lOMoARcPSD|38638208

OLFU Introduction to Diagnostic Bacteriology LEC

2020-2021
2nd SEM
College of Medical CLINICAL BACTERIOLOGY BACT211
Laboratory Science Transcriber: Riyoma Surell 1 LEC

Batch 2023 Date: February 1, 2021

❖ Infection
Outline ➢ Refers to the entry, invasion and multiplication of pathogens in or
At the end of the session, the student must be able to learn: on to the host body system which results to subsequent tissue
• Define Microbiology, • Outline some of the injury and progress to overt disease
Medical Microbiology and contributions of ➢ Types of Infection
Diagnostic Microbiology Leeuwenhoek, Pasteur and ▪ Based on Source of Pathogen
• Define terms related to Koch to Microbiology
• Endogenous Infection
infectious process and • Identify the careers in
disease transmission Microbiology  Infection arising from colonizing flora
• Exogenous Infection
 Infection arising from invading pathogen from the
General Definitions external environment
▪ Based on Clinical Onset of Signs and Symptoms
❖ Microbiology • Acute Infection
➢ A branch of biology which deals with the study of living organisms  Rapid/ sudden onset of signs and symptoms
that are too small to be seen by the naked eye which are usually severe to fatal that may lead to
❖ Medical Microbiology death
➢ A branch of medical science which deals with the study of • Chronic Infection
medically important microorganisms specifically their role in  Gradual onset of signs and symptoms that are
human disease which includes diagnosis, treatment and usually mild to moderate that may progress to
prevention of infectious diseases long standing infection
❖ Diagnostic Bacteriology ▪ Based on Etiologic/ Causative Agent
➢ A branch of medical microbiology that focuses on the laboratory • Nosocomial Infection
identification of medically important bacteria by phenotypical  Infection acquired during hospitalization
(physical characteristic) and genotypical (genes) • Zoonotic Infection (Zoonosis)
characterization including antibiotic susceptibility testing  Is an animal disease which can spread to
humans; animal acquired infection
Microbiology ▪ Based on Clinical Manifestation
➢ Study of living organism (microbes) • Subclinical/ Asymptomatic/ Nonapparent
➢ Study of certain nonliving entities as well as certain living  No obvious appearance of signs and symptoms
organisms and the person is unaware of the infection
❖ Microbes • Clinical/ Symptomatic/ Apparent
➢ Are said to be ubiquitous, meaning they are virtually everywhere  Associated with presence of overt signs and
❖ 4 Groups symptoms of the disease
➢ Viruses ❖ Disease
▪ Very simple microbes, consisting of nucleic acid, a few ➢ An altered health state in an infected host
proteins, and (in some) a lipid envelope ❖ Infectious Disease
▪ Completely dependent on the cells they infect for their ➢ Is an illness caused by a pathogen which invades body tissues
survival and replication and causes damage
➢ Bacteria ❖ Communicable Disease
▪ With both RNA and DNA, metabolic machinery for self- ➢ Is an infectious disease that is capable of spreading from person
replication and a complex cell wall structure. Asexual to person
▪ Prokaryotic – simple unicellular organisms (no nuclear ❖ Symptoms
membrane, mitochondria, endoplasmic reticulum) ➢ Refers to any subjective evidence of disease. These are usually
➢ Fungi perception of the patient having the disease such as headache,
▪ Subdivided into single-celled organisms (yeasts) or multi- dizziness, etc.
celled organisms (molds), with a few medically important ❖ Signs
members existing in both forms (dimorphic fungi) ➢ Refers to readily observable evidence of disease. These are
➢ Parasites usually physical manifestation of the disease such as rashes,
▪ Subdivided into single-celled organisms (protozoa) or multi- bleeding etc.
celled organisms (worms and bugs) ❖ Normal Flora
➢ Bacteria that are in or on different sites of the body that usually
Definition of Terms Related to Infectious Disease Transmission do not harm the host unless the host defense is compromised
❖ Pathogens ➢ Synonyms: Indigenous Flora, Resident Flora, Normal
➢ Disease causing microorganisms such as bacteria, fungi, Microbiota
protozoans and viruses ❖ Colonization
➢ Types of Pathogens ➢ Refers to the establishment of substantial number of
▪ True Pathogen microorganisms usually in the skin or mucosa but there’s no
• Refers to an organism that will cause disease in a penetration of tissues
healthy host
▪ Opportunistic Pathogens Earliest Known Infectious Diseases
• Refers to organisms that will cause disease in an ❖ Pestilence and Plague
immunocompromised host ➢ Fatal epidemic disease caused by a bacteria called Yersinia
❖ Pathogenicity pestis – Black Death
➢ Refers to the ability of an organism to cause disease in a host ➢ Represent the first recorded epidemic
organism ➢ Around 1900 BC, near the end of the Trojan war, the Greek army
❖ Virulence was decimated by an epidemic of what is thought to have been
➢ Refers to the degree of pathogenicity; the power by which a Bubonic Plague
pathogen can cause severe disease ❖ 1500 BC
❖ Pathogenic Determinants/ Virulence Factors ➢ Ebers papyrus
➢ Refers to any genetic, biochemical or structural features that ❖ 1122 BC
enable a pathogen to cause disease in a host organism ➢ Smallpox occurred in China
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[BACT211] 1.01 Introduction to Diagnostic Bacteriology I Prof. Rochelle D. Darlucio, RMT, MPH
❖ Epidemics of plague occurred in Rome in 790, 710, and 640 BC and ▪ Sterile – completely free of all life forms (including spores)
in Greece around 430 BC and virus particles
❖ There are early accounts of rabies, anthrax, dysentery, smallpox,
ergotism, botulism, measles, typhoid fever, typhus fever, diphtheria, The Development of Aseptic Techniques
and syphilis
❖ Syphilis (Treponema pallidum) ❖ Dr. Oliver Wendell Holmes
➢ First appearance in Europe in 1493 ➢ observed that mothers who gave birth at home experienced
▪ Was carried to Europe by Native Americans who were fewer infections than did mothers who gave birth in the hospital
brought to Portugal by Christopher Columbus
▪ Neapolitan Disease ❖ Dr. Ignaz Semmelweis (Father of Hand Hygiene)
▪ French or Spanish Disease ➢ showed quite clearly that women became infected in the
▪ French pox maternity ward after examinations by physicians coming directly
▪ Spanish, German, Polish and Turkish pocks from the autopsy room
❖ Joseph Lister
Pioneers in the Science of Microbiology ➢ first to introduce aseptic techniques aimed at reducing
microbes in a medical setting and preventing wound infections
❖ Anton van Leeuwenhoek (1632 – 1723)
➢ First person to see live bacteria and protozoa PERIOD DEVELOPMENTAL NOTES KEY SCIENTIST/S
➢ “Father of Microbiology, Bacteriology, Protozoology” 1665 Publication of the first description of Robert Hooke
➢ He ground tiny glass lenses, which he mounted in small metal microbes
frames, thus creating what today are known as single-lens 1667 Observation of “little animals” Anton van
microscopes or simple microscopes Leeuwenhoek
➢ He observed various tiny living creatures, which he called 1796 Smallpox Vaccination – first scientific Edward Jenner
“animalcules” (bacteria and protozoa) validation
❖ Louis Pasteur (1822 – 1895) 1850 Advocating handwashing in the Ignaz Semmelweis
➢ Demonstrated that different types of microbes produce different prevention of the spread of disease
fermentation products 1861 Spontaneous generation disproved Louis Pasteur
➢ Disproved theory of spontaneous 1862 Publication of the paper supporting Louis Pasteur
generation/ Abiogenesis the germ theory of disease
▪ Life can arise spontaneously
1867 Practice of Antiseptic Surgery Joseph Lister
from non-living materials
1876 Discovery of Bacillus anthracis which Robert Koch
➢ He introduced the terms “aerobes”
became the first proof of germ theory
(requires oxygen) and
1881 Utilization of solid culture media for Robert Koch
“anaerobes” (does not requires
bacterial growth
oxygen)
➢ Pasteurization 1882 Outlined Koch’s postulate Robert Koch
▪ Heating liquids to 63 – 65’C for Development of Acid-fast stain Paul Erlich
30 minutes or 73 – 75’C for 15 seconds 1884 Gram stain developed Hans Christian Gram
▪ Type of sterilization. Only kills pathogens 1885 First Rabies Vaccination Louis Pasteur
➢ Germ Theory of Disease 1887 Invention of the Petri Dish Richard J. Petri
▪ Specific microbes cause specific infectious diseases 1892 Discovery of Viruses Dmitri losifovich
➢ Developed vaccines to prevent chicken cholera, anthrax and Ivanovski
swine erysipelas 1893 Zoonosis – first described T. Smith, F.I.
❖ Robert Koch (1843 – 1910) Kilbourne
➢ Made many significant contributions to the germ theory of 1899 Viral dependence on living host cells Martinus Beijerinck
disease for reproduction recognized
➢ Discovered that B. anthracis produces spores, capable of 1900 Proof the mosquitoes carry the agent Walter Reed
resisting adverse conditions of yellow fever
➢ Developed methods of fixing, staining, and photographing 1910 Discovered the cure for syphilis Paul Erlich
bacteria, methods of cultivating bacteria on solid media 1928 Discovery of Penicillin Alexander Fleming
➢ Discovered the bacterium (M. tuberculosis) that causes 1953 Proposed and built the DNA model J. Watson, F. Crick
tuberculosis and the bacterium (Vibrio cholerae) that causes 1977 Development of the DNA sequencing W. Gilbert, F. Sanger
cholera method
1983 Invention of the Polymerase Chain Kary Mulis
Reaction (PCR)
1995 Publication of the first microbial The Institute for
genetic sequence genomic Research
(TIGR)

Careers in Microbiology

❖ Bacteriologist
➢ Scientist who specializes in bacteriology – the study of the
structure, functions, and activities of bacteria
❖ Phycologists (or algologists)
➢ Scientists specializing in the field of phycology (or algology) study
The Discovery of Spores and Sterilization the various types of algae
❖ Protozoologists
❖ John Tyndall ➢ Explore the area of protozoology – the study of protozoa and their
➢ Provided the initial evidence that some of the microbes in dust activities
and air have very high heat resistance and that particularly ❖ Mycologists
vigorous treatment is required to destroy them ➢ Those who specialize in the study of fungi, or mycology
❖ Ferdinand Cohn ❖ Virologists and Cell Biologists
➢ Clarified the reason that heat would sometimes fail to completely ➢ Many become genetic engineers who transfer genetic material
eliminate all microorganisms (deoxyribonucleic acid or DNA) from one cell type to another
➢ “Sterile” was established ➢ Virologists also study prions and viroids, acellular infectious
agents that are even smaller than viruses
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[BACT211] 1.01 Introduction to Diagnostic Bacteriology I Prof. Rochelle D. Darlucio, RMT, MPH

References:

• Study Guide on Diagnostic Bacteriology by Mr. Nathaniel

Rañon
• Textbook of Diagnostic Microbiology (6th edition) by Connie
Mahon
• Diagnostic Microbiology (14th edition) by Bailey & Scott
• Burton’s Microbiology for Health Sciences, 9th edition

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OLFU Taxonomy and Bacterial Classification LEC

2020-2021
2nd SEM
College of Medical CLINICAL BACTERIOLOGY BACT211
Laboratory Science Transcriber: Riyoma Surell 2 LEC

Batch 2023 Date: February 8, 2021

Outline
At the end of the session, the student must be able to learn:
• Naming, Classifying and Identifying Microorganisms
• Classification
• Nomenclature
a. Guidelines
• Identification
• Comparison of Prokaryotic and Eukaryotic Cell
• Bacterial Cell Wall Components
• Gram Variability
a. Guiding Rules in the Gram Stain Reaction of Medically
Important Bacteria
b. Acid Fast Cell Wall
c. Summary of Acid-Fast Staining Techniques: Reagents
and Reactions
d. Acid Fast Smear Reporting
• Parts Internal to the Cell Wall

❖ Carl R. Woese
I. Naming, Classifying and Identifying Microorganisms ➢ devised a Three-Domain System of Classification
➢ there are two domains of procaryotes – base on cellular
❖ Taxonomy organization and function (Archaea and Bacteria) and one
➢ The science of classifying living things domain (Eucarya or Eukarya), which includes all eucaryotic
➢ Greek word “taxes” meaning arrangement, “nomos” – law organisms.
❖ Three Categories of Taxonomy
➢ Nomenclature
▪ Is the assignment of scientific names to the various
taxonomic categories and individual organisms
➢ Classification
▪ Attempts the orderly arrangement of organisms into a
hierarchy of taxa (categories)
➢ Identification
▪ Is the process of discovering and recording the traits or
organisms so that they may be recognized or named and
placed in overall taxonomic scheme (genotypic and
phenotypic characteristics)
III. Nomenclature
II. Classification
❖ International Code of Nomenclature of Bacteria (ICNB) or the
❖ Carl von Linné (Linnaeus; 1701–1778) Bacteriological Code (BC)
➢ a Swedish botanist ➢ provides the accepted labels by which organisms are universally
➢ laid down the basic rules for classification and established recognized
taxonomic categories, or taxa ❖ Binomial system of nomenclature
➢ every organism is assigned a genus and a species of Latin or
Greek derivation
➢ Each organism has a scientific “label” consisting of two parts:
▪ Genus – the first letter is always capitalized
▪ Species – first letter is always lower case
▪ Printed in italics or underlined in script (separate line for
genus and species)
A. Guidelines

❖ The first letter of the family name (similar to a human “clan”) is

capitalized and has a suffix -aceae (e.g., Staphylococcaceae,
Streptococcaceae, Micrococcaceae, Enterobacteriaceae)
❖ Robert Whittaker ❖ The first letter of the genus is capitalized followed by the species in
➢ Whittaker’s tree lowercase; both the genus and species should be italicized in print or
➢ based on structural similarities and differences, such as should be underlined when written in script
prokaryotic and eukaryotic cellular organization, and the way ❖ Commonly, the genus is abbreviated with the first letter (capitalized)
these organisms obtained their nutrition of the genus followed by a period and the species (e.g., S. aureus, S.
pneumoniae, S. pyogenes). To avoid confusion, the first two letters of
the first syllable are used when two or more genera begin with the
same first letter (e.g., Staph. aureus, Strept. pyogenes)
❖ The genus followed by the word species may be used to denote the
❖ Monera - Eubacteria (true bacteria) all medical bacteria entire genus as a whole (e.g., Staphylococcus spp., Streptococcus
❖ Protists spp., Enterococcus spp.) The species are abbreviated as “sp.”
❖ Plants (singular) or “spp.” (plural) when the species is not specified
❖ Fungi - Yeast, molds, mushroom ❖ Lastly, when bacteria are referred to as a group, their names (e.g.,
❖ Animals Staphylococci, Streptococci, Enterococci, Micrococci)

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[BACT211] 1.02 Taxonomy and Bacterial Classification I Prof. Rochelle D. Darlucio, RMT, MPH
IV. Identification ❖ Peptidoglycan/ Murein Layer
➢ cell wall of the bacteria
❖ Genotypic characteristics ➢ shape/ rigidity to the cell
➢ relate to an organism’s genetic makeup, including the nature of ➢ 2 alternating sugars
the organism’s genes and constituent nucleic acids 1. N-acetyl-D-glucosamine (NAG)
❖ Phenotypic characteristics 2. N-acetyl-D-muramic acid (NAM)
➢ are based on features beyond the genetic level and include both ❖ All microorganism/ bacteria has cell wall except: Mycoplasma,
readily observable characteristics and characteristics that may Ureaplasma, Spiroplasma, Anaeroplasma
require extensive analytic procedures to be detected
Base on Cell Wall
V. Comparison of Prokaryotic and Eukaryotic Cell

Characteristic Prokaryote Eukaryote

Typical Size 0.4 – 2 micrometer in 10-100 micrometer in
diameter diameter
0.5-5 micrometer in length >10 micrometer in length
Nucleus No nuclear membrane; Classic membrane-
nucleoid region of the bound nucleus
cytosol
GENOME
Location In the nucleoid, at the In the nucleus
mesosome
Chromosomal Circular, complexed with Linear; complexed with
DNA RNA basic histones and other
proteins
Genome: Plasmids, small circular In mitochondria and
extrachromosomal molecule of DNA chloroplasts
circular DNA containing accessory
information; most
commonly found in gram-
negative bacteria; each
carries genes for its own
replication; can confer
resistance to antibiotics
Reproduction Asexual (binary fission – Sexual and Asexual
spontaneous splitting of
bacterial cells given
optimum growth
requirements)
Membrane-bound Absent All
Organelles
Golgi bodies Absent in all Present in some
Lysosomes Absent in all Present in some, contain
hydrolytic enzymes VI. Bacterial Cell Wall Components
Endoplasmic Absent in all Present in all; lipid
Reticulum synthesis, transport ❖ Cell Wall
Mitochondria Absent in all Present in most ➢ also referred to as the peptidoglycan, or murein layer
Nucleus Absent in all Present in all ▪ 2 alternating sugars
Chloroplasts for Absent in all Present in algae and 1. N-acetyl-D-glucosamine (NAG)
photosynthesis plants 2. N-acetyl-D-muramic acid (NAM)
Ribosomes; site of Present in all Present in all ➢ This structure gives the bacterial cell shape and strength to
protein synthesis withstand changes in environmental osmotic pressures that
(nonmembranous) would otherwise result in cell lysis
Size 70s consisting of 50s and 80s consisting of 60s ➢ Protects against mechanical disruption of the cell
30s subunits and 40s subunits
➢ Offers some barrier to the passage of larger substances
Electron transport In the cell membrane; no In the inner membrane of
for energy mitochondria present mitochondria and
chloroplasts
Sterols in Absent except in Present
cytoplasmic Mycoplasma spp.(site of
membrane energy)
Plasma Lacks carbohydrates Also contains glycolipids
membrane and glycoproteins
Cell wall Peptidoglycan in most Cellulose, phenolic
bacteria polymers, lignin (plants)
chitin (fungi), other
glycans (algae)
Glycocalyx Present in most as an Present; some animal
organized capsule or cells
unorganized slime layer
Cilia Absent Present; see description ❖ Gram Positive Cell Wall
of flagella ➢ Has a very thick protective peptidoglycan (murein) layer
Flagella Simple flagella; composed Complex cilia or flagella; ➢ Presence of teichoic acid and lipoteichoic acid
of polymers of flagellin; composed of MTs and ▪ Teichoic acid – provides rigidity to cell wall by attracting
movement by rotary action polymers of tubulin with cations such as Mg and Ca
at the base; spirochetes dynein connecting MTs; ❖ Gram Negative Cell Wall
have MTs movement by ➢ Thin peptidoglycan layer, Periplasmic space
coordinated sliding ➢ Outer membrane: proteins, phospholipids, and
microtubules lipopolysaccharide (LPS)
Pili and fimbriae Present Absent
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[BACT211] 1.02 Taxonomy and Bacterial Classification I Prof. Rochelle D. Darlucio, RMT, MPH
➢ Functions of the Outer Membrane
Primary stain
▪ It acts as a barrier to hydrophobic compounds and
Add crystal violet
harmful substances
Wash with water
▪ It acts as a sieve, allowing water-soluble molecules to
enter through protein-lined channels called porins
▪ It provides attachment sites that enhance attachment to
host cells
Mordant
Strengthen the affinity of the
primary stain
Gram’s iodine
Wash with water

Decolorization
Decolorized using alcohol
Gram (+) Do not decolorize
primary stain and it will remain
purple/ violet
Gram (-) Lipid layer will
dissolve, colorless

Gram (+) Gram Staining Gram (-)

Procedure
Gram (+) cell walls 1. Heat fix cells Gram (-) cell walls have a
have single membrane to slide thin layer of Counterstaining
enclosed by thick, peptidoglycan in the Safranin red
cross linked periplasmic space within Gram (+) remain purple
peptidoglycan its inner and outer lipid Gram (-) pink/red
membranes
Thick peptidoglycan 2. Saturate with Cell wall takes up dye. ❖ REMEMBER VIAS (Crystal Violet, Iodine, Alcohol, Safranin red)
takes up dye. Appears crystal violet dye Appears purple
purple for 60 seconds VII. Gram Variability
Dye and mordant 3. Add iodine Dye and mordant form
complex forms. (mordant) for 60 complex, but does not ➢ A characteristic exhibited by gram positive bacteria
Adheres firmly to thick seconds adhere to the thin layer of ➢ Natural gram variability: Mobiluncus spp., and Gardnerella
peptidoglycan layer peptidoglycan vaginalis
Alcohol cannot wash 4. Rinse slide Dye and mordant ➢ Acquired gram variability (for gram positive bacteria)
out the dye-mordant with alcohol for complex is easily ➢ Contributing factors
complex because it is 20 seconds removed from ▪ Use of old culture
firmly secured in the peptidoglycan layer with
• Fresh culture: incubated for 16-24 hours
thick peptidoglycan alcohol
▪ pH of staining reagents
layer ▪ Bacterial autolysis
Saturated with the 5. Stain slide Colorless cell wall can ▪ Staining reaction time
crystal violet dye, the with safranin easily take up counter
cell counter stain has (counter-stain) stain A. Guiding Rules in the Gram Stain Reaction of Medically Important
little to no effect Bacteria
Cell wall ranges in Cell wall, counter stained
color from mid to dark with safranin, ranges in
❖ All COCCI are Gram Positive except:
purple color from pink to red
➢ Neisseria, Branhamella/ Moraxella, Veilonella
❖ All BACILLI (rod or elongated in shape) are Gram Negative except:
➢ Mycobacterium, Bacillus, Clostridium, Corynebacterium,
Lactobacillus, Listeria, Erysipelothrix, Aerobic Actinomyces,
Rothia, Kurthia (MBCCLLEARK)
❖ Mycoplasma & Ureaplasma usually have a gram-negative reaction
not because it has a gram-negative cell wall but because they do not
have a cell wall
❖ Spirals are very difficult to stain using gram staining however
stainable spirals are usually gram negative
❖ Mycobacterium and Nocardia spp., (acid fast organism) have a
gram-positive cell wall structure however because 60% of the cell wall
is made of hydrophobic lipids mainly mycolic acid, it affects its
permeability this makes it difficult to gram stain

B. Acid-Fast Cell Wall

❖ Acid-Fast Organism
➢ contain a waxy layer of glycolipids and fatty acids (mycolic acid)
➢ >60% of the cell wall is lipid
❖ Acid-Fast Staining
➢ specifically designed for a subset of bacteria whose cell walls
contain long-chain fatty (mycolic) acids.
➢ Mycolic acids
▪ render the cells resistant to decolorization, even with acid
alcohol decolorizers
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[BACT211] 1.02 Taxonomy and Bacterial Classification I Prof. Rochelle D. Darlucio, RMT, MPH
➢ Mycobacteria VIII. Parts Internal to the Cell Wall
▪ are the most commonly encountered acid-fast bacteria,
typified by Mycobacterium tuberculosis, the etiologic agent ❖ Cytoplasmic/ Plasma Membrane
of tuberculosis ➢ a phospholipid bilayer embedded with proteins that envelops the
▪ sputum or phlegm specimen cytoplasm but does not contain sterols (except Mycoplasma/
❖ Partially Acid-Fast Organism Ureaplasma)
➢ Nocardia ➢ site of energy production (prokaryotes)
➢ Rhodococcus ➢ Functions
➢ Legionella micdadei ▪ Separates the intracellular components of the bacterial cell
❖ Distinctly Acid-Fast from the extracellular environment
➢ Cryptosporidium ▪ Acts as an osmotic barrier between the inside and outside
➢ Isospora of the bacterial cell by allowing selective permeability of the
❖ 2 Methods: membrane to macromolecules
➢ Ziehl-Neelsen Method (hot method) ▪ Site of electron chain transport necessary for energy
➢ Kinyoun Method (cold method) production, hence maintaining the viability of the bacterial
cell
❖ Acid-Fast (Ziehl-Nee Isen or Hot Method) ▪ Houses enzymes involved in outer membrane and cell wall
➢ Use heat as mordant synthesis, and the assembly and secretion of
extracytoplasmic and extracellular substances
▪ Present in both gram-positive and gram-negative bacteria
and is the deepest layer of the cell envelope
▪ Serves as an additional osmotic barrier and is functionally
similar to the membranes of several of eukaryotic cellular
organelles
❖ Mesosomes
➢ Folds or invagination along the length of the cytoplasmic/plasma
membrane which serves as a point of attachment for
chromosomes
❖ Free Ribosomes
➢ Sites of protein synthesis in bacterial cells which has a size of
❖ Kinyoun Acid-Fast Method 70s (Svedberg) comprised of two subunits being 50s and 30s
➢ does not require the use of heat or boiling water, minimizing (loss of the surface area)
safety concerns during the procedure. ❖ Inclusion Bodies
➢ Because of a higher concentration of phenol in the primary stain ➢ Serves as depot or storage deposits under certain circumstances
solution, heat is not required for the intracellular penetration of such as limited or excess of a particular nutrient
carbolfuchsin. ➢ These may accumulate, precipitate out, and form an inclusion
➢ Referred to as the “cold method” body which is not bounded by a membrane freely floating in the
➢ Used chemical known as tergitol cytoplasm of the bacterial cell
➢ Inclusion bodies may be in the form of glycogen (carbohydrate
C. Summary of Acid-Fast Staining Techniques: Reagents and reserves, polyphosphates (ATP reserves), and poly-β-
Reactions hydroxybutyric acid (lipid reserves)
❖ Much Granules
co Key Steps Reagent/s Duration/ Time Acid Non-Acid ➢ Contains lipids (Mycobacterium tuberculosis)
de (Ziehl-Neelsen Fast Fast ❖ Volutin/ Babes – Ernst Bodies/ Metachromatic Granules
Method)
➢ Contains polyphosphates or inorganic phosphates
C Primary/ Initial Carbol Fuchsin 4-5 minutes RED RED
Staining (rinse) (Corynebacterium diphtheriae)
H Mordanting Physical: heat RED RED ❖ Bipolar Bodies
A Decolorization Acid Alcohol (3% 2 minutes (rinse) RED Colorless ➢ Prominent staining of each end of the bacilli Yersinia pestis
HCl in 95%
Ethanol) (pestilence/ plague) using Methylene Blue or WAYSON stain
M Counterstaining Methylene Blue 1 minute (rinse) RED Blue giving it a “safety pin appearance” (pardible)
co Key Steps Reagent/s Duration/ Time Acid- Non-Acid ❖ Bacterial Spores/ Endospores
de (Kinyoun Fast Fast ➢ Complex multilayered highly refractile structure that can be found
Method)
C Primary/Initial Carbol Fuchsin 5 minutes (rinse) RED RED within the cytoplasm of the vegetative cell of the bacteria or in the
Staining environment when the bacterial cell has been disintegrated
T Mordanting Chemical: RED RED ➢ Serves as a resting or hibernating stage for bacteria when they
Tergitol
A Decolorization Acid Alcohol (3% 2 minutes (rinse) RED Colorless
are exposed to unfavorable conditions
H2SO4 in 95% ➢ It is highly resistant to desiccation, heat, chemical agents
ethanol) ➢ Main compositions:
M Counterstaining Methylene Blue 1-3 minutes RED Blue
(rinse)
▪ Calcium Dipicolinate or Calcium-Dipicolinic Acid Complex
➢ Two Important Sporulating Bacteria:
D. Acid-Fast Smear Reporting ▪ Bacillus
▪ Clostridium
Number of AFB Number of AFB Number of AFB Report ❖ Pili (Plural) or Pilus (Singular)
seen Fuchsin seen seen ➢ Protein projections that are thinner and shorter than flagella and
Stain (1000x Fluorochrome Fluorochrome are most usually found in gram negative bacteria
magnification) stain (450x stain (250x
magnification) magnification) ➢ The terms Fimbriae (Latin, fringe) and Pili (Latin, hairs) are
0 0 0 No AFB seen commonly used synonymously (Brinton, 1965; Duguid &
1-2/300 fields 1-2/70 fields 1-2/30 fields Doubtful; request Anderson, 1967)
another specimen ➢ Composition: made up of protein material known as Pilin
1-9/100 fields 2-18/50 fields 1-9/10 fields 1+ ➢ Attachment to host cell or bacterial conjugation
1-9/10 fields 4-36/10 fields 1-9/ fields 2+ ➢ Two Types of Pili
1-9/field 4-36/field 10-90/field 3+ 1. Common/ Somatic/ Ordinary Pili
>9/field >36/field >90/field 4+ ▪ usually shorter, numerous, sticky hair-like appendages that
are primarily used for adherence to one another, host cells,
and environment surfaces

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[BACT211] 1.02 Taxonomy and Bacterial Classification I Prof. Rochelle D. Darlucio, RMT, MPH
2. Sex/ Fertility/ F Pilus References:
▪ usually longer and singular, long and hollow protein tubes
• Study Guide on Diagnostic Bacteriology by
that is primarily used for bacterial conjugation (transfer of
Mr. Nathaniel Ranon
genetic material between bacterial cells)
• Textbook of Diagnostic Microbiology (6th
❖ Flagella (Plural) or Flagellum (Singular)
edition) by Connie Mahon
➢ exterior protein filaments or whip-like projections which is
embedded in the cell envelope with a motor attached in a basal • Diagnostic Microbiology (14th edition) by
body responsible for its propeller-like rotation of the flagella which Bailey and Scott
makes bacteria move. Hence, flagellated bacteria are said to be • Burton’s Microbiology for Health Sciences, 9th
moving of motile edition
➢ Composition: made up of protein material known as Flagellin • Powerpoint Presentation and Lecture Notes
➢ Associated with H Antigen (Hauch Antigen) which is very useful of Prof. Rochelle Darlucio
is serologically typing and identifying species of Salmonella

❖ Peritrichous Flagella
➢ Flagella occur around the bacterium
❖ Amphitrichous Flagella
➢ Presence of single flagella at both ends
❖ Lophotrichous
➢ Multiple flagella at one end
❖ Monotrichous Flagella
➢ Presence of single flagella at one end
❖ Glycocalyx
➢ Exterior high molecular weight appendage or structure usually
made up of polysaccharide polymers or sometime polypeptides
which are produced be certain bacteria depending on
environmental and growth conditions surrounding the bacterial
cell
➢ There are two (2) forms:
1. Capsule
▪ uniform and condensed organized material that is firmly
attached to the cell wall of the bacteria
▪ It is associated with K Antigen (Kapsule Antigen) and a
slight change in the capsular
▪ Acts as virulence factor in helping the pathogen evade
phagocytosis
▪ Medically Important Capsulated Bacteria
• Neisseria meningitidis
• Haemophilus influenzae serotype b
• Streptococcus pneumoniae
• Klebsiella pneumoniae
• Bacillus anthracis
2. Slime Layer
▪ Loose or diffused, thick, viscous unorganized material that
appears to be detached from the bacterial or not firmly
attached to the cell wall of the bacteria
▪ Functions
• primarily it also serves as a form of protection from
phagocytosis, or in some instances, it helps the
bacteria to adhere to host tissues or synthetic
implants such as prosthetic heart valves

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OLFU Bacterial Morphology LEC

2020-2021
2nd SEM
College of Medical CLINICAL BACTERIOLOGY BACT211
Laboratory Science Transcriber: Riyoma Surell 3 LEC

Batch 2023 Date: February 15, 2021

Coccus is Tetrad/s
Outline divided by two Coccus in packets of four
At the end of the session, the student must be able to learn: (2) planes of (4)
I. Bacterial Morphology division
a. Cocci E.g., Micrococcus
b. Bacillus tetragena
c. Spirals
II. Staining
a. Direct/ Simple Stain
b. Differential Stain
c. Selective/ Special Stain Coccus is Sarcina/e
d. Indirect/ Negative/ Relief Stain divided by Coccus in cubical packets
III. Methods of Studying Bacteria three (3) of eight (8)
a. Colonial/ Cultural Characteristics planes of
IV. Antigenic Determination by Serological Typing division in a E.g., Micrococcus luteus
regular pattern
I. BACTERIAL MORPHOLOGY Coccus is Cocci in Clusters
divided by two E.g., Staphylocccus spp.
(2) or more (grape-like clusters)
➢ 0.25 to 1um in width and 1-3um in length planes of
(0.4-2um – Mahon) division in an
➢ The staining procedure separates almost all medically relevant irregular
bacteria into two general types: pattern
▪ Gram positive
▪ Gram negative
➢ Common bacterial cellular morphologies:
▪ Cocci (circular)
▪ Coccobacilli (ovoid) B. Bacillus (Bacilli)
▪ Bacillus (rod shaped) ➢ Rod shaped, cylindrical or elongated but it’s interesting to know
▪ Fusiform (pointed end) that this is not always true to all bacilli since some of them also
▪ Curved varies in morphologies
▪ Spiral shapes
❖ Microscopic Shapes Table 9: Bacilli Morphologies
➢ Thiomargarita namibiensis (largest bacteria) Prominent Arrangement Illustration
▪ Found in ocean sediment Single Bacillus – rod shaped
▪ Has a diameter of 0.1 – 0.3mm bacillus
Diplobacilllus – bacillus in pair

Streptobacillus – bacillus in chains

Coccobacillus – bacillus that are

small, short, stout/pump
A. Cocci (Coccus)

➢ Round/ spherical shaped bacteria

➢ The resulting arrangement of cocci depends on the Plane of Small and short bacillus arranged in
school of fish, rail road track or
Division
fingerprint pattern in stained smear

Table 8: Cocci Morphologies E.g., Haemophilus ducreyi –

Plane of Resulting Illustration causative agent of soft chancre/
Division Arrangement chancroid
Coccus is Diplococcus/ Diplococci
divided by one All diplococcic appears to
(1) plane of be two cocci adjacent to Large, square cut-ends, spore
division each other except the forming bacillus, arranged in chains
following:
E.g., Bacillus anthracis – causative
Neisseria spp. – kidney or agent of anthrax
coffee bean shaped
diplococci except:
Neisseria weaver and
Neisseira elongate
Large, rounded ends, non-spore
Streptococcus
forming bacillus arranged in chains
pneumoniae –
flame/lancet shaped
E.g., Fusobacterium spp. –
diplococci
anaerobic bacteria which chiefly
Coccus is Cocci in Chains found as a normal flora in the
divided by one E.g., Streptococcus spp. gastrointestinal tract (GIT)
(1) plane of
division but
continuously
dividing it

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[BACT211] 1.03 Bacterial Morphology I Prof. Rochelle D. Darlucio, RMT, MPH

Slim, slender, threadlike bacillus, II. STAINING
sometimes arranged in serpentine
cord (crawling snake) pattern in
stained smears ➢ Imparts an artificial coloration not only to bacteria but for other
material found on clinical specimen smear that allows them to be
E.g., Mycobacterium tuberculosis – visualized better using the magnification of microscope
causative agent of human ❖ The four categories of staining:
tuberculosis ➢ Direct/ Simple stain
Branching or filamentous bacilli ➢ Differential stain
➢ Selective/ special stain
E.g., Actinomyces spp., Nocardia
spp., Actinomycetes spp. ➢ Indirect/ Negative/ Relief stain

A. Direct/ Simple Stain

Irregular bacilli – club shaped or
barb shaped arranged in palisade, ❖ Usually contains one specific active chromogen in the stain which
fence stick, cigarette packet.
enhances the appreciation of bacterial size, shape and arrangement
Sometimes resembles X, V, Y, Z or
Chinese character ❖ The commonly used simple stains are crystal violet, gentian violet,
methylene blue, malachite green
E.g., Corynebacterium diphtheriae
– causative agent of diphtheria

Curved or comma shaped bacilli

E.g., Vibrio spp.

