Clinical Bacteriology (Lecture)

SECOND TERM

DASH
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science TWO
TWO
PRELIMS
UNIT OUTLINE JOHN NEEDHAM
I. Introduction to Clinical Bacteriology - He observed that boiled mutton broth (tightly
II. ____________________________ sealed) eventually became cloudy (after an hour)
III. ____________________________ with microorganisms.
IV. ____________________________ - He proposed that organic matter possessed a
V. _____________________________ vital force that could give rise to life.
LESSON 1: INTRODUCTION TO CLINICAL
BACTERIOLOGY
INTRODUCTION TO BACTERIOLOGY
MICROBIOLOGY
- Study of organisms that individually are too small
to be seen by the naked eye.
- Study of microorganisms, a large and diverse
group of microscopic organisms that exist as
single cells or cell clusters; it also includes
viruses, which are microscopic but not cellular.
- In microbiology, we are studying about bacteria,
fungi and viruses that can be observed under
the microscope. These microorganisms can • Cloudy means it contains growth or bacteria.
cause disease to humans and there are some • Seal protects the broth so there’s no growth.
microorganisms that we consider as commensal
LAZZARO SPALLANZI
which means they cannot cause any harm even if
they’re inside our body. - He improved the previous experiments of
DISCOVERY OF MICROORGANISMS Needham by using sealed boiled water and
seeds
ROMAN PHILOSOPHER LUCRETIUS AND
- He observed that no growth took place as long as
GIROLAMO FRACASTORO the flasks remained sealed.
- They suggested that a disease was caused by BIOGENESIS
“invisible living creatures” - Living cells can rise only from preexisting living
FRANCESCO STELLUTI cells
- He made the earliest microscopic observations RUDOLF VIRCHOW
on bees and weevils using a microscope probably - He challenged spontaneous generation with the
supplied by Galileo concept of biogenesis
ANTON VAN LEEUWENHOEK THEODORE SCHWANN
- The first true microbiologist - He observed that no growth occurred in a flask
- Father of protozoology and bacteriology containing nutrient solution after allowing air to
- The first person to observe and describe pass through a red-hot tube.
microorganisms accurately GOERG FRIEDRICH SCHRODER AND
- He discovered animalcules
- He used his self-made single lens microscope
THEODORE VON DUSCH
with 50-300x magnification. - Observed that no growth occurred after allowing
SPONTANEOUS GENERATION air to pass through sterile cotton wool placed in a
flask of heat-sterilized medium.
ARISTOTLE
- Schwann, Schroder and Dusch observed that
- Simpler invertebrates could arise from upon application of heat, there will be no
spontaneous generation presence of growth in bacteria. They believe that
FRANCESCO REDI application of heat can lead to killing the
- In 1668, he demonstrated that maggots do not bacteria or can be used as a sterilizing agent..
arise spontaneously from decaying meat LOUIS PASTEUR
- His results were a serious blow to the long-held - He resolved the issue of spontaneous generation
belief that large forms of life could arise from non- - He stated that microorganisms are indeed
life. present in the air and can contaminate seemingly
sterile solutions, however the air itself does not
create microbes.

TUAZON A. 1
 - He showed that microorganisms can be present - He developed the antiseptic system of surgery
in non-living matter (fomites). - He demonstrated the used of phenol for treating
- He started that microbial life can be destroyed by surgical wounds and also sprayed phenol over
heat (basis of the aseptic technique) the surgical area.
- He provided evidence that microorganisms - The main contribution of Lister in the field of
cannot originate from mystical forces present in surgery is the application of phenol as a type of
nonliving materials. wound sterilizer or an aseptic technique in the
wound.
ROBERT KOCH
- He established the first proof that bacteria indeed
cause disease
- He discovered Bacillus anthracis – causative
agent of anthrax
- He discovered Mycobacterium tuberculosis
- He was the first to culture bacteria on boiled
potatoes, gelatin and used meat extracts and
protein digests for cultivation.
- He developed culture media for observing growth
of bacteria isolated from human body.
JOHN TYNDALL KOCH’S POSTULATES:
- He showed that dust carry germs which 1. The microorganisms must be present in every
contaminates sterile broth. case of the disease but absent from healthy
- “Tyndallization” - form of sterilization for three organisms.
consecutive days. 2. The suspected microorganisms must be isolated
ANTOINE LAURENT LAVOISER and grown in a pure culture.
3. The same disease must result when the isolated
- Importance of oxygen to life.
microorganisms is inoculated into healthy host.
FERMENTATION AND PASTEURIZATION 4. The same organisms must be isolated again from
THEODORE SCHWANN the diseased host.
- Stated that yeast cells were responsible for the
conversion of sugars to alcohol, however he said COLLABORATION OF KOCH:
that fermentation was not due to microorganisms 1. Fannie Eilshemius Hesse -suggested the use of
but to a chemical instability that converted agar as a solidifying agent
sugars to alcohol. 2. Richard Petri - developed the petri dish (plate)
- Air is present 3. Martinus Beijernick and Sergie Winogradsky -
PASTEUR developed the enrichment-culture technique and
the use of selective media.
- Described that certain microorganism known as
“yeast” converts sugar to alcohol in the absence
IMMUNOLOGICAL STUDIES – VACCINATION
of air (fermentation) EDWARD JENNER
- There’s no presence of air. - Experimented on how people can be protected
- Souring and spoilage are caused by different against small pox.
microorganisms called bacteria. - He collected scrapings from cowpox blisters and
- In the presence of air, bacteria change the alcohol inoculated a healthy volunteer with the cowpox
in the beverage into vinegar (acetic acid) material by scratching the person’s arm with a
- Heating the beer and wine just enough to kill most pox-contaminated needle.
of the bacteria (pasteurization) LOUIS PASTEUR AND PIERRE PAUL EMIL
ROAX
PASTEUR’S CONTRIBUTION TO SCIENCE:
1. He disproved the theory of spontaneous - Pasteur sed the term “vaccine” - for culture of
generation avirulent microorganisms use for preventive
2. He developed vaccines against anthrax (1881) inoculation
and rabies (1885). - He used attenuated culture known as vaccine
3. He improved the wine industry (theory of (Latin “vacca” - cow)
fermentation) - Pasteur and Roux: Attenuated strains of bacteria.
CHARLES CHAMBERLAND MODERN THERAPY
- Created a porcelain bacterial filter (1884) and CHEMOTHERAPY
developed anthrax vaccine together with Pasteur. - Is the treatment of disease by using chemical
THE GERM THEORY OF DISEASE substances
- Microorganisms might cause the disease - It also refers to chemical treatment of
JOSPEH LISTER noninfectious diseases, such as cancer.

TUAZON A. 2
 o Synthetic drugs - prepared from CLASSIFICATION
• Organization of microorganism
• Classification system is hierarchic
Bacteria Domain
Monera Kingdom
Proteobacteria Phylum
Gammaproteobacteria Class
Enterobacteriales Order
Enterobacteriaceae Family
Escherichia Genus
coli Species
The classification system is hierarchic and consists
of the following taxa designations:
• Life
• Domain
chemicals in the laboratory o Determine the type of cell.
o Antibiotics - produced naturally by
• Kingdom
bacteria and fungi to act against
o Composed of similar phylum / division.
microorganisms
• Phylum / Division
OTHER NOTABLE PERSON o Composed of similar classes.
EMIL VON BEHRING • Class
- Prepared antitoxins for diphtheria and tetanus. o Composed of similar orders.
PAUL EHRLICH • Order
- Discovered salvarsan (arsphenamine) for o Composed of similar families.
treatment of syphilis. • Family
ALEXANDER FLEMING o Composed of similar genus.
- Accidentally discovered penicillin (Penicillium • Genus / Genera
notatum) o Composed of similar species.
HOWARD FLOREY AND ERNST CHAIN o It is the next higher taxon and comprises
different species that have several
- Made the purification process for penicillin
important features in common but differ
Dr. Abelardo B. Aguilar
sufficiently to still maintain their status as
- An Ilonggo physician, discovered the antibiotic
individual species.
erythromycin in soil samples he obtained but
• Species / Epithet
never got the credit and compensation he truly
o Belongs to a similar genus.
deserved.
o It is the most basic taxonomic group and
can be defined as a collection of bacterial
strains that share many common
physiologic and genetic features and as
a group differ notably from other bacterial
species.
NOMENCLATURE
Nomenclature provides naming assignments for each
organism.
The family name is capitalized and has an “-aceae”
ending (e.g., Micrococcaceae).
Writing the Genus Name:
LESSON 1.1: BACTERIAL TAXONOMY
1. Should be capitalized and followed by the species
TAXONOMY epithet, which begins with a lower-case latter.
• Carl Linnaeus, also known after his 2. Both the genus and species should be italicized
ennoblement as Carl von Linné, was a Swedish in print, but underlined when written in script
botanist, zoologist, taxonomist, and physician o Example: Escherichia coli or Escherichia
who formalised binomial nomenclature, the coli
modern system of naming organisms. He is 3. When bacteria are to as a group, their names are
known as the "father of modern taxonomy" neither capitalized nor underlined
• Taxonomy is the orderly classification and o Example: staphylococci, streptococci,
grouping of organisms into taxa (categories). salmonellae
• Three distinct of taxonomy are: Classification, 4. Often the genus name is abbreviated by using the
Nomenclature, Identification. first letter (capitalized) of the genus followed by a
period and the species epithet (e.g., S.aureus).

