is detected better
production of flagella.
E. coli 055, O111, and 0127 serotypes are most frequently
associated with infantile
diarrhea.
brus 4. Antimicrobial Susceptibility Test

The exchange of susceptibility and resistance of the Enterobacteriaceae to antibiotics is one of
the focal points of diagnostic medicine for many years.
The presence of p-lactamase genes like the blaTEM, blaSHV, and blaCTX-M is the subject of
studies to assist the clinicians in the treatment of patients.
Extended-spectrum f-lactamases (ESBLs) and Ampc genes are etiologies of the resistant
strains of E. coli, K.
pneumoniae, P. mirabilis, and other enterobacteria to third-generation
cephalosporins and all
B-lactam antibiotics except carbapenems.
An antibiotic-resistant gene known as blaKPC secretes that inactivate all
carbapenems, anditwas identifiedfromthe isolates of K. pneumoniae.
TOUTES
Carbapenem-resistant Enterobacteriaceae (CRE) is considered as an urgent concern by the
CDC especially
in healthcare settings
since it is often life-threatening.
CRE is characterized by the
presence of carbapenemase causing resistance to imipenem,
meropenem, doripenem, or isolate
any documentation showing that the
possesses I carbapenemase. tatian
911 5.11 Molecular Diagnosis

It is used
o enhance the recovery and identification of enterc bacteria.1013
It involves the
use of DNAprobesandnucleicacid amplification.
ENTEROBACTERIACEAE IC
Multiplex PCR assays such as the

up to 20 organisms in one hour. FilmArray (BioMiérieux) can detect several pathogens, like
t reclassifies the genus Plesiomonas from the group of non-enteric cf pathogens to the family
Enterobacteriaceae
16S ribosomal RNA

sequencing and
determination of
enteric bacteria. I coh
DNA-DNA hybridization are utilized for the
The Xpert Carba-R Assay, which is

a real time PCR, detects several genes coding for
carbapenemases.
Biochemical Test of Enterobacteriaceae

In this
assay, various culture media and reagents ares used for the differentiation and
identification of enterobacteria.
1. Fermentation of sugar - Triple sugar iron agar (TSIA) test

The fermentation of sugar is usually detected by acid production that yields a change of color
in the culture medium.
It is used todetermine whether a Gram-negative rod utilizes glucose and/or lactose

fermentatively or has no reaction on carbohydrate utilization test.
It also identifies bacteria that can produce gas and hydrogen sulfide (H.S). and
nuthat Enzymes essential for enterobacteria to take up lactose and convert it to monosaccharides:
a. Beta-galactoside permease . facilitates the entry of lactose molecule through the bacterial cell
wall
b. Beta-galactosidase - hydrolyzes lactose into glucose and galactose

Characteristics of TSIA
H,S indicators: Ferrous sulfate and sodium thiosulfate

Inoculation technique: Stab and Streak
and it has both slant and butt portion.
Uninoculated TSIA (red color medium) is at pH 7.4,
The ratio of sugars in TSIA is 10:10:1 (1% lactose, 1%sucrose, and 0.1%glucose, respectively).
of incubation because the aerobic
TSIA reactions should not be read beyond 24 hours
oxidation of the fermentation products from lactose and/or sucrose will revert the slant to an
alkaline state.
TSIA Reactions
No fermentation of sugars
Result:. Alkaline slant/Alkaline butt (K/ K or Red/Red)

this reaction are
not members of the Enterobacteriaceae.
Organisms with
b. No lactose and sucrose fermentation (Glucose fermenter)
Red/Yellow)
Result: Alkaline slant/Acid butt (K/A or
concentration is depleted forming acid end products in the butt
In this reaction, glucose
medium.
portion of the agar
BACTERIOLOGY
organism has utilized the peptonesin

to 24 hours of incubation, change of the slant
After completing
the slant,
to red color. Edwardsiella, Morganella, Proteus,
fermenters:
Glucose/Nonlactose
Providencia, Salmonella, Shigella, and Versinia
C. Lactose,
stcrose, and glucose fermentation (Lactose Fermenter
or Yellow/Yellow)
Result: Acid slant/Acid butt (A/A
first and then the disaccharides
The enterobacteria attack the simple sugar (glucose)
(lactose and sucrose).
acid production in the entire TSIA, and the color of
All sugars are fermented resulting in
slant and butt is changed to yellow.
Escherichia coli, Enterobacter, and Klebsiella
Rapid lactose fermenters:
and Serratia
Late lactose fermenters: Citrobacter freundii, Shigella sonnei,
d. Hydrogen sulfide (H,S) production
ferrous sulfate
H,S indicators: Sodium thiosulfate and
() Result:. Formation of black precipitate in the medium pieb
H,S production requires an acidic environment.
e. Gas production
(+) Result: Formation of bubbles (CO, and H.),splitting of the medium in the butt portion
of the tube, or complete displacement of the medium
Quality Control
A/A + gas: Escherichia coli ATCC 25922 mungninial posviowb
K/A + H,S: Proteus mirabilis ATCC 12453

K/A+/-gas H,S: Salmonella serotype Typhimurium ATCC 14028
K/A: Shigella flexneri ATCC 12022
K/K: Pseudomonas aeruginosa ATCC 27853
Interpretations of the TSIA

Bacterial species that are incapable of fermenting glucose cannot utilize lactose.
Lactose fermenters (LFs) secrete both beta-galactoside permease and beta-galactosidase.

Late lactosefermenters
(LLFs) only possess beta-galactosidase.
Glucose and lactose fermenters are
mostly opportunistic enterics.
True enteric pathogens only ferment glucose.
TABLE 19-10. Summary of TSIA

A/A, (+) G, (-) H,S
Reactions of Enterobacteriaceae
E. coli
KA, (+) G, (+)H,S K/A, (-) G, (-)H,S
E. tarda
Enterobacter
M. morganii
C. freundii Providencia
Serratia
Shigella
Salmonella
Note: C. freundii A/A or K/A, (+) gas, (+)HS
Serratia - A/ A or K/A, (+) gas,
(-) H,S
P. vulgaris K/A, (+/-) gas, (+) H,S
S. serotype Typhi - K/A, (-) gas, (+) H,S
2. Sulfide indole motility (SIM) test

Culture
medium: SIM medium (butt tube)
Inoculation Technique: Stab the medium once in the center.
Incubation: 18 hours to 24 hours at 35°C
Indole (Kovac's) Test Reagent: Dimethylamine-benzaldehyde and hydrochloride

Procedure for Indole Test: Add 3 to 5 drops of the Kovac's reagent into SIM after overnight
incubation.
(+) Sulfide: Black color formation

(+) Indole: Presence of pink to "wine-colored" ring after the addition of Kovac's reagent
(+) Motility: Movement away from the stab line creating a hazy appearance
Quality Control
Sulfide Positive: Proteus mirabilis ATCC 12453

Indole (Kovac's) Test - Positive: Escherichia coli ATCC 25922

Hod bivA Vimslatbat
Negative: Klebsiella pneumoniae ATCC 13883
Motility = Positive: Escherichia coli ATCC 25922

Negative: Staphylococcus aureus ATCC 25923
3. Citrate utilization test
It determines the ability of an organism to utilize sodium citrate as its only carbon source
nitrogen source.
and inorganic ammonium salts as its only
If citrate is used as the carbon source, the culture medium will turn alkaline (ammonia and
ammonium hydroxide).
color)
Culture medium: Simmons citrate agar slant (green

(+) Result: Blue-colored slant
Klebsiella and Enterobacter utilize citrate.
slant) in this test.
Escherichia and Edwardsiella have a negative reaction (green
DIAGNOSTIC
BACTERIOLOGY
color slant)
Quality Control
ATCC 13048 (with growth, blue
Positive: Enterobacter aerogenes growth, no color change
(no
on the slant)
coli ATCC 25922
Negative: Escherichia
Lysine iron agar (LIA) test decarboxylates or deaminates lysine.

ne rond to determihe whether a Gram negative rod Providencia since they deaminate amino
4.
Proteus, Morganella, and

B
It aids in the identification of

acids.
and Butt)
Culture Medium: LIA (Slant
Streak
Inoculation Technique: Stab and
Major components
sf LIA: Lysine, peptones, and 0.1% glucose
and sodium thiosulfate
H.S indicator: Ferric ammonium citrate
PH indicator: Bromcresol purple
Quality Control
K/K: Escherichia coli ATCC 25922
K/K with H,S: Salmonella serotype Typhimurium ATCC 14028
R/A: Proteus mirabilis ATCC 12453
TABLE 19-11. Lysine Iron Agar Test

Interpretation Related Bacteria
LIA Reaction
Salmonella
Alkaline slant/Alkaline (-) Lysine deamination and
butt (K/K) (+) Lysine decarboxylation
Alkaline slant/ Acid butt (-) Lysine deamination and Shigella and
(K/A) (-) Lysine decarboxylation
Red slant/Acid butt (+) Lysine deamination and Proteus, Providencia, and
(R/A) (-) Lysine decarboxylation Morganella
ALOES DOTA
Interpretations of LIA reactions
If the organism secretes lysine decarboxylase, cadaverine (purple) is formed.

9811102
If the decarboxylase is not produced by the organisms, the butt remains acidic(yellowcolor)
006600 If deamination of lysine occurs, it will form a burgundy (red) color on the slant.
In the absence of
the deamination, the LIA slant
remains purple in color.
fermented, the butt turns yellow due to acid formation.
If glucose is
Production of hydrogen sulfide is observed in Salmonella and Serratia species.

5. Decarboxylase test (Moeller's method)
decarboxylate (hydrolyze/remove/
the enzymatic ability of an organism to
It measures
amino acid to form an amine (putrescine or cadaverine in an anaerobic environment.

ENTEROBACTERIACEAE (GRAM-NEGATIVE BACILLI) 291
t detects the bacterial secretion of decarboxylase.

Moeller decarboxylase base
medium: Glucose and amino acid (3
lysine, and ornithine) tubes each with arginine,
Commonly usedamino acid for
decarboxylation: Lysine and ornithine
Promote anaerobiosis: 4 mm Mineral oil (on top of the
medium)
pH indicators: Bromcresol purple and cresol red
Uninoculated medium: Yellow color
Incubation condition: 4 days at 35°C to 37°C
(+) Result: Purple color (alkaline end product)
Quality Control
Positive - Arginine: Pseudomonas

Lysine: Klebsiella pneumoniae ATCC 33495
Ornithine: Enterobacter aerogenes ATCC 13048
Negative - Arginine: Escherichia coli ATCC 25922

Lysine: Citrobacter freundii ATCC 8090
Ornithine: Proteus vulgaris ATCC 6380
Glucose Fermenters: Klebsiella pneumoniae ATCC 27736
Enterobacter aerogenes ATCC 13048
Interpretation of Results
Enterobacter species have a positive reaction to the ornithine decarboxylase test while
Klebsiella is negative.
Glucose fermentation results in a yellow color.
6. Ortho-nitrophenyl-B-D-galactopyranoside (ONPG) test

It identifies slow or late lactose fermenters.
It differentiates organisms which can produce p-galactosidase.
Beta-galactosidase acts on ONPG, a colorless compound, and then cleaves it into

galactose
and ortho-nitrophenol.
to 6 hours at 35°C)
(+) result: Yellow color as early as 20 minutes (incubation
is hour
(-) result: Colorless (Non-lactose fermenters)

Quality Control
Positive: Shigella sonnei ATCC 9290
Negative: Salmonella serotype Typhimurium ATCC 14028
7.
Methyl Red/Voges-Proskauer (MRVP) test and maintain stable acid end products
It determines the ability of an organism to produce
(pyruvic acid) from glucose
fermentation.
Culture medium for MR test:
MRVP broth/Peptone glucose broth
KOH
VP reagents: 1% a-naphthol and 40%
BACTERIOLOGY
pH indicator: Methylred
Incubation of MRVP Broth:
days to 5 days at 352C
less
color at pH 4.4 or
MR test () result: Bright red
Red color
VP/Barritt's test (+) result:
MRVP é result: yellow color reaction to VP.
negative
has a positive reaction to MR and
E. coli
with Hafnia group has a positive reaction to VP.
The Klebsiella-Enterobacter-Serratia-Fidr
color reaction is yellow, which indicates a negative
If the medium turns
alkaline, the end
result.
Quality Control
ATCC 25922
MR Positive/VP Negative: Escherichia coli
MR Negative/VP Fositive; Enterobacter aengeres ATCC 13045
8. Nitrate reduction test
to reduce nitrate to nitrite.
It determines the ability of an organism
other organisms.
It is used to detect the nitrate reductase production of enterobacteria and
the reaction will not occur in the
anaerobic condition since
It is best performed in an
presence of oxygen.
Zinc dust is added to confirm a negative reaction.

Reagents: Sulfanilic acid and alpha-naphthylamine
(+) Result: Red
(-) Result: Colorless
Quality Control
Positive: Positive: Proteus mirabilis ATCC 12453

Kingella denitrificans CDC 10,236
Negative: Acinetobacter baumannii ATCC 19606 dout
Neiserria gonorrheae ATCC 43069
9. Phenylalanine deaminase (PAD) test

It helps in the initial differentiation of Proteus, Morganella, and Providencia (PPM).
Itdetermines the ability of an organism to oxidatively deaminate phenylalanine to phery
pyruvic acid.
The PPM group is the only PAD-positive genera in the list of Enterobacteriaceae.
Culture Medium: Agar with 0.2% phenylalanine

Confirmatory reagent: 10% ferric chloride
(t) Result: Green-colored complex on the slant
Quality Control
Positive: Proteus mirabilis

ENTEROBACTERIACEAE (CRAM-NEGATIVE BACILLI 293
10. Urea hydrolysis/Urease

test (Christensen's method)
It detects the ability of an organism to
secrete urease which hydrolyzes urea.
Culture medium: Urea slant (light orange color)
ST
pH indicator: phenol red

End product: Ammonia and
carbon dioxide (alkaline product) IT
(+) Result: Orange to magenta colored slant
(-) Result: No color change or yellow color which indicates acid production
Rapid urease producers: Proteus and Morganella
Other Urease-positive enterobacteria: Serratia, Citrobacter, and Yersinia
Weakly-positive: K. pneumoniae (delayed urease positive, ASM urease test protocol, 2010)
Quality Control
Positive: Proteus vulgaris ATCC 13315
11. IMViC (Indole, Methyl red, Voges-Proskauer, and Citrate) test

The lysis of the substrate tryptophan by the bacterial enzyme forms indole.
Methyl red is pH indicator that detects acid production in the organisms.
The Voges-Proskauer test detects the formation of acetoin.
The utilization of citrate in the medium as the sole source of carbon is observed.
IMViC reaction
E. coli, E. tarda and M. morganii: +
K. pneumoniae, Enterobacter, Serratia:

S. enterica: - +-+
12. Gelatin Liquefaction Test

which hydrolyzes and liquefies the
It determines the production of gelatinase (protease)
gelatin in the medium.
added into the medium instead of an
Culture medium: Nutrient gelatin tube (gelatin was
agar)
culture broth into the gelatin tube (butt/deep)
Procedure: Add four to five drops of pure
and incubate for up to 14 days at 35°C to 37°C or at 25'C (if the organism prefers a lower
temperature) to achieve a positive reaction. Daily monitoring of the

tube at 4°C is required to
validate liquefaction of the medium.
(+) Result: Liquefied gel
(-) Result: Solid gel

Serratia and Proteus show a positive reaction.
Quality Control
Positive: Bacillus subtilis ATCC 9372
Negative: Escherichia coli

ATCC 25922
294 REVIEW HANDBOOK IN DIAGNOSTIC BACTERIOLO0Y
its
n (fetatetn
13. Malonate Test
enties the abliy of the organism to uilizels
sodium malonate as s soe soma y
bic eta
carbon
laid ea
Culture Medium: Malonate broth
aho1 t
pH indicator: Bromothymol blue hiol nuarelnan
(+) Result: Blue color at pH 76tbeeio danunt t
(-) Result: Green colormluiaertewoongutntne
Enterobacter and Salmonella both exhibit a positive result.eabetiq
Pegcl sh
TABLE 19-12. Summary óf Biochemical Reactions of Enterobacteriaceae
H,S|Indole MR |VP |Citrate PAD| Urease
(0tI1ty
ať 35C
LDC
Escherichia coli A/A t(v)
Enterobacter aerogenes A/A++

Enterobacter cloa A/A++
Klebsiella pneumoniae A/A
A/A
Klebsiella oxytoc
Serratia marcescens
Proteus mirabilis K/A
Proteus vulgaris K/A
Providencia rettgeri K/A

Providencia stuartii K|A
Morganella morganii K/A
A/A -(v)
(v)
Citrobacter freundii K/A
Citrobacter koseri K|A +(v)
Salmonella serotype K/A
Typhi
Shigella dysenteriae K|A
Shigella sonnei K/A
Hafnia alvei K|A

Edwardsiella tarda K|A
Yersinia enterocolitico K|A
Plesiomonas shigelloidesK|A
Cronobacter sakazakii A/A (v)
Pantoea agglon K|A-(v)

* LDC - Lysine Decarboxylase t
ju 1Eo
V - variability is either positive or negative
+(v) - greater probability for positive reaction E re
(v) - greater probability for negative reaction
Kligler Iron Agar (KIAN/Double Sugar.Iron Agar os ie

It has the same components as those of TSIA except sucrose.da n
It contains ten times more laciose than glacose as compared with ISIArt
thas the sme reacion as TSIA, hence, it is an alerate medim for the cartelulth
fermentation test of enterobacteria.
Sorbitol Macconkey Agar
B
FIGURE 19-1. SMAC (A) E. coli ATCC 25922 and (B) E. coli 0157:H7
Source; https://microbenotes.com/sorbitol-macconkey-agar/
FIGURE 19-2. Colonies of Escherichia coli on Hektoen enteric agar plate

(Photo from USCDCP)
sheen on EMB agar

greenish metallic
FIGURE 19-3. Escherichia coli with.blue-emb-agar-composition-usearolony
composition-uses-colony-characteristics/
characte
Source: https://microbeontine.com/eosin-methylene-blue-e
BACTERIOLOGY
E. Coli
E. Coli 0157:87
of CC disk under UV (365.nm) light.

FIGURE 19-4. MUG test showing the appearance
E coli 0157.H17 is MUG-negative with no fluorescence white other E. coliare MUG-positive and with
fluorescence.
K. Jinneman)
(Photo by P. Feng, S. D. Weagant, &
FIGURE 19-5. Mucoid colonies of Klebsiella pneumoniae on MAC

(Photo from JJMMC)
FIGURE 19-6.
serratia marcescens on MAC showing red-pigmented colonies
(Photo from
College of Computer, Mathematical & Natural Sciences)
Shigella flexneri Salmonella typhimurium
A B
FIGURE 19-7. Growth on HEA. (A) Non Lactose Fermenters,

(B) Escherichia coli
Source: http://www.medical-labs.net/hektoen-enteric-agar-2204/
enteric agar
FIGURE 19-8. Salmonella serotype Typhimurium on Hektoen
(Photo from USCDCP)
enteric agar
FIGURE 19-9. Shigella
boydii on Hektoen
(Photo from USCDCP)
Salmonella on XLD
Shigella on XLD
limare Source. Faculty of Health and Medical Sciences - University of Copenhagen, Denmark
FIGURE 19-10. Growth of Shigella and Salmonella on XLD agar
characteristics/
FIGURE 19-11. Salmonella (right) and Shigella (left) on SSA

Source: https://www.fishersci.com/shop/products/remel-salmonella-shigella-agar-ss-agar/p-4523398
FIGURE 19-12. Differentiated Growth of Bacteria on CHROMagar Orientation

Source: http://www.chromagar.com/clinical-microbiolnovs
ENTEROBACTERIACEAE (GRAM-NEGATIVE BACILL) 299
FIGURE 19-13. Yersinia enterocolitica bull's eye colonies

Photo by J.S. Virdi)
FIGURE 19-14. Bipolar bodies of Yersinia pest

(Photo from USCDCP)
inanuotn n
ssti
in e
l e
FIGURE 19-15. Yersinia pestis on bloód agar plate

(Photo by P. T. Seidel, A. Moore, T. Parker, &A. Marsh)
tzlp
t
FIGURE 19-16. Yersinia pestis on chocolate agar plate

(Photo from P. Seidel, A. Moore, T. Parker, & A. Marsh)
FIGURE 19-17. Uninoculated tubes [from the left] with triple sugar iron agar, sulfide indole motility medium,
lysine iron agar repectively for the biochemical test of Enterobacteriaceae
citrate slant, and
FIGURE 19-18. Biochemical reactions in triple sugar iron agar: Left tube, A/A with production; Right,tube
gas
K(/A with hydrogen sulfide [H,S]
ENTEROBACTERIACEAE(GRAM-NECATIVE BACILLI) 301
FIGURE 19-19. Uninoculated sulfide indole motility inedium left]

and positive H,5 test with black precipitate fright]
FIGURE 19-20. Sulfide indole motility medium. Right tube is positive for indole and
motility and both tubes are negative for H,S.
indole
SIM
sulfide indole motility medium [left]

FIGURE 19-21. Reaction in
and an uninoculated medium Iright)
(Photo by 1. Reynolds)
FIGURE 19-22. Positive [blue color] and
negative [green color]
biochemical reactions in citrate agar slant
FIGURE 19-23. Positive lysine decarbonylation [purple slantI reaction in lysine iron aear
negative positive
methyl red methyl red
FIGURE 19-24.
Positive [left and negative Irightl methyl red test results
(Photo from Los
Angeles City College)
ENTERO ACTERIACEAE (GRAM-NEGATiVE BACIL) 303
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BACTERIOLOGY
ASSESSMENT QUIZ

1. This Mochemical test differentiates Enterobacter cloacae from Enterobncter aeroge.
a. lysine decarboxylase
b. urease
c. sorbitol fermentation
d. a and b
urgent concern by the CDC since it has been

2. This life-threatening strain is considered
as an
isolated from healthcare settings.

a. CRAB
b.
C.
d. CRE
3. Which significant species of genera does indole test distinguish?

a. Klebsiella and Enterobacter
b. Proteus and Citrobacter
C. Morganella and Providencia

d. Shigella and Salmonella
4. The presence of "rose spots" on the abdomen during the second week of fever is a hallmark of
infection associated with these enterics.
a. Shigella
b. Yersinia
c. Salmonella
d. Proteus
5. It detects production of B-glucuronidase and separates the strains of Escherichia coli.

a. S-MAC test
b. MUG test
C. Sereny test
6.
What are the positive quality control organisms for nitrate reduction test?
gonorrheae ATCC 43069
b.
Klebsiella pneumoniae ATCC 33495 and Enterobacteraerogenes ATCC 13048
C.
Proteus mirabilis ATCC 12453 and Kingella
denitrificans CDC 10,236
d. Bacillus subtilis ATCC 9372 and
Escherichia coli ATCC 25922
ENTEROBACTERIACEAE (GRAM-NECATIVE BACILLI) 305
7.
I is a nonselective medium that distinguishes the entertc urinary pathogen.
a. CHROMagar Orientation
b. CAP with sheep blood
C. BAP
d. CIN
It is the only member of the

8.
Enterobacteriaceae that is positive to all the three decarboxylase tests.
a. Plesiomonas shigelloides
b. Cronobacter sakazakii
C. Pantoea agglomerans
d Klebsiella ornithinolytica
9. Which of the following have the same IMViC reaction?

a. Escherichia and Enterobacter
b. Salmonella serotype Typhi and Yersinia
c. Serratia and Morganella

d. a and b
10. Identify the organism after phenotypic testing.

Lab test results:
TSI: Glucose fermenter, no gas and hydrogen sulfide

Indole: Negative
Urease: Positive
Motility: Positive
ONPG: Positives
Gelatinase: Positive
PAD: negative
a. Edwardsiella tarda
b. Morganella morganii
C. Serratia marcescens
d. Enterobacter cloacae
CHAPTER 20
NON-ENTERIC
GASTROINTESTINAL
PATHOGENS

compare the characteristics of the genera that are
1.
included in this
group;
2. describe the serologic groupings and pathogenecity of Vibrio cholerae;
3. contrast the colony patterns and biochemical reactions of Vibrio
species;
4. discuss the clinical significance of the genus Aeromonas;
5. distinguish the characteristics of Campylobacter from Helicobacter;
6. differentiate the culture media that support the growth of the genera
and species; and
7. create an algorithm of diagnostic tests to identify the significant genera

in the group of gastrointestinal pathogens.
Vibrio
Vibrionaceae.
It belongs to the family
The members are not part of the indigenous human microbiota. ort 10 prutmb innesd i
The species are glucose fermenters, facultatively anaerobic, and

exhibit peritrichous when
monotrichous organisms though some may
suspended in a broth medium.
or salt water and
They are found in brackish water, marine water
even in recreational facilities like pools.
fish, and shellfish.

They can be isolated from algae, plankton,
cholerae and V. mimicus.
The species are halophilic except V.
or undercooked seafood,
Mode of acquisition: Consumption of raw
with shell fish, and ingestion
of
skin trauma due to contact
contaminated water
wound infection,
Cholera,
Related diseases and infections:
septicemia, and necrotizing fasciitis

Common isolates: V. cholerae O1 and non-O1, V. parahaemolyticus, V. vulnificus, and V. alginolyticus

rod ("comma-shaped' bacillus)
Microscopy: Gram-negative, short, and curved
with a greenish
luminous color; may exhibit a- Or
Culture: BAP Colonies are smooth, opaque
B-hemolysis.
MAC - Colonies are colorless or NLF except V. vulnificus.
MHA
Susceptibility test: 150-ug vibriostatic 0/129 disk in
reduction except V. metschnikovii
Biochemical tests: (+) catalase, oxidase, and nitrate
(+) gelatin liquefaction
Motility test: Broth polar-sheated flagella

Solid media - peritrichous, unsheated flagella
TABLE 20-1. Groups of Vibrio Species

Vibrio Species Distinguishing Features
Group 1: V. cholerae and V. mimicus the only group with positive growth in culture media with
0% NaCl and the only group strongly positive with both
lysine and ornithine decarboxylase test
Group 2: V. metschnikovii the only strong positive reaction is the growth in media
with 1% NaCl to 6% NaCI.
Group 3: V. cincinnatiensis the only species with strong positive reaction in Myo-
inositol fermentation
Group 4: Grimontia hollisae (formerly V. hollisae) Negative Decarboxylase and dihydrolase tests
Group 5: V. fluvialis, V. furnisii, and the only group strongly positive with arginine dihydrolase
Photobacterium damsela (formerly V. damsela) test
Group 6: V. alginolyticus, V. paranemolyticus the only group strongly positive with lysine decarboxylase
V. vulnificus, and V. harveyi test
Source: Henry's Clinical Diagnosis by Lab Management, Mc Pherson and Pincus, 23rd ed., 2017
Vibrio cholerae
It is the causative agent of cholera.
It has a rapid darting or "shooting-star" motility (single, sheathed polar flagellum).

Culture: BAP Colonies are smooth and medium- to
large-sized with a greenish hue.
Principal virulence factor: Choleragen (cholera enterotoxin)
Potent enterotoxins: Zot toxin and ace toxin
Antigenic structures: Somatic O and flagellar H
String test: Positive (exhibits a "stringing" reaction)
Biochemical tests: (+) Indole, citrate, and lysine and ornithine decarboxylase
V. cholerae serogroups:
V. cholerae O1, V. cholerae non-O1, and V. cholerae 0139
V. cholerae O1
serotypes: Ogawa (A, B), Inaba (A, C), and Hikojima (A, B, C)
V. cholerae O1 biotype: Classic and El Tor
NON-ENTERIC GASTROINTESTINAL PATHOGENS 309
Agents of epidemiccholera: V. cholerae O1 and V. cholerae

O139
Common cause of epidemic cholera: V. cholerae O1
bi enun neernante-ad.a•egluti
ns iresinatend/chicken
Classical: VP (-); does not oat hiRUCy
matisuscepi
s.comtbletopolymibkin b (8019
b. El Tor: VP
(h agglutinate chicken RECy and resistant to polymixin B
Cholera
It is an
acute diarrheal infection that is mainly spread through contaminated water sources.
It is
acquired from ingesting improperly preserved food like shelfish and dairy products
milk, and ice cream.
such as
It is also acquired via fecal-oral route.

Hallmark of cholera: Rice-watery stool (10 to 30 times of defecation per day) Matho
V. cholerae O1 is the
common cause of the epidemic cholera while the non-O1 strain causes
gastroenteritis.
Choleragen
It is protein enterotoxin.
It is mainly produced by V. cholorae OI strain.
It stimulates the hypersecretion of water and chloride ions and inhibits sodium ion absorption,
and decreased plasma concentration of
which results in a fluid loss of 10 liters to 15 liters daily
electrolytes.
It binds with the adenylate cycles of cells in the small intestine causing over secretion of water
and electrolytes.
All antigens of this toxin are poorly immunogenic leading to recurring infections.
Vibrio parahaemolyticus
It is the second most common Vibrio species that is associated with gastroenteritis.
It is the etiologic agent of "summer diarrhea."
Leading cause of pandemic: Vibrio parahaemolyticus serotype O3:K6
Virulence factor: Heat-stable hemolysin
Pathogenicity: Hemolysin lyses human red blood cells

Selective medium! Wagatsuma agar (high-salt mannitol medium)
to the presence of lateral flagella (same with V. alginolyticus).
Culture: It may exhibit swarming due
Vibrio vulnificus
It is known as the "lactose-positive Vibrio species.
infections.
It is second to V. cholerae as the cause of severe Vibrio-associated
Related infections: Septicemia and wound infection
Vibrio alginolyticus
species.
It is the least pathogenic to humans among the major Vibrio
of 1% to 10% NaCl in the culture media.
It is a strict halophile that requires the addition
Related infections: Eye, ear,
and wound infections (extraintestinal pathogen)
Specimens: Stool, rectal swab, pus, and tissue
in Cary-Blair medium.
Vibrio species should be collected and transported only
1. Microscopy
curved (common-shaped) rods
Gram-negative and straight or slightly
2. Culture Media
Culture media: Thiosulfate-citrate-bile salts-sucrose agar(TCBS),MAC, BAP,and CHROMagal

Transport medium: Cary-Blair medium
Enrichment medium for V. cholerae: Alkaline peptone water (pH 8.4)
The inoculation of stool samples into alkaline peptone water and incubation of the broth for
5 hours to 8 hours at 35 PC promotes the multiplication of vibrios prior to subculture in TCBS.
The media utilized for the isolation of the Vibrio species usually contain sodium chloride to
support the growth requirements.
For best isolation of vibrios, culture media should contain 0.5% NaCI except V. cholerae and
V. mimicus.
V. alginolyticus tolerates up to 10% NaCI.

Pathogenic Vibrio species grow as non-lactose fermenters on MAC except V. vulnificus.
CHROMagar Vibrio, though not routinely used in the clinical laboratories, can identify
V. cholera, V. parahemolyticus, and V. vulnificus in food and water samples.
Colonial growth in TCBS agar
Sucrose fermenters (yellow colonies on TCBS): V. cholerae, V. alginolyticus, V. metschnikovi,

V. fluvialis, V. furnissii, and V. cincinnatiensis
Non-sucrose fermenters (green colonies on TCBS): V. mimicus, V. vulnificus, and

V. parahaemolyticus
pH indicator: Thymol blue and parathymol blue
TCBS inhibits Gram-positive bacteria.
3. Biochemical Test
TSIA
reaction: A/A, (-) gas, (-) H,S
LIA reaction: K/K
(+) Citrate Test: V. cholerae

(+) Indole Test: V. cholerae, V. mimicus,

V. parahemolyticus,
and V. vulnificus
(+) Lysine decarboxylase: V. cholerae, V.
mimicus, V.
V. alginolyticus parahemolyticus, V. vulnificus, and
Differential Test for the Groups of Vitrio Species: Carbohydrate Fermentation
(+) Glucose fermentation
(+) Maltose fermentation except G. hollisae
(+) Mannitol fermentation except P. damsela and G.
hollisae 151
(-) Lactose, Cellobiose and Salicin Fermentation except V. vulnificus Umiqv 162
(-) Arabinose Fermentation except
V. metschnikovii, V. fluvialis, and G. hollisae
String Test
It differentiates Vibrio
spp. (positive result) from Aeromonas (negative result). helow
Reagent: 0.5% sodium deoxycholate
(+) Result: The lysis of bacterial cells after suspending a
loopful of the organism into a drop
of the reagent releases DNA which can then be pulled up into a viscous string ("string line")
using an inoculating loop.
V. cholerae has a positive reaction with this test compared with the other Vibrio species.
4. Serological Test
Strains that phenotypically resemble V. cholerae but fail to agglutinate in 01 antisera are
referred to as V. cholerae non-O1.
V. parahemolyticus can be serotyped by means of its O and K antigens.

Disk diffusion method using MHA and microdilution using MH broth can be used.
ciprofloxacin while other vibrios are treated with
V. cholerae are susceptible to doxycycline or
tetracyclines, chloramphenicol, and monobactams. ru to beepqmteo
Vibriostatic 0/129 Test
It is used to separate vibrios (susceptible) from other oxidase-positive, glucose fermenters

like aeromonads (resistant).
This test utilizes a 150-1g vibriostatic agent 0/129 (2, 4-diamino-6, 7-disopropylpteridine).
Culture medium: MHA or TSA (trypticase soy agar)
6. Molecular Test
16s rRNA sequencing is an accurate
way to identify most Vibrio spp.
used for molecular typing of the members of
Ribotyping and multilocus sequence typing are
the genus Vibrio.
It belongs to the family Aeromonadaceae. like tap water.

The species are found in fresh,
estuarine, and chlorinated water
These are not part of
the indigenous human microbiota.
with single polar flagellum.
The members are facultatively anaerobic and motile
The species are glucose fermenters.
They can typically grow at 4°C to 42°C;
meat and dairy products
apart from seafoods such
as oysters.
The species are isolated from
amphibians and reptiles.
It causes infections not only in humans but also in animals such as
ETEC and may also exhibit

The species may cause traveler's diarrhea which is similar to rice.
watery" stool same with V. cholerac infection. n wwithoog)-gege ofriry selssaretn

mug boe
It is the causative agent of the "red-leg" disease in amphibians.
Mode of transmission: Ingestion of contaminated water, food like meat and dairy products, and
skin trauma.
Extraintestinal human infections: Septicemia, meningitis, keratitis, and wound infections

(cellulitis and necrotizing fasciitis)
Microscopy: Gram-negative straight rods
Culture: BAP - Colonies are large, smooth, round, raised, white, and opaque; with variable
Some colonies have ground glass appearance with fruity odor.
Biochemical test: (+) Oxidase, catalase, and nitrate reduction
Groups of Aeromonas
1. Mesophilic group
This group is composed of motile species and are frequently encountered in clinical samples.
Their optimal growth is at 37'C.
Examples: A. hydrophila complex, A. veronii complex, and A. caviae complex
Most common isolate: A. caviae
Common agent of GI infection: A. cavige (pediatric diarrhea)

Agents of hemolytic uremic syndrome (HUS): A. hydrophila and A. veronii biovar sobria
Common cause of cholera-like disease: A. veronii biovar sobria
Agent of wound infection: A. hydrophila subsp. hydrophilia
producing the enzyme elastase.,

Abbott, Cheung, and Janda (2003) mentioned that strain
stapholysin belongs to the A. hydrophila complex.
2.
Psychrophilic group
This is
the non-motile group.
It grows best at 25 C
and lower temperature.
NON-ENTERIC 313
GASTROINTESTINAL PATHOGENS
Example: A.
salmonicida (fish pathogen)
Donlon, et al (1983) expressed the
isolates of A. salmonicida. melanin-like pigmentation on agar medium of the fish
Characteristics of the Aeromonas Species

MAC: Colonies exhibit a pink
color though other species are colorless.
CIN II: Colonies show a
"bull's-eye" appearance.
Growth
in broth with 6% NaCI: Negative
Sucrose fermenter: All species except A. jandaei and A. schubertii
Mannitol fermenter: All species except A. chubertii
Vibriostatic O/129 test: Resistant
Inositol fermentation: Negative
String test: Negative
Positive indole test: A. caviae, A. hydrophila, and A. veronii
TSIA reaction: A. caviae: A/A, 4) gas, (-) H,S

A. hydrophila and A. veronii: K/A or A/A, (+) gas, (+) H,S
-+ + (A. hydrophilia)/+ + - + (A. caviae)
TABLE 20-1. Differential Characteristics of Aeromonas, Plesiomonas, and Vibrio

Test Plesiomonas Vibrio
Oxidase test +
Resistant Susceptible Susceptible

+
Growth in media with 0% NaCl
Growth in media with 6% NaCl
Fermentation Test
+ + +
Glucose
Inositol
Mannitol
Sucrose
*V. metschnikovii is oxidase-negative

v, either positive or negative reaction
Campylobacter
It belongs to the family Campylobacteraceae
and secretes oxidase.
It is motile
by a single polar flagellum C. curvus, and C. rectus
42 °C and are
microaerophilic except C. concisus,
Its species grow best at
which are strictly anaerobes.

causes sterility and abortion.
It is an animal pathogen (cattle and swine) that
of contaminated water, poultry and dairy products;handling

Mode of acquisition: Ingestion
and birds toccupational hazard); and sexual transmission of
pets like dogs, cats,
or
Microscopy: Faintly staining. Gram-negative, small, curved S-shaped "seagull-wing" rods
Old culture may appear coccobacillary.
asaccharolytic
Biochemical test: (+) Oxidase and catalase;
raised or flat, glistening, and irregular with a
Culture: Campy-BAP - Colonies are gray, "tailing
effect" along the streak line or "runny spreading" growth; nonhemolytic
C. sputorum, C. concisus, C.
Species: C. jejuni subsp. jejuni, C. coli, C. lari, C. fetus subsp. fetus, curvus,
and C. rectus
C. rectus
Enteric campylobacters: C. jejuni, C. coli, C. lari, and
Agents of bacteremia: C. lari and C. fetus subsp. fetus (commonly isolated in blood culture)
Periodontal disease: C. concisus and C. CUTTUS
Campylobacter jejuni subsp. jejuni

It is a slow-growing, fastidious, and asaccharolytic organism.
It has a darting motility and is unable to grow in media with a high salt concentration.
It is the most common cause of bacterial gastroenteritis or the "global diarrhea."