S or C shaped bacilli, sometimes

resembles seagull wing

E.g., Campylobacter spp., B. Differential Stain

Helicobacter spp., Arcobacter spp.
❖ Contains 2 or more chromogens which further differentiate specific
component within the bacterial cell which aids in the differentiation or
grouping of bacteria
C. Spirals ❖ This staining technique also includes a decolorization which is the
➢ Helical or twisted bacteria most critical step in the process
➢ Spirillum spp. Which is helical but rigid while the spirochetes ➢ Gram stain
which are helical as well but more flexible in movement ▪ Differentiates gram positive bacteria which stain purple or
violet from gram negative which stains red or pink
Table 10: Spiral Morphologies ➢ Acid-Fast stain
Prominent Arrangement Illustration ▪ Differentiate acid fast organism such as Mycobacterium
Spiral with two or more curves, tuberculosis which stains red from non-acid fast organisms
quite rigid which stains blue or green depending on the counterstain
used in the process using the Ziehl-Neelsen or Kinyoun
E.g., Spirillum minor/minus staining methods
causative agent of Sodoku (a ➢ Fluorochrome stain
rat bite fever infection)
▪ Uses fluorescent dyes such as auramine or rhodamine or
Loosely twisted spiral
resembling a stretched spiral combination of both. These dyes remain in the cell wall of
acid-fast organism even after decolorization
E.g., Borrelia spp., causative ❖ Other Stains for Acid-fast Organisms
agent of Relapsing fever and ➢ Pappenheim stain
Lyme Disease ▪ Mycobacterium tuberculosis – RED
Tightly twisted spiral ▪ Mycobacterium lacticola (smegmatis) – BLUE
resembling a cork screw ➢ Baumgarten stain – uses rosolic acid as a decolorizer
▪ Mycobacterium tuberculosis – BLUE
E.g., Treponema pallidum
▪ Mycobacterium leprae – RED
causative agent of Venereal
syphilis
C. Selective/ Special Stain

❖ Stains that specifically highlight or emphasize certain bacterial cell

Tightly twisted spiral with one
or both ends bent into a hook,
structures or components which aids in the presumptive identification
sometimes even resembling of the bacteria
an interrogative symbol ❖ Stain for Cell Wall
➢ Victoria Blue Dye – cell wall
E.g., Leptospira interrogans stains blue
causative agent of zoonotic ❖ Stains for Capsule
infection Leptospirosis ➢ HISS – capsule stains pale
brown
➢ TYLER – capsule stains light
violet
➢ MUIR – capsule stains light
blue
➢ GIN – capsule is unstained but the bacteria will be stained with
its margins delineated by the ink
➢ WADSWORTH – capsule stains pinkish and bacteria stains blue
➢ WELCH – capsules stains pale violet

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[BACT211] 1.03 Bacterial Morphology I Prof. Rochelle D. Darlucio, RMT, MPH

❖ Stain for Metachromatic Granules/ Babes Ernst Bodies/ Volutin D. Indirect/ Negative/ Relief Staining
➢ Loeffler’s Alkaline Methylene Blue (LAMB) – granules stain
red ❖ Type of staining which actually provides coloration to the background
➢ Albert – granules appear blue black of the smear while rendering the bacteria and covering structure such
➢ Neisser – granules appear dark blue as capsule unstained
➢ Lindegran – granules appear reddish brown ❖ Useful in the identification of medically important capsulated bacteria
➢ Burke’s Technique – a modified gram’s staining technique as well as capsulated strains of Cryptococcus spp., especially in
➢ Ljubinsky – granules stain dark violet cerebrospinal fluid sample in cases of meningitis
❖ Bacteria/ structure (capsule) – unstained
❖ Background – colored/ stained
➢ India ink or Nigrosin – background is black
➢ Congo red – background is red
➢ Anthony – background is purple

❖ Stains for Bacterial spores/ Endospores

➢ Fulton-Schaeffer – spores are green
➢ Dorner – spores are red
➢ Wirtz-Conklin – spores are green

III. METHODS OF STUDYING BACTERIA

❖ After the standard incubation of 18-24 hours, inoculated plates are

❖ Stains for Flagella retrieved from the incubator and the colonial or cultural characteristics
➢ Tannic Acid of the bacterial colonies that grew in each culture media for each
▪ Important component in flagellar stain which coats, swells, specimen is examined, this is referred to as plate reading
precipitates the flagella enhancing its visualization
• Leifson A. Colonial/ Cultural Characteristics
• Gray
• Silver ❖ Size
• Fisher-Conn ➢ Relative size of the bacterial colony
Colony Size Description
Pinpoint Colonies less than 1mm
Small About the same size of a pinhead
Medium Slightly larger than a pin head
Large Usually 6-8mm in diameter

❖ Margin
➢ Appearance of the edge of the colony
❖ Stains for Rickettsia Edge of the Colony Description
➢ Castaneda – stains blue Smooth or entire Circular without interruption
➢ Machiavelo – stains red Undulate Waxy edge
➢ Giemsa – stains blue Rough or rhizoid Crenated edge
Lobate Lobulated edge
Fringed or filamentous Branchlike edge
❖ Stains for Chlamydia Fingerlike Uneven rounded projections
➢ Gimenez – elementary bodies stains red Irregular Uneven length of projection with no definite
➢ Machiavelo – stains red shape
➢ Giemsa – stains purple
❖ Stains for Spirochetes ❖ Elevation
➢ Fontana-Tribondeau – spirochetes stains dark brown or black ➢ Height of the colony
➢ Levaditi Silver Impregnation – spirochetes stains black Height of the Colony Description
➢ India Ink Negative Stain – spirochetes are unstained; Flat No visible elevation or height
background is black Raised Slight elevation
❖ Stains for Mycoplasma Convex Dome shaped
➢ Dienes – stains blue Umbilicate (innie) Depressed or concaved center
❖ Stain for Bipolar Bodies (Yersinia pestis) Umbonate (outie) Raised or bulging center
➢ Wayson – bipolar bodies stain

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[BACT211] 1.03 Bacterial Morphology I Prof. Rochelle D. Darlucio, RMT, MPH

❖ Density
➢ Optical property to pass light through the bacterial colony
Colony Density Description
Opaque Light won’t shine through the colony
Transluscent Light will shine through the colony
Transparent Light shines through the colony

IV. ANTIGENIC DETERMINATION BY SEROLOGICAL TYPING

❖ O Antigen
➢ Associated with the cell wall
❖ H Antigen
➢ Associated with the flagella
❖ K Antigen
➢ Associated with the capsule
❖ Vi Antigen
➢ Specific capsular antigen of Salmonella typhi

❖ Texture/ Consistency
Texture of the Colony Description
Brittle or splinters Crumbling colony (e.g., Nocardia spp.)
Creamy or butyrous Butterlike (e.g., Staphylococcus spp.)
Dry & Waxy Sticky colony (e.g., Diptheroids)
Rough & Warty Cauliflower appearance (e.g.,
Mycobacterium spp.)
Mucoid Wet & sticky colony (e.g.,
Streptococcus pneumonia)

❖ Hemolytic Pattern
➢ Exhibits the bacteria’s ability to lyse RBCs in the culture media
Hemolysis Type Description
Beta Clear zone around the colony;
complete hemolysis
Alpha Greenish or brownish zone around the
colony; incomplete/ partial hemolysis
Gamma No hemolysis around the colony
Alpha prime Inner alpha hemolysis surrounded by
an outer beta hemolysis

❖ Pigmentation
➢ Ability of the bacteria to produce unique coloration their colony
Colony Color Bacteria Producing the Pigment
Lime yellow Micrococcus luteus
Golden yellow Staphylococcus aureus
Blue green Pseudomonas aeruginosa
Red Serratia marscesens
Porcelain white Staphylococcus albus
Violet Chromobacterium violaceum

❖ Odor
➢ Certain bacteria produce characteristic odor in culture media
Colony Odor Bacterial Producing the Odor
Unwashed stockings Staphylococcus spp.
Rancid potato Serratia odorifera
Com tortilla/ fruity Pseudomonas aeruginosa
Ammonia like Acinetobacter spp.
Freshly plowed field Nocardia spp.
Mousy/ mouse nest Haemophilus spp.
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OLFU Bacterial Cultivation LEC

2020-2021
2nd SEM
College of Medical CLINICAL BACTERIOLOGY BACT211
Laboratory Science Transcriber: Riyoma Surell 4 LEC

Batch 2023 Date: February 15, 2021

❖ Additional/ Special Growth Requirements

Outline ➢ There bacteria that are very difficult to grow which we refer to as
At the end of the session, the student must be able to learn: fastidious (very difficult to grow) which requires special or
• Bacterial Growth additional requirements to grow in culture media
• Nutritional Requirements ➢ E.g., Haemophilus spp. (blood loving organism)
• Bacterial Growth Phase ▪ Requires both X & V factor
• Culture Media ▪ Culture Media: Blood Agar Plate
a. Classification of culture media
• 1st priority: 5% defribinated sheep’s blood
b. Inhibitors in culture media
• Most Commonly and Routinely used Selective & Differential Culture • 2nd priority: horse blood
Media in the Laboratory • 3rd priority: rabbit’s blood
a. Eosin Methylene Blue (EMB) Agar • Least priority: human blood (must be type O – no
b. Mc Conkey (MAC) Agar presence of antigens)
c. Salmonella-Shigella (SSA) Agar ▪ Since blood agar plate lacks V factor, use chocolate agar
d. Hektoen Enteric (HEA) Agar
plate (contains NAD – v factor)
e. Bismuth Sulfite (BSA) Agar
f. Brilliant Green (BGA) Agar ➢ X Factor (Hemin/ Hematin)
g. Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar ▪ Degradation product of Hgb (hemoglobin)
h. Mannitol Salt (MSA) Agar ➢ V Factor (Nicotinamide Adenine Dinucleotide or NAD)
i. Lowenstein Jensen (LJ) Medium ❖ Gaseous Requirement
j. Selective Medium for Neisseria spp. ➢ Aerobe
• Culture Media for Antibiotic Susceptibility/ Sensitivity Testing (AST) ▪ Bacteria that grow, live, and survive in the presence of
a. Characteristic/ Biochemical Culture Media oxygen
▪ Strict/ Obligate Aerobe
I. BACTERIAL GROWTH • Absolutely requires oxygen to grown, live, and survive
• Micrococcus spp., Mycobacterium spp.,
❖ Refers to the increase in the number of bacteria rather than in size Pseudomonas spp., Neisseria spp., Brucella spp.,
❖ Basically, bacteria grow in number and do not grow in size Francisella spp., Bordetella spp., Leptospira spp.
❖ This growth is affected by various factors such as optimum growth ▪ Facultative Anaerobe
requirements, dynamics of growth, including the use of a medium that • Bacteria that have the ability to grow, line and survive
can be artificially prepared in the laboratory in small concentration of oxygen environment
• Staphylococcus spp., Streptococcus spp., Family
Enterobacteriaceae
▪ Microaerophilic
• Bacteria that prefers small concentration of oxygen
environment approximately 2%-10%
• Campylobacter spp., Helicobacter spp., Arcobacter
spp., and some Streptococcus spp.
II. NUTRITIONAL REQUIREMENTS ➢ Anaerobe
❖ Carbon (at least 50% of weight) ▪ Bacteria that grow, live and survive in the absence of
➢ Needed for the synthesis of cellular components oxygen
➢ Carbon Dioxide from the air – autotroph ▪ Strict/ Obligate Anaerobe
➢ Organic compounds in the culture media (glucose) – • Absolutely do not require oxygen to grow, live and
heterotroph survive
❖ Energy Source (4% of weight) • Many Clostridium spp., such as Clostridium noyvi,
➢ Light – phototroph most Bacteroides spp., Fusobacterium spp.,
➢ Chemical energy – chemotroph Peptostreptococcus spp., and Porphyromonas spp.
❖ Electron Source ▪ Aerotolerant Anaerobe
➢ Inorganic molecule (Fe2+) – lithotroph • Bacteria that do not require oxygen but may tolerate or
➢ Organic molecule – organotroph withstand limited exposure to oxygen
➢ NADH reduced from NAD • Some Clostridium spp., such as Clostridium
➢ FADH2 reduced from FAD perfringens, Bacteroides fragilis, most strains of
❖ Nitrogen (14% of weight) Proprionibacterium and Lactobacillus
➢ Needed for the synthesis of proteins ➢ Capnophilic
➢ Free nitrogen from the air ▪ Bacteria that requires 5%-10% CO2 to grow
➢ Nitrogenous compounds in the culture media (e.g., peptone, ▪ (NHACEK GROUP)
yeast, beef extract) • Neisseria spp.,
❖ Water/ Moisture/ Humidity (70% of bacteria) • Haemophilus spp.,
❖ Mineral Elements • Aggregatibacter spp.,
➢ Needed as co-factors in various metabolic process of the
• Cardiobacterium spp.,
bacteria
• Eikenella spp., & Kingella spp.,
❖ Salt
➢ Bacteria can tolerate salt concentration below 6%, however there ▪ Streptococcus pneumoniae
❖ Temperature Requirement
are certain bacteria that can survive high salt concentration
➢ Most pathogenic bacteria would grow at temperature between
environment hence they’re called as Halophilic bacteria or
Halophiles (salt-loving) 35-37C, hence incubator in the laboratory is usually set and
maintained within this temperature range for routine isolation of
▪ Staphylococcus spp.
▪ Enterococcus spp. pathogens
▪ Mesophilic: 20-40C
▪ Vibrio spp. except Vibrio cholerae & Vibrio mimicus
▪ Psychrophilic/ Cryophilic: 0-20C
▪ Bacillus spp.
▪ Thermophilic: 50-60C
▪ Hyperthermophilic/ Extremely Thermophilic: 80-110C
▪ Eurithermophilic: wide range
▪ Sternothermophilic: narrow range
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[BACT211] 1.04 Bacterial Cultivation I Prof. Rochelle D. Darlucio, RMT, MPH

❖ pH Requirement A. Classification of Culture Media
➢ most pathogenic bacteria can grow in a neutral or slightly alkaline
environment (pH 7.0 – 7.5), hence most culture media used in ❖ Classification of Culture Media according to Composition
routine isolation of pathogens is adjusted to this pH range ➢ Synthetic/ Chemically Defined
▪ Acidophilic ▪ Composed of known and exact amounts of pure chemical
• Acid loving bacteria (e.g., Lactobacillus acidophilus) – substances
normal flora of vagina ➢ Non-Synthetic/ Non-Chemically Defined/ Complex
▪ Alkaliphilic ▪ Composed of complex materials that are rich in vitamins
• Alkali loving bacteria (e.g., Gardnerella vaginalis) and nutrients that are not usually represented by a chemical
➢ High Osmotic Pressure formula such as peptones, beef or yeast extract, plant
▪ Osmophilic bacteria (Archaebacteria spp.) extracts etc.
➢ Tissue Culture Media
III. BACTERIAL GROWTH PHASE ▪ Live cells harvested from organs of humans and animals
that supports the growth of obligate intracellular organisms
❖ Generation Time that cannot grow in artificially prepared culture media
➢ Time to replicate
➢ Refers to the stages of bacterial growth Table 7-4 Tissue Culture Media used in Microbiology Laboratory
❖ Fast Growing Bacteria: 20 minutes Tissue Culture Media Source
❖ Slow Growing Bacteria: 24 hours Vero Cell Line Kidney cells of an African Green Monkey
Mc Coy Cell Line Mouse cell line
Chicken Embryo Fertilized chicken egg
A549 Cells Human lung carcinoma
HELA Cell Line Human cervical carcinoma
Hep-2 Cell Line Human epithelial cells of larynx carcinoma

❖ Classification of Culture Media according to Physical State/

Consistency
➢ Liquid
▪ a culture medium that doesn’t contain a solidifying agent
➢ Semi-Solid
▪ A culture medium that contains 0.5% - 1% agar
▪ Sulfide Indole Motility (SIM) Medium
• Used for observation of hydrogen sulfide gas
production, indole production and motility
➢ Solid
▪ A culture medium which contains 1.5% - 3%T (2%-3%) agar
❖ Classification of Culture Media according to Manner of
Growth Phase Key notes Cell Increase in Dispensing/ Formation
Division number ➢ Plated
Lag phase Bacteria are still adjusting to the new NO NO
▪ Usually contained in a container that can be made of glass
environment hence there is no cell
division that occurs in this phase but (pyrex) or disposable plastic (Petridish)
they are activity synthesizing DNA & ➢ Tubed
proteins that are necessary for cell ▪ Usually, container in glass tubes such as Wassermann
division tubes with different volume capacity (3mL, 5mL, 10mL) or
Log/ Phase where there is a sudden YES YES in a tube with a flat bottom and a screw cap
Logarithmic/ increase of bacteria because of rapid
Exponential generation or doubling time. The
phase number of generation per hour is
called growth rate constant. This is
also the phase where bacteria are
most metabolically active hence
most susceptible to the action of
antimicrobial agents
Maximum After essential nutrients are YES NO
stationary/ depleted, toxic products accumulate
Plateau or oxygen becomes limiting, the rate
phase of cell division equates the rate of cell
➢ Bottled
death ▪ Culture media contained in a glass bottle that is usually
Decline/ Due to unfavorable environment for YES NO used for blood culture
Death phase growth, bacterial cell division ❖ Classification of Culture Media according to Function/ Use
decreases while cell death becomes ➢ General Purpose/ Primary/ Basic/ Basal/ Supportive/
more accelerated General Isolation Culture Media
▪ Contains basic nutritional requirements to support the
IV. CULTURE MEDIA growth of non-fastidious microorganisms
▪ This is also used a base medium in the preparation of other
❖ An artificial preparation in the laboratory which contains basic culture media
foundation of nutrients and a solidifying agent (if needed) to support ➢ Enriched Culture Media
the growth of microorganisms ▪ Contains the basic nutritional requirement to support the
❖ Additional substances may be added to enrich the media for growth of growth of non-fastidious microorganisms with additives,
microorganisms that are very difficult to grow (fastidious) enriching substances, or supplements to support the growth
❖ Terminologies of fastidious microorganisms
➢ Culture ▪ E.g., Blood agar, chocolate agar plate
▪ Noun: growth of microorganisms ➢ Enrichment Broth
▪ Verb: to growth/ to cultivate microorganism ▪ A primary media used to support or favor the selective
➢ Inoculate/ Plant/ Cultivate growth of pathogens in a specimen, such as stool or
▪ Introducing the microorganism to the culture media sputum, where the number of normal flora outnumber the
➢ Transplant/ Subculture pathogens
▪ Transfer of microorganisms from one culture media to ▪ Use commonly stool specimen
another
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[BACT211] 1.04 Bacterial Cultivation I Prof. Rochelle D. Darlucio, RMT, MPH

▪ Examples of Enrichment Broth:
• Alkaline Peptone Water (APW)
 Used to selectively favor the growth of Vibrio
while inhibiting all other normal intestinal flora due
its high pH
• Selenite F Broth
 Used to selectively favor the growth of
• RLF: Rapid Lactose Fermenter (able to ferment lactose for 18-
Salmonella while inhibiting all other normal
24 hours incubation)
intestinal flora
• LLF: Late Lactose Fermenter (able to ferment lactose for 36-72
• Thioglycolate Broth (THIO)
hours incubation)
 An enrichment broth for anaerobic bacteria but
should be used solely in the isolation of anaerobic
Differentiation of Family Enterobacteriaceae based on lactose
bacteria since it can also grow aerobes and
fermentation
facultative anaerobes
• GN Broth (Gram Negative Broth)
❖ RLF
 Used to selectively favor the growth of
➢ Escherichia
Salmonella and Shigella while inhibiting all other
▪ E. coli – medium sized dark violet colonies with greenish
normal intestinal flora
metallic sheen
• Todd-Hewitt Broth
➢ Enterobacter
 A liquid enrichment recommended for the ▪ Appears medium sized dark violet colonies with/without
production of Streptococcal haemolysin and the dark center
cultivation of Streptococci prior to serological ➢ Klebsiella
grouping ▪ Large sized mucoid dark violet colonies with or without dark
➢ Transport Culture Media center
▪ A primary isolation culture media which maintains the
viability of bacteria allowing rapid multiplication if there is an
anticipated delay in bringing the specimen collected
bedside or remotely to the laboratory
❖ Selective and Differential Culture Media
➢ Selective Culture Media
▪ Favors the growth of the organism of interest using
inhibitors added in the culture media
➢ Differential Culture Media
▪ Contains indicators which changes in color as a result of a
product produced be a chemical reaction in the components Escherichia Enterobacter
of the media such as glucose

Inhibitor Indicator
Gram + Gram -
Gram - Gram +
Klebsiella
➢ Note: it is important to remember and understand that not all
selective culture media are differential but all differential culture
media are selective

INHIBITORS IN CULTURE MEDIA ❖ LLF

Inhibitors for Gram Positive Bacteria ➢ Hafnia, Serratia, Citrobacter
Dyes Crystal violet, eosin, methylene blue, brilliant green, ➢ Salmonella arizonae
etc., ➢ Shigella sonnei
Chemicals Bismuth sulfite, bile salts (sodium desoxycholate), ➢ Yersinia enterocolitica
thiosulfate, citrate, etc., ❖ NLF (appear colorless colonies)
Antibiotics Vancomycin ➢ All Salmonella except S. arizonae
Inhibitors for Gram Negative Bacteria ➢ All Shigella except S. sonnei
Dyes Basic fuchsin and thionine for Brucella abortus ➢ All Yersinia except enterocolitica
Chemicals Potassium tellurite, sodium azide, phenylethyl alcohol ➢ Proteus, Providencia, Morganella, Edwardsiella
Antibiotics Colistin, Nalidixic Acid, Trimethoprim (Proteus)
Inhibitors for Fungi B. Mc Conkey (MAC) Agar
Antibiotics Nystatin, Anisomycin, Ampothericin B.
Indicators in Culture Media ❖ Original Color: Light Pink
Dyes and chemical substances such as pH indicators ❖ Selective:
➢ For: Gram Negative Enteric bacilli
➢ Inhibitors: Crystal violet, Bile salts, Citrate
V. MOST COMMONLY AND ROUTINELY USED SELECTIVE &
❖ Differential:
DIFFERENTIAL CULTURE MEDIA IN THE LABORATORY
➢ Indicators: Neutral Red
A. Eosin Methylene Blue (EMB) Agar

❖ Original Color: Dark Violet

❖ Selective for: Gram Negative Enteric bacilli
❖ Inhibitors: Eosin and Methylene Blue
❖ Differential Indicators: Eosin and Methylene Blue

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[BACT211] 1.04 Bacterial Cultivation I Prof. Rochelle D. Darlucio, RMT, MPH

E. Bismuth Sulfite (BSA) Agar

❖ Selective:
➢ For: Salmonella spp. (Salm. Typhi has distinct appearance)
➢ Inhibitors: Bismuth sulfite
➢ CHO Incorporated: Glucose
❖ Salmonella typhi colonies appear as black colonies with silver
metallic sheen

C. Salmonella-Shigella (SSA) Agar

❖ Original Color: Light Orange

❖ Selective
➢ For: Salmonella and Shigella spp.
➢ Inhibitors: Brilliant Green, Bile Salts, Citrate
❖ Differential:
➢ Indicators: pH indicator: Neutral red
➢ H2S Indicator: Ferric ammonium citrate
F. Brilliant Green (BGA) Agar
➢ Sulfur Source: Sodium thiosulfate
❖ Selective:
➢ For: Salmonella spp. except for Salmonella typhi
➢ Inhibitors: Brilliant green
➢ CHO Incorporated: Lactose
❖ Salmonella spp., colonies appear as white colonies resembling a
snowflake surrounded by brilliant red medium

G. Thiosulfate Citrate Bile Salts Sucrose (TCBS) Agar

D. Hektoen Enteric (HEA) Agar ❖ Original Color: Light Green/ Olive Green
❖ Selective:
❖ Original Color: Dark Green ➢ For: Vibrio spp.
❖ Selective: ➢ Inhibitors: Thiosulfate, Citrate, Bile Salts
➢ For: Gram Negative Enteric bacilli ❖ Differential:
➢ Inhibitors: Bile Salts, Citrate ➢ Indicators: Bromthymol Blue (BTB)
❖ Differential:
➢ Indicators: pH Indicator: Bromthymol Blue (BTB)
➢ H2S Indicator: Ferric ammonium citrate
➢ Sulfur Source: Sodium thiosulfate

H. Mannitol Salt (MSA) Agar

❖ Original Color: Light/ Salmon Pink

❖ Selective:
➢ For: Staphylococcus spp.
➢ Inhibitors: High concentration of salts (7.5%)
❖ Differential:
➢ Indicators: Phenol Red

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[BACT211] 1.04 Bacterial Cultivation I Prof. Rochelle D. Darlucio, RMT, MPH

I. Lowenstein Jensen (LJ) Medium

❖ Original Color: Light Green

❖ Selective:
➢ For: Mycobacterium spp.
➢ Inhibitors: Malachite Green
❖ Sputum Sample needs to be:
1. Decongested/ Digested – To dissolve the thick mucus/mucin
❖ Voges-Proskauer (VP)
that might be trapping the bacteria in the sample. N-acetyl-L-
➢ Used for the detection of bacterial pathogen that metabolizes
cysteine (NALC) is usually used
glucose using the Butylene Glycol Pathway
2. Decontaminated – To eliminate normal flora that contaminates
▪ Add alpha-naptol as reagent
the sample. NaOH is usually used
➢ Note: Positive MR = Negative VP or Negative MR = Positive VP
(to determine what pathway of glucose metabolism)
❖ Simmon Citrate Agar (SCA)
➢ Used for the detection of bacterial pathogen that can utilize
citrate as a sole source of carbon
▪ Green slanted media (original color)
▪ Positive result (blue)

J. Selective Medium for Neisseria spp. ❖ Triple Sugar Ion Agar (TSI)
➢ Used for the determination of bacterial pathogen’s ability to
❖ Neisseria spp., Gram negative cocci ferment glucose, sucrose or lactose. It can also detect sulfide
❖ Usually composed of chocolate agar base with antibiotics production (blackening of the agar) and gas production (+
result = bubbles, cracks or spaces on the culture media)
Culture Gram + Inhibitor Gram - Fungal Proteus spp.
Media Inhibitor Inhibitor Inhibitor a. Red/ red (no sugar
Thayer Vancomycin Colistin Nystatin fermentation)
Martin Agar
(TM) b. Control
Modified Vancomycin Colistin Nystatin Trimethoprim c. Red/ yellow (Glucose
Thayer fermented but lactose and
Martin Agar
(MTM) sucrose not fermented)
Martin- Vancomycin Colistin Anisomycin Trimethoprim d. Yellow/ yellow (Glucose
Lewis Agar fermented. Lactose and/or
(MLA)
New York Vancomycin Colistin Amphotericin Trimethoprim
sucrose fermented)
City Agar B e. Red/ yellow with H2S
(NYC)
GC-LECT Decreased Colistin Amphotericin Trimethoprim
concentration of B
Vancomycin; added
0.1% dextrose, 1.0% sucrose, 1.0% lactose
with Lincomycin

❖ Lysine Iron Agar (LIA)

VI. CULTURE MEDIA FOR ANTIBIOTIC SUSCEPTIBILITY/ ➢ Used for the determination of bacterial pathogen’s ability to
SENSITIVITY TESTING (AST) decarboxylate or deaminate lysine. It can also detect glucose
fermentation, sulfide production and gas production
❖ Most Bacteria
➢ Mueller Hinton Agar (MHA) and Mueller Hinton Broth (MHB)
❖ Haemophilus spp. (fastidious organism – difficult to grow)
➢ Mueller Hinton with Chocolate Agar Base or Haemophilus Test
Medium (HTM) Agar
❖ Mycobacterium spp.
➢ Middlebrook 7H10 or 7H11 Medium

A. Characteristic/ Biochemical Culture Media

❖ Sulfide Indole Motility Medium (SIM) ❖ Moeller’s Broth

➢ Used for observation of hydrogen ➢ Used to detect lysine decarboxylation, ornithine decarboxylation,
sulfide gas production (there will be and arginine dihydrolysis
presence of black precipitate), indole ❖ Stuart’s Urea Broth or Christensen Urea Agar
production (can be observed by the ➢ Used to detect bacterial pathogen that hydrolyze urea substrate
appearance of red or pink ring
formation) and motility (presence of
turbidity or spread of colonies along the
side of inoculation)
❖ Methyl Red (MR)
➢ Used for the detection of bacterial
pathogen that metabolize glucose using the Mixed Acid
pathway
▪ Add methyl red as reagent

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[BACT211] 1.04 Bacterial Cultivation I Prof. Rochelle D. Darlucio, RMT, MPH

References:
• Study Guide on Diagnostic Bacteriology by
Mr. Nathaniel Ranon
• Powerpoint Presentation and Lecture Notes
of Prof. Rochelle Darlucio

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OLFU Sterilization and Disinfection LEC

2020-2021
2nd SEM
College of Medical CLINICAL BACTERIOLOGY BACT211
Laboratory Science Transcriber: Riyoma Surell 5 LEC

Batch 2023 Date: February 23, 2021

3. Concentration of Disinfecting Agent

Outline ➢ The amount needed to destroy microorganisms varies based on
At the end of the session, the student must be able to learn: the agent to be used. It is therefore important to follow, the correct
• Sterilization vs Disinfection preparation and dilution as prescribed by the manufacturer.
a. Factors that affect the degree of killing Microorganisms Simply, concentrated agents do not necessarily mean that it
• Methods of Sterilization would work better
a. Physical Methods ▪ 1:10 bleach (common disinfectant)
b. Chemical Methods
4. Presence of Organic Material
• Methods of Disinfection
a. Physical Methods ➢ Blood, puss, and mucus are examples of organic materials that
b. Chemical Methods may prevent the full contact of the agent to the organisms, hence
limiting its action
➢ Example: is bleach (sodium hypochlorite) that is easily
I. STERILIZATION VS DISINFECTION inactivated by organic material.
5. Nature of Surface to be Disinfected
➢ Some instrument that we use in the laboratory sometimes are
❖ Sterilization
made up of biomaterial which exempts them to disinfection or
➢ Refers to the destruction of all forms of life, including bacterial
sterilization due to possible damage
spores
➢ Example: is endoscopic instruments which can’t be autoclaved
➢ Originated more than 100 years ago
6. Contact Time
➢ Complete removal of microorganisms including spores
➢ It is critical to observe proper contact time of the agent and the
➢ Physical and chemical methods may be used
object to be disinfected or sterilized. In principle, contact time
❖ Disinfection
may be affected by all previous factors already mentioned as well
➢ Refers to a process that eliminates a defined scope of
as temperature
microorganisms, including some spores
➢ Example: Alcohol and betadine has to be in contact for about 1-
➢ Reduces the number of microorganisms
2 minutes to work properly. Spore forms may need more contact
▪ Physical or chemical methods may be used, but most
time than its vegetative counterpart
disinfectants are chemical agents applied to inanimate
7. Temperature
objects
➢ Generally, disinfectants are usually used at room temperature
❖ Antiseptic
(20C to 220C). However, their activity may increase at a certain
➢ Substance applied to the skin for the purpose of eliminating or
degree by a corresponding increase in temperature or may
reducing the number of bacteria present
decrease when temperature is decreased. Too high or low
▪ E.g., Alcohol
temperature may inactivate disinfectants and sterilants
▪ Do not kill spores and cannot be used as disinfectants
8. pH
➢ It is also important to consider the pH of the material to be treated
A. Factors that Affect the Degree of Killing of Microorganisms
and he agent itself. Manufacturers usually optimize this factor to
achieve maximum activity
9. Biofilms
➢ Certain bacteria have to ability to form communities of layers of
bacteria with protective shield which is called as biofilm. It is
important to consider that biofilm formation may require longer
contact time or increase in the concentration of the agent
▪ E.g., Hospital – Catheter (lots of bacteria)
➢ Inanimate and animate objects
10. Compatibility of Disinfectants
1. Types of Organisms ➢ Some disinfectants may inactivate the action of another hence it
➢ Different organisms have varying ability in withstanding and is also important to consider the compatibility of the disinfectants
chemical and physical treatment due to the different biochemical ➢ Example is bleach and quaternary ammonium compounds
composition of these organisms and various mechanisms that which may negate each other
they use to protect themselves
➢ Examples II. METHODS OF STERILIZATION
▪ Spore forming Bacteria
• Spores are coated with proteins, lipids and
A. Physical Methods
carbohydrates as well as dipicolinic acid calcium
▪ Mycobacterium spp.
• cell wall is high in lipid which enables them to become 1. Moist Heat
resistant to most environmental stress such as ➢ coagulation of bacterial proteins including bacterial enzymes
desiccation a. Autoclave
▪ Biofilm forming Bacteria ▪ operates based on the principle of steam under pressure
• certain bacteria can aggregate into communities of Effective indication:
bacteria which makes then resistant to chemical and STERILIZATION: 121C to 15lbs/ in2 for 15 minutes
physical means of destruction DECONTAMINATION: 135C for 30lbs in2 for 30 minutes
▪ Prions Biological indicator: Bacillus stearothermophilus
b. Tyndallization
• naked pieces of proteins, similar to viruses but without
▪ fractional discontinuous sterilization
the nucleic acid, that is most resistant to the action of
Effective indication: 100C for 30-60 minutes
heat, radiation, and chemicals
Instrument: Arnold’s Sterilizer
2. Number of Organisms
c. Inspissation
➢ this factor basically refers to the amounts of organisms present
▪ thickening through evaporation
in the object to be treated referred to as microbial load
Effective indication: 75C to 80C for 2 hours
(bioburden). In principle, the higher the number of organisms, the
Instrument: Inspissator
longer the exposure time needed to eliminate 99.9% of the
microorganisms

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[BACT211] 1.05 Sterilization and Disinfection I Prof. Rochelle D. Darlucio, RMT, MPH
Table 4-2 Control of Microorganisms using Heat Methods III. METHODS OF DISINFECTION
Method Temperature Time Applications
(C) Required A. Physical Methods
Boiling Water 100 15min Kills microbial 1. Boiling
(steam) vegetative forms; ➢ Destroys vegetative cells of bacteria but not their spores
endospores Effective indication: 100C for 15-30 minutes
survive 2. Pasteurization
Autoclave 121.6 15min at Sterilizes and kills ➢ Used for the preservation of alcoholic beverages such as beers,
(steam under 15psi endospores wines, and also dairy products such as milks and yogurt
pressure) a. Batch: 62.5C for 30 minutes
Pasteurization 63 30min Disinfects and b. Flash: 72C for 15 seconds
Batch Method kills milk-borne c. Ultra-High Temperature (UHT): 72C to 110C for 5
pathogen and seconds
vegetable forms; 3. Non-Ionizing Radiation
endospores ➢ Uses low energy long wavelength ultraviolet rays to disinfect heat
survive sensitive materials as well as large spaces
Pasteurization 72 15s Same, but shorter B. Chemical Methods
Flash Method time at higher 1. Alcohol
temp. ➢ MOA: Dehydration, Lipid dissolution and Protein denaturation
Over (Dry 160-180 1.5-3hrs Sterilizes; keeps ➢ 70% Alcohol not 90%
Heat) materials dry ➢ Minimum Contact Time: 1-2 minutes or until completely
evaporated
2. Dry Heat ➢ Ethyl and Isopropyl alcohol. Kills Mycobacterium tuberculosis
➢ oxidation of bacterial components 2. Halogens
➢ MOA: inhibits protein function and acts as strong oxidizing
a. Direct Flame agents
▪ direct application of flame in aseptic technique ➢ Chloride (Cl) IN NaOCl: used as disinfecting agents in many
b. Dry/Hot Air Oven laboratory and hospitals spaces, surfaces, and also in treating
▪ used in the sterilization of heat resistant materials water for portability Iodine (I2) in Betadine used as a household
Effective indication: 160-180C for 1.5 to 2 hours antiseptics and surgical antiseptics
Biological indicator: Bacillus subtilis var. niger ➢ Sodium hydrochloride commonly used in household
c. Incineration 3. Heavy Metals
▪ burns materials into ashes; used in the disposable of ➢ MOA: Denaturation of enzymes and other essential bacterial
biological wastes proteins
Effective indication: 870-980C for 2 seconds ➢ Mercury (Hg): active ingredient or merthiolate but this is already
banned in the market due to its known toxicity
3. Ionizing Radiation ➢ Cooper (Cu): CuSO4 crystals are used as algaecide in
➢ works by alkylation of nucleic acid of bacteria using high energy swimming pools and aquarium
short wavelength deep penetrating gamma rays; used for heat ➢ Silver (Ag): 1% AgNO3 – used as prophylactic agent in Crede’s
sensitive materials Prophylaxis in suspected cases of Ophthalmia neonatorum
Biological indicator: Bacillus pumilis (caused by Neisseria gonorrhoeae) (replaced with erythromycin)
4. Quaternary Ammonium Compounds (QUATS)
4. Filtration ➢ Gram negative bacteria are resistant with QUATS
➢ Based on membrane gradient by differences in particle size ➢ MOA: enzyme inhibition, protein denaturation, and disruption of
➢ Used for the sterilization of heat sensitive materials plasma membrane
a. Zephiran: Benzalkonium chloride
a. Water/ Liquid Solutions/ Antibiotics/ Vaccines b. Cepacol: Cetylpyridium chloride
▪ usually uses a thin membrane filter of cellulose acetate with 5. Phenol/Phenolic Compounds/Bisphenols
different pore size depending on the intended purpose: ➢ Germicidal soaps
▪ 0.45 – 0.80um ➢ MOA: Plasma membrane destruction and enzyme denaturation
• most bacteria, yeasts, and molds are retained but may Table 4-3 Chemical Agents Commonly used as Disinfectants and Antiseptics
allow passage of Pseudomonas- like organisms Type Agent Action Applications and
▪ 0.22um Precautions
• used to filter Pseudomonas- like organisms; used for Alcohols Ethanol, Denature proteins; Skin antiseptics
critical sterilization of parenteral solutions (50%-70%) isopropanol, make lipids soluble
benzyl alcohol
▪ 0.01um
Aldehydes Formaldehyde React with NH2+, -SH Disinfectants; kill
• able to retain small viruses (in (8%), and -COOH groups endospores; toxic to
solution) glutaraldehyde humans
b. Air: High Efficiency Particulate Air Filter (HEPA) (2%)
▪ Has a pore size of 0.3 μm; usually used in Biological Safety Halogens Tincture of iodine Inactive proteins Skin disinfectants
Cabinet (BSC) and rooms of immunocompromised patients (2% in 70%
alcohol) Reacts with water to
Chlorine and form hypochlorous acid Used to disinfect
B. Chemical Methods
chlorine (HClO); oxidizing agent drinking water;
1. Peracetic Acid compounds surface disinfectants
➢ for surgical instruments Heavy Silver nitrate Precipitates proteins Eye drop (1%
2. Formaldehyde Vapor/ Vapor Phase H2O2 Metals (AgNO3) Reacts with -SH groups; solution)
➢ for HEPA filters and large spaces Mercuric chloride lyses cell membrane Disinfectant; toxic at
3. Glutaraldehyde (HgCl2) high concentrations
➢ for medical instruments (e.g., bronchoscopes, etc.) Detergents Quaternary Disrupt cell membranes Skin antiseptics;
ammonium disinfectants
4. Ethylene Oxide (ETO) Gas compounds
➢ The recommended concentration is 450 to 700mg of ethylene Phenolics Phenol, carbolic Denature proteins; Disinfectants at high
oxide per liter of chamber space at 55C to 60C for 2 hours acid, Lysol, disrupt cell membranes concentrations; used
➢ This method is also used extensively by the manufacturing hexachlorophen in soaps at low
industry for the sterilization of low-cost thermoplastic products e concentration
➢ For large spaces and spaceships and other heat sensitive Gases Ethylene oxide Alkylating agent Sterilization of heat-
materials sensitive objects
Biological indicator: Bacillus subtilis var. globijii
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[BACT211] 1.05 Sterilization and Disinfection I Prof. Rochelle D. Darlucio, RMT, MPH

References:
• Study Guide on Diagnostic Bacteriology by
Mr. Nathaniel Ranon
• Textbook of Diagnostic Microbiology (5th
edition) by Connie Mahon
• Powerpoint Presentation and Lecture Notes
of Prof. Rochelle Darlucio

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OLFU Specimen Collection and Processing LEC 2020-2021

2nd SEM
College of Medical CLINICAL BACTERIOLOGY 6 BACT211
LEC
Laboratory Science Transcriber: Riyoma Surell MIDTERMS
Date: March 16, 2021 TRANS 6
Batch 2023

III. SPECIMEN COLLECTION GUIDELINES

Outline
At the end of the session, the student must be able to learn: Specimen Patient Preparation Container/
I. Basic Principles of Specimen Collection
Maximum Quantity
II. Collection Procedures
III. Specimen Collection Guidelines Blood culture Disinfect skin with alcohol Blood culture media set
IV. Patient-Collected Specimens and iodine or chlorhexidine (aerobic and anaerobic
a. Urine bottles) or vacutainer
b. Sputum tube with SPS/adults
c. Stool 20mL per set; children
V. Labeling and Requisitions 5-10mL per set
VI. Preservation, Storage and Transport Body fluids (abdominal, Disinfect skin before needle Sterile, screw-cap tube
a. Specimen Storage amniotic, ascites, bile, aspiration or anaerobic transport
b. Preservatives joint, pericardial, pleural) system/ >1mL
c. Anticoagulants Catheter tips, IV (Foley Disinfect skin before Sterile, screw-cap
VII. Specimen Receipt and Processing catheters not cultured) removal container
a. Specimen Priority Cerebrospinal fluid Disinfect skin before Sterile, screw-cap tube/
VIII. Unacceptable Specimens and Rejection aspiration bacteria >1mL, fungi
>2mL, AFB >2mL, virus
>1mL
I. BASIC PRINCIPLES OF SPECIMEN COLLECTION Ear
Inner Clean ear canal with mild Sterile, screw-cap tube
❖ If possible, collect the specimen in the acute phase of the infection soap, aspirate fluid with or anerobic transport
and before antibiotics are administered needle if eardrum intact; system
❖ Select the correct anatomic site for collection of the specimen use swab if eardrum
ruptured
❖ Collect the specimen using the proper technique and supplies with
Outer Remove debris or crust Swab transport system
minimal contamination from normal biota (normal flora) from ear canal with saline-
❖ Collect the appropriate quantity of specimen moistened swab; rotate
❖ Package the specimen in a container or transport medium designed swab in outer canal
to maintain the viability of the organisms and avoid hazards that result Eye
from leakage Conjunctiva Sample both eyes; use Swab transport system
❖ Label the specimen accurately with the specific anatomic site and the separate swabs moistened
patient information: with sterile saline
➢ patient’s name Corneal Scrapings Instill local anesthetic, Agar available at
scrape with sterile spatula bedside
➢ unique identification number and inoculate directly to
➢ date and time of collection agar
❖ Transport the specimen to the laboratory promptly or make provisions Feces Collect directly into Clean, leakproof
to store the specimen in an environment that will not degrade the container, avoid container or enteric
suspected organism(s) contamination with urine transport system
❖ Notify the laboratory in advance if unusual pathogens or agents of Fungal Scrapings Wipe nails or skin with Clean, screw-cap
bioterrorism are suspected Hair/nails/skin alcohol container
❖ The laboratory can make accurate and useful determinations only if Hair: 10-12 hairs with shaft
intact
the specimen has been collected properly Nails: clip affected area
❖ The specimen to be analyze are likely to contain living organism and Skin: scrape skin at outer
the goal of the specimen collector must be to maintain the viability of edge of lesion
this organisms with minimal contamination Genitalia
Cervix/ Vagina Remove mucus before Swab transport system
II. COLLECTION PROCEDURES collection; do not use or JEMBEC transport
lubricant or speculum; swab system
endocervical canal or
❖ Instructions should be written so that specimens collected by the vaginal mucosa
patients are handled properly Urethra Flexible swab inserted 2- Swab transport system
❖ Most urine or stool collection kits contains instructions in several 4cm into urethra for 2-3s or or JEMBEC transport
languages collect discharge system
❖ Specimens for microbiology cultures should be collected in sterile Lesion/wound/abscess Wipe are with sterile saline
containers except for stool specimens, which can be collected in or alcohol
Superficial Swab along outer edge Swab transport system
clean, leakproof containers
Deep Aspirate with needle and Anaerobic transport
❖ Swabs are not recommended for collection and generally they are syringe system
poor specimens if tissue or needle aspirates can be obtained because Respiratory Tract:
they do not provide sufficient quantity, they are easily contaminated Lower Bronchial
and they can become dried out leading to a lost of microorganisms Specimens
❖ Swabs are appropriate for specimens from the upper respiratory tract, Sputum Rinse mouth or gargle with Sterile, screw-cap
external ear, eye, and genital tract water, instruct to cough container
❖ Cotton tip swabs tend to have excessive fatty acids that maybe toxic deeply into container
to certain bacteria Respiratory Tract:
Upper
❖ The tips of swabs may contain cotton, Dacron, rayon or calcium
Nasal Insert premoistened swab Swab transport system
alginate with sterile saline 1 inch into or direct inoculation to
❖ Dacron or rayon polyester swabs have a wide range of uses nares media
❖ Before the specimen is collected, the area should be cleansed to Nasopharynx Insert flexible swab through Swab transport system
eliminate as much of the commensal flora (normal flora) as possible nose into posterior
❖ The specimen should be collected by needle aspiration whenever nasopharynx, rotate for 5
possible, rather than by swab from the advancing margin of the lesion seconds
❖ Aspirated material should be placed into a sterile tube or transport vial Throat Swab posterior pharynx, Anerobic transport
tonsils and inflamed areas system or sterile screw-
and not “squirted” onto a swab
cap container