TUAZON A. 3
 IDENTIFICATION LESSON 1.2: BACTERIAL STRUCTURE AND
FUNCTION
GENOTYPIC CHARACTERISTICS
PROKARYOTES
• Genotype refers to genetic makeup of an
- “Pro” meaning before and “karyon” meaning
organism, or combinations of forms of one or a
nucleus, nut or kernel.
few genes under scrutiny in an organism’s
- These are organisms that do not contain a true
genome.
nucleus surrounded by nuclear membrane,
o Examples: Base sequencing of DNA or
characteristic of lower forms such as bacteria
RNA and DNA base composition ratio to
- They do not contain organelles and all functions
measure the degree of relatedness of
take place in the cytoplasm or cytoplasm or
two organisms.
cytoplasmic membrane.
PHENOTYPIC CHARACTERISTICS
• Readily observable physical and functional
features of an organism expressed by its
genotype
• Features
• Morphology, staining, nutritional requirements,
biochemical and susceptibility test.
o Examples: macroscopic (colony
morphology on media) and microscopic
(size, shape, arrangement into groups or
chains of organisms) morphology,
staining characteristics (gram-positive or
gram-negative), nutritional requirements,
physiologic and biochemical
characteristics, and susceptibility or
resistance to antibiotics or chemicals.
TERMINOLOGY
• Biovars – are variant prokaryotic strains
CELL ENVELOPE
characterized by biochemical or physiological - Outermost structure
difference - Composed of outer membrane and periplasm
(gram negative only), cell wall and plasma
• Epithet – is the proper word for the name of the
membrane.
species
• Morphovars – are variant prokaryotic strains
which differ morphologically. Differentiate
bacteria base on their morphology.
• Serovars – are strains with distinctive antigenic
properties
• Strain – is population of organisms that is
differentiated from population with a particular
taxonomic category.

CELL WALL
- Also referred to as peptidoglycan or murein layer
- It is composed of disaccharide-pentapeptide
subunits; also made up of teichoic acid or
lipoteichoic acid.
- Is a rigid structure that maintains the shape of the
cell

TUAZON A. 4
 MAJOR FUNCTION: Phototrophic,
• To prevent bacterial cells from rupturing when the Chemoorganoh
Chemolithoautot
osmotic pressure inside the cell is greater than Metabolism rophic,
eterotropic
outside the cell Chemoorganoh
• It also serves as a point of anchorage for flagella eterotropic
• Its synthesis and structure have been the primary Teichoic Acid Present Absent
target of antimicrobial agents. Endospore Present in some Absent
GRAM POSITIVE CELL WALL Periplasmic
Absent Present
- Composed of a very thick protective space
peptidoglycan (murein) layer Flagellar 2 rings in the 4 rings in the
- Consist of glycan chains of alternating N-acetyl- structure basal body basal body
D-glucosamine and N-acetyl-D-muramic acid. Resistance to
- Teichoic acid is negatively charged and physical High Low
contributes to the negativity of the cell wall; it may disruption
also bind and regulate the movement of cations Resistance to
Low High
into and out of the cell. lysozyme
- Penicillin acts by preventing the synthesis of Inhibition by
High Low
peptidoglycan. basic dye
GRAM NEGATIVE CELL WALL Reproduction Binary fission Binary fisiion
- Composed of outer and inner (thin peptidoglycan)
membranes ACID-FAST CELL WALL
- Outer membrane – proteins, phospholipids and - It has a Gram-positive cell wall structure
lipopolysaccharide. - Contain a waxy layer of glycolipids and fatty acids
o Lipid A – major constituent; toxic (mycolic acid) that is bound to the exterior of the
o Core polysaccharide cell wall
o Antigenic O-specific polysaccharide - Mycolic acid has a strong hydrophobic structure
- LPS have the following important function: o Example:
o They are vitals in evading the host ▪ Mycobacterium
defense ▪ Nocardia
o They contribute to the negative charge of ABSENCE OF A CELL WALL
the bacterial surface, which stabilizes the - Prokaryotes that do not have a cell wall contain
membrane structure sterols in their cell membrane
o LPS considered as an endotoxin. o Example
▪ Mycoplasma
▪ Ureaplasma
PLASMA MEMBRANE
- Deepest layer of the cell envelope and the
internal matrix of the cell.
- It consists of a phospholipid bilayer that
surrounds the cytoplasm.
- It functions as the mitochondria, golgi complexes
and lysosomes of eukaryotic cells.
- Act as an osmotic barrier
- Regulates the transport of solute across the
membrane as well as the generation of chemical
energy
PROPERTIES GRAM + GRAM - - Site of respiration and photosynthesis
The outer
The thick cell CYTOPLASMIC STRUCTURE
membrane is
wall is
composed of
RIBOSOME (NON-MEMBRANOUS STRUCTURE)
composed of - Site of protein biosynthesis and gives the
lipids, proteins,
Cell Wall peptidoglycan cytoplasm a granular structure.
LPS, and thin
and teichoic GENOME
inner
acids may also - Consist of single, circular chromosome
peptidoglycan
be present. - Appears diffused nucleoid or chromatin body that
layer.
Spherical, oval, is attached to a mesosome or a sac like structure.
Spherical, rod- straight or PLASMID
Shape shaped or curved rods, - Extrachromosomal, double stranded element of
filamentous. helical or DNA that is associated with virulence.
filamentous.

TUAZON A. 5
 - Located in the cytoplasm and serves as a site for CAPSULE
the gene to code for antibiotic resistance and - Organized material that is firmly attached to the
toxin production. cell wall
- Not essential for bacterial growth so a bacterial - Made up of polysaccharide polymers
cell may or may not contain a plasmid - Protects the bacteria from the attacks of human
- It sometimes disappears during cell division and defense system. Resist phagocytosis and
it can make bacteria pathogenic. desiccation.
TYPES OF PLASMIDS SLIME LAYER
• Large plasmid - production of B-lactamases that - Unorganized material that is loosely attached to
provide resistance to B-lactam antibiotics like the cell wall
penicillin and oxacillin. - Also made up of polysaccharide
• Small plasmid - is resistant to tetracyclines and - It can either inhibit phagocytosis or aid in the
chloramphenicol. adherence of the bacteria to the host tissue or
INCLUSION BODIES synthetic implants.
- Serves as the energy source or food reserve of FLAGELLUM
the bacteria - It is an exterior protein filament (flagellin) that
- Composed mainly of polysaccharide; they lessen rotates and thus, causes bacteria to be motile.
osmotic pressure - Important in the survivability and the pathogenic
- Examples: ability of bacteria
o Glycogen - The arrangement of the flagella is as follows:
o Cyanophysin granules o Atrichous - without flagellum
o poly-B-hydroxybutyrate granules o Monotrichous - single flagellum on one
o carboxysomes end
o Gas vacuoles o Amphitrichous - single flagellum on
o Polyphosphate granules both ends
▪ Corynebacterium diphtheriae – o Lophotrichous - tuff/group of flagella on
babes-ernst bodies one end or both ends
▪ Yersinia pestis – bipolar bodies o Peritrichous - spread over the whole
▪ Mycobacterium tuberculosis – surface
Much’s granule
ENDOSPORES/ASEXUAL SPORES (RESISTANT
STRUCTURES)
- They are small, dromant structures located inside
the bacterial cell
- They aid in survival of bacteria against external
conditions
- They are produced within vegetative cells of
some Gram-positive bacteria.
- They are composed of dipicolinic acid and
calcium ions (calcium dipicolinate)
o Examples PILI (FIMBRIA)
▪ Bacillus - These are hair-like, proteinaceous structures that
▪ Clostridium extend from the cell membrane to the external
TYPES OF SPORE ACCORDING TO LOCATION environment (2 um in length).
1. Terminal spore - Clostridium tetani - Aid in the attachment of bacteria to surface
2. Subterminal spore - Clostridium botulinum o Example:
3. Central spore - Bacillus anthracis ▪ Neisseria gonorrhoeae
TERMINOLOGY: ▪ Pseudomonas
• Sporogenesis/Sporulation - process of spore COMMON OR SOMATIC PILUS
formation • Aids the virulence factor/organ of attachment
• Germination - is the end of spore’s dormant SEX PILUS
stage. • Essential part of the genetic transfer/conjugation
GLYCOCALYX process.
- It is an outward complex of polysaccharides on
the bacterial surface and other cells.
- It helps the bacteria to attach to the surface of
solid objects or tissue.
- It appears as capsule or a slime layer