It is themost recognized antecedent cause of Guillain-Barré syndrome because many afflicted
patients are found positive for its antibodies.
It causes septic arthritis among AIDS patients.
Virulence factor:
Cytolethal distending toxin, LPS, SOD, and flagellin (motility)
Biochemical test: (+)
Nitrate reduction and hippurate hydrolysis; (-) urease
Mode of acquisition: Eating contaminated chicken and turkey (does not multiply in food)
Infective dose: > 10,000 organisms
Specimen: Feces, rectal swab, and blood
Rectal swab is a less-preferred specimen for the isolation of enteric

campylobacters.
1. Microscopy
Campylobacters are difficult to
observe in blood culture especially after staining the
samples.
If safranin is used as a
secondary stain, it should be applied for 2 to 3 minutes.
Recommended counterstain: Carbolfuchsin (darker color
compared to safranin red)
Alternate to Carbolfuchsin: Acridine orange
Hanging drop preparation: To observe the darting motility
pattern
Typical motility of
campylobacters is usually seen if the organisms are suspendedin
trypticase soy broth(TSB).
2. Culture
Selective
media: Campy-BAP
Deoxycholate agar (CCDA) agar, Skirrow's medium, and Cefoperazone
Transport medium: Cary-Blair medium
Campy-BAP contains Brucella agar
base with 10% sheep blood.
Antibiotics added to
Campy-BAP and Skirrow's medium.
vancomycin, trimethoprim, and
contains charcoal and antimicrobial agents such as cefoperazone and is selective for
CCDA
Campylobacter species.
Butzler medium contains antibiotics
such as bacitracin, colistin, and novobiocin to suppress
the growth of other bacteria.
Amphotericin B is an
antifungal agent incorporated in CAMPY-BAP and CCDA while
cycloheximide is added to Butzler medium.
In blood culture, two-week incubation may be needed since the natural turbidity affects
"detection"; thus, blind cultures may also be necessary. 9107910
Campylobacter can be detected effectively by a CO, monitoring system. 01009291 23050
Initial reading of culture plates and tubes is best after 48 hours of
incubation.
3. Immunodiagnosis
Enzyme immunoassay is used for direct identification of Campylobacter species in stool
specimens.

Campylobacters are sensitive to azithromycin; though susceptibility test is not commonly
performed for this organism.
Helicobacter
It is found in thegastrointestinal tract of mammals and birds.

It is motile by monopolar flagellum or multi-bipolar flagella.
Most species have a strong urease activity and are microaerophiles.

with increased carbon dioxide.
The growth is better observed in an environment
Campylobacter
Microscopy: Gram-negative, helical (S-shaped) rods that resemble
Culture: CAP - Colonies are gray and translucent.
Biochemical test: (t) Oxidase and catalase
fecal-oral route
Routes of transmission: Oral-oral route and
H. bilis, and H. felis
Species: H. pylori, H. cinaedi, H. fennelliae, H. canadensis,
Agents of gastritis: H. pylori and H. felis
Agents of colitis: H. cinaedi and H. fennelliae

Helicobacter pylori gastric carcinoma.

It is the major cause of type B gastritis, peptic ulcer,fundus
and
of the stomach but does not penetrate
the antrum and
It is found in the mucous layer of
the gastric epithelium.
blood group antigens) and the monosaccharide sialic
It binds with the Lewis antigen (part of the
acid.
Helicobacter that may utilize carbohydrate (glucose)
the only significant member of the genus
It is
either fermentatively or oxidatively.

Virulence factor: urease, adhesins,
and flagellin (motility)
Primary habitat: Human gastric mucosa

and fecal-oral route
Routes of transmission: Oral-oral route
42°C (sometimes variable)
Optimum temperature for growth:
Biochemical test: Strong urease producer (vital in the survival and growth in the gastric mucosa
Diagnostic test: (+) Urea breath (sensitive and specific test)
Genes responsible for virulence: IceA (peptic ulcer) and BabA (attachment to blood group
antigens)
Helicobacter rappini (formerly Flexispira rappini)

It is a Gram-negative, motile, fusiform bacterium with periplasmic fibers and bipolar tufts of
sheathed flagella.
It is phylogenetically related to the genus Helicobacter, and it shares morphological features with
some of its species.
It has been reported as an agent of bacteremia in homosexual men positive with HIV.
Helicobacter cinaedi and Helicobacter fenneliae

These species have been isolated from patients with bacteremia and homosexual males with or
without HIV.
Specimen: Gastric tissue, urine, feces, saliva, and dental plaque
Gastric tissue is the best specimen for the culture of H. pylori.
Tissue specimens should be maintained at 4 °C and processed within 2 hours of collection.
Urine specimen is utilized for ammonia testing.

Fecal samples are not used as routine samples for the isolation of Helicobacter.
ONL
1. Microscopy
The use of 0.1% basic fuchsin as a counterstain enhances
morphology.
Stains for biopsy specimens: Warthin-Starry stain (silver stain) or Giemsa stain (eosin and
methylene blue)
The use of Giemsa or silver
stain increases the microscopic sensitivity.
NON-ENTERIC
- GASTROINTESTINAL PATHOGENS 317
2. Culture
Culture media: CAP, BHI, MIM, Skirrow's agar, and Brucella agar with 5% sheep blood
Transport media: Stuart medium, brucella broth with 20% glycerol, cysteine Albimi broth
with 20% glycerol, and isotonic saline with 4% glucose
A
gastric tissue biopsy specimen should be placed in a Stuart medium
Helicobacters may require more than five days of incubation
environment.
in a microaerophilic

Agar dilution using MHA with 5% sheep blood incubated at microaerophilic condition for
3 days is utilized.
H.
pylori is susceptible to macrolides with metronidazole as a secondary drug of choice.
Nucleic amplification (polymerase chain reaction or PCR) is a sensitive method for detecting
H. pylori.
TABLE 20-2. Differential Characteristics of Campylobacter and Helicobacter

Biochemical/Susceptibility test Campylobacter jejuni Helicobacter pylori
Catalase + +
Nitrate Reduction
Urease
Hippurate Hydrolysis
Indoxyl Acetate hydrolysis
Susceptibility to:
Resistant Susceptible
Cephalothin (30 pg)
Susceptible Resistant
Nalidixic acid (30 1g)
Growth at:
V - variable
318 REVIEW HANDBOoK IN DIAGNOSTIC BACTERIOLOGY
ue
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)
liditqeant isdoroinin
ne guiar noillo ngA
T li noino2 pilnino DinE baisn hssiltua 2b
prioro 2nb sue 9e 6 FIGURE 20-1. Vibriodhiz

3s plasobinontam ptlemicroscopy
species
(Photo from Public Health Source) tpiaousi opeil
fod) colisctilqa piobu
gntbapt 1o tiearihare2 nai (o ne
dang
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haue ant
pae
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FIGURE 20-2. String test for Vibriod ubydeoA con
|PI0 0 I U DCP
p hh
21 5 ntolb
r ti hdRte
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a
Ss
sdaiu
FIGURE 20-3. This image depicts Campylobacter jejuni magnified 9,951k.

Source: https://www.cdc.govcampylobacter/technical.html
eNON-ENTERIC GASTROINTESTINAL PATHOGENS 319
Helicobacter t n s
rue i mtiol adt ombn

ki
cftu wit aharul
(e lel
hGp
unng ta
d n 5
brom01e e a
FIGURE 20-4, Helicobacter Gram s

(Photo from Universidad Autónoma de Zacatecas)ienotpg piad
n siewe▇
16gs amuaigoW
Ssandama9Ln aitalita ei gniwolloi cl to rbms
tnlaoub naoiqao1
dent o sed
ais
adnoio ieg
b sil as goilbrind bb
tbeqarle wv▇
tizo1 yiltaM
tinpaLntinanl
2 Dlan gninadatioenclcD
on thiosulfate-citrate-bile salts-sucrose agarno
ihrio dholerae (
(Photo from USCDCP)

svitiaolacianb 1 ci
puitizoa0nagu
(ustaizel idaloia
Vibrio
quuonunoh5
parahaemolytlcus
(NSF) eebndiy d
e Thabge
plnhnbd
v c e f tl
onlb
(SF)
Vlbrio cao
cholerae ittE
FIGURE 20-6. Vibrio species on thiosulfate-citrate-bile salts-sucrose agar,.

Green colonies are non-sucrose fermenters(Photo(NSF/fromandQuizlet)
yellow colonies are sucrose fermenters (SF).
ASSESSMENT QUIZ

of Helicobacter pylori in the gastric mucosa.
It supports the growth and survival
a. flagella
b. urease
C. gastrin
d. a and b
Vibrio parahemolyticus?
2. What is the recommended culture medium to isolate
b. Alkaline peptone broth

C.
d Wagatsuma agar
Which of the following is NOT yet considered as

a mode of transmission of Campylobacter?
3.
a. respiratory droplet
b. sexual contact
C. ingestion of contaminated poultry meat

d. handling pets like dogs and cats

Microscopy: S shaped rods
Motility: Positive
Incubation requirement: 42°C, microaerophilic
Culture: Glistening colonies with runny spreading pattern
Oxidase: Positive
Nitrate reduction: Positive
Hippurase: Positive
Cephalothin: Resistant
a. Aeromonas hydrophila
b. Campylobacter jejuni
C. Campylobacter curvus
d. Helicobacter pylori
5. What is the principal virulence factor of Vibrio cholerae?

a. choleragen
b. zot toxin
C. ace toxin
d. a and b
NON-ENTERIC GASTROINTESTINAL 321
PATHOGENS
6. Which genes are responsible for the
attachment to Lewis
genetic make-up of Helicobacter
blood group antigens? pylori that promotes its
a. BabA
b.
C. Tox R
d.
7. It is considered as a second
agent of traveler's diarrhea but the stool
a. Vibrio cholerae
sample appears "rice watery."
b. Campylobacter jejuni subsp. jejuni

C. Aeromonas veronii
d. Helicobacter pylori
8. What is the preferred staining method

to identify Helicobacter pylori in gastric biopsy samples?
a. Warthin-Starry
b.
C. 0.1% Carbol-fuchsin
d. a and b
9. It is added into the culture medium for isolation of vibrios

to support and enhance their growth.
a. bile salt
b. citrate
C. sucrose
d. sodium chloride

Microscopy: Pink, straight rods
Motility: Positive
Incubation requirement: 42°C, facultative anaerobic
Culture: Fruity odor
Oxidase: Positive
Nitrate reduction: Positive
Sucrose fermentation: Positive
0/129 test: Resistant
a. Vibrio cholerae
b. Helicobacter cinaedi
C. Aeromonas caviae
d. Campylobacter coli
NON-FERMENTATIVE
GRAM-NEGATIVE BACILLI

1. discuss the general characteristics of non-fermentative bacilli and the
metabolic pathways;
2. describe the distinct features and pathogenecity of Pseudomonas
3. outline the clinical relevance of Burkholderia species;
compare the clinically significant nonfermentative bacilli as to colony taknomobrs * load

appearance, biochemical properties, and related diseases and
infections; and
5. develop an algorithm of diagnostic tests to identify the significant
nonfermentative bacilli. I1
species in this group of
General Characteristics of Non-Fermentative

Gram-Negative Bacilli
These are opportunistic pathogens that are not usually found
as
indigenous microbiota of the human body.

They commonly found in
are
the environment such as water, soil, and
act as contaminants in food
vegetables (plant pathogens), and they
and
hospital devices.
such as
They are able to resist treatments with disinfectants
compounds.
Chlorhexidine and quaternaryammonium
ventilators, catheters, dialysate
They can be isolated from nebulizers,
fluid, and other hospital instruments and equipment.
MAC with some species
There are non-lactose fermenters on
exhibiting variable growth.

no acid production even in the
They fail to acidify the TSI agar and
anaerobic portion of the medium (butt).
DIAGNOSTIC BACTEI
and produce very

carbohydrates by
purdation and not by enzymatic reaction
respiratory and
fermentative
Weak
They utilize is primarily

acid end products - their metabolism
allie formentative (OF) media if it is overlaid with
mineral
oxidat
production is also absent
in
Acid
oil.
Most species secrete oxidase.
Pseudomonas
It belongs to the family Pseudomonadaceae. non-fermentative bacilli.

The species of this genus are the most commonly isolated
and motile.
The species areobligately aerobic, non-spore-forming,
The species usually grow on primary
plated media like MAC and BAP.
Their members are usually oxidizers with few asaccharolytic.
pools, hot tubs, clothing, and foot wear.
The species can be found in cosmetics, swimming
Biochemical test: (+) Oxidase and catalase

TSIA reaction: K/K, (-) gas, (A) H,S
Groups of Pseudomonas
P. aeruginosa, P. fluorescens, P. putida, P veronii, P. monteilii, and
1. Pseudomonas fluorescent group:
P. mosselii
2. Pseudomonas non-fluorescent group: P. stutzeri, P. alcaligenes, P. pseudoalcaligens, and P. luteoa
Pseudomonas aeruginosa (Agent of Blue Pus)

It is the most commonly isolated species of the genus.
It is the most commonly encountered Gram-negative bacterium that is not a member of the
Enterobacteriaceae.
It is a monotrichous organism that exhibits good growth at 42°C.

Virulence factors: Endotoxin, pili, alginate, exotoxin A, lecithinase, elastase, and protease
Culture: BAP Colonies are flat, blue-green or red, or brown in color, spreading with ground
glass or metallic sheen appearance, and "grape-like or corn tortilla odor." Some isolates have
mucoid appearance (observed in patients with cystic fibrosis) with p-hemolytic pattern.
Modes of Acquisition
through the ingestion of contaminated food or contaminatedmedical
It is acquired
water, use of
devices, and open skin wounds.
It has been isolated from contact lenses that are rinsed with a contaminated solution; the
contamination may also come from the distilled
water which is utilized in preparing the solution
It is also found in hot tubs and whirlpool baths.
Tap water is the reservoir of P. aeruginosa.
GRAM-NEGATIVE BACILLI 325
clinical Significance
ahi hohw
It is one of the major

causes of Gram-negative 1
bacillary bacteremias hulony
It is the causative agent of
(necrotizing skin rash). ecthyma gangrenosum, swimmer, ear, and Jacuzzi syndrome
It is the most common cause of
acquired pneumonia. hospital-acquiredrespiratoryinfection and less of community-

It is an etiologic agent of lung infection in patients with
cystic fibrosis and chronic obstructive
pulmonary disease.
It causes hospital-acquired infections that are related to antibiotic resistance.

rwond-wolloy
Pigments that are Produced by P

aeruginosa
a. Pyoverdin yellow-green or yellow-brown pigment
b. Pyocyanin blue pigment (only produced by P. aeruginosa)
c. Pyorubin - red pigment
d. Pyomelanin brown or black pigment
Distinguishing Characteristics of Pseudomonas aeruginosa nth oi Bart al

(+) Growth at 42°C
(+) Acetamide and citrate utilization (both reactions produce blue color) torq ydstors
(+) Gluconate production
(+) Arginine dihydrolase (ADH)
Pseudomonas fluorescens and Pseudomonas putida

These species are isolated from contaminated blood products, cosmetics, hospital equipment,
urine, and respiratory specimens.
These organisms have been linked to transfusion-associated septicemia.
They can grow at 4°C but not in 42°C. 101 59 281s bateduont ons sodas diod q8
Biochemical test: (+) Xylose fermentation

Differential test: (+) Gelatin hydrolysis for P. fluorescens
Laboratory Diagnosis for Pseudomonas digo ninolos wollaY-notabh O evitle 1

fluids ay-nortctharmot ewiahen t
Specimens: Blood, wound discharge, sputum, and sterile
1. Microscopy
Straight and slender Gram-negative rods
9001
2.
Culture
Cetrimide agar (cetyltrimethylammonium
Culture media: BAP, CAP, MAC, Sellers agar, ether), and C390 (9-chloro-9[4-
hydroxydiphenyl
bromide), Irgasan (2,4,4'-trichloro-2 hyar
diethylaminopheny!]-10 phenylacridan)
the uppermost layer and sides of

Pseudomonas species produce scum or a "floating layet" on
the thioglycolate tube.
Some isolates of Pseudomonas have wrinkled-looking colonies
Sellers agar promotes pigment production. enhances
medium that also the pigment
agar is a differential and selective
P. aeruginosa creating yellow-green colonies to
production (pyoverdin and pyocyanin) of
blue-green colonies.
Pseudomonas fluorescent group (P. aeruginosa, P. fluorescens and P. putida) has yellow-green or
3. Biochemical test
TSIA reaction: K/N or K/K (alkaline slant/neutral butt)

Motility test: Positive
(+) Oxidase
(-) H,S production
Oxidative-Fermentative (OF) Test

It is used to differentiate Enterobacteriaceae from Pseudomonas.
It determines whether the isolate is oxidative or fermentative, after utilizing the substrate,
thereby producing acid by-products in the presence or absence of oxygen. mslon
OF medium: Hugh and Leifson medium with 1% carbohydrate
Substrate: 1% Carbohydrate (glucose)

Oxygen barrier: Mineral oil
Uninoculated OF medium: Green color
Procedure: An isolated colony from an 18- to 24-hour old culture plate is inoculated into each
tube, with one tube overlaid with mineral oil while the other is covered with a loosen screw
cap. Both tubes are incubated at 35 'C for 14 days and must be examined daily for any color
change.
Hugh and Leifson medium is a semi-solid medium with peptones aside from glucose.
Quality Control
Positive Oxidation-Yellow color in open tube only: Pseudomonas aeruginosa ATCC 27853
Positive Fermentation-Yellow color in both tubes: Escherichia coli
ATCC 25922 ARGOL SLE
Interpretation
Yellow color in both tubes: Fermenter (Enterobacteriaceae)

Yellow color in open tube but green/blue color in closed tube: Oxidizer but nonfermente
(Pseudomonas)
Green/blue color in open tube but yellow color in closed tube: Nonoxidizer but
(Obligate anaerobe)
NON-FERMENTATIVE GRAM-NEGATIVE BACILLI 327
Green/blue color in both tubes: Nonoxidizer

and
Hugh and Leifson medium detects production ofNonfermenter (Asaccharolytic)
acid in small quantity, either aerobically
(open tube/oxidation) or anaerobically
closed tube/fermentation)."
A yellow color in
blue or green color anyishube indicates an acid production and that is a positive reaction
a negative test
while a
result and it suggests an alkaline end product.

Growth of P. aeruginosa is inhibited by
however, it exhibits resistance aminoglycosides, carbapenems, and the quinolones;
to sulfamethoxazole-trimethoprim and tetracyclines.
Broth microdilution or Kirby-Bauer disk diffusion is recommended for P aeruiginosa while
former is for the
the
other species of the genus.
P. aeruginosa can develop resistance to polymyxin as a consequence of mutations in the
PhoPQ regulatory system, mediated by covalent lipid A modification (Gutu, et al, 2013).
The antibiotic resistance is higher in nosocomial strains of P. aeruginosa.
This test allows the identification of acteria by testing their vulnerability to bacteriophage.
1601
It is a member of the family Moraxellaceae.

It is the second most commonly isolated, non-fermentative, Gram-negative bacilli.
It is an obligate aerobe and non-motile organism (some isolates demonstrate variable mobility).
It is isolated from hospital equipment such as catheters, ventilators, and humidifiers.
It is acquired through contacts with contaminated medical instruments where the organisms
due to prolonged exposure.
become a part of the skin and respiratory microbial flora
a member of the genus Neisseria while other
Some species may be mistaken microscopically as
alcohol decolorization and appear as "Gram-positive cocci."
species have the tendency to resist
Growth may also be observed at 42°C.

Microscopy: Gram-negative coccobacilli which may appear in pairs; may also
appear as "Gram-
positive bacilli" from blood culture bottles

Culture: BAP _ Colonies are small and creamy-looking. a ibsm
MAC - Colonies exhibit a purple color.
Biochemical test: (+) Catalase and () oxidase peepileit brunf
Related Ventilator-associated pneumonia, endocarditis, meningitis, nosocomial
infections:
septicemia, cellulitis, and UTI

and pleural fluid, and wound
Samples for isolation: Blood, CSF, sputum, urine, pericardial
discharge
BACTERIOLOGY
328 REVIEW HANDBOOK
IN DIAGNOSTIC
Types of Acinetobacter Species

non-hemolytic strains (A. baumannii, common isolate)
Acinetobacter spp. glucose-oxidizing,1
a.
non-hemolytic strains (A. Iwoffii, representad
b. Acinetobacter spp. - non-glucose-oxidizing
species) strains (A. haemolyticus;
non-glucose-oxidizing, representative
C. Acinetobacter spp.
species)
Acinetobacter baumannii
It is an alarming cause
of nosocomial infection globally due to its multi-antibiotic resistant
property.
infected persons, unwashed hands of healthcare staff.
It is acquired through direct exposure to
or prolonged stay in the hospital or homecare facilities.
It can cause lung infection, septicemia, and UTI.
Blaschke, Skiebe, and Wilharm (2020)
described this organism as a critical priority 1 pathogen
the WHO in 2017. They mentioned that A. baumannii
for the development of new antibiotics by
can move along wet surfaces
in 2 different ways: via twitching motility and surface-associated
is known to depend on type IV
motility. They have also presented that while twitching motility
pili, the mechanism of surface-associated motility is poorly understood.
It has shown drug resistance specifically the carbapenem-resistant A. baumanii (CRAB) strain.
It belongs to the family Xanthomonadaceae.

It is the third most commonly isolated non-fermentative, Gram-negative bacillus.
It is strictly aerobic and motile, and it can grow at 42°C.
Apart from water (including bottled water) and sewage, it is found in contact lens solutions,
handwash soaps, hemodialysis water, iced machines, and nebulizers (Brooke, 2012).
It can contaminate blood-drawing equipment, antiseptics, and disinfectants.
Maltophilia came from the Greek words malthum (malt) and philia (affinity) that collectively
means "affinity to malt" referring to the acid production of S. maltophilia from maltose and not
glucose.
Microscopy: Short- to medium-sized, Gram-negative, straight rods

Culture: BAP Colonies are large and glistening with lavender-green to light purple
pigmentation and distinct "ammoniacal odor." has gestore
MAC - Colonies exhibit a blue color. bewsioneas-TotaliinoV enoig
Biochemical tests: (+) Catalase,
esculin, gelatinase, DNAse, and LDC; (-) oxidase
Antimicrobial susceptibility test: Broth microdilution and E-testo
Related infections: Endocarditis, pneumonia, osteomyelitis, septicemia, eye infection,
UTI, and wound infection
Samples
for isolation: Blood, CSF, wound discharge, sputum, urine, and pericardial and pleural
fluid
It belongs to the family Burkholderiaceze Tbnls

It is an obligate aerobe and is
motile by polar flagella except B. mallei.
It is generally non-pathogenic
to healthy individuals but may contribute to the severity of
infections
when persistently exposed especially to the immunocompromised individuals.
It is
acquired through contact with heavily contaminated medical deviod" tike bigerial)
It grows well on BAP, CAP, and MAC.
Microscopy: Medium-sized,
Biochemical test: (+) Catalase; variable reaction in oxidase test

Species: B. cepacia, B. pseudomallei, B. mallei, and B. gladioli
Human Pathogen: B. cepacia and B. pseudomallei
Samples for isolation: Blood, discharge, sputum, and biofluids like pleural, peritoneal, and
Burkholderia cepacia complex

It
is a nosocomial pathogen that is isolated from anesthetics, nebulizers, detergents, and
disinfectants.
It grows well on most culture media like BAP and MAC but may lose its viability on the former in
three to four days.
Selective Medium: Bacitracin lactose agar (OFBL) and Burkholderia cepacia selective agar
Culture: BAP - Colonies exhibit a non-wrinkled yellow or yellow-green color with "soil-like odor."
OFBL - Colonies are nonwrinkled with yellow color. n bris 2AM no Llewe #WOTg
Biochemical tests: Weak-positive oxidase reaction -h bala limcogeon animoned at 81
and endocarditis
Related infection: Pneumonia in patients with cystic fibrosis, septic arthritis,
among users of prohibited drugs

Species: B. cepacia, B. cenocepacia, B. multivorans and B. vietnamene 2)-test
Burkholderia mallei
a severe infection that affects horses and
It is the agent of glanders or farcy disease which is
donkeys.
It is a potential bioterrorism agent.
It is the only non-motile member of the genus.
It has a variable growth on MAC.
infected animals and through skin cuts
Mode of transmission: Exposure from
Culture: BAP - Colonies are non-pigmented.
Biochemical test: (+) Nitrate reduction
BACTERIOLOGY
Burkholderia pseudomallei disease.

It is the agent of melioidosis, which is glanders-like
It is motile with a polar tuft of flagella, with optimum growth at 124C.
isolated from muddy soil and rice paddies. ticod of 315
to
It is
inhalation of contaminated
debris or direct
inoculation through
Modes of acquisition: By
damaged skin or mucous membranes i1s
Microscopy: Presence of bipolar bodies
orange in color with mucoid-like
appearance on young
Culture: BAP Colonies are smooth,
growth.
Colonies are dry, wrinkled, deep pink or purple color
Ashdown medium with colistin
with "earthy or soil-like odor."
Other Non-Fermentative Gram-Negative Bacilli
Alcaligenes faecalis 46.00
It belongs to the family Alcaligenaceae.

fLaSEn It is an obligate aerobic, Gram-negative bacillus.
It is isolated in soil and water and in moist hospital environments.
medical devices and solutions and
Infection is acquired through exposure to contaminated
moloD- 1A8010100
respiratory droplets.sg-woul
It grows well on MAC and motile by peritrichous flagella.
It is becoming a nosocomial alert due to the isolation of antibiotic resistant strain.nenboki
Culture: BAP - Colonies are feather-edged, non-pigmented, with "fruity odor" similar to apple
or strawberry; some isolates exhibit alpha-hemolysis.
Biochemical test: (+) Oxidase and catalase; grows in 6.5% NaCl broth; (-) nitrate reduction
Related infection: Meningitis, endocarditis, bacteremia, UTI, and lung and wound infection
dord voter

It is an aerobic rod that may appear coccoid in shape.
It may colonize the distal urethra and cause a serious and active infection.
Microscopy: Small, paired, Gram-negative bacilli or coccobacilli

Culture: BAP - Colonies are small, opaque, and white. 1 1-.
MAC - No growth
Biochemical tests: (+) Catalase, oxidase, and PAD; gas producer, and assaccharolytic
Species: O. urethralis - non-motile; (+) oxidase and (-) urease
O. ureolytica - motile; (+) oxidase and urease
NON-FERMENTATIVE CRAM-NEGATIVE BACILLI 331
Moraxella lacunata Morax-Axenfeld bacillus)

It belongs to the family Moraxellaceae
It is obligately aerobic, assacharolytic, and non-motile.
Itis the agent of blepharoconjunctivitis or angular conjunctivits.
Microscopy: Gram-negative with coccobacillary to bacillary forms
Culture: BAP - Colonies
are small and pit the agar.
MAC - No
visible growth
Catalase, oxidase, nitrate reduction
Chromobacterium violaceum
It belongs to the family Neisseriaceae.
It is the only species in the genus Chromobacterium.
It is facultative anaerobic, motile with polar flagella.
It is an
opportunistic pathogen that causes neutrophil deficiency to immunocompromised
slaw patients. endsh
It grows on MAC at 42°C.
Microscopy: May appear as curved bacilli
Culture: BAP Colonies are smooth, and they exhibit violet pigmentation (violacein pigment)
with "ammonium cyanide" odor.
Biochemical test: (+) Catalase; variable oxidase
Psychrobacter
It belongs to the family Moraxellaceae.
The species have diplococcus-like morphology.
They have been isolated from processed meat, poultry products, and seafoods. *wtte
their best growth is observed between 5° to
They are aerobic, nonmotile, asaccharolytic, and
25° C ("cold-loving" bacteria).
It causes nosocomial eye infection among neonates.
as TMA.
It has the ability to grow on gonococcal media such
that sometimes appear as cocci in pairs.
Microscopy: Gram-negative coccobacilli
Culture: BAP . Colonies are smooth with "rose petal odor."
Biochemical test: (+) Oxidase and nitrate reduction
and P. immobilis
Species: P. sanguinis, P. phenylpyruvicus,
Common isolates: P sanguinis and P phenyipyruvicus

from immunocompromised patients (AIDS)
and those with fatal
P. immobilis has been isolated

sepsis.
DIAGNOSTICBACTERIOLOGY
Shewanella putrefaciens
it belongs to the family Shewanellaceae.
It is isolated from water, dairy products, petroleum gas, and other environmental sources.
is a strong H,S producer. dgold to Mmos entr
It is aerobic, motile, nonhalophilic; and it
a characteristic"sulfur.
It has been associated with food spoilage, mostly marine products with
like odor or foul odor of fish."
It is isolated from humans with ocular infections, otitis media, and septicemia.
Microscopy: Gram-negative rod with tendency to form filaments
Culture: BAP - Colonies appear mucoid and tan producing greenish discoloration of the medium.
Biochemical test: (+) Oxidase, catalase, citrate, nitrate reduction, and ODC; (-) indole and LDC
Related infection: Wound infection (leg ulcer) and pneumonia
Elizabethkingia meningoseptica
It is a member of the Weeksellaceae.
It is an environmental organism that can be isolated from soil, water, saline solution, as well as
from medical equipment.
It is aerobic, nonmotile, and it ferments glucose, maltose, and mannitol.
It has variable growth in primary media such as MAC.

It is associated with meningitis and septicemia in premature neonates and also in adults.
Ratnami and Rao (2013) mentioned that bacteremia and pneumonia are the other common
manifestations in neonates. Prematurity is a primary risk factor for this bacterial infection.
It has been isolated as "contaminant" in tissue allograft. 006001
As cited by Shinha and Ahuya (2015), this organism has been detected among individuals with
renal pathology which necessitates chronic hemodialysis therapy.
It shows resistance to antimicrobials such as colistin.
Culture: BAP - Colonies are typically
circular with shiny appearance.
Biochemical test: (+) Oxidase, indole, gelatin liquefaction, and esculin hydrolysis
TABLE 21-1.
Differential Characteristics of the Non-fermentative Gram-negative bacili
Organisms Oxidase Growth Growth Glucose
Motility oxidation
on MAC at 42°€
Pseudomonas aeruginosa
+
+ +
Stenotrophomonas maltophilia
- variable
eNON-FERMENTATIVE GRAM-NEGATIVE BACILL 333
i
s Escherichia col
ilbs
Wtk Hi
FIGURE 21-1. Growth of Pseudomonas aeruginosa and Escherichia coli on MacConkey Agar
Source: http:/lantimicrobe.org/ClinicMicro/Pseudomonas%20aerugi.htr
äal
eriti tce
FIGURE 21-2. Pseudomonas aeruginosa on Blood Agar Plate

Source: https:/latlas.sund,kudk/microatlas/food/bacteria/Pseudomonas_aeruginosa/
a
Burkholby dT,eriParkera pseudomal
FIGURE 21-3, Colonies of(Photo lei on chocolate agar plate
&A. Marsh)
BACTERIOLOGY
on Ashdown's medium
FIGURE 21-4. Burkholderia pseudomallei on
(Photo from Academic)
Stenotrophomonas maltophia
Columbn sheep blood agan
24 h. 37 C. 54 COS
FIGURE 21-5. Stenotrophomas maltophilia on Sheep Blood Agar
maltophilia-7.html
triduRE 71-6; Chromobacterium violaceum from a blood culture sample

ASSESSMENT QUIZ
1. It is listed as an "urgent threat" by the WHO due

common.
to its
antibiotic strains, where CRAB is the most
a. Acinetobacter baumannii
C. Elizabethkingin meningoseptica
d. Psychrobacter immobilis
2. Which of the following non-fermenters is considered as a blood transfusion hazard?

a. Oligella
b. Pseudomonas putida
C. Burkholderia pseudomallei
d. Moraxella lacunata
3. This medium enhances the pyocyanin production of Pseudomonas aeruginosa.

a. Sellers
b. Ashdown
c. Cetrimide

Microscopy: Rods with filamentous formation
Motility: Positive
Growth at 42°C: Negative
Culture: Greenish discoloration on BAP with sulfur-like odor
Ornithine decarboxylase: Positive
Hydrogen sulfide: Positive

a. Chromobacterium violaceum
b. Shewanella putrefaciens
C. Moraxella lacunata
d. Burkholderia mallei
336 REVIEW HANDBOOK IN DIAGNOSTIc BA TERIOLOGY
5.
hie vinine fiat serosd by Baitmnnr ampinos comituts io the seaihy s pln
infection in patients with cystic fibrosis.
a.exotoxin A
na
b. lecithinase
c. polysaccharide capsul
d. alginate
6. That is the preferred culture medium for oxidative and fermentative test?l
Eu
a. MAC
b. OFBL
s )
c. Hugh and Leifson
d.
Krigler double agar
7. Identify the organism after phenotypic testing. mh
Microscopy: Straight rods
Motility: Positive
Culture: Lavender green colonies with ammoniacal odor
Growth at 42°C: Positive o.
Oxidase: Negative Ewth

Lysine decarboxylase: Positive Ahe)
DNAse: Positive
Gelatinase: Positive
O t
a. Pseudomonas aeruginosa
ste
b.Stenotrophomonas maltophilia
S in
C. Alcaligenes fecalis
d. Burkholderia cepacia
8. It has been isolated from tissue allograft and an agent of neonatal septicemia particulanly amng
premature infants tnoa
a. Elizabethkingia meningoseptica
uer
b. Psychrobacter immobilis
Oligella ureolytica
s Na
d. Pseudomonas fl orescens
p .
Which of the following is the

only non-motile species in the genus Burkholderia and with variable
9.
growth on MAC?
a. Burkholderia cepacia
b Burkholderia gladioli
C. Burkholderia pseudomallei
d. Burkholderia mallei
10.
Identify the organism after phenotypic testing.
Microscopy: Pleomorphic shape
Motility: Negative
Culture: Smooth with rose-petal odor
Growth at 42°C: Negative
Oxidase: Positive
Nitrate Reduction: Positive
DNAse: Negative
a. Alkaligenes
b.
C.
d. Shewanella
CHAPTER 22
SMALL, PLEOMORPHIC
GRAM-NEGATIVEBACILLI

compare the distinct traits of the
1.
genera and relevant species of
pleomorphic bacilli;
2. explain the growth requirements, colony patterns, and pathogenesis of
3. outline the members of HACEK and related diseases;

4. discuss the clinical significance of Bordetella pertussis and Legionella
pneumophilo;
5. contrast the modes of acquisition and transmission of the recognized

potential agents of bioterrorism such as Brucella and Francisella;
6. cite the samples and culture media used for isolation of the
pleomorphic rods ; and

7. create an algorithm of diagnostic tests to determine the clinically
significant species in the group of Gram-negative pleomorphic bacilli.
It belongs to the family Pasteurellaceae.