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[BACT211] TRANS 6: Specimen Collection and Processing I Prof. Rochelle D. Darlucio, RMT, MPH
Tissue Disinfect skin; do not allow Anaerobic transport
tissue to dry out; if system or sterile screw- V. LABELING AND REQUISITIONS
necessary, moisten with cap container
sterile saline
Urine ❖ Proper identification of each specimen includes a label firmly attached
Clean-Catch Clean external genitalia; Sterile, screw-cap to the container with the following information:
Midstream begin voiding; after several container or urine ➢ Name
mL have passed. Collect transport kit/ 2-3mL ➢ Identification number
midstream without stopping ➢ Room number
flow of urine
➢ Physician
Catheter Clean urethral area, insert Sterile, screw-cap
catheter, and allow first container or urine
➢ Culture site
15mL to pass; collect transport kit ➢ Date of collection
remainder ➢ Time of collection
Indwelling Catheter Disinfect catheter collection Sterile, screw-cap ❖ The requisition form should provide the following information:
port, aspirate 5-10mL with container or urine ➢ Patient’s name
needle and syringe transport kit ➢ Patient’ age (or date of birth) and gender
Suprapubic Aspirate Disinfect skin, aspirate with Sterile, screw-cap ➢ Patient’s room number or location
needle and syringe through container or anerobic ➢ Physician’s name, address, and phone number
abdominal wall into full transport system
bladder
➢ Specific anatomic site (where the specimen has been collected)
➢ Date and time of specimen collection
➢ Clinical diagnosis or relevant patient history
IV. PATIENT-COLLECTED SPECIMENS ➢ Antimicrobial agents (if patient is receiving any)
➢ Name of individual transcribing orders
A. Urine
VI. PRESERVATION, STORAGE AND TRANSPORT
❖ Clean-Catch Midstream Urine Specimen
➢ The patients are asked to void without collecting the first portion A. Specimen Storage
of the urine flow instead to collect the middle portion
➢ The first portion of the urine flow, washes contaminants from the
urethra and the midstream or the middle portion is more ➢ If specimens cannot be processed as soon as they are receive
representative of the urinary bladder they must be stored
➢ Person who collects catheterized specimen should also follow ➢ Specimen should be transported to the laboratory ideally within
this technique to eliminate the organism carried up the urethra 30 minutes of collection, preferably within 2 hours
during catheterization ➢ Urine, stool, sputum, bronchial secretions, swabs (not for
❖ First morning specimen is preferred because it provides a more anaerobes), foreign devices such as catheters, and viral
concentrated samples specimens can be maintained at refrigerator temperature (4C) for
❖ The patient collects this specimen following cleansing of the external 24 hours
genitalia to reduce the presence of the indigenous flora or the normal ➢ Fecal specimens submitted in preservatives can be maintained
flora at room temperature
➢ If cerebrospinal fluid is not processed immediately, it can be
stored in a 35C incubator for 6 hours
B. Sputum
Specimen Storage Guidelines
❖ Are often collected for the diagnosis of bacterial pneumonia
Refrigerate Room Temperature
❖ First early morning specimen is preferred
❖ Lower respiratory tract specimens are among the most difficult Catheter tips (IV) Abscess, lesion, wound
specimen to collect adequately because they are contaminated with CSF for viruses Body fluids
oropharyngeal flora Ear: outer CSF for bacteria
❖ Patients should rinse their mouth with water and expectorate with the Feces (unpreserved) Ear: inner
aid of a deep cough directly into a sterile container (expectorated Feces for Clostridium difficile Feces (preserved)
sputum) toxin (up to 3 days; >3 days
❖ Respiratory therapy technicians may also assist the patients who are store at -70C)
unable to expectorate a respiratory specimen. This specimen can be Sputum Genital
collected through aerosol induction in which the patient breath Urine (unpreserved) Nasal, N/P, throat
aerosolized droplets of a solution that stimulates cough reflex Tissue
(induced sputum) Urine (preserved)
❖ Once sputum specimen is submitted to microbiology, the laboratory CSF, Cerebrospinal fluid, IV, intravenous; NIP, nasopharynx
should be informed whether the specimen was expectorated or
induced B. Preservatives
❖ Patients with dentures should remove the dentures first ❖ Common preservatives:
❖ A single specimen should be adequate for detection of bacterial lower ➢ Boric Acid for urine
respiratory tract infection ▪ Used to maintain appropriate colony count
❖ If fungal or mycobacterial infections are suspected, three separate ➢ Buffered Formalin for stool (ova and parasite)
early morning specimens are appropriate ▪ To maintain the integrity of the trophozoite and cyst
❖ Stool specimens for bacterial culture that are not transported
C. Stool immediately to the laboratory can be refrigerated; if the delay is longer
than 2 hours, the specimen can be added to Cary-Blair transport
❖ Specimen of choice for the detection of gastrointestinal pathogens media
❖ A rectal swab can be submitted for bacterial culture as long as fecal ❖ Transport or Holding Media
material is visible on the swab ➢ Maintain the viability of microorganisms present in the specimen
❖ Patients should also be instructed to defecate directly into the without supporting the growth of any organisms
collection devices ➢ This maintains the organisms in a state of suspended animation
❖ Specimen should never be taken from the toilet and should not be so that no organism overgrows another or dice out
contaminated with urine ➢ Commonly used transport media:
❖ A single specimen that has yielded a negative result is not usually ▪ Stuart’s & Amie’s
sufficient to exclude bacteria or parasites • Charcoal is added to these media to absorb fatty acids
❖ If a bacterial infection is suspected, three specimens should be present in the specimen that could kill fastidious
collected – one per day for 3 days (fragile) organisms such as Neisseria gonorrheae &
Bordetella pertussis
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[BACT211] TRANS 6: Specimen Collection and Processing I Prof. Rochelle D. Darlucio, RMT, MPH
C. Anticoagulants ❖ More than one specimen from the same source was submitted from
the same patient on the same day; blood cultures are an exception
➢ Used to prevent clotting of specimens such as blood, bone ❖ One swab was submitted with multiple requests for various organisms
marrow and synovial fluid ❖ Gram stain of expectorated sputum reveals fewer than 25 white blood
➢ The type and concentration of anticoagulant is very important cells (WBCs) and more than 10 epithelial cells per low-power field
because many organisms are inhibited by some of these and mixed bacterial flora
chemicals ❖ In a microbiology laboratory, take note:
➢ Sodium polyanethol sulfonate (SPS) at a concentration of ➢ Specimen collection
0.025% (w/v) is usually used because Neisseria spp. and some ▪ Proper collection, preservation and storage of the specimen
anaerobic bacteria are particularly sensitive to higher ➢ Macroscopic observation
concentrations ▪ This will give you the gross appearance and the physical
➢ Heparin for viral cultures (but it inhibits Gram + bacteria and appearance of the specimen that may provide useful
yeast) information to both the microbiologists and physician
➢ Citrate, EDTA and Other Anticoagulants can’t be used for ➢ Direct microscopic observation
microbiology because their efficacy has not been demonstrated ▪ It can be used to determine the quality of the specimen
for most organisms ▪ It can give the microbiology technologist and the physician
an indication of the infectious process involved
VII. SPECIMEN RECEIPT AND PROCESSING ▪ The routine culture workup can be guided by the results of
the smear
▪ It can dictate the need for non-routine and additional testing
A. Specimen Priority
➢ Primary inoculation
▪ This involved the type of culture media, and the culture
Levels of Specimen Prioritization media to be used
Level Description Specimens ➢ Isolation techniques
1 Critical/ invasive Amniotic fluid ▪ To yield a semi-quantitative estimate of the growth
(represent potentially life- Blood
➢ Incubation
threatening illnesses, Brain
invasive source, require Cerebrospinal fluid ▪ Incubation conditions: temperature and environmental
immediate processing) Heart valves atmosphere and they are determined by the type of
Pericardial fluid specimen and the pathogens that may be detected
2 Unpreserved Body fluids (not listed for level ▪ Most bacterial cultures are incubated 35-37C
(quickly degrade or 1)
overgrowth contaminating Bone
flora, provide optimal Drainage from wounds
growth requirement for Feces
the fastidious organisms Sputum
found in these specimens) Tissue

3 Quantitation required Catheter tip

(delay processing may Urine
adversely affect the Tissue for quantitation
accuracy of the
quantitation)
4 Preserved Feces in preservative
Urine in preservative
Swabs in holding medium
(aerobic and anaerobic)

VIII. UNACCEPTABLE SPECIMENS AND REJECTION

➢ Upon the receipt in the laboratory the specimen needs to be

examined to ensure that it has been properly selected, collected
and transported
➢ This is very important because performing test on specimens that
are of poor quality would yield misleading information that might
result in misdiagnosis and inappropriate therapy
➢ It is also important to always talk to the requested physician or
another member of the health care team before discarding
unacceptable specimens. This is particularly important if the
specimen was collected using an invasive technique for example
is the surgical biopsy and the collection of a new specimen would
be difficult or impossible
❖ The information on the requisition does not match the information on
the specimen label. If the patient’s name or source does not match,
the specimen should be collected again
❖ There is no patient identification on specimen container
❖ The specimen is not submitted in the appropriate transport container
or the container is leaking
❖ The quantity of the specimen is inadequate to perform all tests
requested
❖ The specimen transport time is more than 2 hours and the specimen
has not been preserved
❖ The specimen is received in a fixative such as formalin; stools for O &
P examinations are an exception
❖ An anaerobic culture is requested on a specimen in which anaerobes
are indigenous
❖ Microbiology processing of a particular specimen results in
questionable data (e.g., Foley catheter tip)
❖ The specimen is dried up

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OLFU Staphylococci and Micrococci LEC 2020-2021

2nd SEM
College of Medical CLINICAL BACTERIOLOGY 6 BACT211
LEC
Laboratory Science Transcriber: Riyoma Surell MIDTERMS
Date: March 16, 2021 TRANS 7
Batch 2023

Outline II. CLINICALLY SIGNIFICANT SPECIES

At the end of the session, the student must be able to learn:
I. Staphylococci and Micrococci
A. Staphylococcus aureus
a. Staphylococci
b. Micrococci
II. Clinically Significant Species ❖ Epidemiology: (mahon)
a. Staphylococcus aureus ➢ Primary reservoir for staphylococci is the human nares with
a. Virulence Factors colonization also occurring in the vagina, pharynx, axillae and
b. Infections caused by S. aureus other skin surfaces
b. Staphylococcus epidermidis ➢ Transmission of S. aureus may occur by direct contact with
c. Staphylococcus saprophyticus
unwashed, contaminated hands and by contact with inanimate
d. Staphylococcus lugdunensis
e. Other Coagulase-Negative Staphylococci objects (fomites)
III. Laboratory Diagnosis ❖ Definition:
a. Specimen Collection and Handling ➢ Most clinically significant species
b. Microscopic Examination ➢ Causes various cutaneous infections and purulent abscesses
c. Isolation (mahon)
d. Identification Methods ➢ Responsible for numerous infections (relatively mild to life-
a. Catalase Test threatening infections)
b. Coagulase Test ➢ An important cause of nosocomial infection
c. Modified Oxidase Test ➢ 3 Types of Nasal Carrier States associated with the
d. Oxidation-Fermentation Test
colonization of S. aureus
e. Pyrrolidonyl Arylamidase
f. Vogue-Proskauer Test ▪ Persistent Carriers harbor single strain for an extended
period of time
▪ Intermittent Carriers harbor different strains overtime
▪ Noncarriers individuals do not harbor any organism
I. STAPHYLOCOCCI AND MICROCOCCI
A. Virulence Factors
A. Staphylococci
A. Enterotoxins
▪ Heat-stable exotoxins that are able to exhibit symptoms
➢ Common isolates in the clinical laboratory and are responsible such as vomiting and diarrhea. Resistant to hydrolysis by
for several suppurative infections (mahon) the gastric and intestinal enzymes
➢ Normal inhabitants of the skin and mucous membranes of ▪ Stable at 100C for 30 minutes
humans and other animals (mahon)
• Reheating contaminated food does not prevent the
❖ General Characteristics
disease
➢ Under the family Staphylococcaceae
▪ Staphylococcal found poisoning is most commonly used by
➢ Gram positive cocci, catalase producing (upon the use of 3%
enterotoxins A, B and D
hydrogen peroxide, exhibit bubbles), coagulase positive
▪ Enterotoxins B and C and sometimes G and I are
➢ “staphle” – brunches of grapes (gram positive cocci in clusters)
associated with Toxic Shock Syndrome (TSS)
➢ Nonmotile, non-spore-forming, and aerobic or facultatively
▪ Enterotoxin B has been linked to staphylococcal
anaerobic except for S. saccharolyticus (obligate anaerobe)
pseudomembranous enterocolitis
➢ Spherical cells mainly 0.5 – 1.5micrometers in diameter that
▪ Superantigens (TSST-1) and have the ability to interact with
appears in singly, in pairs or clusters
many T cells, activating an aggressive, over reactive
➢ Colonies produced after 18 to 24 hours of incubation:
immune response
▪ Medium sized (4 to 8mm)
B. Toxic Shock Syndrome Toxin-1
▪ Cream-colored, white or rarely light gold
▪ Previously referred to as enterotoxin F
▪ “buttery-looking”
▪ At low concentrations, TSST-1 causes leakage by
▪ Some species are B-hemolytic
endothelial cells, and it is cytotoxic to these cells at higher
➢ Small Colony Variants (SCVs) (mahon)
concentrations
▪ Rare strains of staphylococci are fastidious, requiring a
▪ TSST-1 is absorbed through the vaginal mucosa leading to
carbon dioxide, hemin or menadione for growth
the systemic effects seen in TSS associated with tampon
▪ Grown on media containing blood, forming colonies about
use
one tenth the size of wild-type strains even after 48 hours
• Associated with approximately 50% of non-
or more of incubation
menstruating associated TSS cases
❖ Staphylococcal species can be initially differentiated by the
C. Exfoliative Toxin
coagulase test (mahon)
▪ Also known as epidermolytic toxin
➢ Positive result: clot formed in a tube containing plasma due to
▪ Two Types of Toxin
staphylocoagulase
B. Micrococci • Exfoliative toxin A
• Exfoliative toxin B
▪ It causes the epidermal layer of the skin to slough off and is
❖ General Characteristics
known to cause staphylococcal Scalded Skin Syndrome
➢ Under the family Micrococcaceae
(SSS), sometimes referred to as Ritter Disease
➢ Catalase-producing, coagulase-negative, gram-positive cocci
• Blister formation and sloughing at the top layer of the
➢ Found in the environment and as members of the indigenous skin
skin
microbiota
➢ Some micrococci have a tendency to produce a yellow pigment • Most common in infant and newborns
• Most reported cases less than 5 years old
Other gram-positive cocci that are occasionally recovered ▪ This toxin has also been implicated in bullous impetigo
with staphylococci: (formation of large blisters)
▪ Rothia mucilaginosa
▪ Aerococcus
▪ Alloiococcus otitis (recovered from the
human middle ear fluid)
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[BACT211] TRANS 7: Staphylococci and Micrococci I Prof. Rochelle D. Darlucio, RMT, MPH
D. Cytolytic Toxins
▪ Produces other extracellular proteins that affect red blood
cells and leukocytes (mahon)
▪ Hemolysins and Leukocidins
• a-hemolysin, in addition to lysing erythrocytes, can
damage platelets and macrophages and cause severe
damage
• b-hemolysin, (sphingomyelinase C) acts on ❖ Scalded Skin Syndrome
sphingomyelin in the plasma membrane of ➢ Caused by staphylococcal exfoliative or epidermolytic toxin
erythrocytes and is also called the “hotcold” lysin produced by S. aureus which is probably present at a lesion
 exhibited in the Christie, Atkins and Munch- distant from the site of exfoliation
Petersen (CAMP) test performed in the laboratory ➢ Bullous exfoliative dermatitis that occurs primarily in newborns
to identify group B streptococci (mahon) and previously healthy young children
• g-hemolysin, although found in a higher percentage ➢ The severity of the disease varies from a localized skin lesion in
of S. aureus stains and some CoNS, is considered less the form of a few blisters, pemphigus neonatorum, to a more
toxic to cells than either a-hemolysin or b-hemolysin extensive generalized condition affecting 90% of the body,
• Panton-Valentine leukocidin (PVL) an exotoxin known as ritter disease
lethal to polymorphonuclear leukocytes ➢ Cases of SSS in adults occur most commonly in patients with
 Contributing to the invasiveness o the organism chronic renal failure and in patients with compromised immune
by suppressing phagocytosis and has been systems
associated with severe cutaneous infections and ➢ The toxin is metabolized and secreted by the kidneys. It is
necrotizing pneumonia believed an immature or compromised renal or immune system
E. Enzymes (coagulase, protease, hyaluronidase, and lipase) contributes to why the incidence of SSS is higher among children
▪ Staphylocoagulase is produced mainly by S. aureus younger than 5 years and among adults
• Although the exact role of coagulase in pathogenicity ➢ Toxic Epidermal Necrolysis (TEN)
is uncertain, it is still considered a virulence marker ▪ Associated with drug reactions and has been linked to
▪ Hyaluronidase, hydrolyzes hyaluronic acid present in the antimicrobials and anticonvulsives
intracellular ground substance that makes up connective ▪ Can be resolved by the administration of steroids early in
tissues, permitting the spread of bacteria during infection the initial stages of presentation, whereas steroids
▪ Lipases are produced by both coagulase-positive and aggravate SSS
CoNS
• Lipases act on lipids present on the surface of the skin,
particularly fats and oil secreted by the sebaceous
glands
F. Protein A
▪ One of the several components that have been identified in
the cell wall of S. aureus
▪ Has the ability to bind the Fc portion of immunoglobulin G
(IgG)
▪ Binding IgG in this manner can block phagocytosis and
negate the protective effect of IgG

B. Infections Caused by Staphylococcus aureus

❖ Skin and Wound Infections ❖ Toxic Shock Syndrome

➢ Abscess is filled with pus and surrounded by necrotic tissues and ➢ Rare but potentially fatal, multisystem disease characterized by
damaged leukocytes a sudden onset of fever, chills, vomiting, diarrhea, muscle aches,
▪ Folliculitis and rash, which can quickly progress to hypotension and shock
• Is a relatively mild inflammation of a hair follicle or oil ➢ Associated with use of highly absorbent tampons
gland; the infected area is raised and red ➢ The initial clinical presentation of TSS consists of high
▪ Furuncles (boils) temperature, rash and signs of dehydration, particularly if the
• Can be an extension of folliculitis, are large, raised, patient has had watery diarrhea and vomiting for several days
(mahon)
superficial abscesses
▪ Carbuncles
• Occur when larger, more invasive lesions develop
from multiple furuncles, which can progress into
deeper tissues
▪ Bullous Impetigo
• Larger and surrounded by a small zone of erythema. A
highly contagious infection that is easily spread by
direct contact, fomites or autoinoculation
➢ These opportunistic infections usually occur as a result of
previous skin injuries such as cuts, burns and surgical incisions
➢ It can be suppurative or toxin mediated
❖ Toxic Epidermal Necrolysis
➢ A life-threatening disorder with erythema, necrosis and bullous
detachment of the epidermis resulting in possible sepsis and or
death
➢ Cause is unknown but symptoms appear to be due to a
hypersensitivity reaction
➢ A clinical manifestation with multiple causes; it is most commonly
drug induced, but some cases have been linked to infections and
vaccines
➢ The cause is unknown, but symptoms appear to be due to a
hypersensitivity reaction

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[BACT211] TRANS 7: Staphylococci and Micrococci I Prof. Rochelle D. Darlucio, RMT, MPH
C. Staphylococcus saprophyticus

➢ Associated with UTIs in young women

➢ Second most common cause after E. coli of uncomplicated
cystitis in this population
➢ This species adheres more effectively to the epithelial cells lining
the urogenital tract than other Coagulase-negative staphylococci
(CoNS)
▪ Includes S. epidermidis, S. saprophyticus, S. haemolyticus,
S. lugdunensis
➢ It is rarely found on other mucous membranes or skin surfaces

D. Staphylococcus lugdunensis

➢ Can cause both community-associated and hospital acquired

infections
➢ This organism can be more virulent and can clinically mimic S.
aureus infections
➢ Known to contain the gene mecA that encodes oxacillin
resistance
➢ It is an important pathogen in infective endocarditis, septicemia,
meningitis, skin and soft tissue infections, UTIs and septic shock

Other Coagulase-Negative Staphylococci

❖ Food Poisoning
➢ S. aureus enterotoxins most commonly A (78%), D (38%), B ➢ A wide range of infections have been associated with these
(10%) have been associated with gastrointestinal disturbances organisms including endocarditis, septicemia and wound
➢ A type of intoxication resulting from ingestion of a toxin formed infections
outside the body ➢ Other species of coagulase negative staphylococci are found as
➢ Foods that are incriminated in Staphylococcal food poisoning normal biota in humans and animals although they are not
include: commonly seen as pathogens, their role in some infections is well
• Salads containing mayonnaise and eggs established and they cannot be automatically discarded as
• Meet or meet products contaminants
• Poultry, egg products, bakery products with cream ❖ S. warneri
fillings ❖ S. capitis
• Sandwich fillings and dairy products ❖ S. simulans
➢ Disease occurs when food becomes contaminated with ❖ S. hominis
enterotoxin-producing strains of S. aureus by improper handling ❖ S. schleiferi
and is then improperly stored, which allows growth of the bacteria
and resulting toxin production III. LABORATORY DIAGNOSIS
➢ Foods kept at room temperature are specially susceptible to
higher levels of toxin production when contaminated with toxin A. Specimen Collection and Handling
producing staphylococci and they are more commonly
associated with food poisoning ❖ Clinical materials collected from infected sites should be transported
to the laboratory without delay to prevent drying, maintain the proper
❖ Other Infections environment, and minimize the growth of contaminating organisms
➢ Staphylococcal pneumonia ❖ Specimens should be taken from the site of infection after appropriate
▪ Has been known to occur secondary to influenza virus cleansing of the surrounding area to avoid contamination by the skin
infection microbiota
▪ Characterized by multiple abscesses and focal lesions in ❖ Normal skin biota contamination can be further reduced by the
the pulmonary parenchyma physician submitting secretion aspirates, tissue samples, or blood
➢ Staphylococcal bacteremia culture specimens instead of swabs
▪ Leading to secondary pneumonia and endocarditis has
been observed among intravenous drug users B. Microscopic Examination
➢ Staphylococcal osteomyelitis
▪ Occurs as a manifestation secondary to bacteremia
▪ The infection develops when the organism is present in a
wound or other focus of infection and gains entrance into
the blood

B. Staphylococcus epidermidis

➢ It is considered as normal skin biota but is a common source of ❖ Staphylococci appears as gram positive cocci in clusters
hospital acquired infections and often a contaminant in
improperly collected blood culture specimens C. Isolation
➢ Infections are predominantly hospital acquired
➢ Some predisposing factors for HAI are instrumentation ❖ General Culture Media
procedures such as catheterization, medical implantation and ➢ Sheep blood agar (SBA)
immunosuppressive therapy ❖ Selective Culture Media
➢ A common cause of health care-acquired UTIs ➢ Mannitol salt agar (MSA)
➢ Prosthetic valve endocarditis is most commonly caused by S. ▪ Contains high concentration of
epidermidis salt atleast 10%
▪ Also contains sugar mannitol
▪ pH Indicator: Phenol red
▪ Staphylococcus aureus ferments mannitol and produces a
yellow halo on MSA as a result of acid production altering
the pH

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[BACT211] TRANS 7: Staphylococci and Micrococci I Prof. Rochelle D. Darlucio, RMT, MPH
▪ Not typically used in the clinical identification, it may still be ❖ 2 Types of Coagulase Test
used to purify staphylococci from contaminating organism ➢ Slide coagulase test
for further characterization ▪ Used for the determination of bound coagulase or clumping
❖ Columbia colistin-nalidixic acid agar (CNA) factor
❖ Phenylethyl alcohol (PEA) agar ➢ Tube coagulase test
❖ CHROMagar ▪ To detect the free coagulase
➢ is a selective and differential media for the identification of ❖ Reagent:
methicillin resistant staphylococcus aureus ➢ Rabbit’s plasma
❖ Staphylococci produce round, smooth, white, creamy colonies on SBA ❖ False Positive Reactions:
after 18 to 24 hours of incubation at 35C to 37C ➢ Can be done with the used of citrate
Cultural Characteristics ➢ Colonies from high salt media concentrations
S. aureus Can produce hemolytic zones around the ➢ Some strains are able to produce fibrinolysin which dissolves the
colonies and may rarely exhibit pigment clot after 4 hours of incubation at 35C and it may appear to be
production (yellow) with extended incubation negative if allowed to incubate longer than 4 hours
S. epidermidis Small to medium sized, nonhemolytic gray to ➢ Citrate utilizing organisms may yield false positive results,
white colonies
Some are weakly hemolytic
plasma containing EDTA rather than citrate should be used
S. saprophyticus Forms slightly larger colonies with about 50%
of the strains producing a yellow pigment
S. haemolyticus Produced medium sized colonies with
moderate or weak hemolysis and variable
pigment production
S. lugdunensis Often hemolytic and medium sized, although
small colony variants can occur

D. Identification Methods

A. Catalase Test
❖ This test differentiates catalase-positive micrococcal and Coagulase-Positive Staphylococci and their Clinical Source and
staphylococcal species from catalase-negative streptococcal species Significance
❖ Principle: the enzyme, catalase, is capable of converting hydrogen Staphylococcus species Source
peroxide to water and oxygen. The presence of enzyme in bacterial S. aureus Humana, Animal
isolate causes rapid elaboration of bubbles S. aureus subsp. anaerobius Animal
❖ Reagent: 3% hydrogen peroxide
S. hyicus Animala
❖ Results:
S. agnetis Animal
➢ Positive: rapid bubbling formation
S. intermedius Animala
➢ Negative: no rapid bubble formation
S. pseudointermedius Animala, human
❖ False positive results S. schleiferi subsp. coagulans Animala
➢ Are caused by some organism such as enterococci that produces S. delphini Animal
a peroxidase (slowly catalyses the breakdown of hydrogen S. lutrae Animal
a
peroxide) Common in human or veterinary disease
➢ From the sample contaminated from the blood agar and the used C. Modified Oxidase Test
of platinum wire
❖ Can be used to rapidly differentiate staphylococci from micrococci
❖ Most staphylococci are negative, whereas micrococci are positive
❖ Positive result is color purple

B. Coagulase Test
D. Oxidation-Fermentation Test
❖ The test is used to differentiate Staphylococcus aureus (positive) from
coagulase-negative staphylococci (negative)
❖ Another staphylocoagulase producing staphylococci aside from S. ❖ Staphylococci ferment glucose, whereas micrococci fail to produce
aureus: acid under anaerobic conditions
➢ S. intermedius ❖ Staphylococci that fail to grow or produce acid anaerobically:
➢ S. pseudointermedius ➢ S. saprophyticus
➢ S. delphini ➢ S. auricularis
➢ S. lutrae ➢ S. hominis
➢ Some strains of staphylococcus hyicus ➢ S. xylosus
❖ Principle: ➢ S. cohnii
▪ Bound coagulase or “clumping factor” is bound to the
bacterial cell wall and reacts directly with fibrinogen
• Some species or some isolates such as S.
lugdunensis, S. schleiferi can also be occasionally
mistaken for coagulase-positive staphylococci
because of the presence of clumping factors
▪ The presence of bound coagulase correlates with free
coagulase, an extracellular protein enzyme that causes the
formation of a clot when S. aureus colonies are incubated
with plasma

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[BACT211] TRANS 7: Staphylococci and Micrococci I Prof. Rochelle D. Darlucio, RMT, MPH
E. Pyrrolidonyl Arylamidase

❖ Can be used to differentiate S. aureus (negative) from S. lugdunensis,

S. intermedius, S. schleiferi (positive)
❖ The substrate, pyroglutamyl-B-naphthylamide (pyrrolidonyl-B-
naphthylamide; PYR), is hydrolyzed to pyrrolidone and B-
naphthylamine, which combines with p-
dimethylaminocinnamaldehyde to form a red compound

F. Vogue-Proskauer (VP) test

❖ Differentiates S. aureus (positive) from S. intermedius (negative)

❖ A positive result is the formation of acetoin from glucose or pyruvate
❖ Other VP positive Staphylococci:
➢ S. lugdunensis
➢ S. haemolyticus
➢ S. schleiferi

References:
I. Study Guide on Diagnostic Bacteriology by
Mr. Nathaniel Ranon
II. Textbook of Diagnostic Microbiology (5th
edition) by Connie Mahon
III. Diagnostic Microbiology (12th edition) by
Bailey and Scott
IV. Powerpoint Presentation and Lecture Notes
of Prof. Rochelle Darlucio
Differentiation between Staphylococci and
Micrococci in the Routine Laboratory
Test Staphylococci Micrococci
Modified oxidase - +
Anaerobic acid + -*
production from
glucose
Growth on - +
Furoxone-Tween
80-oil red O agar
Anaerobic acid + -
production from
glycerol in the
presence of
erythromycin
Resistance to R+ S
bacitracin (0.04
units)
Lysosome (50- R S
mg disk)
Lysostaphin test S+ R
R, resistance ; S, sensitive
*Micrococcus kristinae and Micrococcus varians are positive
+Some stains show opposite reaction

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2020-2021
Streptococcus and Enterococcus, Other LEC 2nd SEM
OLFU Catalase-Negative, Gram-Positive Cocci BACT211
College of Medical
CLINICAL BACTERIOLOGY
7 LEC
MIDTERMS
TRANS 8
Laboratory Science
Transcriber: Riyoma Surell
Batch 2023 Date: March 16, 2021

Outline
At the end of the session, the student must be able to learn:
I. General Characteristics
▪ Streptococcus and Enterococcus
▪ Cell Wall Structure of Streptococci
▪ Types of Hemolysis
II. Clinically Significant Streptococci and Streptococcus-like Organisms
• Virulence Factors
• Clinical Infections
• Laboratory Diagnosis
A. Streptococcus pyogenes
B. Streptococcus agalactiae
C. Groups C and G Streptococci
D. Streptococcus pneumoniae
E. Viridans Streptococci
F. Enterococcus
III. Streptococcus-like Organisms ❖ Types of Hemolysis
A. Abiotrophia and Granulicatella ➢ Alpha (a)
B. Aerococcus
▪ Partial lysis of RBCs around colony
C. Gemella
D. Lactococcus ▪ Greenish discoloration of area around colony
E. Leuconostoc ➢ Beta (β)
F. Pediococcus ▪ Complete lysis of RBCs around colony
IV. Laboratory Diagnosis ▪ Clear area around colony
A. Classification Schemes ➢ Nonhemolytic
V. Biochemical Tests ▪ No lysis of RBCs around colony
A. Bacitracin Susceptibility Test
▪ No change in agar
B. CAMP Test
C. Hippurate Hydrolysis Test ➢ Alpha-prime (a’) or Wide Zone
D. Pyrrolidonyl-a-Naphthylamide Hydrolis (PYR) ▪ (some isolates belonging to the viridans group)
E. Bile Esculin Hydrolysis ▪ Small area of intact RBCs around colony
F. Leucine Aminopeptidase ▪ Surrounded by a wider zone of complete hemolysis
G. Voges-Proskauer Test (VP)
H. B-D-Glucuronidase
I. Salt Tolerance Test
J. Optochin Susceptibility
K. Bile Solubility

I. GENERAL CHARACTERISTICS

❖ Streptococcus and Enterococcus

➢ Belong to the family Streptococcaceae
➢ Catalase-negative (but sometimes they exhibit weak false-
positive catalase reaction when the growth is taken from media
containing blood, owing to the peroxidase activity of
hemoglobin), gram positive cocci that are usually arranged in
pairs or chains
➢ Facultative anaerobes, should be considered aerotolerant
anaerobes
➢ Some species are capnophilic compared with other gram-
positive cocci the cells of enterococci and some streptococci
appear more elongated than spherical

II. CLINICALLY SIGNIFICANT STREPTOCOCCI AND

STREPTOCOCCUS-LIKE ORGANISMS

❖ Cell Wall Structure of Streptococci

➢ The cell wall structure of streptococci possessed a typical gram-
positive cell wall consisting of peptidoglycan and teichoic acid
➢ Most streptococci except for many of viridans group have a group
or common C carbohydrate (polysaccharide) which can be use
to identify an isolate serologically
➢ Developed by Rebecca Lancefield (1930s)
➢ Lancefield was able to divide the streptococci into serologic
groups, designated by letters
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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH
Notes: ❖ Streptolysin S
❖ S. pyogenes ➢ Oxygen stable
➢ Most important clinically lance-field group A that produces ➢ lyses leukocytes, and is nonimmunogenic hemolysin capable of
several factors that contribute to its virulence lysin erythrocytes, leukocytes and platelets in the presence of
➢ One of the aggressive pathogens encountered in the clinical room air
microbiology laboratories ❖ Hyaluronidase or Spreading Factor
➢ Among these factors are the streptolysin O and S which not only ➢ Enzyme that solubilizes the ground substance of mammalian
contribute to virulence but are also responsible for the Beta connective tissues (hyaluronic acid)
hemolytic pattern on blood agar plates as used to guide to ➢ It was postulated that the bacteria use this enzyme to separate
identify these species the tissue and spread the infection, however, no evidence to
❖ S. agalactiae support this hypothesis exists
➢ Infections usually associated with neonates and are acquired ❖ Streptococcal Pyrogenic Exotoxins
before and during the birthing process ➢ Causes a red spreading rash, referred to as scarlet fever
❖ In the most recent classification of beta hemolytic streptococci, ➢ Erythrogenic toxins
isolates from humans that belong to lancefield group C and G are ➢ The four immunologically distinct exotoxin types found in S.
subdivided into large colony and small colony forms pyogenes are:
➢ Large Colony Forms ▪ SpeA
▪ Isolates with group C and G and sometimes group A and L ▪ SpeB
antigens are classified with the pyogenic streptococci a. ▪ SpeC
▪ SpeF
A. Streptococcus pyogenes ▪ These toxins function as superantigens

❖ The infections cause by S. pyogenes maybe localized or systemic. B. Clinical Infections

Other problems may arise a result of the host-bodies response the
infection caused by these organisms ❖ Bacterial Pharyngitis
❖ Localized Infections: ➢ Pharyngitis and tonsilitis
➢ Acute pharyngitis (S. pyogenes most common bacterial cause) ➢ “strep throat”
➢ Impetigo and erysipelas ➢ Most often seen in children between 5 and 15 years of age
❖ Lancefield group A ➢ After an incubation period of 1 to 4 days an abrupt onset of illness
❖ M protein is attached to the peptidoglycan of the cell wall and extends ensues, with sore throat, malaise, fever and headache
to the cell surface ➢ The tonsils and pharynx are inflamed
❖ Colonizes the throat and skin on humans, making these sites the
primary sources of transmission

❖ Pyodermal Infections
➢ Impetigo
▪ A localized skin disease, begins as small vesicles that
progress to weeping lesions
▪ Usually seen in young children (2 to 5 years) and affect
A. Virulence Factors exposed areas of the skin
❖ M protein
➢ Essential for virulence
➢ Encoded by the genes emm.
➢ Causes the streptococcal cell to resist phagocytosis and plays a
role in adherence of the bacterial cell to mucosal cells
❖ Lipoteichoic Acid and Protein F
➢ Are adhesion molecules that mediate attachment to host
epithelial cells
➢ Lipoteichoic acid which is affixed to proteins of the bacterial
surface in concert with M proteins and fibronectin-binding protein,
secures the attachment of streptococci to the oral mucosal cells ➢ Erysipelas
❖ Hyaluronic Acid Capsules ▪ Rare infection of the skin and subcutaneous tissues
➢ Weakly immunogenic observed frequently in elderly patients
➢ The capsule prevents opsonized phagocytosis by neutrophils or ▪ Characterized by an acute spreading skin lesion that is
macrophages intensely erythematous with a plainly demarcated but
➢ The capsule also allows the bacterium to mask its antigens and irregular edge
remain unrecognized by its host
❖ Streptolysin O
➢ Responsible for hemolysis on SBA plates incubated
anaerobically
➢ Oxygen labile
➢ lyses leukocytes, platelets and other cells as well as RBCs
➢ SLO is highly immunogenic capable of lysin the same cells and
cultured cells is broken down by oxygen and will produce
hemolysis only in the absence of room air
➢ Inhibited by the cholesterol in skin lipids resulting in the absence
of the development of protective antibodies associated with skin
infections
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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH
➢ Cellulitis ❖ Poststreptococcal Sequeiae
▪ Painful, first appear as red swollen area that feels hot and ➢ Rheumatic fever and acute glomerulonephritis
tender to touch ➢ Rheumatic fever
▪ Can develop following deeper invasion by streptococci ▪ typically follows S. pyogenes pharyngitis
▪ The infection can be serious or life-threatening with ▪ It is characterized by fever and inflammation of the heart,
bacteremia or sepsis joints, blood vessels and subcutaneous tissues
▪ In patients with peripheral vascular disease or diabetes, ▪ Attacks usually begin within 1 month after infection
cellulitis may lead to gangrene ▪ The most serious result is chronic, progressive damage to
the heart valves. Repeated infections can produce further
valve damage
➢ Acute Glomerulonephritis
▪ Sometimes occurs after a cutaneous or pharyngeal
infection
▪ It is most common in children than in adults
▪ The pathogenesis appears to be immunologically mediated

C. Laboratory Diagnosis

➢ An essential step in the diagnosis of streptococcal pharyngitis is

➢ Scarlet Fever proper sampling
▪ Appears 1 to 2 days after bacterial infection. The rash ❖ Specimen Collection
disappears over the next 5 to 7 days and is followed by ➢ The tongue should be depressed and the swab rubbed over the
Butterfly rash desquamation posterior pharynx and each tonsillar area
▪ Characterized by a diffuse red rash that appears on the ➢ Transport media are not required for normal conditions
upper chest and spreads to the trunk and extremities ➢ The organism is resistant to drying and can be recovered from
swabs several hours after collection
➢ If exudate is present, it should also be touched with the swab
➢ Care should be taken to avoid the tongue and uvula
❖ Gram Staining
➢ Gram stain reveals gram-positive cocci with some short chains
❖ Culture
➢ An SBA (Sheep’s blood agar) plate is inoculated and streaked for
isolation
➢ Incubation should be at 35C either in ambient air or under
anerobic conditions
➢ The normal respiratory microbiota tends to overgrow β-hemolytic
streptococci when incubated in increased concentration of CO2
❖ Necrotizing Fasciitis ➢ On the surface of the SBA plate are small, transparent and
➢ “flesh-eating disease” smooth with a well0defined area of β-hemolysis
➢ An invasive infection characterized by rapidly progressing
inflammation and necrosis of the skin, subcutaneous fat and
fascia
➢ Occurs most frequently in individuals who have other health
problems
➢ It may be categorized as type 1, 2 or 3
▪ Type 1 NF
• A polymicrobial infection from which aerobic and
anaerobic bacteria are recovered
▪ Type 2 NF
• Consist of only gas
▪ Type 3 NF ❖ Biochemical Tests
• Gas gangrene or clostridial myonecrosis ➢ Bacitracin susceptibility or pyrrolidonyl a-napthylamide (PYR)
➢ Although uncommon, NF is a life-threatening infection hydrolysis
➢ Hippurate hydrolysis
➢ CAMP test
➢ Bile esculin test
➢ Growth in 6.5% sodium chloride (NaCl) broth

B. Streptococcus agalactiae

➢ All strains of S. agalactiae have the group B-specific antigens, an

acid-stable polysaccharide located in the cell wall
➢ The only species that expresses group B antigen

❖ Streptococcal Toxic Shock Syndrome

➢ Condition in which the entire organ system collapses, leading to
death
➢ The initial streptococcal infection is often severe (e.g.,
pharyngitis, peritonitis, cellulitis, wound infections) and the
symptoms that develop are similar to symptoms of
staphylococcal TSS
➢ The exact portal of infection is unknown for most cases of
streptococcal TSS, although minor injuries or surgical
procedures have been implicated

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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH
A. Virulence Factors C. Groups C and G Streptococci

❖ Capsule ➢ In the most recent classification of β-hemolytic streptococci,

➢ Prevents phagocytosis but is ineffective after opsonization isolates from humans that belong to Lancefield groups C and G
❖ No evidence exists that any of these products plays a role in the are subdivided into large-colony and small-colony forms
virulence of this organism ▪ Large-Colony-Forming Isolates
➢ Hemolysin • Isolates with group C and G and sometimes group A
➢ CAMP factor and L antigens are classified with the pyogenic
➢ Neuraminidase streptococci
➢ Dnase • The large colony forming β-hemolytic isolates with
➢ Hyaluronidase group A, C, G or L antigens belong to S. dysgalactiae
➢ Protease subsp. Equisimilis
▪ Small-Colony-Forming β-hemoytic
B. Clinical Infections • Isolates with group A, C, F or G-antigens belong to the
S. anginosus group which is included in the viridans
➢ Group B streptococci were known for many years as the cause streptococci
of mastitis in cattle ➢ Clinical infections by S. dysgalactiae subsp. equisimilis although
➢ GBS are the leading cause of death in infants in the United uncommon, have involved several body sites and are thought to
States, although the incidence decreased dramatically from the be common in domestic animals
1990s to 2008 ➢ The spectrum of infections resembles S. pyogenes and includes
➢ The drug of choice for treating GBS infection is penicillin, upper respiratory tract infections, skin infections, soft tissue
although GBS are less susceptible to penicillin than GAS infections, and invasive infections such as NF
➢ Some clinician recommend a combination of ampicillin and an ➢ S. equi subsp. zooepidemicus
aminoglycoside for treating GBS infection ▪ Which also express C antigen, is primarily an animal
❖ Neonatal GBS disease pathogen rarely isolated from humans
➢ Early-onset infection (<7 days old)
▪ Manifest pneumonia and sepsis D. Streptococcus pneumoniae
▪ Presence of GBS in the vagina of the mother. It is
recommended that all pregnant women be screened for ➢ Also known as pneumococcus
GBS at 35 to 37 weeks of gestation ➢ Isolated from a variety of clinical specimens
➢ Late-onset infection (at least 7 days old to about 3 months old) ➢ A member of the S. mitis group, but it is much more virulent
▪ Manifests as meningitis and sepsis than other members of the group
➢ Early onset disease accounts for about 80% of the clinical cases ➢ The cell wall contains an antigen, referred to as C substance,
in newborns and is caused by vertical transmission of the which is similar to the C carbohydrate of the various Lancefield
organism from the mother groups
➢ Colonization of the vagina and rectal area with GBS is found in
10% to 30% of pregnant women

C. Laboratory Diagnosis

❖ Specimen Collection
➢ Collecting vaginal and rectal material with swabs between 35 and
37 weeks of gestation
❖ Gram Stain
➢ Gram-positive cocci that form short chains in clinical specimens
and longer chains in culture
❖ Culture
➢ SBA- grayish white mucoid colonies surrounded by a small zone
of β-hemolysis A. Virulence Factors