TUAZON A. 6
 PRESENCE OR ABSENCE OF APPENDEGES COMPARISON BETWEEN PROKARYOTIC AND
EUKARYOTIC CELL
NON-MOTILE
MOTILE BACTERIA
BACTERIA CHARACTER
PROKARYOTES EUKARYOTES
Alcaligenes Bacillus anthracis ISTICS
Bacillus cereus Bordetella Size of the
0.20 – 2.0 um 10 – 100 um
Bacillus subtilis Clostridium perfringens cell
Corynebacterium Usually absent
Campylobacter
diphtheria except in fungi
Clostridium tetani Erysipelothrix (chitin); if
Cell wall Usually present
Escherichia coli Haemophilus present, it is
Helicobacter Klebsiella pneumonia chemically
Mycobacterium simple.
Listeria True nucleus
tuberculosis Absence of
Pseudomonas Neisseria with nuclear
Nucleus nuclear membrane
Vibrio Staphylococci membrane and
or nucleoli
nucleoli.
BACTERIA WITH BACTERIA WITH Genome In the nucleoid or
In the nucleus.
CAPSULES SPORES location at the mesosome
Bacillus anthracis Bacillus Single, singular Multiplier linear
Klebsiella pneumonia Clostridium Chromosome chromosome; chromosomes;
Streptococcus without histone. with histones.
pneumoniae Membrane-
bound Absent Present
BACTERIA WITH INCLUSION BODIES OR organelles
GRANULES Present, smaller Present, larger
Ribosomes
Corynebacterium Babes-Ernst bodies size (70s) size (80s)
Mycobacterium Much’s granules Pili and
Present Absent
Nocardia and fimbriae
Sulfur granules Simple; consists of Complex
Actinomyces
Pasteurella and Flagella two protein- (multiple
Bipolar granules building blocks microtubule)
Bordetella
Lysosomes
and Absent Present
EUKARYOTES
peroxisomes
- These are microorganisms that contain a true
Absent (except in
nucleus (with chromosome bound by a nuclear Sterols Present
Mycoplasma spp.)
membrane)
- These are cells of higher plants, animals, fungi, Introns in
Absent Present
protozoa and other more morphologically genes
complex and larger organisms than prokaryotes. With
Cytoskeleton
- They contain many membrane-bound organelles No cytoskeleton cytoskeleton
and
such as nucleus, endoplasmic reticulum, golgi and cytoplasmic and
cytoplasmic
body, mitochondria and lysosomes, in which streaming cytoplasmic
streaming
cellular functions are performed. streaming
Gas vesicles Present Absent
Asexual (binary Sexual and
Cell division
fission) Asexual

ARCHEABACTERIA (ARCHEA)
• Archea - from greek word “archaics”, which
means ancient.
• The cellular structures of archaea include the cell
wall, plasma membrane, ribosomes and flagella.
• They do not contain a nucleus and membrane
bound organelles
• Cell wall of archaebacteria never contain
peptidoglycan, but they mostly contain a protein
or glycoprotein wall structure known as the “S-
layer”

TUAZON A. 7
 • Classified aerobes, facultative anaerobes or REPLICATION AND EXPRESSION OF GENETIC
obligate anaerobes INFORMATION
• Either Gram positive or Gram negative • Replication is the duplication of chromosomal
LESSON 1.3 :BACTERIAL GENETICS DNA for insertion into a daughter cell.
HISTORY • It results from division of one parent cell. The
• Frederick Miescher each of the resulting daughter cells receives the
o First discovered DNA in 1869 full and identical genetic complement contained
• Phoebus Levine in the original parent cells.
o In 1920, discovered that DNA is TRANSCRIPTION
composed of Phosphate, five carbon - It is the synthesis of single-stranded RNA using
sugars and nitrogen bases one strand of the DNA template
• Rosalind Franklin - Converts DNA base sequence of a gene into an
o Discovered the helical structure using X- mRNA molecule
ray crystallography - RNA polymerase is the enzyme that is central to
• James Watson and Francis Crick the transcription process.
o Described in 1950’s the three- TRANSLATION
dimensional structure of DNA molecule.
- It is the actual synthesis of a specific protein from
DEOXYRIBONUCLEIC ACID (DNA) the mRNA
- It is a double stranded helical chain of nucleotides - The genetic code with mRNA molecules is
- The helix formed from the twisted double translated into a specific amino acid sequence
stranded structures appears like a spiral that is responsible for the protein structure and
staircase. function.
- The information contained in the DNA is The mechanisms by which genetic information is
determined primarily by the sequence of letters changed and exchanged among bacteria.
along the staircase.
• Mutation
- It is involved in the RNA synthesis. Usually it
• Recombination
contains adenine, thymine, guanine, and
cytosine. MUTATION
- Change in the original nucleotide sequence of a
RIBONUCLEIC ACID (RNA)
gene or genes within an original genome
- It is a single-stranded and short nucleic acid and
(genotype)
contains the sugar ribose instead of deoxyribose.
- May be induced by chemical or physical factors in
GENETICS the environment or by biologic factors such as
- It is the process of hereditary and variation. introduction of foreign DNA into the cells.
- It is the starting point from which all other cellular - Occurs as a result of error during DNA replication.
pathways, function and structures originate.
RECOMBINATION
MAJOR ASPECTS OF GENETICS - Process by which genes are transferred or
STRUCTURE AND ORGANIZATION OF exchanged between homologous regions on two
GENETIC MATERIALS DNA molecules.
• The bases (adenine, guanine, cytosine and - Provides a way for organisms to acquire and copy
thymine), which are covalently linked to each new combinations of biochemical pathways from
deoxyribose sugar. the changes in their environment.
• DNA sequence that encodes for specific product - It occurs when a potion of genetic material that
(RNA or Protein) is define as gene. originates from one bacterial cell which is the
• All genes taken together within an organism donor is transferred into the second bacterial cell
comprise a genome. which is the recipient. It involves a number of
• Genome organized in discrete elements is known biding proteins with direct a protein which is
as chromosome. essential for DNA repair and maintenance playing
• Bacterial chromosome - contains all the genes a central role.
and exists as a double-stranded, closed, circular,
naked macromolecule; most eukaryotes have
linear DNA.
o Information for protein synthesis is
encoded in the bacterial DNA and
transmitted in the chromosome to each
generation
o mRNA act as “blueprint” for protein
construction.

TUAZON A. 8
 MECHANISMS OF GENE TRANSFER
TRANSFORMATION
- Involves the recipient cell uptaking free DNA that
is released into the environment when bacterial
cell (donor) dies and undergoes lysis or cell
disintegration caused by a rupture in the cell wall.
TRANSDUCTION
- It is the transfer of bacterial genes by a
bacteriophage from one cell to another.
- In this mechanism, DNA from two bacteria may
come together in one cell.
THREE TYPES:
• Generalized
o Process in which the bacterial DNA may
be randomly incorporated with the viral
DNA
• Specialized
o Process in which the bacterial DNA
adjacent to the viral DNA in the bacteria
is packaged into a new virus particle
LESSON 2: BACTERIALBIOCHEMISTRY AND
• Lateral
METABOLISM
o It occurs through a temperate phage that
excises late in its atypical lytic cycle METABOLISM
(Chiang et al., 2019). Unusually, phage - Sum of all chemical processes that take place in
replicates first, then, starts the excision a living organism and results in its growth, energy
process from the bacterial genome. This generation, waste disposal and other functions in
type is distinguished from the other types relation to cell distribution.
as being able to transfer large DNA BACTERIAL METABOLISM:
stretches at high frequencies in addition • Anabolism (Constructive phase)
to the prophage (Chen et al., 2018) o There is a synthesis of complex
molecules and energy production.
• Catabolism (Destructive phase)
o The large complex molecules are broken
down into simpler smaller molecules
accompanied by energy utilization.
- Bacterial Metabolism also consists of
biochemical reactions that breaks down organic
compounds and produce new bacterial structure
from the resulting carbon skeleton. These
biochemical reactions in the cell will depend in the
presence and activity of a specific enzyme.
ENERGY PRODUCTION
- Breakdown of chemical substrates (chemical
energy) through the degradative process of
CONJUGATION catabolism that is coupled with oxidation
- The transfer of genetic material from a donor cell reduction reaction.
to a recipient cell. GLUCOSE
- Occurs between two living cells - Is an essential nutrients for energy production in
- Requires the mobilization of the donor’s organisms.
chromosome - Compound such as glucose that have many
- The sex pilus (donor) establishes a conjugative hydrogen atoms are highly reduced compounds,
bridge that serves as the channel for DNA and thus contain a large amount of potential
transfer to the recipient cell. energy.
- To produce energy from glucose,
microorganisms use these two general
processes: Fermentation and Respiration.
FERMENTATION AND RESPIRATION
- Biochemical pathways to catabolize (break down)
carbohydrates and produce energy.