The genus name is derived from the Greek words haima and philos,
which means "blood lover."
on the mucous
The species are obligate opportunistic parasites
membranes of humans and animals.
humans except
They normally inhabit the upper respiratory tract of
H. ducreyi.
The members are fastidious, non-motile, and facultatively anaerobic
bacteria.
The species die rapidly in clinical specimens and are very susceptible
to drying and extreme temperatures.
IN DIAGNOSTIC
BACTERIOLOGY
340 REVIEW HANDBOOK
BAP.
"blood lovers," most species cannot grow on pure
Though they are
or rods
Microscopy: Gram-negative, small, pleomorphic coccobacilli
and some are mucoid with distinct odor while others
Culture: CAP - Colonies are tan-colored
appear dry.
Biochemical test: (+) Catalase and oxidase except H. ducreyi

(+)
Growth factors: X (hemin) and V (nicotinamide adenine dinucleotide or NAD)
Species: H. influenzae, H. ducreyi, H. aegyptius, H. hemolyticus, H. pittmaniae, H. parainfluenzae,
H. parahaemolyticus, and H. paraphrohemolyticus
H. aegyptius
Major human pathogens: H. influenzae, H. ducreyi, and
Haemophilus influenzae
It is the main cause of meningitis in children.
the blood and meninges.
It spreads from the nasopharynx and the regional lymph nodes to
It is very fastidious and can be rapidly killed by phagocytes.
It is nonhemolytic, and it is the only member of the genus that produces IgA protease.
It does not secrete any form of endotoxin.
Currently, it has eight (8) biotypes, with biotype I having the most positive results in biochemical
tests (indole, ornithine and urea) as opposed to the negative results of biotype VIII.
Principal virulence factor: Polysaccharide capsule

Other virulence factors: IgA protease, fimbrae, and lipopolysaccharide
Mode of transmission: From person to person (contaminated respiratory droplets)
Culture: CAP - Colonies are translucent, convex, tan-colored, and mucoid with a "mousy" or
"bleach-like" odor.
Serotypes: A to F
Biochemical test: (+) Xylose fermentation; (-) porphyrin
Clinically Significant Biotypes Snic

Biotype I: Common cause of meningitis and epiglottis and associated with severe infection
Biotype I, II and IIII: Ear infection and conjunctivitis
Categories of Haemophilus influenzae

1. Typeable form
It is based on the capsular characteristics of H. influenzae.
Encapsulated strains: Types A, B, C, D, E, and F (capsular serotypes)

H. influenzae type b (Hib) is the leading cause of meningitis in unvaccinated children.
Other related infections: and
Septicemia, arthritis, epiglottitis, tracheitis, osteomyelitis,
pneumonia
SMALL
Non-typeable form (Non-typeable

2.
H. influenzae-NTHi
It does not
produce a capsule (non encapsulated straing).
It is part
of the indigenous microbiota of the
epithelial cells. respiratory tract, and it adheres to human
It is the second prevalent etiologic agent of otitis media with effusion

after Streptococcus pneumoniae. (middle ear infection)
Other related infections:
neonatal sepsis
Conjunctivitis, sinusitis, cystic fibrosis, meningitis in adults, and
Haemophilus ducreyi
It is not part of the indigenous human flora.
It is the only asaccharolytic member in the clinically
significant group of the genus Hemophilus.
It is the agent of chancroid or "soft chancre," which is a highly communicable sexually
transmitted disease.
It infects the mucosal epithelium, genital, and non-genital skin and regional lymph nodes.
Characteristics of chancroid: painful and tender genital lesions that advance to ulcers with
satellite lesions
Hallmarks of chancroid: Buboes or suppurative, enlarged, draining, inguinal lymph nodes

Culture: CAP - Colonies are transparent, small, smooth, with tan or yellow color
Biochemical test: (-) Catalase and oxidase
TABLE 22-1. Differential Characteristics of Haemophilus Species s1 Yod To nimed anT

Associated infection/
Distinguishing characteristics Growth factor
Species disease
H. influenzae Mousy/bleach-like odor; non- Meningitis, epiglottis,

arthritis
(Pfeiffer's bacillus)
Pink eye
Genetically related to us not evilssion bolmoo x, Vin0 b
conjunctivitis
(Koch-Weeks bacillus) H. influenzae
Brazilian purpuric
H. influenzae
Non-typeable fever
biogroup aegypticus
H. haemolyticus
(+) Urease
Chancroid or soft
School of fish chancre
Tan and dry colonies; f-hemolytic

(+) Urease Pharyngitis
H. parahaemolyticus Strong carbohydrate fermenter
(elucose, fructose, sucrose, and
maltose)
fermenter
Strong carbohydrate 1 Endocarditis
H. (glucose, fructose, sucrose, and
parainfluenzae maltose)
Non-hemolytic
BACTERIOLOGY
or ulcer, joint
fluid, vaginal swab, abscess
drainage,
Specimens: CSF, sputum, genital lesion
and blood
conjunctival swab, bronchial washing,
blood and CSF.
Isolation of H. influenzae uses
CSF samples should be centrifuged.

and extreme temperature, hence, the need for
Haemophilus species are very susceptible to drying
an immediate transport and processing for their isolation is a must.
a sterile gauze that is pre-
ducreyi, the ulcer should be cleansed with
For the recovery of H.
moistened with sterile phosphate-buffered saline.
Bedside plating is preferred instead of using a transport medium for the isolation of H. ducreyi.
1. Microcopy
Haemophilus species resemble an "amorphous serous material* because of their pleomorphic
appearance.
H. ducreyi cells have a "school-of-fish" arrangement. mawarius esopum onf elsoim
2. Culture
Culture media: CAP, BAP, BHI, gonococcal (GC) medium and thioglycolate - 1 2
Horse blood is the recommended source of the supplement to allow better visualization of
the growth of the Haemophilus spp. over SBA (sheep blood agar); rabbit blood is the alternate.
Most strains will not grow on pure BAP because it only contains hemin.
The hemin or the X factor is directly utilized by the Haemophilus species though both growth
factors are located in the red blood cells.
All clinically significant Haemophilus species except H. ducreyi require NAD

Incubation in a 5% to 10% carbon dioxide environment supports H. influenzae, H. ducreyi, and
H. paraphrohemolyticus.
Plates should only be reported negative for culture after 7 days.
also
Although not routinely differentiated through this pattern, Haemophilus spp. are
recognized as carbohydrate fermenters.

Importance of CAP
After autoclaving, the horse blood should be added immediately into the blood agar based
medium since the heat causes lysis of the red blood cells releasing the growth factors (X and
V factors) while inactivating the nicotinamide adenine dinucleotidase (NADase).
CAP is the preferred medium for Haemophilus because it contains both growth factors
(X and V factors).
of
CAP with bacitracin (300 mg/L) is an excellent selective medium for the isolation
Haemophilus spp. from respiratory specimens.
Selective media for isolation of Haemophilus species
a.
H. influenzae - Horse blood-bacitracin agar for respiratory secretions of patients with cystic
fibrosis
b. H. aegypticus - CAP with 1% IsoVitaleX or Vitox

SMALL, PLEOMORPHIC
C. H. ducreyi - Nairobi
supplemented with vancomycin (a combination of GC medium
Growth patterns of Haemophilus specior

Seolom nl
Haemophilus
species grow best at 354C to 37•C, except H. ducreyi at 33 •C.
Y-factor-dependent Haemophilus spp. like H. influenzac grow as "Satellitesf on
disk incorporated with NAD or bacterial
BAP around
colonies-producing-V-factor such as S. aureus.
Hemophilns species are grown anaerobically only NAD, not hemin, is required.
When
H. aegyptius requires four days of incubation while H. ducreyi takes longer up to 7 days.
H. haemolyticus and H.
parahaemolyticus create $-hemolytic pattern on horse blood agar
though some isolates may be
No growth
occurs on MAC. domin inin auonogibn odliohbeth ametnc wen f
3. Porphyrin Test (Delta-aminolevulinic acid test)
It detects the presence of bacterial enzymes that converts delta-aminolevulinic acid (ALA)
into porphobilinogen or porphyrin.
It identifies the heme-producing species of Haemophilus.
It
can be performed in broth, solid medium, or by using a disk impregnated with the reagent.
Porphyrins reddish-orange color at UV of 360 nm as bake/boist
(+) Result: Red color (H. parainfluenzae, H. parahaemolyticus, H. paraphrophilus, and H. aphrophilus)
(-) Result: No color change (H. influenzae, H. haemolyticus, H. aegyptius, and
H. ducreyi)m9sh
Interpretation of results: Haemophilus species that need the X factor are unable to synthesize
porphyrin from delta-ALA.
4. Immunodiagnosis
identification of the distinct capsular antigen by
can be determined through the
latex agglutination, capsular swelling or immunofluorescence test.
Neufeld-Quellung reaction is a rapid direct identification test of the capsular antigen of
H. influenzae.

only for B-lactamase
Due to its fastidious nature, Haemophilus species are usually determined
amoxicillin.
activity to ampicillin and Test Medium (HTM) is utilized
to erythromycin, and Haemophilus
H. influenzae is susceptible
for this assay.
effective against life-threatening H. influenzae illness
while
ceftriaxone is used for non-life-threatening
amoxicillin-clavulanate or trimethoprim
infections.
H. ducreyi.
tilized for the therapy of
BACTERIOLOGY
tr inrhtincae can be distinguished by PCR asays detecting the liyd gene (coding for Protein
D) and the fuck (fuculose kinase) gene. eniga sulingonash mans,
HACEK Group
the following organisms:
This group of fastidious Gram-negative bacteria includes
Source: (https://asm.org/Articles/2019/February/Wh
Characteristics of the HACEK Group bituorusdpon adecm istslodioppo

These organisms are part of the indigenous oral microbiota and are considered as opportunistic
pathogens.
They are small, facultative anaerobes, pleomorphic, and non-motile, that cannot grow on MAC
The members are glucose fermenters except Eikenella corrodens. neger loonen
They are fastidious, and most of the species grow slowly (dysgonic) on BAP and CAP and require
in blood culture bottles can be evident in days.
days to 14 days of incubation while growth
They cause slow and progressive bacterial endocarditis (vegetation of fibrinous clots in heart
valves).
The organisms utilize delta-aminolevulinic acid.I Dim Pinii velonim and bismia
Microscopy: Gram-negative bacilli to coccobacilli
Biochemical test: (+) Glucose and maltose fermentation except Eikenella corrodens
Haemophilus parainfluenzae
It is one of the most common HACEK pathogens linked to endocarditis.
It also causes otitis media.
Biochemical test: Variable indole reaction
Aggregatibacter aphrophilus ("Foam-loving" bacterium)

It belongs to the family Pasteurellaceae.
It is formerly known as Haemophilus paraphrophilus.
It represents the "H" in HACEK until it was transferred to the genus Aggregatibacter.
Its nomenclature came from the Greek words aphros and philos, which collectively means"foam-
loving."
It is the most common HACEK species that causes endocarditis. MILIMQn1S D.
It is isolated from dental

plaques and gingival scrapings.
the only lactose-fermenteramong the members of HACEK -
It is fermente
a strong carbohydrate
compared with the other
species in the group.
Culture: CAP -
Colonies are raised, convex, granular, and yellowish.
Biochemical
test: (+) Nitrate
reduction and x-factor dependent
SMALL, PLEMORPHIC GRAMNEGATIVE BACILLI 345
gregatibacter actinomycetemcomitanc
It belongs to the familyPasteurellaceae.
It is formerly known as
Actinobacillus actinomycetemcomitans.
It is a common cause of periodontitis.
from a polymicrobial intection together with. Actinonnes.

It has been isolated
It is the only catalase-positive HACEKspecies.

Virulence factors: Collagenase and leukotoxin
Serotypes: A to F
Culture: BAP:
Small colonies, sticky with greenish appearance.
BHIA: Colonies have "star-shaped" centers after 48 hours of incubation.
Biochemical test: (+) Nitrate Reduction
Cardiobacterium hominis
It belongs to the family Cardiobacteriaceae.
It infects the aortic valve more
frequently than the other HACEK species.
It shows "false Gram-positive" staining reactions in some parts of the cells.
It is the only indole-positive HACEK member.
Microscopy: Rosette formation with sticklike projections (rose petal arrangement)
Culture: BAP - Colonies are smooth and capnophilic, and they may exhibit "pitting."
Biochemical test: (+) Oxidase
Eikenella corrodens (Corroding bacilli) mon onshobil ols auinin ahl in aalss
It belongs to the family Neisseriaceal. cinicauolbnache

It is the least common isolate of the HACEK group in adult infectious endocarditis. arg
It is the only assacharolytic member of HACEK.
infections due to human bites.
It causes various infections such as clenched fist
injuries and skin
persontransmission.
It is acquired through skin trauma and from person to
BAP _ Colonies exhibit a yellow colot, fuzzy-edged with an umbonate center, pit
or
Culture:
odor.
corrode the agar; and have "sharp bleach' and arginine dihydrolase
Biochemical test: (+) oxidase and ornithine decarboxylase; (-) catalase
It belongs to the family Neisseriaceae. rods to coccobacilli that are arranged in pairs
Microscopy: Plump, square-ended, Gram-negative
or short chains
Species: K. kingae, K. oralis, K. denitrificans, and K. potus

Major pathogen: K. kingae
K. denitrificans
Agents of endocarditis: K. kingae and
BACTERIOLOGY
Culture: BAP - Colonies exhibit white

to yellowish-brown pigmentations and may pit the agar.
of N. gonorrhoeae sh
TMA - K. denitrificans resembles the growth
Biochemical test: (+) Oxidase
Kingella kingae
It has a tendency to resist decolorization.
It is associated with decenerative joint and bone infections or osteoarthritis in children.
It exhibits 6-hemolysis same with K. denitrificans.
It secretes phosphates (ACP and ALP).
Biochemical test: (+) Maltose fermentation
TABLE 22-2. Differential Characteristics of the HACEK Groupediv

Oxidase Glucose Lactose Maltose Sucrose
Species
H. parainfluenzae + +
+ +
A.aphrophilus
+
A actinomycetemcomitans
C. hominis
E. corrodens
K. kingae +
v - variable
Brucella (Bang's Bacillus)

It belongs to the family Brucellaceae.
The species of this genus are important human and animal pathogens. an9D01ion slionsli
The
species are obligate aerobes, fastidious, intracellular parasites, and a Class B bioterrorism
agent.
They are non-motile, assacharolytic, and non-encapsulated; some species require an increased
supply of CO, for growth. T edt bortonolo as dous
The members are localized in tissues
that are rich in erythritol (placental tissue) and induces
spontaneous abortion among animals.
Preferred specimen for isolation: Blood and bone marrow
Microscopy: Small coccobacilli that are arranged singly, in pairs, or in short chains with "sandy
appearance"
Culture: BAP - Colonies small,
convex, translucent, yellowish, and non-hemolytic; they may
are
become brownish in color with age.

Biochemical test:
(+) Catalase and oxidase; rapid urease producer
Disease: Malta/Crimean/Mediterranean fever or undulant fever
Human pathogens: B. abortus, B. canis, B. suis, and B.
melitensis
Common isolate: B. melitensis
Most virulent species: B. melitensis and B. suis
SMALL, PLEOMORPHIC GRAM-NEGATIVE BACILLI 347

undulant fever (Brucellosis)
It is characterized by a normal
temperature in the morning and then followed by high
temperature in the afternoon and evening,
a
It is a systemic disease with low mortality rate.
It has three stages namely, acute (onset

of symptoms), sub-chronic (undulant form), and chronic
(late symptoms such as chronic fatigue syndrome) forms.
Primary routes of human infection (brucellosis)
a.
Ingestion of unpasteurized and contaminated milk or cheese from infected animals
Inhalation of air
b.
around animal carcasses (acquired through aerosol)
c. Penetration of ocular or oral mucosa
Direct inoculation into the

d.
bloodstream through abrasions in the skin or needlestick injuries
Specimens: Blood, bone marrow, skin lesions, CSF, and placental tissues
Brucella species should be handled as a biosafety level 3 agent in a class III BSC due to aerosol
transmission.
1. Microscopy
Carbol fuchsin should be used as a substitute for safranin O to improve the Gram stain.
2. Culture
Culture media: BAP, trypticase soy agar (TSA), and Castañeda medium
it can be inhibited by adding
B. abortus requires niacin or nicotinic acid for growth, but
thionine dye.
Modified TMA or Martin Lewis.
Brucella species may be grown on gonococcal media such as
Brucella isolates can be recovered within seven days, but they may require prolonged
incubation of up to 30 days.
blood culture system, and it is incubated for up to
Castañeda medium is used in the manual
6 weeks at 35 'C + 2°C with subsequent subculture; automated blood culture system holds
specimens up to 2 weeks.
is a normal occurrence in positive culture bottles.
Turbidity of the specimen
3. Immunodiagnosis
Serum agglutination test (SAT):
Positive result if with 2 1:160 titer
with Francisella tularensis,
SAT shows cross-reactivity
with SAT is suggested to increase the sensitivity of the
Indirect Coombs test in combination
result and prevent misdiagnosis.
B. suis is not detected by SAT.

4. Antimicrobial Susceptibility Test not suggested for

classification as a
hioterrorism agent, sensitivity test is
Due to the
species.
It is used to subtype Brucella spp. into biovars. p*054
TABLE 22-3. Differential Characteristics of Brucella Species Inhibition by Dyes

H.S Production
Growth in Urease
Natural (Lead Acetate Fuchsin
Species Hosts 5%-10% CO, Method)
+
+
B. abortus Cattle
B. canis Dog
Goat or
B. melitensis
sheep
Swine
v - variable

Gram-negative coccobacilli.
The species in this genus are obligately aerobic, fastidious
bronchiseptica.
They are non-carbohydrate fermenters and are non-motile except B.
humans.
They replicate on ciliated respiratory epithelial cells of
biochemical test systems.
They are mostly inactive in
Culture: Bordet-Gengou agar - Colonies are smooth, glistening with a silver color.
Biochemical test: (+) Catalase; (-) indole

Growth factors: Nicotinic acid, cysteine, and methionine
Species: B. pertussis, B. parapertussis, B. bronchiseptica, B. avium, B. petrii, B. trematum, and B. hinzii
Bordetella pertussis (Bordet-Gengou bacillus)

ablo
It is the etiologic agent of whooping cough.
It only infects and causes diseases in humans.
It does not survive well outside the host.
Principal virulence factor: Pertussis toxin (protein toxin)

Culture: Bordet-Gengou agar Colonies are small and shiny, and they resemble "mercury
droplets."
Growth inhibitors: Fatty acids, metal ions, sulfides, and peroxides

Growth protectors: Charcoal, blood, and starch alchiom.
Other virulence factors: Filamentous hemagglutinin, endotoxin, tracheal cytotoxin, and pertactin
Preferred specimen for isolation: Nasopharyngeal aspirate (young children)
Nasopharyngeal swab (adults)
SMALL, PLEOMORPHIC GRAM-NECATIVE BACILL 349
It is a highly contagious, acute infection of the

affects children.
upper respiratory tract (URT) that primarily
Mode of acquisition: Inhalation of infected droplets

Incubation period: 7 to 14 days nine
Stages of whooping cough

a. Catarrhal stage
It is a
highly communicable phase that is characterized by mucous membrane
inflammation and mild coughing with runny nose.
b. Paroxysmal stage
It is associated
with vomiting and "whooping" or hurried, deep respiration that may last
for six weeks.
Infected individuals experience severe coughing for at least 15 to 25 times in 24 hours.
Convalescent stage
This is the stage in which the symptoms slowly decline. This period may last for six
months after infection.
Laboratory Diagnosis for Bordetella

Specimens: Nasopharyngeal swab and bronchoalveolar lavage0f to nobmaltilm
A nasopharyngeal swab is preferred for B. pertussis.
into a transport
Nasopharyngeal swabs should be inoculated directly into the culture media or
medium at the bedside.
the collection of specimens.
Calcium alginate or a Dacron swab is used for
1.
Microscopy
0.2% basic fuchsin as counterstain enhances its visibility.
The use of a two-minute safranin or
2. Culture
Culture media: Bordet-Gengou potato infusion agar, Modified Jones-Kendrick charcoal agar
and Stainer-Scholte medium
the commonly used medium composed of potato infusion, sheep
The Bordet-Gengou agar is
or methicillin as inhibitors.
blood, glycerol, and cephalexin
extract and cephalexin antibiotic.
The modified Jones-Kendrick charcoal agar contains yeast
medium composed of horse blood and cephalexin.1511
Regan Lowe agaris a transport
present a hemolytic reaction on Bordet-Gengou potato
B. pertussis and B. parapertussis may
infusion agar.
an increased CO,.
Plates are incubated for seven days at 35°C without
medium is the Regan-Lowe agar with
The favorable selective

transport and enrichment
charcoal, horse blood, amphotericin B, and cephalexin
transport medium for swab specimens.

The Casamino broth (1% casein hydrolysate) is
a
B. bronchiseptica because
The Bordetella species will not grow on MAC agar except
and less fastidious.
biochemically it is the most active member
Culture plates are observed using &
stereomicroscope for typical colonies.
3. Immunodiagnosis it is sometimes
for an agglutination test; thus,
greater concentration of bacteria required
is
A
from the primary isolation plate.
necessary to perform subcultures
a hallmark of seroconversion
increase in the serum level of antibodies, such as the IgG,
is
An
against whooping cough.

of Bordetella species
Clinical specimens can bemicroscopically examined for the presence
using direct fluorescent antibody (DFA) stains.
ELISA is reliable test for Bordetella species.

agent of whooping cough
It is not routinely performed for Bordetella species especially for the
due to the known sensitivity of the organism to macrolide antibiotics. neglevnol
Aside from macrolides, B. pertussis and B. parapertussis are susceptible to the penicillin,
tetracycline, and ketolides.
considered that best method
The polymerase chain reaction (PCR) assay is a rapid test and is
for identification of Bordetella species.
TABLE 22-4. Differential Tests for Bordetella Species

Motility Nitrate BAP
Species Oxidase Urease
Test Reduction Growth
B. bronchiseptica
B. parapertussis
Francisella
It belongs to the family Francisellaceae.

It is a very small coccobacillus, fastidious; and it is transmitted by a vector. brod odi
It is obligately aerobic, non-motile, intracellular bacteria.
Francisella tularensis
It is a category A potential bioterrorism agent that requires a biosafety level 3 containment for
sample processing.
3
subspecies: F. tularensis subsp.tularensis(type A), F.tularensis subsp.holarctica(type B), and
F. tularensis subsp. mediasiatica.
SMALL.
virulent subspecies:
Most
F. tularensis subsp. tularensto
Virulence factor: Capsule
Vector: Deer flies and ticks grit
Reservoir: Cottontail rabbit
Microscopy: Gram-negative bacilli

with faint bipolar bodies
Culture: CAP - Colonies are round, smooth, bluish-gray to white, and slightly mucoid.
Growth factors: Cysteine or
cystine and thiosulfate
Biochemical test. Weak positive in catalase and /-lactamase reactions; () oxidase

Standard
Serological Test: Tube Agglutination/Microagglutination - 1:160/1.128 titer is diagnostic
Direct Fluorescent Antibody Test: Positive
Disease: Tularemia
Tularemia
It is also known as the deer fly or rabbit fever.

It is a zoonotic disease which can be acquired through ingestion of undercooked or contaminated
animal meat, arthropod bite, or inhalation of the infected animal carcasses.
Specimens: Scrapings from infected ulcers, blood, gastric aspirates, and sputum slowstarg
Best specimen: lymph node biopsy endsmurl mheoibege betsloai vinommoo lapm ond at i1
1. Microscopy
It requires an acridine orange stain to visualize organisms that are obtained from a blood
culture bottle.
2. Culture
Charcoal Yeast Extract (BCYE)
Culture media: CAP, TSB, MTM, and Buffered
growing organism which requires two four days prior to colony

to
F. tularensis is a slow
formation.
dioxide.
Growth is not enhanced by incubation at an increased carbon
No growth observed on MAC due to the fastidious nature of the organism.
is
medium with L-cysteine and glucose as
It is best recovered from a liquid blood culture
supplements. bne phooula a rod af it
3.
Antimicrobial Susceptibility Test recommended; however,
method using
Mueller Hinton broth is
The broth microdilution

fularensis in a routine clinical laboratory facility is not ideal since
susceptibility testing of F.
this is a bioterrorism agent.
BACTERIOLOGY
Pasteurella
It belongs to family Pasteurellaceae.

the
facultatively
aerobic, non-motile, and zoonotic organisms.
The species of this genus are
wounds.
from cats) or scratch
They are isolated from animal bites (mainly
with variable to no growth on MAC.
The species grow well on BAP and CAP but
Actinobacillus.
genus.
It has similar phenotypic characteristics with the
Virulence factor: Endotoxin and capsule
ovoid, Gram-negative bacilli with a "safety-pin or bipolar
Microscopy: Small, straight or
staining" appearance
with narrow green-to-brown halo
Culture: BAP and CAP - Colonies are gray and non-hemolytic
around the colony after 48 hours.
Biochemical test: (+) Oxidase, catalase, and
indole; weak glucose fermenter
Species: P. multocida, P. stomatis, P. bettyae, and P. dagmatis

Common isolate: P. multocida
Related Infection: soft tissue infection (animal bite), septicemia, osteomyelitis, pneumonia, and
meningitis
Antimicrobial Susceptibility Test: Susceptible to Penicillin
boold erosin belbom mod eg nigstod :anominogd

Pasteurella multocida .eststlgg.5 2*7125g
It is the most commonly isolated species in humans.
It is found in the respiratory and gastrointestinal tract of wild animals.
It is a common isolate in dog and cat bite infections.
book
It grows only on culture medium enriched with blood and is susceptible to penicillin.
Culture: BAP - Colonies are smooth to rough appearance, mucoid with "mushroom odor."
It has five serogroups A, B, D, E, and F) differentiated by capsular antigens.

Virulence factor: Endotoxin, adhesins, and capsule
Biochemical test: (+) Oxidase, indole, ornithine decarboxylase, and urease; (-) ONPG
Pasteurella bettyae
It is isolated from amniotic fluid, blood, and genitourinary tract of humans.
It can be sexually transmitted since it has been isolated from the cervix and vagina.
It is both glucose and fructose fermenter.
It has variable growth on MAC.
Catalase; variable indole production cotlulibomim mlou
SMALL, PLEOMORPHIC GRAM-NEGATIVE BACILLI 353
Legionella
It is the only genus in
the family Legionnellaceze.
The members are
fastidious, aerobic, motile, and non-carbohydrate fermenter
The species
are primarily acquired through inhalation or exposure to contaminated water from
faucets and public water station (aerosolized water particles)yet without a documented human to
human transmission.
Microscopy: Faintly staining, thin,

Gram-negative bacillary, or coccobacillary in form
Culture: BCYE - Young colonies are sticky with "ground centers and exhibit "rainbow
glass"
colors'.
Biochemical test: (+) Catalase and gelatinase; weakly positive in oxidase reaction
Major reservoirs: Hot water system, cooling towers, and evaporative condensers
Species: L. pneumophila, L. micdadei (Pittsburg pneumonia agent), L. bozemanii (WIGA agent), and
Major Human Pathogen: L. pneumophila
Distinguishing characteristics of Legionella species:

a. They can infect and multiply within some free-living amoebae (species of Hartmanella,
b. They can be isolated from lakes, rivers, hot springs, mud, and piped water systems.
C. They can tolerate up to 3 mg/L of chlorine, and thus resist water treatments.
d. They will not grow on routine primary plated media.
Legionella pneumophila
It is the most commonly isolated human pathogen in the genus Legionella.
and Pontiac fever.
It is the agent of Legionnaire's disease
macrophage.
It is a facultative intracellular pathogen which invades the bronchoalveolar
It invades the bronchoalveolar macrophage, which is a facultative intracellular pathogen.
humidifiers, and nebulizers.
It is isolated in air-conditioned units, cooling towers,
Serogroups: 1 to 7
Serogroups associated to the Legionnaire's disease: 1 4, and 6
Most common serogroup causing Legionnaire's disease: Serogroup 1

Preferred medium: BCYE with L-cysteine that is buffered to pH 6.9
Preferred specimens: Respiratory secretions
and convex.
Culture: BCYE - Colonies are blue-green, glistening,
light gray in color while periphery is
Young colonies have ground-glass appearance,
pink and furrowed.
BACTERIOLOGY
Legionnaire's disease
febrile and pneumonic illness.
It is also known as legionellosis which is
Direct physical contact does
ransmission: Airborne spread or inhalation of infectious aerosols
Mode
Symptoms: High fever, non-productive cough, headache, neurological,

and
severe
bronchopneumonia
wood
Diagnostic test. Antigen testing in urine and antibody titer
Pontiac fever paibnot biof
disease but exhibits the symptoms
It is a non-fatal respiratory infection that resembles an allergic
bns. fin of pneumonia.
Other specimens: Urine, pleural fluid, blood, and lung tissue

Urine is an important sample for antigen detection of L. pneumophila. brus uisomanmsh
1. Microscopy
Microscopy: Faint staining and sometimes undetectable through Gram staining
Prolonging the contact of smear with safranin for 10 minutes enhances the staining of the
cells.
Legionella is intracellularly and extracellularly located in the phagocytes.

L. micdadei is weakly acid-fast bacterium when using the modified Kinyoun method.
2.
Culture is the most important test for the Legionella species. Asorionond onl abbown l
Selective Media: BCYE with L-cysteine, ferric salt, and a-ketoglutarate or BCYE with
antibiotics
Legionella will not grow on BAP and MAC.

The application of acid reagent (KCI-HCI) in contaminated specimens enhances the isolation
of Legionella species.
Saline or buffer should not be used in processing because of
or transporting the specimens
the inhibitory effect of sodium to Legionella.
the
The addition of antibiotics to BCYE, such as polymyxin and vancomycin, would make
medium selective.
visible.
Legionella species grow slow in media requiring days or more for colonies to become
SMALL,PLEOMORPHIC GRAM-NEGATIVE BACILI 355
3.
Immunogdiagnosis
a. Indirect fluorescent antibody (IFA)
It is the most
termon method usedfortheserologiediagnosisofLeglonnatre's

CDC (+) result: Greater than
4-fold rise in
disease.
L. pneumophila serogroup 1 between

IFA antibody titer to greater than 1:128 against
specimens. paired acute- and convalescent-phase serum
b.
Direct fluorescent antibody (DFA)
tas lused
has cor detecting
low sensitivity common Legionel species in the lower respirationy tract though it
rate.
A positive result exhibits yellow- or green-colored bacilli.
4. Antimicrobial Susceptibility Testing
it is not recommended for Legionella due to the intracellular nature of the organism.
Antibiotics
of infection.
such as macrolides and fluoroquinolones show positive response in the treatment
5. Rapid methods
a. Molecular Diagnosis - DNA test (PCR)

b. Urine antigen test - detects L. pneumophila serogroup 1 as early as three days from infection
using immunochromatographic method; enzyme immunoassay is the alternate method.
It belongs to the family Flavobacteriaceae.

It is found as indigenous microbiota of the oral cavity of humans and animals (dogs and cats).
enhance the
It resembles the HACEK group as to requirements for carbon dioxide to growth and
isolation from blood cultures.
reaction to most biochemical tests.
It is facultatively anaerobic with a negative
fermenter).
It is a carbohydrate fermenter (glucose, maltose, lactose, and sucrose
It is atrichous but may exhibit a gliding motility on solid surfaces.
oral ulcers utilizing blood
It has been isolated from individuals who have neutropenia with
cultures as specimens.
Microscopy: Cram-negative rods to filamentous or spindle-shaped bacteria (fusiform rod)
shiny and
exhibit pigmentations with spreading pattern
Culture: BAP or CAP Colonies are
away from the center of the colonies.
Biochemical test for human isolates: () Oxidase and catalase

Related infection. Periodontitis, endocarditis, and septicemia canimorsus, and
C. sputigena, C. haemolyticus, C.
Human isolates: C. ochracea, C. gingivalis,
C. granulosa
Common isolate: C. ochracea

Agent of dog-bite infection: C.
(causes septicemia in humans particularly those with
asplenia or alcoholism after dog or cat bite)
BACTERIOLOGY
FIGURE 22-1. Gram stain of Haemophilus influenzae

(Photo from Fujita Health University)
FIGURE 22-2. X and V factor test for Haemophilus

(Photo from Wikimedia Commons) Banurdlurs
FIGURE 22-3. Haemophilus influenzae showing "satellitism" growth

around the Staphylococcus aureus streak line
(Photo by M. Miller)
SMALL,PLEOMORPHIC CRAN-NEGATIVE BACILL 357
FIGURE 22-4. Gram staining of Brucella melitensis

(Photo from USCDCP)
FIGURE 22-5. Brucella abortus on blood agar plate

(Photo by T. Parker & A. Marsh)
olonies on sheep's blood agar

FIGURE 22-6. Brucella
melitensis
(Photo by L.Stauffer)
358 REVIEW HAND OOK IN DIAGNOSTIC BAC ERIOLOGY
FIGURE 22-7. Colonies of Francisella(Photo

tularensis on brain-heart infusior
by L. Stauffer)
FIGURE 22-8. Bordetella pertussis on Regan Lowe agar

Source:https://www.who.int/immunization/sage/meetings/2014/april/2_Laboratory_manual_WH0_2013Update pdt
t
SMALL,
ASSESSMENT QUIZ

correct answer.
1. What is the
most common member of HACEK that causes endocardifis?
a. Haemophilus parainfluenzae
b. Aggregatibacter aphrophilus
C.
d. Kingella
2. Which of the following is the best specimen for isolation

of Bordetella pertussis in children?
a. nasopharyngeal swab
b. saline-induced sputum
c. nasopharyngeal aspirate
d. throat swab
3. Which of the following statements is INCORRECT about Francisella tularensis?

a. It grows on Modified TMA
b. Cysteine is preferred over cystine as a growth enhancer
C. It is isolated from BCYE medium
d. Endotoxin is the principal virulence factor

Microscopy: Ovoid, closed-safety pin
Motility: Negative
mushroom smell
Culture: Green to brown halo around colonies,
Oxidase: Positive
Indole: Positive:
Ornithine decarboxylase: Positive

ONPG: Negative
Variable
Carbohydrate Fermentation:
a.
Hemophilus aegypticus
b. Pasteurella multocida
C. Legionella
d.
Capnocytophaga
5. This selective culture medium contains potato glycerol and sheep blood.
a. Stainer-Scholte
b.
Bordet-Gengou
C. Jones-Kendrick
d. Trypticase soy agar

IN DIAGNOSTIC
BACTERIOLOGY
360 REVIEW HANDBOOK
6. It grows as satellites
around colonies producing nicotinamide adenine dinucleotide.
a. Pasteurella
b. Francisella
C.
d. Legionella

rosette formation
Microscopy: Small, coccobacilli with
Motility: Negative
Culture: Small colonies with pitting
Oxidase: Positive
Indole: Positive
Ornithine decarboxylase: Negative

Carbohydrate Fermentation: Positive
a. Eikenella corrodens
b. Brucella abortus
c. Francisella tularensis
d. Cardiobacterium hominis
8. It can be utilized to isolate Legionella pneumophila.
b. Modified TMA
c. Skirrows
d. a and b
9. What are the routes of transmission of Brucella?

a. aerosolized through inhalation of animal carcasses
b. skin cut
C. oral-fecal
d. a and b
10. Identify the possible organism after phenotypic testing.

Microscopy: Small coccobacilli with sand appearance
Motility: Negative
Culture: Translucent, nonhemolytic
Catalase: Positive
Oxidase: Positive ouaule bae

Urease: Positive
Inhibition by dye: Positive
Carbohydrate Fermentation: Negative

a. Brucella abortus
b. Bordetella bronchiseptica
c. Capnocytophaga
d. Haemophilus ducreyi
CHAPTER 23
AEROBIC GRAM-POSITIVE
BACILLI

differentiate the characteristics
and virulence factors of the species of
medical interest;
2. distinguish between the traits and clinical significance of Bacillus

anthracis and Bacillus cereus;
explain the pathogenesis of Corynebacterium species, Listeria, and

Erysipelothrix;
4. describe the aerobic actinomycetes and related diseases and infections;

5. outline the culture media used in the isolation of this group of bacilli;
and
6. design an algorithm of diagnostic tests to identify the aerobic Gram-olsnt brt

positive bacilli.
Bacillus
It belongs to the family Bacillaceae.

The species of this genus are spore-forming, aerobic or facultatively israli
anaerobic, and rod-shaped bacteria; and they can be isolated from
the
soil.
also be
The species form endospores aerobically but may
anaerobically.
B. anthracis
The species are motile with peritrichous flagella except
and
B. mycoides.
conditions due to their
They can survive in extreme environmental
endospores.
Microscopy: Large, boxcar-shaped,
Gram-positive rods with clear,
unstained, central spore or "empty space"
Biochemical test: (+)Catalase and
Voges-Proskauer; some species may
exhibit glucose fermentation
DIAGNOSTIC
BACTERIOLOGY
B. subtilis, B. thuringiensis, B. pumilus, and B. licheniformis

B. cereus,
Common isolates: B. anthracis, thuringiensis, and B. mycoides
B.
anthracis, B. cereus,
Clinically significant species: B.
B. cereus
Common human pathogens: B. anthracis and
Three (3) Major groups of the Genus Bacillus

1. Bacillus cereus Group
B corcus type species, B anthracis, B cytotosicus, B. megaterium, 3. myeorde, and B, thuringiensis
2. Bacillus circulans Group
lentus
B. circulans (type species), B. coagulans, B. firmus, and B.
3. Bacillus subtilis Group

B. pumilus
B. subtilis (type species), B. amryloliquifaciens, B. licheniformis, and
Source: Henry's Clinical Diagnosis and Management by Laboratory Methods, McPherson and Pincus, 23rd ed., 2017.
Bacillus anthracis (Anthrax bacillus)

It is the causative agent of anthrax.
It is not part of the indigenous human microbiota.

It is the most pathogenic species in the genus Bacillus.
It is not highly contagious and can grow aerobically or anaerobically.
It is non-motile and a halophilic organism that can withstand up to 7% NaCl.
It is a potential biological weapon of mass destruction (select agent).

It grows in a low pH (< 6.0) environment, produces lecithinase, and ferments glucose.
"string-of-pearl" appearance when penicillin (10 U/mL) is added into the culture
It exhibits a
medium, describing its susceptibility to such antibiotic.

Virulence factor:
D-glutamic acid capsule and protein exotoxins (edema factor, protective
antigen, and lethal factor)
Growth factor: Thiamine (Vitamin B,) Md0196
Modes of acquisition: Inhalation of and

spores during exposure to infected animals
contaminated animal products, through skin cuts, and ingestion of food and water with spores.
Related disease: Anthrax velv
Microscopy: Gram-positive, large, encapsulated, and square-ended rod; has "bamboo fishing
rod" appearance with an
unstained central spore
Culture: BAP -
Colonies have "medusa head" and non
appearance with swirling projections
They also exhibit a "beaten egg white" used
to pull-up the colonies. appearance when an inoculating loop is
AEROBIC GRAM-POSITIVE BACILLI 363

Anthrax
It is a disease that primarily affects animals, such as
contaminated goat and sheep, by
with the spores and not through animal-to-person feeding on plants that are
transmission.
The protein exotoxins
cause the signs and symptoms of anthrax
Person-to-person transmission has not been documented
Meningitis is reported as one of the complications of
anthrax.
A serum sample can be used to detect the lethal
factor toxin.
Vaccination is recommended to those
persons who are at risk for occupational exposure.
Types of Anthrax
a. Cutaneous Anthrax
It is the most common

type of anthrax and the least fatal. fom ad sh
It is acquired through skin cuts and abrasions.

A small papule appears at the site of the spore inoculation two to five days after exposure.
It is characterized by the appearance of a "black eschar," which is necrotic and painless
central area that does not produce pus.
Preferred sample: Vesical fluid or swab specimens from the edge of the eschar
b. Pulmonary Anthrax/Woolsorter's Disease
It is the most severe form of anthrax.
It is acquired when spores are inhaled into the pulmonary parenchyma.