➢ The characteristic of S. pneumoniae that is clearly associated

with virulence is the capsular polysaccharide
➢ Strains that have lost the ability to produce a capsule are
nonpathogenic
➢ In addition, opsonization of the capsule renders the organism a
virulent
❖ Several toxins are produced including:
➢ Hemolysin
➢ Immunoglobulin A protease
▪ Destroys IgA in the respiratory tract assisting in colonization
➢ Neuraminidase
▪ Enhance adherence to mucous membrane surfaces by
cleaving sialic acid exposing molecules enhancing
adhesion
➢ Hyaluronidase
❖ Biochemical Tests
➢ Hippurate hydrolysis B. Clinical Infections
➢ CAMP tests
➢ S. pneumoniae is a common isolate in the clinical microbiology
laboratory
➢ Pneumonia, Sinusitis and Otitis Media, Bacteremia, Septicemia,
Meningitis, Endocarditis, Septic Arthritis, Soft Tissue Infections
❖ Pneumococcal Pneumonia
➢ Organism must be present in the nasopharynx, and the individual
must be deficient in the specific circulating antibody against the
capsular type of the colonizing strain of S. pneumoniae

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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH
❖ Pneumonia
➢ Is not usually a primary infection but is rather a result of ❖ Colonial Morphology
disturbance of the normal defense barriers ➢ Round, glistening, wet, mucoid, dome-shaped appearance, a-
➢ Predisposing conditions such as alcoholism. Anesthesia, hemolytic on SBA
malnutrition and viral infections of the upper respiratory tract, can ➢ Some isolates require increase CO2 concentration for growth
lead to pneumococcal disease in the form of lobar pneumonia during primary isolation
➢ May be complicated by a pleural effusion that is usually sterile ➢ As the colonies become older, autolytic changes result in a
(empyema) collapse of each colony’s center, giving it the appearance of a
➢ An infected effusion contains many white blood cells and coin with a raised rim
pneumococci, which are visible on gram stain ❖ Biochemical Tests
❖ Meningitis ➢ Optochin susceptibility
➢ Pneumococcus causes bacterial meningitis in all age groups ➢ Bile solubility
➢ Usually follows other S. pneumonia infections such as otitis
media or pneumonia E. Viridans Streptococci
➢ The course of the disease is rapid, and the mortality rate is near
40% ➢ Constituents of the normal microbiota of the upper respiratory
❖ Bacteremia tract
➢ Often occurs during the course of a serious infection ➢ Viridans means “green” referring to the a-hemolysis many
➢ Samples for blood culture are often taken simultaneously with species exhibit
sputum or a fluid aspirate ➢ However, B-hemolytic and nonhemolytic species are also
❖ Note: classified as viridans streptococci
➢ 2 pneumococcal vaccines are available ➢ Fastidious, with some strains requiring CO2 for growth
▪ PCV13 ➢ Oropharyngeal commensals that are regarded as opportunistic
• Pneumococcal Vaccine protecting against 13 pathogens
serotypes commonly affecting children is composed of
purified polysaccharides conjugated to a diphtheria
protein and approved for use in children
▪ PPSV 23
• Composed of 23 purified polysaccharides is used for
adults
• Successful in reducing the incidence and severity of
pneumococcal disease
❖ Five Groups
C. Laboratory Diagnosis ➢ S. mitis group
▪ Including S. mitis, S. pneumoniae, S. sanguis, S. oralis
❖ Gram Stain
➢ Gram-positive cocci in pairs (diplococci)
➢ The ends of the cells are slightly pointed, giving them an oval or
lancet shape

➢ S. mutans group
▪ Including S. mutans, S. sobrinus

❖ Culture
➢ Brain-heart infusion agar
➢ Trypticase soy agar with 5% sheep RBCs
➢ Chocolate agar ➢ S. salivarius group
➢ SBA ▪ S. salivarius and S. vestibularis

➢ S. bovis group
▪ S. equinus, S. gallolyticus, S. infantarius, S. alactolyticus
➢ S. anginosus group
▪ S. anginosus, S. constellatus, S. intermedius

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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH
A. Clinical Infections F. Enterococcus

➢ Viridans streptococci are oropharyngeal commensals that are ➢ The enterococci consist of gram-positive cocci that are natural
regarded as opportunistic pathogens inhabitants of the intestinal tracts of humans and animals
➢ Most member of the S. anginosus group are considered part of ➢ Previously classified as group D streptococci
the normal oral and gastrointestinal microbiota, however, they ➢ The most commonly identified species in clinical specimens are
have been associated with abscess formation in the oropharynx E. faecalis and E. faecium
➢ The S. mitis group is normally found in the oral cavity, ➢ Everyone has Enterococcus in their digestive system but few
gastrointestinal tract, and female genital tract. Members also can people get sick from the endogenous strains
be found as transient normal microbiota of the skin. Although ➢ Typically, Enterococcus isolates are not as virulent as other
they can be isolated from the blood of asymptomatic individuals, gram-positive cocci and often they are seen in polymicrobial
they are the most common isolates associated with bacterial infections in immunosuppress host
endocarditis in native valves and less frequently, in prosthetic ➢ Clinical manifestations include UTI, bacteremia, endocarditis,
valve infections and intraabdominal pelvic wound and soft tissue infections
➢ S. salivarius and S. vestibularis have been isolated from human ➢ Most enterococci are nonhemolytic or a-hemolytic, although
specimens. S. salivarius has been linked to bacteremia, some strains show B-hemolysis
endocarditis, and meningitis, whereas S. vestibularis has not ➢ Enterococci sometimes exhibit a pseudocatalase reaction –
been associated with disease week bubbling in the catalase test
➢ Members of the S. bovis group are often encountered in blood ➢ Identification to the species level is based on biochemical
cultures of patients with bacteremia, septicemia, and characteristics. In contrast to streptococci, enterococci have the
endocarditis ability to grow under extreme conditions
▪ Presence of bile or 6.5% NaCl
❖ Subacute Bacterial Endocarditis ▪ 45C or alkaline pH
➢ Viridans streptococci are the most common cause ➢ The ability of enterococci to hydrolyze PYR is useful for
➢ A condition associated with a transient bacteremia differentiating them from group D streptococci
❖ Oral infections such as gingivitis and dental caries (cavities)

B. Virulence Factors

❖ Polysaccharide capsule and cytolysin have been identified in some S.

anginosus group
❖ Extracellular dextran and cell surface-associated proteins (adhesins)
have been implicated in adherence and colonization of these
organisms in endocarditis
❖ Group C and G streptococci possess M proteins with similarity to
those of GAS
❖ Some of them also produce extracellular enzymes such as SLO,
hyaluronidase, and DNase A. Virulence Factors
C. Laboratory Diagnosis
❖ Extracellular surface adhesin proteins, extracellular serine protease,
gelatinase, cytolysin
❖ Gram Stain ➢ Play a role in the colonization of the species and adherence to
➢ Typical streptococcus characteristics on gram stain (they also heart valves and renal epithelial cells
appear as gram positive cocci) ❖ E. faecalis produces a two-subunit toxin termed cytolysin
❖ Colonial Morphology ➢ This toxin shows similarity to bacteriocins produced by other
➢ Small and are surrounded by a zone of a-hemolysis, some gram-positive bacteria and is expressed by a quorum-sensing
isolates are B-hemolytic or nonhemolytic mechanism
❖ Biochemical Tests
➢ Voges-Proskauer (VP) B. Clinical Infections
➢ B-D-glucuronidase activity
➢ Hippurate hydrolysis
➢ PYR ➢ Enterococcus is the frequent causes of nosocomial infections
➢ Leucine aminopeptidase (LAP) ❖ Urinary Tract Infections (UTI)
➢ Most common followed by bacteremia
➢ Often associated with urinary catheterization or other urologic
manipulations
❖ Bacteremia
➢ Prolonged hospitalization is a risk factor for acquiring
enterococcal bacteremia
➢ Often observed in patients receiving hemodialysis,
immunocompromised patients with a serious underlying disease,
or patients who have undergone a prior surgical procedure
➢ Rare cases of enterococcal infection of the central nervous
system in patients who have had neurosurgery or head trauma
and in immunosuppressed patients
❖ Endocarditis
➢ Seen in elderly patients with prosthetic valves or valvular heart
disease
➢ Enterococci account for about 5% to 10% of infections in patients
with bacterial endocarditis
❖ In burn patients, enterococcal wound infection and sepsis resulting
from contaminated xenografts have been reported

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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH
C. Laboratory Diagnosis

❖ Specimen Collection
➢ Standard procedures for collection and transport of blood, urine
or wound specimens should be followed
➢ The specimens should be cultured as soon as possible with
minimum delay
❖ Gram Staining
➢ Gram stain reveals gram-positive cocci with some short chains

B. Aerococcus

➢ A common airborne organism

➢ These organisms sometimes show a weak catalase or
pseudocatalase reaction
➢ They grow in the presence of 6.5% NaCl and can easily be
confused with enterococci
➢ It is a widespread, opportunistic pathogen associated with
bacteremia, endocarditis, and UTI in immunocompromised
❖ Culture patients
➢ Enterococci grow well at 35C in the presence of CO2 but do not ➢ Resemble viridans streptococci on culture but are
require a high level of CO2 for growth microscopically similar to staphylococci in that they occur as
➢ Trypticase soy or brain-heart infusion agar supplemented with tetrads or clusters
5% sheep blood ➢ Aerococcus urinae
➢ Bile esculin ▪ Bile esculin negative and PYR negative
➢ Colistin-nalidixic acid ▪ is a cause of UTI, endocarditis, lymphadenitis and peritonitis
➢ Phenylethyl alcohol
➢ Chromogenic substrates
➢ Cephalexin-aztreonam-arabinose agar

➢ Aerococcus sanguinicola have been associated with invasive

diseases, such as sepsis, endocarditis, lymphadenitis, and
peritonitis, often originating from the urinary tract
➢ Aerococcus viridans
▪ Are bile esculin positive and PYR positive
❖ Biochemical Tests ▪ has been linked to cases of bacteremia and endocarditis
➢ Identified based on several biochemical characteristics: and UTI in immunocompromised patients
▪ Ability to produce acid in carbohydrate broth
▪ Ability to hydrolyze arginine
▪ Tolerance of 0.4% tellurite
▪ Utilization of pyruvate
▪ Ability to produce acid from methyl-a-D-glucopyranoside
▪ Growth around 100-ug efrotomycin and acid disk
▪ Motility
➢ E. faecalis is easily identified by its ability to grow in the presence
of tellurite
➢ 16S ribosomal ribonucleic acid (rRNA) sequencing and
III. Streptococcus – like Organisms MALDI-TOF testing seem to accurately identify A. urinae, A.
sanguinicola, A. viridans
A, Abiotrophia and Granulicatella
C. Gemella
➢ Formerly known as the nutritionally variant streptococci
➢ Should be suspected when gram-positive cocci resembling ➢ Similar in colony morphology and habitat to viridans streptococci
streptococci are observed in positive blood cultures that ➢ a-hemolysis or are nonhemolytic
subsequently fail to grow on subculture ➢ The bacteria easily decolorize on gram staining and often appear
➢ These bacteria grow as satellite colonies around other bacteria as gram-negative cocci in pairs, tetrads, clusters or short chains
and require sulfhydryl compounds for growth ➢ Isolated from cases of endocarditis, wounds, and abscesses
➢ Part of the human oral and gastrointestinal microbiota ➢ The most significant species is Gemella haemolysans
➢ Significant cause of bacteremia, endocarditis, and otitis media
➢ Have been linked to a few cases of osteomyelitis,
endophthalmitis after cataract extraction, brain abscess, chronic
sinusitis, septic arthritis, meningitis and breast implant
associated infections
➢ G. adiacens, G. elegans, G. balaenopterae have been isolated
from blood cultures and tissue samples

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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH
D. Lactococcus IV. LABORATORY DIAGNOSIS

➢ Gram-positive cocci that occur singly, in pairs, or in chains and A. Classification Schemes
are physiologically similar to enterococci
➢ On SBA, a-hemolysis or are nonhemolytic
➢ Previously classified as group N streptococci ❖ Hemolytic pattern on SBA
➢ Isolated from patients with UTI and endocarditis ❖ Physiologic characteristics
➢ Production of acid from carbohydrates is useful in distinguishing ➢ Pyogenic streptococci
Lactococcus spp. from enterococci ▪ Produce pus
➢ These microorganisms do not react with the genetic probe in the ▪ Mostly B-hemolytic and constitute most of the Lancefield
AccuProbe Enterococcus culture confirmation test groups
➢ Lactococci
▪ Nonhemolytic organisms with Lancefield group N antigen
and are found in dairy products
➢ Viridans Streptococci
▪ Widely found as normal biota in the upper respiratory tract
of humans
▪ Most strains lack a C carbohydrate and are not part of the
Lancefield classification
▪ a-hemolytic or nonhemolytic and are often seen as
opportunistic pathogens
❖ Serologic grouping or typing of C carbohydrate (Lancefield
classification), capsular polysaccharide or surface protein such as the
M protein of S. pyogenes
❖ Biochemical characteristics
E. Leuconostoc
V. BIOCHEMICAL TESTS
➢ Catalase-negative, gram-positive microorganisms with irregular
coccoid morphology A. Bacitracin Susceptibility Test
➢ These organisms share several phenotypic and biochemical
characteristics with Lactobacillus spp., viridans streptococci, ➢ Used as an inexpensive test for presumptive identification of S.
Pediococcus spp., and Enterococcus spp., and are sometimes pyogenes
misidentified ➢ This method is helpful in screening for GAS (group A
➢ Some species cross-react with the Lancefield group D antiserum streptococci) in throat cultures
➢ Resistant to vancomycin ➢ The throat swab is used to inoculate SBA containing SMZ and a
➢ In nature, they are frequently found on plant surfaces and bacitracin disk is placed directly onto the agar
vegetables and milk products ❖ Purpose
➢ They are recognized as opportunistic pathogens ➢ To differentiate Streptococcus pyogenes from other B-hemolytic
➢ These microorganisms have been isolated from cases of streptococci
meningitis, bacteremia, UTIs and pulmonary infections ❖ Principle
➢ Species associated with infection include: ➢ Group A streptococci are susceptible to low levels (0.04 units) of
▪ Leuconostoc citreum bacitracin, whereas other groups of B-hemolytic streptococci are
▪ Leuconostoc cremoris resistant
▪ Leuconostoc dextranicum ❖ Specimen
▪ Leuconostoc lactis ➢ Isolated colonies of test organism on sheep blood agar
▪ Leuconostoc mesenteroides ❖ Media
▪ Leuconostoc pseudomesenteroides ➢ 5% sheep blood agar plate
❖ Biochemical Identification ❖ Reagent
➢ Biochemical identification is based on the absence of catalase, ➢ Bacitracin disk, 0.04 units
PYR, and LAP activities ❖ Procedure
➢ Hydrolysis of esculin in the presence of bile 1. Streak surface of agar plate to obtain isolated colonies
➢ Growth in the presence of 6.5% NaCl 2. Aseptically place bacitracin disk onto inoculated surface. Press
➢ Production of gas from glucose down gently on the disk to ensure complete contact with the agar
surface
F. Pediococcus 3. Incubate the plate at 35C for 18 to 24 hours in a CO2 incubator
➢ Facultatively anaerobic, gram-positive cocci (arranged in pairs, ❖ Interpretation
tetrads, and clusters) ➢ Susceptible = any zone of inhibition around the bacitracin disk
➢ Grows at 45C and resistant to vancomycin ➢ Resistant = uniform lawn of growth up to the edge of the disk
➢ They may be misidentified as viridans streptococci or enterococci ❖ Controls Used
➢ Associated with patients who have underlying gastrointestinal ➢ S. pyogenes
abnormalities or who have previously undergone abdominal ▪ Positive
surgery ▪ Susceptible to bacitracin and resistant to SMZ
➢ The following species have been associated with infection: ➢ S. agalactiae
▪ Pediococcus acidilactici ▪ Negative
▪ Pediococcus damnosus ▪ Resistant to both bacitracin and SMZ
▪ Pediococcus dextrinicus
▪ Pediococcus parvulus
▪ Pediococcus pentasaceus
❖ Biochemical Characteristics:
➢ Positive bile esculin test
➢ Presence of LAP activity
➢ Absence of PYR activity
➢ Do not produce gas from glucose
➢ Some strain are able to grow in the presence of 6.5% NaCl

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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH
C. Hippurate Hydrolysis Test

➢ A useful test for differentiating S. agalactiae from other B-

hemolytic streptococci
➢ S. agalactiae possesses the enzyme hippuricase (Hippurate
hydrolase) which hydrolyzes sodium Hippurate to form sodium
benzoate and glycine
➢ A 2-hour rapid test is available to detect the presence of
Hippurate hydrolase
❖ Purpose
B. CAMP Test ➢ To differentiate Streptococcus agalactiae from other B-hemolytic
streptococci
➢ Presumptive identification of Group B streptococci ❖ Principle
➢ CAMP is an acronym derived from the first letters of the ➢ The enzyme hippuricase hydrolyzes hippuric acid to form sodium
surnames of the individuals who first described the reaction: benzoate and glycine. Subsequent addition of ninhydrin results
Christie, Atkins and Munch-Petersen in the release of ammonia from the oxidative deamination of the
❖ Purpose a-amino group in glycine as well as the reduced form of
➢ Differentiate Streptococcus agalactiae from other B-hemolytic ninhydrin, hydrindantin. The ammonia reacts with residual
streptococci ninhydrin and hydrindantin to produce a purple-colored
❖ Principle complex
➢ Streptococcus agalactiae produces CAMP factor, which ❖ Reagent
enhances the lysis of sheep red blood cells by staphylococcal B- ➢ Sodium Hippurate
lysin. ➢ Ninhydrin Reagent
➢ A positive reaction can be observed in 5 to 6 hours with ❖ Procedure
incubation in CO2 (18 hours with incubation in ambient air) 1. Inoculate the solution of sodium Hippurate heavily with colonies
❖ Specimen 18 to 24 hours old until a milky suspension is obtained
1. Isolated colonies of test organism on sheep blood agar 2. Incubate tube for 2 hours at 35C
2. B-lysin producing S. aureus on sheep blood agar 3. Add 0.2mL of ninhydrin reagent
❖ Medium 4. Mix and incubate for 10 to 15 minutes at 35C
➢ Sheep blood agar plate ❖ Interpretation
❖ Procedure ➢ Positive Result = deep purple color
1. Inoculate S. aureus along a line down the center of the agar plate indicates Hippurate hydrolysis
2. Inoculate the streptococcal isolates along a thin line about 2cm ➢ Negative Result = no color change or very
long and perpendicular to, but not touching the S. aureus streak slight purple color
3. Incubate plate at 35C for 18 hours
❖ Interpretation
➢ Positive Result = arrowhead-shaped area of enhanced
hemolysis
➢ Negative Result = no enhanced hemolysis
➢ CAMP Inhibition Reaction (reverse CAMP positive) =
inhibition of hemolysis by S. aureus where the two streaks
approach each other (Arcanobacterium haemolyticum)

D. Pyrrolidonyl-a-Naphthylamide Hydrolis (PYR)

➢ Provides a high probability for the presumptive identification of

the B-hemolytic GAS and the nonhemolytic group D streptococci
➢ S. pyogenes is the only species of streptococcus that is PYR
positive
➢ Other genera that are PYR positive include Enterococcus,
Aerococcus and Gemella
➢ The PYR test detects the activity of L-pyrrolidonyl arylamidase,
also called pyrrolidonyl aminopeptidase
❖ Purpose
➢ To differentiate gram-positive cocci that will hydrolyze the
substrate L-pyrrolidonyl a-naphthylamide (PYR) from those that
are PYR negative
❖ Principle
➢ PYR-impregnated disks serve as the substrate to produce a-
naphthylamine, which is detected in the presence of D-
dimethylaminocinnamaldehyde (DMCA) by the production of a
red color
❖ Procedure
1. Lightly moisten a PYR-impregnated disk with sterile water
2. Using a sterile loop, rub one or more isolated colonies onto the
surface of the disk
3. Note: incubation time and temperature vary slightly by
manufacturer. Incubate disk as indicated in the manufacturer’s
instructions, generally 2 to 15 minutes
4. Add a drop of color developer and observer for a red color on the
disk within 5 minutes

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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH
❖ Interpretation
➢ Positive Result = red color
➢ Negative Result = colorless

G. Voges-Proskauer Test

➢ The VP test detects acetoin production from glucose

E. Bile Esculin Hydrolysis ➢ A heavy suspension of bacteria is made in 2mL of VP broth. After
about 6 hours of incubation at 35C a few drops of 5% a-naphthol
❖ Purpose and 40% potassium hydroxide added
➢ To differentiate group D streptococci and enterococci from other ➢ The tube is shaken vigorously to increase the concentration of
catalase-negative, gram-positive cocci dissolved oxygen and the broth is incubated at room temperature
❖ Principle for 30 minutes
➢ Group D streptococci and enterococci grow in the presence of ❖ Purpose
bile and also hydrolyze esculin to esculetin and glucose. ➢ Used to distinguish the small-colony forming B-hemolytic
Esculetin diffuses into the agar and combines with ferric citrate anginosus group containing groups A or C antigens from large-
in the medium to produce a black complex colony-forming pyogenic strains with the same antigens
❖ Media ❖ Positive Reaction
➢ Bile esculin agar ➢ Red or pink color
❖ Procedure
1. Pick one or two isolated colonies from the sheep blood agar plate
and inoculate to bile esculin agar medium. A single plate can be
divided into several-pie shaped sections for inoculation of
multiple test organisms
2. Incubate plate or slant at 35C for 18 to 24 hours. A positive
result is often seen within 4 hours. A negative result should be
incubated for an additional 24-hour period
❖ Interpretation
➢ Positive Result = blackening of the agar
➢ Negative Result = no blackening of the agar
➢ Note: growth alone does not constitute a positive result

H. B-D-Glucuronidase

➢ Detects the action of B-D-glucuronidase, an enzyme found in

isolates of large-colony-forming B-hemolytic groups C and G
streptococci but not in the small-colony-forming B-hemolytic
anginosus group
➢ A fluorogenic assay using methylumbelliferyl-B-D-glucuronide
has also been described

I. Salt Tolerance Test

➢ Growth in 6.5% NaCl broth is used to identify Enterococcus and

F. Leucine Aminopeptidase Aerococcus organisms
➢ Some species of Pediococcus and Leuconostoc grow in 6.5%
➢ A peptidase that hydrolyzes peptide bonds adjacent to a free NaCl broth when incubated for 24 hours. However, group D
amino group. Because LAP reacts most quickly with leucine, it is streptococci do not grow in a 6.5% NaCl broth
called leucine aminopeptidase ❖ Purpose
➢ The LAP test is often used with PYR test and is most helpful in ➢ Organisms positive for bile esculin are separated into group D
differentiating Aerococcus and Leuconostoc spp. from other streptococci or Enterococcus
gram-positive cocci ❖ Medium
❖ Principle ➢ 6.5% Sodium Chloride Broth
➢ The substrate, leucine-B-naphthylamide, is hydrolyzed to B-
naphthylamine. After the addition of
paradimethylaminocinnamaldehyde reagent, a red color
develops
❖ LAP Positive
➢ Streptococcus
➢ Enterococcus
➢ Pediococcus spp.
❖ LAP Negative
➢ Aerococcus
➢ Leuconostoc spp.

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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH
J. Optochin Susceptibility

➢ In the optochin test, a filter paper disk containing optochin

(ethylhydrocuprein hydrochloride) is added to the surface of an
SBA plate that has just been inoculated with an a-hemolytic
Streptococcus
➢ The plate is incubated overnight at 35C in a CO2 incubator
❖ Purpose
➢ Determine the effect of Optochin (ethylhydrocupreine
hydrochloride) in an organism
➢ Optochin lyses Pneumococci (positive)
❖ Principle
➢ Optochin interferes with the ATPase and production of ATP in
microorganisms
❖ Result
➢ A zone of inhibition greater than 14mm with a 6mm disk or a zone
of inhibition greater than 16mm with a 10mm disk is considered
susceptible and presumptive identification of S. pneumoniae
➢ Isolates producing smaller zones should be tested for bile
solubility to confirm their identity

K. Bile Solubility

➢ Confirmatory test for Streptococcus pneumoniae

➢ Used to differentiate S. pneumoniae from viridans strep
➢ Correlates well with optochin susceptibility
➢ The test for bile solubility takes advantage of the S. pneumoniae
autocatalytic enzyme amidase
➢ Under the influence of a bile salt or detergent, the organisms cell
wall lyses during cell division
➢ A suspension of S. pneumoniae in a solution of sodium
deoxycholate lyses, and the solution becomes clear. Other a-
hemolytic organisms do not undergo autolysis, and the solution
remains cloudy
➢ Suspension of bacteria made in saline serve as negative
controls
❖ Principle
➢ sodium deoxycholate (bile salts) rapidly lyses pneumococcal
colonies. Lysis depends on the presence of an intracellular
autolytic enzyme
❖ Reagents
➢ Plate (10% sodium desoxycholate)
➢ Tube (2% sodium desoxycholate)
❖ Results
➢ Tube
▪ + no turbidity
▪ - remains turbid
➢ Plate
▪ + lysed colonies
▪ - intact colonies

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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH

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[BACT211] TRANS 8: Streptococcus and Enterococcus I Prof. Rochelle D. Darlucio, RMT, MPH

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OLFU Neisseria spp. and Moraxella catarrhalis LEC 2020-2021

2nd SEM
College of Medical CLINICAL BACTERIOLOGY 8 BACT211
LEC
Laboratory Science Transcriber: Riyoma Surell MIDTERMS
Date: April 09, 2021 TRANS 9
Batch 2023

❖ Cell Outer Membrane Proteins

Outline ➢ Protein I (por) porin
At the end of the session, the student must be able to learn: ▪ (porA, porB both expressed in Neisseria meningitidis,
I. General Characteristics porB only for Neisseria gonorrhoeae)
II. Pathogenic Neisseria Species ▪ Forms channels for nutrients to pass into and waste
a. Neisseria gonorrhoeae products to exit the cell
b. Neisseria meningitidis ➢ Protein II (opa) opacity
c. Moraxella catarrhalis ▪ Facilitate the adherence to phagocytic and epithelial cells
III. Commensal Neisseria Species ➢ Protein III (reduction modified protein, Rmp)
a. Neisseria cinerea ▪ Blocks host serum bactericidal (IgG) action against the
b. Neisseria flavescens organism
c. Neisseria lactamica
d. Neisseria mucosa A. Neisseria gonorrhoeae
e. Neisseria polysaccharea
f. Neisseria sicca ❖ Agent of gonorrhea
g. Neisseria subflava ➢ Gonorrhea, meaning a “flow of seed”
h. Neisseria elongata ➢ Also called the “clap” from the French word clapoir meaning
i. Neisseria weaveri “brothel”
IV. Differentiation of Family Neisseriaceae ➢ An acute pyogenic infection of non-ciliated columnar and
transitional epithelium
I. GENERAL CHARACTERISTICS ➢ Infection can be established at any site where these cells are
found
➢ Acquired by sexual contact and occur primarily in the urethra,
❖ The family Neisseriaceae contains the genus Neisseria as well as
endocervix, anal canal, pharynx and conjunctiva (most common
Kingella, Eikenella, Simonsiella, Alysiella, and several other genera
in infants)
❖ Moraxella catarrhalis is in the family Moraxellaceae with other
Moraxella spp., and Acinetobacter
❖ Aerobic, non-motile, non-spore-forming, gram-negative diplococci
except: Neisseria elongata, Neisseria weaveri, and Neisseria
bacilliformis (arranged in rod shape)
❖ All species are cytochrome oxidase and catalase-positive except for
Neisseria elongata and Neisseria bacilliformis
❖ Many Neisseria spp. are capnophilic requiring carbon dioxide (CO2)
for growth, and have optimal growth in a humid atmosphere

❖ Natural Habitat:
➢ Mucous membranes of the respiratory and urogenital tracts
❖ Fastidious organisms and are sensitive to unfavorable environmental
conditions
❖ Both N. gonorrhoeae and N. meningitidis require iron for growth
(pathogenic Neisseria species)
❖ Primary Pathogens: ❖ Humans are the only natural host
➢ Compete with their human host by binding human transferrin ❖ Five Morphologically Distinct Colony Types
(iron-bonding blood plasma glycoprotein) to specific surface ➢ Types T1 and T2
receptors ▪ Which possess pili, are virulent forms
➢ Neisseria gonorrhoeae (gonococci) sexually transmitted ➢ Types T3 through T5
➢ Neisseria meningitidis (meningococci) respiratory droplets ▪ Devoid of pili and are avirulent
▪ May be found as a commensal inhabitant of the upper
respiratory tract of carriers, but it can also become as A. Clinical Infections
invasive pathogen
❖ Neisseria weaveri
➢ A commensal in the upper respiratory tract of dogs, has been ❖ Gonorrhea: incubation period 2 to 7 days
isolated from dog bites in humans
❖ All other Neisseria spp., are considered opportunistic pathogens Men Women
Acute urethritis, usually resulting Dysuria
in purulent discharge
II. PATHOGENIC NEISSERIA SPECIES
Dysuria (painful urination) Cervical discharge
Only 3% to 5% of cases are Lower abdominal pain
❖ Virulence Factors
asymptomatic
➢ Receptors for human transferrin
Complications in male patients However, 50% of cases in women
➢ Capsule (N. meningitidis)
include ascending infections may be asymptomatic leading to
➢ Pili (fimbriae)
such as prostatitis and complications such as pelvic
➢ Cell membrane proteins
epididymitis inflammatory disease, which may
➢ Lipooligosaccharide (LOS) or endotoxin:
cause sterility, ectopic pregnancy
▪ Lipid A moiety and core LOS that differentiates it from the
or perihepatitis (Fitz-Hugh-Curtis
lipopolysaccharide found in most gram-negative bacilli and
Syndrome)
is loosely attached to the underlying peptidoglycan
▪ Major in vivo virulence factor that mediates damage to body
tissues and elicits an inflammatory response ❖ Anorectal and Oropharyngeal Infections
➢ Immunoglobulin A (IgA) protease ➢ More common in men who have sex with men but can also occur
▪ Cleaves IgA on mucosal surfaces in women
➢ Asymptomatic or have nonspecific symptoms

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[BACT211] TRANS 9: Neisseria species and Moraxella catarrhalis I Prof. Rochelle D. Darlucio, RMT, MPH
❖ Ophthalmia Neonatorum ➢ Cultures are examined daily for growth and held for 72 hours
➢ A gonococcal eye infection, during vaginal delivery through an ➢ Use only white unscented candles
infected birth canal
➢ Can result in blindness if not treated immediately ❖ Presumptive Identification
➢ Before:1% silver nitrate Now: erythromycin, neomycin and ➢ Colony Morphology
chloramphenicol ▪ Small, gray to tan, translucent and raised after 24 to 48
hours if incubation
❖ Presumptive Identification Oxidase Test
➢ Reagent:
▪ 1% dimethyl-p-phenylenediamine dihydrochloride
▪ tetramethyl-p-phenylenediamine dihydrochloride
▪ Positive Result: purple color within 10 seconds
B. Laboratory Diagnosis
❖ Specimen Collection
➢ Specimens collected may come from genital sources or from
other sites such as the rectum, pharynx and joint fluid (2cm
inserted into the urethra)
➢ Specimen of Choice:
▪ Men: urethra
▪ Women: endocervix
❖ Transport
➢ Calcium alginate and cotton swabs are inhibitory to N.
gonorrhoeae ❖ Definitive Identification
➢ Dacron or Rayon Swabs are preferred ➢ Carbohydrate utilization
➢ Transport Systems: ▪ Cystine trypticase agar (CTA)
▪ James E. Martin Biological Environmental Chamber ▪ pH Indicator: 1% of individual carbohydrate and phenol red
(JEMBEC) plates ➢ Positive Result: Yellow color (acid) within 24 to 72 hours
➢ Neisseria gonorrhoeae – positive for glucose only

▪ Gono-Pak C. Treatment
▪ Transgrow
▪ Amies Medium with charcoal
❖ Direct Microscopic Examination ❖ Fluoroquinolones
➢ Gram stains is not recommended for pharyngeal specimens ➢ No longer be used as therapy for gonorrhea and associated
➢ Gram-negative intracellular diplococci (kidney or coffee bean gonococcal infections
shape) ❖ Cephalosporins (e.g., ceftriaxone, cefixime)
➢ Are currently recommended

B. Neisseria meningitidis

❖ Only found in humans

❖ Can be found as a commensal as well as an invasive pathogen
❖ An important etiologic agent of endemic and epidemic meningitis and
meningococcemia and rarely pneumonia, purulent arthritis or
endophthalmitis
❖ Serogroups A, B, C, Y and W-135
❖ Culture ➢ Account for most cases of disease in the world
➢ Does not grow on SBA ❖ Close contact with respiratory droplets secretion
➢ Medium of choice: chocolate agar
➢ All specimens received in the laboratory for recovery of Neisseria
spp., should be held at room temperature and plated as soon as
possible
➢ Because Neisseria spp., are susceptible to cold, media should
be warmed to room temperature before inoculation

❖ Incubation
➢ 35C in a 3% to 5% CO2 atmosphere
➢ Use of a CO2 incubator
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[BACT211] TRANS 9: Neisseria species and Moraxella catarrhalis I Prof. Rochelle D. Darlucio, RMT, MPH
A. Clinical Infections

❖ Fulminant meningococcemia or meningitis

❖ Meningococcemia or Sepsis
➢ May occur with or without meningitis and carries a 25% mortality
rate, even if treated
❖ Purpura
➢ (hemorrhaging of blood into the skin and mucous membranes
producing bruises) with petechial skin rash (pinpoint red spot III. COMMENSAL NEISSERIA SPECIES
caused by hemorrhage)
A. Neisseria cinerea

❖ Glucose in CTA sugars

❖ Grows on SBA
❖ Useful observation for differentiation from Neisseria flavescens is lack
of yellow pigment production

B. Neisseria flavescens

❖ Yellow-pigmented
❖ Disease becomes fulminant and spreads rapidly, causing: ❖ Asaccharolytic
➢ Disseminated intravascular coagulation ❖ It can be differentiated from N. cinerea by its ability to grow on SBA or
➢ Septic shock CHOC agar at 22C and its yellow colonies
➢ Hemorrhage in the adrenal glands (Waterhouse-Friderichsen
Syndrome) C. Neisseria lactamica

B. Treatment ❖ Commonly found in the nasopharynx of infants and children and

similar to Neisseria polysaccharea is commonly encountered in
❖ Drug of Choice: Penicillin meningococcal carrier surveys
❖ Meningococcemia is best treated with third-generation ❖ Only Neisseria species that uses lactose
cephalosporins
❖ Chemoprophylaxis with rifampin or ciprofloxacin is recommended D. Neisseria mucosa
for contacts
❖ Azithromycin can be used in areas where ciprofloxacin resistance is ❖ Large, often adherent to the agar and very mucoid
a problem ❖ Usually isolated from the nasopharynx of children or young adults
❖ Isolated from the airways of dolphins
❖ Causes pneumonia in children

E. Neisseria polysaccharea

❖ Produces large amounts of extracellular polysaccharide when grown

in media containing 1% to 5% sucrose – hence the species name

F. Neisseria sicca

❖ Dry, wrinkled, adherent and breadcrumb-like

G. Neisseria subflava
C. Moraxella catarrhalis
❖ “less yellow”
❖ Family Moraxellaceae
❖ Although it is considered to be a nonpathogen, it has been reported to
❖ Contains three genera:
cause serious infections, such as bacteremia, meningitis, and
➢ Moraxella, Acinetobacter and Psychrobacter
septicemia
❖ Isolated only from humans
❖ Commensal of the upper respiratory tract
H. Neisseria elongata
❖ Opportunistic pathogen and is recognized as a cause of upper
respiratory tract infection in otherwise healthy children and the elderly
❖ Neisseria elongata, N. weaver and N. bacilliformis are unique among
❖ The third most common cause of acute otitis media and sinusitis in
the members of the genus Neisseria in that they are rodshaped
children
❖ N. elongate contains three subspecies, elongata, glycolytica and
❖ Endocarditis, meningitis, and bacterial tracheitis
nitroreducens

I. Neisseria weaveri

❖ Normal oral microbiota in dogs and can be found in humans in

infections following dog bites

A. Laboratory Diagnosis

❖ Intracellular, gram-negative diplococci

❖ Smooth, opaque, gray to white colonies
❖ Hockey Puck
➢ Has been used to describe the colony because it remains intact
when pushed across the plate with a loop
❖ Older colonies may give a “wagon wheel” appearance

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[BACT211] TRANS 9: Neisseria species and Moraxella catarrhalis I Prof. Rochelle D. Darlucio, RMT, MPH
IV. DIFFERENTIATION OF FAMILY Neisseriaceae

Note: Kingella (NO3 Reduc. Test and IndoleTest)

• K. kingae (-, -)
• K. denitificans (+, -)
• K. indologenes (-, +)

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OLFU Enterobacteriaceae LEC 2020-2021

2nd SEM
College of Medical CLINICAL BACTERIOLOGY 9 BACT211
LEC
Laboratory Science Transcriber: Riyoma Surell MIDTERMS
Date: April 13, 2021 TRANS 10
Batch 2023

Outline I. Obesumbacterium
At the end of the session, the student must be able to learn: J. Photorhabdus
I. General Characteristics K. Rahnella
II. Virulence and Antigenic Factors L. Trabulsiella
A. Antigens M. Yokenella
B. Plasmids VII. IMVC Reactions
III. Clinical Significance
IV. Opportunistic Members of Enterobacteriaceae
A. Escherichia coli I. GENERAL CHARACTERISTICS
A. 5 Major Categories of Diarrheogenic E. coli
1. Enterotoxigenic E. coli (ETEC)
❖ Large heterogenous group of gram-negative rods whose natural
2. Enteropathogenic E. coli (EPEC) habitat is the intestinal tract of humans and animals. Often referred to
3. Enteroinvasive E. coli (EIEC) as enterics
4. Enterohemorrhagic E. coli (EHEC) ❖ Gram-negative bacilli and coccobacilli
5. Enteroadherent E. coli (EAEC)
B. Extraintestinal Infections
C. Other Escherichia Species
1. Escherichia hermannii
2. Escherichia vulneris
3. Escherichia albertii
B. Klebsiella and Raoultella
A. Klebsiella pneumoniae
B. Klebsiella oxytoca
C. Klebsiella pneumoniae subsp. ozaenae
D. Klebsiella pneumoniae subsp. rhinoscleromatis
E. Raoultella (Klebsiella) ornithinolytica
❖ Do not produce cytochrome oxidase except for Plesiomonas
F. Raoultella (Klebsiella) planticola ❖ All ferment glucose, oxidase negative
G. Klebsiella variicola ❖ Reduce nitrate to nitrite except for Photorhabdus and Xenorhabdus
C. Enterobacter, Cronobacter and Pantoea ❖ Motile at body temperatures except for Klebsiella, Shigella and
A. Enterobacter cloacae Yersinia
B. Enterobacter taylorae ❖ All members can grow rapidly, aerobically and anaerobically on variety
C. Cronobacter (Enterobacter) zakazakii of non-selective culture media such as blood agar plate. Appear large,
D. Enterobacter gergoviae
E. Pantoea (Enterobacter) agglomerans
moist, and gray on sheep blood agar (SBA), chocolate (CHOC) agar,
F. Enterobacer hormaechei and most nonselective media
G. Enterobacter asburiae ❖ They can also grow on selective culture media such as MacConkey
H. E. dissolvens and E. nimipressuralis (MAC) agar and Eosin-methylene blue agar (EMB)
I. Enterobacter cancerogenus (E. taylorae) ❖ All members of the group generally aerogenic except Shigella
D. Serratia ❖ The following may or may not be aerogenic:
A. Serratia odorifera ➢ Salmonella
B. Serratia liquefaciens
C. Serratia rubidaea
➢ Serratia
E. Hafnia ➢ Proteus
A. Hafnia alvei ➢ Providencia
F. Proteus ➢ Klebsiella
A. Proteus mirabilis and Proteus vulgaris ❖ Can be differentiated based on lactose fermentation:
B. Proteus penneri ➢ Rapid Lactose Fermenter
G. Morganella ▪ Escherichia
H. Providencia
A. Providencia rettgeri
▪ Enterobacter
B. Providencia stuartii ▪ Klebsiella
C. Providencia alcalifaciens ➢ Late Lactose Fermenter
D. Providencia rustigianii ▪ Hafnia
E. Providencia heimbachae ▪ Serratia
I. Edwardsiella ▪ Citrobacter
A. Edwardsiella tarda ▪ Salmonella arizonae
B. Edwardsiella hoshinae
C. Edwardsiella ictaluri
▪ Shigella sonnei
J. Erwinia and Pectobacterium ▪ Yersinia enterocolitica
K. Citrobacter ➢ Non-lactose Fermenter
A. Citrobacter freundii ▪ All Salmonella except S. arizonae
B. Citrobacter koseri ▪ All Shigella except S. sonnei
C. Citrobacter braakii ▪ All Yersinia except enterocolitica
D. Citrobacter amalonaticus ▪ Proteus
V. Primary Intestinal Pathogens of Family Enterobacteriaceae
A. Salmonella
▪ Providencia
A. Salmonella typhi ▪ Morganella
B. Salmonella enteritidis ▪ Edwardsiella
B. Shigella ❖ Some produces H2S gas (blackening of the colony or medium)
C. Yersinia ➢ Triple Sugar Iron Agar (TSI)
A. Yersinia pestis ▪ Salmonella
B. Yersinia enterocolitica ▪ Proteus
C. Yersinia pseudotuberculosis
VI. Other Genera of the Family Enterobacteriaceae
▪ Arizona
A. Budivicia ▪ Citrobacter
B. Buttiauxella ▪ Edwardsiella
C. Cedecea ➢ Lysine Iron Agar (LIA)
D. Ewingella ▪ Salmonella
E. Kluyvera ▪ Arizona
F. Leclercia ▪ Citrobacter
G. Leminorella
H. Moellerella
▪ Edwardsiella