TUAZON A. 9
 RESPIRATION
- Is an efficient energy-generating process in which
molecular oxygen is the final electron acceptor.
- In this process, the glucose is completely broken
down and results in high energy production. In
the presence of oxygen, glucose will turn into
carbon dioxide and water. It is carried out by
obligate aerobes and facultative anaerobes.
AEROBIC RESPIRATION
- Oxygen is the final electron acceptor
ANAEROBIC RESPIRATION
- Nitrate, sulfate and fumarate is the final acceptor
FERMENTATION
• It does not require oxygen (anaerobic), the use of
Krebs cycle or electron transport chain
• Release energy from sugars or other organic
molecules such as (through) amino acids and
purines.
• Forms a mixture of end products: lactate,
butyrate, ethanol and acetoin).
• Carried out by both obligate and facultative
anaerobes.

• Alcoholic fermentation: The major end product

is ethanol. This is the pathway used by yeasts
RESPIRATION when they ferment glucose to produce ethanol.
• Glycolysis (Embden-Meyerhof-Parnas • Homolactic fermentation: The end product is
pathway) almost exclusively lactic acid. All members of the
o Oxidation of glucose —--> to Pyruvic Streptococcus genus and many members of the
Acid Lactobacillus genus ferment pyruvate using this
o Major route of glucose metabolism in pathway. This is also used when making yogurt
most cells. and pickles.
• Krebs Cycle (Tricarboxylic acid)
o Enzyme system converts PYRUVATE —
--> to CARBON DIOXIDE and ACID
o Generate energy in the form of
adenosine triphosphate (ATP).
o Substrate for this process is acetyl
coenzyme A.

• Heterolactic fermentation: Some lactobacilli

use this mixed fermentation pathway, of which, in
addition to lactic acid, the end products include

TUAZON A. 10
 carbon dioxide, alcohols, formic acid, and STAINING TECHNIQUES
acetic acid. SIMPLE STAINING
• Propionic acid fermentation: Propionic acid is - Single stain is used
the major end product of fermentations carried - Directed towards coloring the forms and shape of
out by Propionibacterium acnes and some the cells
anaerobic non–spore-forming, gram-positive - Example: Methylene blue
bacilli DIFFERENTIAL STAINING
- Divide bacteria into separate groups
- Directed towards coloring the components of the
elements present.
- Example: Gram staining and Acid-fast bacilli
(AFB) staining
STEPS IN DIFFERENTIAL STAINING ARE AS
FOLLOWS:
1. Application of Primary stain
2. Application of the mordant
3. Application of the decolorizing agent\
4. Application of the secondary stain/counterstain
• Mixed acid fermentation: It involves the NEGATIVE STAINING
production of ethanol and acids, such as lactic, - Demonstrate presence of diffuse capsule
acetic, succinic and formic acid surrounding some bacteria
- Excellent technique for studying bacterial gas
• Butanediol fermentation: pyruvate is converted
vacuole and viral morphology
in acetoin then reduced to 2,3-butanediol with
- Appearance: bacteria as light-colored bodies
NADH; small amounts of ethanol and mixed acids
against dark background
are also synthesized.
- Example: India Ink or Nigrosin dye
• Butyric acid fermentation: it involves the
conversion of pyruvate into butyric acid along GRAM STAIN
with acetic acid, carbon dioxide and hydrogen. - Most commonly used differential stain
- Utilizes crystal violet as the primary stain, while
safranin is the secondary stain or counterstain.
- Iodine - act as the mordant
- Acetone alcohol - act as decolorizing agent

LESSON 2.1: BACTERIAL STAINING

KINDS OF IONIZABLE DYES USED IN STAINING
BACTERIA
BASIC DYES
- Commonly used in bacteriology
- Cationic dyes with positively charged groups that
adhere to negatively charge molecules like
nucleic acids and proteins. (Basic dye: positively
charge or cationic that stains negatively charge
e.g. nucleic acid and protein)
- Example: Methylene blue, crystal violet, safranin
and malachite green.
ACIDIC DYES
- Anionic dyes with negatively charged groups
that bind to positively charge cell structures.
(Negatively charged that stains positively
charged)
- Example: Eosin and acid fuchsin

TUAZON A. 11
 GRAM STAIN PROCESS o Nocardia
Gram o Corynebacterium
Staining Cell effects Gram + Gram - o Streptomyces
Steps o Erysipelothrix
Step 1: o Trophyrema whipplei
Crystal Violet
Primary stain Stains cells REASON WHY GRAM-POSITIVE BECOME
added to purple or blue. GRAM-NEGATIVE BACTERIA
specimen
smear • Removal of MgRNA
Step 2: Iodine • Aged, dying and autolyzing cells
Mordant o Old cells may lose their ability to retain
makes dye less Cells remain strains
soluble so it purple or blue. o Antibiotic-treated bacterial cells have
adheres to cell atypical staining reaction
walls. • Using acidic iodine during staining
Step 3:
Gram positive • Due to a technical error or the wrong use of stains
Alcohol
cells remain
Decolorizer EXCEPTION IN GRAM STAINING
purple or blue.
washes away
Gram negative • Organisms that exists almost exclusively within
stain from
gram negative
cells are host cells (Chlamydia)
cell walls.
colorless. • Organisms that lack cell walls (Mycoplasma and
Step 4: Ureaplasma)
Gram positive
Safranin • Organisms with insufficient dimension to be
cells remain
Counterstain resolved by light microscopy (Spirochetes).
purple or blue.
allows dye ACID-FAST STAINING
Gram negative
adherence to - Used to stain bacteria that have high lipid
cells appear
gram negative
cells.
pink or red. contents in their cell wall
- Utilizes carbol fuchsin as the primary stain and
methylene blue or malachite green as the
GRAM + GRAM -
secondary stain.
- Cell wall of acid-fast bacteria resists the acid-
Primary stain
alcohol in decolorizing step.
(Crystal Violet)
- Heat - applied as a mordant in Ziehl-Neelsen
method
Mordant - Tergitol - applied in Kinyoun method (Cold
(Iodine) method)

Decolorization
(Acetone)

Secondary
Stain
(Safranin)

• V - crystal Violet
• I – Iodine
• A – Acetone-Alcohol
• S – Safranin
GENERAL RULE OF GRAM STAINING
• All cocci are Gram positive except:
o Neisseria
o Veilonella
o Branhamella (Moraxella)
• All bacilli are Gram negative except:
o Arcanobacterium
o Listeria
o Bacillus
o Mycobacterium
o Clostridium

TUAZON A. 12
 • Aerotolerant Anaerobes
- Don’t use oxygen and can’t survive if it is
present.
• Microaerophile
- Use oxygen but only at very low
concentrations.