It resembles an upper respiratory tract infection.
The clinical manifestations appear within a week of exposure, however in severe cases, the
onset of symptoms to fatality may take within 24 hours.
dyspnea
Signs and symptoms: Mild fever, fatigue, malaise, and
Preferred sample: Sputum
c. Gastrointestinal Anthrax
Spores are inoculated into a lesion on the intestinal mucosa following their ingestion.
Infection occurs after 1 to 7 days of exposure.
bloody diarrhea
symptoms: Abdominal pain, nausea, anorexia, vomiting,
and
Signs and
Preferred sample: Stool
d. Injection Anthrax
of prohibited drugs.
This type of anthrax has been identified among users
It is associated with severe soft tissue infection.
the affected areas and blood culture
Preferred sample: Swab specimens from
364 REVIEW HANDBOOK IN DIAGNOSTIC BACTERIOLOOY
Laboratory Diagnosis for Bacillus anthracis and blood

Specimens: Malignant pustule, sputum, stool, CSt, blood, lung tissue, and CSF.
B. anthracis is typically
isolated from normally sterile sites such as
in a biological safety cabinet with
anthracis should only be done
Processing of samples for
B.
(weaponized anthrax) and high-risk disinfection

of the work
provisions for biosafety level 3
station.
Testing of B. anthracis is only conducted in reference laboratories.

won en borioges
CDC Recommendation
each for the culture and molecular assay (PCR).
It is important to collect 2 separate swabs, one
sterile saline.
The swab for collection of the samples should be pre-moistened with
For a locale without access to a clinical laboratory facility, samples intended for both culture
and RT-PCR should be transported at 20C to 8'C while -70 °C is the best storage condition if the
specimens will be for molecular assay only.
1. Microscopy
The presenceof large, boxcar-shaped Gram-positive bacilli with unstained central spore in
any specimens should be considered significant and presumptive.
of direct samples.
Isolates from culture plate can be utilized for staining aside from the use
The use of old cultures in staining may result in gram-variable cells.
Fluorescent microscopy can provide rapid presumptive diagnosis.
Spore stains: Malachite green and McFadyean stain
Capsule stain: India ink (for blood or CSF specimens)
both B. anthracis
CDC: Demonstration of B. anthracis in immunohistochemical staining using
cell wall and capsule monoclonal antibodies.
2. Culture
The gold standard for the laboratory diagnosis of B. anthracis is cultivation/culturing.

Culture media: BAP CAP, Egg yolk agar (EYA), Phenylethyl alcohol (PEA), Polymyxin-
lysozyme-EDTA-thallous acetate (PLET), Bicarbonate agar, and Nutrient broth
Enrichment and selective technique: Application of heat or alcohol shock technique before
plating
PEA is recommended for identification of B. anthracis in fecal specimens.
PEA and PLET are used in isolating Bacillus species from contaminated specimens.
EYA is used to determine if the B. anthracis produced lecithinase, in which the medium will
have an opaque zone.
A
gelatin medium is utilized to observe the"inverted-pine-tree" appearance of B. anthracis.
Cultures can also be incubated with an increased
CO, to stimulate capsule formation.
3.
CDC: Four-fold rise in antibodies to

or a fourfold
change in antibodies toprotective antigen between acute and convalescent sera
CDC-Quantitative anti-PA protective antigen in pairedconvalescent sera usingthe
immunoglobulin ELISAtestingin an unvaccinated person.
fluorescent antibody (DFA) assay is also suggested, and it serves another point of
Direct
confirmation for the presence of B. anthracis. as
Ascoli Test (Precipitin Test)
It detects thermostable anthrax antigens.

It uses rabbit antiserum to
observe precipitin formation.
Sample: A 2-g sample in a 5-mL saline that
is placed in a 1/100 final concentration of acetic
acid
(+) Result: Formation of precipitin band after less than 15 minutes
4. Antimicrobial Susceptibility Testing

CDC: Ciprofloxacin or
doxycycline should be used for initial intravenous therapy until
results for the AST are released.
B. anthracis are susceptible penicillin and can be utilized for therapeutic management
to
together with other antibacterials such as tetracycline and clindamycin as cohort drugs.
5. MALDI-TOF Mass Spectrometry
This method has been very useful for the accurate differentiation of the genus Bacillus from
CDC: Detection of B. anthracis or anthrax toxin genes by the LRN-validated PCR and/or
sequencing in clinical specimens collected from a normally sterile site (such as blood or CSF)
or lesion of other affected tissue (skin, pulmonary, reticuloendothelial, or gastrointestinal).
Bacillus cereus ("Fried rice" bacillus)

of contaminated cooked rice dishes (typical source)
It causes food poisoning due to the ingestion
or other food products.
infections that causes eye
It is the most commonly encountered
Bacillus species in opportunistic
and ear infections.
to penicillin,
but it is susceptible to vancomycin.
It exhibits motility and resistance
with spreading growth and a
Culture: BAP Colonies are large, feathery, beta-hemolytic
"frosted-glass' appearance.
and salicin fermentation; glucose fermenter
Biochemical test: (+) Lecithinase
(heat-stable and heat-labile), cerelysin, phospholipase C, and
Virulence factors: Enterotoxins
Pyogenic toxin
105 cells/gram)
Best specimen for
isolation: Suspected contaminated food (
BACTERIOLOGY
Tynes of food polsoning or gastroenteritis that are caused by B. cercus -orgaloneurninl

blot tous 000
a. Diarrheal type
vegetables.
It is associated with the ingestion of contaminated poultry, meat, and
Incubation period: 8 to 16 hours (long incubation)
diarrhea without fever
Symptoms: Abdominal pain and watery
Production of heat-labile enterotoxin
b. Emetic type
It is associated with the ingestion of improperly stored cooked/fried rice.
It is caused by B. cereus type
Incubation period: One to six hours (short incubation)

Abdominal cramps, nausea, and profuse
vomiting but no fever
Symptoms:
(+) Production of heat-stable enterotoxin
TABLE 23-1. Differential Characteristics of Bacillus Species

Bacillus anthracis Bacillus cereus
Characteristics
Non-motile Motile
Motility
Capsule Encapsulated Non-encapsulated
Non-hemolytic
Growth on PEA
Growth at 45°C Zero to slight growth mast Rapid growth

Penicillin sensitivity Sensitive Resistant
"String-of-pearl" pattern
Salicin fermentation
Gelatin hydrolysis
Lecithinase production
v - variable
Bacillus subtilis (Hay bacillus)

It is the most commonly encountered laboratory contaminant.
It is halophilic organism that can tolerate up to 7% NaCl.
It is the source of the bacitracin antibiotic.
It can cause
an eye infection among prohibited drug users.
Culture: BAP - Colonies are large, flat, and dull with a ground glass appearance, and beta-
hemolytic; they may exhibit pigmentation (pink, yellow, orange, or brown).
Biochemical test: (t) Mannitol, xylose, and arabinose fermentation
Bacillus pumilus
It is used as a
biological indicator in sterilization
methods.
Culture: BAP
- Colonies are large and
Biochemical
moist with a blister-like appearance, and beta hemolysis.
test: (+) Mannitol and xylose fermentation
Bacillus thuringiensis
It is an insect pathogen.
It produces
parasporal crystals that can be utilized as a pesticide.
It secretes cereulide toxin
which serves as a virulence factor.
It causes food poisoning and ocular and wound infection.
Corynebacterium
It belongs to the family Corynebacteriaceae.
Majority of the species are found as indigenous microbiota on the skin and mucous membranes
of humans and animals.
Some of the members

are pathogenic to animals, plants, and humans.
The species are found in fresh water, salt water, soil, and air.
They are facultatively anaerobe, non-motile, non-encapsulated, non-spore-forming, and highly

pleomorphic rods.
This genus is closely related to Mycobacterium and Nocardia - the cell walls of Corynebacteria
contain mesodiaminopimelic acid (m-DAP) as the diamino acid and short-chain mycolic acids.
They are glucose and maltose fermenters except C. urealytcum and C. pseudodiphtheritic.
Microscopy: Gram-positive rods, slightly curved with unparallel sides, and wider ends that
produce a "club-shaped" appearance and palisade arrangement. Cells are highly pleomorphic,
arranged in pairs and create X, V, Y, and L formations that closely resembles "Chinese letters".
Culture: BAP - Small, smooth to granular appearance, moist-like to matte, with white to gray
colored colonies
Colonies have a small zone of -hemolysis although some strains are non-hemolytic.
Biochemical test: (+) Catalase and oxidase
Groups of Corynebacteria
a. Lypophilic corynebacteria
The members of this group are fastidious corynebacteria that require 48 hours of incubation
before any growth can be observed.
The addition of lipids in the culture medium enhances the bacterial growth.
Examples: C. jeikeium and C. urealyticum

b. Non-lipophilic corynebacteria
This group exhibits fermentative or oxidative metabolism.
Examples: C. amycolatum, C. diphtheriae, C. pseudotuberculosis, C. pseudodiphtheriticum,
C. striatum, C. ulcerans, and C. xerosi
Corynebacteria that are associated with human infections and diseases:

C. diphtheriae, C. jeikeium, C. pseudotuberculosis, C. pseudodiphtheriticum, C.
ulcerans, and
C. urealyticum
It is also known as the diphtheria bacillus or Kleb-Loffler bacillus.
It is a non-lypophilic, facultative anaerobe that grows best under aerobic conditions.
It is not part of the indigenous microbiota of the respiratory tract, and it only inhabits the human
nasopharynx in a carrier state.

It is acquired through inhalation of contaminated respiratory droplets where the pharynx and
tonsils are the common sites of infection or direct contact with infected cutaneous lesions (hand-
to-mouth transmission) and contaminated materials.
It rarely enters the bloodstream.
It is readily killed by heat and by most of the usual disinfectants.
It is a glucose and maltose fermenter.
Virulence factor: Diphtheria toxin
Preferred medium: Enriched medium with serum, cystine, and potassium tellurite
Biochemical test: (+) Nitrate reduction; (-) urease

Best specimens: Nasopharyngeal and throat swabs
Biotypes of Corynebacterium diphtheriae

a. Intermedius
small, shiny, and grayish-black colonies; small zone of $-hemolysis
b. Mitis medium-sized colonies, convex, smooth with black pigmentation and "fried-egg'
-
appearance; p-hemolytic
C. Belfanti - medium-sized colonies, grayish-black in color, and opaque; small zone of B-hemolysis
d.
Gravis - large, flat, and glossy smooth grayish-black colonies with "daisy-head" appearance; non-
Diphtheria Toxin
It is a heat-labile
polypeptide secreted by C. diphteriae.
It is produced by strains with a lysogenic $-phage that carries
the TOX gene.
It inhibits protein synthesis.
It causes tissue necrosis and exudate

formation (pseudomembrane lining) over the tonsils,
larynx, and pharynx.
It favors an alkaline pH (7.8 to 8.0), an aerobic environment, and a sufficient amount of iron in the
medium for production to facilitate in
vitro survival.
It is released when the iron in

the culture medium is consumed.
It is also produced by C. ulcerans
and C. pseudotuberculosis, hence they belong to the
"Corynebacterium diphtheriae" group.
Types of Toxin Fragments: Fragment A (active site) and Fragment B (receptor binding site)
1. Respiratory diphtheria
It is an acute, infectious disease that is characterized
by the production of a systemic
false membrane lining (pseudomembranous formation) of the throat that may
toxin and
eventually lead
to respiratory obstruction.
Signs and symptoms: Low-grade fever, thick mucopurulent nasal discharge, and cough
Control measure: Immunization (DPT vaccine)
Diphtheria antitoxin is administered to neutralize any unabsorbed exotoxin in the patient's
tissues.
2. Cutaneous or skin diphtheria (Veldt sore or Barcoo rot)

It is characterized by slow, non-healing ulcers and membrane formations.
The toxin is absorbed systematically but is less severe.
Corynebacterium amycolatum
It is part of the indigenous flora of the skin and nasopharynx.
It is isolated from various human biofluids such as blood, urine, prostatic secretion, and CSF.
It can cause ear infection and bloodstream and prosthetic joint infections especially in
immunocompromised patients.
It exhibits multiple antibiotic resistance.
Sengupta et al (2015) reported the presence of metachromatic granules in this organism which
can be demonstrated by Albert stain.
Culture: P - Colonies are flat and dry with waxy appearance.
(+) Metachromatic granules

in nitrate reduction
Biochemical test: (+) Urease and glucose fermentation; variable result
Corynebacterium jeikeium
It is a skin microbiota that is found in inguinal, axillary, and rectal
sites.
(B-lactams) bacterium.
It is an obligate aerobe and multi-antibiotic resistant
It is isolated from immunocompromised individuals (AIDS) and patients with meningitis.
adults.
diphtheroid prosthetic valve endocarditis
in
It is a common cause of
and arranged in V-shaped forms
Microscopy: Gram-positive, pleomorphic, club-shaped,
and the addition of 1% Tween 80 produces
Culture: BAP Colonies appear with metallic sheen,
larger growth.
Biochemical test: (-) Urease and nitrate reduction
Corynebacterium pseudodiphtheriticum (Hoffman bacillus)

of the human nasopharynx.
It is an indigenous microbiota
UTI, and cutaneous wound infections in
It causes diphtheria-like respiratory infection,
immunocompromised patients such as those who have AIDS.
palisades and do not exhibit any other
Microscopy: Cells are arranged in parallel rows or
characteristic "pleomorphism" that is similar to other corynebacteria.
Biochemical test: () Urease and nitrate production
Corynebacterium pseudotuberculosis
It is an animal pathogen where humans can contract through direct exposure with infected
animals.
It produces a dermonecrotic toxin that causes death of cells.

It can also secrete diphtheria toxin.
Culture: CTBA - Colonies exhibit a black color surrounded by a brown halo.
BAP - Colonies are small and yellowish-white.
Biochemical test: (+) Urease; (-) gelatinase
Corynebacterium ulcerans
It is acquired through animal contact or consumption of unpasteurized dairy products. 11100
It is associated with diphtheria-like sore throat since a significant number of isolates have shown
to produce the same potent toxin of C. diphtheriae.
It is also isolated from skin ulcers and exudative
pharyngitis.1 535 921145 1125
It exhibits growth in Loeffler's serum agar.
Culture: BAP - Colonies have a narrow zone of beta hemolysis.
CTBA - Colonies are surrounded by a brown halo.
Biochemical test: (+) Urease, glycogen fermentation, and gelatinase; (-) nitrate reduction
Corynebacterium urealyticum
It is one of the most frequently isolated and most clinically significant Corynebacteria.
It is a urinary pathogen, a strict aerobe, and lipophilic.
It does not ferment glucose and maltose.
Microscopy: Gram-positive slightly curved rods, arranged in V-shaped forms and palisades
Culture: BAP - Colonies are pinpoint, white, smooth, and are non-hemolytic.
Biochemical test: Rapid urease producer; () nitrate
Specimens: Nasopharyngeal and throat swabs, urine, blood, and wound
discharge(aspirate)
Nasopharyngeal and throat swabs are the best specimens for isolation of C. diphtheriae, obtaining
samples
from the inflamed regions of the nasopharynx and below the pseudomembrane.
A calcium alginate swab is preferred for collection.
1. Microscopy
Corynebacteria appear
as highly pleomorphic, Gram-positive, short or slightly curved rods
The
various arrangements of C. diphtheriae are due to
multiplication.
its incomplete fission during
Methylene blue staining: Bacterial cells exhibit a beaded formation.

Neisser and Albert stains are used
for staining metachromatic granules.
Neisser staining method is composed mainly of either methylene blue or crystal violet.
Malachite
green and toluidine are the components of Albert stain.
Culture
Culture media:
BAP, CAP, CTBA, Tinsdale agar, and Loeffler serum agar
The primary inoculation of throat swabs to a Loeffler slant and the overnight incubation and
subculture of any growth to CTBA may
respectively improve the recovery of C. diphtheriae
for both media.
CTBA and Tinsdale agar should be incubated for at least 48 hours at 35°C.
The multiplication of corynebacteria occurs within the range of 15°C to 40 °C.
Tinsdale agar
It is composed of sheep blood, cystine, potassium tellurite, and sodium thiosulfate.
(+) Result: Colonies exhibit black color surrounded by a brown halo.
All the biotypes of C. diphtheriae have brown haloes around their colonies.
Cystine tellurite blood agar (CTBA) 036210512 to 9R012757100 aris

It is the preferred medium for the isolation and identification of corynebacteria.
It is both a selective and differential medium.usuibege udi
It contains sheep blood, bovine serum, cystine, and potassium tellurite.

(+) Result: Colonies of corynebacteria exhibit a black or brown color
after 48 hours of
incubation.
brown halo: C. diphtheriae, C. ulcerans, and
(+) Black or brown colonies surrounded by
a
C. pseudotuberculosis
Pai's slant or Loeffler's serum agar

metachromatic granules of
It is useful for observing the microscopic morphology and
C. diphtheriae.
(+) Result: C. diphtheriae colonies exhibit a "poached-egg appearance.
are called Babes-Ernst bodies.
The metachromatic granules of C. diphtheriae
Christensen urea slant

C. urealyticum and other species that are
This is used to observe the urease production of
secreting the same enzyme.
3. Schick test
to diphtheria.
this a skin test that is used to determine the susceptibility of a person
of a small amount of the diphtheria
The procedure involves the intradermal introduction
be harboring the disease.
the individual who is suspected to
toxin into the arm of
site
(+) Result: Redness and swelling around the
4. Toxigenicity test
a. Immunodiffusion test Elek plate test (in vitro test)
(+) Result: 4-mm to 5-mm fine

white precipitin lines at a 45° angle to the streaks of the
after 18 hours to 48 hours incubation at
test organism and control organism is observed
test (in vivo test)

b. Guinea pig lethal test - also known as the diphtheria antitoxin
(in vitro test)
C. Tissue culture test . is used for the detection of diphtheria antitoxin
d. Polymerase chain reaction (PCR) test - is used for the detection of toxin genes
5. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)

It can be utilized for the detection of the toxic strains of corynebacteria.
6. Antimicrobial Susceptibility Tet

C. diphtheriae is susceptible to beta lactam drugs like penicillin.
Antibiotic therapy may be ineffective in the treatment of diphtheria due to the very lethal
property of the toxin; however, it assists in preventing the dissemination of infection.
Immunization is the cornerstone of eradication and prevention of diphtheria. rev
The use of nucleic acid amplificationand sequencing techniques is very important for the
accurate taxonomy of the species under the genus Corynebacterium. 8a s food erdi
PCR is utilized to detect the tox gene of C. diphtheriae.
Results of 16S ribosomal ribonucleic acid (rRNA) sequencing shows that corynebacteria have
similarities with mycobacteria and nocardiae.
TABLE 23-2. Differential Tests for Corynebacterium Species

Species Esculin hydrolysis Gelatinase Nitrate production Urease
C. diphtheriae
C. ulcerans
C. pseudotuberculosis
C. urealyticum
C. pseudodiphtheriticum
+
room temperature; v- variable
Listeria
Listeria monocytogenes
It belongs to the family Listeriaceae
It is both a human and animal pathogen.
It is aerobic or facultatively
It is recovered from the
soil, dust, water, dairy products, and processed meats.
It is both human and
animal pathogen.
It is motile with peritrichous flagella,
and it exhibits a characteristic "tumbling motility."
It can grow in a
high salt medium with up to 10% NaCl.
It has an optimal growth between 30°C and 35°C but can also
grow at refrigerated temperature.
It causes miscarriage or stillbirth in humans.
Primary Mode of Transmission: Ingestion of contaminated food
Principal Virulence Factor: Listeriolysin O (hemolytic and cytotoxic) red antrom PW

Other Virulence Factors: Siderophores, phospholipase C, internalin, E-cadherin (placental
receptor), and surface proteins (p60 and ActA)
Microscopy: Coccobacillarry in form and are arranged singly or in short chains that resemble
streptococci
Culture: BAP Colonies are smooth, translucent, small, grayish-blue, and are surrounded by a
narrow zone of B-hemolysis.
Related infection: Listeriosis
Listeriosis
It is a serious infection that affects neonates, pregnant women, and immunocompromised hosts.
It is acquired through ingestion of contaminated food such as meat, chicken, dairy products, and
vegetables.
Processed meat products should be thoroughly cooked or heated before consumption as a
primary preventive measure.
Immunocompromised individuals and pregnant women are predisposed to listeriosis after
like cheese.
ingesting contaminated dairy products
An infected pregnant woman may pass the organisms onto the fetus (vertical transmission).
host defenses against listeriosis.
Macrophages and T-lymphocytes are the most important
Lypes of Listeriosis
1.
Maternal disease
It usually occurs during the third trimester of pregnancy.
It leads to miscarriage or stillbith (granulomatosis infantisepticum).
fever, headache, and myalgia
Signs and symptoms: Flu-like illness,
BACTERIOLOGY
2. Neonatal disease
It is associated with an intrauterine infection due to the aspiration of infected amniotic fluid
(early-onset neonatal listeriosis).
The infected infants are at full term and appear healthy at birth, then meningitis develops
and is usually seen by the third week of life (late-onset neonatal listeriosis).
000196
3. Disease of immunocompromised hosts p non
develops through the ingestion of contaminated dairy products and processed
meat
It
products.
Specimens: Blood, CSF, and swabs of lesions
It is commonly isolated from blood and CSF.

62760 11
1. Motility test
Wet mount/hanging drop method: Exhibits a "tumbling motility" at room temperature

SIM test: Has an "umbrella-shaped" or "inverted-Christmas-tree" pattern at 25 °C but not at
2. Culture
Culture media: BAP, CAP, BHI, thioglycollate medium, McBride agar, and nalidixic acid
medium
Enrichment technique: Cold enrichment (4'C using broth, then incubated for weeks)
Colonies and hemolytic patterns may be confused with those of group B streptococci.
Growth occurs at a wide temperature range, from 0.5°C to 45°C.
It requires slightly increased amount of CO,.
3. Biochemical test i Bib montane
(+) Catalase and motility

(+) CAMP reaction "block-type" hemolysis
(+) Hippurate and bile esculin hydrolysis

(+) Growth in 6.5% NaCl
(+) Glucose fermentation
(+) Voges-Proskauer and methyl red tests
(-) H,S production, nitrate reduction, and urease
4.
Antimicrobial susceptibility test
L. monocytogenes is sensitive to
antibiotics like aminoglycosides and penicillin.
It has
less pattern of antimicrobial resistance compared to other Gram-positive bacilli.
There are three (3) species in this genus, namely, E.

thusiopathiae, E. tonsillarum, and E. inipinata.
tysipelothrix rhusiopathiae
It belongs to the family Erysipelothrichaceae.
It is not part of the indigenous human microbiota.
It is facultatively anaerobic, non-motile, and non-spore-forming.
It is the only catalase-negative, non-spore-forming, Gram-positive, rod-shaped bacterium that
produces hydrogen sulfide.
It is acquired by direct contact with infected excreta, blood, and flesh of animals through skin
breaks.
It is isolated from wild and domestic animals like birds and fish.
Microscopy: Gram-positive, thin, pleomorphic rods that are arranged singly, sometimes in short
chains with a V-shaped formation
Culture: BAP Colonies are pinpoint with a-hemolytic or non-hemolytic zone.
Gelatin stab culture: Has: pattern of a "pipe cleaner" or "test tube brush" at 22°C
Virulence factors: hyaluronidase and neuraminidase
Major reservoir: Domestic swine
and fish handlers
Predisposed individuals: Veterinarians
- Related infections: Erysipeloid (red-skin infection), endocarditis, and septicemia
.m verl) elavloms/f slod
Best specimen: Tissue biopsies or aspirates from skin lesions
The organism is localized in
the deeper layer of the subcutaneous tissue.
site for collecting specimens.
For skin lesions, the outward margin of the lesion is the best
1.
Microscopy
E. rhusiopathiae has a tendency to form filaments.
2. Culture
Culture media BAP CAP, nutrient broth, Columbia

when specimens are first
in
issue and aspirates is enhanced incubated
Isolation of E. rhusiopathiae with glucose supplement and
at 35 °C in an
suspended into nutrient broth
increased CO, environment. to seven days.

on BAP and CAP for up
The organism can grow
ypes of colonies after 48 hours of incubation slender, filamentous with a tendency to over-
colonies flat, curled,

a. Large and rough
deco ore and become Gram-negative bacilli and slender rods
b. Small and smooth colonies - transparent, glistening,
BACTERIOLOGY
3. Biochemical tests
(+) H,S production in a TSI medium

(+) Glucose and lactose fermentation
nitrate reduction, VP, and urease
(-) Catalase, oxidase, esculin hydrolysis,
TABLE 23-3. Differential Tests for Gram-positive Non-spore-forming Bacilli
Esculin Nitrate
Gram-positive bacilli Urease
Catalase Motility hydrolysis . reduction
(Non-spore-forming)
teris Corynebacteriuma bor .oilieo tanD
+ +
Listeria monocytogenes +
+
Fid. Erysipelothrix rhusiopathiae orto Bold 115700
v - variable
bre bliw mot betsloal at fI
Other Gram-positive Aerobic Bacilli
Arcanobacterium
It belongs to the family Actinomycetaceae.
The species of this genus are classified as pleomorphic rods, non-motile, non-sporeforming, and
catalase negative.
The species are microbiota of the skin and upper respiratory tract.Bubivibrd bsaogprbonkin
They can cause soft tissue infections, pharyngitis, and endocarditis. d enoisiru baisls!
Microscopy: Gram-positive rods with faintly staining reaction
Culture: BAP - Colonies have smooth to rough appearance, grayish white with narrow zone of
beta hemolysis; they may exhibit pitting of the agar with black opaque dots under each colony
Biochemical test: (+) Lipase, Lecithinase, and Reverse CAMP; (-) Catalase
Significant species: A. haemolyticum noteal ofd to nigmam buswiuo odll anolesl mislool
It belongs to the family Planococcaceae.

The members of this genus are obligately aerobic, non-sporeforming, Gram-positive rods that are
motile by peritrichous flagella.
Goen and MacLea (2018) discussed that the type species have been isolated from meat and dairy
products, water and air samples, and from the intestines of poultry.
The members of this genus have not been associated yet
with any serious human infections or
diseases.
Culture: BAP Colonies exhibit

large "medusa-head"appearance.
Nutrient agar Colonies exhibit a rhizoid growth.
Catalase;(-) gelatinase and oxidase
Species: K. zopfii (type species), K. gibsonii, K. sibirica, K.
massiliensis, and K. huakuii (as cited by
Fang et al, 2015)
It belongs to the family Micrococcaceae, hence the description coccal to rod appearance."
The species
are
normal flora of the oropharyngeal which can be isolated in saliva and in dental
plaque.
The members are nonmotile bacilli with tendency to become filamentous like the actinomycetes.
The species are related to bacteremia and endocarditis.
Microscopy: Gram-positive bacillary form resembling corynebacterial and may transform to
cells when grown in broth culture
Culture: Colonies are small creamy-white color with smooth to rough appearance.
Biochemical test: (+) Bile esculin hydrolysis and nitrate reduction; (-) urease niapearmoond
Species: R. mucilaginosa and R. dentocariosa
It belongs to the family Paenibacillaceae.

The members of this genus are Gram-positive aerobic rods.
They have been isolated from the environment and human samples.
The species have also been isolated from contaminated blood culture bottles and intensive care
unit.
They are potential sources of antimicrobial agents and

the species secrete enzymes and
substances that support plantations and cosmetic and fuel production.
Distinguishing Characteristics: Endospore-forming bacilli
Species: P. alvei, P. macerans, P. pasadenensis, and P. polymyxa 2:1:5 1:tos 1iabrge plovi,5/ a
Aerobic Actinomycetes
are composed of Gram-positive, nonsporeforming
The members of this large and diverse group
bacilli.
They are classified as aerobes with a branching filamentous growth that extends along the agar
due to the aerial hyphae
due to the substrate hyphae and into the agar
one week to two weeks of incubation or
They are slow-growing bacteria, and
they may require
even
longer.
They can be solated from culture media which supports the growth of fungi.
They are omnipresent in the soil and organic material
They cause diseases in humans
and many animals.
Microscopy Filamentous Gram-postive rods with a beaded appearance
filamentous forms while some organisms form
branching,
Culture: Cells elongate to form
hyphae on the agar surface or into the agar.
BACTERIOLOGY
Acid-Fast Aerobic Actinomycetes
It belongs to the family Nocardiaceae. and non-motile.

acid-fast bacilli, obligate aerobic,
The species of this genus are partially
Their cell wall contains peptidoglycan,
meso-diaminopimelic acid (DAP), and the sugars,
01 arabinose and galactose.

recover fungi.
The species grow on media that are used to
Microscopy: Cram-positive bacili with long, thin, beaded, branching flaments; oid
cultures may
mammogroom
fragment into short, rod-shaped, coccoid forms.

Culture: Colonies exhibit wrinkled,
"chalk-like" appearance, sometimes waxy, with varied
"plowed field" odor; with aerial hyphae
pigmentation such as orange, pink, and yellow, and
Most isolated species: N. brasiliensis, N. nova, N. cyriageorgica, and N. farcinica
adli ohegnoled)
Biochemical test: (.) Catalase, urease, and nitrate reduction1--591
Differential test: Resistant to lysozyme nos ovificoq-isid 91s auni
Related Infections and Diseases ossnimsino mont betsloat noed oali oved etooge ent
that are present in dust and soil or
Infection is acquired through the inhalation of the organisms
soil or ingestion of the same material can also initiate a

Contact with the contaminated
gastrointestinal infection.
Nocardia species can cause cutaneous infection and invasive pulmonary infections with
hematogenous dissemination but have not been associated yet with human-to-human
transmission.
1. Pulmonary Nocardiosis
It is a confluent bronchopneumonia where the sputum is thick and purulent although the
encapsulation of the abscess is absent and without granuloma formation.
The affected tissues do not have sulfur granules. trt bms ssnqyil shsriadure oft of aub
10 nodded
Causative agent: N. cyriacigeorgica and N. farcinica
2. Cutaneous Nocardiosis (Actinomycotic/bacterial mycetoma)

chronic, localized, painless subcutaneous infection that is the
It is a characterized by
presence of draining pus and sulfur granules in the affected tissue.
Most common causative agent: N. brasiliensis
Other bacteria causing actinomycetoma: Actinomadura madurae and Streptomycessomaliens1s

Specimens: Biopsy or drainage material from actinomycetoma,
sputum, bronchoalveolar '
lung tissue, transthoracic aspirate of a nodule or abscess, CSF, and blood
Pus and tissue specimens are the best
samples in performing a wet mount and staining.
1. Microscopy
To observe the
branching
filaments
modified Kinyoun method should and partially acid-fast characteristics of Nocardia,
be utilized.
methenamine-silver (C
(GMS) stain is used for
specimens. histopathogical examination of tissue
Culture media: BAP, CAP, BHI, TMA, Sabouraud dextrose

agar (SDA), Middlebrook (MB),
potato dextrose agar (PDA), Lowenstein-Jensen, litmus milk, and
tap water agar s1s/
Some Nocardia species are beta-hemolytic.
Initial growth of nocardiae are observed after 48 hours of incubation to 2
weeks.
The species ferment carbohydrates as shown in the
type of media used for the isolation of
Nocardia species.
Nocardia grow in a variety of culture media, provided they do not contain inhibitory agents
like antibiotics, except for TMA since the incorporated antimicrobials prevent the growth of
microbial flora.
The SDA utilized for cultivation of Nocardia spp. does not contain chloramphenicol; SDA and
PDA are known antifungal media.
Inoculating the samples in litmus milk broth or MB 7H10 agar improves the acid-fastness of
Incubation at 10% CO, promotes the growth of Nocardia species. d0.0 ostiowill
Nocardia species may not always survive the decontamination procedures for mycobacteria.
Tap water agar is observe the morphology of actinomycetes, and to differentiate
used to
the branching with aerial hyphae Nocardia species from non-branching (or very minimal
branching) without aerial hyphae Rhodococcus species.
3. Biochemical Test
Tests that will assist in the identification of the pathogenic Nocardia

a. Casein and tyrosine hydrolysis: (+) N. brasiliensis
b. Growth at 45 °C: (+) N. farcinica
brasiliensis
C. Gelatin hydrolysis: (+) N.
d. Opacification of Middlebrook agar: (t) N. farcinica inte Blow
4.
Antimicrobial Susceptibility Testing
while most members are resistant to beta
Nocardia species are susceptible to sulfonamides
lactam drugs like penicillin.
5.
Molecular Assay
The 16S rDNA sequencing
is currently considered the most accurate method for the
identification of the Nocardia species, compared to MALDI-TOR.

antimicrobial susceptibility tests owing to its
replaced
the biochemical and
PCR testing has
various species.
false results in distinguishing the
BACTERIOLOGY
Rhodococcus, Gordonia, and Tsukamurella

Catalase-positive, branching, filamentous, Gramn-positive
bacteria that can
These are aerobic, bonELO!
fragment into rods and cocci.
They can be isolated from the soll, fresh and marine water, and organic matter,
They are primarily acquired through inhalation.
They can grow on most of the non-selective media for bacterial, mycobacterial, and fungal
and SDA, respectively.
isolation such as the brain-heart infusion agar, Lowenstein-Jensen,
Related infections and diseases. Skin infections, pneumonia, peritonitis, and catheter-associated
sepsis
Rhodococcus equi
It belongs to the family Nocardiaceae.
cell wall composed of long-chain
It is asaccharolytic, non-motile, and partially acid-fast, with its
mycolic acid.
It is an intracellular organism which can persist and replicate within macrophages.
It can be acquired through inhalation or accidental ingestion of soil and animal carcasses and
excreta, and exposure to infected animals and poultry, hence, it is a zoonotic organism.
It can infect immunocompromised individuals (HIV patients) and cause slowly progressive,
granulomatous pneumonia.
Microscopy: Coccobacilli with a "zigzag" and scanty branching patterns. nordsdnont

Culture: BAP Colonies exhibit a pale/salmon pink or yellow color; nonhemolytic
Biochemical test: (+) CAMP, lipase, and catalase; variable urease production
Differential test: Variable result to lysozyme test barqur iambs nliw amtrbabu arb n tech
Gordonia
It belongs to the family Gordoniaceae.
The species of this genus vary from Gram-positive to Gram-variable rods.
They are partially acid-fast and non-motile.
It has similar characteristics with Rhodococcus, and the use 16S rRNA sequencing serves as the
differential marker.
It has been isolated from surgical (sternal) wound, coronary artery infection, and pulmonary
disease.
Culture: Colonies are smooth and "slimy" with irregular edges; but they may appear as dry or
rough without mycelia.
Biochemical
test: ($) Nitrate reduction, urease and catalase; (6) arysulfatase
Differential test: Susceptible to lysozyme
Species: G. bronchialis and G. sputi
It belongs to the family Tsukamurellaceae

It is isolated from soil
and water, same with the other Actinomycetes.
The species of this genus
into three parts.
are Gram-positive, nonmotile, and shaped like long rods that fragments
The members are
slightly acid-fast when the Kinyoun method is used.
Microscopy: Rod shaped or coccobacilli, arranged in
pairs or clusters; no aerial hyphae or spore-
like bodies
Culture: Colonies are small, circular, dry with rhizoid

white or orange pigmentation
edges, have no aerial hyphae, and exhibit
Related infection: Cutaneous infection and

Biochemical test: (+) Catalase and urease
Differential test: Resistant to lysozyme

Some of the
species are T. paurometabola, T. sputi, and T. ocularis
rento
Non-Acid Fast, Aerobic Gram-Positive Actinomycetes an 1swye cht ned
Streptomyces
The species are Gram-positive rods with branching filaments but variable hyphae.
They are omnipresent in the environment like the soil and gain access to humans through skin
penetration (cuts and abrasions).
Human pathogen: S. somaliensis (agent of actinomycotic mycetoma)
Culture media for isolation: BHI agar and SDA
Microscopy: Atypical rods with "spore-like bodies" and extensive branching but does not
fragment
Culture: Colonies are grayish-white, dry to chalky and heaped with a "musty basement" odor
Biochemical test: Variable urease production and nitrate reduction
Distinguishing feature: (+) Extensive branching with aerial hyphae (Tap Water Agar Test)
Actinomadura
of the species of this genus are very similar to the
The microscopic and colony morphology
Nocardia species.
The mode of acquisition is same with the genus Streptomyces since both organisms inhabit the
soil.
with Nocardia.nAur
It causes bacterial mycetoma ("madura foot"or "madura belt") same
Microscopy: Branching, fragmented, filamentous rods with intertwining branches and "spore-
like" structures
Culture: Routine agar - Colonies are waxy with varied colors from yellow, pink to red and
exhibit
a "molar tooth" appearance after 14 days of incubation
Biochemical test: (+) Nitrate reduction; cellobiose and xylose fermentation

Differential Test: Susceptible to Lysozyme
-81002
Species: A. madurae and A. pelletieri
Tropheryma whipplei
It belongs to the family Tropherymataceae. bas noboelai euosnefdD :nolbotat bstale$
It is a Gram-positive actinomycete and a facultative intracellular pathogen that resides in the
macrophages.
It is the etiologic agent of Whipple's disease that affects the gastrointestinal tract, joints, and
muscles.
It is isolated from gastric secretions, saliva, blood, human feces, and other human organs other
than the small intestine.
It grows in axenic culture medium apart from the living cell lines (as cited by Dolmans, et al.,
2017).
It is a slow-growing bacterium, which requires an average of 30 days to observe visible colonies
(as cited by Dolmans, et al., 2017).
Microscopy: It has tendency to become Gram-variable due to its intracellular property. og

Preferred specimen: Duodenal biopsy wrno
Gold standard test for identification: Periodic acid-Schiff-staining (they appear as rods within the
vacuoles)
elsa4AEROBIC GRAM-PosITIVE BACILLI 383
23-1.http://textbookofbacteriology.net/Anthrax.html
FIGURESource: Gram-staining reaction of Bacillus anthraci
i l
FIGURE 23-2. McFadyean staining of Bacillus anth

(Photo by L. Stauffer)
FIGURE 23-3. Capsule of Bacil us anthracis through direct fluorescent antibody testing
BACTERIOLOGY
FIGURE 23-4. Capsule of Bacillus anthracis visualized through India ink staining
(Photo from L. Stauffer)
FIGURE 23-5. Capsule production of Bacillus anthracis on bicarbonate agar