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[BACT211] TRANS 10: Enterobacteriaceae I Prof. Rochelle D. Darlucio, RMT, MPH

❖ Some produces Urease Enzyme III. CLINICAL SIGNIFICANCE
➢ Rapid Urease Producers
▪ Proteus ❖ Members of the family Enterobacteriaceae resides in the
▪ Providencia gastrointestinal tract (GIT)
▪ Morganella ❖ They are often commensals, causing no harm, and yet they can be
➢ Slow Urease Producers responsible for a large number of opportunistic infections when
▪ Citrobacter introduced into inappropriate body sites
▪ Klebsiella ❖ 2 Categories:
▪ Enterobacter except E. gergoviac ➢ Opportunistic pathogens
▪ Yersinia ➢ Primary pathogens
▪ Serratia ▪ Shigella spp.
❖ Some are Deaminase producing: ▪ Yersinia spp.
➢ Proteus • Considered true pathogens, they are not present as
➢ Providencia commensal biota in GIT of humans. These organisms
➢ Morganella produce infections resulting from ingestion of
❖ Can be differentiated based on Lysine Decarboxylation (LDC) contaminated food and water or from other sources
➢ LDC positive (+)
IV. OPPORTUNISTIC MEMBERS OF ENTEROBACTERIACEAE
▪ Klebsiella
▪ Escherichia
▪ Edwardsiella A. Escherichia coli (IMVC ++--)
▪ Serratia
▪ Salmonella except S. paratyphi A.
▪ Hafnia
➢ LDC negative (-)
▪ Proteus
▪ Providencia
▪ Morganella
▪ Citrobacter
▪ Yersinia
▪ Enterobacter except E. aerogenes and E. gergoviae
▪ Shigella ❖ Most common and important member of the family
Enterobacteriaceae
II. VIRULENCE AND ANTIGENIC FACTORS ❖ Colon bacillus/ golden bacillus
❖ It has an IMVC reaction of positive, positive, negative, negative
A. Antigens
➢ IMVC (Indole, Methyl Red, Vogues Proskauer, Citrate)
❖ O antigen or Somatic antigen ❖ Associated with variety of diseases including gastroenteritis and
➢ This is a heat-stable antigen located on the cell wall extraintestinal infections such as cystitis, pyelonephritis,
❖ H antigen or Flagellar antigen intraabdominal infection, meningitis and sepsis
➢ This is a heat-labile antigen found on the surface of flagella, ❖ Used as a primary marker of fecal contamination in water quality
structures responsible for motility testing
❖ K antigen or Capsular antigen ❖ Motile and generally possess adhesive fimbriae and sex pili and O, H
➢ This is a heat-labile polysaccharide found only in certain and K antigens
encapsulated species ❖ MAC Agar: It usually appears as a lactose-positive (pink) colony with
➢ E.g., K1 antigen of E. coli and the Vi antigen of Salmonella a surrounding area of precipitated bile salts
enterica subsp. enterica serotype typhi ❖ EMB Agar: it has a green metallic sheen
➢ Detection of these various antigens has important clinical
significance beyond epidemiologic investigations and some
pathogenic species of bacteria are associated with specific O and
H serotypes
❖ These are useful in the identification of the following species:
➢ E. coli
➢ Klebsiella
➢ Shigella
➢ Salmonella

❖ E. coli is associated with the following properties:

➢ Fermentation of glucose, lactose, trehalose and xylose
➢ Production of indole from tryptophan
➢ Glucose fermentation by the mixed acid pathway:
B. Plasmids ▪ Methyl Red: Positive
▪ Voges-Proskauer: Negative
❖ Can provide antimicrobial resistance genes ➢ Does not produce H2S DNase, urease or phenylalanine
❖ Produce plasmid-mediated extended spectrum B-lactamases deaminase
(ESBLs) including carbapenemases cephalosporinases, or metllo-B- ➢ Cannot use citrate as a sole carbon source
lactamases which can inactivate extended-spectrum cephalosporins
(e.g., cefotaxime), penicillins, and aztreonam ❖ Uropathogenic Escherichia coli
➢ E. coli ➢ Most common cause of UTIs in humans
➢ K. pneumoniae ➢ The E. coli strains that cause UTIs usually originate in the large
➢ Klebsiella oxytoca intestine as resident biota and can exit either as the predominant
❖ Strains harboring these plasmids have been found in healthy E. coli population or a small part of the E. coli strains in the large
volunteers and hospitalized patients intestine
➢ Primary Virulence Factor associated with the ability of E. coli to
cause UTIs

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[BACT211] TRANS 10: Enterobacteriaceae I Prof. Rochelle D. Darlucio, RMT, MPH

▪ Production of pili (allows ➢ Nucleic acid-based assays to detect EPEC adherence factor
uropathogenic strains to adhere plasmid
to epithelial cells and not be ➢ Serologic typing with pooled antisera (for epidemiologic studies
washed out with urine flow) rather than diagnostic purposes)
▪ Cytolysins (characterized as
hemolysins, can kill immune 3. Enteroinvasive Escherichia coli (EIEC)
effector cells and inhibit
phagocytosis and chemotaxis of ❖ Produce dysentery with direct penetration, invasion and destruction of
certain WBCs) the intestinal mucosa
▪ Aerobactins (allows the ❖ Produces Shiga-like toxin hence causing Shigella-like diarrhea
bacterial cell to chelate iron; free ❖ Stool: watery with blood, pus, and mucus
iron is generally unavailable ❖ Virulence Factors:
within the host for use by bacteria) ➢ Uncertain
➢ Organism invades enterocytes lining the large intestine in a
❖ Gastrointestinal Pathogens manner nearly identical to Shigella spp.
➢ E. coli may also cause several gastrointestinal syndromes base ❖ Disease and Infection:
on virulence factors, clinical manifestations, epidemiology, and ➢ Dysentery (i.e., necrosis, ulceration and inflammation of the large
different O and H serotypes bowel) usually seen in young children living in areas of poor
sanitation
A. 5 Major Categories of diarrheogenic E. coli
4. Enterohemorrhagic Escherichia coli (EHEC)
1. Enterotoxigenic Escherichia coli (ETEC)
❖ Also known as Verotoxigenic E. coli (VTEC)
❖ Associated with diarrhea of adults and especially children in tropical ❖ The EHEC strain serotype O157:H7 has since been associated with
and subtropical climates, especially in developing countries, where it hemorrhagic diarrhea, colitis and hemolytic uremic syndrome (HUS)
is one of the major causes of infant bacterial diarrhea ❖ Hemolytic Uremic Syndrome (HUS) characterized by low platelet
❖ Most common cause of a diarrheal disease sometimes referred to as count, hemolytic anemia and kidney failures
traveler’s diarrhea ❖ Virulence Factor:
❖ Virulence Factors: ➢ Toxin similar to shiga toxin produced by Shigella dysenteriae
➢ Pili (that permit gastrointestinal colonization) ❖ Disease and Infection:
➢ It also contains heat-labile and heat-stable enterotoxins that ➢ Inflammation and bleeding of the mucosa of the large intestine
mediates secretion of water and electrolytes into the bowel lumen (i.e., hemorrhagic colitis)
➢ Heat-labile toxin (LT) ➢ Hemolytic uremic syndrome, resulting from toxin-mediated
▪ Similar in action and amino acid sequence to cholera toxin damage to kidneys
from Vibrio cholerae ❖ MOT:
▪ Consists of two fragments A and B ➢ Ingestion of undercooked ground beef or raw milk
▪ B portion binds to the GM1 ganglioside of the intestinal ❖ Watery diarrhea that progresses to bloody diarrhea with abdominal
mucosa, providing entry for the A portion cramps and low-grade fever or an absence of fever
▪ A portion activates cellular adenylate cyclase, causing an ❖ Stool: watery with excessive blood
increase in the conversion of adenosine triphosphate to
cyclic adenosine monophosphate (cAMP) 5. Enteroadherent Escherichia coli
➢ Heat-stable toxin (ST)
▪ Stimulates guanylate cyclase, causing increased ❖ Diarrheal syndromes and UTIs
production of cyclic guanosine monophosphate, ❖ 2 Types:
accumulation of which also causes hypersecretion ➢ Diffusely Adherent Escherichia coli (DAEC)
❖ Disease: ▪ May be associated with UTIs and diarrheal diseases
➢ watery diarrhea, abdominal cramps, and sometimes nausea ➢ Uropathogenic Diffusely Adherent Escherichia coli strains
usually with no vomiting or fever. It causes travelers and ▪ Closely associated with cystitis in children and acute
childhood diarrhea characterized by profuse watery stools pyelonephritis in pregnant women
❖ MOT: Spread commonly via consumption of contaminated food and ❖ Enteroaggregative Escherichia coli (EAEC)
water ➢ Causes diarrhea by adhering to the surface of the intestinal
❖ Colonization of ETEC on the proximal small intestine is recognized as mucosa
being mediated by fimbriae that permit ETEC to bind to specific ❖ These strains are found to adhere to Hep2 cells, packed in an
receptors on the intestinal microvilli aggregative “stacked-brick” pattern on the cells and between the cells
❖ Diagnosis: by means of fimbriae
➢ Isolation of solely lactose-fermenting organisms on differential ❖ Virulence Factors:
media ➢ Probably involves binding by pili, ST-like, and hemolysin-like
➢ Immunologic assays to detect two toxins from culture toxins; actual pathogenic mechanism is unknown
supernatants ❖ Disease and Infection:
➢ Multiplex PCR to detect diarrheagenic strains ➢ Watery diarrhea that in some cases can be prolonged
❖ MOT:
2. Enteropathogenic Escherichia coli (EPEC) ➢ Not well understood

❖ Known to cause infantile diarrhea B. Extraintestinal Infections

❖ Virulence Factors:
➢ Bundle-forming pilus, intimin, and other factors that mediate ❖ Most common causes of septicemia and meningitis among neonates,
organism attachment to mucosal cells of the small bowel, accounting for about 40% of the cases of gram-negative meningitis
resulting in changes in cell surface (e.g., loss of microvilli) ❖ Capsular antigen K1: most documented virulence factor associated
❖ Disease and Infection: with neonatal meningeal infections
➢ Diarrhea in infants in developing, low-income nations; can cause
a chronic diarrhea
❖ O serogroups: cause of diarrhea
❖ H antigenic: intestinal infections
❖ Stool: watery with mucus but without blood
❖ Diagnosis:

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[BACT211] TRANS 10: Enterobacteriaceae I Prof. Rochelle D. Darlucio, RMT, MPH

C. Other Escherichia Species B. Klebsiella oxytoca

1. Escherichia hermannii ❖ The only indole positive Klebsiella species

❖ Has been isolated in stool and blood cultures
❖ A yellow-pigmented organism that has been isolated from ❖ Isolates have been linked to antibiotic-associated hemorrhagic colitis
cerebrospinal fluid (CSF), wounds and blood ❖ Biochemically, K. oxytoca is identical to K. pneumoniae except for its
❖ Reports of isolating E. hermanni from foodstuffs such as raw milk and production of indole, and there are reports of ornithine-positive
beef, the same sources as E. coli O157:H7, have been published. isolates as well
However, its clinical significance is not yet established

2. Escherichia vulneris

❖ Has been isolated from humans with infected wounds

❖ More than half of the strains of E. vulneris also produce yellow-
pigmented colonies

3. Escherichia albertii

❖ Associated with diarrheal disease in children

C. Klebsiella pneumoniae subsp. ozaenae
B. Klebsiella and Raoultella (IMVC --++)
❖ Highly associated with the presence of plasmid-mediated ESBLs,
❖ Members of the genus are covered with a prominent mucoid capsule contributing to the large numbers of antimicrobial-resistant hospital
which makes recognition of the bacteria by gram stain and culture acquired infections seen today
relatively easy ❖ Have been isolated from nasal secretions and cerebral abscesses
❖ The bacteria in this genus caused both community and hospital- ❖ This organism causes atrophic rhinitis, a tissue-destructive disease
acquired pneumonia with destruction of the alveolar spaces, formation restricted to the nose
of cavities and production of blood sputum prominent
❖ Usually found on the intestinal tract of humans and animals or free-
living in soil, water, and on plants
❖ Associated with various opportunistic and hospital-acquired
infections, particularly pneumonia, wound infections and UTIs
❖ It has an IMVC reaction of negative, negative, positive, positive
➢ Indole negative except for Klebsiella oxytoca
➢ Methyl Red negative
➢ Voges Proskauer Positive
➢ Citrate Positive
❖ Biochemical Reactions:
➢ Most grow on Simmons citrate and in potassium cyanide broth
➢ None produce H2S D. Klebsiella pneumoniae subsp. rhinoscleromatis
➢ A few hydrolyze urea slowly
➢ All give a negative reaction with the methyl red test and a positive ❖ Has been isolated from patients with rhinoscleroma, an infection of the
reaction with the Voges-Proskauer test nasal cavity that manifests as an intense swelling and malformation of
➢ With a few exceptions, no indole is produced from tryptophan the entire face and neck
➢ Motility is variable

A. Klebsiella pneumoniae

❖ Prominent cause of pneumonia

❖ Friedlander’s bacillus
❖ Most commonly isolated species and has the distinct feature of
possessing a large polysaccharide capsule
❖ The capsule offers the organism protection against phagocytosis and
antimicrobial absorption, contributing to its virulence
❖ The capsule is also responsible for the moist, mucoid colonies
characteristic of K. pneumoniae E. Raoutella (Klebsiella) ornithinolytica
❖ Pneumonia is very necrotic and hemorrhagic causing currant jelly like
sputum ❖ Indole and ornithine decarboxylase-positive
❖ Other infections commonly associated with K. pneumoniae involving
immunocompromised hosts are wound infections, UTIs, liver F. Raoultella (Klebsiella) planticola
abscesses and bacteremia
❖ Capsulated: mucoid colonies or tend to “string”
❖ Have been isolated from the urine, respiratory tracts and blood of
humans

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[BACT211] TRANS 10: Enterobacteriaceae I Prof. Rochelle D. Darlucio, RMT, MPH

G. Klebsiella variicola

❖ Has been isolated from primarily sterile sites

D. Enterobacter gergoviae

❖ Resembles Enterobacter aerogenes

❖ Rapid Urease Producer (RUP)
❖ Causes infection of the urinary tract, and blood
Test K.pneumoniae K. K. pneumoniae ❖ Found in respiratory samples and is rarely isolated from blood cultures
Subsp. oxytoca subsp.
pneumoniae ozaenae
Indole - + - E. Pantoea (Enterobacter) agglomerans
Methyl Red Most strains – - +
Cultures + ❖ Gained notoriety with a nationwide outbreak of septicemia resulting
VP + + - form contaminated intravenous fluids
Citrate + + V ❖ Lysine, ornithine, and arginine negative or “triple decarboxylases
Urease + + V negative”
Lysine Most strains – ❖ More than 13 hybridization groups (HGs) have been described in this
+ + complex
Decarboxylase Some cultures +
Gas from glucose + + V ❖ P. agglomerans HG XIII, which may produce a yellow pigment is
Lactose V primarily a plant pathogen
+ +

C. Enterobacter, Cronobacter, and Pantoea (IMVC --++)

❖ Motile bacilli
❖ IMVC reaction of negative, negative, positive, positive
❖ Resembles Klebsiella when growing on MAC agar
❖ Enterobacter spp., grow on Simmons citrate medium and in
potassium cyanide broth
❖ The methyl red test is negative, and Voges-Proskauer test is
positive. F. Enterobacter hormaechei
❖ Usually produce ornithine decarboxylase
❖ Lysine decarboxylase is produced by most species but not by E. ❖ Isolated from human sources such as blood, wounds and sputum
gergoviae or E. cloacae
G. Enterobacter asburiae
A. Enterobacter cloacae
❖ Similar biochemically to E. cloacae
❖ Predominant clinical isolate ❖ Has been isolated from blood, urine, feces, sputum and wounds
❖ Associated with bacteremia, respiratory tract infections, UTI and
wound infection in burn patients
❖ Also contaminated IV fluids and other hospital instrumentation H. E. dissolvens and E. nimipressuralis

❖ Newly recognized species with unknown clinical significance

I. Enterobacter cancerogenus (formerly E. taylorae)

❖ Associated with osteomyelitis after traumatic wounds

D. Serratia (IMVC -V++)

❖ Opportunistic pathogens associated with outbreaks in health care

B. Enterobacter taylorae settings
❖ Ferment lactose slowly and are positive for the o-nitrophenyl-B-D-
❖ Unique member because it is lactose negative but ONPG positive galactopyranoside (ONPG) test except S. fonticola
❖ Ability to produce extracellular DNase
❖ Also known for their resistance to a wide range of antimicrobials.
C. Cronobacter (Enterobacter) zakazakii Susceptibility tests must be performed on each isolate to determine
appropriate antimicrobial therapy
❖ Biochemically similar to Enterobacter cloacae ❖ S. marcescens, S. rubidaea, S. plymuthica
❖ Differentiated by its yellow pigment production intensifies at 25C ➢ Often producing a characteristic pink-to-red pigment, prodigiosin,
❖ Has been associated with neonatal sepsis and meningitis especially when the cultures are incubated at room temperature
❖ Typically produces a yellow pigment ➢ Pigment production is typically a characteristic in those strains of
❖ Has been documented as a pathogen in neonates causing meningitis environmental origin
and bacteremia, often coming from powdered infant formula
❖ Isolated from cultures taken from brain abscesses and respiratory and
wound infections

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[BACT211] TRANS 10: Enterobacteriaceae I Prof. Rochelle D. Darlucio, RMT, MPH

A. Serratia odorifera F. Proteus

❖ Normal inhabitants of the gastrointestinal tract
❖ They are motile, non-lactose fermenters capable of deaminating
❖ Dirty, musty odor resembling that of rotten potatoes
phenylalanine
❖ Two biogroups:
❖ Easily identified by their classic “swarming” appearance on culture
➢ S. odorifera biogroup 1
media, however, some strains lack the swarming phenotype
▪ Isolated predominantly from the respiratory tract and is
❖ Has a distinct odor that is often referred to as :chocolate cake” or “burn
positive for sucrose, raffinose and ornithine. In addition,
chocolate” smell
biogroup 1 may be indole-positive (60%)
❖ Widely disseminated in the environment, are normal intestinal
➢ S. odorifera biogroup 2
microbiota and are recognized as opportunistic pathogens
▪ Negative for sucrose, raffinose and ornithine and has been
❖ Ability to deaminate the amino acid phenylalanine
isolated from blood and CSF
❖ Did not ferment lactose
▪ Biogroup 2 may also be indole-positive (50%)
❖ Unique Characteristics:
➢ Rapid Urease Producers (RUP-RXN within 4 hours)
➢ Typical swarming motility (growth in waves or swarms)
➢ H2S producing in TSI but not in LIA

A. P. mirabilis and P. vulgaris

❖ Can produce “swarming”
❖ Colonies on nonselective media such as SBA
❖ “burnt chocolate”
❖ Thought to play a role in the ascending nature of Proteus associated
UTIs
❖ Both species have been isolated from urine, wounds, and ear and
B. Serratia liquefaciens bacteremic infections
❖ Both species hydrolyze urea and produce H2S
❖ Very similar to S. marscesens but differentiated to S. marscesens by ❖ P. mirabilis
its ability to ferment arabinose ➢ Does not produce indole from tryptophan and is ornithine-
positive
❖ P. vulgaris
➢ Produces indole and is ornithine-negative
➢ Ferment sucrose and gives an acid/acid reaction in triple sugar
iron (TSI) agar

B. Proteus penneri
❖ Has been isolated from patients with diarrhea and UTIs, although the
organism’s role in diarrheal disease has not been proven
❖ It can also swarm on nonselective media

Differentiation of Proteus Species

Species Diseases Indole ODC Chlorampenic OL
C. Serratia rubidaea susceptibility
P. mirabilis Wound infection, UTI, - + S
❖ Also produces red pigment prodigiosin pneumonia,
septicemia
P. vulgaris UTI usually + - R
E. Hafnia (IMVC -V+-)
nosocomial
P. penneri UTI/ wound infection - - R
❖ Has been linked to gastroenteritis and is occasionally isolated from
P. Rare human isolate - - S
stool cultures
myxofaciens
❖ A delayed positive citrate reaction is a major characteristic of Hafnia
❖ Proteus spp., are the source of antigen for the Weil-Felix Reaction
❖ Has been isolated from many anatomic sites in humans and in the
(serologic test for the diagnosis of Rickettsial diseases) because it
environment
shares common polysaccharide
➢ P. vulgaris – source of OX2/ OX19
A. Hafnia alvei ➢ P. mirabilis – source of OXK (Kingsbury strain)

❖ Has been associated with gastrointestinal infections G. Morganella (IMVC ++--)

❖ The organism resides in the gastrointestinal tract of humans and many
animals
❖ Are found ubiquitously throughout the environment and are normal
❖ In addition, infections are associated with consumption of
microbiota of the gastrointestinal tract
contaminated food such as meat and dairy products
❖ They are often associated with stool specimens collected from
❖ It is a motile, non-lactose fermenter and is often isolated with other
patients with symptoms of diarrhea
pathogens
❖ Motile but does not swarm
❖ Representative organism of this genus; formerly Enterobacter alvei
❖ Lactose, citrate, H2S and LDC negative (-)
❖ Although it has been recovered from stool and wound specimens, its
❖ Urease and Deaminase, ODC positive (+)
clinical significance is still questionable
❖ M. morganii is the only known species in this genus
❖ Resembles Enterobacter. To differentiate:
❖ Infections include nosocomial infections of UTI and wound
➢ Late Lactose Fermenter
➢ Citrate Negative
❖ To differentiate from Serratia:
➢ DNAse negative
➢ Lipase negative
➢ Gelatinase negative
❖ Treatment is based on antimicrobial susceptibility testing

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[BACT211] TRANS 10: Enterobacteriaceae I Prof. Rochelle D. Darlucio, RMT, MPH

H. Providencia (IMVC ++-+) J. Citrobacter
❖ Are most commonly associated with urinary tract infections and
isolated from the feces of children with diarrhea ❖ Are inhabitants of the intestinal tract
❖ These organisms may be associated with health care associated ❖ The most common clinical manifestation in patients as a result of
outbreaks infection occurs in the urinary tract. However, additional infections
❖ Implicated in diarrheal disease among travelers including wound infections, respiratory tract infections, bacteremia,
❖ Lactose negative, deaminase positive, H2S negative, motile but no endocarditis, septicemia meningitis, brain abscess and neurologic
swarming, ferments mannose and citrate positive complications
❖ Has IMV reaction of positive, positive, negative, positive ❖ Resembles Salmonella but differs because it is:
➢ ONPG positive
A. Providencia rettgeri ➢ LDC negative
➢ Slow Urease Producer (SUP)
❖ Important human pathogen which are commonly associated with UTIs
❖ Is a documented pathogen of the urinary tract and has caused
and respiratory tract infections
occasional outbreaks in health care settings
❖ Most Citrobacter spp., hydrolyze urea slowly and ferment lactose,
❖ It has also been implicated in diarrheal disease among travelers
producing colonies on MAC agar that resemble those of E. coli.
❖ All species grow on Simmons citrate medium (hence the genus name)
and give a positive methyl red test

A. Citrobacter freundii

❖ Can be isolated in diarrheal stool cultures, and although it is a known

extraintestinal pathogen, its pathogenic role in intestinal disease is not
established
B. Providencia stuartii ❖ Has been associated with infectious diseases acquired in hospital
settings; UTIs, pneumonias and intraabdominal abscesses have been
❖ Has been implicated in outbreaks in burn units and has been isolated reported
from urine cultures ❖ Associated with endocarditis in intravenous drug abusers
❖ Most (80%) C. freundii produce H2S and some strains (50%) fail to
C. Providencia alcalifaciens ferment lactose, the colony morphology of C. freundii on primary
❖ Most commonly found in the feces of children with diarrhea selective media can be mistaken for Salmonella when isolated from
stool cultures
❖ Indole and sucrose vaiable and positive for melibiose
D. Providencia rustigianii
❖ Rarely isolated, and its pathogenicity also remains unproven

E. Providencia heimbachae

❖ Has yet to be isolated from any clinical specimens

H. Edwardsiella (IMVC ++--)

❖ Negative for urea and positive for lysine decarboxylase, H2S, and
indole and do not grow on Simmons citrate
❖ Resembles E. coli but differs because it is H2S positive and lactose B. Citrobacter koseri
negative
❖ A pathogen documented as the cause of nursery outbreaks of
A. Edwardsiella tarda neonatal meningitis and brain abscesses
❖ Variable for dulcitol and sucrose and negative for melibiose and all
❖ An opportunistic, causing bacteremia and wound infections other reactions are positive

B. Edwardsiella hoshinae

❖ Has been isolated from snakes, birds and water

C. Edwardsiella ictaluri

❖ Causes enteric septicemia in fish

C. Citrobacter braakii

❖ A rare human pathogen associated with community-acquired

infections including a septicemia in a patient with cervical cancer
❖ Positive for ODC and variable for indole, dulcitol and melibiose

I. Erwinia and Pectobacterium

❖ Plant pathogens and are not significant in human infections

❖ Erwinia organisms grow poorly at 37C and fail to grow on selective
media, such as EMB and MAC, and other differential media typically
used for the isolation of enterics

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[BACT211] TRANS 10: Enterobacteriaceae I Prof. Rochelle D. Darlucio, RMT, MPH

D. Citrobacter amalonaticus B. Salmonella enteritidis

❖ Frequently found in feces, but no evidence has been found that it is a ❖ Associated with infections acquired from the ingestion of eggs or
causative agent of diarrhea chicken
❖ Has been isolated from sites of extraintestinal infections such as blood
and wounds Salmonella Nomenclature Summary
❖ Positive for indole and ODC Salmonella Former Subspecies Key notes
Differentiation of Citrobacter species subgroup genus
Species Diseases Indole H2S ODC 1 Salmonella Choleraesuis Usually isolated from humans
C. Septicemia Highly pathogenic to humans
- + V Almost all clinical isolates of
freundii Wound infection
Gastroenteritis Salmonella are from this group
C. Meningitis Includes S. typhi, S. paratyphi, S.
+ - + cholerasuis, S. gallinarum, S.
diversus
pullorum
2 Salmonella Salamae Usually isolated from cold blooded
V. PRIMARY INTESTINAL PATHOGENS OF FAMILY 3a Arizona Arizonae animals and not from human or other
ENTEROBACTERIACEAE 3b Arizona Diarizonae warm blooded animals
4 Salmonella Houtenae
A. Salmonella (IMVC -+--) 5 Salmonella Bongar
❖ Facultatively anaerobic bacilli, motile, gram-negative rods commonly 6 Salmonella Indica
isolated from the intestines of humans and animals
❖ Identification is primarily based on the ability of the organism to use A. Virulence Factors
citrate as the sole carbon source and lysin as a nitrogen source in
combination with hydrogen sulfide production ❖ Regulated by genes on 2 pathogenicity islands that facilitate the
❖ Serotypes are differentiated based on the characterization of the heat- attachment, engulfment and replication of bacteria in intestinal cells
stable O antigen, included in the lipopolysaccharide; the heat-labile H and macrophages
antigen flagellar protein, and the heat-labile Vi antigen, capsular ❖ Bacteria are transported from the intestines to liver, spleen and bone
polysaccharide marrow by macrophages
❖ Salmonella spp., can colonize all animals particularly poultry and ❖ Fimbriae
cause disease in a variety of host including humans. However, ❖ Enterotoxin
Salmonella typhi is a strict human pathogen that can cause severe ➢ Produced by certain Salmonella strains that cause gastroenteritis
disease and survive in the gall bladder establishing chronic carriage
❖ Produce clear, colorless, non-lactose fermenting colonies B. Antigenic Structures
❖ Colonies with black centers are seen if the media (e.g., HE or XLD)
contain indicators for H2S production ❖ Somatic O Antigens
❖ Biochemical Features: ➢ Heat stable
➢ Do not ferment lactose ➢ Located on the outer membrane of the cell wall
➢ They are negative for indole, Voges-Proskauer test, ❖ Flagellar H Antigens
phenylalanine deaminase and urease ➢ Heat labile
➢ Most produce H2S; a major exception is Salmonella paratyphi A, ❖ Capsular K Antigens
which does not produce H2S ➢ Heat-labile Vi (is a surface polysaccharide capsular antigen
➢ Does not grow in medium containing potassium cyanide found in Salmonella typhi and few strains of Salmonella
❖ Pathogenic for humans and animals cholerasuis)
❖ Anima sources includes: ➢ Plays a significant role in preventing phagocytosis oof the
➢ Poultry (chicken, ducks and turkeys) organism
➢ Pets (dogs, cat, hamsters, turtles) ➢ Blocks the O antigen during serologic typing but may be removed
➢ Others (pigs, sheep, cows, donkeys, snakes, parrots) by heating
❖ S. typhi (most common cause of typhoid fever)
❖ S. cholerasuis (most common cause of bacteremia) C. Clinical Infections
❖ S. typhimurium (most common cause of enterocolitis and
gastroenteritis)
❖ Salmonella-associated gastroenteritis is typically accomplished by ❖ Acute gastroenteritis or food poisoning characterized by vomiting and
diarrhea, fever, and abdominal cramps diarrhea
❖ Cases of gastroenteritis may cause extraintestinal infections such as ❖ Typhoid fever, the most severe form of enteric fever, caused by
bacteremia, urinary tract infection or osteomyelitis Salmonella serotype Typhi, and enteric fevers caused by other
❖ Transmission may be fecal-oral, person-to-person, or contact with Salmonella serotypes (e.g., Salmonella paratyphi and choleraesuis)
infected animals ➢ The clinical features of enteric fevers include prolonged fever,
bacteremia, involvement of the reticuloendothelial system
particularly the liver, spleen, intestines and mesentery,
A. Salmonella typhi dissemination to multiple organs
❖ Nontyphoidal bacteremia
❖ The primary identifiable Salmonella serotypes are Salmonella
serotype typhi associated with a severe disease called typhoid fever B. Shigella (IMVC ++--)
❖ Diarrhea and vomiting are not associated with typhoid fever
❖ The symptoms are often headache, abdominal cramping, constipation ❖ Cause bacillary dysentery
and high fever ❖ Inert bacilli (yields negative reaction to most tests)
❖ The patient may present with a rash and appear confused ➢ They are nonmotile
❖ Human carriers have been identified. The disease is transmitted ➢ Except for certain types of S. flexneri, they do not produce gas
person-to-person or through contaminated food and water from glucose
➢ They do not hydrolyze urea
➢ They do not produce H2S
➢ They do not decarboxylate lysine
❖ S. dysenteriae, S. flexneri, S. boydii, S. sonnei are nonmotile, lysine
decarboxylase negative; citrate, malonate, and H2S negative; non-
lactose fermenting; gram-negative rods that grow well on MacConkey
agar
❖ All strains ferment glucose without gas production except a few strains

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[BACT211] TRANS 10: Enterobacteriaceae I Prof. Rochelle D. Darlucio, RMT, MPH

Subgrouping of Shigella species ➢ Pneumonic Form
Subgroup Species #of Synonym ▪ Occurs secondary to bubonic plague or septicemic form
Subtypes/ when organisms proliferate in the bloodstream and
Serotypes respiratory tract
A S. 1-12 Shiga’s ▪ Can be a primary infection if the bacteria are inhaled
dysenteriae Bacillus ▪ Fatality rate is high – essentially 100% in untreated patients
B S. flexneri 1-8 Strong’s ❖ Gram-negative, short, plump bacillus
bacillus ❖ When stained in methylene blue or Wayson stain, it shows intense
C S. boydii 1-18 New castle staining at each end of the bacillus, referred to as bipolar staining
Manchester which gives it a “safety pin” appearance
bacillus ❖ May be isolated on routine culture medium
D S. sonnei 1 Duval’s ❖ Although it grows at 37C, it has a preferential growth temperature of
Bacillus 25C to 30C

A. Antigenic Structures

❖ Each subgroup has several serotypes. Serotyping is base on the

somatic lipopolysaccharide O antigen
❖ Shigella resembles E. coli but are lactose negative and non-motile
❖ All Shigella spp. posses O antigens, and certain strains can possess
K antigens
❖ Nonmotile; therefore, they lack H antigens
B. Yersinia enterocolitica
❖ The Shigella spp. attached invade and replicate in the cell’s lining in
the colony. They replicate the cytoplasm phagocytic cells and move
cell-to-cell without extracellular exposure, thus, protected from ❖ Found in gastrointestinal tract of swine, rodents and dogs and cats
immune mediated clearance ❖ Several Forms
❖ Shiga toxin can also mediate damage the glomerular endothelial cells ➢ Acute enteritis
resulting in renal failure called hemolytic uremic syndrome (HUS) ➢ Appendicitis-like syndrome
➢ Arthritis
B. Clinical Infections ➢ Erythema nodosum (inflammation of fat cells under the skin,
tender red nodules)
❖ Shigellosis/ Bacillary Dysentery
➢ Vary from asymptomatic to severe forms of the disease
➢ The initial symptoms, marked by high fever, chills, abdominal
cramps and pain accompanied by tenesmus, appear
approximately 24 to 48 hours after ingestion of the organisms
➢ Early Stage
▪ Incubation period for 1-7 days
▪ Fever, abdominal cramping and pain, diarrhea
➢ Diarrheic Stage
▪ Watery diarrhea for 3 days
➢ Dysenteric Stage
▪ Frequent stools with blood, puss and mucus C. Yersinia pseudotuberculosis
▪ Bacteria had invaded the lining of the GIT
❖ Found in a variety of wild and domesticated animals including rodents,
C. Yersinia birds and rabbits
❖ The mode of transmission is by contact with infected animals ir the
❖ The best known for human pathogen for this genus are Y. pestis ingestion of contaminated food or water
(highly fatal systemic disease known as plague) and Y. enterocolitica ❖ Pathogen primarily of rodents, particularly guinea pigs
(responsible for gastroenteritis in cold weather climates in northern ❖ Causes a disease characterized by caseous swellings called
European and north American as well as two additional diseases: pseudotubercles
chronic inflammation of the terminal ileum with enlargement of the ❖ Appears as a typical-looking plague bacillus
mesenteric lymph nodes resulting in pseudoappendicitis primarily a ❖ It can be differentiated from Y. pestis by its motility at 18C to 22C,
disease in children. Blood transfusion related bacteremia) production of urease and ability to ferment rhamnose
❖ Y. enterocolitica can grow at 4C so this organism can multiply to high
concentrations in blood products stored in a refrigerator. Late lactose
fermenter, ONPG positive, weakly fermentative, requires cold
enrichment. On selective media, it appears as bull’s eye colonies
❖ Are gram-negative; catalase, oxidase and indole positive; non-lactose
fermenting; facultative anaerobes capable of growth at temperatures
ranging from 4C to 43C

A. Yersinia pestis
❖ Causative agent of the ancient disease plague Differentiation of Yersinia Species
❖ Plague is a disease primarily of rodents Species Motility (C) Disease Fermentation
❖ It is transmitted to humans by bites of fleas, which are its most 22 25 37 Sucr. Rham. Sorb.
common and effective vectors Y. pestis + - - Plague - - -
❖ Plague in 3 Forms: Y. entero + + - Entero + - +
➢ Bubonic or Glandular Form colitica colitis
▪ Most common, usually results from an infected flea Y. pseudo + + - Lympha - + -
▪ Symptoms appear 2 to 5 days after infection tuberculosis denitis
▪ Symptoms include high fever with painful regional lymph
nodes known as buboes (swollen lymph nodes)
➢ Septicemic Form
▪ Bacteria spread to bloodstream

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[BACT211] TRANS 10: Enterobacteriaceae I Prof. Rochelle D. Darlucio, RMT, MPH

VI. OTHER GENERA OF THE FAMILY ENTEROBACTERIACEAE H. Moellerella

A. Budivicia ❖ The genus Moellerella contains one species, M. wisconsensis

❖ Positive for citrate, methyl red, lactose and sucrose
❖ Based on DNA hybridization, Budivicia aquatica is a group of related ❖ Negative for lysine, ornithine, arginine decarboxylase and indole and
organisms; however, they are not as closely related to the other it resembles E. coli growing on enteric media
members of Enterobacteriaceae ❖ The clinical significance of this organism has not been established,
❖ Usually found in water, however, they occasionally occur in clinical although it has been isolated from feces in two cases of diarrhea,
specimens infected gallbladders and a bronchial-aspirates

B. Buttiauxella I. Obesumbacterium

❖ Consists of seven species isolated from water ❖ Fastidious, slow-growing organisms at 37C and have not been found
❖ Only B. agrestis and B. noackiae have been isolated from human in human specimens
specimens
❖ Biochemically, these organisms are similar to both Citrobacter and J. Photorhabdus
Kluyvera species, but DNA hybridization distinctly differentiates
Buttiauxella from both genera ❖ Three species:
➢ P. luminescens
C. Cedecea ➢ P. asymbiotica
➢ P. temperate
❖ Composed of five species: C. davisae, C. lapagei, C. neteri and ❖ Their natural habitat is the lumen of entomopathogenic nematodes,
Cedecea species types 3 and 5 but strains have occasionally been isolated from human specimens
❖ Most have been recovered from sputum, blood, and wounds ❖ They occur in two phases with the property of luminescence in phase
❖ C. davisae is the most commonly isolated species 1 only
❖ Most strains produce pink, red, orange, yellow or green-pigmented
D. Ewingella colonies on nutrient agar and especially on nutrient-rich media such
as trypticase soy agar and egg yolk agar
❖ Ewingella americana is the only species of the genus Ewingella ❖ They are also negative for nitrate reduction
❖ Most isolates have come from human blood cultures or respiratory
specimens and exhibit resistance to multiple antimicrobial agents K. Rahnella
❖ Ewingella was first thought to be related to Cedecea, however, DNA
hybridization confirmed the placement of these organisms in separate ❖ Rahnella aquatilis is the name given to a group of water bateria that
genera are psychrotolerant, growing at 4C
❖ These organisms have no single characteristics that distinguishes
E. Kluyvera them from the other members of the Enterobacteriaceae
❖ They resemble E. agglomerans, however, they can be distinguished
❖ Three closely related species: K. ascorbata (the most common clinical by a weak phenylalanine deaminase reaction; the fact that they are
isolate), K. cryocrescens, K. georgiana negative for potassium cyanide (KCN), gelatin, lysine, ornithine and
❖ They have been found in respiratory, urine and blood cultures motility; and their lack of yellow pigmentation
❖ Most strains are nonpigmented but occasional isolates may produce
a reddish-blue or violet pigment L. Trabulsiella
❖ All species resemble E. coli colonies growing on MAC agar
❖ Cephalothin and carbenicillin disk susceptibility tests separate the first ❖ Trabulsiella guamensis is the only species in this genus known to be
two species: K. cryocrescens shows large zones of inhibition and K. associated with humans, and although it is very rarely isolated, it is
ascorbata has small zones biochemically similar to Salmonella
❖ In addition, K. ascorbata does not ferment glucose at 5C, whereas K. ❖ The type strain was isolated from vacuum-cleaner contents on the
cryocrescens ferments glucose at this temperature island of Guam when environmental indoor dirt samples were being
collected
F. Leclercia
M. Yokenella
❖ Proposed in 1986 for 58 isolates from human clinical specimens,
including blood, urine, sputum and feces and 27 isolates from ❖ Yokenella regensburgei was first thought to be another species of
nonhuman species Hafnia, but DNA hybridization showed a 15% relatedness, which was
❖ It has been isolated more recently in pure culture from a septicemia not sufficient to include these organisms in that genus
and wounds ❖ They are biochemically similar to Hafnia but differ primarily by yielding
❖ L. adecarboxylata, which can have a yellow pigment but only on initial negative Voges-Proskauer test results
isolation ❖ Yokenella strains have been isolated from human specimens, but
❖ Similar IMViC reactions to E. coli, negative for lysine and ornithine further study is required to determine their significance in human
decarboxylase and arginine dihydrolase disease

G. Leminorella VII. IMVC REACTIONS

❖ Was proposed as a genus with two species: L. grimontii and L. IMVC Reactions (Escherichia)
richardii I M V C
❖ Produce H2S and have shown weak reactions with Salmonella E. coli + + - -
antisera
❖ However, complete biochemical testing differentiates Leminorella
from Salmonella; Leminorella spp. are relatively inactive IMVC Reactions (Klebsiella spp.)
❖ The clinical significance of these organisms is unknown; however, I M V C
they have been isolated from patients with hospital-acquired infections K. pneumoniae - - + +
K. oxytoca + v + +
K. ozaenae - + - V

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[BACT211] TRANS 10: Enterobacteriaceae I Prof. Rochelle D. Darlucio, RMT, MPH

IMVC Reactions (Enterobacter spp.)
I M V C
E. aerogenes - - + +
E. cloacae - - + +
E. agglomerans V V V V

IMVC Reactions (Citrobacter spp.)