ACID-FAST STAINING METHOD

1. Ziehl-Neelsen/Hot staining Method
2. Kinyoun’s/Cold staining Method
3. Pappenheim method - differentiate
Mycobacteriuam smegmatis from
Mycobacteriumtuberculosis
4. Baumgarten method - differentiate
PHYSIOLOGIC REQUIREMENTS OF BACTERIA
Mycobacterium leprae from Mycobacterium ACCORDING TO OXYGEN REQUIREMENT
tuberculosis. 1. AEROBES
5. Auramine-rhodamine method - selective for the - These organisms require oxygen and
cell wall of AFB. grow well with room air
- Air contains 15% to 21% oxygen and 1%
MODIFIED ACID-FAST STAINING METHOD CO2
(MODIFIED KINYOUN) - Example:
• Useful for the identification of intestinal coccidian o Bordetella, Brucella,
oocysts Mycobacteria and Pseudomonas
• Ideal for cryptosporidia and cyclospora parasites 2. ANAEROBES
• Specimen : stool - These organisms do not require oxygen to grow
• Reagent: same with conventional acid-fast - Three different types:
except the concentration of acid-alcohol (1% o Obligate anaerobes
H2SO4) ▪ Absolutely do not require the
• Result: oocysts appear as magenta-stained presence of oxygen because
organisms against a blue background they die after prolonged
Bacteria – Acid-fast or Kinyoun only exposure to air
Parasite – Modified acid-fast or modified kinyoun ▪ Examples: Clostridium and
Bacteroides
SPECIAL STAINING METHOD o Facultative anaerobes
▪ Clinically significant bacteria
CELLULAR BACTERIA ▪ Organisms grow either in the
STAINING TECHNIQUE
STRUCTURE presence or absence of oxygen
Dyar Cell wall ▪ Examples: Enterobacteriaceae
Anthony’s, Hiss and Gin’s Capsule o Aerotolerant anaerobes
Nigrosin Capsule ▪ These organisms can survive in
Neisser Metachromatic granules the presence of oxygen but will
Albert Metachromatic granules be unable to perform metabolic
Domer Endospore processes unless situated in an
Schaeffer-Fulton Endospore anaerobic environment
Gray Flagella ▪ Example: Propionibacterium
Leifson Flagella acne.
Feulgen DNA Obligate aerobes and Facultative anaerobes contain
protective enzymes such as your superoxide
Levaditi’s Spirochetes
dismutase and catalase that counter the toxic effect of
Fontana-Tribondeau Spirochetes
oxygen.
LESSON 2.2: MICROBIAL NUTRITION
BACTERIA BASED ON OXYGEN 3. MICROAEROPHILES
REQUIREMENTS
- It is an organism that requires 2% to 10% oxygen
• Obligate Aerobes for growth
- Require oxygen and can’t survive without - Example: Campylobacter and Treponema
it. pallidum.
• Facultative Aerobes
- Use oxygen but can survive without it.

TUAZON A. 13
 ACCORDING TO CARBON DIOXIDE
REQUIREMENT
CAPNOPHILES
- Require an increased CO2 (5% to 10% CO2)
- Example: Haemophilus influenzae, Neisseria
gonorrhoeae and Streptococcus pneumoniae
ACCORDING TO NUTRITIONAL REQUIREMENT
1. AS TO THE CARBON SOURCE
- Autotrophs use CO2 as the sole source of
carbon
- Heterotrophs use reduced, preformed, organic
molecules from other bacteria. ACCORDING TO TEMPERATURE
2. AS TO THE ENERGY SOURCE REQUIREMENT (35 C TO 37 C)
- Phototrophs are organisms that use light as their 1. PSYCHROPHILES/CRYOPHILES
energy source
- These organisms grow well at 0C (zero degree)
- Chemotrophs are organisms that utilize the
to a maximum of 20 C.
energy produced by organic or inorganic
- Example: Listeria monocytogenes and Yersinia
compounds oxidation.
enterocolitica.
3. AS TO THE ELECTRON SOURCE
2. MESOPHILES
- Lithotrophs reduce inorganic molecules to be
- These organisms grow 20 C to 45 C
used in biosynthesis or energy conservation.
- The most commonly encountered pathogenic
- Organotrophs require organic substances
bacteria in the clinical laboratory.
(CHO, lipids) for growth and multiplication.
Fastidious bacteria – this requires additional
3. THERMOPHILES/HYPERTHERMOPHILES
substance such as vitamins, purines, pyrimidines and - These organisms grow 50 C to 125 C
hemoglobin for growth and survival. - Examples: Bacillus stearothermophilus,
Saprophytes – This requires dead organic Sulfolobus, Pyrococcus, Pyrodictium and
substances. Thermus aquaticus.
Parasites - requires organic substances from living 4. EXTREMOPHILES
tissues. - Prokayotes that are able to survive in unusual
condition like the absence of oxygen, increased
NUTRITIONAL CLASSES OF MICROORGANISMS temperature and living below the earth’s surface
Nutritional Energy/Electron/C (Bacillus infernus).
Organisms
class arbon source ACCORDING TO PH
Cyanobact • pH scale is a measure of the hydrogen ion
Light energy, eria, Purple concentration of an organism’s environment.
Photoautotroph
s
Inorganic electron and Green • Three different types of organisms according to
donor, CO2 Sulphur pH tolerance.
bacteria o Acidophiles - grow at pH 0 to 5.5
Light energy, Purple and o Neutrophiles - grow between pH 5.5 and
Photoheterotro Organic electron Green 8.0 (most clinically bacteria)
phs donor, Organic Nonsulfur o Alkalophiles - - grow between pH 8.5 and
carbon source bacteria 11.5
Inorganic chemical • Note: Optimum pH 6.5 to 7.5
Nitrifying
compounds as
Chemoautotrop bacteria,
energy source,
hs Iron
Inorganic electron
bacteria
donor, CO2
Most
Organic compounds pathogenic
Chemoheterotr
as energy, electron bacteria,
ophs
and carbon source fungi and
protozoa

ACCORDING TO MOISTURE
• Moisture is vital for bacterial growth and
susceptibility testing.

TUAZON A. 14
 ACCORDING TO SALT CONCENTRATION - Ex: Brain heart infusion, trypticase soy broth
• Halophiles - require and grow in increased (TSB) and thioglycollate.
concentration of sodium chloride. 2. SEMI-SOLID MEDIUM
• Example: Staphylococcus aureus and Listeria - It contains 0.5% to 1% Agar
monocytogenes. - It is used to observe bacterial motility and
o Listeria monocytogenes can be classified detect indole and sulfide production
as psychrophiles or cryophiles because it - Ex: Sulfide Indole Motility (SIM) Medium
can grow in cold temperature. It can also 3. SOLID MEDIUM
be classified as halophiles because it - It contains 2% to 3% agar
requires sodium chloride or salt for it to - Ex: Triple sugar Iron (TSI) agar, MacCOnkey
be able to grow. (MAC) agar, Blood agar plate (BAP) and
ACCORDING TO PRESSURE Chocolate agar plate (CAP)
• Barophiles - organisms that grow rapidly in
highpressure environments (600 to 1100 atm
pressure)
• Example: Photobacterium, Shewanella and
Colwellia
ACCORDING TO GROWTH FACTORS
• Growth factors are substances that are required
by fastidious bacteria for their growth and
multiplication.
• Example of growth factors: amino acids, purines,
pyrimidines and vitamins. ACCORDING TO COMPOSITION
LESSON 3: CULTURE AND CULTURE MEDIA 1. SYNTHETIC OR DEFINED MEDIUM
CULTURES - Medium in which all the components are known
- Used for research purposes
- Growth of microorganisms in a culture media.
- Preferred for isolation of cyanobacterium and
Utilizing effective and appropriate culture media
chemoorganotrophs
for growth, transport, and storage facilitates the
- Ex: BG-11 medium
study in Bacteriology
CULTURE MEDIA
- Composed of a mixture of nutrients such as
carbon, nitrogen, sulfur, phosphorus, hydrogen,
oxygen and buffers
TYPES:
• Liquid, Semi-solid and Solid Medium
TYPES OF CULTURE
PURE CULTURE
- It is composed of only one species
MIXED CULTURE
- It is composed of more than one species
STOCK CULTURE 2. NON-SYNTHETIC OR COMPLEX MEDIUM
- It is composed several culture species contained - Medium in which some substances are unknown
in a separate culture medium (one species per - Peptones, Meat and Yeast Extract
culture medium). - Useful for isolation of medically significant
- It is used for academic and industrial purposes. bacteria
- Reserved bacteria - Ex: Nutrient broth (NB) broth medium, TSB and
CLASSIFICATION OF CULTURE MEDIA MAC agar
1. According to Consistency 3. TISSUE CULTURE MEDIUM
2. According to Composition - Used for obligate intracellular bacteria (Rickettsia
3. According to Dispensing or Distribution Method and Chlamydia).
for the Medium - Example: W138 cells, HeLA 299 Cells and
4. According to Use McCoy cells
ACCORDING TO CONSISTENCY HeLa 299 cells Human cervical tissue
1. LIQUID MEDIUM McCoy cells Fibroblast
- It does not contain any amount of agar W138 cells Fibroblast
- It allows the growth of aerobes,
anaerobes and facultative anaerobes HeLa cell came from Henrietta Lacks which is a cancer
patient. She has cervical cancer and is admitted to