FIGURE 23-6. Bacillusanthracis on blood agar plate

(Photo by J.Feely)
2 > AEROBIC GRAM-POSITIVE BACILLI 385
plate showing hon-hemolytic pattern

FIGURE 23-7.
Bacitus anthracis on blood agar
IP.Seidel, Moore,
A. Marsh
T. Parker, & A.
FIGURE 23-8. Gram stain of Corynebacterium diphtheriae

(Public Health Image Library)
FIGURE 23-9. The characteristics"test tube brush" growth pattern of Erysipelothrixthusiopathiae

386 EVIEW HANDB0OK IN DIAGNOSTIC BACTERIOLO
FIGURE 23-10. Gram stain of Nocardia asteroides

(Dhoto from IHCWorld)
e r1ea
ASSESSMENT QUIZ
Encircle the letter that corresponds to the correct answer

1. It differentiates the morphology of actinomycetes,
including the presence of aerial hyphae.
a. Tap water agar
b. Brain heart infusion
agar
C.
d. Middlebrook
2. It is best
isolated after cold enrichment technique. Sobnucmita esinolos doull outie
a. Bacillus anthracis
b. Listeria monocytogenes
C. Streptomyces
d. Actinomadura
3. Identify the possible organism after phenotypic testing:

Microscopy: Thin, pleomorphic rods
Motility: Negative
Culture: Black precipitate in the agar, test tube pattern
Catalase: Negative
Oxidase: Negative
Nitrate reduction: Negative

Carbohydrate Fermentation: Positive
Identify the possible organism.
a. Erysipelothrix rhusiopathiae
110 99/T6 1 b. Rhodococcus equi
C. Corynebacteria
d. Kurthia
of Bacillus anthracis?
4. What is the principal virulence factor
a. exotoxin
b. D-glutamic acid capsule

c. lecithinase
d. a and
5. All are partially acid-fast bacilli, EXCEPT:

a.
b. Gordonia
c. Rhodococcus
d. Trophyrema
6. Identify the possible organism after phenotypic testing:

Microscopy: Highly pleomorphic rods, palisade arrangement
Motility: Negative
Culture: Black colonies surrounded by brown halo
Catalase: Positive
Oxidase: Positive
Urease: Variable
Nitrate reduction: Variable
Carbohydrate fermentation: Variable
Identify the possible organism.

a. Bacillus
b.
c. Corynebacteria
d. Listeria
7. All of the following are considered as a culture medium for isolation of actinomycetes, EXCEPT:
a.
b. SDA
C. litmus milk
d. Pai slant
8. Which of
the following biotypes of Corynebacterium diphtheriae has with fried egg appearance on
BAP and with B-hemolysis?
a. mitis
b. gravis
C.
d. intermedius
AEROBIC
GRAM-POSITIVE BACILLI 389
•. This antimicrobial susceptibility test exhibits the reaction ofBacillus anthracis

a. to penicillin.
b. Schick
C. String of pearl
d. Tinsdale
10.
Identify the organism after phenotypic testing.
Microscopy: Thin, beaded, and filamentous
Motility: Negative
Culture:
Chalk-like colonies, plowed field odor
Tap water agar:
Branching with acrial hyphae
Catalase: Positive
Urease: Positive
Lysozyme treatment: Resistant
a.
b. Rhodococcus
c. Erysipelothrix
d.
CHAPTER 24
MYCOBACTERIA
(ACID-FAST BACILLI)

1. discuss the characteristics of the genus Mycobacterium compare with a ngonnglod
other aerobic bacilli;
2. differentiate Mycobacterium tuberculosis from other members of MTC;

3. explain the clinical significance of nontuberculous mycobacteria;
4. compare Mycobacterium leprae from other mycobacterial species;
5. outline the culture media and growth requirements used in the
isolation of the acid-fast bacilli;

6. describe the automated procedures utilized in mycobacterial
identification; and
7. create an algorithm of diagnostic tests to distinguish the members of
Mycobacterium tuberculosis complex and nontuberculous

mycobacteria.
Mycobacterium
It belongs in the family Mycobacteriaceae.
The species are strictly aerobic, slow-growing, and non-spore-
forming bacilli.
The members are non-motile, catalase-positive, non-encapsulated,
and produce Much's granules.
The cell wall contains N-glycolylmuramic acid mycolic acid that resist
are known as the "acid-
decolorization with acid ethanol, hence they
fast" bacilli (AFB).
than two
Most pathogenic species grow very slow requiring more
weeks of to 379C while the colonies of a group
incubation at 35 •C
in three days at
known as the rapid growing mycobacteria appear
Some 10% CO; for better cultivation.

species require 5% to
AFB Microscopy: Slender, slightly curved or straight rods that have a tendency to "clump!"
Culture: Egg-based are smooth and soft or with a rough and friable growth.
medium - Colonies
pH requirement: 6.5 to 6.8 (culture media)
Generation time: > 12 hours
ulcerans, and M.
Notorious Pathogens: M. tuberculosis, M. bovis, M.
Major Groups of Mycobacteria

1. Mycobacterium tuberculosis complex (MTC)
2. Non-tuberculous mycobacteria (NTM)
Mycobacterium tuberculosis Complex (MTC)

1. Mycobacterium tuberculosis (Koch's bacillus) ales
It is theprincipal etiology of human tuberculosis. unagoratoes
It has the longest replication time among the mycobacteria.
Virulence factor: Cord factor
Replication time: 20 to 22 hours
Specific gravity: 0.79 to 1.07

Growth enhancement: Incubation at 5% to 10% CO,
Biochemical tests: (+) Niacin, nitrate reduction, pyrazinamidase, and urease

(-) T2H inhibition test and catalase test
Growth Patterns of M. tuberculosis on Egg-based Media
Colonies are buff color (nonphotochromogen)

Colonies are raised and dry with "cauliflower-like" appearance.
Rough colonies exhibit "cording" (curved strands of bacilli).
Mycobacterium bovis
It is the cause of tuberculosis in humans and animals (cattle, dogs, cats, and swine).
Itsattenuated or the BCG (Bacillus-Calmette-Guérin) strain is used for the vaccination of
newborns against tuberculosis.
It is acquired by humans through the ingestion of contaminated milk from infected cows or
exposure to these animals including goats, dogs, cats and pigs and their carcasses. bris
Culture:
Egg-based media - Colonies are slow-growing, small, smooth, rounded, and non-
pigmented.
MB 7H10 - Colonies
are rough and dry.
Biochemical tests: (+) T2H inhibition test (do
not grow in medium with T2H) and urease
() Niacin, nitrate reduction, catalase test, and
It has a negative test result in most of the biochemical tests for mycobacteria, with variable
result in the urease test.
MYCOBACTERIA (ACID-FAST BACILLI) 393
3. Mycobacterium africanum
It is associated with
human cases of tuberculosis in tropical Africa or in individuals who had
resided in Africa.
The detection
of this organism requires the use of spoligotyping (spacer oligotyping).
It has variable result in T2H inhibition.

Urease; (-) catalase test, niacin, nitrate reduction, and pyrazinamidase
4. Mycobacterium canettii
It is the smooth strain of M. tuberculosis.
It grows more rapidly than M. tuberculosis (six days on solid media).
It is isolated from children with
acquired immunodeficiency syndrome (AIDS) and
mesenteric tuberculosis.
Biochemical tests: (+) Niacin and nitrate reduction
5. Mycobacterium microti
It has been isolated from TB patients in both immunocopetent and immunocompromised
individuals.
It is found in rodents and is considered an agent of tuberculosis in animals.
6. Mycobacterium pinnipedii and Mycobacterium caprae

These bacteria are first isolated from animals, then further studies explained that they can
also be transmitted to humans.

1. Tuberculosis
It is a disease of the respiratory tract.
It is a chronic granulomatous infection which is acquired through the inhalation of infected

droplets from coughing, sneezing, or talking.
to 5 um airborne droplet of nuclei
Mode of acquisition: Inhalation of 1um
fever, night sweats, fatigue, anorexia, and weight loss
Signs and symptoms: Low-grade
alcoholism, and immunosuppression
Predisposing factors: Malnutrition,
It is usually limited to the detection of a positive tuberculin
Clinical diagnosis of primary TB:
test (PPD)
2. Pott's disease
It is also known as
the tuberculosis spondylitis or skeletal tuberculosis of the spine.
invasion of M. tuberculosis into the
that is caused by the
It is serious form of tuberculosis
spinal vertebrae.
and tissues.
It is confirmed through culture of bones
bones
Common sites of the disease: Hip and knee
Common symptom: Severe back pain

3. Miliary tuberculosis
that affects
is caused by M. tuberculosis many
It is an extrapulmonary tuberculosis that
organs through hematogenous spread.
Common sites of dissemination: spleen, liver, lungs,

bone marrow, kidneys, adrenal gland,
and eyes
Notes to Remember
An individual develops tuberculosis based on the degree of

exposure to M. tuberculosis and the other
members of the MTB complex, the virulence nature of
the strain, and the cellular immune response of
the person against the tubercle bacilli.
M. tuberculosis cellsare phagocytized by alveolar macrophages after initial infection and are able to
replicate intracellularly instead of killing the tubercle bacilli.
The Gohn lesion, which is located in the lungs, is the site of the pulmonary tuberculosis and possible
"storage source" of the organism.
bacilli transit to other
During the period of active proliferation, the macrophages carrying the tubercle
parts of the human body such as the lymph nodes and sensitize the immunocompetent T cells, or to
lymphatics and blood then to lungs, bones, kidneys and brain.
A hypersensitive reaction to mycobacterial antigen causes the pathologic symptoms of tuberculosis.
Tubercle bacilli may not be completely eradicated after recovery from the disease and can remain
viable or dormant for months or years in granulomas, which signifies a possibility of reactivation.
Reactivation of tuberculosis occurs when there is an alteration of the cellular immune system.
Multidrug-Resistant Mycobacterium tuberculosis (MDR-TB)

It develops as a result of the resistance of the M. tuberculosis complex to anti-TB drugs
batootnt to n caused by spontaneous mutation, close contact with a person diagnosed with MDR-TB, after
receiving treatment for tuberculosis (previous TB patient), and residing in a locale with high
prevalence of resistance to antimycobacterial drugs.
Spontaneous mutation related to MDR-TB is due to
noncompliance to proper intake of
antibiotics.
Types of MDR-TB:
a. Primary MDR-TB is defined as without a previous history of tuberculosis and is showing

resistance
at least two anti-TB drugs such as isoniazid and rifampicin.
to
b.
Extensively resistant TB (XDR-TB) - is the resistance to isoniazid, rifampicin, and
fluoroquinolone, and at least one of the three
parenteral second-line anti-TB drugs (amikacin
kanamycin or capreomycin).
Non-tuberculous Mycobacteria (NTM)

These organisms also
known as the atypical mycobacteria or mycobacteria other than
are
tubercle bacilli (MOTT).

These are found in the environment

(soil, dust, and water) and they sometimes colonize the
skin, respiratory tracts, and GIT of healthy individuals.
Chronic pulmonary disease resembling tuberculosis is the usual clinical presentation
associated with these opportunistic pathogens.
Infections that are caused
by these organisms are not usually transmissible from person to
person.
The isolation of this

group of bacteria among individuals with AIDS have contributed greatly
to
the awareness of infections and diseases related to NTM.
Major causes of NTM human infection:
Mycobacterium avium complex (MAC) and
Mycobacterium kansasii
TABLE 24-1. Non-tuberculous Mycobacteria (NTM)

Runyon Classification Color produced Growth rate Species
Group I - Photochromogens Non-pigmented unless 10 to 21 days M. kansasii
exposed to light M. asiaticum
Grow in the dark: M. marinum
cream or buff colonies
Exposure to light:
Orange or yellow colonies
Group II -Scotochromogens Pigmented both in the dark 10 to 21 days
and under light exposure
Yellow- to orange-colored
M. scrofulaceum
colonies
M. xenopi*
Group III - Non-photochromogens Non-pigmented both in the 10 to 21 days M. avium complex

dark and light M. ulcerans
M. terrae complex
M. gastri
Group IV - Rapid growers Varying pigmentation 3 to' days M. fortuitum group

M. chelonae-abscessus
group
M. smegmatis group
NTM - Slow Growing Mycobacteria (NTM-SGM)

1.
Mycobacterium avium complex (MAC)
in humans that is similar
cause of pulmonary infection to
This is the most common
MAC resembles the clinical picture of the pulmonary
tuberculosis - the disease caused by
tuberculosis.
pathogen in immunocompetent and in immunocompromised

individuals - the
This is a
gastrointestinal tract
is the most common site of colonization and dissemination in persons
with AIDS.
It can be isolated from the sputum, blood, and bone marrow aspirates.
with acid-fast bacilli in
It is characterized by the presence of caseating granulomas
pulmonary examination.
Reservoir: Natural water
Portal of entry: Respiratory tract and GIT

Microscopy: Pleomorphic, short, coccobacilli without beading
Periodic acid-Schiff (PAS) staining: Positive
Culture: Transparent or opaque, smooth colonies with mostly nonpigmented
Biochemical test: (+) Tellurite reduction and pyrazinamidase; variable in heat-stable catalase
M. intracellulare
Major Species of MAC: M. avium and
M. timonense
Other species of MAC: M. colombiense, M. marseillense, and
Subspecies of Mycobacterium avium: M. avium subsp. avium, M. avium subsp. paratuberculosis,
and M. avium subsp. silvaticum (wood pigeon bacillus), and M. avium subsp. hominissuis (agent
of most human infections)
Mycobacterium avium
It is a zoonotic bacterium causing diseases in cattle, swine, and poultry.
cause severe diseases in humans due to uncommon transmission

been reported
It has not to
from animal to human.
Mycobacterium avium subsp. paratuberculosis

It is the causative agent of Johne's disease, a zoonotic intestinal disorder or a chronic diarrhea
in cattle, sheep and goat.
It has very slow growth rate requiring three months to four months of incubation.
Mycobactin is added into the culture medium to enhance colony formation.
2. Mycobacterium gordonae (Tap water bacillus)

It contaminates the tap water that is used by patients in rinsing their mouths prior to the
procedure for sputum collection.
It is a contaminant in the preparation of bacteriologic smears.
It rarely causes infection in humans.
Culture: Colonies are smooth with
yellow-orange color (scotochromogen).
Biochemical tests: (+) Tween 80 hydrolysis and catalase test
3. Mycobacterium kansasii (Yellow bacillus)

It is second to M. avium
complex as the cause of NTM lung diseases (chronic cavitary
pulmonary lesions resembling tuberculosis) in humans.
It is
not normally contagious from person to person.
Microscopy: Long rods with distinct crossbanding
and catalase test
Tween
80 hydrolysis (rapid hydrolyzer), nitrate and tellurite reduction,
Culture: MB 7H10 - Colonies are

smooth to rough with dark centers and waxy edges.
Photochromogenic coloniesexhibit dark red crystals of 10-B-carotene.
4. Mycobacterium marinum
It causes diseases on
fishes and can be isolated from aquariums.
It is the
agent of "swimming pool granuloma," which is a red or bluish-red nodule on the
elbow, knee, toe, or finger.
Mode of acquisition: When an

open wound comes in contact with contaminated fresh water
or salt water.
It grows best at lower temperature (28°C to

32°C) compared to other clinically significant
Natural reservoir: Fresh water and salt water
Microscopy: Long rods with cross-banding o nonciorioris

Culture:
Colonies are smooth to rough, wrinkled, and with yellow color (photochromogenic).
Biochemical tests: (+) Tween 80
hydrolysis, urease and pyrazinamidase
5. Mycobacterium terrae complex
It is normally saprophytic and rarely causes human infections.
Species: M. terrae, M. triviale, and M. nonchromogenicum
Microscopy: Short to medium coccobacilli

Biochemical tests: (+) Tween 80 hydrolysis and heat-stable catalase; (+) growth in 5% NaCl for
M. triviale
Culture: M. triviale - Colonies are rough and dry.

M. terrae - Colonies are smooth.
M. nonchromogenicum - Colonies are smooth to rough with white to buff color.
6. Mycobacterium ulcerans
It is the third most common Mycobacterium species after M. tuberculosis and M. leprae.
It is a rare cause of Buruli ulcer which is a painless nodule under the skin after a previous
trauma.
Microscopy: Moderately long rods without cross-banding or beading

are smooth, rough, and non-pigmented (6 to 12 weeks of incubation).
Culture: Colonies
Biochemical test: (+) Heat-stable catalase
7.
Mycobacterium xenopi
from water storage tanks of hospitals.
It is recovered from hot and cold water taps, especially
in adults mostly those with chronic
It is a potential pathogen of pulmonary infection
obstructive pulmonary disease.
It can be either non-photochromogenic or scotochromgeni.
filamentous rods
Microscopy: Long and
BACTERIOLOGY
Biochemical tests: th) Heat stable catalase and aryisulfatase tests f.abd sunte?
and arylsulfatase tests.
It is only strongly positive in the heat-stable catalase
Optimal Growth Temperature: 42°C
Culture: MB 7H10 - Colonies are small with filamentous edges.
Colonies are round with
branching filaments
Cornmeal glycerol agar
1000
Rough colonies have aerial hyphae
TABLE 24-2. Characteristics of Other NTM-Slow Growers

Distinguishing
Biochemical tests
Species Microscopy Culture characteristics
M. asiaticum Acid-fast Dysgonic, smooth, and (+) SQ and heat-stable

rarely causes
coccoid cells pigmented colonies taois
human infections
Photochromogen
Fastidious
M. genavense Distinct acid- Dysgonic colonies; requires (t) SQ and heat-stable
fast cells an extended incubation (six catalase mycobacteria;
to eight weeks)
(+) Pyrazinamidase and on routine
Nonphotochromogen
media; has been
recovered from
BACTEC culture
M. haemophilum Distinct acid- Colonies are rough to 15905 (+) Pyrazinamidase Requires
fast cells; short smooth and non- hemoglobin or
pigmented; the
without recommended media enor growth
beading, include CAP, MHA with 5%
appears in Fildes enrichment, and LJ Negative result
clusters or medium with 2% ferric mil birdios in most
cords ammonium citrate. biochemical tests
Nonphotochromogen
Short Non-pigmented, smooth, (+) Tween 80 hydrolysis, Growth at 22°C
glistening, and opaque telurite reduction, and requires 7 to
without colonies with dense centers
crossbands 12 weeks of
Nonphotochromogen incubation
M. scrofulaceum Medium to Light yellow to deep

orange (+) SQ and heat-stable
long rods smooth colonies with dense catalase
centers
M. simiae Short
Filamentous colonies; (+) Niacin It is one of the
yellow and smooth colonies
after an extended incubation (+) SQ and heat-stable that produce
catalase, tellurite
Photochromogen reduction
niacin.
Medium to Yellow to
orange and (+) sQ and heat-stable
long rods with smooth to rough colonies Strong nitrate
cross-barring catalase, nitrate reducer
reduction
NTM - Rapid Growing Mycobacteria (NTM-RGM)

These
group of mnycobacteria is considered as potentially pathogenic.
They are found in dust,
soil, water system, liquid reagents, and even from hospital wastewater.
They enter their hosts through breaks in
the skin and subcutaneous tissues by trauma,
parenteral, surgery, or animal contact.
They can
grow in routine bacteriologic media and mycobacterial media01
Microscopy: Weakly-staining Gram-positive rods that resemble diphtheroids
Culture: Colonies appear in solid media in seven days or less.
Distinct Feature: (+) Growth in MAC without crystal violet

Agents of human diseases: M. fortuitum group (mostly M. fortuitum), M. chelonae and M. abscessus
subsp. abscessus
Other NTM-RGM: M. smegmatis, M. goodii, M. cosmeticum, M. mucogenicum, and M. peregrinum
jor Groups of NTM-RGM: smpron STA esindioD Lihom beeed
1. M. fortuitum Group: M. fortuitum, M. peregrinum, M. septicum, M. houstonense, M. porcinum,

M. senegalense, M. setense, M. neworleansense, M. benickei, and M. brisbanes
m11SO 2. n M. chelonae-abscessus Group: M. chelonae subsp. chelonae, M. chelonae subsp. abscessus,
M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. salmoniphilum
Mycobacterium fortuitum
It is the most common rapidly growing mycobacteria that are associated with localized
cutaneous and soft tissue infections.
Microscopy: Pleomorphic - long to short, thick rods

Old cultures - may appear partially AFB
Culture: MB 7H11 - Colonies are branching and filamentous with aerial hyphae.
and catalase test
Biochemical test: (+) 3-day arylsulfatase, nitrate reduction, iron uptake,
(+) Growth in L-J medium with 5% NaCl
Mycobacterium chelonae
It is mostly associated with cutaneous infections in immunocompromised persons.
M. fortuitum.
It exhibits greater resistance to antimicrobial agents than
Microscopy: Strongly acid-fast with pleomorphism in young cultures
filamentous branching.
rough or smooth, non-pigmented, and
have no
Biochemical test: (+) 3-day arylsulfatase and growth in sodium citrate medium
in L-J medium with 5% NaCl
(-) Nitrate reduction, iron uptake, and growth
Mycobacterium abscessus subsp. abscessus

It is associated with chronic lung disease and otitis media.
is the most common respiratory pathogen among the NTM-

Griffith (2014) discussed that it
to its inducible erythromycin methylase
GRM with trails of antibiotic resistance owing
abscessus was recognized as a distinct species two

(erm) gene. He further explained that M.
from the M. chelonae/abscessus or M. fortuitum/chelonae
decades ago since it was separated this complex.
include M. abscessus in
complexes, but still laboratories would
Reservoir: Tap water
short rods)
Microscopy: Pleomorphic acid-fast bacilli (long to
without branching filaments rasT
Culture: Rough or smooth, nonpigmented colonies
L-J medium with 5% NaCl
Biochemical test: (+) 3-day arylsulfatase and growth in
in sodium citrate medium
A-) Nitrate reduction, iron uptake, and growth
Mycobacterium smegmatis Group

Related infections: Pulmonary, skin, and bone infections
may be beaded or ovoid in
Microscopy: Long and tapered rods with partial acid-fastness;
Culture: Egg-based media - Colonies are nonpigmented, rough and wrinkled to sometimes
smooth and butyrous.
MB 7H10 - Colonies are smooth or rough.

Biochemical test: (+) Nitrate reduction, iron uptake, and growth in 5% NaCl and sodium
citrate medium
(-) 3-day arylsulfatase test

Species: M. smegmatis and M. goodi
Virulence Test
It detects latent tuberculosis infection (LTBI).
It does not differentiate latent tuberculosis from active tuberculosis disease.
Tuberculin Skin Test • Purified Protein Derivative (PPD) Test
It detects exposure of individuals specifically to M. tuberculosis.

It uses PPD antigen which is a protein extracted and purified from the cell wall of culture-
grown M. tuberculosis.
Procedure: A standardized amount of PPD
antigen is injected intradermally into the forearm
and results are observed after 48 hours.
Interpretation: A reactive or positive result indicates

past exposure to M. tuberculosis.
Types of Tuberculin Skin Test:

a. Mantoux Test
. It is the preferred skin test.

It utilizes
0.1 mL intermediate-strength PPD.
(+) Result: Erythema and induration around the site of
hours
injection after 48 hours to 72
Interpretation of Result Bi
Source: (https://www.cdc.gov/tb/publications/factsheol
tsheets/testing/skintesting.htm)
recent close contact with a person infected with pulmonary tuberculosis;
organ transplant recipients; persons with history of tuberculosis (old healed TB); and
STIP SLOND immunosuppressed individuals
10 mm residing in
areas/countries with high prevalence of TB; use of prohibited drugs;
with
comorbidities; working in a
mycobacterial laboratory or occupational exposure;
working in high-risk places like nursing homes; and those with low body weight
15 mm - no known risk factors
Cause of
False-Positive Result: Presence of NTM infection; immunized with BCG; and
incorrect test antigen
Cause of False-Negative Result: anergy; recent antiviral vaccination; and improper
administration of PPD
b. Von Pirquet's test (Scratching Method)

od bluoc. Vollmer Tuberculin Patch Test (for infants)
Commonly submitted samples for M. tuberculosis and NTM: Sputum and bronchial aspirates
573
Other samples for mycobacteriology: Urine, aspirates of tissue and organ, biopsy specimen,
blood, fecal material, and CSF
Ideally, specimens for mycobacteriology should be processed upon arrival in the laboratory to
prevent false-negative results and overgrowth of contaminants.
If samples cannot be processed immediately, they should be refrigerated. Overnight preservation

at 2°C to 8'C is acceptable.
Precautions in handling and processing samples for mycobacteriology:

1. Prior to actual work, a PPD test should be administered across personnel handling samples for
TB assay with provisions for regular monitoring.
Compliance to proper use of personal protective equipment
2. (PPE) with fit-testing of the
respirator is a must.
have negative air pressure in which the air
3.
The laboratory where specimens are processed should
movement should be from clean areas (corridors) to less
clean areas (mycobacteriology lab) and
without air recirculation.
be done in class II or class IlI biological safety cabinet.
The processing of specimens should
aerosolization, centrifugation of samples should only be made inside the
5. BSC
To prevent
including sterilization of wire loops incineration

by in case a disposable loop is not available.
BSC and during transport.

6. Samples should always be tightly capped when outside the
It is recommended that slides be fixed using a slide-warming tray rather than heat-fixing (flame).
of MDR-TB should be manipulated following BSL-

8. Samples from known or suspected individuals
2 with full BSL-3 practices.
low risk if the laboratory is well-designed,
9. Working in a mycobacteriology laboratory, poses a
proper equipment is accessible, and precautions are strictly observed. lenene
actual receiving and processing
10. A reliable system of work orientation should be in placed prior to
Samples for Mycobacteriology

1. Sputum
Method of collection: Spontaneously produced sputum
for 3 consecutive days, with one early
Number of samples: 3 sputum samples collected 10
morning sample
Required volume: 5 to 10 mL expectorated or aerosol-induced sputum

pus cells
Microscopy prior to culture: < 10 epithelial cells and > 25
If none or only one of the sputum samples is tested positive,
another specimen should be
submitted for culture.
increased
sputa are unacceptable for culture because of the high probability
of
Pooled
contamination.p roes
Aerosol-induced (use of hypertonic saline) collection is done on ambulatory patients who are
unable to follow instructions.
ed blunts potoheladooym Tot anamlonge vllsabl

2. Gastric lavage diwonmovo bae allues ovilsgen-eeld ineven
It is preferred over the bronchoalveolar lavage for the detection of mycobacteria in children.
It is used to isolate mycobacteria that may have been swallowed overnight.
Advantage: This method is for senile, non-ambulatory patients, and children younger than 3
years-old.
Procedure: Three specimens should be collected in the morning, within three days after an
overnight fasting.
Required volume: 20 mL to 25 mL of gastric secretions
116 3. Urine
ban (dsl
Preferred specimen: First morning midstream urine collected on three consecutive days
Required volume: 15 mL or the entire volume of voided urine
The specimen may be collected through an indwelling catheter with a sterile needle and
syringe.
4. Bronchoscopy samples
It is the specimen of choice for detecting non-tuberculous mycobacteria.

Specimens: Bronchoalveolar lavage (BAL), bronchial washings, and transbronchial biopsies
GOJOIRETAS MYCOBACTERIA (ACID-FAST BACILLI) 403
5. Feces
It is used
to identify patients with possible M. avium complex diseases, especially those with
AIDS.
Stool specimens should be submitted without preservative.

It is utilized in
automated equipment such as MGIT 960 for identification of mycobacteria.
Blood
It is collected to assist in the detection

of tuberculosis among individuals with AIDS.
Culture medium: BACTEC 13A vial
Advantage: It is used for the isolation of M. avium complex.

ford anomioggs Mrburge to motfsnimetnopab edt rol bsen
7. Wound and skin lesion aspirates, and tissue specimens
For wound and skin aspirates, the specimens are transferred to a liquid medium.
specimens cannot be processed immediately, 10 mL to 15 mL of sterile saline
If the tissue
should be added to prevent dehydration. 5 85* hobeosdb-nodanumsl1pooh 21
8. Body fluids
Specimens: Pleural, pericardial, peritoneal, joint aspirates and CSF
Pleural, pericardial, and peritoneal fluids are collected in sterile tubes with anticoagulant
such as ethylenediaminetetraacetic acid (EDTA) or heparin.
Spinal fluid is utilized for the diagnosis of TB meningitis though difficult to detect.
Required volume:
a.
b. pericardial, and synovial fluid - 3 mL to 5 mL hue

Exudates,
C. Abdominal and pleural fluid - 10 mL to 15 ml isnidnos nominos tec
Digestion and Decontamination

to dissolve mucous
substances and to kill all contaminating
These procedures are performed
organisms (non-mycobacteria).
utilize the nutrients in
inoculation allows the mycobacteria to
The digestion of sputum prior to
the media.
decontamination, specimens are concentrated, usually through centrifugation, to

enhance
After
the detection of mycobacteria.
from the disruptive action of the
The high lipid cell wall of mycobacteria protects the cells
influence the action of the
contact time, and temperature
Concentration of the reagent,
decontaminating agent.
sputum, gastric and bronchial washing,
Specimens that require digestion and decontamination;
bronchoalveolar lavage, and transtracheal aspirate synovial fluid, and biopsy samples from
Specimens that do not require
decontamination: CSF,
innermost organs
If the quantity of CSF is insufficient, the following performed:

can be
gril
a. The specimen should be used directly for smear and culture. reabi
b. The sample can be concentrated by centrifugation and directly inoculated;
Reagents for Digestion and Decontamination Procedure
roni besidberf
1. 2% to 4% NaOH
It is the most common decontamination agent (2-mL sputum + 2-mL NaOH).

It serves as both a decontaminant and digestant.
2. 5% Oxalic acid
It is used for the decontamination of sputum specimens that contain Gram-negative rods like
Pseudomonas aeruginosa.
3. Zephiran-trisodium phosphate (Z-TSP)
Zephiran is an effective decontaminant with minimal bactericidal action on M. tuberculosis.
TSP requires a longer time to completely decontaminate the specimen though it is a rapid
digestant.
The addition of phosphate buffer increases the isolation of mycobacteria.
4.
It is utilized to prolong the shelf life of sputum up to days.

It is ideal for the transport of specimen.
5. N-acetyl-L-cystein- Sodium hydroxide (NALC-NaOH) labish yasmbwr

It is the most common combination of
digestion and decontamination agent.
NALC is also known as dithiothreitol.
It is utilized in automated equipment such as MGIT 960.
6. Sulfuric acid (H,SO,)

It can be utilized for "thin" specimens such as the urine sample.
It is an alternate decontamination
reagent for NALC-NaOH when processing urine
specimens.
7. Chlorhexidine
It is recommended for respiratory samples from children and adolescents with cystic fibrosis.
Advantage: It improves the isolation of NTM such as M. abscessus.
Disadvantage: It may not be used in MGIT system.

Microscopy
be
Smears can
prepared from specimens with
procedures. or without the decontamination and digestion
Bacterial smears should
not be prepared directly from specimens with possible low
fast bacilli such as CSF and urine, instead it should yield of acid-
utilized.
be concentrated and the sediments should be
To obtain a positive AFB staining, 5,000 to 10,000

organisms/mLare required.
10' AFB/mL is essential to detect mycobacteria in concentrated specimens.
Acid-fastness is affected by
colonial age, culture media, and exposure to UV light. 1s710 262ucD
AFB Staining Methods
1. Ziehl-Neelsen (ZN)/Hot Stain Procedure*
2. Kinyoun/Cold Stain Procedure*
3.
Auramine-Rhodamine Fluorochrome Staining
*See Chapter 4 for the discussion.
Auramine-Rhodamine Fluorochrome Staining (Truant Method)

It is a sensitive, reliable, and specific method compared to the ZN and Kinyoun methods.
Auramine is more sensitive than carbol fuchsin in the detection of the acid-fast bacilli.
AFB are examined at 250x and 400x magnification using a fluorescent microscope.
Potassium permanganate is the preferred counterstain over methylene blue.
(+) Result: Bright yellow-orange bacilli against a dark background
with a Ziehl-Neelsen stain
It is recommended that all positive fluorescent smears are confirmed
or examined by another laboratory technologist.
Quality Control
Positive-AFB: Mycobacterium tuberculosis ATCC25177 (H37Ra)
25922
Negative-NonAFB: Escherichia coli ATCC
Classification of AFB after Ziehl-Neelsen or Kinyoun Staining

1. Distinct AFB: Mycobacterium
2. Partially ARB. Nocardia, Gordonia, Rhodococcus, Tsukamurla, and Legionella

Precautions during AFB Staining and Microscopy
1. Smears should not come
in contact with one another during staining.
Staining jars should not be used.
2.
3. View a minimum of 300 fields

before a slide is called negative.
next smear slide.
4.
Wipe the immersion objective before reading the
BACTERIOLOGY
Causes of False-Positive AFB Microscopy
101. Changes
2. Insufficient decolorization
the oil immersion
microscopy (through
3. Cross contamination of slides during staining or
orl blito objective)
of other bacteria
4. Delayed processing and overgrowth
5. Altered stains (formation of precipitants)
Causes of False-Negative AFB Microscopy

1. Use of unclean slides causing the samples to be washed off during staining
use of thin smear
2. Organisms obscured by a very thick smear or
3. Incorrect temperature of the slide warmer®
4. Over-decolorization of the smear
5. Poor counterstaining
6. Lack of staff proficiency in reading AFB smear
TABLE 24-3. Acid-fast Smear Preparation - Number of AFB Observed

Ziehl-Neelsen/ Fluorochrome Stain (450x) Quantitave Report
Kinyoun Method (1000x)
No AFB observed seen
Doubtful AFB observed;
1-2/300 fields 1-2/70 fields
1-9/100 fields 2-18/50 fields 1+
1-9/10 fields 4-36/10 fields 2+
1-9/field 3+
> 9/field 36/field 4+
Source: McPherson and Pincus, Henry's Clinical Diagnosis and Management by Laboratory Methods, 23rl ed., 2017. ISES
TALE 24-4. Direct Sputum Smear Microscopy (DSSM)
Bright Field Microscopy Fluorescence Microscopy

Ziehl-Neelsen Method 200x-250x Magnification 100x Magnification
WHO Scale 1,000x Magnification
length - 30 fields
length - 40 fields
(Oil Immersion Field)
= 300 HPF
WHO WHO
Negative/No Zero AFB/100 fields No AFB/ Zero AFB/ Zero AFB/
No AFB/
AFB observed length length length length
*For
1-4 AFB/ 1-2 AFB/
Confirmation length
length
Scanty 1-9 AFB /100 fields
5-49 AFB/ 1-29 AFB/ 3-24 AFB/ 1-19 AFB/
length length length
length
1+ 1-9 AFB/10 fields 3-24 AFB/ 30-299 AFB/ 20-199 AFB/
1-6 AFB/
field
length field length
Bright Field Microscopy Fluorescence Microscopy

Ziehl-Neelsen Method
WHO Scale 1,000x Magnification 200x-250x Magnification 400x Magnification
(Oil Immersion Field) length 30 fields
length = 40 fields
WHO WHO
2+ 1-10 AFB/field 25-250 AFB/ 10-100 AFB/ 7-60 AFB/ 5-50 AFB /
field field field field
> 10 AFB/field > 250 AFB/ > 100 AFB/ > 60 AFB/ > 50 AFB/
field field field field
Source: https://www.who.int/tb/laboratory/mycobacteriology-laboratory-manual.pdf
#WHO - For confirmation by another technician or prepare another smear and read againiohdim
IUATLD - International Union Against Tuberculosis and Lung Diseases
WHO - World Health Organization
HPF - High Power Field
AFB Acid-Fast Bacilli
10 nirabbedooyM) 08Q TIOM bris DATDA8 od ni boan ef 1l

Types of Culture Media for Mycobacteriology ysn shmogedsidomimi
I. Solid Media
The use of solid media allows observation of colony morphology and pigmentation, which is
important for differentiating the isolates of M. tuberculosis from those of NTM.
The malachite green in the culture media is an inhibitory agent for non-mycobacteria.
1. Egg-based media
These are mainly composed of fresh whole eggs, potato, glycerol, and malachite green.
The egg yolk (lipid source) promotes the growth of mycobacteria.
source of energy.
Glycerol is added as a
Shelf-life: One year
medium the most commonly used egg-based medium that

Lowenstein-Jensen
contains lipids (egg yolk).
b. American Thoracic Society (ATS) medium - used for sterile specimens like CSF and
bone marrow
for the recovery of mycobacteria from heavily contaminated

C. Petragnani medium
specimen and with a higher concentration of malachite green (0.052%)
growth of M. avium complex
d. Wallenstein medium - to promote the
2. Serum Agar-based media (Transparent media) malachite green, agar,

These are mainly composed of bovine albumin, glycerol,
acid, and beef catalase.
growth.
dissecting microscope for early detection of
They can be examined using & produced in the
Aliburntin protects the AFB against toxic agents that are sither utilized
or
culture media.
Oleic acid is important for the metabolism of the mycobacteria.

of isoniazid-resistant (drug-resistant) strains
Casein hydrolysate improves the recovery
of M. tuberculosis
a. Middlebrook (MB) 7H10 and 7H11

to four weeks.
It can produce positive cultures in three
MB 7H11 contains 0.1 % casein hydrolysate.
blon
Inhibitory agents: Nalidixic acid, carbencillin, lincomycin, amphotericn B, and
b. Mitchison 7H11
It is recommended for heavily contaminated specimens like sputum.