I M V C
C. freundii V + - V
C. diversus/ koseri + + - +

IMVC Reactions (Serratia spp. & Hafnia alvei)

I M V C
S. marcescens - V + +
S. liquifaciens - + + +

IMVC Reactions (H. alvei)

I M V C
H. alvei - V V -

IMVC Reactions (PPM Organisms)

Proteus spp. I M V C
P. mirabilis - + V V
P. vulgaris + + - V
P. penneri - + - -
Providencia spp. I M V C
P. rettgeri + + - +
P. stuartii + + - +
Morganella I M V C
M. morganni subsp. + + - -
morgani

IMVC Reactions (some species & Shigella)

Salmonella spp. I M V C
Most Serotypes - + - +
Shigella spp. I M V C
ABC V + - -
D - + - -

IMVC Reactions (Yersinia spp. & Edwardsiella tarda)

Yersinia spp. I M V C
Y. enterocolitica V + - -
Y. frediriksenii + + - V
P. penneri + + - -
E. tarda + + - -

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OLFU Non-Enteric Gastrointestinal Pathogens LEC 2020-2021

2nd SEM
College of Medical CLINICAL BACTERIOLOGY 10 BACT211
LEC
Laboratory Science Transcriber: Riyoma Surell FINALS
Date: May 04, 2021 TRANS 11
Batch 2023

➢ 3 major subgroups of V. cholerae

Outline ▪ Share a common flagellar (H) antigen and somatic (O)
At the end of the session, the student must be able to learn: antigen
I. Vibrio ▪ V. cholerae O1
A. Vibrio cholerae ▪ V. cholerae O139
1. Virulence Factors ▪ V. cholerae non-O1
B. Vibrio parahaemolyticus
➢ V. cholerae O1 organisms are divided into the following
1. Clinical Manifestations
C. Vibrio vulnificus serotypes:
D. Vibrio alginolyticus ▪ Ogawa (A, B) – India
E. Laboratory Diagnosis ▪ Inaba (A, C) – Philippines
1. Specimen Collection and Transport ▪ Hikojima (A, B, C) – Japan
2. Culture Media
II. Aeromonas Optimum Temperature for Growth
A. General Characteristics
1. Mesophilic Group 15C 25C 42C
2. Psychrophilic Group - - +
B. Clinical Manifestations
C. A. caviae
D. A. hydrophila and A. veronii
E. Laboratory Diagnosis
1. Selective Culture Media
2. Presumptive Identification
III. Plesiomonas
A. Clinical Manifestations
B. Laboratory Diagnosis
1. Culture Media
2. Identification
IV. Campylobacter
A. Laboratory Diagnosis
1. Specimen Collection and Transport
2. Incubation
3. Colony Morphology A. Vibrio cholerae

I. VIBRIO

❖ Family Vibrionaceae
❖ Commonly found in a wide variety of aquatic environments, including
fresh water, brackish or estuarine water, and marine or salt water
❖ Temperature-sensitive
❖ Can easily be isolated from water, suspended particulate matter,
algae, plankton, fish and shellfish
❖ Risk of infection from all Vibrio spp. can be reduced by the avoidance ❖ Vibrio cholerae O1
of eating raw or undercooked shellfish, particularly in warm summer ➢ Causative agent of cholera, also known as Asiatic cholera or
months epidemic cholera
❖ Gram negative (-) comma shaped, curved, straight bacilli ➢ Agglutinate O1 anti-sera and produces a very potent enterotoxin
❖ Facultative anaerobe ➢ Cholera Toxin or Choleragen responsible for the massive
❖ Catalase negative, oxidase positive, able to reduce nitrate to nitrite amount of water and electrolytes loss
except V. metschnikovii ➢ Characterized by “Rice Water Stool” watery stool with gray
❖ Motile – “shooting start motility” – monotrichous flagella mucin
❖ Halophilic except V. cholerae and V. mimicus ➢ Dehydration is usually the cause of death
❖ Non-Inositol fermenter ➢ Patient exhibits sunken eyes, washerwoman’s hands, and pallor
❖ Susceptible to vibriostatic agent O/129 (2,4 diamino-6,7-
diisopropylpteridine) V. cholerae 01 BIOTYPE DIFFERENTIATION
➢ Antibiotic Disk: 150ug vibriostatic disk Biotype Key Note BAP Chicken RBC VP Polymyxin
❖ String Test Positive Hemolytic Agglutination Test B Suscept.
➢ Emulsify the colony of Vibrio spp. to 0.5% sodium deoxycholate Pattern
(formation of string) Classical Pandemics Non- - - S
of the past hemolytic
El Tor Recent B + + R
pandemics hemolytic

❖ Non-O1 serogroup
➢ Strains have been implicated in a variety of extraintestinal
infections, including cholecystitis, ear infections, cellulitis and
septicemia
➢ No enterotoxin
➢ Implicated in a variety of extraintestinal infections, including
cholecystitis, ear infections, cellulitis and septicemia
❖ V. cholerae serogroup O139
➢ first V. cholerae non-O1 strain producing epidemic disease
▪ Share cross-reacting antigens with Aeromonas trota

❖ Antigenic Structure

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[BACT211] TRANS 11: Non-Enteric Gastrointestinal Pathogens I Prof. Rochelle D. Darlucio, RMT, MPH

1. Virulence Factors ➢ Thiosulfate Citrate Bile Salt Sucrose (TCBS) agar –

recommended
➢ Alkaline peptone water with 1% NaCl (pH 8.5)
❖ Cholera toxin (CT)
➢ CHROMagar Vibrio (CHROMagar Microbiology, Paris) for the
➢ Water and electrolyte loss
isolation and identification of V. cholerae, V. parahaemolyticus,
❖ Zonula occludens (Zot) toxin (enterotoxin)
and V. vulnificus
❖ Accessory cholera enterotoxin (Ace) toxin
❖ O1 and O139 somatic antigens
❖ Hemolysin/ cytotoxins
❖ Motility and chemotaxis
➢ Mediate the distribution of microorganism
❖ Mucinase
➢ Allow the penetration of the mucous layer
❖ Toxin coregulated pili (TCP) pili
➢ Provide

B. Vibrio parahaemolyticus

❖ Second most common Vibrio species implicated in gastroenteritis after

V. cholerae
❖ Primary cause of so-called summer diarrhea in Japan
➢ V. parahaemolyticus serotype O3:K6
▪ has been implicated in numerous foodborne outbreaks
❖ Found in aquatic environments, but appears to be limited to coastal or
estuarine areas Sucrose-fermenting Non-sucrose-fermenting (green)
❖ Halophilic (requirement of 1% to 8% NaCl (yellow colonies) species species
❖ V. cholerae ❖ V. mimicus
1. Clinical Manifestations ❖ V. alginolyticus ❖ V. parahaemolyticus
❖ V. fluvialis ❖ P. damsela
❖ Gastrointestinal disease (self-limited) ❖ V. furnissii ❖ Most V. vulnificus strains
❖ Watery diarrhea, moderate cramps or vomiting and little if any fever ❖ V. cincinnatiensis ❖ **not all Vibrio spp. grow on TCBS,
❖ Kanagawa phenomenon ❖ V. metschnikovii especially Grimontia (formerly Vibrio)
➢ Heat-stable hemolysin produced by V. parahaemolyticus that is ❖ Some V. vulnificus hollisae
able to lyse human erythrocytes in a special, high-salt mannitol
medium (Wagatsuma agar) Differentiation of Pathogenic Vibrio Species
Pathogenic TCBS Lact. Cholera String Kanagawa
Spp. Ferm. Red React. Test Pheno.
C. Vibrio vulnificus
V. cholerae SF- - + + -
yellow
❖ Can be found in marine environments on the Atlantic, gulf, and pacific V. SF- - - - -
coasts of north America alginolyticus yellow
❖ Lactose positive among Vibrio spp. V. para NSF- - - - +
❖ Infections include: haemolyticu blue
➢ Primary septicemia s green
➢ Wound infections V. vulnificus V + - - -

D. Vibrio alginolyticus ❖ Nitroso indole reaction – double indicator test (to detect indole and
nitrate reduction, positive: red)
❖ Least pathogenic for humans and is the one most infrequently isolated
❖ It is a common inhabitant of marine environments II. AEROMONAS
❖ A strict halophile, requiring at least 1% NaCl; it is able to tolerate up to
10% NaCl ❖ Aeromonadaceae
❖ Almost all isolated originate from extraintestinal sources, such as eye ❖ Ubiquitous, oxidase-positive, glucose-fermenting, gram-negative rods
and ear infections or wound and burn infections ❖ Widely distributed in freshwater, estuarine and marine environments
worldwide
E. Laboratory Diagnosis ❖ Frequently isolated from retail produce sources and animal meat
products
1. Specimen Collection and Transport ❖ Responsible for a diverse spectrum of disease syndromes in warm
and cold-blooded animals, including fish, reptiles, amphibians,
❖ Swabs are acceptable if they are transported in an appropriate holding mammals and humans
medium, such as Cary-Blair, to prevent desiccation ❖ Key Characteristics
❖ Buffered glycerol Saline is not recommended as a transport or ➢ Gram negative straight bacilli
holding medium ➢ Facultative anaerobe
❖ Feces is preferable, but rectal swabs are acceptable during the acute ➢ Fermentative
phase of diarrheal illness ➢ Indole positive
➢ Oxidase positive
2. Culture Media ➢ Motile
➢ Resistant to vibriostatic agent O/129 (2,4 diamino-6,7-
❖ Salt Concentration (0.5%) diisopropylpteridine)
➢ In most commonly used laboratory media, such as nutrient agar ➢ Non-Inositol Fermenter
or sheep blood agar (SBA) ➢ A. hydrophila most common human isolate
❖ SBA or Chocolate (CHOC) Agar
➢ Medium to large colonies that appear smooth, opaque and A. General Characteristics
iridescent with a greenish hue
❖ Selective Differential Media ❖ Straight rods (1.0 to 3.5 um long by 0.3 to 1.0um wide)
➢ MAC
➢ Cefsulodin-Irgasan-Novobiocin (CIN) agar

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[BACT211] TRANS 11: Non-Enteric Gastrointestinal Pathogens I Prof. Rochelle D. Darlucio, RMT, MPH

1. Mesophilic Group (Optimal Growth Around 37C) 1. Selective Culture Media

❖ A. hydrophila complex ❖ MAC Agar

➢ A. hydrophila ➢ Ferment lactose (A. caviae)
➢ A. bestiarum ➢ A. trota
➢ Certain motile strains of A. salmonicida ▪ Unusual universal susceptibility to ampicillin
❖ A. veronii complex ❖ CIN Medium
➢ A. veronii biovar sobria (formerly misidentified as A. sobria) ➢ Pink-centered colonies from the fermentation of mannitol, with an
➢ A. veronii biovar veroni uneven, clear apron resembling Yersinia enterocolitica
➢ A. jandaei ➢ Alkaline peptone water
➢ A. trota
➢ A. schubertii
❖ A. caviae complex
➢ A. caviae
➢ A. media
➢ A. eucrenophila

2. Psychrophilic Group

❖ A. salmonicida
➢ Which is a fish pathogen with several subspecies
➢ Nonmotile 2. Presumptive Identification
➢ Grows best at 22C to 25C
➢ Not considered human pathogens ❖ Oxidase Test (Aeromonas = positive)
❖ All aeromonads, in general, can typically grow from 4C to 42C ❖ Spot indole test on suspicious colonies on SBA, especially B-
hemolytic colonies
B. Clinical Manifestations ❖ Ability to grow in the presence of NaCl

❖ Intestinal Infections
1. Acute secretory diarrhea
▪ Often accompanied by vomiting
2. Acute dysenteric form of diarrhea
▪ Similar to shigellosis, with blood and mucus
3. Chronic diarrhea
▪ Usually lasting more than 10 days
4. Cholera-like disease
▪ Including rice water stools
5. Nebulous syndrome
▪ Commonly referred to as traveler’s diarrhea (similar to
enterotoxigenic E. coli)
❖ Extraintestinal Infections
➢ Septicemia, meningitis and wound infections III. PLESIOMONAS
➢ Osteomyelitis, pelvic abscesses, otitis, cystitis, endocarditis, ❖ Oxidase-positive
peritonitis, cholecystitis ❖ Glucose fermenter
➢ Keratitis associated with contact lens wear ❖ Facultatively anaerobic
➢ Endophthalmitis in healthy and immunocompromised individuals ❖ Gram-negative bacilli that occur in singly, in pairs or in short chains or
➢ Aeromonad wound isolates: filamentous
▪ A. hydrophila ❖ Motile by monotrichous or two to five lophotrichous flagella
▪ A. veronii biovar sobria ❖ String test negative
▪ A. schubertii ❖ Inositol fermenter
❖ Can be serotyped by somatic O antigens and their flagellar H antigen
C. A. caviae
A. Clinical Manifestations
❖ Most frequently associated with gastrointestinal infections, especially ❖ Gastroenteritis
in neonate and pediatric populations ➢ The more common watery or secretory diarrhea
❖ It has been associated with inflammatory bowel disease ➢ A subacute or chronic disease that lasts from 14 days to 2 to 3
months
D. A. hydrophila and A. veronii (biovars sobria and veronii) ➢ A more invasive, dysenteric form that resembles colitis
❖ Extraintestinal Infection
❖ Hemolytic-uremic syndrome or kidney disease that might require ➢ Bacteremia and meningitis usually occur only in severely
kidney transplantation immunocompromised patients or neonates
❖ A. veronii biovar sobria
➢ Linked to cholera-like characterized by abdominal pain, fever and B. Laboratory Diagnosis
nausea

E. Laboratory Diagnosis 1. Culture Media

❖ Shiny, opaque, nonhemolytic colonies appear, with a slightly raised
❖ Large, round, raised, opaque colonies with an entire edge and a center and a smooth and entire edge
smooth, often mucoid surface ❖ Inositol Brilliant Green Bile Agar
❖ Extremely strong odor is present ➢ Can enhance the isolation of plesiomonads
❖ Pigmentation ranges from translucent and white to buff-colored ➢ White to pink on this medium, and most coliform colonies are
❖ Hemolysis is variable on SBA, but most major clinical species, such green or pink
as A. hydrophila, A. veronii biovar sobria, and A. jandaei, display ➢ Plesiomonas shigelloides
strong B-hemolysis ❖ Will not grow on TCBS agar
❖ CIN
➢ Opaque (non-mannitol-fermenting) colonies with an opaque
apron
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[BACT211] TRANS 11: Non-Enteric Gastrointestinal Pathogens I Prof. Rochelle D. Darlucio, RMT, MPH

2. Identification 2. Incubation

❖ Positive Oxidase Activity ❖ C. jejuni and other enteric campylobacters grow optimally at 42C
➢ Separates it from other Enterobacteriaceae ❖ Campylobacter and Helicobacter spp.
❖ Sensitivity to the Agent O/129 ➢ Require a microaerophilic and capnophilic environment
➢ Separates it from Aeromonas ➢ For Campylobacter spp:
❖ Ability to ferment Inositol ▪ 5%
➢ Separates it from all Aeromonas and almost all Vibrio spp. ▪ 10%CO
❖ Ability to grow in nutrient broth with 0% NaCl ▪ 85% N
❖ Inability to grow in nutrient broth with 6%NaCl ➢ For Helicobacter spp:
➢ To separate from the halophilic Vibrio spp. ▪ 5% to 10% O
▪ 5% to 12% CO
IV. CAMPYLOBACTER ❖ The incubation time should be extended to 72 hours to isolate enteric
Campylobacter spp. more efficiently
❖ Key Characteristics
➢ Gram negative comma, curved, S-shaped, seagull wing shaped 3. Colony Morphology
bacilli
➢ Microaerophilic (5% O2) ❖ C. jejuni and other enteric Campylobacters
➢ Capnophilic ➢ Moist, runny looking and spreading, usually nonhemolytic; some
➢ Motile (one polar flagellum) are round and raised and others may be flat
➢ Oxidase positive ❖ C. fetus subsp. fetus
➢ Non-fermentative ➢ Produces smooth, convex, translucent colonies
➢ Sodium Hippurate Hydrolysis positive ❖ C. mucosalis and C. hyointestinalis
➢ Urease negative ➢ Can produce a dirty yellow pigment
➢ Exhibit a characteristic ❖ Definitive Identification
➢ Motility on hanging drop preparations or when visualized under ➢ Oxidase positive
phase contrast microscopy ➢ Can grow at 42C in a microaerophilic environment
❖ Have been known to cause abortion in domestic animals, such as ➢ Positive Hippurate Hydrolysis for C. jejuni
cattle, sheep and swine and are primarily zoonotic organisms ➢ H. pylori – urease positive
❖ Patients suffering from Guillain-Barre Syndrome (GBS) often test ➢ Christensen’s urea medium and incubated at 37C for 2 hours
positive for Campylobacter antibodies ➢ Non invasive indirect test
❖ Campylobacter jejuni ▪ Urea Breath Test
➢ Most common cause of bacterial gastroenteritis
➢ Cramps and bloody diarrhea
❖ Campylobacter fetus subsp. fetus
➢ Has been isolated most frequently from blood cultures
➢ Rarely associated with gastrointestinal illness
❖ Helicobacter pylori
➢ Strongly associated with gastric, peptic and duodenal ulcers as
well as with gastrointestinal carcinoma
➢ Major cause of type B gastritis
❖ Helicobacter cinaedi and Helicobacter fennelliae
➢ Have been associated with human gastroenteritis, generally in
immunocompromised patients

A. Laboratory Diagnosis

1. Specimen Collection and Transport

❖ Specimen Collection and Transport

➢ Can be recovered in several routine blood culture media
➢ Transport medium: Cary-Blair
➢ Buffered Glycerol Saline
▪ Toxic to enteric campylobacters and should therefore be
avoided
❖ H. pylori can be recovered from gastric biopsy materials
➢ Transport medium: Stuart Medium
➢ For tissue samples: cysteine-brucella broth with 20% glycerol
▪ Frozen at -70C

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OLFU Aerobic Gram-Positive Bacilli LEC 2020-2021

2nd SEM
College of Medical CLINICAL BACTERIOLOGY 11 BACT211
LEC
Laboratory Science Transcriber: Riyoma Surell FINALS
Date: May 11, 2021 TRANS 12
Batch 2023

A. Corynebacterium diphtheriae
Outline
At the end of the session, the student must be able to learn:
I. Non-spore Forming, Nonbranching, Catalse-Positive Bacilli
A. Corynebacterium
A. Corynebacterium diphtheriae
B. Corynebacterium amycolatum
C. Corynebacterium jeikeium
D. Corynebacterium pseudodiphtheriticum
E. Corynebacterium pseudotuberculosis
F. Corynebacterium striatum
G. Corynebacterium ulcerans
H. Corynebacterium urealyticum
B. Rothia
C. Listeria monocytogenes
II. Nonspore Forming, Nonbranching, Catalase Negative Bacilli 1. Virulence Factor
A. Erysipelothrix rhusiopathiae
B. Arcanobacterium and Trueperella
❖ Diphtheria toxin
A. A. haemolyticum
C. Gardnerella vaginalis ➢ Major virulence factor
III. Nonspore Forming, Branching, Aerobic Actinomycetes ➢ This toxin is produced by strains of C. diphtheriae infected with a
A. Nocardia lysogenic B-phage, which carriers the gene tox for diphtheria
B. Actinomadura toxin
C. Streptomyces ▪ Nontoxigenic strains can be converted to tox positive by
D. Gordonia infection with the appropriate B-phage
E. Rhodococcus
▪ Only toxin-producing C. diphtheriae causes diphtheria,
F. Tropheryma whipplei
IV. Spore Forming, Nonbranching, Catalase Positive Bacilli however C. ulcerans and C. pseudotuberculosis which
A. Bacillus belong to the C. diphtheriae group can also produce the
B. Bacillus anthracis toxin when they become infected with the tox-carrying B-
C. Bacillus cereus phage
D. Other Bacillus Species ➢ Proteins of 62,000 daltons (Da)
➢ Composed of 2 fragments:
I. NON-SPORE FORMING, NONBRANCHING, CATALASE-POSITIVE ▪ Fragment A responsible for the cytotoxicity. Disrupts
BACILLI protein synthesis, splits nicotinamide adenosine
dinucleotide to form nicotinamide and adenosine
A. Corynebacterium diphosphoribose (ADPR)
▪ Fragment B binds to receptors on human cells and
mediates the entry of fragment A into the cytoplasm
➢ The toxicity is caused by the ability of diphtheria toxin to block
protein synthesis in eukaryotic cells

2. Clinical Infections

❖ Two different forms of disease in humans:

➢ Respiratory Diphtheria
▪ Sudden onset with exudative pharyngitis, sore throat, low-
grade fever, and malaise
▪ A thick pseudomembrane develops over the pharynx
▪ In critically ill patients, cardiac and neurologic complications
are most significant
❖ General Characteristics ▪ Exotoxin: Bull’s neck appearance
➢ Kleb Loeffler’s bacillus
➢ On the basis of 16S ribosomal ribonucleic acid (rRNA)
sequencing, corynebacterial are closely related to mycobacteria
and nocardiae
➢ Slightly curved, gram-positive rods with nonparallel sides and
slightly wider ends, producing the described “club-shape” or
coryneform
➢ Diphtheroid, meaning “diphtheria-like”, is sometimes used in ➢ Cutaneous Diphtheria
reference to this gram staining morphology ▪ A papule can develop on the skin, which progresses to a
➢ Can be divided into non-lipophilic and lipophilic species nonhealing ulcer
▪ Lipophilic corynebacteria are often considered fastidious ▪ Systemic signs can develop
and grow slowly on standard culture media; cultures often
must be incubated for at least 48 hours before growth is
detected. Growth is enhanced if lipids are included in the
CM
➢ Nondiphtheria Corynebacterium spp. are frequently isolated from
clinical specimens, and they are often dismissed as commensals
➢ Corynebacterium spp. isolated from various body sites can be
opportunistic pathogens, especially in immunocompromised
patients

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[BACT211] TRANS 12: Aerobic Gram Positive-Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH
3. Laboratory Diagnosis

❖ Microscopy
➢ Highly pleomorphic gram-positive bacillus
➢ Appears in palisades (cells lie in parallel rows) or as individual
cells lying at sharp angles to another in “V” and “L” formations
➢ Club-shaped swellings and beaded forms are common
➢ Often stain irregularly, especially when stained with methylene
blue
➢ The metachromatic areas of the cell, which stain more intensely
than other parts, are called Babes-Ernst granules
❖ Culture Characteristics
➢ Facultative anaerobe
➢ It grows best under aerobic conditions and has an optimal growth
temperature of 37C, although multiplication occurs within the
range of 15C to 40C
➢ Grows on nutrient agar, better growth is usually obtained on a
medium containing blood or serum, such as Loeffler Serum or
Pai Agars

4. Treatment

❖ Diphtheria is treated by prompt administration of antitoxin

❖ Drug of Choice: Penicillin
❖ Erythromycin: used for penicillin-sensitive individuals

B. Corynebacterium amycolatum
➢ Have a very small zone of B-hemolysis
➢ Cystine-tellurite Blood Agar (CTBA) a modification of Tinsdale
medium, contains sheep red blood cells, bovine serum, cystine
and potassium tellurite (inhibits many noncoryneform bacteria)

❖ Most frequently recovered from human specimens

❖ It is part of the normal skin microbiota and has often been misidentified
by clinical laboratories as C. striatum, C. xerosis and C. minutissimum
❖ Flat and dry, have a matte or waxy appearance, and are non-lipophilic
❖ Resistant to a wide range of antimicrobials, including B-lactams,
fluoroquinolines, macrolides, clindamycin, and aminoglycosides

➢ When grown on CTBA, corynebacteria form black or brownish

colonies from the reduction of tellurite
❖ Identification
➢ Catalase positive
➢ Nonmotile
➢ Brown halo on CTBA
▪ C. diphtheriae
▪ C. ulcerans
▪ C. pseudotuberculosis
➢ C. diphtheriae
▪ Lacks urease production
▪ Ferments glucose and maltose
▪ Producing acid but not gas
▪ Reduces nitrate to nitrite
❖ Test for Toxigenity
➢ In vitro
▪ ELEK TEST – Immunodiffusion test
• Positive: lines of precipitation

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[BACT211] TRANS 12: Aerobic Gram Positive-Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH

C. Corynebacterium jeikeium G. Corynebacterium ulcerans

❖ Named after Johnson and Kaye ❖ Has been isolated from humans with diphtheria-like illness
❖ Most common cause of Corynebacterium-associated prosthetic ❖ A veterinary pathogen, causing mastitis in cattle and other domestic
valve endocarditis in adults and wild animals
❖ Also causes septicemia, meningitis, prosthetic joint infections and skin ❖ Produces a brown halo around colonies on CTBA
complications such as rash and subcutaneous nodules
❖ Lipophilic and a strict aerobe that is nonhemolytic, does not produce
urease, and is nitrate reduction negative
❖ Resistant to a wide range of antimicrobials, including penicillin,
cephalosphorins, macrolides and aminoglycosides
❖ Drug of Choice: vancomycin

D. Corynebacterium pseudodiphtheriticum

❖ Part of the normal biota of the human nasopharynx

❖ Respiratory tract infection can mimic respiratory diphtheria
❖ Does not show the characteristic pleomorphic morphology
❖ Often appear in palisades ❖ Grow well on SBA and produce a narrow zone of B-hemolysis
❖ Grows well on standard laboratory media, reduces nitrate and ❖ Does not reduce nitrate, differentiating it from C. diphtheriae, and it is
produces urease urease positive

E. Corynebacterium pseudotuberculosis H. Corynebacterium urealyticum

❖ Most commonly associated with UTIs

❖ Primarily a veterinary pathogen ❖ Lipophilic and is a strict aerobe
❖ Causes a granulomatous lymphadenitis in humans ❖ Pinpoint, nonhemolytic, white colonies that have characteristic
❖ Produces a dermonecrotic toxin that causes death of various cell coryneform microscopic morphology
types and it can produce diphtheria toxin ❖ Nitrate negative, catalase positive, and urease positive within minutes
❖ Also produces a brown halo on CTBA after inoculation on a Christensen Urea Slant
❖ Produces urease and on SBA forms small, yellowish-white colonies ❖ Drug of Choice: vancomycin
F. Corynebacterium striatum
B. Rothia

❖ Often considered a commensal or skin contaminant. However, ❖ Gram-positive cocci that can appear rodlike, belong to the family
nosocomial infections have been reported Micrococcaceae
❖ Nonlipophilic and pleomorphic and it often produces small, shiny, ❖ Nitrate positive
convex colonies in about 24 hours ❖ Nonmotile
❖ Shown resistance to penicillin and other B-lactams, macrolides and ❖ Esculin hydrolysis positive
fluoroquinolones but is typically susceptible to vancomycin ❖ Urease negative
❖ Approximately two thirds of the isolates are catalase positive
❖ Contains 6 spp. but only 2 are significant
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[BACT211] TRANS 12: Aerobic Gram Positive-Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH
➢ Rothia mucilaginosa ❖ Cultural Characteristics
▪ Linked to bacteremia, endocarditis, pneumonia ➢ Grows well on SBA, chocolate agar, nutrient agars, brain-heart
infusion medium and thioglycolate broth
➢ Prefers a slightly increased carbon dioxide (CO2)
➢ Colonies are small, round, smooth and translucent, surrounded
by a narrow zone of B-hemolysis, which may be visualized only
if the colony is removed
➢ Optimal growth temperature for L. monocytogenes is 30C to 35C,
but growth occurs over a wide range (0.5C to 45C)
➢ Because L. monocytogenes grows at 4C, a technique called cold
enrichment may be used to isolate the organism from
polymicrobial clinical specimens

➢ Rothia dentocariosa
▪ Member of the normal human oropharyngeal flora and
found in saliva

❖ Identification
➢ Hippurate hydrolysis positive like S. agalactiae
➢ Catalase positive
➢ Bile esculin hydrolysis positive
❖ Microscopic Characteristics
➢ Motile at room temperature
➢ Resembles coryneform bacilli, forming short gram-positive bacilli
➢ Wet mount preparations “tumbling motility” (end-over-end
but also branching filaments that resembles of facultative
motility)
actinomycetes
➢ In motility medium: “umbrella pattern” RT
➢ When placed in broth, the species produces coccoid cells, a
characteristic differentiating it from actinomycetes

C. Listeria monocytogenes

❖ Has been recovered from soil; water; vegetation; and animal products,
such as raw milk, cheese, poultry and processed meats
❖ It also has been isolated from crustaceans, flies and ticks
❖ Listeriosis is recognized as an uncommon but serious infection
primarily of neonates, pregnant women, older adults and
immunocompromised hosts

1. Virulence Factors
❖ Hemolysin (listeriolysin C)
➢ Damages the phagosome membrane
❖ Catalase
➢ Produces a positive CAMP reaction
❖ Superoxide dismutase
❖ Phospholipase C
❖ Surface protein (p6o) II. NON-SPORE FORMING, NONBRANCHING, CATALASE-NEGATIVE
➢ Induces phagocytosis through increased adhesion and BACILLI
penetration into mammalian cells
A. Erysipelothrix rhusiopathiae
2. Clinical Infections
❖ Listeriosis 1. General Characteristics
➢ Newborns and immunocompromised adults – most common
➢ For healthy individuals, particularly in pregnant women, also ❖ Three species in the genus Erysipelothrix:
occurs 3rd ➢ Erysipelothrix rhusiopathiae (caused disease in humans)
➢ Erysipelothrix tonsillarum
3. Laboratory Diagnosis ➢ Erysipelothrix inopinata
❖ Gram-positive, catalase-negative
❖ Microscopy ❖ Non-spore forming
➢ Gram-positive coccobacillus ❖ Pleomorphic rod that has a tendency to form long filaments
➢ Found singly, in short chains, or in palisades ❖ It is found worldwide and is a commensal or a pathogen in a wide
variety of vertebrates and invertebrates
❖ Usual route of infection is through cuts or scratches on skin

2. Clinical Infections

❖ Erysipeloid
❖ Septicemia
❖ Generalized, diffuse cutaneous infection

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[BACT211] TRANS 12: Aerobic Gram Positive-Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH

3. Laboratory Diagnosis ❖ Trueperella (Arcanobacterium) pyogenes

❖ Microscopy
➢ Thin, rod-shaped, gram-positive organism that can form long
filaments
➢ Arranged singly, in short chains, or in a “V” shape
➢ Decolorizes easily, so it may appear gram variable

❖ Trueperella (Arcanobacterium) bernardiae

❖ Catalase negative

A. A. haemolyticum

❖ Produces small colonies on SBA that demonstrate a narrow zone of

B-hemolysis after 24 to 48 hours of incubation
❖ Culture Characteristics ❖ A black opaque dot is observed on the agar when the colony is
➢ Grows on SBA and chocolate agar scraped away
➢ On SBA, the colonies are usually nonhemolytic and pinpoint after ❖ Pitting of the agar beneath the colony has also been reported
24 hours of incubation ❖ Lipase and lecithinase positive
❖ Exhibits a reverse CAMP reaction (CAMP inhibition reaction)

C. Gardnerella vaginalis

❖ Normal biota in the human urogenital tract

❖ Short, pleomorphic gram-positive rod or coccobacillus that often stains
gram variable or gram negative
❖ Known for its association with bacterial vaginosis (BV) in humans
❖ Microscopy:
➢ “Clue cells”
▪ aids the diagnosis of BV
▪ large squamous epithelial cells with gram positive bacilli and
coccobacilli

➢ After 48 hours of incubation, two distinct colony types are seen:

▪ A smaller, smooth form is transparent, glistening, and
convex with entire edges
▪ Larger, rough colonies are flatter with a matte surface,
curried structure, and irregular edges
➢ Often appear a-hemolytic
❖ Identification
➢ Catalase-negative
➢ Nonmotile
➢ Pleomorphic
➢ Aerobic or facultatively anaerobic
➢ Hydrogen sulfide positive
➢ Voges-Proskauer test negative ❖ Amsel’s Clinical Criteria: (Positive: if 3/4 criteria are found)
➢ A gelatin stab culture “Test tube brush-like” pattern at 22C ➢ Homogenous, thin, white discharge that smoothly coats the
vaginal walls
➢ Clue cells
➢ pH of vaginal fluid greater than 4.5
➢ fishy odor of vaginal discharge before or after addition of 10%
potassium hydroxide, the whiff test

1. Culture Characteristics

❖ Grows best in 5% to 7% CO2 at a temperature of 35C to 37C

❖ Grows on SBA as pinpoint, nonhemolytic colonies
❖ Medium of Choice: Human Blood Bilayer Tween (HBT) agar

B. Arcanobacterium and Trueperella

❖ A. haemolyticum (formerly Corynebacterium haemolyticum)

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[BACT211] TRANS 12: Aerobic Gram Positive-Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH
III. NON-SPORE FORMING, BRANCHING, AEROBIC B. Actinomadura
ACTINOMYCETES

A. Nocardia

1. General Characteristics

❖ Aerobic, branched, beaded, gram-positive bacilli

❖ Partially acid fast
❖ True bacteria

2. Virulence Factors

❖ No virulence factors have been identified, although virulence has been

correlated with alterations in the components in the cell wall
➢ Superoxide dismutase and catalase ❖ Actinomadura madurae
▪ Provides resistance to oxidative killing by phagocytes ❖ Actinomadura pelletieri
➢ Ironchelating compound called nocobactin ❖ Causes mycetomas (identical to nocardia)
❖ The microscopic morphology and colony morphology are very similar
to those of Nocardia spp.
3. Clinical Infections ❖ A. madurae
➢ is cellobiose and xylose positive, whereas Nocardia spp. do not
❖ Pulmonary Infection produce acid from these two carbohydrates
➢ Most common manifestation; confluent bronchopneumonia
➢ Sputum is thick and purulent C. Streptomyces
➢ No sulfur granules develop, and no sinus tract formation occurs
❖ Cutaneous Infection ❖ Primarily saprophytes found as soil inhabitants and resemble other
➢ N. brasiliensis is the most frequent cause of this form of aerobic actinomycetes with regard to morphology and the diseases
nocardiosis and actinomycotic mycetomas they cause
➢ Pus may be pigmented and contain “sulfur granules” ❖ Streptomyces somaliensis
➢ Granules often appear yellow or orange and have a distinct ➢ Associated with actinomycotic mycetoma
granular appearance ❖ Streptomyces anulatus (formerly Streptomyces griseus)
➢ Specimens has been increasingly isolated from many clinical
specimens, including sputum, wound, blood and brain

4. Laboratory Diagnosis

❖ Microscopy
➢ Gram-positive, beaded, branching filaments

D. Gordonia

❖ Aerobic
❖ Catalase positive
❖ Gram positive to gram variable
❖ Culture Characteristics ❖ Partially acid fast
➢ Modified Thayer-Martin agar, may enhance recovery of Nocardia ❖ Nonmotile
spp., by inhibiting the growth of contaminating organisms ❖ Grow with mycelial forms that fragment into rod-shaped or coccoid
➢ Grow on nonselective buffered charcoal-yeast extract agar elements-hence the term nocardioform
➢ A chalky, matte, velvety or powdery appearance and may be ❖ They differ from rapidly growing mycobacteria by their weak acid
white, yellow, pink, orange, peach, tan or gray pigmented fastness and the absence of arylsulfatase
➢ Dry, crumbly appearance similar to breadcrumbs

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[BACT211] TRANS 12: Aerobic Gram Positive-Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH
B. Bacillus anthracis

1. Virulence Factors

❖ Capsule – D-glutamic acid

❖ Anthrax toxin
➢ Consists of three proteins:
▪ Protective antigen (PA)
• Binding molecule for EF and LF
▪ EF
• Increases the concentration of cyclic adenosine
G. polyisoprenivorans monophosphate (cAMP) in host cells
▪ LF
E. Rhodococcus • Protease that kills host cells by disrupting the
transduction of extracellular regulatory signals
❖ Rhodococcus equi
➢ Found in soil and causes respiratory tract infections in animals 2. Clinical Infections
➢ Human infection is rare
➢ Demonstrate filaments, some with branching ❖ Anthrax
➢ Partially acid fast or acid fast ➢ Cutaneous Anthrax
▪ Occur when wounds are contaminated with anthrax spores
acquired through skin cuts, abrasions or insect bites
▪ Eschar or Black Eschar or Malignant pustule

➢ Inhalation Anthrax
F. Tropheryma whipplei ▪ Woolsorter’s disease
▪ Acquired when spores are inhaled into the pulmonary
❖ Agent of Whipple disease parenchyma
❖ Facultative intracellular pathogen first identified in 1991 by using PCR
from a duodenal biopsy specimen ➢ Gastrointestinal Anthrax
❖ Gram-positive actinomycete ▪ Occurs when the spores inoculated into a lesion on the
❖ Detected in human feces, saliva, and gastric secretions and is intestinal mucosa after ingestion of the spores
apparently ubiquitous in the environment

➢ Injectional Anthrax
▪ Characterized by soft tissue infection associated with “skin
popping” or other forms of injection drug use and results
IV. SPORE-FORMING, NONBRANCHING, CATALASE-POSITIVE from the direct injection of the spores into tissue
BACILLI

A. Bacillus

A. General Characteristics

❖ Gram positive or gram variable

❖ Aerobic or facultative anaerobic bacilli that form endospores
❖ Some are thermophiles that grow best at 55C or higher

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[BACT211] TRANS 12: Aerobic Gram Positive-Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH

3. Laboratory Diagnosis

❖ Microscopic
➢ Large, square-ended, gram-positive or gram-variable rod found
singly or in chains
➢ Appearance of bamboo rods
➢ As the bacteria are subcultured, capsule production cases
➢ Incubation in an atmosphere containing increased CO2 can
stimulate capsule production

D. Other Bacillus Species

❖ Bacillus subtilis
❖ Bacillus licheniformis
❖ Bacillus circulans
❖ Bacillus pumilus
❖ Cultural Characteristics ❖ Bacillus sphaericus
➢ SBA: Nonhemolytic, large, gray and flat with an irregular margin
because of outgrowths of long, filamentous projections
➢ “Medusa Head”
➢ “beaten egg whites”

❖ Identification
➢ Catalase positive
➢ Nonmotile
➢ Grows aerobically or anaerobically
➢ Ferment glucose
➢ Produce lecithinase
➢ Opaque zone can be seen around colonies growing on egg-yolk
agar
➢ High-salt (7% sodium chloride) and low pH
➢ Susceptible to penicillin (10U/mL)
➢ Capsule production can be detected by India ink staining on
blood or CSF

C. Bacillus cereus

1. General Characteristics

❖ Common cause of food poisoning and opportunistic infections in

susceptible hosts
❖ Grown aerobically at 37C on SBA
❖ B-hemolytic frosted glass-appearing colony
❖ Spore-forming
❖ Gram-positive bacilli
❖ Motile
❖ Able to ferment salicin, and lecithinase positive

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Nonfermenting and Miscellaneous LEC 2020-2021

2nd SEM
OLFU Gram-Negative Bacilli 11 BACT211
College of Medical LEC
CLINICAL BACTERIOLOGY TRANS 13
FINALS
Laboratory Science
Transcriber: Riyoma Surell
Batch 2023 Date: May 11, 2021

❖ Gram-negative bacillus or coccobacillus

❖ Strictly aerobic metabolism
Outline ❖ Motile usually with polar flagellum or polar tuft of flagella
At the end of the session, the student must be able to learn: ❖ Oxidase positive (except P. luteolus and P. oryzihabitans)
I. General Characteristics of Nonfermenters ❖ Catalase positive
II. Biochemical Characteristics and Identification ❖ Usually grows on MAC agar
III. Clinically Significant Nonfermentative, Gram-Negative Bacilli ❖ Usually an oxidizer of carbohydrates, but some species are
A. Pseudomonas
asaccharolytic
B. Pseudomonas Fluorescent Group
A. Pseudomonas aeruginosa
C. Pseudomonas Non-Fluorescent Group B. Pseudomonas Fluorescent Group
A. Pseudomonas stutzeri
B. Pseudomonas mendocina A. Pseudomonas aeruginosa
C. Pseudomonas pseudoalcaligenes and Pseudomonas
alcaligenes
D. Pseudomonas luteola and Pseudomonas oryzihabitans
D. Acinetobacter
E. Stenotrophomonas maltophilia
F. Burkholderia
A. Burkholderia cepacia Complex
B. Burkholderia mallei
C. Burkholderia pseudomallei
D. Burkholderia gladioli
IV. Less Commonly Encountered Nonfermentative Gram-Negative Bacilli
A. Alcaligenes and Achromobacter
B. Brevundimonas
A. B. diminuta
B. B. vesicularis ➢ Found in moist environment
C. CDC Groups EO-3, EO-4 and Paracoccus
➢ Reservoir: Plants, soil and tap water
D. Chromobacterium
E. Comamonas and Delftia
F. Flavobacteriaceae ❖ General Characteristics
G. Methylobacterium and Roseomonas ➢ Large, mucoid spreading colonies
H. Ralstonia and Cupriavidus ➢ Cetrimide Agar
I. Shewanella ▪ Transparent selective culture media that aids in pigment
J. Sphingomonas production visualization

I. GENERAL CHARACTERISTICS OF NONFERMENTERS

❖ Fail to acidify an oxidative-fermentative (OF) medium when it is

overlaid with mineral oil
❖ Fail to acidify triple sugar iron agar (TSIA) butts
❖ Prefer and grow much better in an aerobic environment
❖ Most are oxidase positive