TUAZON A. 15
John Hopkins University Hospital. Physicians saw that - Example: Alkaline peptone water, Selenite F,
Henrietta Lacks’ cells are continuously dividing. They Thioglycollate, Tetrathionate, Gram-negative
stole her cells without her permission and used it for (GN) broth and Lim broth
research purposes. They found out that Henrietta’s 3. ENRICHED MEDIA AND NON-SELECTIVE
cells can be used to grow / have chlamydia. Until now, MEDIA
HeLa cells are still in use. - Media with additional supplement such as blood,
vitamins and yeast extract.
McCoy cells came from a patient that has arthritis. - Solid type of media
- Example: Blood agar plate and Chocolate agar
W138 cells came from fibroblast. plate
o Blood agar plate - hemolytic pattern of
bacteria (extract 10ml of blood and mix it
to the base TSB or Tryptone soy broth)
o Chocolate agar plate – recovery of
Haemophilus (boil blood agar plate to
make chocolate agar)
4. DIFFERENTIAL MEDIA
- These media allow the visualization of metabolic
HeLa cells W138 cells for Rickettsia differences between groups of bacteria
- Example: MAC, BAP, eosin methylene blue
ACCORDING TO THE DISPENSING OR (EMB) and Hektoen Enteric Agar (HEA)
DISTRIBUTION o MacConkey Agar
PLATE MEDIA ▪ Indicator: Neutral red
- Distributed into the dish or plate ▪ Pinkish
o Blood Agar Plate
TUBE MEDIA
▪ Differentiate hemolytic pattern of
- Prepared as either liquid, slant, butt and slant or streptococci
butt
5. SELECTIVE MEDIA
• EXAMPLES:
o Triple sugar iron (TSI) - These media are incorporated with antibiotics,
o SIM dyes or chemicals to inhibit the growth of other
o Simmon’s citrate agar organisms.
o Lysine iron agar (LIA) - Example: HEA, MAC, Xylose lysine
desoxycholate (XLD) agar, Bismuth sulfate agar
(BSA), Mannitol Salt Agar (MSA) and Thayer-
Martin Agar (TMA)
o Other selective media:
▪ Gentamicin blood agar:
Streptococcus
▪ Bacitracin chocolate agar:
Haemophilus
▪ Blood agar plate with ampicillin:
Aeromonas
ACCORDING TO USE ▪ Phenylethyl alcohol: Gram
1. SIMPLE MEDIA, GENERAL PURPOSE MEDIA positive bacteria
▪ Colistin-Nalidixic acid (CNA)
AND SUPPORTIVE MEDIA
agar: Gram positive bacteria
- Routinely used in the laboratory and without - Inhibitory substances
additional supplement Gram Positive Bacteria Crystal/Gentian violet,
- Support the growth of most non-fastidious basic/carbol fuchsin and
bacteria (Simple media, General-purpose media, bile salt
Supportive media) Gram Negative Bacteria Potassium tellurite and
- Usually composed of meat and soybean extract sodium
- Ex: Nutrient agar, Nutrient broth and Tryptone azide
soy broth (TSB)
For Swarming Bacteria Alcohol and chloral
2. ENRICHMENT MEDIA (LIQUID-TYPE MEDIA) hydrate
- Used to propagate the growth of certain group of
bacteria from a mixture of organisms. MEDIA DESCRIPTION
- Contain specific nutrients and without additional Bile salt and dyes: Inhibit
supplement Hektoen Enteric Agar
indigenous microbiota of
(HEA)
LGIT;

TUAZON A. 16
 used for recovery of fecal • Specimen require direct or bedside inoculation
bacteria such as blood, genital specimen, corneal
pH indicator: Bromthymol scraping, sterile body fluids (synovial), peritoneal
blue fluid and nasopharyngeal swab.
Bile salts and crystal INOCULATION TECHNIQUES
violet: inhibit gram- • Streaking
MacConkey Agar (MAC positive bacteria; - most common manner of inoculation
used for recovery of fecal • Specimen collected through swab
bacteria - Inoculate gently by rolling the tip of the swab
Xylose, lysine, sucrose, to the upper portion of the plate.
0.25% sodium • Placement of fluid specimen
Xylose Lysine desoxycholate - Broth or liquid media
Desoxycholate Agar and sodium thiosulfate; • Stabbing of the medium
(XLD) X for fecal bacteria - To perform Group A Streptococci to observe
Differentiate: Shigella and its anaerobiosis
Salmonella • Overlapping inoculation
Mannitol Salt Agar Support growth of - Used for anti-microbial sensitivity test
(MSA) Staphylococcus aureus
Thayer-Martin Agar
Selective for Neisseria sp.
(TMA)

XYLOSE LYSINE DESOXYCHOLATE AGAR

INOCULATION OF SPECIMEN
6. SPECIAL MEDIA
- Used to isolate bacteria with specific growth MANNER OF INOCULATION
requirements • The inoculating loop is sterilized and allowed to
- Example: Lowenstein-Jensen (LJ) medium and cool thoroughly before use
Thiosulfate citrate–bile salts-sucrose (TCBS) • Inoculating loop should be flamed (red hot)
agar between different media plates, except if
o Lowenstein-Jensen Medium specimen is collected with swab
▪ Composed of Whole eggs and • Urine specimen are inoculated using a
malachite green quantitative isolation technique with calibrated
▪ Used for Mycobacterium loop (usually 0.01ml or 0.001ml).
tuberculosis
o Thiosulfate citrate–bile salts-sucrose INCUBATION CONDITIONS
(TCBS) Type of Bacteria O2 CO2 H2 N2
▪ Vibrio cholerae on TCBS agar Aerobes 21% 0.03% -- --
0% 5-10% 5- 80-
Anaerobes
10% 90%
Capnophiles 15% 5-10% -- --
5- 8-10% -- --
Microaerophiles
10%
MANNER OF REPORTING (GRADING) OF
GROWTH
GRADE INTERPRETATION
LESSON 3.1: INOCULATION AND CULTIVATION Many, heavy growth; if growth is up to 4th
4+
INOCULATION OF SPECIMEN quadrant
• Specimen: Sterile body fluids, pus, urine and Moderate growth; if the growth is up to 3rd
3+
sputum quadrant
Few or light growth; if growth is in the 2nd
• Specimen received on swan that can be 2+
quadrant
inoculated directly into culture media

TUAZON A. 17
 Rare growth; if growth is in the 1st quadrant
1+
only
ANAEROBIC CULTIVATION
WAYS TO FACILITATE ANAEROBIC
CULTIVATION
1. Use of special culture: with thioglycolate and
cystine as reducing agent.
2. Boiling of culture media to remove excess
oxygen.
3. Use of anaerobic chamber system: with vacuum
pump and nitrogen gas to remove residual
oxygen,
4. Use of gas-pack jar: with palladium catalyst FORM ELEVATION MARGIN DENSITY
5. Plastic bags or pouches: with calcium carbonate Punctiform Flat Entire (even) Transparent
and catalyst (for small volumes) Undulate
Circular Raised Transluscent
COMPONENTS OF AN ANAEROBIC CHAMBER (wavy)
Serves as filler for the Filamentous Convex Filamentous Opaque
Nitrogen gas remaining percentage of Lobate
Irregular Pulvinate
(lobes)
anaerobic atmosphere
Erose
Used to remove residual Rhizoid Umbonate
Palladium pellets (serrated)
oxygen Spindle
Silica gel (dessicant) Used to absorbed water Curled
(lens)
Oxygen reduction 2. GROWTH ON AGAR SLANT
Methylene blue or indicator
• Amount: scanty, moderate or abundant
reazurin Colorless: means
• Margin or edge: Evenly circular or with
absence of oxygen
irregularities
CULTURAL CHARACTERISTICS • Consistency: butyrous or butter-like, viscous or
MANNER OF REPORTING stingy, dry and brittle
1. AGAR-PLATE COLONIES • Chromogenesis: pigmented or colored
• Size: small (pinpoint) or large; mucoid or slimy
(example is Pseudomonas and Proteus which
spread across the entire agar surface or what we
call, swarming)
• Margin or edge of colonies
• Chromogenesis
• Optical features

• Growth patterns on slants:

o Filiform (thread-like)
o Arborescent (tree-like)
o Beaded
o Effuse (spreading)
o Rhizoid
o Echinulate (spiny)

TUAZON A. 18
3. GROWTH IN NUTRIENT BROTH • This is also the phase where microorganisms
• Turbid utilize the physiological and biochemical testing.
• Confined as film (pellicle) or accumulated STATIONARY/PLATEAU PHASE
(sediment) • It is the period when there is a balance between
• Granular or viscous cell division and dying organisms although the
• Odor: produced putrid, fruity or aromatic odor number of visible microorganisms remain
constant.
• This is also where the metabolic activity of
surviving cells slows down. Nutrients are also
limited in this phase. This is also the phase where
dead debris starts to accumulate.
DEATH OR DECLINE PHASE
• It is the period when there is a cessation of
bacterial growth as the number of dead cells
exceed the number of living organisms.
• This is also the stage where there is a loss of
4. GROWTH IN GELATIN SLANT
nutrients and an increasing amount of toxic
• Growth along the line of inoculation waste.
• Confined within the zone of inoculation tree and it
has a varying degree of threading outside the
inoculation tree
• Liquefaction of Gelatin starts evenly from the top
or various liquified design may also occur.