Inhibitory agents: Polymyxin B, amphotericin B, carbenicillin, and trimethoprim
lactate
Liquid/Broth Media
1. BACTEC 12B
It is used in the BACTEC and MGIT 960 (Mycobacteria Growth Indicator Tube) systems.
Antimicrobial agents may be added to the medium such as polymyxin B, amphotericin
B, nalidixic acid, trimethoprim, and azlocillin.
MGIT (Mycobacteria Growth Indicator Tube) is modified Middlebrook 7H9 broth with
a fluorescence quenching-based oxygen sensor for detection.
Advantage: It reduces the processing time for the isolation of AFB and it can also be used
for the antimicrobial susceptibility testing of M. tuberculosis
M. tuberculosis: Detected in 9 days to 14 days
NTM: Detected in less than 7 days deal to besognop vinitm STR
2. Middlebrook 7H9 broth and Dubos Tween Albumin Broth

These are used as non-selective liquid media.
They are also utilized for subculturing stock strains.
Biphasic Medium
Septi-Chek AFB
It is utilized for rapid growth and identification of mycobacteria.
Incubation Requirements of Mycobacteria

Growth of mycobacteria depends on the
type of species, culture media, and incubation
requirements.
Cultures are incubated at 33 °C to 37°C and in 5% to 10%
CO,.
The tubed media are incubated at a
slanted position with the screw caps loose for one week to
allow
the evaporation of excess fluid and entry of CO,,
Specimens from skin lesions suspected for M.
mycobacteria grow best at a lower temperature. marinum,
M. ulcerans, and the rapidly growing
Cultures are examined weekly for growth until

at least six weeks because most
mycobacteria appear between three to six weeks. slow-growing
Grow best at 30°C to 329C: M. marinum, M. ulcerans, and M. haemophilum
Grow best at 42 °C: M. xenopi
TABLE 23-5. Quality Control Organisms for the Mycobacterial Culture Media
Organism/ATCC
Incubation Requirement Result
Mycobacterium tuberculosisH37Ra ATCC 25177 Up to 21 days at 33°C-37°C with CO, (+) Growth
Group 1- Mycobacterium kansasii ATCC 12478
Up to 21 days at 33°C-37°C with CO, (+) Growth
Mycobacterium fortuitum ATCC 6841 Up to 21 days at 33 'C-37°C with CO, (+) Growth
Escherichia coli ATCC 25922 18 hours-24 hours at 33°C-37°C with CO, (-)Growth
Pseudomonas aeruginosa ATCC 27853 18 hours-24 hours at 33°C-37°C with CO, (-)Growth
Candida albicans ATCC 10231P 18 hours-24 hours at 33°C-37•C with CO, (-)Growth
Source: Clinical and Laboratory Standards Institute (CLSI), Quality Control of Commercially Prepared Microbiological Culture
Media, 2004
Pigment Production
It is used to differentiate mycobacteria as photochromogen, non-photochromogen and
scotochromogen (according to Runyon's classification).
M. szulgai is scotochromogenic at 35° and non-pigmented at 25 °C to 30°C
Automated Detection System for Mycobacteria

Radiometric detection system: BACTEC 460 TB System
VersaTREK culture
Non-radiometric detection system: MGIT 960 system, BacT/Alert MB,
and
system
Culture bottles contain a lysing agent
and enrichment medium and are incubated for up to 6
weeks.
1. BACTEC 460 TB System

It has radiometric detection system.
It utilizes MB 7H12 for

the isolation of mycobacteria.
(+) Result: Presence of radioactive CO
Disadvantage: Use of
radioisotopes and no colonial morphology
(MGIT) 960 System
2.
Mycobacterial Growth Indicator Tube 7H9 broth.
which utilized a modified MB
It is a continuous monitoring system which is sharply detected
of free oxygen
leads to consumption
The presence of mycobacteria
by MGIT system. negative (no change) for six
tube negative if it remains
The instrument declares a sample
weeks. NAOH-NALC
CDC Resinimended Digestion and Decontamination Reagent

and catalase) L0 ReTtInD

Growth supplements: OADC (oleic acid, albumin, dextrose,
Inhibitors for nonmycobacteria: PANTA (polymyxin B, amphotericin B, nalidixic acid,
trimethoprim, and azlocillin)
and the production of carbon dioxide which
(+) Result: Depletion of free oxygen in the tube
results in fluorescence within the MGIT tube
Biochemical Test
1. Semi-Quantitative Catalase (SQC) Test
Reagent: 30% H2O2 with 10% Tween 80 (polyoxyethylene sorbitan monooleate)
column of
Procedure: Using a 2-week old test organism, add the reagents and measure the
bubbles after minutes. Do not shake or invert the tubes.
(+) Result: > 45mm column of bubbles - M. terrae complex, scotochromogens, NTM-RGM, and
photochromogens except M. marinum
(-) Result: < 45 mm column of bubbles
Quality Control
Positive: Mycobacterium flavescens ATCC 14474

Negative: Mycobacterium tuberculosis ATCC 25177
2. 68°C/Heat-stable Catalase Test

It differentiates the members of the photochromogens.
Reagent: 0.5 mL of 30% H,O, with 10% Tween 80 (polyoxyethylene sorbitan monooleate)
Incubation Temperature: 68 'C for 20 minutes
Procedure: Transfer isolated colonies into a test tube with catalase buffer then incubate for 20
minutes at 68°C. Add the reagents and observe for the reaction.
(+) Result: Bubble formation - M. terrae complex, M. fortuitum group, M. smegmatis, M. ulcerans,
M. xenopi, scotochromogens, and photochromogens except M. marinum
This is the only biochemical test wherein M. ulcerans has a strong positive reaction
Mycobacterium avium complex (MAC) has variable result.
Quality Control
Positive: Mycobacterium flavescens ATCC 14474 040rb lo
Negative: Mycobacterium tuberculosis ATCC 25177
Notes to Remember
M. terrae complex is positive in both catalase

test amongthe nonphotochromogens. 520914q OrCT
M. fortuitum group and M. smegmatis are the only NTM-RGM that are strongly positive in both catalase
test.
Mycobacterium tuberculosis complex (MTC) has a negative result in both catalase test.
3. Niacin (nicotinic acid) Test

It is the most commonly used test between M.
tuberculosis and the members of the MTC.
This test detects the deficiency of the
into niacin ribonucleotide.
niacin-connecting enzyme that converts free niacin
performed with cultures in a Lowenstein-Jensen medium (egg-based medium)

It should be
that are 3 weeks old and show at least 50 colonies.
Bhalla, et al (2018) explained that nearly all Mycobacterium species produce niacin
ribonucleotide.
Reagents: Cyanogen bromide and aniline reagent

(+) Result: Yellow color M. tuberculosis (accumulation of niacin in the medium, the only
mycobacteria that is strongly positive)
M. simiae (photochromogen) has variable result in this test.
Quality Control
Positive: Mycobacterium tuberculosis ATCC 25177
4. Nitrate Reduction Test (Broth method)

It detects the ability of the isolates to reduce nitrate to nitrite, including the further
conversion of nitrite to nitrogen gas.
It also determines the presence of nitroreductase, the enzyme which reduces nitrates into
nitrites.
It distinguishes M. tuberculosis from other members of MTC and MAC.

It separates M. fortuitum complex from other NTM-RGM.
Culture Medium: Nutrient broth with 0.1% potassium nitrate
Indicator reagent: Zinc (detects nitrate or true-negative result)

M. szulgai, M. flavescens, M. terrae complex,
(+) Result: Red color - M. tuberculosis, M. kansasii,
M. fortuitum group
M. terrae complex is the only nonphotochromogen which is strongly positive with this test,
while M. kansasii for the photochromogens.
Quality Control
tuberculosis ATCC 25177
Positive: Mycobacterium
Negative: Mycobacterium marinum ATCC 927

5.
Arylsulfatase test
It separates the
NTM-RGM from other mycobacteria.
free phenolphthalein.
This enzyme breaks down phenolphthalein disulfate into
sodium bicarbonate ha) tisly

Reagents: Potassium phenolphthalein SO, and
(+) Result: Pink color
(+) 3-day and 14-day arylsulfatase test: M. fortuitum group, M. chelonae group, and M. xenopi
M. triviale
14-day arylsulfatase test: M. marinum, M. gastri, and
(+)
tests.
MTC has negative reaction in 3-day and 14-day arylsulfatase
Quality Control
ATCC 6841
Positive-3 days or 2 weeks: Mycobacterium fortuitum
tuberculosis ATCC 25177
Negative-3 days or 2 weeks: Mycobacterium
6. Tween 80 Hydrolysis
It is useful for separating species of non-photochromogens and scotochromogens.
Tween 80
PH indicator: Neutral red (amber color) bound to
Test procedure: Tubes are incubated at 37'C for 1 day to 10 days
End product: Oleic acid and polyoxyethylated sorbitol
(+) Result: Pink color in 10 days M. flavescens,
M. gordonae, M. gastri, M. malmoense, M. terrae
complex, M. smegmatis, and photochromogens except M. simiae
(-) Result: Amber color (no color change)
M. kansasii exhibits a positive result in three days (rapid tween 80 hydrolyzer).
Quality Control (10 days incubation)

Positive: Mycobacterium kansasii ATCC 12478
Negative: Mycobacterium intracellulare ATCC 13950
7. Tellurite Reduction
It differentiates M. avium complex from other slow-growing NTM.

All NTM rapid growers reduce tellurite (positive reaction) in three days.
Reagent: Potassium tellurite (colorless)
and
(+) Result: Black metallic color precipitate (tellurium) - MAC, NTM-GRM, M. simiae,
MAC is rapid tellurite reducer producing : black color precipitate in 3 days same with the
Quality Control
Positive: Mycobacterium intracellulare ATCC 13950
Negative: Mycobacterium kansasii ATCC 12478
8. Growth Inhibition by Thiophene-2-Carboxylic Acid Hydrazide (T2H)

It is used to distinguish M. bovis from M. tuberculosis.
The procedure is due after 3 weeks of incubation at 379C with increased
Most mycobacteria grow in culture medium with T2H.
(+) Result: M. bovis exhibits no growth at 10 mg/mL TCH

9. Pyrazinamidase Test
Reagent: Ferrous ammonium sulfate
End products: Pyrazinoic acid and ammonia
(t) Result: Red or pink-colored band in 4 days - M. tuberculosis, MAC, M. marinum, M. simiae,
M. flavescens and M. szulgai.
(-) Result: Colorless or grayish color

Except for M. ulcerans, non-photochromogens have either a strong positive reaction or a
variable result.
NTM-RGM usually not differentiated by the pyrazinamidase test though the members of
are
M. fortuitum and M. chelonae exhibit positive reaction.
Quality Control
Positive: Mycobacterium intracellulare ATCC 13950

Negative: Mycobacterium bovis ATCC 19210
10. Iron Uptake

It detects the ability of mycobacteria to convert ferric ammonium citrate into iron oxide.
It differentiates the rapid growing mycobacteria. 01 b93 /516
Reagent: 20% ferric ammonium citrate
Procedure: Add a drop® of the reagent into a 2-week old L-J culture. Incubate for 21 days at
1291 m
28°C with at least 5% CO,"102
colonies and medium (egg-based medium) - M. fortuitum and
(+) Result: Rusty brown
M. smegmatis
Most mycobacteria do not require iron for growth,
M. chelonae and M. abscessus have variable result.
Quality Control
Positive: Mycobacterium fortuitum ATTC 6841
Negative: Mycobacterium kansasii ATCC 12478
11. Urease Test
and 5 days minha
It is performed in 3 sets: 1 day, 3 days,
urease producers.
Rapid growing NTM are
at 35°C to 37°C
Reagent: 4 mL urea broth incubated kansasii, M. marinum, NTM-RGM, and
MTC, M.
(+) Result: Pink
to red color in three days
scotochromogens except M. gordonae
M. gordonne exhibits a variable reaction.
and M. gastri (variable), nonphotochromogens do not
Except for M. genavense (positive)
secrete urease.
12. Sodium Chloride Tolerance Test
Reagent: 5% NaCl added to egg-based culture media wire mints BuicT9

(+) Result: Growth on the media (salt-tolerant) - M. flavescens and most NTM-RGM
Only M. flavescens among the NTM-SGM is able to withstand high salt concentration.
Quality Control:
Positive: Mycobacterium fortuitum ATTC 6841
Negative: Mycobacterium mucogenicum ATCC 49649
Immunodiagnosis
Interferon-Gamma Release Assay (IGRA)

It detects the interferon-gamma released by the white blood cells infected with the M. tuberculosis.
It measures the cell-mediated immune response of the individual to M. tuberculosis antigens.

It does not monitor the success of treatment.
It is not recommended for individuals less than 17 years old.

Advantage: Preferred for those immunized with BCG and no cross reactions with other
mycobacteria
15 Disadvantage: Not utilized for immunosuppressed patients such as those with AIDS, cannot
differentiate active TB from latent infection, and costly compared to the tuberculin skin test
Types of IGRA: QuantiFERON-TB Gold In-Tube Test (QFT-GIT) and T-SPOT TB Test (T-Spot)
Specimen: Whole blood (heparinized)
M. tuberculosis antigens (reagent): QFT-GIT - ESAT-6, CFP-10 and TB7.7ibsdooynr 1a0M
T-Spot - ESAT-6 and CFP-10 a .M bas gwolsdo .M
CDC Interpretation of Result

Positive: M. tuberculosis infection is likely
Negative: M. tuberculosis infection is unlikely
Indeterminate: Uncertain likelihood of M. tuberculosis infection
Borderline (T-Spot only): Uncertain likelihood of Mtb infection ri bomtoiton ar il
Chromatographic Analysis
This procedure is possible due to the high lipid
content (mycolic acid) of the cell wall of
mycobacteria.
Some examples are thin layer chromatography
(TLC), high-performance liquid chromatography
(HPLC), and gas chromatography (GC).
HPLC is commonly used and it differentiates mycobacterial colonies in two to four hours; it can
speciate the mycobacteria
based on the concentration of the mycolic acid.
2191298
Loop-Mediated Isothermal Amplification for

the detection of
M. tuberculosis (TB-LAMP) Assay 106d6ob
manual-based commercial molecular assay that involves amplification of the
It is
DNA from a
sputum sample.
It has been recommended
to nou
by the WHO as
a rapid tool for the detection of the Mycobacterium
tuberculosis complex specifically the technique developed by Eiken
(Tokyo, Japan).
Chemical Company Ltd
It is a respond to
the World Health Organization End TB Strategy which aims for the carly
diagnosis of tuberculosis
and of
MTB/RIF.
and universal drug-susceptibility testing without displacing the Xpert
It may bereplacement for sputum microscopy for diagnosing pulmonary TB in adults with
a
signs and symptoms consistent with TB, most importantly in locale and regions with low
prevalence of HIV and drug resistance, and in areas where the operation of the NAAT equipment
have limitations.
In this test, the final result is released in less than one hour through a visual display of the
In the study of Van Anh Thi Nguyen et al (2018), the findings do not support the use of
Loopamp™MMTBC detection kit (TB-LAMP) as a replacement test for smear microscopy in
peripheral laboratories. On the contrary, Sok Heng Peng et al (2020) concluded that TB-LAMP can
the AFB microscopy for rapid diagnosis of new TB patients, in areas
be a better choice in lieu of
with relatively low prevalence of drug-resistant Mtb, and in areas with difficulties to perform the
Xpert assay.
It is mostly performed on M. tuberculosis and not usually a routine procedure for slow-growing
CDC recommendation for M. tuberculosis: Isolates should be tested against isoniazid, rifampin,
ethambutol, and streptomycin (primary drugs for treatment).
Manual Methods for Susceptibility Testing of
M. tuberculosis: Agar proportion, broth micro
dilution absolute concentration (for MIC), and resistance ratio
BACTEC MGIT 960, VersaTREK, and MycoTB panel
Automated AST for M. tuberculosis:
diffusion, and Etest
Manual Methods for NTM-RGM: Broth microdilution, Disk
dilution
CLSI Reference Method for NTM: Broth
tormodfoal botriboM-goof
Molecular Diagnosis rolibutsb Bilf
It is the most rapid method
for detection of mycobacteria. "A. (414A 1-41T) eieohamodint M
but in the high-risk category.
It is recommended for testing individuals with negative AFB smear
It determines resistance to anti-tuberculosis drugs.
for the identification of
Automated DNA sequencing is considered as the most accurate method
mycobacterial isolates. It targets the gene coding for 16S 1RNA which is present in all bacteria
and contains conserved and variable gions.
in the M. tuberculosis complex,
not identify separately and differentially the species
It does
the
however, molecular typing methods, such as spoligotyping can identify such group
to
species level.
Non-cultivable NTM
Mycobacterium leprae (Hansen's bacillus) decalet
It is not cultivated in vitro and in any synthetic mycobacterial media. & pt1 h ponht-s
It invades peripheral nerves and skin cells and becomes an obligately intracellular parasite.
Microscopy: Rod-shaped and exhibits "cigar-pocket" or pocket-fence" arrangement
Culture: Colonies exhibit growth in living tissues of the footpads of mice and armadillos.
Optimal growth: 30°C

Skin test: Fernandez and Mitzuda reaction
Leprosy (Hansen's Disease)

It is a chronic disease of the skin, mucous membranes, and nerve tissues.
It is not considered a highly contagious disease resistance to the disease is through a cell-
mediated immune response to the Hansen's bacillus.
Cardinal signs: Skin lesions, cutaneous anesthesia, and enlarged peripheral nerves
Incubation period: year to years, sometimes reaching 10 years
Modes of transmission: Person-to-person contact through inhalation of the infected nasal
secretions, contact with infected skin, arthropod bite, ingestion of breast milk, and transplacental
transmission
Forms of leprosy: Tuberculoid leprosy (localized form) and Lepromatous leprosy (anergic form)
Comparison Between Tuberculoid Leprosy and Lepromatous Leprosy

TABLE 24-5.
Indicator
Distribution
Tuberculoid leprosy Lepromatous leprosy
Localized and benign Disseminated and malignant
Cell-mediated immune response Effective Ineffective
Presence in skin scrapings and
biopsy specimens Rare and often negative
Abundant and positive
Symptoms Skin lesions and damaged
nerves Facial and nasal deformities
Lepromin skin test Positive

Negative
M. leprae has higher detection if collected from biopsy samples of patients with lepromatous
leprosy compared to samples from individuals with tuberculoid leprosy.
Specimens: Nasal mucosal smears and skin snips from eyebrows and other sites
1. Acid-fast staining
M. leprae has a weak acid-fast
staining reaction compared with other mycobacteria.
Common specimen: Biopsy specimen
The bacteriologic index (BI) and morphologic index (MI) aid in identifying the progress of
the disease.
The number of organisms per immersion field is reported as the bacteriologic index (BI).
The number of solid-staining cells per 100 total bacilli is reported as the morphologic
index (MI).
2. Culture
M. leprae is non-cultivatable in routine bacteriologic media and will only grow in living cells.
It is the definitive test for M. leprae.
Specimen: Biopsy material from the infected individual

Culture medium: Footpads of mice
the Hansen's disease in animal medium
(+) Result: The development of
3.
Serological tests
Fluorescent leprosy antibody absorption test
DNA amplification
Enzyme-linked immunosorbent assay (ELISA)

4.
Molecular Techniques
identify the M. leprae.
PCR assays are used to rapidly
Molecular
FIGURE 24-1. Acid-Fast bacilli smear (Tubercle bacilli are shown in red color)
FIGURE 24-2. The aggregation of acid-fast bacilli into the suprastructure known as cording, is highly
this was derived contained M. tuberculosis
213 suggestive of M. tuberculosis complex. The culture from which
complex. The image is of a Ziehl-Neelsen stain at a magnification of 500x.
Source: https://journals.asm.org/doi/10.1128/microbiolspec.TNMI7-0022-2016
FIGURE 24-3. Colonies of two Mycobacterium species on L-J agar slants. M. tuberculosis isolate (left) is
'rough and buff," with colonies that have a cauliflower-like appearance.
M. marinum isolate (right) demonstrates pigment development after exposure to light.
A MOISETOAS MYCOBACTERIA
(ACID-FAST BACILLI) 419
FIGURE 24-4. The MGIT 960 tube on the right contains growing mycobacteria and is fluorescent when exposed
to UV light. In contrast, the tube on the left contains no mycobacteria.
0.60 -
0.55 - MA
0.50.
0.45.
-d (F2)/d T
0.40•
0.35.
0.25 -
F l u o re s c n e
0.20.
0.15 -
0.10 -
-0.15 - 60.0 62.0 64.0 66.0 68.0 70.0 72.0 74.0 76.0 78.0 80.0 82.0
50.0 52.1 54.0 56.0
42.0 44.0 46.0 48.0
Temperature ('C)
resonance energy transfer
FIGURE 24.5. These postamplification melting curves, derived using florescence
probes rollowhes crost range mycobacterial PCR, demonstrate that M. uberciloss complenbarei
kansasii (MK), M. avium (MA), and M. intracellulare (MI).
differentiated from the nontuberculous mycobacteria M.
he nonthttps:/iournals.asm.org/doi/10.1128/microbiolspec.T117-0oz- 2010
FIGURE 24-6. This Ziehl-Neelsen-stained photomicrograph of an unknown species demonstrates the cross-
barred pattern that is also typically exhibited in Mycobacterium kansasii. Image courtesy of the CDC-Public
Health Image Library/Ronald W. Smithwick (ID#14600).
FIGURE 24-7. Mycobacterium fortuitum on blood agar. Cultivated 9 days in an aerobic atmosphere, 37°C.
FIGURE 24-8.
Acid-fast smear with numerous red-colored Mycobacterium leprae are visible inside a chronic
inflammatory lesion
Source: https://www.cdc.gov/leprosy/health
theatth-care-workers/laboratory-diagnostics.html
ASSESSMENT QUIZ
1. Which of the following is the preferred digestion-decontamination reagent for automated

equipment?
a. NALC-NaOH
b. Z-TSP
C. Cetylpyridium chloride
d. Oxalic acid
2. What secondary stain is used for Truant method? nmpigcon bnnot Jame swlue
a. malachite green evils2oid dest vablolco sloste tael
b. potassium permanganate
c. acridine orange
d. calcofluor white
3. What is the principal virulence factor of Mycobacterium tuberculosis?

a. cord factor
b. mycolic acid
c. endospore
d. exotoxin A
4. What is the recommended sample for isolation of Mycobacterium avium complex aside from
a. blood
b. feces
c. pleural fluid
d. a and b
1056
5. It is added into culture medium to improve the recovery of isoniazid-resistant (drug-resistant)
strains of Mycobacterium tuberculosis.
a. cycloheximide
b.
C. casein hydrolysate
d. albumin
Tween 80 hydrolysis test?

6. What is the positive quality control organism for
a. Mycobacterium intracellulare ATCC 13950
b. Mycobacterium fortuitum ATCC 6841
C. Mycobacterium kansasii ATCC 12478
d. Mycobacterium marinum ATCC 927

Gram staining: Gram ghost
AFB Microscopy: Deep pink beaded rods
Motility: Negative
Culture: Small, round, nonpigmented
Heat-stable catalase test: Negative
Niacin test: Negative
Nitrate Reduction: Negative
Inhibition by T2H: Positive
Salt Tolerance: Negative
a. Mycobacterium avium
b. Mycobacterium ulcerans
c. Mycobacterium fortuitum
d. Mycobacterium bovis
8. This culture medium differentiates the growth pattern of rapid growing mycobacteria, EXCEPT:
a. MAC
b. Sodium citrate agar
c. L-J
d. Middlebrook
m.9. It is known as the smooth strain of Mycobacterium tuberculosis with a positive result in both niacin
and nitrate reduction.
a. Mycobacterium africanum
b. Mycobacterium canettii
c. Mycobacterium microti
d. Mycobacterium caprae

Gram staining: Gram ghost
AFB Microscopy: Deep
pink beaded rods
Motility: Negative
Culture: Branching, filamentous with aerial hyphae
MAC without CV: Positive
Heat-stable catalase test: Positive
Niacin test: Negative
Inhibition by T2H: Negative
Arylsulfatase test: Positive (3 days)
Salt Tolerance: Positive
a. Mycobacterium chelonae
b. Mycobacterium fortuitum
c. Mycobacterium xenopi
d. Mycobacterium tuberculosis
CHAPTER 25
ANAEROBIC BACTERIA

1. discuss the general characteristics of the anaerobic bacteria including
habitat and mode of acquisition;
2. differentiate the virulence factors of the clinically significant clostridia;
3. distinguish the characteristics of the nonsporeforming anaerobic bacilli
and cocci;
4. explain the distinct sample management in anaerobic culture to ensure

growth and isolation;
5. outline the related diseases and infections of anaerobic bacteria; and
6.
develop an algorithm of diagnostic tests to distinguish the significant
species in the group of anaerobic bacteria.
Anaerobes can be isolated in humans and the environment such as in

soil and and high
freshwater. They require a condition with reduced oxygen
concentration of nitrogen to promote the growth of organisms. They are major
indigenous flora of humans especially of the skin, and the mucosal surfaces of
the oral cavity, gastrointestinal tract, and genitourinary tract.
Anaerobic bacteria can be opportunistic, causing endogenous and
contact with
eogenous infections and intoxications. Skin trauma, surgery, and
soil through cuts and abrasions are portals of entry and sources of exposure.
This and
food
group of organisms cause surgical wound infection, endocarditis,
granules, and
Poisoning. Moreover, the presence of foul odor, sulfur
brick-red
I fluorescence are some of the indicators of anaerobic bacteriology.
Gram-Positive Anaerobic Sporeforming Bacilli
Clostridium
It belongs to the family Clostridiaceae.
The species of this genus are obligate anaerobes and catalase-negative.
ramosum (Gram-negative or
The members are Gram-positive, spore-forming bacilli except
C.
Gram-variable, non-sporeforming species, Lavigne et al, 2013).

They are frequently encountered in exogenous anaerobic infections or intoxications.
The infections are acquired when spores are ingested or through open wounds that have been
contaminated with soil.
Virulence factors: Collagenase, hyaluronidase, lecithinase (cell destruction), and phospholipase

Species: C. perfringens, C. tetani, C. botulinum, C. novyi, C. septicum, C. histolyticum, C. bifermentans,
C. sordellii, and C. innocuum
Histotoxic clostridia causing myonecrosis: C. perfringens, C. novyi, C. septicum, C. histolyticum, and

C. bifermentans
Related infections: Bacteremia, colitis, genitourinary tract, and perirectal infections
Distinguishing characteristics of the Clostridium species

1. Form endospores anaerobically
2. Motile with peritrichous flagella except C. perfringens, C. ramosum, and C. innocuum
3. With swollen sporangia except C. perfringens and C. bifermentans

4. Non-encapsulated except C. perfringens
5. With a single hemolytic reaction except C. perfringens
6. Carbohydrate fermenters except C. tetani and C. histolytica enemud ot botsloal ad nED BOdOTSENA
7.
It is the most common agent of myonecrosis.

It is the frequently isolated member of Clostridium in blood cultures. .
Microscopy: "Boxcar-shaped" bacilli with subterminal spores which are seldom seen
Culture: BAP - Colonies are
dome-shaped and grayish-white with double zone of hemolysis
Litmus milk - Colonies exhibit
a stormy fermentation of milk.
Differential Biochemical tests of C. perfringens:

Very fermentative
(+) Lecithinase production on egg yolk agar (EYA)
ANAEROBIC BACTERIA 427
(+) Gelatinase
(+)
Reverse CAMP test, formation of an "arrowhead-shaped" zone of hemolysis towards the test
organism

1. Gas gangrene (Myonecrosis)
It is a life-threatening destruction of muscle and other tissues.

moni som
It develops when bacteria gain entry into cuts and
abrasions or infect wounds.
It is accompanied
by bullae (fluid-filled blisters), pain, swelling, serous discharge,
discoloration, and tissue necrosis.
Alpha toxin contributes to the severity of myonecrosis.
2. Clostridial Type C Food Poisoning or Necrotizing Enteritis

It is also known as the enteritis necroticans.
It is caused by the ingestion of beta-toxin (enterotoxin) in contaminated food.

Improperly stored food allows the germination of the spores and growth of
vegetative
bacteria - heating only kills the vegetative bacteria and not the spore-forming organisms.
Symptoms: abdominal pain, vomiting, and bloody diarrhea
Clostridium tetani (Tack head bacillus)

It is a soil and environmental inhabitant. (nimofowush mello/ud Thoblakor melluion
The endospores are found in the soil, dust, and feces of many farm animals.
Virulence factor: Tetanospasmin (neurotoxin)iaav sigs
Microscopy: Cells are with terminal spores and swollen sporangia that have a "drumstick" or
tennis-racket" appearance.
Culture: BAP _ Colonies exhibit a slow, heavy but "smooth and swarming" growth
Distinct matte surface with a narrow zone of B-hemolysis
Biochemical test: (+) Gelatinase and indole; (-) Lecithinase and lipase
Related infection: Tetanus
It is an endopeptidase that selectively cleaves the synaptic vesicle membrane protein known as
synaptobrevin.
It causes tension or twisting in skeletal muscles that surround the wound.
may lead to respiratory arrest.
It also causes tightness of the jaw muscles that
Tetanus
It is characterized by trismus and risus sardonicus.

open
wound and spreads a potent toxin that
occurs when the organism (spore) enters an
It
mediates generalized muscle spasms.
Symptoms: Muscular rigidity and difficulty in swallowing

related to the distance from
Incubation period: 3 days to 21 days (the long incubation period
is
the injury to the central nervous system).

Tetanus neonatorum is contracted through the use of contaminated instruments on newborns.
Clostridium botulinum (Canned food bacillus)

It is found in soil and aquatic sediments.
It is a potential bioterrorism agent.
It is characterized by the presence of subterminal spores.
Virulence factor: Botulism toxin noilsninton
Culture: BAP - Colonies exhibit a beta hemolytic reaction.
Related infection: Botulism
Biochemical test: (+) Lipase, gelatinase, and esculin hydrolysis
Botulism Toxin (BoNT- Butolism Neurotoxin)

It is considered as one of the most potent natural toxins known to man.
It selectively cleaves the synaptic vesicle membrane protein (synaptobrevin) preventing
exocytosis and the release of the neurotransmitter (acetylcholine), thereby resulting to paralysis
and death.
It requires only a small amount of this neurotoxin to cause fatality, produce paralysis, and death.
Botulism antigens: A to G (7 antigenic types)
Antigens associated with human diseases: A, B, and E
Peck (2009) and Hill and Smith (2012) as cited by Pellette (2013) explained that BoNTs are
produced also by some strains of Clostridium argentinense, and rare strains of Clostridium baratii
and Clostridium butyricum.
Botulism
characterized by double or
It is blurred vision,
impaired speech, difficulty in swallowing,
weakness, and paralysis.
It occurs when spores are ingested, then germinate
and secrete toxin.
while the vegetative cells migrate to the colon
Symptoms usually appear between 18 hours and 36 hours after ingestion of contaminated food.
Types of Botulism:
1. Foodborne botulism
It results from the
ingestion of preformed botulism toxin in
preserved or meat-based food.
It is commonly caused by botulism toxin A.
2. Infant botulism
It is an infection that
by ingesting the organism from honey products added to
is caused
milk.
Clostridiodes difficile (formerly Clostridium difficile)

It is a former member of the genus Clostridium.

antibiotic-associated diarrhea and pseudomembranous colitis
(bloody diarrhea with necrosis of colonic mucosa).
It is part of the gastrointestinal flora
of very few individuals while higher yield is observed in
persons staying in homecare facilities - during antibiotic treatment, where majority of the GI
microbiota is suppressed and destroyed, it multiplies secreting potent toxins.
It is an "infection control dilemma" among hospitalized patients receiving antimicrobial
treatment due to the onset of nosocomial diarrhea.
It ferments fructose producing formic acid that changes the color of medium from pink to yellow.
Preferred culture medium: Cycloserine-cefoxitin-fructose agar (CCFA)
Virulence factor: Toxin A (enterotoxin) and Toxin B (cytotoxin)
Microscopy: Cells appear in chains and aligned from end to end with oval, subterminal
endospores
Culture: BAP Colonies are flat with a "horse stable" odor, non-hemolytic, and produce a
fluorescent chartreuse under a long UV light
CCFA _ Colonies exhibit a yellow color and a "ground-glass" appearance
Biochemical test: (+) Gelatinase and lipase
TABLE 25-1. Differetial Characteristics of Anaerobic Bacilli and Cocci

Distinguishing characteristics
Organism Gram stain reaction
rods, with Young colonies "spider-like" or
Actinomyces Gram-positive banded or beaded 'wooly" appearance.
israelii branching filaments • Old colonies - "molar tooth"
appearance.
(+) Yellow granules

Grayish-white, circular, smooth, and
Bacteroides fragilis Pale-staining, pleomorphic, Gram-negative non-hemolytic
rods with a "safety pin" appearance
Colonies corrode (pit) the agar.
Bacteroides Pale-staining, thin, Gram-negative rods
coccoid or Small, white, shiny, and convex
Gram-positive diphtheroids; that are
bifurcated (forked) ends
colonies
pointed in shape with 'dog bone"
which resemble the shape of
Distinguishing characteristics
Organism Gram stain reaction
Gram-negative rods
May exhibit filamentous growth; with
translucent colonies with black centers
resembling 'fish eye" colonies
nixo! mellulo(+) H.S production, bile-resistant
Clostridium Formation of rhizoid margins that
Gram-positive rods in young cultures that turn
septicum resemble "Medusa head"
Gram-negative with age; have subterminal
Diphtheroid-like, Gram-positive rods; that have Small, grayish-white colonies

a palisade arrangement
Eubacterium Pleomorphic, Gram-positive rods that are ar Fluorescent chartreuse color0ial

seagull wing-shaped
Spindle-shaped, Gram-negative rods that The medium exhibits a green color
nucleatum resemble Capnocytophaga when expose to air; colonies have
renbomn ambloa to "breadcrumbs-like" appearance.
Gram-variable rods or short coccobacilli that Pinpoint colonies Toiled andl
Large-fusiform Gram-negative rods1 1i bevw! "Raspberry-like" colonies ,131

Peptococcus niger Gram-positive cocci that are paired singly, nippl Small, black, and shiny colonies
pairs, or in tetrads
Peptostreptococcus Large, Gram-positive coccobacilli in chainsJurah01 Grayish-white colonies that emits a foul
anaerobius odor
Porphyromonas Gram-negative coccobacilli Brown, mucoid colonies with brick red

in ato fluorescence marant
Prevotella disiens Gram-negative rods White, shiny colonies with a brick-red

fluorescence
Tiny Gram-negative diplococci Red fluorescence
broil V0 anola tatinn Nonmotile; asaccharolytic

Note: Anaerobic BAP is the medium used for culture.
10100 wollow s fidirke cotmolos
Anaerobic Non-Sporeforming Bacilli and Cocci
Bacteroides fragilis (Bile-Tolerant Anaerobe)

It belongs to the family Bacteroidaceae.
It is a beta-lactamase producer, non-motile, and saccharolytic.

It is significant cause of intra-abdominal abscesses and bacteremia.
It is the most common cause

of anaerobic bacteremia (frequently isolated from blood culture).
It is also considered an agent for brain and liver abscess.
Virulence Factor: Polysaccharide capsule and fimbriae
Preferred Culture
Medium: Bacteroides Bile Esculin (BBE) Agar
Culture: BBE agar - Colonies exhibit gray color against the black slant
Biochemical test: (+) Esculin hydrolysis and catalase; () indole itsss tes
Differential test: (+) Bile

tolerance(also known asthe biledisk test)
Bacteroides fragilis complex: B. fragilis, B. distasonis, B.
thetaiotaomicron, B. vulgatus, and B. ovatus
Actinomyces israelii
It belongs to the
family Actinomycetaceae.
actinomycosis.
It is
non-motile, saccharolytic, and non-AFB
It is part of the indigenous microbiota of the oral cavity.
Preferred specimen: Surgical biopsy or discharge (pus) mit9g1o nogu bollima al tobo luod
Selective media: Phenylethyl alcohol and mupirocin-metronidazole blood agar ro b.e
The
species of this genus are pleomorphic, Gram-positive anaerobic rods (sometimes appear as
Gram-variable).
It is non-motile, and may also be considered as an aerotolerant anaerobe. 16 onos aldood
It is part of the indigenous microbiota of the mouth, GIT, and vaginal canal. l4pmk3
It contributes in maintaining the female genital health by protecting against urogenital
infections.
Culture: Colonies are small, pinpoint, gray with rough appearance.

vaginalis, and L. salivarius
Species: L. acidophilus, L. fermentum, L.
Related infection: Bacterial vaginosis, dental caries, and intraabdominal abscess

Biochemical test: Glucose, maltose and sucrose fermenter To moribloel odd bruess of
It protects the female genital tract from urogenital infections.
Differential medium: Tomato juice agar (pH 3 to 4) sve arht then

at pH 6.0 will produce white or yellow
Selective Medium: MRS (de Man, Rogosa and Sharpe) agar
colonies
Biochemical test: (+) Litmus milk, clot formation

esculin hydrolysis
(-) Catalase, H2S, and
Notes to Remember
while Finegoldia
reptostreptococci are the frequently encountered anaerobic Gram-
often associated
positive cocci
endocarditis,
with meningitis, and
magna is the most pathogenic in the group and
Pneumonia.
cocci in lung and brain abscess

Veillonella is the most commonly isolated anaerobic Gram-negative
and infections involving the oral cavity, while the B. fragilis group, Fusobacterium, Porphyromonas, and
clinical samples.
Prevotella are the predominant Gram-negative anaerobic rods in
genera Actinomyces,
The Gram-positive non-sporeforming anaerobic bacilli include the
Mobiluncus, Cutbacterium, and Lactobacillus.
Most anaerobic infections are caused by the endogenous microbiota.
Indicators of the Presence of Anaerobic Bacteria

1. Foul odor is emitted upon opening an anaerobic jar or pouch
Example: Clostridium difficile, Fusobacterium, and Porphyromonas
2. Presence of sulfur granules
Example: Actinomyces, Cutibacterium, and Eubacterium nodatum
3. Presence of brick-red fluorescence when exposed to ultraviolet light
Example: Prevotella and Porphyromonas
4.
Example: Clostridium perfringens
5. Absence of superoxide dismutase

6. Growth on the anaerobic culture plate
Requirements for Anaerobic Bacteriology

1. Sample collection
To ensure the isolation of anaerobic clostridia, the specimens must ber collected from the
actual site of the infection; swabbing of the mucosal surface is insufficient.
Needle aspiration is the

best specimen for anaerobic culture; biopsy sample is also
acceptable.
In extreme cases where a swab is used, placed the swab into a 0.5-mL sterile thioglycollate
and pressed the swab against the
broth, spun wall of the tube and the liquid suspension is
utilized for inoculation.
Stool sample is commonly used for the isolation of C. difficile.