II. BIOCHEMICAL CHARACTERISTICS AND IDENTIFICATION

❖ Oxidase positive reaction, although reaction can be weak and

variable
❖ Nonreactivity in 24 hours in commercial multi-test kit systems used
primarily for the identification of Enterobacteriaceae
❖ No acid production in the slant or butt of TSIA or KIA
❖ Resistance to a variety of classes of antimicrobial agents, such as ➢ Oxidase positive
aminoglycosides, third-generation cephalosporins, penicillin, and ▪ To differentiate it from family Enterobacteriaceae and other
fluoroquinolones enterics
➢ Oxidative
III. CLINICALLY SIGNIFICANT NONFERMENTATIVE, GRAM- ▪ Dextrose (glucose) but not maltose
NEGATIVE BACILLI ➢ Fermentative
▪ TSI Reaction: K/K
➢ Growth at 42C (to differentiate it from the rest of the
A. Pseudomonas
pseudomonads)
➢ Acetamide Utilization + (blue slant) after 7 days of standard
incubation

❖ Causes the following infections:

➢ Severe wound infections in burn patients
➢ UTI and nosocomial pneumonia
➢ Septicemia in immunosuppressed patients and infants
➢ Chronic lung infections in cystic fibrosis patients
➢ Septic arthritis in intravenous drug abusers
➢ Destructive eye infection (keratitis and corneal ulcers) in contact
lens wearers
➢ Swimmer’s ear – especially in athletes
➢ Jacuzzi/ Hot Tub Syndrome – shanghai fever
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[BACT211] TRANS 13: Nonfermenting and Miscellaneous Gram-Negative Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH
C. Pseudomonas Non-Fluorescent Group

A. Pseudomonas stutzeri

❖ Virulence Factors

Virulence Factors Function ❖ Wrinkled, leathery, adherent colonies that may produce a light-yellow
Lipopolysaccharide Antiphagocytic activity, cytotoxicity or brown pigment
Pili Adhesion ❖ ADH negative
Flagella Motility, adhesion ❖ Starch hydrolysis positive
Type II secretion system Cytotoxic activity ❖ Responsible for diseases that include septicemia, meningitis in the
human immunodeficiency virus infected patient, pneumonia,
Phospholipases Cytotoxicity
endocarditis, postsurgical wound infections, septic arthritis,
Proteases Cytotoxicity, proteolytic activity
conjunctivitis and UTIs
Exotoxin A Cytotoxicity
Capsule Antiphagocytic activity
B. Pseudomonas mendocina
❖ Identifying Characteristics
➢ Produce pyoverdine (a yellow-green or yellow-brown pigment) ❖ Nonwrikled, flat colonies that may appear with a yellowish- brown
▪ P. aeruginosa pigment
▪ P. fluorescens ❖ Smooth buttery appearance
▪ P. putida ❖ Oxidase and ADH positive, and is acetamide negative
▪ P. veronii ❖ Motile by means of a single polar flagellum
▪ P. mosselii ❖ Oxidizes glucose and xylose
▪ P. monteilii ❖ Nonproteolytic
➢ P. aeruginosa also produce pyocyanin (blue) ❖ Does not hydrolyze starch

C. Pseudomonas pseudoalcaligenes and Pseudomonas alcaligenes

❖ Oxidase positive
❖ Grows on MAC agar
❖ Variable in the reduction of nitrates to nitrites or nitrogen gas
❖ Motile by means of a polar flagellum

D. Pseudomonas luteola (Chryseomonas luteola) and Pseudomonas

oryzihabitans

B. Pseudomonas fluorescens and Pseudomonas putida

❖ Oxidase negative
❖ Catalase positive
❖ Motile
❖ Oxidize glucose
❖ Produce pyoverdin, but neither produces pyocyanin ❖ Grow on MAC agar
❖ Grows at 42C ❖ Often produce an intracellular non-diffusible yellow pigment
❖ Cannot reduce nitrate to nitrogen gas, but they can produce acid from ❖ Wrinkled or rough colonies at 48 hours
xylose ❖ ONPG and Esculin test to differentiate P. luteola and P. oryzihabitans
❖ Gelatin hydrolysis can be used to differentiate the two species from
each other: D. Acinetobacter
➢ P. putida is negative
➢ P. fluorescens is positive
❖ Usually susceptible to the aminoglycosides, polymyxin, and
piperacillin but are resistant to carbenicillin and SXT

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[BACT211] TRANS 13: Nonfermenting and Miscellaneous Gram-Negative Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH

C. Burkholderia pseudomallei
❖ Aerobic gram-negative coccobacilli or even gram-negative cocci
❖ Resist decolonization and retain the crystal violet stain, leading to
misidentification
❖ Appear as gram-positive cocci in smears made from blood culture
bottles
❖ Oxidase negative, catalase positive, and nonmotile
❖ A. baumannii is saccharolytic and A. lwoffii is asaccharolytic
❖ Produces purple pigment

E. Stenotrophomonas maltophilia

❖ Cream to tan wrinkled colonies in BAP

❖ Motile via polar tuft flagella
❖ ADH +
❖ Highly oxidative – glucose, maltose, lactose, mannitol
❖ Causes Melioidosis
➢ glanderslike disease which has a long latent period.
Manifestation of symptoms takes years hence the synonym
Vietnam Time Bomb
❖ Before: member of Pseudomonas Now: Xanthomonas
❖ Oxidase negative, non-fermentative, gram negative bacillus D. Burkholderia gladioli
❖ Colonies may appear bluish on MAC agar
❖ Positive for catalase, DNase, esculin and gelatin hydrolysis and lysine
❖ Produces after 48-72 hours of incubation
decarboxylase
❖ Motile by means of one or two polar flagella
❖ Usually susceptible to SXT
❖ Catalase and urease positive
❖ Grows on MAC agar
F. Burkholderia
❖ Oxidizes glucose
❖ Mannitol positive and decarboxylase negative, and is negative for
A. Burkholderia cepacia Complex oxidase, although some strains are weakly positive

IV. LESS COMMONLY ENCOUNTERED NONFERMENTATIVE, GRAM-

NEGATIVE BACILLI

A. Alcaligenes and Achromobacter

❖ Often produces a weak, slow positive oxidase reaction

❖ Almost all strains oxidize glucose and many will oxidize maltose,
lactose and mannitol
❖ Most strains are lysine decarboxylase and ONPG positive
❖ Most strains are ornithine decarboxylase negative and fail to reduce
nitrate to nitrite
❖ Motile by means of polar tufts of flagella
❖ Causes onion bulb rot in plants and in humans ❖ Presence of peritrichous flagella
❖ Resistant to some disinfectants***
B. Burkholderia mallei (Pseudomonas mallei)
Differentiation of Alcaligenes Species
A. A. xylosoxidans
faecalis
Oxidase + +
Glucose/Xylose Oxidation - +
Nitrate Reduction - +
Nitrite Reduction to N. gas + -

B. Brevundimonas

A. B. diminuta
❖ Smooth and cream to white colonies in BAP and weakly oxidase (+)
❖ The only non-motile among all pseudomonads ❖ Motile and possess a single polar flagellum
❖ Can’t grow in 42C ❖ Oxidize glucose
❖ Causes Glander’s Disease ❖ Oxidase positive
➢ infectious disease of horses, goats, sheep and donkey. Rare ❖ Most strains grow on MAC agar
cause of human infection acquired by direct contact, trauma or
inhalation
❖ Farcy
➢ disseminated form of glander’s disease

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[BACT211] TRANS 13: Nonfermenting and Miscellaneous Gram-Negative Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH

B. B. vesicularis F. Flavobacteriaceae

❖ Balaneatrix, Bergeyella, Chryseobacterium, Elizabethkingia,

❖ Slender rod, with polar flagella Empedobacter, Myroides, Weeksella, Wautersiella,
❖ Only about 25% grows on MAC agar Sphingobacterium spp.
❖ Most strains of B. vesicularis produce an orange intracellular pigment
❖ Oxidase positive and oxidizes glucose and maltose ❖ Elizabethkingia meningoseptica (Chryseobacterium
❖ Esculin Hydrolysis: to differentiate B. diminuta (rarely positive) and meningosepticum)
B. vesicularis (positive) ➢ Meningitis or septicemia in a newborn, especially in conjunction
with prematurity
❖ Balaneatrix alpaca
C. CDC Groups EO-3, EO-4 and Paracoccus ➢ First isolated in 1987 during an outbreak of pneumonia and
meningitis linked to individuals attending hot springs spa
❖ Eugonic oxidizer G. Methylobacterium and Roseomonas
❖ Oxidase positive
❖ Nonmotile, saccharolytic coccobacilli that grow weakly, if at all, on
MAC agar
❖ All oxidize glucose and xylose
❖ EO-3 and many EO-4 isolates have a yellow nondiffusible pigment
❖ P. yeei (EO-2) is further characterized by the production of
characteristic coccoid or O-shaped cells on gram stain

D. Chromobacterium
❖ Chromobacterium violaceum
➢ Motile, facultative anaerobe, oxidase positive
➢ Rare cause of human infection ❖ Pink pigmented colonies
➢ Found in soil and water
H. Ralstonia and Cupriavidus

❖ Linked to meningitis, endocarditis and osteomyelitis

I. Shewanella

❖ Often mucoid and can produce a tan to brown pigment causing

discoloration of SBA

J. Sphingomonas

➢ Violacein ❖ Isolated from many water sources, including swimming pools as well
▪ Unique because of the violet pigment that it produces as from hospital equipment and laboratory supplies

E. Comamonas and Delftia

➢ Straight to slightly curved rods

➢ Produce alkalinity in OF media
➢ Catalase and oxidase positive
➢ Motile by multitrichous polar flagella
➢ Reduce nitrate to nitrite
❖ Comamonas testosteroni and Comamonas terrigena
➢ Have been reported to cause nosocomial bacteremia
❖ Delftia acidovorans (Comomonas acidovorans)
➢ Associated with keratitis in soft contact lens wearers and
nosocomial infections including bacteremia and endocarditis
❖ Delftia tsuruhatensis
➢ Associated with catheter-related bacteremia

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[BACT211] TRANS 13: Nonfermenting and Miscellaneous Gram-Negative Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH

’
References:
❖ Powerpoint presentation of Prof. Rochelle Darlucio
❖ Diagnostic Microbiology by Mahon (6th edition)
❖ Diagnostic Microbiology by Bailey’s and Scotts (14th edition)
❖ Study Guide on Diagnostic Bacteriology by Mr. Nathaniel Ranon

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Haemophilus, HACEK, Legionella, Other LEC 2020-2021

2nd SEM
OLFU Fastidious Gram Negative Bacilli 12 BACT211
College of Medical LEC
CLINICAL BACTERIOLOGY TRANS 14
FINALS
Laboratory Science
Transcriber: Riyoma Surell
Batch 2023 Date: May 18, 2021

➢ Philus – loving
Outline ➢ Blood lover
At the end of the session, the student must be able to learn: ❖ Require performed growth factors present in blood:
I. HACEK Group ➢ X Factor (hemin or hematin)
A. Haemophilus ➢ V Factor (nicotinamide adenine dinucleotide, NAD), V for
A. General Characteristics vitamin, or both
B. Haemophilus influenzae
1. Virulence Factors
❖ Media of Choice: CHOC Agar
2. Clinical Manifestations ❖ Satellitism: it is a phenomenon which occurs when an organism
C. Haemophilus aegyptius produces V factor as a by-product of metabolism (e.g.,
D. Haemophilus influenzae biogroup aegyptius Staphylococcus aureus, Streptococcus pneumoniae or Neisseria
E. Haemophilus ducreyi spp.)
F. Haemophilus parainfluenzae
G. Laboratory Diagnosis
1. Specimen Processing and Isolation B. Haemophilus influenzae
2. Culture Media
3. Colony Morphology
4. Microscopic Morphology
5. X Factor and V Factor Requirements
6. Porphyrin Test
B. Aggregatibacter aphrophilus
C. Aggregatibacter actinomycetemcomitans
D. Cardiobacterium hominis
E. Eikenella corrodens
F. Kingella
G. Capnocytophaga
H. Pasteurella
I. Brucella
J. Francisella (Picture 2: Haemophilus influenzae satellitism around and between large,
K. Legionella white, hemolytic staphylococci. The small, gray, glistening colony is H.
L. Bordetella
influenzae)
A. Virulence Factors
B. Clinical Manifestations
C. Laboratory Diagnosis 1. Virulence Factors
II. ANAEROBES OF CLINICAL IMPORTANCE
A. Gram-Positive, Spore-Forming Anerobic Bacilli ❖ Capsule
A. Clostridium spp.
❖ Immunoglobulin A (IgA) proteases
1. Clinical Infections
B. Gram-Positive, Non-Spore Forming, Anaerobic Bacilli ❖ Adherence by fimbriae and other structures
A. Actinomyces spp. ❖ Outer membrane proteins and lipopolysaccharide (LPS)
B. Bifidobacterium spp.
C. Lactobacillus spp. 2. Clinical Manifestations
D. Proprionibacterium and Cutibacterium spp.
C. Anaerobic Gram-Negative Bacilli
A. Bacteroides spp. Infections caused by H. influenzae
B. Bilophila spp. Encapsulated Strains Non-Encapsulated Strains
C. Prevotella spp. Septicemia Otitis media with effusion
D. Porphyromonas spp. Septic arthritis Conjunctivitis
E. Campylobacter ureolyticus Group
Meningitis Sinusitis
F. Fusobacterium
Osteomyelitis Bacteremia
Cellulitis Pneumonia
Pericarditis
I. HACEK GROUP Pneumonia
Epiglottitis
❖ Haemophilus spp., (e.g., H. paraphrophilus)
❖ Aggregatibacter actinomycetemcomitans (formerly Actinobacillus
actinomycetemcomitans) and A. aphrophilus (formerly H. aphrophilus)
❖ Cardiobacterium hominis C. Haemophilus aegyptius
❖ Eikenella corrodens
❖ Kingella spp. ❖ Koch-Weeks bacillus
❖ Associated with an acute, contagious conjunctivitis, commonly
A. Haemophilus referred to as “pinkeye”

A. General Characteristics D. Haemophilus influenzae biogroup aegyptius

❖ Gram-negative, pleomorphic coccobacilli or rods ❖ Conjunctivitis primarily in pediatric populations

❖ Nonmotile ❖ Non-encapsulated
❖ Facultatively anaerobic ❖ First caused a severe systemic disease known as Brazilian purpuric
❖ Adapted in the respiratory tract of humans and animals except fever (BPF) in Brazil in 1984
Haemophilus ducreyi (causes sexually transmitted diseases – ➢ Presence of conjunctivitis, fever and presence of petechial rash
chancroid) or purpura
❖ Ferment carbohydrates
❖ Oxidase and catalase positive
❖ Reduce nitrates to nitrites
❖ Haemophilus
➢ Haemo – Blood
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[BACT211] TRANS 14: Haemophilus, HACEK, Legionella, Fastidious Gram (-) Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH

E. Haemophilus ducreyi 4. Microscopic Morphology

❖ Varies from small, gram-negative coccobacilli to long filaments

❖ Haemophilus ducreyi
➢ gram-negative coccobacilli arranged singly or in groups
(clusters)
➢ commonly referred to as “school of fish” or “railroad tracks”
➢ loosely coiled clusters of organisms lined up in parallel or
appearing as “fingerprints”

❖ Strictly human pathogen

❖ Causative agent of chancroid
➢ Commonly referred to as soft chancre (incubation period of 4 –
14 days)
❖ Not part of the normal microbiota

5. X Factor and V Factor Requirements

❖ using impregnated strips or disks

F. Haemophilus parainfluenzae

❖ Found in the oral cavity

❖ Causes a few cases of otitis media and acute sinusitis (This organism would be identified as Haemophilus influenzae because it
❖ Rarely implicated as causative agent of endocarditis requires both X and V factors)
❖ Para – only requires V factor for growth

G. Laboratory Diagnosis

1. Specimen Processing and Isolation

❖ Genital sites first should be cleaned with sterile gauze moistened with
sterile saline before specimens are collected for the isolation of this
organism
❖ Next, a swab premoistened with sterile phosphate-buffered saline
should be used to collect material from the base of the ulcer
❖ As an alternative, pus can be aspirated from buboes if they are present (This organism requires V factor only and would be identified as
Haemophilus parainfluenzae)
2. Culture Media
6. Porphyrin Test
❖ is a commonly used medium incubated between 33 and 37C in an
atmosphere of 5% to 10% carbon dioxide (CO2) ❖ based on the ability of the organism to convert the substrate Delta-
❖ CHOC agar supplemented with bacitracin (300mg/L) aminolevulinic acid (ALA) into porphyrins or porphobilinogen, which
➢ Haemophilus spp. from respiratory specimens are intermediates in the synthesis of X factor
❖ Enriched CHOC medium or Nairobi biplate
➢ Haemophilus ducreyi
❖ Nairobi biplate:
➢ GC agar base with 2% bovine hemoglobin and 5% fetal calf
serum
➢ Mueller Hinton Agar (MHA) with 5% chocolatized horse blood
➢ Both sides contains 3mg/L of vancomycin

3. Colony Morphology

❖ Translucent, tannish, moist, smooth, and convex with a distinct

“mousy” or bleachlike odor

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[BACT211] TRANS 14: Haemophilus, HACEK, Legionella, Fastidious Gram (-) Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH
Common Other X V B BAP/ Por-
Species Name Factor Factor A CAP phyrin
P
H. Pfeiffer + + + - -
influenza bacillus
H. Koch- + + - - -
aegyptius Week
bacillus
H. Ducrey + - - - -
ducreyi bacillus
H. Normal + + - + -
haemolyticus Flora
H. URT - - + - +
aprophilus
H. - + - - +
paraprophilus (Aggregatibacter actinomycetemcomitans on sheep blood agar. The star-
H. - + + - +
parainfluenza
shaped centers of the colonies are not usually evident until after 48 hours
H. - + - + + of incubation and are best observed by using x100 magnification – light
parahaemolyticus microscope or a stereomicroscope)

B. Aggregatibacter aphrophilus D. Cardiobacterium hominis

❖ A. aphrophilus
➢ Greek aphros and philia: foam loving or desiring high
concentration of CO2
➢ One of the most prevalent species in the HACEK group involved
in endocarditis
➢ Linked to bone and joint infections
➢ Found in dental plaque and gingival scrapings
(Gram stain of Cardiobacterium hominis showing typical “rosettes” –
x1000)

❖ Pleomoprhic
❖ Nonmotile
❖ Fastidious
❖ Gram-negative bacilli
❖ Found as normal microbiota of the nose, mouth and throat and may
be present in the gastrointestinal tract
❖ Organisms tend to form rosettes, swellings, long filaments or sticklike
structures in yeast extract
❖ On agar “pitting” can be produced

(Aggregatibacter aphrophilus isolate that is not X factor dependent and is E. Eikenella corrodens
growing over the entire surface of a trypticase soy agar plate)

C. Aggregatibacter actinomycetemcomitans

❖ Animal pathogens or animal endogenous biota that in general do not

routinely cause infections in humans
❖ Grows better with increased CO2
❖ A distinctive with four to six points in the center of the colonies is often
seen at 48 hours (Growth of Eikenella corrodens on chocolate agar)
❖ Catalase positive and oxidase variable
❖ Do not grow on MAC agar ❖ A member of the normal biota of the oral and bowel cavities
❖ Negative for X and V growth factor requirements, urease, indole, ❖ Fastidious, gram-negative coccobacilli that grow best under conditions
esculin and citrate of increased CO2 with hemin
❖ Nonmotile, oxidase positive, and asaccharolytic
❖ Catalase negative and often produce a yellow pigment
❖ “pit” (make a depression) or corrode the surface of the agar

F. Kingella

(Kingella kingae gram stain)

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[BACT211] TRANS 14: Haemophilus, HACEK, Legionella, Fastidious Gram (-) Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH
❖ Coccobacillary to short bacilli with squared ends that occur in pairs or
short chains
❖ Resist decolorization in gram stains
❖ Nonmotile
❖ Fastidious
❖ Oxidase positive
❖ Catalase-negative fermenters of glucose and other sugars but with no
gas

G. Capnocytophaga
(Pasteurella multocida growing on sheep blood agar and chocolate agar.
The MacConkey agar plate is negative for growth)

P. multocida - + + + + - -
P. - + + + + V +
pneumotropica
P. - + + - + - +
dagmatis
(Growth of Capnocytophaga organisms on chocolate agar. Note the P. - + + - + - -
spreading away from the center of the colony. Compare this growth with stomatis
Eikenella) P. - + + + + - -
canis
P. - V + - + V -
➢ Belongs to the family Flavobacteriaceae Bettyae
➢ Fastidious
➢ Facultatively anerobic I. Brucella
➢ Gram-negative bacilli and require increased CO2 for growth and
isolation from blood cultures
➢ Thin and often fusiform (pointed ends) resembling
Fusobacterium spp., spindle-shaped, coccoid and curved
filaments may be also seen
➢ “gliding motility”
❖ C. ochracea
➢ Most common clinical isolate

❖ Brucellosis, infection with bacteria from the genus Brucella

❖ Characteristics by chronic and recurring fever with weight loss and
anorexia
➢ Undulant fever
➢ Malta fever
➢ Gibraltar fever
➢ Mediterranean fever
❖ C. canimorsus and C. cynodegmi ➢ Bang’s disease
➢ Normal inhabitants of the oral cavity of dogs and cats ❖ Requires biohazard level III in handling specimens suspected with
❖ C. canimorsus Brucella
➢ Cause a fulminant, life threatening septicemia in humans, ❖ Mononuclear Phagocytic Cells – Facultative intracellular pathogens
particularly in patients with asplenia or alcoholism after dog or cat ❖ Small gram-negative, appear as coccobacilli or bacilli
bite through continuous contacts ❖ Aerobic, nonmotile, unencapsulated bacteria
❖ Smooth, raised and translucent colonies

B. + - V - - -
melitensis
B. + + +<2hrs +/- + -
abortus
B. suis + + +<0.5hr - - +
B. canis - - +<0.5hr - - +
H. Pasteurella

❖ Zoonosis
❖ Animal bites – most common presentation Brucella + - + + + -
❖ Gram-negative, nonmotile, facultative, anaerobic coccobacilli that B. - + + + + -
appear ovoid, filamentous or as bacilli bronchiseptica
Acinetobacter - - V - + -
❖ Bipolar staining (safety pin appearance)
P. + - + V + -
❖ Catalase and oxidase (most isolates) positive phenylpyruvicus
❖ Ferment glucose with weak to moderate acid production without gas O. ureolytica + V + + +
❖ Grow on SBA and CHOC agar, producing grayish colonies H. influenzae V - V + - +
❖ P. multocida
➢ Nonhemolytic colonies on SBA
➢ Appear mucoid after 24 hours of incubation at 37C
➢ Production of a around the colony after 48 hours

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[BACT211] TRANS 14: Haemophilus, HACEK, Legionella, Fastidious Gram (-) Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH
J. Francisella K. Legionella

❖ Small, nonmotile, gram-negative bacilli or coccoid bacteria and are

(L. pneumophila gram stain)
strictly aerobic
❖ Facultative intracellular parasites
❖ Ubiquitous gram-negative bacilli
❖ Fastidious and require supplementation with cysteine, cystine or
❖ Acquired primarily through inhalation from environmental sources
thiosulfate
❖ Slow growth (3-5 days)
❖ CHOC, MTM and buffered charcoal yeast extract (BCYE) agars and
❖ Characteristic
thioglycolate broth may be used
➢ “ground glass” colony morphology
❖ Broth cultures are not recommended
❖ Lightly staining, gram-negative bacillus
❖ Tularemia
❖ Requires L-cysteine for primary isolation
➢ Zoonotic disease and has many other names including:
❖ No growth on unsupplemented sheep blood agar
▪ Rabbit fever
❖ Asaccharolytic
▪ Deerfly fever
❖ Catalase or oxidase
▪ Lemming fever
▪ Water rat trapper;s disease
➢ Tularemia can be contracted through ingestion, inhalation, A. Clinical Infections
arthropod bite (e.g., ticks, biting flies) or contact with infected
tissues ❖ Legionnaire’s Disease
➢ Febrile disease (associated with fever) with pneumonia
➢ Incubation period: 2 to 10 days
❖ Pontiac Fever
➢ Influenza-like febrile disease
➢ Non-pneumonic form of legionellosis
➢ Incubation period: 2 days
➢ L. pneumophila is responsible for most cases of this illness

B. Laboratory Diagnosis

❖ Pleomorphic, weakly staining, gram-negative bacilli that are

❖ Common Species: approximately 1 to 2 um x 0.5um in size
➢ Francisella tularensis subsp. tularensis ❖ Extending the safranin counterstaining time to at least 10 minutes can
▪ Type A enhance the staining intensity of the organisms
▪ Most severe, all forms of tularemia ❖ L. micdadei is weakly acid fast in tissue and stains best with the
➢ Francisella tularensis subsp. holartica modified Kinyoun procedure
▪ Type B ❖ Will not grow on standard Sheep Blood Agar
▪ Least severe, all forms of tularemia ❖ Buffered Charcoal Yeast Extract Agar (BCYE)
➢ Francisella tularensis subsp. mediasiatica ➢ Cysteine is essential for growth
▪ Severe ➢ Iron is essential for growth
➢ On BCYE agar, colonies appear as grayish white or blue-green,
Different Types of Tularemia convex, and glistening, measuring approximately 2 to 4mm in
Types of Tularemia Clinical Manifestations diameter
Infection ➢ “ground glass” appearance
Ulceroglandular Common; ulcer & lymphadenopathy, rarely
fatal
Glandular Common, lymphadenopathy, rarely fatal
Oculoglandular Conjunctivitis and lymphadenopathy
Oropharyngeal Ulceration in the oropharynx
Systemic (typhoidal) Acute illness with septicemia, no ulcer or
tularemia lymphadenopathy
Pneumonic tularemia Pneumonia, most serious form of tularemia

A. Ideal Media and Methods of Identification

❖ Culture Media of Choice:

➢ Blood-Cystine-Glucose Agar with Thiamine
➢ Modified/ Buffered Charcoal Yeast Extract Agar (BCYE)
➢ Chocolate Agar with ISOVITALEX
❖ Direct Fluorescent Antibody (DFA) Stain
❖ Antibody titers as low as 1:40 in the absence of previous disease is (colony morphology of L. pneumophila on BCYE agar)
diagnostic and may rise up to 1:640 or greater with the 1st 3 weeks
❖ Forshay Test
➢ Susceptibility test for tularemia

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[BACT211] TRANS 14: Haemophilus, HACEK, Legionella, Fastidious Gram (-) Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH
L. Bordetella ❖ Isolation Methods
➢ Bordet-Gengou Potato Infusion Agar with glycerol and horse
or sheep blood
➢ Regan-Lowe Medium contains charcoal, starch, horse blood,
cephalexin, and amphotericin B
➢ Stainer-Scholte Medium contains casamino acids
❖ Colony Morphology
➢ Young cultures-smooth, glistening and silver, resembling
mercury droplets
➢ Turn whitish gray as they age

(B. pertussis gram stain)

❖ Small, gram-negative bacilli or coccobacilli

❖ Obligate aerobic bacteria, grow best at 35 to 37C
❖ Do not ferment carbohydrates
❖ B. pertussis is inhibited by fatty acids, metal ions, sulfides and
peroxidases, constituents found in many media (B. pertussis on charcoal horse blood agar with cephalexin)
❖ Media for the isolation of B. pertussis require protective substances ❖ Identification Methods
such as charcoal, blood or starch, to bind and neutralize inhibitory ➢ Gram Stain: tiny gram-negative coccobacilli
substances ➢ Because of the fastidious nature of these organisms,
❖ B. pertussis and B. parapertussis identification by biochemical testing is difficult
➢ Primary human pathogens of the respiratory tract, causing B. pertussis B. B.
whooping cough or pertussis parapertussis bronchioseptica
Charcoal-Horse + + +
Blood
A. Virulence Factors (3-5 days) (2-3 days) (1-2 days)
Blood Agar - + +
MacConkey - - +
❖ Filamentous hemagglutinin (FHA) Agar
❖ Pertussis toxin (PT) Catalase + + +
❖ Adenylate cyclase toxin Oxidase + - +
❖ Tracheal cytotoxin Urease - + (24hrs) + (4hrs)
Nitrate - - +
reduction
B. Clinical Manifestations Motility - - +

1. Pertussis (1 to 3 weeks) II. ANAEROBES OF CLINICAL IMPORTANCE

A. Gram-Positive, Spore-Forming Anerobic Bacilli

A. Clostridium spp.

(Infant with pertussis)

❖ Catarrhal Phase
➢ Symptoms are insidious and nonspecific and include sneezing,
mild cough, runny nose and perhaps conjunctivitis
➢ Infants can develop apnea or respiratory distress or both (C. perfringes and C. tetani gram stain)
➢ Highly communicable
➢ Cultures are not performed because in this stage it is nonspecific ❖ Gram positive large spore forming bacilli
❖ Paroxysmal Phase ❖ Oval subterminally located spores
➢ Hallmark: sudden onset of severe, repetitive coughing followed ❖ C. tetani
by the characteristic “whoop” at the end of the coughing spell ➢ Round terminally located
➢ B. parapertussis ❖ Drumstick, tack head, lollipop, tennis racket bacillus
▪ Causes a similar disease with milder symptoms ❖ No spores – box or car shaped
❖ Convalescent Phase ➢ Terminal spores located at the end
➢ Begins with 4 weeks of onset with a decrease in frequency and ➢ Subterminal spores located other than the end
severity of the coughing spells ➢ Central spores located at the center

C. Laboratory Diagnosis

❖ Specimen Collection and Handling

➢ Specimen of Choice: Nasopharyngeal aspirates or swabs of
Dacron polyester
➢ Transport Media:
▪ Casamino acid (1% casein hydrolysate)
▪ Amies with charcoal
▪ Regan-Lowe transport medium

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[BACT211] TRANS 14: Haemophilus, HACEK, Legionella, Fastidious Gram (-) Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH
1. Clinical Infections
C. C. C. C.
perfringens botulinum tetani difficile
❖ Clostridium perfringens (Food Poisoning) Motility - + + +
➢ Type A – mild and self-limited GI illness BAP B-hemolysis B- B- B-
➢ Type C (enteritis necroticans) – more serious but rarely seen (double hemolysis hemolysis hemolysis
zone)
disease Glucose + + - +
❖ Botulism Lactose + - - -
➢ Ingestion of preformed botulinum toxin, produced in food by C. Lecithinase + - - -
botulinum Lipase - + - -
Reverse + - - -
➢ Botulinum Toxin Type A (Botox) is also used medically to treat CAMP
strabismus (wandering and chronic migraines, and as a beauty Stormy + - - -
enhancer by temporarily improving facial wrinkles) Fermentation
of Milk
➢ Food Botulism
▪ Ingestion of improperly canned goods
➢ Wound Botulism B. Gram-Positive, Non-Spore Forming, Anaerobic Bacilli
▪ Severe wound infection
A. Actinomyces spp.

➢ Infant Botulism
▪ Ingestion of contaminated home-made honeys

(A. israelii gram stain and colony morphology on blood agar plate)

❖ Straight to slightly curved bacilli of differing lengths, from short rods to

long filaments
❖ Some are fastidious, requiring special vitamins, amino acids and
hemin for adequate growth
❖ Young Actinomyces colonies are frequently spider like or wooly
❖ Older colonies of A. israelii usually have a molar tooth appearance

❖ Tetanus
➢ Attributed to the neurotoxin tetanospasmin produced by
Clostridium tetani
❖ Myonecrosis or Gas Gangrene
➢ Usually occurs when organisms contaminate wounds, through
trauma or surgery
➢ C. perfringens – most common cause
➢ C. histolyticum
➢ C. septicum
➢ C. novyi
➢ C. bifermentans

(A. israelii molar tooth appearance)

B. Bifidobacterium spp.

❖ Bacteremia
➢ Clostridium perfringens – most common
➢ Clostridium septicum (present in the bloodstream)
▪ marker organism for a malignancy in the GI tract
❖ Clostridioides difficile Associated Disease
➢ C. difficile ❖ Variable in shape, ranging from coccobacilli to long branching rods
▪ Most common but not the sole cause of antibiotic- ❖ Ends of the cells may be pointed, bent, club-shaped, spatulated or
associated diarrhea and pseudomembranous colitis bifurcated (forked)
▪ ❖ Singly or in chains and as starlike aggregates, V arrangements or
palisade clusters
❖ Colonies of are convex, entire and cream to white, smooth, glistening
and soft
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[BACT211] TRANS 14: Haemophilus, HACEK, Legionella, Fastidious Gram (-) Bacilli I Prof. Rochelle D. Darlucio, RMT, MPH

C. Lactobacillus spp. C. Prevotella spp.

❖ Pinpoint a-hemolytic colonies on SBA

❖ Medium in size, with a rough appearance and gray color
❖ Catalase negative

(P. corporis gram stain and P. nigrescens colony morphology)

❖ Gram-negative coccobacilli or bacilli
❖ Will grow on KVLB agar but no on BBE agar
❖ Resistant to vancomycin and kanamycin but are variable in colistin
❖ Produce protoporphyrin a dark pigment that causes their colonies to
(Colony morphology and gram stain of Lactobacillus plantarum) become brown to black with age

D. Propionibacterium and Cutibacterium spp. D. Porphyromonas spp.

❖ Produce brick red fluorescence under UV light, similarly to Prevotella,

but some species do not fluoresce
❖ Not grow on KVLB agar
❖ Resistant to colistin
❖ Most are spot indole positive

E. Campylobacter ureolyticus Group

❖ Pleomorphic rods with a diphtheroid appearance

❖ C. acnes is a common member of the skin microbiota, it is frequently
isolated from blood culture bottles as a contaminant
❖ Propionibacterium propionicum can cause actinomycosis
❖ Propionic Acid is a major metabolic end product of
Propionibacterium spp.
C. Anaerobic Gram-Negative Bacilli

A. Bacteroides spp.

❖ Pitting anaerobes of the C. ureolyticus group:

➢ C. ureolyticus – bile sensitive
➢ C. gracili
➢ C. curvus
➢ C. rectus
➢ Sutterella wadsworthensis
▪ Bile tolerant
▪ Microaerophiles rather than obligate anaerobes
F. Fusobacterium
B. Bilophila spp.
❖ Long, thin, tapered rods, a morphology characteristically referred to as
❖ Bilophila wadsworthia is a bile-resistant anaerobe fusiform
❖ Grow on BBE agar with a characteristic fish-eye appearance ❖ Fusobacterium nucleatum has cells that are consistently fusiform in
(Bacteroides Bile Esculin) shape
❖ Also grow on KVLB agar (Kanamycin and Vancomycin with Laked
Sheep Blood)
❖ The organism is strongly catalase positive and nitrate positive

❖ Fusobacterium mortiferum appear pleomorphic and exhibit globular

forms, swellings and other bizarre shapes

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Mycobacterium tuberculosis and LEC 2020-2021

2nd SEM
OLFU Nontuberculous Mycobacteria 13 BACT211
College of Medical LEC
CLINICAL BACTERIOLOGY TRANS 15
FINALS
Laboratory Science
Transcriber: Riyoma Surell
Batch 2023 Date: May 25, 2021

➢ Incubation:
Outline ▪ Rapid Growing Mycobacteria growth is apparent sooner
At the end of the session, the student must be able to learn: than 7 days
I. Developmental Notes ▪ Slow Growing Mycobacteria require more than 7 days to
II. Mycobacteria produce colonies on solid media
A. Mycobacterium tuberculosis
A. Identification of Mycobacterium Tuberculosis
B. Mycobacterium bovis
A. Mycobacterium tuberculosis
III. Clinical Significance of Non-tuberculous Mycobacteria
A. Slow-Growing Species
A. Mycobacterium avium
B. Mycobacterium intracellulare
C. Microscopic Examination
D. Mycobacterium avium subsp paratuberculosis
E. Mycobacterium kansasii
F. Mycobacterium genavense
G. Mycobacterium haemophilum
H. Mycobacterium marinum
I. Mycobacterium scrofulaceum
J. Mycobacterium simiae
K. Mycobacterium szulgai
L. Mycobacterium ulcerans
M. Mycobacterium xenopi
B. Rapidly Growing Species
A. Mycobacterium chelonae-Mycobacterium abscessus group ❖ First described by Robert Koch in 1882 (known as Koch’s bacillus)
B. Mycobacterium foruitum group ❖ TB is one of the oldest documented communicable diseases and
C. Mycobacterium smegmatis group
C. Mycobacterium leprae
remains a leading cause of morbidity and death globally
IV. Isolation and Identification of Mycobacteria
A. Specimen Collection ❖ Primary Tuberculosis
B. Digestion and Decontamination of Species ➢ Tubercle bacilli are acquired from persons with active disease
A. Decontamination and Digestion Agents who are excreting viable bacilli by coughing, sneezing or talking
C. Staining for Acid-Fast Bacilli ➢ Positive purified protein derivative (PPD) skin test result
D. Culture Media and Isolation ➢ Children: nonproductive cough and fever, with or without
E. Media for Antibiotic Susceptibility Testing
F. Preliminary Identification of Mycobacteria
shortness of breath
V. Biochemical Identification
A. Niacin Accumulation ❖ Reactivation Tuberculosis
B. Nitrate Reduction ➢ Occurs when there is an alteration or suppression of the cellular
C. Catalase immune system in the infected host that favors replication of the
D. Hydrolysis of Tween 80 bacilli and progression to disease
E. Iron uptake ➢ Symptoms:
F. Arylsulfatase
G. Pyrazinamidase
▪ Slow in developing (insidious) and consist of fever
H. Tellurite Reduction ▪ Shortness of breath
I. Urease ▪ Night sweats and chills
VI. Inhibitory Tests ▪ Fatigue, anorexia
A. Thiophene-2-Carboxylic Acid Hydrazide ▪ Weight loss
B. Sodium Chloride Tolerance ❖ Extrapulmonary Tuberculosis
C. Growth on MacConkey Agar ➢ Common presentation in individuals with HIV infection, although
it is most often associated with pulmonary disease
I. DEVELOPMENTAL NOTES ➢ Miliary Tuberculosis
❖ First isolated by Robert Koch as the cause of tuberculosis ▪ Life threatening
❖ 1886 – named as Bacterium tuberculosis by Zoppf ▪ Refers to the seeding of many organs outside the
❖ Later named as Mycobacterium tuberculosis by Neumann because of pulmonary tree with AFB through hematogenous spread
the organisms “fungus-like” characteristics (myco) ▪ Most common sites of spread of M. tuberculosis are:
❖ Mycobacterium tuberculosis identified as cause of tuberculosis (TB) • Spleen
in man • Liver
❖ Mycobacterium bovis identified as cause of tuberculosis (TB) in • Lungs
cattle • Bone marrow
❖ Mycobacterium avium identified as cause of tuberculosis (TB) in • Kidney
chickens • Adrenal gland
❖ 1959 – Runyon classified Mycobacteria other than tuberculosis
(MOTT) bacilli on pigment production and growth characteristics –
arising to Runyon’s Classification A. Identification of Mycobacterium Tuberculosis
II. MYCOBACTERIA
❖ Colonies Appearance
❖ General Characteristics ➢ Raised, with a dry, rough appearance, nonpigmented and
➢ Slender, slightly curved or straight, rod-shaped organisms 0.2 to classically described as being buff colored “cauliflower
0.6um x 1 to 10um in size appearance”
➢ Nonmotile and non-spore forming ➢ Cord factor can result in characteristic cord formation
➢ Cell Wall: has extremely high lipid content (mycolic acid), ➢ Optimal growth occurs 35C to 37C
contains N-glycolylmuramic acid instead of N-acetylmuramic ➢ Positive for niacin accumulation
acid ➢ Reduces nitrate to nitrite
➢ Strictly aerobic ➢ Production of catalase (which is destroyed after heating – heat
stable catalase negative)

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[BACT211] TRANS 15: Mycobacterium tuberculosis and Nontuberculous Mycobacteria I Prof. Rochelle D. Darlucio, RMT, MPH
❖ Mycobacterium intracellulare
➢ Grow slowly
➢ Producing thin, transparent or opaque, homogenous smooth
colonies
➢ A small proportion may exhibit rough colonies
➢ Nonpigmented, but they may become yellow with age
➢ Optimal growth temperature is 37C
❖ Microscopic Examination
➢ Short, coccobacillary, and uniformly stained, without beading or
banding
➢ Long, thin, beaded bacilli resembling Nocardia spp., may be seen
in stains of very young cultures or under certain other conditions
❖ Mycobacterium avium subsp. paratuberculosis
➢ Causative agent of Johne disease, an intestinal infection
❖ Treatment occurring as a chronic diarrhea in cattle, sheep, goats and other
➢ Pulmonary TB: 9-month course of therapy with isoniazid and ruminants
rifampin, usually once per day in the first month ➢ Difficult to cultivate because of its very slow growth rate (3 to 4
➢ BCG (Bacillus Calmette-Guerin) Vaccine: an attenuated form months)
of M. bovis ➢ Need for a mycobactin supplemented medium for primary
isolation
B. Mycobacterium bovis ➢ Mycobactin is an iron-binding hydroxamate compound
produced by other mycobacterial species
❖ Produces TB primarily in cattle but also in other ruminants as well as ❖ Mycobacterium kansasii
in dogs, cats, swine, parrots and humans ➢ Second to MAC as the cause of NTM lung disease
❖ In Humans: closely resembles that caused by M. tuberculosis and is ➢ Chronic Pulmonary Disease: most common manifestation
treated similarly ➢ Slow-growing organism
❖ It grows very slowly on egg-based media ➢ Appears as long rods with distinct cross-banding
❖ Produces small, granular, rounded, nonpigmented colonies with ➢ Optimal growth temperature: 37C
irregular margins after 21 days of incubation at 37C ➢ Colonies appear smooth to rough, with characteristic wavy edges
❖ On Middlebrook 7H10 medium, colonies are similar to those of M. and dark centers when grown on Middlebrook 7H10 agar
tuberculosis but slower to mature ➢ Colonies are photochromogenic
❖ Most strains are niacin negative, do not reduce nitrate and do not grow ➢ Most strains are strongly catalase positive
in the presence of T2H, characteristics that distinguish the species
from most strains of M. tuberculosis