LESSON 3.2: BIOSAFETY IN MICROBIOLOGY

LABORATORY
❖ In your clinical laboratory, specimens are
potential hazards. Since they may contain
infectious agents like bacteria, viruses and fungi.
That’s why universal precaution is needed and
BACTERIAL GROWTH CURVE this is recommended by the Center for Disease
STAGES OF BACTERIAL GROWTH Control and Prevention.
1. Lag phase or Period of Rejuvenescence ❖ All specimen must be treated as infectious.
2. Log or Exponential phase or Balance growth BIOLOGICAL SAFETY CABINET
3. Stationary/Plateau phase - Device that encloses a working area to protect
4. Death or Decline phase workers from aerosol exposure and infectious
LAG PHASE OR PERIOD OF disease agents.
REJUVENESCENCE - Air that contains the infectious material is
sterilized thru:
• Period when there is no cell division or an abrupt
o Heat
increase in the cell number.
o UV light
• This is also where biosynthesis starts but there is
o Passage through a high-efficiency
no increase in cell mass because the bacteria are
particulate (HEPA) resistance filter
still adjusting in the environment that’s why this is
also called Adjustment phase.
LOG OR EXPONENTIAL PHASE (BALANCE
GROWTH)
• It is the period when microorganisms are actively
growing and dividing.
• It is also the stage in which the bacteria increase
logarithmically since cellular production is most
active during this period.

TUAZON A. 19
CLASS I CABINET BIOSAFETY LEVEL 3 AGENTS
• Open-fronted cabinet with negative pressure • Potential agent for aerosol transmission
• Room air —---- > sterilized using HEPA filter (respiratory)
• Only air to be exhausted is sterilized by HEPA • In processing this lethal pathogen, the air
filter movement in the laboratory must be controlled to
• Used for biosafety levels (BSL) 2 and 3 agents contain the infectious material.
CLASS II CABINET • Must have extra precaution in processing lethal
• Also known as Laminar flow BSC pathogen
• Most commonly used BSC • Ex: Mycobacterium tuberculosis, Francisella
tularensis, Brucella spp., Coxiella burnetti, St.
• Sterilized air using HEPA filter flows over the
Louis encephalitis and systemic fungi
infectious material and the air to be exhausted
• Used for biosafety levels (BSL) 2 and 3 agents BIOSAFETY LEVEL 4 AGENTS
• 2 types of Class II cabinet • Life-threatening infections
o A. Class IIA - has fixed opening; 70% of • Needs maximum containment and
the air recirculated decontamination of all personnel and materials
o B. Class IIB - used for chemicals, before leaving the area.
radioisotopes and carcinogens. • These agents could also be transmitted through
• Most hospital clinical microbiology laboratory aerosol.
technologist use Class II BSC • Example: arbovirus, arenavirus, filovirus and
smallpox virus
CATEGORIES OF POTENTIAL INFECTIOUS
AGENT OF BIOTERRORISM
CATEGORY A
• Agent that poses the greatest public health threat
• Easily transmitted and highly infectious
• Examples: smallpox, Bacillus anthracis and
Francisella tularensis
CATEGORY B
• Moderate morbidity and low mortality
• Not easily transmitted
• Examples: Coxiella burnetti, Burkholderia
CLASS III CABINET pseudomallei and Rickettsia sp.
• Provides the highest level of safety to the worker CATEGORY C
• Air coming into and going out of the cabinet is • Emerging pathogens
sterilized using HEPA filter.
• Examples: viruses that causes yellow fever and
• Infectious material within is handled with rubber dengue hemorrhagic fever.
gloves that are attached and sealed to the
LABORATORY AIR-HANDLING SYSTEM
cabinet.
• Should be under negative pressure (ideal clinical
• Used for biosafety levels (BSL) 4 agents
microbiology laboratory) and the air should not be
CLASSIFICATION OF BIOLOGIC AGENTS recirculated after passing through a laboratory.
BIOSAFETY LEVEL 1 AGENTS • Move air from lower to higher risk areas
• No known potential for infecting healthy people • Maximum precaution
• For academic purposes • Ex: Mycobacterium tuberculosis
• Ex: Bacillus subtilis and Naegleria gruberi PERSONAL PROTECTIVE EQUIPMENT (PPE)
BIOSAFETY LEVEL 2 AGENTS PPE:
• Agents acquired by ingestion and exposure to • Laboratory coat
percutaneous and mucous membrane • Gloves
• All common agents of infectious diseases • Mask
• In handling this agent, access to the laboratory is • Goggles
limited. It is also required for the personnel to • Face shield
change their clothes with the recommended *MUST REMOVED BEFORE leaving the work area and
laboratory clothing before going to their specific MUST BE DISPOSED in PPE-assigned areas.
stations. The personnel handling the agent INFECTIOUS CONTROL
should receive immunization. ❖ All laboratory related accidents must be reported
• Ex: HIV, Bacillus anthracis, Yersinia pestis, immediately to the head of the laboratory and
Salmonella sp. and Shigella sp. safety officer. For HIV testing, serum samples
must be examined at an interval of 6 weeks – 3
months – 6 months.

TUAZON A. 20
HAZARDOUS WASTE handwashing. Handwashing is the corner stone
• These are substances, which singly or in of modern infection control program.
combination, have a significant threat or hazard 4 COMMON TYPES OF NOSOCOMIAL
to human or to the environment and require INFECTION
special handling, processing or disposal. • Urinary tract infection
• Flammable, explosive, reactive, corrosive, • Lung infection (Pneumonia)
infectious, carcinogenic, bioconcentrative • Surgical site infection
persistent in nature, lethal, irritant, oxidizer, or • Blood stream infection
strong sensitizer. ❖ There are also predisposing factor for nosocomial
DISPOSAL OF HAZARDOUS WASTE infections.
• All materials contaminated with potentially o First is there are varieties of
infectious agents must be decontaminated before microorganisms that circulates in the
disposal using an autoclave, incinerator or hospital environment. So, to be always
alternative waste treatment methods. protected, you must wear masks.
• Pipettes, swab and other glass objects should be o Second factor is the patients are weak or
placed inside a firm cardboard container before immunocompromised. Their immune
disposal. system is somehow defective.
• Sharp objects (needles and scalpels) place in o Third factor is the chain transmission. It
containers that are autoclaved and incinerated. can either be direct or indirect.
LESSON 4: PATHOGENESIS o Fourth is fomites or the inanimate objects
like catheter, needle, dressing, bed, or
❖ Pathogenesis is the development of infection and wheelchair.
disease. There are certain virulence agents with o Fifth factor is the disease can be airborne
mechanism of resistance against the host such as tuberculosis. It can also be
protective factors are involved in the proliferation pertussis.
of microorganisms and the progress of disease. o Lastly, vector borne transmission.
INFECTION Anopheles can carry/give malaria. Aedes
❖ Infection involves growth and multiplication of can carry dengue. Cockroaches when
microorganisms that causes damage to our host they perch on food and cause diarrhea.
cell. TYPES OF INFECTION ACCORDING TO HOST
❖ This is also known as bodily invasion of DISTRIBUTION
pathogenic microorganism to reproduce, multiply,
and cause disease through local cellular injury,
LOCAL INFECTION
secrete toxins through antigen antibody reaction • Confined in one area
of our body. • The signs and symptoms are confined in one
TYPES OF INFECTION area. (localized)
AUTOGENOUS INFECTION • Ex. boils and abscesses, pimples, pigsa, pus.
• Infection from the host
FOCAL INFECTION
• It is caused by microorganisms from the • From local spreads to other part
microbiota of the host. • Starts from local infection then spread to other
• Auto = self parts of the body.
IATROGENIC INFECTION • Ex. Tonsillitis, tooth, wound infection by
Clostridium tetani, etc.
• From medical treatment or procedure
SYSTEMIC INFECTION
• It is a type of infection that occurs as the result of
some medical treatment or procedure. • Infection spreads through blood and lymph
OPPORTUNISTIC INFECTION • The microbes spread throughout the body
through blood and lymph.
• Affects immunocompromised hosts but not the
individual with a normal immune system. 4 TYPES OF SYSTEMIC INFECTION
• This happens when the immune system of the BACTEREMIA
host is compromised or the host has a weak • Presence of bacteria in the blood
immune system. • Bacteria = bacteria, emia = blood
• Ex: chemotherapeutic drugs, overusing • This organism invades the blood stream without
antibiotics, immune suppressive drugs, active multiplication. The highest concentration of
undergoing chemo bacteria in the blood occurs before the fever
NOSOCOMIAL INFECTION spikes.
• Hospital-acquired infection SEPTICEMIA
• It is a type of infection that is acquired at a health • Active multiplication in the blood (sepsis)
care facility. This can be avoided with proper
PYEMIA
• Pus producing organisms invade blood