Food and fecal specimens that are suspected of C. perfringens should be transported at 4°C.
The anaerobic Gram-negative bacteria are the major microbiota in the colon, as compared
with aerobes in a 1000:1 ratio.
Other specimens acceptable for culture: sterile fluids like CSF,

peritoneal, and joint; biopsy
samples; and bone marrow
2. Transport, processing and storage of specimens

The specimen
must be transported promptly to the laboratory under anaerobic conditions or
with minimal exposure to oxygen.
Samples upon receipt in the laboratory should

be
placed immediately in an
the anaerobic chamber or use appropriate holding mediathoxygen-free
environment such as
of such equipment. the absence
In case it cannot be
processed immediately, all specimens for anaerobic culture should be
kept at room temperature for a minimal time.
Avoid refrigerating the samples because it
exposes the specimen to oxygen.
3. Unacceptable specimens for anaerobic culture

a. Swabs
b. Sputa
C. Bronchial washings
d. Voided or catheterized urine
e. Feces and effluents from ileostomy and colostomy

f. Gastric and small bowel contents
1. Gram stain
Spores are only observed in Gram-stained smears of clinical specimens or colonies from an
agar plate, if the culture will be incubated for several days.
C. ramosum and C. clostridioforme may be stained as Gram-negative bacilli.
Gram-negative anaerobes are typically pleomorphic morphologically.
The presence of leukocytes implies an inflammation at the site of infection.
Carbol fuchsin may be used as an alternate counterstain to enhance the morphology of the
Gram-negative anaerobes.
2.
Culture and Antimicrobial Susceptibility Test
Brucella broth, EYA, CCFA, PYG, PEA,
Culture media: Anaerobic blood agar, thioglycollate,
and CNA-BAP
Brucella blood agar (BRU/BA), Bacteroides bile esculin (BBE)
Primary Media for Anaerobes:
agar, and Laked Kanamycin Vancomycin with Blood (LKVB) agar.
Amies medium, and
Transport media. Pre-reduced anaerobically sterilized (PRAS) medium,
Cary-Blair
prepared and have not been exposed to oxygen prior
Ideally, culture media should be freshly
to inoculation or the least is
used within one week of preparation.
Gram-negative bacteria.
BBE agar contains gentamicin and bile salts as inhibitors mostily to
fecal sample intended for C. difficile assay.
Cary-Blair medium is recommended for
thioglycolate, cysteine, and dithiothreitl are added into the
culture
Reducing agents such as
media to obtain low redox potential.

used for susceptibility testing
of B. fragilis group,
Brucella broth with lysed horse blood is
with an inoculum size of 10° CFU/mL.
are the preliminary antibiotics for testing anaerobes,
Kanamycin, colistin, and vancomycin
at-4 Purpose of the Anaerobic culture media

for culture.
PRAS provides a semi-solid agar and an anaerobic environment
PRAS medium is used for aspirates and tissue/biospy
An oxygen-free transport tube with
samples.
EYA detects lecithinase and lipase production.
the formation of double zones of
BRU/BA is used to observe swarming growth and
hemolysis. It supports nearly all obligate and facultative anaerobes,
Cycloserine and cefoxitin in CCFA inhibit Gram-negative coliforms while neutral
red is the
pH indicator.
anaerobes by
utilized to differentiate the
Peptone-yeast extract glucose (PYG) broth is
chromatography.
anaerobes such as Bacteroides and Prevotella
selective media for
species.
identification of
Kanamycin inhibits Gram-negative bacilli while laked blood aids in the
pigmented species of Prevotella (brownish-black colonies).
Inoculation of culture media
where agar plates and
Inoculation and incubation should be done in an anaerobic chamber
1S mICMERA broths are also kept until use.
The inoculation of the organism should begin with the use of CAP followed by BAP.
For blood culture, at least culture bottle should be utilized and immediately
one anaerobic
transported to the laboratory in cases where the use of two anaerobic bottles are not possible.
Anaerobic (Gas pack) jar and pouch are used in the absence of the anaerobic chamber and
then placed inside the routine incubator.

Anaerobic cultures are incubated at 35°C to 37°C for five to seven days.- 17
EYA is used to detect lecithinase and lipase activity while PYG identifies volatile fatty acid.
PEA allows the isolation of both anaerobic cocci and rods.
3. Aerotolerance Test
It differentiates obligate anaerobe from other bacteria such as the aerobes and the facultative
anaerobes.
Culture media: BAP, CAP, and anaerobic BAP
Incubation: 35° C for up to 48 hours
BAP with 5% sheep blood is for strict aerobic culture while CAP is for facultative anaerobes
requiring increased carbon dioxide for growth.
(+) result: Obligate anaerobes will only grow in culture media that are incubated without
oxygen.
4. Enzyme-based Test (Manual and Automated Procedures)

It allows identification of anaerobes from pure culture after 4 hours of incubation.
A
positive reaction is obtained if the colorless substance forms a yellow or red end products.
Examples: BBL Crystal, RapID ANA-II, MicroScan, and Vitek ANI
212046710 ANAEROBIC BACTERIA 435
5. MALDI-TOF and Molecular Test
Both methods are considered rapid

tests for the identification of bacteria including
anaerobes.
The nucleic acid amplification test (NAAT) is utilized to detect the toxins produce by
C. difficile in fecal material.
TABLE 25-2. Differential Tests for Anaerobes

Tests Description Positive Result
Catalase It is used to differentiate the enus Bacillus Formation of bubbles
from the aerotolerant strains of Clostridium.
Bacillus: Positive
Reagent: 15% H,O,
Clostridium: Negative
Direct Nagler It is performed using an EYA plate and Inhibition of the lecithinase reaction that is
C. perfringens type A antitoxin. produced by C. perfringens
Mouse It detects the presence of neurotoxins in Survival of the guinea pig after introducing
Neutralization serum, feces, and gastric aspirate after 1 day the specific antitoxin.
to 4 days of exposure to the organism.
Definitive test to identify the specific

C. botulinum toxin causing botulism
Reverse CAMP It confirms the presence of C. perfringens. BAP: Formation of an "arrowhead-shaped"
zone of hemolysis at the intersection of the
two streaks toward the Clostridium isolates
Cell Culture It determines the cytotoxic activity of Rounding of cells in tissue culture and
Cytotoxicity C. difficile in stool samples after 2 to days neutralization of the toxin activity
of incubation.
Gold standard test for the detection of the

C. difficile toxin (Alcala, 2013)
Lipase and It identifies certain clostridia isolates. Lecithinase reaction: Formation of an
Lecithinase opaque zone in the medium that surrounds
Medium: Egg yolk agar (EYA) colonies
Lecithinase-positive species: C. perfringens, Lipase reaction: Colonies exhibiting a

C. bifermentans, C. sordellii, and C. novyi " mother-of-pearl' or 'gasoline-on-water"
appearance
Lipase-positive species: C. botulinum and
Sodium Susceptible: P. anaerobius

For rapid detection and separation of
Polyanethol Peptostreptococcus anaerobius from Resistant: P. asaccharolyticus
Sulfonate (SPS) Peptoniphilus asaccharolyticus
Disk
Metronidazole
Disk
It distinguishes the anaerobic Gram-positive Susceptible: Anaerobic Gram-positive cocci
cocci from the microaerophilic Gram- Resistant: Microaerophilic Gram-positive
positive cocci. cocci
436 REVIEW HANDBOOK IN DIAGNOSTIc BACTERIOLOGY
EnM bas 12 1AM 2

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-stained specimen, depicted

FIGURE 25-1. Under a magnification of 956 , this photomicrograph of a Gram-의
numbers of Gram-positive, Clostridium tetani bacteria, which had been cultivated on a blood agar plate (BAP),
and incubated over a 48 hour time period. Note that several of these organisms had entered their endospo
phase, assuming the characteristic tennis racket-type morphology.
Source: https://phil.cdc.gov/Details.asp ?pid=12056h aBthof
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FIGURE 25-2. Gram stain of Clostridium pefringens grown on Schaedler broth

t
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FIlGURE 25-3. Gram stain of Clostridium botulinu

(Photo by G. Lombard)
2시
Ooca ASb2a ACANAERO IC BACTERIA 437
5
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FIGURE 25-4. Nagler test reaction of Clostridium perfringens on egg yolk agar
(Photo by S. E. Starr)
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. M. Kiser)
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ASSESSMENT QUIZ
1. What is the selective medium for the isolation of Lactobacillus acidophilus?

a. MRS
C. PYG
d. BBEA
2. Which of the following detects the neurotoxin produced by Clostridium botulinum in serum and
fecal samples?
a. cell culture toxicity
b. Nagler test
C. mouse neutralization
d. EYA test

Microscopy: Large rods, subterminal spores seldom seen
Swollen sporangia: Negative
Motility: Negative
Culture: Double zone of hemolysis
Nagler test: Positive
Reverse CAMP test: Positive
EYA test: Positive
Bile tolerance: Negative
a. Bacteroides fragilis
b. Clostridium perfringens
C. Actinomyces israelii
d. Clostridium tetani
4. The following are reducing agents added to culture media to promote low redox potential or
oxygenation, EXCEPT:
a. cysteine
b.
C.
d. methylene blue
5. What is the purpose of adding "laked blood"

in LKVA medium?
a.
identifies pigmented species of Prevotella
b. observes hemolytic patterns of anaerobes
C. provides supplements
d. promotes swarming growth of Clostridia tochisiom entmolos
Microscopy: Gram-negative rods, safety pin appearance 1291 SMA
Swollen sporangia: Negative

Motility: Negative
Culture: Smooth colonies, nonhemolytic
Nagler test: Negative
Reverse CAMP test: Negative
EYA test: Negative:
Bile Tolerance: Positive
a. Cutibacterium acnes
b. Lactobacillus
C. Bacteroides fragilis
d. Clostridium difficile
7. It is a semisolid transport medium used for anaerobic bacteriology.

a. Cary-blair
b. Stuart
c. Amies
d.
8. distinguishing characteristics of Clostridium difficile?

What are the
sardonicus
a. drumstick appearance, raspberry colonies, risus
b. streptobacilli, horse stable odor, fluorescent chartreuse
fermentation, myonecrosis
c. boxcar-shaped rods, stormy
fluorescence
d. dog bones, fish eye colonies, red
440 KEVIEV HANDB00K IN DIAGNOSTIC BACIERIOLU Y
9. IdentiMifyctheroscopy: e organismtivafter
possiblGram-posi phenotypi c testing. pai b e hetomdprtt
e filamentous rods, yellowish sulfur granulesnsh
aadi
udaans o raog btzbrerzevnnde
Swollen sporangia: Negative heelkgue cajuro1ę
Motility: Negative
Culture: Web-like colonies, molar toothnn o rzon guuNa zotosig
shn5o sit virsh
Nagler test: Negative rtleni biq donere stin t
Reverse CAMP test: Negative ( ni daine abo7 Svigen-meo qosenmilV
EYA test: Negative: svidngs cdigaore rullow
Nitrate Reduction: Positive viisga vilioM
a. Prevotella bdyplonsdnon 2sinolos donme1eutlD
vin ike) ielgo7
b. Bifidobacterium
c. Actinomyces svidep dest MN ssgey-
d. Bilophila pvins de91 AYa
10. A foul odor when examining plates after incubation indicates the presence of this organism
S
in the
sample.
a.Veillonella rllonole
b. Eubacterium od abiorston
c. Leptotrichia ph mnhhieds
d. Porphyromonas dsrt biioainea a air
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HAPTER 26
RELATED ORGANISMS

1. describe the characteristics of the genus Rickettsia;
2. discuss the modes of transmission of rickettsiae and types of vectors;
3. differentiate the genera Orientia, Anaplasma, Coxiella, and Bartonella;

4. explain the methods used in the identification of rickettsiae and related
organisms; and
5. discuss the infections and diseases associated with this group of
bacteria.
Rickettsia
It belongs to the family Rickettsiaceae.

The species of thisgenus have the simplest bacterial form and are
considered as transitional organisms between bacteria and viruses.
The members are fastidious bacteria, short and obligate intracellular
organisms.
not survive in cell-
The species have Gram-negative cell wall and will
free media.
They grow in the cytoplasm and multiply through binary fission.
They do not undergo any intracellular developmental cycle.

They survive briefly outside of their host.
The attachment to the host cell is mediated by proteins such OmpA
as
and OmpB which are found on the outer membrane of the organism.
Microscopy: Small (0.5-2 m), pleomorphic, and with bacillary forms
Culture: Require living cells for growth
of
Mode of acquisition: Humans become infected following the bite
aninfected arthropod vector.
Mode of vectors.
prevention: Avoid contact with the
Accidental host: Humans
Possible agents of bioterrorism: R. prowazeki, R. rickettsia, and R. conorit
Transmission and Spread of Rickettsiae A BABDAI2TTENDI

The organisms gain access into humans through arthropod bites and skin abrasions by
disseminated through hematogenous, thus causing
scratching the site. The organisms are
vasculitis in the human blood vessels.
Transovarian and horizontal transmission are modes to maintain the viability of the organisms.
is also a mode of transmission of Rickettsia
Inhalation of aerosol from dried infected flea feces
typhi to humans.
Rickettsia feis is maintained transovarially in cat flea, Ctenocephalide feli.
Pathogenesis of Rickettsia
Early-onset: Presence of rickettsiae in the surface of the blood vessels but without any cellular
damage
Late-onset: Presence of the lymphohistiocytic perivascular infiltrate in organs such as in the
lungs, brain and heart and even in the dermis
Clinical manifestations: Fever, rashes, vomiting, muscle pain, and diarrhea
Rickettsial diseases are diagnosed based on the clinical manifestations and epidemiological
investigations.
Major Groups of Rickettsia

1. Spotted Fever Group
R. conorii, R. rickettsii, R. africae, R. honei, R. japonica, R. parkeri, R. sibirica, and R. tamurae
Common human pathogens: R. conorii, R. rickettsii, and R. africae
2. Transitional Group
R. akari, R. felis, and R. australis
3. Typhus Group
R. prowazekii and R. typhi
Spotted fever group

Rocky Mountain spotted fever (RMSF) is the most serious rickettsial infection.
stivie vell l
For RMSF,
humans are the accidental hosts and ticks are the main vector and reservoir.
The rashes develop on the palms of the hands and soles of
the feet of RMSF patients.melons
The boutonneuse fever exhibits rashes that are similar
with that of RMSF but on the face of the
individuals.
Tache noires (black spots) are present in patients with boutonneuse fever. wnheit
In rickettsialpox, the rashes are observed on
the face and extremities y bon
RICKETTSIACEAE AND RELATED ORGANISMS 443
Typhus group
Epidemic typhus is characterized by rashes on the face,
palms, and soles of the feet of the sick.
Rashes are not
commonly observed in patients with endemic typhus.
Recrudescence following years from the onset of infection is a hallmark in areas identified with
the Brill-Zinsser
disease or the epidemic typhus.
Humans serve as the reservoir for
the R. prowazekii. coninios faotd ante ikln dasbaue
Orientia
Orientia tsutsugamushi
It belongs to the family Rickettsiaceae.
It was categorized as a separate genus due to the absence of lipopolysaccharide and
peptidoglycan, and the presence of 54- to 58-kDa major surface proteins.
It replicates in the cytoplasm of its host cell and is released through a process that involves
"pinching off" the host cell.
It is the causative agent of the scrub typhus.

Mode of acquisition: Bite of an infected arthropod vector )espai1s 24021519t1 TOV192021
Vector: Leptotrombidium deliense (chigger)
Clinical manifestation: Presence of tache noires at the site of infection.
Accidental host: Humans and rats
There are five genera in this family, namely, Ehrlichia, Anaplasma, Neorickettia, Neoehrlic, and
divide by binary fission.

The species are obligate intracellular organisms which
The relevant species are considered as both human and veterinary agents of diseases.
and reticulate
Dense-cored elementary body (infectious form)
Two (2) developmental stages:
body (dividing form)
Human pathogens: E. chaffeensis, E. ewingi, and A. phagocytophilum
This genus belongs to the family Anaplasmataceae.

intracellular organism with Gram-negative coccobacillary
The species are nonmotile and obligate
cell wall.
The species are not transmitted transovarially.

vacuole of leukocytes.
The
replication of its species occurs in the cytoplasmic bodies, and morulae.
Developmental forms; elementary body (infective form), initial
"mulberries" or a
Microscopy: Presence of intravacuolar microcolony of bacteria that resembles
morula mostly in monocytes
Target cells: White blood cells (mostly monocytes) and macrophagesarmbe lon
Natural hosts: Humans and animals (dogs and deer)
Primary vector: Amblyomma americanum (lone star tick)

Species: E. chaffeensis (most common agent of HME) and E. ewingii
Related infection: Human monocytic ehrlichiosis (HME)
Anaplasma phagocytophilum
with Ehrlichia (but both
Itis nonmotile and has a Gram-negative coccobacilli cell wall same
genera lack the distinct lipopolysaccharide and peptidoglycan).

It causes humangranulocytotropic anaplasmosis (GA).
DUBUORW EI
Microscopy: Morula-containing neutrophils in peripheral blood
Target cells: White blood cells (neutrophils)
Natural hosts: Humans and animals (deer, horses and rodents)
Vector: Ixodes pacificus (Western black-legged tick) and Ixodes scapularis (deer tick)
Reservoir: Peromyscus leucopus (white-footed mouse)
Bartonellaceae
of Rickettsia
Bartonella
The species of this genus are Gram-negative bacilli, facultative, and intracellular organisms.
They live within the red blood cells in their natural mammalian hosts.
The species can be cultivated in charcoal yeast extract agar and incubate for
CAP or in 3 weeks at
35 °C to 37°C in an atmosphere with elevated CO, levels.
Some species exhibit a "twitching motility" in wet mounts (B. bacilliformis and B. henselae).
Target cells: Red blood cells and endothelium
Human pathogens: B. quintana, B. henselae, B. elizabethae, and B. bacilliformis

Reservoir: Humans
of Bartonella and
TABLE 26-1. Species Their Associated Infections and Diseases
Species Infections and Diseases
B. quintana Trench fever (louse-borne disease)
Cat scratch disease
B. elizabethae Infective endocarditis
bacilliformis Oroya fever (chronic

verruga peruana) and febrile acute hemolytic anemia
B.
Cat scratch disease (secondary agent)

YOD IO
RICKETTSIACEAE AND RELATED ORGANISMS 445
Coxiella burnetii
It is the only species in the genus Coxiella.
It is extremely contagious and considered as potential bioterrorism agent (Category B select
agent).
the causative agent of Q
It is
(Query) fever which is a systemic infection of the lungs.
is described as the most
It
common obligate intracellular fastidious bacterium causing
endocarditis.
It can survive extracellularly because of its endospore-like body.

It can infect birds and rodents, which in turn excrete the organisms via their urine, feces, and
birth products.
It is not transmitted by arthropod vectors.
It does not multiply in routine bacteriologic culture media.

bonof Modes of acquisition: Inhalation of contaminated aerosols from dried animal excreta and
has ingestion of contaminated unpasteurized milk

Microscopy: Gram-negative coccobacillary form
Target cells: Macrophage
Animal reservoir: Cattle, goat, and sheep
Laboratory Diagnosis for Rickettsiaceae and Related Organisms

blood, buffy coat, lymph node
Specimens: Biopsy of skin tissues (including eschar), peripheral
aspirate, and CSF
in a biosafety level 3 laboratory.
Processing of specimens should only be done
1. Microscopy
visualize Rickettsia using antibodies specific for the target

is a sensitive and specific test to
group of organisms. oditns digin of elles
immunomagnetic beads coated with
Immunocytologic studies using blood specimens and
immuronaiantibody reveal K, conori in detached, circulating endothelial clls.
are utilized for microscopic analysis.
TheWarthin-Starry and Giemsa stains
is utilized for the histopathologic diagnosis of
Modified Brown-Hopps staining method
"thin rods" within endothelial cells.
Rickettsia where the organisms appear as
for the detection of morulae in
or buffy coat is used
Giemsa staining of peripheral blood
febrile stage of ehrlichiosis.
and B. henselae.
dentification of B. quintana
Warthin-Starry stain is for the
2. Culture
Culture media: Yolk sacs of embryonated eggs and tissue/cell cultures

humans in an antibiotic-free,
Rickettsia, Ehrlichia, and Anaplasma can be isolated from
centrifugation-enhanced shell vial cell culture.
Rickettsia can be inoculated in the yolk sacs of embryonated eggs.
Anaplasma and Ehrlichia utilized HL-60 cells and DH-82 cells for culture, respectively.
Bartonella species can be isolated in blood cultures using the lysis centrifugation method and
later plating the concentrated blood onto freshly prepared culture media with 5% horse or
rabbit blood.
Columbia blood agar with 5% defibrinated blood has been utilized to recover B. henselae and
B. quintana from patients (Tierno et al, 1995).
Lung tissue cell line is the preferred culture medium for C. burnetti.
3. Immunodiagnosis
It is the only test that is performed for the diagnosis of rickettsial diseases by demonstrating
bas 6191 increase in antibody titer especially during period of convalescence: it is usually performed
after the acute phase of the illness since antibodies are usually undetectable during the early
onset of the disease (antibodies to Rickettsia, except for R. rickettsii, would not be detected
until at least two weeks after infection).
Antibodies against Bartonella henselae, the agent of the cat scratch disease, can be confirmed
by serodiagnosis.
It is the method of choice for the detection of C. burnetti if the biosafety facility is not
available and for Bartonella species.
It is the gold standard serologic test for antibody detection.

It is the reference method for the identification of rickettsioses
It is useful for the diagnosis of scrub typhus and Query fever.
An IFA titer of 1:64
is diagnostic for Rickettsia and Ehrlichia infections while a titer of
199161 edt noli oft 2 1:12,800 is for scrub typhus. A axileuaiv of 1ea! aitioogs ons evifienna s ol
Query fever caused by C. burnetti results to high antibody titer, usually an anti-
phase II
b.
It is a presumptive test for rickettsioses though not

routinely perform. rT
The agglutination of certain strains of Proteus vulgaris when
treated with the serum
sample from patients with rickettsial diseases is diagnostic. orive Belsw
It utilizes
group-specific antibody and is a non-specific test. Dininta camid)
Individuals with Q
antibody. Mela
fever, ehrlichiosis, and rickettsialpox do not produce Weil-Felix
bm cuathiat
20 D-RICKETTSIACEAE AND RELATED ORGANISMS 447
C. Microimmunofluorescent Dot
D
Test
an excellentsensitivity for detectingantibodies to Rickettsia.

It has
It is used for
the early diagnosis of RMSF after the
d.
onset
of symptoms.
Immunohistochemistry is utilizedin diagnosing rickettsioses, ehrlichiosis.

related to Bartonella. and diseases
It is used
for the detection of the agent
of RMSF in skin biopsy samples.
e.
Other Serological Tests: Latex Agglutination, Enzyme Immunoassay and Line Blot
4. Nucleic Acid Amplification
It is useful for the detection of
the relevant rickettsiae such as R. rickettsii, R. conorii, R. typhi,
and R. prowazekii from clinical samples.
It is
also a diagnostic tool for ehrlichiosis using species-specific primers.
Cat scratch
disease can be confirmed through PCR testing of lymph node aspirates and
biopsies.
This method
may not identify early-onset of the rickettsial disease as well as recovery from
the illness.
Samples other than peripheral blood can be used such as biopsy and necropsy specimens,
plasma, buffy coat and even the actual arthropod vectors recovered from the patients.
Target genes for Rickettsiae: 17-kDa, lipoprotein gene, gItA, rrs, groEL, omp A and B
Target genes for Anaplasmataceae: 16S rRNA (rrs), TRP 32 and 120, dsb, ank- and msp2
Target genes for Coxiella: 16S rRNA and 23S rRNA

TABLE 26-2. Differential Characteristics of Rickettsiaceae and Related Organisms

Infection Disease
Vector/Mode of Transmission
Organism
Spotted fever group
a. Rickettsia conorii Boutonneuse fever or
Ticks (Rhipicephalus sanguineus)
Mediterranean spotted fever
Rickettsia rickettsii
Wood ticks (Dermacentor andersoni)
b. Rocky mountain spotted fever
Dog ticks (Dermacentor variabilis)
Brown dog ticks (Rhipicephalus sanguineus
and Amblyomma cajennense)
Transitional group
a. Rickettsia akari Rickettsialpox Mouse mite (Liponyssoides sanguineus)
b. Rickettsia felis Flea-borne spotted fever Flea bite or feces
Typhus group
a. Rickettsia prowazekii Epidemic typhus Body louse (Pediculus humanus corporis)
Brill-Zinsser disease Squirrel flea (Orchopeas howardi)
Squirrel louse (Neohematopinus sciuriopter)
b. Rickettsia typhi Endemic murine typhus Rat flea (Xenopsylla cheopis)
Scrub typhus Group
Orientia tsutsugamuchi Scrub typhus Chigger (Leptotrombidium deliense) bite
a. Ehrlichia chaffeensis Human monocytic ehrlichiosis Lone star tick (Amblyomma americanum)
ELE
Human granulocytotropic Deer tick (Ixodes scapularis and
phagocytophila Ixodes pacificus)
Coxiella burnetti Q fever Inhalation of aerosol from infected animals
Bartonella quintana Trench fever Feces of body louse (Pediculus humanus

corporis)
Bartonella henselae Cat scratch disease Kitten scratch or bite
Bacillary angiomatosis
Bartonella bacilliformis Oroya fever and verruga peruana Sandfly (Lutzomyia) bite
CHAPTER 27
CHLAMYDIACEAE

1. discuss the general characteristics of the genus Chlamydia;
differentiate the reticulate body from elementary body;
3. outline the serovars of Chlamydia trachomatis;
4. explain the clinical significance of chlamydiae; and
5. cite the various laboratory tests for the identification of Chlamydia
species.
Chlamydiaceae
The species of this genus are non-motile, small (0.2 to 1.5 um), and
have a Gram-negative cell wall.
They are obligate, intracellular organisms that require living cells
for
growth.
The members are once called "energy parasites" because they depend
on the eukaryotic cells of their hosts for metabolism, growth, and
reproduction; however recent molecular studies reveal that the
ATP by utilizing D-glucose
chlamydiae generate their own
can
6-phosphate from the host cell.

the cytoplasm of infected
The species replicate by binary fission in
cells.
Most species are considered human pathogens.

Genera: Chlamydia and Chlamydophila
psittaci, and
Species: Chlamydia trachomatis, Chlamydophila
Chlamydophila pneumoniae
Chlamydia has two (2) morphologic forms:

d. Reticulate body (RB) replicative and non-infectious form
It is the intracellular and metabolically active form.
needs SO they multiply by
It directs the reproduction of host cells to their own metabolic
binary fission.
b. Elementary body (EB) - infectious form
It is the extracellular form and spherical in shape.
It resembles Gram-negative bacilli with a rigid cell wall.
It infects the host cell by inducing active phagocytosis.
components, the major outer membrane protein (MOMP)

and the
It has two
lipopolysaccharide antigen.
Chlamydia trachomatis
It is one of the major sexually transmitted pathogens.
It is one of the principal causes of pelvic inflammatory disease (PID) and ocular trachoma.
It can travel through the birth canal where infants can be infected during birth.
It is associated with infertility and ectopic pregnancy.
It has 20 known serovars and all are associated with human infections and diseases.
Natural host: Humans
Unique feature: 10 stable plasmids

Biovars: 2 (LGV and Trachoma)
WHO Elimination Strategy

SAFE: Surgery for advanced disease, Antibiotics to clear C. trachomatis infection, Facial
cleanliness, and Environmental improvement to reduce transmission hTi
Serovars of C. trachomatis based on the MOMP Antigenic Differences n enlin ons arodimam onf
1.
2. LGV serovars: L1, L2, L2a, L2b, and L3

nwo fiedl simonog nso ssibymsirb
3. Inclusion conjunctivitis and Urogenital infection serovars: D to K, Da, la, Janoi sadlonodg-d

The epidemiological categories trachomatis infection include classic trachoma, sexually
of C.
transmitted infections of adults, and perinatal ocular and respiratory tract infections.
Chlamydia infection is transmitted sexually, as well as during birth, from the mother to the infant.
C. trachomatis is also transmitted through contaminated objects and by laboratory aerosols
causing pneumonitis and hilar lymphadenopathy.
The intraperitoneal spread may cause peritonitis or perihepatitis (Fitz-Hugh-Curtis syndrome).
CHLAMYDIACEAE 451
1.
Trachoma
It is a
chronic inflammation of the conjunctiva that leads to blindness vid ism
It can cause distortion
of the eyelids (eyelashes become misdirected and turn inward).
Modes of Transmission: Contact with contaminated objects (fomites), hand-to-hand contact
with carriers, and through vectors (flies)
2.
It is a sexually transmitted disease which has a multi-system involvement.

A small, painless ulcer or papule appears initially at the site of infection such as the penis
cispesou and cervix, and then nodules (buboes) develop after several weeks.
lisp 3. Inclusion conjunctivitis lo\entbe

It is characterized by an abundant eye discharge and a swollen conjunctiva.
It affects infants who acquire it through aspiration or ocular exposure during birth.
It is the most common expression of C. trachomatis infection in infants.
Infantile chlamydiae infection acquired at birth may lead to interstitial pneumonitis,
serum, urine, and
Specimens: Urethra and cervical secretions, conjunctiva discharge, rectal swab,
materials aspirated from fallopian tubes and epididymis
are recommended.
For infantile pneumonia, nasopharyngeal swab, throat swab, and lung tissue
If the case is a suspected LGV, the first morning urine is the best sample for male patients while
vaginal swab is for the females.
Due to the intrinsic nature of C. trachomatis, recovery from the clinical specimen and sometimes
lentborli from asymptomatic carriers takes several weeks to months. nigm
The use of Dacron or rayon-tipped swabs is preferred.
1. Culture (Living Tissue Cell Culture)

229 cells, HEp-2 cells, McCoy cells,
Culture media: Buffalo green monkey kidney cells, HeLa
and cycloheximide-treated McCoy cells
McCoy Cells
Most commonly used cell lines: Buffalo Green Monkey Kidney Cells and
Growth enhancer: Cycloheximide (0.5 to 1.5 pL/mL)
the specimen onto the cell monolayer is a
Procedure Prior to culture, centrifugation of
requirement. n
facilitates the adherence
of elementary bodies.
The shell vial technique
Di
Cytologic Examination bodies in smear specimens taken
It allows direct
evomination of C. trachomatis elementary
and oropharynx), genital (vagina), and anal
from conjunctiva, respiratory (nasopharynx
(Kuo, et al 1984).
the monolayers are treated with iodine or

the cell lines,
After 48 to 72 hours of incubating
immunofluorescent stain to identify the presence of inclusion
It is not routinely recommended for genital samples. arti
3. Immunodiagnosis
become less
The relevance of the serological tests for the diagnosis of genital chlamydiae has
the antibodies. morgues f
significant through the years due to nonspecific nature of
The extractable LPS or elementary body with keto-deoxyoctonate is the primary antigen that
can be idenified in genus-specific tests.
fluorescein.
A direct fluorescent antibody (DFA) staining method that utilizes the
isothiocyanate-conjugated monoclonal antibodies is best over iodine and Giemsa stain in
detecting the organisms (presence of inclusion bodies/elementary bodies) in infected cell
lines.
DFA utilizes monoclonal antibodies and monospecific polyvalent antisera against the outer
membrane lipopolysaccharide (LPS) or the MOMP to detect chlamydial antigen in ocular
specimens.
a. Complement fixation
It identifies family-reactive antibodies.
It is utilized in the presumptive diagnosis of LGV.
Genus-specific antigens can be detected by using this method.
LPS is the major antigen that is detected in genus-specific serologic tests for chlamydial
infection.
(+) Result: 2 1: 256

b. Microimmunofluorescence (Micro-IF) assay
It is the recommended method for the
detection of antibodies (type-specific antibodies)
to C. trachomatis.
It determines LGV, trachoma, inclusion conjunctivitis, and chlamydial neonatal

infection.
(+) Result: IgM titer of ≥ 1:32 venbid yetnom nen ollted
It is the most sensitive method for detecting C, trachomatis especially in genital specimens.
reliable method in
It is
the identification of symptomatic patients with LGV.
It is also utilized for the detection of non-LGV C. trachomatis in urogenital and non-
urogenital (rectal and oropharyngeal) specimens in both
individuals.
symptomatic and asymptomatic
Preferred Molecular Techniques:

Nucleic Acid Amplification (NAA) and Nucleic Acid
Hybridization (NAH)
Advantage: It determines in one sample other bacteria causing STD such as N. gonorrheae.
NAAT is considered the gold
standard for the identification of C. trachomatis.
Specimens: Urine, and endocervical and urethral swabs
CHLAMYDIACEAE 453
amydophila psittaci
It is the causative agent of psittacosis or ornithosis.
It is an endemic pathogen of avian species such as parrots, parakeets, chickens, and ducks.
It has 10 serovars causing human and animal diseases.
Modes of acquisition: Handling of infected birds and inhalation of infectious aerosols from dried
bird excreta
Major reservoir: Psittacine birds

Genotypes: A, B, C, D, E, F and E/B (all infect humans)
Primary target of infection: Lungs ("hacking cough")
CDC recommended sample for culture: Sputum and nasopharyngeal/oropharyngeal swabs
Other sample for detection and isolation: Pleural fluid, bronchial lavage, and lung tissue
CDC recommended culture media: Mice or chick embryo
CDC Immunodiagnosis: Complement Fixation (titer 2 1: 256)
Microimmunofluorescence Antibody (IgM ≥ 1:16 or IgG ≥ 1:512)
CDC Molecular Diagnosis: Nucleic Acid Amplification
Precautionary measures: Only laboratories with biosafety level 3 facilities can perform the
isolation and cultivation of this organism.
Chlamydophila pneumoniae
It is formerly known as the TWAR (Taiwan Acute Respiratory Agent) strain.
It is a human pathogen that is transmitted through aerosol droplets.
It has one known serovar and is one of the major causes of infectious respiratory diseases.
It is associated with community acquired-pneumonia, bronchitis, pharyngitis, and sinusitis.

viral
transport media
CDC recommended samples: Nasopharyngeal/Oropharyngeal swabs in
sputum, throat swabs,
(VTM) or universal transport media (UTM), nasopharyngeal aspirates,
bronchial lavage and washings, and CSF
Other sample for detection and
isolation: Lung tissue
Preferred Swab: Dacron/Rayon/Nylon swab with plastic shaft

Culture media: HeLa cells or Hep-2 cell lines
(Fluorescein-conjugated monoclonal
Microscopic Examination: Direct Fluorescent Antibody
antibodies)
CDC Primary Laboratory Method: Nucleic Acid Amplification
256)
CDC Recommended Immunodiagnosis: Complement Fixation (21:
or IgG
Microimmunofluorescent assay (IgM ≥ 1:16
2 1:512)
TABLE 27-1. Differential Characteristics of Chlamydia Species

C. psittaci C. pneumoniae
Properties C. trachomatis
Humans
Host range Human
Round Pear-shaped
Elementary body Round
Variable, dense neiiiel
Round, vacuolar
Inclusion morphology and Levinthal-Cole- Round, dense.
Halberstaedter-
inclusion bodies
Prowazek bodies Lillie bodies
Macchiavello stain Giemsa stain

Stains used Lugol's iodine stain and Giemsa stain
Absent Absent
Glycogen-containing inclusions Present
Resistant Resistant
Susceptibility to sulfonamides Susceptible
Trachoma, LGV, and rotel Psittacosis be100
Pneumonia, pharyngitis
Diseases
inclusion conjunctivitisM Pneumonia
Number of serovars 20 10
FIGURE 27-1. Chlamydia trachomatis inclusion bodies (brown) in cell culture (MO
Source: Dr. E. Arum, Dr. N. Jacobs/Centers for Disease Control and Prevention (CDC) (Image Number: 6427) https://www.britannica.
com/science/Chlamydia-trachomatis
CHAPTER28
MYCOPLASMATACEAE
(CELL WALL-DEFICIENT
BACTERIA)
1. describe the distinct characteristics of Mycoplasma and Ureaplasma;

2. differentiate the colony and biochemical features of the clinically
significant species;
3. discuss the related infections and diseases; and
outline the diagnostic tests to identify the cell wall-deficient bacteria.
Mycoplasma and Ureaplasma (Mollicutes)

The speciesof these genera are considered as the smallest free-living
artificial culture media.
organisms that are capable of growing in
wall.
They are pleomorphic bacteria that lack a cell
The species are found mainly in the oropharyngeal, upper
mycoplasmas have distinct
respiratory, and genitourinary tracts - the
the respiratory and urogenital tracts
affinity with the epithelium in urine
and are not eliminated by formation of mucous secretions and
flow.
tract and their transmission
They are common parasites of the genital
is related to sexual activity.
replicate by binary fission,
Most species are slow-growing,fastidious,
and faculalieanaerobes except the aerobic M. pneumonae.
They require sterols (cholesterol) in the culture media for membrane
function and growth. at the
immediate inoculation
They are very sensitive to drying, hence,
bedside is recommended.
M. fermentans, M. genitalium,
Species: M. pneumoniae, M. hominis,
M. faucium, M. salivarium, and U.
Mycoplasma pneumoniae (Eaton agent)