III. CLINICAL SIGNIFICANCE OF NONTUBERCULOSIS

MYCOBACTERIA

A. Slowly Growing Species ❖ Mycobacterium genavense

A. Mycobacterium avium complex

❖ Mycobacterium avium ➢ Reported as a cause of disseminated infections in patient with

➢ A cause of disease in poultry, cattle and swine but animal to AIDS
human transmission has not been shown to be an important ➢ Slow-growing
factor in human disease ➢ Growth was obtained when Middlebrook 7H11 agar was
➢ M. avium subsp. hominissuis responsible for most human supplemented with mycobactin
infections ➢ Isolates yield positive test results for semi-quantitative and heat-
stable catalase, pyrazinamidase and urease

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[BACT211] TRANS 15: Mycobacterium tuberculosis and Nontuberculous Mycobacteria I Prof. Rochelle D. Darlucio, RMT, MPH
❖ Mycobacterium haemophilum ➢ Most common cause of cervical lymphadenitis in children
➢ Unique characteristic: requirement for hemoglobin or hemin for ➢ Microscopic Examination
growth ▪ Uniformly stained, acid-fast, medium to long rod
➢ Culture Media: ➢ Grows slowly (4 to 6 weeks) at incubation temperature ranging
▪ Chocolate (CHOC) agar from 25C to 37C
▪ Mueller-Hinton agar with 5% Fildes enrichment ➢ Colonies are smooth with dense centers and pigmentation from
▪ LJ medium containing 2% ferric ammonium citrate light yellow to deep orange
➢ Optimal Growth Temperature: 28 C to 32C ➢ Scotochromogenic
➢ Colonies are rough to smooth and nonpigmented ➢ Do not hydrolyze tween 80 or reduce nitrate, but do produce
➢ Strongly acid-fast, short, occasionally curved bacilli without urease and are high (>45mm) catalase producers
banding or beading, and arranged in fight clusters or cords ❖ Mycobacterium simiae

❖ Mycobacterium marinum

➢ Short coccobacilli
➢ When they are grown on inspissated egg medium at 37C, smooth
colonies appear in 10 to 21 days
➢ Colonies on Middlebrook 7H10 agar are thin, transparent or tiny,
and filamentous
➢ Photochromogenic
➢ Development of the yellow pigment may require prolonged
➢ Implicated in diseases of fishes and is isolated from fishes in incubation, whereas some strains may fail to produce pigment on
aquariums exposure to light
➢ Outbreaks of cutaneous lesions in lifeguards have been reported ❖ Mycobacterium szulgai
▪ Tender red or blue-red subcutaneous nodule, or ➢ Most common manifestation is pulmonary disease similar to TB
“swimming pool granuloma” usually occurring on the ➢ Medium to long rods, with some crossbarring
elbow, knee, toe or finger ➢ When the organism is cultured on egg-based medium at 37C,
smooth and rough colonies are observed
➢ At 37C, yellow to orange pigment develops in the absence of light
and intensifies with exposure to light
➢ Colonies grown at 22C are nonpigmented or buff in the absence
of light and develop yellow to orange pigment with light exposure
❖ Mycobacterium ulcerans (Inert Bacillus)
➢ Disease manifests itself as a painless nodule under the skin after
previous trauma
➢ A shallow ulcer, also referred to as Buruli Ulcer develops that
may be quite severe

➢ Cells are moderately long to long rods with cross-barring

➢ Colonies of this slowly growing organism are smooth to rough
and wrinkled on inspissated egg medium but may be smooth
when grown on Middlebrook 7H10 or 7H11 agar ➢ Long, without beading or cross-banding
➢ Photochromogenic ➢ Optimal growth temperature is 30 to 33C with little growth at 25C
➢ Young colonies grown in the dark may be nonpigmented or buff, and usually none at 37C
whereas colonies exposed to light develop a deep yellow color ➢ Grows slowly, often requiring 6 to 12 weeks of incubation before
➢ Growth is optimal at incubation temperatures of 28C to 32C colonies are evident
➢ Hydrolyze tween 80 and produce urease and pyrazinamidase ➢ Colonies are smooth or rough and nonpigmented or lightly buff
and they do not develop pigment with exposure to light
❖ Mycobacterium scrofulaceum ➢ Produces a heat-stable catalase but is inert in most other
conventional biochemical tests
❖ Mycobacterium xenopi
➢ Recovered from hot and cold water taps (including water storage
tanks of hospitals) and birds
➢ Long filamentous rods
➢ Colonies of this slow-growing mycobacterium on Middlebrook
7H10 agar are small, with dense centers and filamentous edges
➢ Cornmeal-glycerol Agar: distinctive round colonies with
branching and filamentous extensions; aerial hyphae are usually
seen in rough colonies
➢ Young colonies grown on cornmeal agar have a “bird’s nest”
appearance, with characteristic sticklike projections
➢ Optimal growth temperature is 42C; the organism grows more
rapidly at this temperature than at 37C and fails to grow at 25C
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[BACT211] TRANS 15: Mycobacterium tuberculosis and Nontuberculous Mycobacteria I Prof. Rochelle D. Darlucio, RMT, MPH
B. Rapidly Growing Species IV. ISOLATION AND IDENTIFICATION OF MYCOBACTERIA

❖ Mycobacterium chelonae – Mycobacterium abscessus group A. Specimen Collection

➢ Found in the environment and is associated with many of the
same opportunistic infections as those associated with M. Respiratory Body Fluids Body Tissues
fortuitum Specimens
➢ Most likely isolated from disseminated cutaneous infections in Spontaneously Pleural fluid Blood
immunocompromised patients
expectorated sputum Pericardial fluid Bone marrow
➢ Strongly acid fast, with pleomorphism ranging from long, tapered Normal saline-nebulized, Joint aspirate biopsy/aspirate
to short, thick rods induced sputum Gastric aspirate Solid organ
➢ Produces rough or smooth, nonpigmented to buff colonies within
Transtracheal aspirate Peritoneal fluid Lymph node
3 to 5 days of incubation at 37C Bronchoalveolar lavage Cerebrospinal Bone
❖ Mycobacterium fortuitum Group Bronchoalveolar fluid Skin
➢ Isolated from water, soil and dust
brushing Stool
➢ After 3 to 5 days of incubation at 37C, colonies of M. fortuitum Laryngeal swab Urine
appear rough or smooth and nonpigmented, creamy white or buff Nasopharyngeal swab Pus
➢ Pleomorphic, ranging from long and tapered to short, thick rods
➢ Positive 3-day arylsulfatase test result and reduction of nitrate
B. Digestion and Decontamination of Specimens
❖ Mycobacterium smegmatis Group
❖ Purpose:
➢ To liquefy the sample through digestion of the proteinaceous
material
➢ To allow the chemical decontaminating agent to contact and kill
the non-mycobacterial organisms
❖ Specimens require digestion and decontamination include sputum,
gastric washing, BAL, bronchial washing and transtracheal aspirate
specimens

A. Decontamination and Digestion Agents

❖ Sodium Hydroxide
➢ Usual concentration: 2%, 3% or 4%
➢ Serves as digestant and decontaminating agent
➢ Contains two species: M. smegmatis and M. goodii ➢ It is a commonly used decontaminant but must be used with
➢ Occasionally, rods are curved with branching or Y-shaped forms; caution because it is only slightly less harmful to the
swollen, with deeper staining, beaded or ovoid forms are mycobacteria than to the contaminating organisms
sometimes seen ❖ N-Acetyl-L-cysteine
➢ Colonies appearing on egg medium after 2 to 4 days are usually ➢ A combination of a liquefying agent, such as N-acetyl-L-cysteine
rough, wrinkled or coarsely folded, smooth, glistening, butyrous (NALC) or dithiothreitol, plus NaOH is also commonly used
colonies may also be seen ❖ Benzalkonium Chloride
➢ Colonies on Middlebrook 7H10 agar are heaped and smooth or ➢ Benzalkonium chloride (Zephiran) combined with trisodium
rough with dense centers phosphate (TSP)
➢ Pigmentation is rare or late: colonies appear nonpigmented, ➢ TSP rapidly liquefies sputum but requires a long exposure time
creamy white, or buff to pink in older cultures to decontaminate the specimen
➢ Negative arylsulfatase reaction, positive iron uptake, ability to ➢ Shortens the exposure time and effectively destroys many
reduce nitrate and growth in the presence of 5% sodium chloride contaminants, with little bactericidal effect on tubercle bacilli
(NaCl) an don MacConkey agar without crystal violet ❖ Oxalic Acid (5%)
C. Mycobacterium leprae ➢ Used to decontaminate specimens contaminated with P.
aeruginosa

C. Staining for Acid-Fast Bacilli

❖ Acid Fast Stain

➢ Ziehl Neeisen Technique (Hot Method)
➢ Kinyoun Technique (Cold Method)
❖ Fluorescent Stain – more sensitive but more difficult and expensive
to perform
➢ Auramine O – primary stain
➢ Rhodamine – counterstain
➢ MTB – yellow green bacilli

❖ Causative agent of Hansen Disease (Leprosy) No. of Acid-Fast Bacilli Seen

❖ Two major forms: Carbolfuchsin Fluorochrome Quantitative Report
➢ Tuberculoid leprosy (one part of the body) Stain x1000 Stain, x450
➢ Lepromatous leprosy 0 0 No acid-fast bacilli seen
❖ Has not been grown on artificial media 1-2/300 fields 1-2/70 fields Doubtful acid-fast
❖ Not as acid fast or alcohol fast as in the case of other mycobacteria; bacilli seen; resubmit
as such, a weaker decolorizer consisting of 10% sulfuric acid is another specimen for
recommended instead of the standard acid-ethanol decolorizer examination
❖ Rod shaped, usually 1 to 7um long and 0.3 to 0.5um wide 1-9/100 fields 1-2/70 fields 1+
1-9/10 fields 2-18/50 fields 2+
1-9/field 4-36/field 3+
>9/field >36/field 4+

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[BACT211] TRANS 15: Mycobacterium tuberculosis and Nontuberculous Mycobacteria I Prof. Rochelle D. Darlucio, RMT, MPH
D. Culture Media and Isolation Methods ❖ Temperature
➢ M. marinum, M. ulcerans, and M. haemophilum grow best at 30C
❖ Primary Isolation Media (egg-based media) to 32C and poorly, if at all, at 35C to 37C
➢ Lowenstein-Jensen ➢ M. xenopi grows best at 42C
▪ Coagulated whole eggs and malachite green as inhibitor ❖ Photoreactivity
(0.025g/dL) ➢ Photochromogens
➢ Petragnani ▪ Produce carotene pigment on exposure to light are
▪ Coagulated whole eggs and malachite green as inhibitor ▪ Color ranges from pale yellow to orange
(0.052g/dL)
▪ More inhibitory than the other
▪ Used for heavily contaminated specimens
➢ American Thoracic Society
▪ Coagulated whole eggs and malachite green as inhibitor
(0.02g/dL)
▪ Less inhibitory than the other
▪ Used for sterile specimens

E. Media for Antibiotic Susceptibility Testing (transparent media-agar

based)

❖ Middlebrook 7H10
➢ Salts, vitamins, cofactors, oleic acid, albumin, catalase, biotin,
glycerol, glucose, malachite green as inhibitor (0.0025g/dL) ➢ Scotochromogens
▪ Produce pigment in the light or the dark
▪ Growth temperature may influence the photoreactive
characteristics of a species

❖ Middlebrook 7H11
➢ Salts, vitamins, cofactors, oleic acid, albumin, catalase, biotin,
glycerol, casein hydrolysate, malachite green as inhibitor
(0.0025g/dL)

F. Preliminary Identification of Mycobacteria ➢ Nonchromogenic or Nonphotochromogenic

▪ Other species, such as M. tuberculosis
❖ Colony Morphology ▪ Buff (tan) color and are nonphotoreactive
▪ Exposure to light does not induce pigment formation

Nonchromogens Photochromogens Scotochromogens

Slow Growers Slow Growers Slow Growers
M. tuberculosis M. asiaticum M. gordonae
M. avium complex M. kansasii M. szulgai
M. bovis M. marinum M. scrofulaceum
M. celatum M. simiae M. xenopi
M. gastri Rapid Growers
M. genavense M. phlei
M. haemophilum M.smegmatis group
M. malmoense
M. terrae complex
M. ulcerans
Rapid Growers
M. chelonae
M. fortuitum group

➢ Smooth and soft or rough and friable appearance

➢ M. tuberculosis: rough, often exhibit a prominent patterned
texture referred to as cording (curved strands of bacilli)
➢ Colonies of MAC have a variable appearance, with glossy whitish
colonies often occurring with smaller translucent colonies
❖ Growth Rate
➢ Rapid Growers: Visible growth in fewer than 7 days
➢ Slow Growers: producing colonies in more than 7 days

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[BACT211] TRANS 15: Mycobacterium tuberculosis and Nontuberculous Mycobacteria I Prof. Rochelle D. Darlucio, RMT, MPH
V. BIOCHEMICAL TESTS D. Hydrolysis of Tween 80

A. Niacin Accumulation ❖ Results are recorded as positive after 24 hours, 5 days or 10 days
❖ This test is helpful in distinguishing between scotochromogenic and
❖ NIACIN + Cyanogen bromide (toxin reagent) = yellow colored nonphotochromogenic mycobacteria
solution ❖ Key reaction for M. kansasii = Positive as quickly as 6 hours
❖ All Mycobacterium species produce niacin and most passes another ❖ Important test to differentiate M. gordonae (tap water bacillus)
enzyme to convert free niacin to niacin ribonucleotide (positive) and M. scrofulaceum (negative)
❖ Important test for identifying MTB because it lacks the enzyme
❖ MTB: Positive Niacin Test

E. Iron Uptake

❖ Some mycobacteria can convert ferric ammonium citrate to an iron

B. Nitrate Reduction
oxide
❖ Positive Reaction: Rusty brown colonies on the addition of 20%
❖ Test to detect nitrosoreductase enzyme production which reduces
aqueous solution of ferric ammonium citrate
Nitrate to Nitrite
❖ Most useful in distinguishing M. chelonae, which is generally negative
❖ Broth (inoculated with the organism then added with HCl,
for iron uptake, from other rapid growers, which are positive
sulfanilamide, and N-naphthylenediamine dihydrochloride = pink to
red color (positive)
F. Arylsulfatase
❖ Positive: M. kansasii, M. szulgai, M. fortuitum, M. tuberculosis
❖ The M. fortuitum complex. M. chelonae, M. xenopi and M. triviale have
rapid arylsulfatase activity that can be detected in 3 days
❖ M. marinum and M. szulgai exhibit activity within 14 days of incubation

G. Pyrazinamidase

❖ This reaction occurs in about 4 days and may be useful in

distinguishing M. marinum (+) from M. kansasii and M. bovis (-) from
M. tuberculosis (+)

H. Tellurite Reduction

❖ Reduction of colorless potassium tellurite to black metallic tellurium in

3 to 4 days is a characteristic of MAC and thus is useful in
distinguishing MAC from other nonchromogenic species
❖ In addition, all rapid growers are able to reduce tellurite in 3 days

C. Catalase

❖ Most mycobacterium species are catalase

❖ Slide is warmed at 68C for 20 mins + 30% H2O2 = bubbling or
effervescence
❖ M. tuberculosis complex are all heat stable catalase (-) which includes:
➢ M. gastri
➢ M. haemophilum
➢ M. marinum

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[BACT211] TRANS 15: Mycobacterium tuberculosis and Nontuberculous Mycobacteria I Prof. Rochelle D. Darlucio, RMT, MPH
I. Urease

❖ Used to distinguish M. scrofulaceum, which is urease positive from M.

gordonae, which is urease negative
❖ A loopful of test organism is grown in 4mL of urea broth at 37C for 3
days
❖ A pink to red color is indicative of a positive reaction

VI. INHIBITORY TESTS

A. Thiophene-2-carboxylic acid Hydrazide

❖ T2H distinguishes M. bovis from M. tuberculosis

❖ M. bovis is susceptible to lower concentrations of T2H than M.
tuberculosis

B. Sodium Chloride Tolerance

❖ M. flavescens, M. triviale, and most rapidly growing Mycobacterium

spp. are exceptions that do grow in the presence of 5% NaCl

C. Growth on MacConkey Agar

❖ Mycobacterium fortuitum chelonae complex an grow on MacConkey

agar without crystal violet, whereas most other mycobacteria cannot
❖ Skin Testing
➢ Tuberculin Skin Test
▪ To determine an individual’s exposure to M. tuberculosis
▪ A reactive tuberculosis skin test indicates past exposure to
M. tuberculosis; other Mycobacterium spp. generally result
in an induration smaller than 10mm

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OLFU Spirochetes LEC 2020-2021

2nd SEM
College of Medical CLINICAL BACTERIOLOGY 13 BACT211
LEC
Laboratory Science Transcriber: Riyoma Surell FINALS
Date: May 25, 2021 TRANS 16
Batch 2023

A. Serovites
Outline
At the end of the session, the student must be able to learn:
I. Spirochetes
❖ Leptospira interrogans Serovar icterohaemorrhagiae – Weil’s
A. Leptospires syndrome
A. Serovites ❖ Leptospira interrogans Serovar canicola – Infectious jaundice
B. Important Laboratory Tests ❖ Leptospira interrogans Serovar autumnalis – Fort bragg/pretibial
B. Borreliae fever
A. Agents of Relapsing Fever ❖ Leptospira interrogans Serovar hebdomadis – Seven day fever
B. Important Laboratory Tests ❖ Leptospira interrogans Serovar grippotyphosa – Marsch fever
C. Agents of Lyme Disease
D. Laboratory Diagnosis
❖ Leptospira interrogans Serovar mitis/Pomona – Swine herd disease
C. Treponemes
A. Treponema pallidum subspecies pallidum B. Important Laboratory Tests
1. Stages of Venereal Syphilis
B. Laboratory Diagnosis
1. Specimen Collection and Handling ❖ Culture Media of Choice
2. Serological Tests ➢ Fletcher’s/ Stuart’s Medium or Ellinghausen-McCullough –
C. Other Treponemal Diseases Johnson – Harris (EMJH) Medium
▪ Incubation of the media in the dark at room temperature
I. SPIROCHETES ▪ Growth may be examined using Darkfield Microscopy
❖ Serologic Tests
❖ Slender, flexuous, helically shaped, unicellular bacteria ranging in size ➢ IgM (detected during acute phase of illness) antibodies are
from 0.1 to 0.5um wide and form 5 to 20um long, with one or more detected within 1 week after onset of disease and may persist in
complete turns in the helix high titers for many months
❖ Have a flexible cell wall ▪ IgG (past infection)
❖ Periplasmic flagella (also known as axial fibrils, axial filaments, ➢ Enzyme-linked immunosorbent assay (ELISA)
endoflagella and periplasmic fibrils) are responsible for motility ➢ Macroscopic slide agglutination test for rapid screening
❖ Treponema spp. reproduce via transverse fission ➢ Gold standard: microscopic agglutination testing
❖ Leptospira and Borrelia divide by the more common binary fission
B. Borreliae
A. Leptospires ❖ General Characteristics
➢ Highly flexible organisms ranging in thickness from 0.2 to 0.5um
❖ General Characteristics and in length from 3 to 20um
➢ Tightly coiled, thin, flexible spirochetes, 0.1um wide and 5 to ➢ Loosely twisted resembling a stretched spiral
15um long ➢ Mode of Transmission: Tick/ Lice bite
➢ One or both ends of the organism have hooks ➢ Microaerophilic
➢ Motion is rapid translational (back and forth) and rotational ➢ Stains well with Giemsa or Wright Stain = Blue in color using
either stain

❖ Important Members
➢ Leptospira biflexa A. Agents of Relapsing Fever
▪ non-pathogenic, found in soil and water
➢ Leptospira interrogans
▪ causes of human and animal leptospirosis, a zoonosis ❖ Borrelia recurrentis
▪ primarily parasitic on vertebrates other than humans such ➢ Agent of louse borne relapsing fever characterized by fever
as rodents, cattle, dogs, cats, raccoons and bats – sheds muscle and bone pain, and confusion
the organism in the urine ➢ Patient appears recovered 6 days after the fever episodes only
▪ Modes of Acquisition of the Infection to relapse few days or weeks after
• Direct contact with the urine of animals carrying the ➢ Relapse attributed to the ability of the organism to after its
organism antigenicity
➢ Vector: Human louse – Pediculus humanus subspecies
• Indirect contact occurs when humans come in close
humanus
contact with contaminated soil and water
❖ Borrelia hermisii and Borrelia parkerii
• Leptospirosis
➢ Agent of tick borne relapsing fever
 Involved the kidney, liver, or CNS. Infection may ➢ Vector: Omithodoros hermisii and Omithodoros parkerii
be mild or severe accompanied by myalgia,
nausea, vomiting, fever, headache, and chills in
acute phase

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[BACT211] TRANS 16: Spirochetes I Prof. Rochelle D. Darlucio, RMT, MPH

B. Important Laboratory Tests ❖ Clinically Significant Species

➢ T. pallidum subsp. Pallidum – the causative agent of syphilis
➢ T. pallidum subsp. Pertenue – the causative agent of yaw
❖ Grows well in modified Kelly Medium – Barbour Stoenner-Kelly
➢ T. pallidum subsp. Endemicum – the causative agent of endemic
Medium (BSK-II) in 7-14 days between 30C to 35C
syphilis
❖ Successfully cultivated using Kelly Medium:
➢ Treponema carateum – the causative agent of pinta
➢ B. recurrentis
➢ Borrelia hermsii
➢ Borrelia parkeri A. Treponema pallidum subspecies pallidum
➢ Borrelia turicatae
➢ Borrelia hispanica ❖ Also known as Great pox/ Evil pox/ French disease/ Italian disease/
❖ Thick Blood Films Great imitator/ Social Disease/ LUES
➢ Preferred due to low number of organisms in the blood. Best ❖ Agent of Veneral Syphilis (STD)
collected during febrile episodes. May be stained sing aniline ❖ Mode of Transmission: direct sexual contact, blood transfusion,
dyes (Wright’s or Giemsa) transplacental route
❖ Syphilis has a wide variety of clinical manifestations, which gave rise
C. Agents of Lyme Disease to the name the “great imitator”

1. Stages of Venereal Syphilis

❖ Borrelia burgdorferi Sensu Lato
➢ Agent of Lyme Disease: transmitted via the bite of infected
ixodes ticks ❖ Primary Stage
❖ Stages of Lyme Disease ➢ Develops 10 to 90 days after infection
➢ Stage One (Localized): ➢ The lesion, known as chancre (hard chancre) is typically a
▪ Erythema migrans (EM), the classic skin lesion that is single erythematous lesion that is nontender but firm, with a clean
normally found at the site of the tick bite surface and raised border
▪ Begins as a red macule and expands to form large annular
erythema with partial central clearing, sometimes described
as having a target appearance
➢ Stage Two
▪ Disseminated early and produces widely variable symptoms
that include secondary skin lesions, migratory joint and
bone pain, alarming neurologic and cardiac disease,
splenomegaly and severe malaise and fatigue
➢ Stage Three (Late Persistent Infections)
▪ Also known as chronic stage
▪ Occur mainly in the cardiac, musculoskeletal and neurologic
systems

D. Laboratory Diagnosis

❖ Specimen Collection:
➢ Serum for serology
❖ Serologic Tests
➢ Immunofluorescence antibody (IFA) or enzyme immunoassay ❖ Secondary Stage
(EIA) screening ➢ Approximately 2 to 12 weeks after development of the primary
➢ Positive or equivocal results are confirmed with IgM and/or just ➢ With clinical symptoms of fever, sore throat, generalized
IgG Western blot lymphadenopathy, headache, lesions of the mucous membranes
❖ Treatment and rash
➢ Macrolides, doxycycline and amoxicillin ➢ All secondary lesions of the skin and mucous membranes are
highly infectious
C. Treponemes

❖ General Characteristics
➢ Thin, spiral organisms about 0.1 to 0.2um in thickness and 6 to
20um in length
➢ Difficult to visualize with a bright-field microscope
➢ Can be seen easily by using dark-field microscopy
➢ The ends are pointed and covered with a sheath
➢ The cells exhibit graceful flexuous movements in liquid
➢ Tightly and Helically Twisted organisms which resembles a
corkscrew appearance

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[BACT211] TRANS 16: Spirochetes I Prof. Rochelle D. Darlucio, RMT, MPH

❖ Latent Syphilis ❖ Treponemal Test
➢ Absence of clinical symptoms with a positive serological test ➢ Uses specific treponemal antigens in the test systems
❖ Tertiary (Late) Syphilis ➢ Treponema pallidum Immobilization (TPI)
➢ May include CNS involvement and neurological abnormalities ▪ Uses live treponemes
➢ Paralysis, delusions, blindness, deafness, cardiovascular ▪ After incubation with patient’s serum, a positive test is
abnormalities and appearance of granulomatous lesions known indicated by Immobilization of the spirochetes due to the
as Gummata/Gummas attachment of specific antibodies to the organisms
❖ Congenital Syphilis
➢ Treponemes can be transmitted from an infected mother to her
fetus by crossing the placenta ➢ Fluorescent Treponemal Antibody-Absorbed (FTA-
Absorbed)
▪ Uses a sorbent (non-pathogenic treponemal strain) to
remove cross reacting antibodies
▪ Treponemes staining with the fluorescent dye is a positive
reaction
➢ Microhemagglutination Test for T. pallidum (MHATP)
▪ RBC sensitized with T. pallidum antigens are mixed with
patient’s serum
▪ Agglutination is the positive reaction if the patient’s serum
has antibodies to T. pallidum
▪ Treatment: Penicillin is the drug of choice for treating
patients with syphilis

C. Other Treponemal Diseases

❖ Yaws
➢ Caused by T. palldum subsp. pertenue
B. Laboratory Diagnosis ➢ Resembles that of syphilis, but the early-stage lesions are
elevated, granulomatous nodules

1. Specimen Collection and Handling

➢ Surface of primary or secondary lesions is cleaned with saline

and gently abraded with dry, sterile gauze; induction of bleeding
should be avoided
➢ Demonstration of spirochetes using dark field microscopy using
sample from skin lesions during primary and secondary syphilis
only

2. Serological Tests

❖ Non-Treponemal Tests
▪ Detects Reagin or Wasserman Antibodies (non-specific)
antibodies produced in response to the infection ❖ Endemic Syphilis (“bejel”)
▪ Uses cardiolipin-lecithin antigen to detect reagin ➢ Caused by T. pallidum subsp. endemicum
➢ Rapid Plasma Reagin (RPR) Test ➢ The primary and secondary lesions are usually papules that often
▪ Antigen is coated with carbon and reaction is observed go unnoticed
against white card to aid visibility of black flocculation ➢ They can progress to gummas of the skin, bones, and
▪ Positive Reaction: Reactive nasopharynx
▪ Negative Reaction: Non-Reactive ➢ Dark-field microscopy is not useful because of normal oral
➢ Veneral Disease Research Laboratories (VDRL) spirochetal biota
▪ Uses cardiolipin-lecithin-cholesterol antigen in a flocculation ❖ Pinta
procedure ➢ Caused by T. carateum
▪ More sensitive than RPR, hence recommended for ➢ Acquired by person-to-person contact and is rarely transmitted
diagnosis and follow up of neurosyphilis through sexual intercourse
▪ Procedure is standardized and antigen must be titrated ➢ Lesions begins a scaling, painless papules and are followed by
an erythematous rash that becomes hypopigmented with time

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LEC
TRANS 11 NON-ENTERIC GASTROINTESTINAL PATHOGENS
10

I. Vibrio Spp.

❖ Oxidase ❖ Ornithine Decarboxylase

➢ All POSITIVE except V. metschnikovii Positive Negative Variable
❖ Indole V. cholerae G. hollisae V. alginolyticus
Positive Negative Variable V. mimicus V. cincinnatiensis
Grimontii hollisae Photobacterium V. alginolyticus V. parahaemolyticus P. damsela
V. cholerae damsela V. cincinnatiensis V. vulnificus V. fluvialis
V. harveyi V. fluvialis V. fumissii
V. mimicus V. fumissii V. harveyi
V. parahaemolyticus V. metschnikovii V. metschnikovii
V. vulnificus
❖ Growth in 0% NaCl
❖ Gas from Glucose Positive Negative Variable
➢ All NEGATIVE except V. fumissii V. cholerae G. hollisae
❖ Lactose V. mimicus V. alginolyticus
Positive Negative Variable V. cincinnatiensis
V. vulnificus Grimontii hollisae V. cholerae P. damsela
V. alginolyticus V. metschnikovii V. fluvialis
V. cincinnatiensis V. mimicus V. fumissii
P. damsela V. harveyi
V. fluvialis V. metschnikovii
V. fumissii V. parahaemolyticus
V. harveyi V. vulnificus
V. parahaemolyticus
❖ Growth in 6% NaCl
❖ Sucrose Positive Negative Variable
Positive Negative Variable G. hollisae V. cholerae
V. alginolyticus Grimontii hollisae V. harveyi V. alginolyticus V. metschnikovii
V. cholerae Photobacterium damsela V. cincinnatiensis V. mimicus
V. cincinnatiensis V. mimicus P. damsela
V. fluvialis V. parahaemolyticus V. fluvialis
V. fumissii V. vulnificus V. fumissii
V. metschnikovii V. harveyi
V. parahaemolyticus
❖ Lysine Decarboxylase V. vulnificus
Positive Negative Variable
V. alginolyticus G. hollisae V. cincinnatiensis ❖ Colony on TCBS
V. cholerae V. fluvialis P. damsela Green Yellow
V. harveyi V. fumissii V. metschnikovii G. hollisae V. alginolyticus
V. mimicus s P. damsela V. cholerae
V. parahaemolyticus V. mimicus V. cincinnatiensis
V. vulnificus V. parahaemolyticus V. fluvialis
V. vulnificus V. fumissii
❖ Arginine Dihydrolase V. harveyi
Positive Negative Variable V. metschnikovii
P. damsela G. hollisae V. metschnikovii
V. fluvialis V. alginolyticus ❖ Nitrate to Nitrite
V. fumissii V. cholerae Positive Negative Variable
V. cincinnatiensis V. cincinnatiensis
V. harveyi G. hollisae
V. mimicus P. damsela
V. parahaemolyticus V. fluvialis
V. vulnificus V. alginolyticus
V. parahaemolyticus
V. vulnificus
V. harveyi

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Summary for Vibrio spp.

❖ Arginine Dihydrolase
❖ Grimontii hollisae: Positive Oxidase, Indole, 6% NaCl, Green
TCBS, Nitrate to Nitrite Positive Negative Varia.
❖ Vibrio alginolyticus: Positive Oxidase, Sucrose, Lysine A. caviae complex A. veronii biovar veronii
Decarboxylase, 6%NaCl, Yellow TCBS, Nitrate to Nitrite A. hydrophila complex
❖ Vibrio cholerae: Positive Oxidase, Indole, Sucrose, Lysine A. jandaei
Decarboxylase, Ornithine Decarboxylase, 0% NaCl, Yellow TCBS A. schubertii
❖ Vibrio cincinnatiensis: Positive Oxidase, Sucrose, 6% NaCl, A. veronii biovar sobria
Yellow TCBS, Nitrate to Nitrite C. violaceum
❖ Photobacterium damsela: Positive Oxidase, Arginine
Dihydrolase, 6% NaCl, Green TCBS, Nitrate to Nitrite ❖ Ornithine Decarboxylase
❖ Vibrio fluvialis: Positive Oxidase, Sucrose, Arginine Dihydrolase, Positive Negative Varia.
6% NaCl, Yellow TCBS, Nitrate to Nitrite A. veronii biovar veronii A. caviae
❖ Vibrio fumissii: Positive Oxidase, Gas Glucose, Sucrose, Arginine A. hydrophila
Dihydrolase, 6% NaCl, Yellow TCBS A. jandaei
❖ Vibrio harveyi: Positive Oxidase, Indole, Lysine Decarboxylase, A. schubertii
6% NaCl, Yellow TCBS, Nitrate to Nitrite A. veronii biovar sobria
❖ Vibrio metschnikovii: Positive Sucrose, Yellow TCBS C. violaceum
❖ Vibrio mimicus: Positive Oxidase, Indole, Lysine Decarboxylase,
Ornithine Decarboxylase, 0% NaCl, Green TCBS ❖ Growth in 0% NaCl
❖ Vibrio parahaemolyticus: Positive Oxidase, Indole, Lysine Positive Negative Variable
Decarboxylase, Ornithine Decarboxylase, 6% NaCl, Green TCBS, A. caviae complex
Nitrate to Nitrite A. hydrophila complex
❖ Vibrio vulnificus: Positive Oxidase, Indole, Lactose, Lysine A. jandaei
Decarboxylase, Ornithine Decarboxylase, 6% NaCl, Green TCBS, A. schubertii
Nitrate to Nitrite A. veronii biovar sobria
A. veronii biovar veronii
II. Aeromonas spp. C. violaceum

❖ Oxidase ❖ Growth in 6% NaCl

Positive Negative Variable Positive Negative Variable
A. caviae complex Chromobacterium A. caviae complex A. hydrophila
A. hydrophila complex violaceum A. jandaei
A. jandaei A. schubertii
A. schubertii A. veronii biovar sobria
A. veronii biovar sobria A. veronii biovar veronii
A. veronii biovar veronii C. violaceum

❖ Indole ❖ TCBS Growth

Positive Negative Variable Positive Negative Variable
A. hydrophila complex A. caviae complex A. caviae complex
A. jandaei A. schubertii A. hydrophila complex
A. veronii biovar sobria C. violaceum A. jandaei
A. veronii biovar veronii A. schubertii
A. veronii biovar sobria
A. veronii biovar veronii
❖ Gas from Glucose
Positive Negative Variable ❖ Voges Proskauer
A. jandaei A. caviae A. hydrophila Positive Negative Variable
A. veronii biovar sobria A. schubertii A. hydrophila
A. veronii biovar veronii C. violaceum A. veronii biovar sobria
A. veronii biovar veronii
❖ Esculin Hydrolysis A. schubertii
Positive Negative Variable
A. caviae complex A. jandaei A. hydrophila
A. veronii biovar veronii A. schubertii
C. violaceum

❖ Lysine Decarboxylase
Positive Negative Variable
A. jandaei A. caviae A. hydrophila
A. schubertii C. violaceum
A. veronii biovar sobria
A. veronii biovar veronii

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❖ Fermentation III. Campylobacter spp.

Sucrose
Positive Negative Variable ❖ Growth at
A. caviae complex A. jandaei A. hydrophila 15C
A. veronii biovar sobria A. schubertii C. violaceum Positive Negative Variable
A. veronii biovar veronii C. jejuni subsp. jejuni
Arabinose C. jejuni subsp. doylei
Positive Negative Variable C. coli
A. hydrophila A. veronii biovar sobria C. lari
A. caviae A. veronii biovar veronii C. fetus subsp. fetus
A. schubertii C. hyointestinalis
A. jandaei C. upsaliensis
C. concisus
Cellobiose C. curvus
Positive Negative Variable C. rectus
A. caviae A. hydrophila A. veronii biovar sobria 25C
A. schubertii A. veronii biovar veronii Positive Negative Variable
A. jandaei C. fetus subsp. Fetus C. coli
Mannitol C. concisus
Positive Negative Variable C. curvus
A. hydrophila A. schubertii C. jejuni subsp. jejuni
A. veronii biovar sobria C. jejuni subsp. doylei
A. veronii biovar veronii C. lari
A. caviae C. rectus
A. jandaei C. sputorum
C. upsaliensis
❖ Susceptibility 42C
Ampicillin Positive Negative Variable
Resistant Susceptible Variable C. coli C. hyointestinalis
A. hydrophila C. concisus
A. veronii biovar sobria C. curvus
A. veronii biovar veronii C. jejuni subsp. jejuni
A. caviae C. lari
A. schubertii C. rectus (slight)
A. jandaei C. sputorum
Cephalothin C. upsaliensis
Resistant Susceptible Variable
A. hydrophila ❖ Hippurate Hydrolysis
A. veronii biovar sobria Positive Negative Variable
A. veronii biovar veronii C. jejuni subsp. jejuni C. coli
A. caviae C. jejuni subsp. doylei C. concisus
A. schubertii C. curvus
A. jandaei C. fetus subsp. fetus
C. hyointestinalis
C. lari
Summary for Aeromonas spp. C. rectus
C. sputorum
❖ A. caviae: Positive Oxidase, Esculin Hydrolysis, Sucrose, Arginine C. upsaliensis
Dihydrolase, 0% NaCl, Arabinose, Cellobiose, Mannitol, Sucrose
❖ A. hydrophila: Positive Oxidase, Indole, Arginine Dihydrolase, ❖ Catalase
0% NaCl, VP, Arabinose, Mannitol, Sucrose Positive Negative Variable
❖ A. jandaei: Positive Oxidase, Indole, Glucose, Lysine C. coli C. concisus
Decarboxylase, Arginine Dihydrolase, 0% NaCl, Mannitol C. fetus subsp. fetus C. curvus
❖ A. schubertii: Positive Oxidase, Lysine Decarboxylase, Arginine C. hyointestinalis C. rectus
Dihydrolase, 0% NaCl, VP C. jejuni subsp. jejuni
❖ A. veronii biovar sobria: Positive Oxidase, Indole, Glucose, C. jejuni subsp. doylei
Sucrose, Lysine Decarboxylase, Arginine Dihydrolase, 0% NaCl, (weak)
VP, Mannitol, Sucrose C. lari
❖ A. veronii biovar veronii: Positive Oxidase, Indole, Glucose, C. upsaliensis (weak)
Esculine Hydrolysis, Sucrose, Lysine Decarboxylase, Ornithine
Decarboxylase, 0% NaCl, VP, Mannitol, Sucrose
❖ C. violaceum: Positive Arginine Dihydrolase, 0% NaCl

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❖ H2S in TSI Agar Summary for Campylobacter

Positive Negative Variable
C. concisus C. coli ❖ C. coli: Positive 42C, Catalase, Indoxyl Acetate Hydrolysis,
C. curvus C. fetus subsp. fetus Nitrate to Nitrite, Nalidixic Acid
C. hyointestinalis C. jejuni subsp. ❖ C. concisus: Positive 42C, H2S in TSI, Nitrate to Nitrite
C. rectus jejuni ❖ C. curvus: Positive 42C, H2S in TSI, Indoxyl Acetate Hydrolysis,
C. sputorum C. jejuni subsp. Nitrate to Nitrite, Nalidixic Acid
doylei ❖ C. fetus subsp. fetus: Positive 25C, Catalase, Indoxyl Acetate
C. lari Hydrolysis, Nitrate to Nitrite, Cephalothin
C. upsaliensis ❖ C. hyointestinalis: Positive Catalase, H2S in TSI, Nitrate to Nitrite,
Cephalothin
❖ Indoxyl Acetate Hydrolysis ❖ C. jejuni subsp. jejuni: Positive 42C, Hippurate Hydrolysis,
Positive Negative Variable Catalase, Indoxyl Acetate Hydrolysis, Nitrate to Nitrite, Nalidixic
C. coli C. concisus Acid
C. curvus C. hyointestinalis ❖ C. jejuni subsp. doylei: Positive Hippurate Hydrolysis, Indoxyl
C. fetus subsp. fetus C. lari Acetate Hydrolysis, Cephalothin, Nalidixic Acid
C. jejuni subsp. jejuni C. sputorum ❖ C. lari: Positive 42C, Catalase, Nitrate to Nitrite
C. jejuni subsp. doylei ❖ C. rectus: Positive 42C, H2S in TSI, Indoxyl Acetate Hydrolysis,
C. rectus Nitrate to Nitrite, Nalidixic Acid
C. upsaliensis ❖ C. sputorum: Positive 42C, H2S in TSI, Nitrate to Nitrite,
Cephalothin
❖ Nitrate to Nitrite ❖ C. upsaliensis: Positive 42C, Indoxyl Acetate Hydrolysis, Nitrate
Positive Negative Variable to Nitrite, Cephalothin, Nalidixic Acid
C. coli C. jejuni subsp. doylei
C. concisus
C. curvus
C. fetus subsp. fetus
C. hyointestinalis
C. jejuni subsp. jejuni
C. lari
C. rectus
C. sputorum
C. upsaliensis

❖ Urease
Positive Negative Variable
C. jejuni subsp. jejuni C. lari
C. jejuni subsp. doylei
C. coli
C. fetus subsp. fetus
C. hyointestinalis
C. upsaliensis
C. concisus
C. curvus
C. rectus

❖ Susceptibility
Cephalothin 30ug
Positive Negative Variable
C. fetus subsp. fetus C. coli
C. hyointestinalis C. concisus
C. jejuni subsp. doylei C. jejuni subsp. jejuni
C. sputorum C. lari
C. upsaliensis
Nalidixic Acid 30ug
Positive Negative Variable
C. coli C. concisus
C. curvus C. fetus subsp. fetus
C. jejuni subsp. jejuni C. hyointestinalis
C. jejuni subsp. doylei C. lari
C. rectus
C. upsaliensis

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