It is the etiologic agent of primary atypical pneumonia or walking pneumonia.
and is extremely
It has gliding motility, aerobic, not included in the normal human commensals,
fastidious.
It has strong attachment to the mucosal cells of the respiratory tract, and can escape
phagocytosis.
Mode of acquisition: Inhalation of contaminated aerosol droplets
Culture: SP4 broth - Colonies exhibit a yellow color
Biochemical test: (+) Glucose fermentation
Primary Atypical Pneumonia

It can occur as separate incidents or as outbreaks in closed populations such as in school, military
camps, and within family members.
It is usually diagnosed through observation of the signs and symptoms because of the limitations
in the laboratory procedures caused by the unique physiological characteristics of M. hominis.
Mycoplasma hominis and Ureaplasma urealyticum (Genital Mycoplasmataceae)

The species can be recovered from the genital tract of healthy adults both males and females.
They are opportunistic pathogens but recent studies reveal that U. urealyticum is not associated
with the disease of the female genital tract but has been isolated in men with nongonococcal
urethritis.
Colonization among infants occurs mostly during passage through an infected birth canal and
results in the isolation of these organisms from their nose and throat, however, both
species have
been identified as well from newborns delivered by caesarian section.
Growth of M. hominis is best observed in an anaerobic condition.
They cause neonatal infections such as sepsis, meningitis and pneumonia.
Culture: M. hominis and

U. urealyticum - Colonies exhibit a "fried-egg" appearance on a plated
medium.
U.
urealyticum - Colonies exhibit dark-brownish lumps on a A7 or A8 agar medium.
Biochemical test: M. hominis = (+) Arginine deamination
U. urealyticum = (+) Urease
Related infection for M. hominis: Pelvic
inflammatory disease (PID), bacterial vaginosis, renal
stone, post-abortal fever, and postpartum fever
alon
Related infection for U.
realyticum: Non-gonococcal urethritis (NGU) and chorioamnion
MYCOPLASMATACEAE (CELL WALL- DEFICIENT BACTERIA) 457
Specimens for general testing include blood, wound aspirates, CSF, amniotic fluid, and serum
(for serology).
-STUDE
Specimens for M.
pneumoniae: Throat swab, bronchoalveolar lavage, sputim, and lung tissue
Specimens for Cenital Mycoplasma and . urealyticumnt urethral, vaginal or endocervical swab,
urine, prostatic secretions, and seminal fluid
1. Microscopy
No direct method
or Gram staining can be used for identification of Mycoplasmataceae
Mycoplasmas have positive reaction with Dienes staining method.
2. Culture
Culture media: Beef

or soybean protein with serum, fresh yeast extract, biphasic
pleuropneumonia-like organism (PPLO).broth or agar with yeast extract and horse serum,
A8 agar, modified New York City medium and anaerobic BAP
Swabs for sample collection: Calcium alginate and Dacron swabs ae fnldonblinA
Transport medium: Shepard 10B arginine broth and SP4 mciulibonin derd erfT
Urea and arginine are added into the media to promote the growth of U. urealyticum and
M. hominis, respectively, producing red color in Shepard 10B broth.
Ureaplasma species are pH-dependent, they require media at pH 6.0.
Overlaying suspicious colonies with 0.5% guinea pig RBC in PO,-buffered saline is the
o nousmino
definitive identification method for M. pneumoniae. m
Blood culture is not usually performed for mycoplasmas due to the inhibitory property of
sodium polyanethol sulfonate (SPS).
24 hours, it should be kept at -70°C.
If samples cannot be processed within
Growth patterns of Mycoplasma and Ureaplasma

M. pneumoniae requires a biphasic culture system and incubation of up to three weeks in a
chamber with 5% to 10% CO,.

is capable of growing on BAP and CAP producing
M. hominis is the only species that
it grows better in the
absence of oxygen using anaerobic
nonhemolytic, pinpoint colonies;
BAP.
of M. hominis is observed on A7 agar.
The typical fried egg colonies
turbidity in broth media.
Mycoplasmas do not normally create
slowly than most of the other bacteria due to lack of cell
Mycoplasma and Ureaplasma grow
wall.
458 REVIEW HANDBOOK IN DIAGNOSTIC BACTERIOLOGY YM
3. Immunodiagnosis
It is the common diagnostic procedure for the M. pneumoniae infection utilizing both
acute-phase and convalescent serum samples - acute infection caused by M. pneumoniae is
confirmed by the presence of IgM in single serum sample with a fourfold rise in titer.
dows ELISA is the routinely used serological method for the diagnosis of Mycoplasma and
Immunofluorescence is also utilized with anti-M. pneumoniae antibody reagent placed over
the colonies.
4. Manganese Chloride Urea Test

diiw notbboor sulleng aved esmaslqoovin
It is a rapid identification test for U. urealyticum.
The reaction for the test is observed under a dissecting microscope.
(+) Result: Dark brown precipitate of manganese oxide around the colonies r f
u. urealyticum utilizes the manganese chloride and urea in the culture medium.
5. Antimicrobial Susceptibility Test ns stehigls hhflalhD trtohbollos alci

The broth microdilution is the routine method but agar dilution is the reference method.
6. Molecular Diagnosis (Nucleic Acid Amplification)

It is the best method for the diagnosis of infection caused by M. pneumoniae.
Preferred specimen: Nasopharyngeal swab has
Confirmation of M. pneumoniae requires collection of respiratory specimens and detection of

nucleic acid by amplification or identification of the specific antibodies (IgM).
TABLE 28-1. Comparison Between the Common Bacteria and Atypical Bacteria®
Characteristic Bacteria Rickettsieae Chlamydiae Mycoplasmas
Obligate intracellular
DNA and RNA
Ribosomes
Peptidoglycan in the cell wall

Replication by binary fission e
Growth on non-living media +
Sensitivity to antibiotics
0 or to faci
MYCOPLASMATACEAE (CELL WALL-DEFICIENT BACTERIA) 459
FIGURE 28-1. Mycoplasma on culture media showing the "fried egg" colonies
CHAPTER 29
SPIROCHETES AND
MISCELLANEOUS
BACTERIA

1. describe the general characteristics of spirochetes;
2. differentiate the distinct features and mechanism of transmission of
Treponema, Borrelia, and Leptospira;
3. explain the different stages of syphilis;
4. discuss the serological tests for the identification of Treponema
pallidum;
5. describe the pathogenesis of Borrelia and Leptospira;
6. cite the methods for the detection of spirochetes; and
7. outline the miscellaneous bacteria and related infections and diseases...
General Characteristics of Spirochetes

This group of bacteria belongs to
the order Spirochaetales.
features with Gram-
The organisms have unusual morphologic
negative bacterial characterization.
The species can be facultatively anaerobic or aerobic organisms.
patterns.
They exhibit various types of motility
They have fibrils or axial filaments which are flagella-like organelles
wall and facilitate motility
that wrap around the bacterial cell
(exhibiting a 'corkscrew-like" winding) of the organism.
General microscopy: Slender,
helical, and unicellular bacteria
Genera: Treponema, Borrelia, and Leptospira
Varying Characteristics of the Genera Within the Order Spirochaetales:

1. Number of axial fibrils or periplasmic flagella
2. Number of insertion disk (plate-like structure where fibrils are
of the cell)
attached; found near the terminal
3. Biochemical and metabolic features
Treponema
It belongs to the family Spirochaetaceae.

which means "to turn," and nema,
The name of the genus came from the Greek words trepein,
which means "thread," that when combined means "turning thread."
The species are best observed with a dark field microscope.
be cultivated after multiple passages in
They only infect humans and have not been observed to
vitro.
Most species stain poorly with Gram or Giemsa stains.

Reproduction: Transverse fission
Clinically Significant Species: T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, T. pallidum
subsp. endemicum, and T. carateum
1610
Microscopic Characteristics of Treponema species:

a. Thin, spiral organisms (4 to 14 spirals/organism) with three axial filaments and one insertion
disk
b. Cell ends are pointed and covered with a sheath

C. (+) Corkscrew motility
Treponema pallidum subsp. pallidum

It is the causative agent of syphilis.
It is a microaerophile which survives longer in the presence of 3% to 5% oxygen.
It is killed rapidly at 42°C, sO it is used as a basis for syphilis therapy.
It remains visible in whole blood or plasma for at least 24 hours, which is of potential importance
in blood transfusion.
It can cross intact mucous membranes and the placenta, and spread throughout the body.
Dark field microscopy: Appears white against a dark background and long with fine spirals that
three fibrils/periplasmic flagella
have 10 to 13 coils and
Generation time: 30 hours
Types of antibodies that are used in diagnostic tests: Reagin and non-treponemal reagin
Related Infection and Diseaseamo ai-slogalt w noidw ethomelhy leixs
Syphilis (French disease/Italian disease/The Great Pox)5 (anibnitw

It is a disease of the blood vessels and the perivascular areas.
It is also known as the "great imitator" because it can copy and assume many clinical
manifestations.
Some untreated syphilitic patients exhibit biologic cure and loses serologic activity. Bond goins
Transmission: Sexual contact;
congenital/vertical transmission (congenital syphilis); skin contact
lesion (primary or secondary syphilitic lesion); transfusion of fresh blood; injuries
with an active
from contaminated needle sticks; and handling of

specimens far
Symptoms: Chancre, fever, sore throat, headache, rashes (palms and soles), and gummas on skin
SPIROCHETES AND MISCELLANEOUS BACTERIA 463
Stages of Syphilis mohabied

a. Primary syphilis
It is characterized by theappearance of hunterian or hard chancre, which is an infectious
primary lesion that is painless and is usually seen at the site of inoculation (most commonly
on the genitalia).
It develops 10 to 90 days after infection though patient is asymptomatic.
No systemic involvement is observed in this stage, i1 ydll lo notte villas ovity nie.
b. Secondary syphilis
It develops 2 to 12 weeks after the appearance of chancre with possible relapse.
All lesions that are seen in this phase are highly infectious.
In this stage, the chancre heals but the organisms are still disseminated in various tissues via
the bloodstream.
Symptoms: Fever, sore throat, weight loss, headache, and rashes (palms and soles)
C. Latent stage
It is the period in which the disease becomes subclinical but not necessarily dormant.
It occurs within more than year of infection.
In this stage, diagnosis can be made only by serological tests. a h nabort
2 Types: Early latent syphilis (within 1 year of infection) and late latent syphilis (more than
one year of latency)
d. Tertiary stage/Late syphilis

It is the tissue-destructive phase.
It appears 10 years to 25 years after the initial infection. aslq DIGEY

botbannu
In this stage, individuals are not usually infectious. nigser momise
Complications: Central nervous disease (neurosyphilis), cardiovascular abnormalities, eye
disease, and granuloma-like lesions (gummas)
Laboratory Diagnosis nudti

bits
Specimen: Skin lesions
sterile saline
Collection of specimen: Primary and secondary syphilitic lesion is cleaned with (not
fluid from the lesion is transferred onto clean
1s Dalis with antiseptics) and following gently abrasion, the
slide and sealed with a cover slip.
Oral lesions should not be examined because non-pathogenic spirochetes may lead to false-
positive results.
1.
Microscopy
dark field microscopy is
Direct microscopic examination of the sample (unstained) utilizing
recommended.
if the sample is too thick.
Saline is added in the preparation of the smear
motile treponemes from the chancre specimen is diagnostic
for
The demonstration of
primary syphilis.
The fluorescent antibody staining of the specimen can also

be performed. dov210p2aMg
Definitive test: Dark field microscopy for the observation of motility dlilg
Special staining technique: Levaditi and Fontana-Tribondeau bexj1or2691941
Direct detection in lesions: Fluorescein isothiocyanate-labeled (FITC) antibodies
2. Culture
due to its poor growth
In vitro cultivation of the treponemes is not routinely performed
outside the host coupled with oxygen requirement.
protocols on the culture of
Edmonson and Norris (2021) mentioned about the latest
treponemes particularly for the T. pallidum utilizing standard laboratory environment, tissue
culture cells, and isogenic strains of the agent of Great Pox.
3. Immunodiagnosis (Serological Test)

It is the basis of diagnosis especially in the secondary and tertiary stages of syphilis.
Non-Treponemal Test (NTT)
It is a screening test and considered non-specific.
presence of non-specific antibodies or antibody-like proteins (reagin or

It detects the
Wassermann antibodies) to lipoprotein materials from damaged cells and cardiolipin from
treponemes.
It is used to monitor the treatment of syphilis.

Principle: Reagin binds to the test antigen cardiolipin-lecithin-coated cholesterol particles
and causes it to flocculate.
Examples: Rapid plasma reagin (RPR), venereal disease research laboratory (VDRL),
unheated serum reagin (USR), toluidine red unheated serum test (TRUST), and enzyme-
linked immunosorbent assay (ELISA)
Sample: Serum or CSF
Reagent: Combination of cardiolipin (phospholipid), lecithin, cholesterol, choline chloride,

and charcoal
RPR does not require the heating of the serum and is not recommended for CSF.
Reagents for the VDRL must befreshly prepared and the patient's serum must be heated at
56 °C for 30 minutes (complement inactivation).
(+) result: Flocculation
Cause of false-positive RPR and VDRL test results: Old age, pregnancy, hepatitis, rheumatic
fever, systemic lupus erythematosus (SLE), and infectious mononucleosis
Treponemal Test
It provides specificity, hence it is the confirmatory test.

It detects the presence of antibodies to treponemal antigens.
method in identifying late-stage infections since the titers remain high
It is the preferred
even during therapy.

In
'reverse syphilis screening test" if
treponemal test is
are positive, a nontreponemal test with titer should be used for screening and the results
guide patient management decisions performed to confirm diagnosis and
Source: (https://www.cdc.gov/std/syphilis/stdfack.ov
ct-syphilis-detailed.htm).
Types of Treponemal Tests
a.
Treponema pallidum Particle Agglutination (TP-PA)
It uses gelatin particles that are
sensitized with T. pallidum antigens.
(+) Result: Agglutination in the presence of
anti-treponemal antibodies
b.
Microhemagglutination T. pallidum (MHA-TP)
It
utilizes sheep red blood cells sensitized with T. pallidum (Nichols strain) and the
patient serum
Recommendation: If positive or reactive by MHA-TP, sample is tested again using EIA to

detect the presence of IgG antibodies to
T. pallidum (Syphilis-G-EIA).
(+) Result: Agglutination of red blood cells
C.
Indirect Fluorescent Antibody Test/Fluorescent Treponemal Antibody Absorption (FTA-
ABS)
1900091
It uses fluorescent-labeled antihuman antibody against the patient antitreponemal
antibodies.
The antitreponemal antibodies are bound to treponema affixed to a commercially

prepared slide.
It confirms positive VDRL or RPR tests.

Procedure: The whole treponemes (T. pallidum Nichols strain) fixed to a slide are treated
with a heated serum from possible syphilis patients.
(+) Result: Fluorescence of the treponemes
3. Enzyme Immunoassay (EIA)

It is commonly used next to TP-PA.
The results are comparable with other treponemal tests.
used as prenatal syphilis screening test.
The Syphilis-IgG test (SYPGN) may be
reactive SYPGN implies exposure to T. pallidum.
Interpretation: A positive or
4. Point of Care Test/POCT (Immunochromatography)

similar with treponemal test.
It has specificity range
It detects the presence of antibodies (IgG and IgM).
blood
Specimen: Skin-punctured
Reagent: Recombinant T. pallidum antigens
5.
Molecular Diagnosis
of syphilis.
PCR is considered to determine the primary stage
the detection of neurosyphilis (AIDS patients).
PCR is used for
RPR and VDRL usually detect the primary syphilis, including the
using CSF specimens.
Charcoal particles are utilized for the visualization of flocculation. 51 1222

Western blot is designed for the detection of congenital syphilis.
Cultivation of is not a routine procedure for
identification of the organisms.
treponemes
a00025
Treponemal antibodies appear earlier than nontreponemal antibodies and usually remain detectable
for life, even after successful treatment
Source: (https://www.cdc.gov/std/syphilis/stdfact-syphilis-detailed.htm)
A positive EIA for syphilis test, requires a quantitative RPR test for counseling purposes to establish
current infection status ("reverse syphilis screening test).s Dchin yanse
Parenteral penicillin G is the recommended freatment in all stages of syphilis. Les (*)
Treponema pallidum subsp. pertenue

It is the causative agent of yaws or frambesia tropica.
It is acquired by direct contact through skin breaks with an infected lesion.

Yaws is a non-venereal infection with random bodily chronic ulcerative sores which eventually
lead to tissue and bone destruction and then to crippling if left untreated.
It is the causative agent of endemic non-venereal syphilis or bejel.

It is transmitted by direct contact with active lesions and contaminated fingers and utensils.
Bejel is a non-venereal syphilis that is characterized by the appearance of a primary lesion
usually on or near the mouth and followed by the development of pimple-like sores on the trunk,
arms, and legs,
Treponema carateum
It is the causative agent of pinta or carate.
It is acquired by contact with infected skin. thinn onbmimh
Pinta is an infection of the skin that is represented by a primary lesion with enlargement of the
regional lymph nodes, followed by a generalized red to
slate-blue macular rash in 1 to 12 months.
Treponema denticola and Treponema socranski

They cause ulcerative gingivitis and chronic periodontitis.
They are related to T. palllidum.

Borrelia (Blood Spirochetes)

925980 6014
the family Spirochaetaceae same with the genus Treponema.

It is in
The species are slow-growing spirochetes that multiply by binary fission.Hobbmlled s

They are composed of 3 to 10 loose coils and is
actively motile.
The species have 15 to 20 axial
filaments and 2 insertion disks.
They stain well with Giemsa
technique and routinely observe using brightfield microscopy.
Species that have been grown in vitro are
microaerophilic.
Species: B. recurrentis, B. dutoni, B. hermsii, B. turicatae, B. parkeri, B. afzelii, B. burgdorferi, and
B. garinii
Borrelia recurrentis
It is the
agent of louse-borne/epidemic/european relapsing fever. -2r 3 19m
Vector: Louse (Pediculus humanus)
Reservoir: Humans
B. hermsii, B. turicatae, B. duttoni, and B. parkeri Boors

These are the agents of tick-borne relapsing fever/endemic/American relapsing fever.
Vector: Soft ticks (Ornithodoros)
Transmission: Attachment of ticks onto skin while the saliva is infected with the organism
B. burgdorferi Sensu Lato

These are the agents of Lyme disease.
Vector: Hard ticks (Ixodes) - Trodes pacificus, Ixodes persulcats, Ixodes dammini, and Ixodes scapularis
Transmission: Bite of the Ixodes ticks
Natural hosts of ticks: Deer and rodents (Peromyscus leucopus or white footed mouse)
adult) can harbor the organisms and transmit disease.
All stages of ticks (larval, nymph, and
maintained in
the environment by horizontal transmission from infected
Theorganisms are
nymphal ticks.
stricto, B. garinii, B. afzelii, B. lonestari, and B.
miyamotoi
Species: B. burgdorferi sensu

Relapsing fever febrile episodes (2 to 10 relapses).
recurring
It is an acute infectious disease with
Symptoms: Fever, headache, and

myalgia (2 to 15 days after infection)
Lyme disease
of the knees.
It is an acute, recurring inflammatory infection involving the large joints like those
The hallmarks of infection are erythema
lesion on the skin) and swelling
migrans (bull's eye
Stages of Lyme disease

a. First stage
The presence of erythema migrans or a red, ring-shaped lesion with a central clearing or a
"bull's-eye-like" lesion at the site of the tick bite is diagnostic of Lyme disease.
Signs and symptoms: Headache, fever, muscle, joint pain, and malaise
b. Second stage
It may start weeks to months after infection.
There is dissemination of the organisms to the other parts of the body.
Symptoms: Neurologic disorders (meningitis) and nerve palsy

C. Third stage
In this phase, the presence of recurring chronic arthritis (acrodermatitis chronica
atrophicans) may continue for years.
Infected individuals may also develop demyelination of neurons with symptoms of
Alzheimer's disease and multiple sclerosis.
Dark field microscopy is the choice for the detection of organism in blood cultures after two
to three weeks of incubation at 35°C.
a. Relapsing fever
Specimen of choice: Peripheral blood
Spirochetes in peripheral blood are stained with Giemsa or Wright's stains where the
organism appears blue-colored.
b. Lyme disease
Specimens: Blood, CSF, and biopsy specimens
For tissue sections, it is treated with Warthin-Starry stain.
For blood and CSF, acridine orange or Giemsa stain is used.
2. Culture
Culture media: Barbour-Stoenner-Kelly medium or chick embryo

The organism is a slow-grower and requires 7 to 14 days of incubation at 35°C.
3.
a. Relapsing fever
Serological test reveals increased titers to
Proteus OXK antigens (up to 1:80).
9 SPIROCHETES AND MISCELLANEOUS BACTERIA 469
b. Lyme disease
Serologyis the standard method for the diagnostic testing of this disease. almotal
Ig and IgG antibodies are detected in
Serology test: ELISA and IFA
ELISA and IFA are used as the first-line of tests for the
identification of the antibody
against the Lyme disease.
4. Molecular tests
PCR
is important in the diagnosis of B. burgdorferi DNA in urine samples.
Leptospira
This genus belongs to the family Leptospiraceae under the order of Spirochaetales.
The species of this genus are obligate aerobes which can be grown in artificial media.
It can be acquired in home and recreational settings (swimming pools) through contact with
contaminated water.
Virulence factor: Hemolysin (L. interrogans)
exhibit a
Microscopy: Tightly coiled, thin, flexible organisms with two long axial filaments
that
spinning motility
Living tissue for cultivation (culture media): Hamsters and guinea pigs
Reproduction: Binary fission
Growth factor: Hemoglobin and thiamine
Generation time: 6 to 16 hours nobelopon losids onlint
Animal reservoirs: Rats (murids) and dogs

Species: L. interrogans (pathogenic species), L. parva, and L. biflexa ME ban tororl
Related disease: Leptospirosis
Leptospirosis or Infectious Jaundice

caused by L. interrogans. eb and nt
It is a zoonotic disease in humans that is
in the lumen of the renal tubules and shed into the
The causative agent of
this disease lives
invade the bloodstream and spread
urine. Upon entry
into the human body, leptospires rapidly
throughout the CNS and kidneys.
Symptoms: Fever, headache, myalgia, anorexia, and vomiting
Modes of acquisition of leptospirosis:
a,
Entry through breaks in
the skin, mucous membranes, or conjunctiva
of carriers like rats
Direct contact with the urine the urine of the carriers
C. Contact with bodies
of water that are contaminated with
Types of Leptospirosis
1. Icteric leptospirosis or Weil syndrome
and causes vascular
It is a severe form of illness that affects the liver and kidney,
dysfunction.
Incubation period: days to 30 days (average of 10 to 12 days) bars ARIUd
2. Anicteric leptospirosis
severe headache (three to seven
Symptoms: Septicemic stage of infection, high fever, and
days) followed by the immune stage
Hallmark of immune stage: Aseptic meningitis
Specimen for the bacteremic phase: Blood, CSF and tissues (first week)
Specimen for the immune phase: Urine (second week)
1. Microscopy
Dark field microscopy can be used for the detection of motile leptospires in the specimens.
2. Culture
Culture media: Fletcher medium, Ellinghausen-McCullough-Johnson-Harris (EMJH)

medium, bovine serum albumin, Stuart's broth, and Noguchi's medium
best recovered in the early stage the disease and before the onset of
Leptospira species are or
symptoms.
The culture media are used in the direct inoculation of leptospires from the blood, CSF, and
urine specimens.
Fletcher and EMJH media are semi-solid media where up to 3 drops of freshly collected
blood is inoculated into 5 mL of Fletcher medium while 0.1 mL heparinized blood is
transferred onto EMJH medium.
Diluted and undiluted urine sample should be inoculated immediately because the acidity
may harm the spirochetes within the specimen.
The culture tubes and plates are incubated at room temperature up to 30°C for one to three
months in the dark and examined weekly by dark-field microscopy; organisms can grow
below the surface.
3. Immunodiagnosis
Immunoglobulin antibodies (IgM) are identified within the first week of the disease or until
several months later.
The diagnosis of leptospirosis is made by demonstrating seroconversion.

Methods for antigen detection: ELISA, Radio immunoassay (RIA), and Immunomagnetic
capture
Methods for antigen detection in tissues: Immunoluorescence and Immunohistochem
Reference method: Microscopic agglutination (MA) using living cells to detect leptospiral
antibodies
4.
Antimicrobial Susceptibility Test
It
is not routinely requested for the
genus Leptospira though the species have shown
sensitivity to streptomycin, doxycycline, tetracycline, and macrolides.9
It detects leptospiral DNA in infected patients.
It is a sensitive method in the rapid
identification of the organism. leat olmerfoonk
Methods: Polymerase chain reaction (PCR) and Hybridization techniques
Miscellaneous Bacteria sodl Hg loniges Bris

26
Streptobacillus moniliformis
It is the etiologic agent of rat-bite fever and Haverhill fever in humans.
It is normally found in the oropharynx of wild and laboratory rats.
It is a Gram-negative bacillus that requires culture media with blood, serum, or ascites fluid. de
It is facultatively anaerobic, non-motile, non-encapsulated, and non-hemolytic. namibeg?
It is known to spontaneously develop "L forms.
contact with infected rats and by ingestion of
It isacquired through bite or by direct
contaminated food like unpasteurized milk or milk products and water.
the L-form colonies of this organism. TameoiM
Dienes stain is required to demonstrate
Microscopy: Chains of bacilli with a "yeast-like" shape (string-of-pearl arrangement)
Culture: Broth culture - "Fluff-balls" or "bread-crumbs" appearance
with a dark center
BAP - "Fried-egg' appearance
indole, and lysine decarboxylase
Biochemical test: (-) Catalase, oxidase, nitrate,
discharge and joint fluid
Preferred specimen: Blood, wound
with 15% sheep blood
Recommended culture medium: BAP
Spirillum minus
sodoku in humans.
It causes a rat-bite fever known as
It cannot be grown
on artificialculture media.
related to the genus Neisseria.
and is closely
It is strictly aerobic, motile,
Direct visualization exudates,of lymph node tissues) using
ofspecimens (blood,recommended. Giemsa stain,
microscopy is
Wright's stain, or dark field and helical with two to three coils
thick,
Microscopy: Gram-negative bacilli,
of adult humans, and part of the vaginal

It is part of the indigenous microbiota of the anorectum
flora.
It is one of the etiologic agents of bacterial vaginosis and is considered as sexually transmitted.
It is small, pleomorphic, and both glucose and maltose fermenter. 1p12 0f vilsthense
environment with
and grow best in
an
It is non-motile, Gram-variable coccobacillus or rod,
increased carbon dioxide.
Culture: BAP- Colonies are small and nonhemolytic in media that contain rabbit or human blood.
Biochemical test: (+) Hippurate hydrolysis; (-) oxidase and catalase
Bacterial vaginosis
It is characterized by a malodorous discharge and vaginal pH that is higher than 4.5. hilleDiM
that is followed
It results from the reduction of normal flora such as the lactobacilli in the vagina
by a rise in the vaginal pH.

It can be caused by other organisms such as Mobiluncus, Mycoplasma hominis, and
Peptostreptococcus.
Culture media: CAP, colistin-nalidixic agar, and human blood Tween (HBT) bilayer agar
HBT is used for the isolation G. vaginalis from female genital
1. Microscopy
a. The Nugent scoring system is designed to confirm bacterial vaginosis by scanning the
stained smears.
b. Direct saline wet mount of vaginal secretions (Definitive Method)

(+) Result: Appearance of "clue cells" or large, squamous, epithelial cells with
numerously attached small rods
2. Whiff test or KOH test
Specimen: Vaginal secretions

Reagent: 10% KOH
(+) Result: Exhibit a "fishy amine odor"

FIGURE 29-1. Using the dark field visualization technique, this photomicrograph revealed the presence of
Treponema pallidum bacterial spirochetes, which exhibited their characteristic corkscrew shape. T. pallidum
caused the sexually transmitted disease known as syphilis.
Source: https://phil.cdc.gov/Details.aspx?pid=20488ldchn
FIGURE 29-2. Microscopic agglutination test of Leptospira using dark field microscopy
(Photo by M. Gatton)
FIGURE 29-3. Silver staining of Leptospira (leptospirosis) from kidney tissue

(Photo by M. Hicklin)
ASSESSMENT QUIZ
for metabolism and

1. They are called the energy parasites because they depend on their hosts
survival.
a. Rickettsia
b. Treponema
C. Mycoplasma
d. Chlamydia

Microscopy: Negative
Dienes stain: Positive
Motility: Variable (Pseudomotility)

Culture: Fried egg colonies, slow-growing
Growth on BAP: Positive
Growth on routine selective medium: Positive
Guinea pig RBC: Negative, no attachment

Arginine deamination: Positive
a. Mycoplasma hominis
b. Ureaplasma urealyticum
C. Bartonella henselae
d. Borrelia burgdorferi
3. These atypical bacteria are cultivatable using Barbour-Stoenner-Kelly medium and charcoal yeast
agar, respectively.
a. Coxiella and Ehrlichia
b. Rickettsia and Anaplasma

C. Borrelia and Bartonella
d. Chlamydia and Mycoplasma
4. What is the most serious rickettsial infection caused by Rickettsia rickettsia?

a. Rocky mountain spotted fever
b. Cat scratch disease
c. Trench fever
d. Rickettsial pox
DO SPIROCHETES AND MISCELLANEOUS BACTERIA 475
5. It is the agent of Q fever

without "hacking cough" and best isolated using lung tissue cells.
a. Rickettsia prowazekii
b. Coxiella burnetti
c. Chlamydia psittaci
d. Mycoplasma pneumoniae o bris R

Microscopy: Gram-negative, slender, helical gninda allis avllet n YotiM
Motility: Positive by axial filaments, corkscrew, dark field
Levaditi stain: Positive
Giemsa stain: Positive
Culture: Not routinely perform

Reagin test: Positive
FTA-ABS test: Positive
a. Orientia tsutsugamuchi
b. Leptospira interrogans
C. Treponema pallidum
d. Anaplasma
of rickettsioses?
7. What is the reference method for the identification
a. IFA test
b. Microimmunofluorescent dot test

C. Complement fixation
d. Weil-Felix reaction
the causative agent of the sexually transmitted disease,

8. Which of the followings is
oldiety at stid doll

a. Treponema pertenue
b. Chlamydia trachomatis
c. Ureaplasma urealyticum
d. Treponema pallidum
for Bartonella henselae?

the vector
9. Which of the following is
a. chiggers
b. louse
C. ticks
d. none
of Chlamydia?
10. What is the recommended culture medium for isolation
a. Shephard 10B
b. A8
C. McCoy cells
d. a and b
11. Identify the possible organism.

Microscopy: Yeast-like cells, string of pearl arrangement
Motility: Negative
Culture: Fluff balls, fried egg appearance
Growth in BAP: Positive
Dienes stain: Positive, L-forms

Catalase: Negative
Oxidase: Negative
Indole: Negative
a. Streptobacillus moniliformis
b. Chlamydia psittaci
c. Spirillum minus
d. Mycoplasma hominis
12. Which stage of syphilis can serological test be performed?

a. primary
b. secondary
C. tertiary
d. latent
13. This is the stage of infection, disease, and organism wherein the presence of erythema migrans or
"bull's-eye-like" lesion at the site of the tick bite is visible.
a. first stage, Lyme disease, Borrelia burgdorferi
b. second stage, Pinta, Treponema carateum
C. first stage, Bejel, Treponema pallidum subsp. endemicum
d. second stage, HME, Ehrlichia chaffeensis
14. Which semi-solid culture medium is used to observe the

growth patterns of Leptospira interrogans?
a. Fletcher
b. Ellinghausen-McCullough-Johnson-Harris
c. HeLa cells
d. a and b
15. Identify the possible organism.

Microscopy: Intravacuolar microcolony of bacteria "mulberries" in monocytes
Elementary body and reticulate body: Positive
Giemsa staining: Positive
Motility: Negative
Culture: Positive, shell vial culture
IFA titer: 1:64
Vector: Lone star tick
a. Anaplasma phagocytophilum
b. Chlamydia pneumoniae
C.
d. Streptobacillus moniliformis
THEAUTHOR
Maria Teresa Tablante-Rodriguez earned her bachelor's

degree in medical technology from San Juan De Dios College
and her master's degree in the same discipline from the
Philippine Women's University. She served as the dean of

the College of Medical Technology at the Trinity University
of Asia, where she also obtained her doctorate degree in

education. She worked at the San Juan De Dios Hospital and
the Riyadh Central Hospital, Kingdom of Saudi Arabia, as a
laboratory staff prior to her journey as an academician.

to education and research in the field
Her contributions
of medical laboratory science for 23 years, specifically in
have been
clinical chemistry and diagnostic bacteriology,
recognized by schools of medical technology and professional
organizations. She has received an Outstanding Alumni and
Also, she
Outstanding Faculty Award from her alma maters.
Federation of
was given travel grants by the International
Association for Clinical
Clinical Chemistry and the American
Chemistry as support to her research studies
new on kidney
function tests such as the beta 2-microglobulin and cystatin C.
has joined
the University of Santo Tomas
Recently, she
of Medical Technology
Faculty of Pharmacy, Department
she is a certified
as a full-time faculty member. Moreover,
scientist by the American
international medical laboratory
Society for Clinical Pathology.
THE BOOK
Using a reader-friendly approach, Review Handbook in Diagnostic Bacteriology

(Third Edition) provides students with a simplified understanding of Microbiology.
It also offers fundamental information on the clinically significant bacterial species
and their morphological and cultural characteristics, related infections and
diseases, and associated laboratory diagnoses. Presented in an outline form, each
chapter gives a straightforward reading to an otherwise comprehensive study.
excello
Excello™ a continuously expanding aggregation of high-quality learning
is
assessments designed to help students master the Philippine curriculum's

key
skills and provide teachers with easy-to-use test creation tools.
Published by:
C & E Publishing, Inc.
839 EDSA, South Triangle
Quezon City, Philippines
Tel. No.: (02) 8929-5088
E-mail: [email protected] EMPOWERED ] INSPIRE

Website: www.cepublishing.com MEMBER FOR THE WORLD CREATE

Rodriguez Bacte

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niteddh sess dntgp9.

benubqp1 od a otscidug ci to sq ol aE ca t 1atn

sbni bns c aerclin iesidnolid eabubrl

Chapter 6 Assessment Quiz. . 41

Biosafety and Quality Control in a Clinical Microbiology Laboratory . 63

Sterilization and Disinfection 81

Specimen Collection, Transport, and Processing .

Culture and Culture Media 115

Methods for Bacterial Identification® 155

Antimicrobials (Antibiotics) 171

Antimicrobial Susceptibility Testing 183

Gram-positive Cocci. 199

Gram-negative Cocci 247

Enterobacteriaceae (Gram-negative Bacilli) 263

Non-enteric Gastrointestinal Pathogens 307

Non-fermentative Gram-negative Bacilli 323

Small, Pleomorphic Gram-negative Bacilli 339

Aerobic Gram-positive Bacilli. 361

Anaerobic Bacteria 425

Review Handbook in Diagnostic Bacteriology is conceptualized and designed for Medical

- Maria Teresa T. Rodriguez

I extend my sincerest thanks and appreciation to:

inspiration, and valuable contributions.

INTENDED LEARNING OUTCOMES

3. discuss the contributions of the early scientists and microbiologists.

Microbiology is defined as the study of organisms that are too small

The Discovery of Microorganisms

Francesco Stelluti (1577-1652)

He made the earliest observations on bees and weevils using a

microscope supposedly supplied by Galileo.

Antonie van Leeuwenhoek (1632-1723)

magnification to study bacteria and protozoa.

Aristotle (384-322 B.C.)

John Needham (1731-1781)

Lazzaro Spallanzani (1729-1799)

Rudolf Virchow (1821-1902)

Theodor Schwann (1810-1882)

Heinrich Schroder (1810-1885) and Theodore von Dusch (1824-1890)

Louis Pasteur (1822-1895)

consecutive days to eradicate vegetative cells and endospores.

Ferdinand Cohn (1828-1898) hoabsatbris8o

Antoine-Laurent Lavoisier (1743-1794)

Fermentation and Pasteurization

alcohol. glbemn ennhiun in ead onl booubommiaH

Ignaz Semmelweis (1816-1865)

Joseph Lister (1827-1912)

TABLE 1-1. Important Events in the Development of Microbiology

1590-1595 Hans Jansen developed the first compound microscope.

1867 Joseph Lister published his work on antiseptic surgery.

1910 Paul Ehrlich developed chemotherapeutic agent for syphilis.

si tolhuing csmuijnte paihits kaeiizepaliadl zaM

ae hign nar ant ho ns ynivtaee

slnane guipploh n "bnenstte" uneodi pu alailod aaw odl

wHie ine t0 ssBoeLbta ipt n d sueoten tnboedouaanas

Who introduced the concept of amplification of microorganisms through the use

5. He first demonstrated the

7. Based on the experiment of Cohn, which cellular structures are heat-resistant?

9. Who introduced the concept of aseptic technique?

BACTERIAL TAXONOMY Aslimic to boaogmop

INTENDED LEARNING OUTCOMES

2. explain how microorganisms are classified;

differentiate phenotypic from genotypic characteristics.

onten auring orl pndinw n

It is a formal system of organizing, classifying, and naming living

It is the organization of microorganisms that have similar™z8T

The classification system is hierarchic and consists of the following taxa

3. Division/Phyla - composed of similar classes

edion blachemizal and mumumolog

960D alvosned adt ewollot sisloed to gnirach/

Polyphasic Taxonomy - is a modern system of bacterial classification and identification combining

8. Strain - is an altered or a variant microorganism within the same species evlovn # .b

estoage smee onlt midtiw mainsgroomimn tnshsv s et it

INTENDED LEARNING OUTCOMES

1. describe the cellular structures of bacteria and their functions;

4. distinguish the characteristics of eukaryotes from those of prokaryotes;