TUAZON A. 21
 • Py = pus, emia = blood symptoms which are attributable to hereditary,
• This pus producing bacteria will localized in the infection, diet or environment.
different parts of the body. ❖ Disease is a result when the infection produces
TOXEMIA notable changes in human physiology specifically
those that can cause damage to our body’s organ
• Presence of toxin in the blood.
system.
• Bacteria are able to localized in one area but will
produce a toxin that spreads and tit will be CLASSIFICATION OF INFECTIOUS DISEASE
absorbed by the cells COMMUNICABLE OR CONTAGIOUS DISEASE
EXTENT OF INFECTION • Spread from one host to another (directly or
PRIMARY INFECTION indirectly)
• Ex. tuberculosis, herpes, flu and chickenpox
• Initial infection that causes the illness.
• Ex: common colds, runny nose. NON-COMMUNICABLE DISEASE
SECONDARY INFECTION • Does not spread from one host to another
• It is usually caused by external microbes or
• Caused by opportunistic pathogen after the
opportunistic pathogen living inside the body.
primary infection has weakened the host’s
immune system. • Ex. tetanus and botulism
• Ex: pneumonia, bronchitis (developed from CLASSIFICATION OF DISEASE ACCORDING TO
common colds) OCCURRENCE
LATENT INFECTION (SILENT PHASE) SPORADIC DISEASE
• Clinically silent inside the body and cause no • Infection occurs occasionally
noticeable illness in the host. Then a severe and ENDEMIC DISEASE
acute infection will manifest. • Constantly present in a particular location and
• Ex: asymptomatic type of infection population.
MIXED INFECTION EPIDEMIC DISEASE
• Two or more infection or organism. • Affects large number of people in a short span of
• Ex: wound infection time
ACUTE INFECTION PANDEMIC DISEASE
• Infection develops and progress slowly • Affects population across large region around the
• Ex: cough world.
CHRONIC INFECTION EFFECTS OF INFECTIOUS DISEASE
• Infection develops slowly(milder) but long lasting. SIGNS
• Ex: tuberculosis • Objective changes that can be measured
ROUTE OF INFECTION • Ex. Fever, redness, swelling and paralysis
DIRECT TRANSMISSION SYMPTOMS
• Congenital • Subjective indications/complaints of the disease
o From mother to fetus or baby. in a person
o S. agalactiae (UTI), N. gonorrhoeae • Ex. Pain and malaise
(eyes, Ophthalmia Nneonatorum) and SYNDROME
syphilis. • Group of signs and symptoms that are associated
• Sexual contact - C. trachomatis, N. gonorrhoeae with the disease.
and syphilis • Ex. Acquired immune deficiency syndrome
• Infectious respiratory droplets - N. meningitidis (AIDS)
(cough, sneezing) S. pyogenes PHASES OF INFECTIOUS DISEASE
• Hand to hand transmission – Rhinovirus INCUBATION PERIOD
(causes common colds)
• Time between the exposure to pathogen and
INDIRECT TRANSMISSION
onset of symptoms
• Fomites • interval
o Inanimate objects. PRODROMAL PERIOD
• Water
o Salmonella, Shigella and Vibrio (diarrhea • Appearance of signs and symptoms
or cholera) CLINICAL OR ILLNESS PERIOD
• Arthropod vectors • Peak of characteristic sign and symptoms of an
o Borrelia (tick), Francisella and Yersinia infection
(rodents, mouse) DECLINE PERIOD
DISEASE • Period in which the signs and symptoms begin to
❖ Disease is a specific illness or disorder that is subside
characterized by a recognizable set of signs and CONVALESCENCE OR PERIOD OF RECOVERY

TUAZON A. 22
 • Period in which the surviving host is recuperating ENZYMATIC FACTORS
towards full recovery. • Produced by the bacteria that aid in the spread of
❖ Predisposing factor on having an illness or infection.
disease. • Ex. Hyaluronidase, coagulase, leukocidin,
o Gender, genetic factors, nutritional, collagenase, streptokinase, hemolysin and
stress or fatigue, environment, lifestyle, resitenase
work place. CELLULAR STRUCTURE
GENERAL CLASSES OF PATHOGENIC
• Capsule resist phagocytosis (engulfment of
MICROORGANISMS microorganisms)
TRUE PATHOGENS • Encapsulated bacteria
• Organisms are able to invade the tissue of HOST RESISTANCE FACTORS
healthy individual through some inherent ability PHYSICAL BARRIER
causing various diseases.
• Normally found outside the host • Skin serves as the physical and chemical barrier
to microorganisms
OPPORTUNISTIC PATHOGENS
• First line of defense.
• Normally do not cause disease in their natural • Ex. Stricture of urethral opening, the flushing
habitat to a healthy person. action of urination and thick mucus plug in the
• Cause disease if the host is cervical opening
immunocompromised or if they enter a different CLEANSING MECHANISM
part of the body.
• Ex. Neisseria meningitidis (usually harmless) and • Nasal hair, cough-sneeze reflex and cell lining of
trachea (contains cilia)
Escherichia coli (urinary tract, colon, intestine
infection) ANTIMICROBIAL SUBSTANCE
HOST MICROBE RELATIONSHIP • Lysozymes - destroy bacterial cell wall
SYMBIOSIS • Bile salts - disrupt bacterial membranes
• Association of two organisms living in close INDIGENOUS/NORMAL MICROBIAL FLORA OR
proximity. MICROBIOTA
MUTUALISM • Microorganisms commonly found on or in the
• Symbiotic relationship in which both organisms body sites of healthy person. They serve as
benefit from each another normal flora. Also known as microbiota.
COMMENSALISM • Ex: producing vitamin K through E. coli,
Bacteroides, Lactobacillus acidophilus, vagina
• One organism benefit while there is no beneficial pH.
or harmful effect to the other.
• One way relationship. TWO TYPES OF MICROBIOTA
PARASITISM • Resident microbiota
• One organism (parasite) benefits at the expense o temporarily inhabit, multiply in and
of its host. colonize an area for months or years.
VIRULENCE • Transient microbiota
o inhabit (do not multiply) and colonize an
• Ability of microorganisms to cause disease
area until they are eliminated.
• It is the degree of pathogenicity
• Organisms that can establish infection with a DIFFERENT BODY SITES AND THIER
relatively low infection dose. More virulent than RESPECTIVE MICROBIOTA
those that requires high dose for infection. • Skin
• Every pathogenic microorganism or rapidly o staphylococci, Propionibacterium and
progressive condition is considered when there is Corynebacterium
a high virulence factor. • Mouth and oral cavity
FACTORS INFLUENCING MICROBIAL o Viridans streptococci
VIRULENCE • Upper respiratory tract
• Toxic factors o Viridans streptococci, diphtheroids and
• Enzymatic factors epidermidis
• Cellular structure • Nasopharynx
TOXIC FACTORS o S.aureus, S.epidermidis and
• Toxins are substance produced by pathogenic N.meningitidis
microorganisms causing tissue and cellular • Colon
damage o E. coli, bacteroides and lactobacilli
• Ex. Diphtheria toxin, tetanospasmin, botulism • Urethra
toxin, canned goods, enterotoxin, streptococcal o diphtheroids, S.epidermidis, alpha and
erythrogenic toxin that causes scarlet fever. non hemolytic streptococci.

TUAZON A. 23
PHAGOCYTOSIS • In order to establish and cause disease a
• Process by which certain cells called phagocytes pathogen must multiply, following its attachment
engulf and dispose of microorganisms and cell to host cells.
debris. • Inhibitors: antibody, lactoferrin and lysozymes
• Endocytosis - engulf microorganisms and digest that inhibits prefoliation.
• Cell eating bacteria such as neutrophils, • Evasion of phagocytosis is essential for most
eosinophils, basophil, macrophages EXCEPT pathogen to survive and reproduce.
Bacillus klebsiella pneumoniae, S. pneumonia TISSUE DAMAGE
that has a capsule. • Results of either through preformed toxins or
• Phago = eat, cyto = cell disruption of the normal functioning of the
INFLAMMATION intestinal cells
• Serves as a reinforcement mechanism against • A disease is usually noticeable when it has a
microbial survival and proliferation in tissue and tissue damage.
organs. • Ex: botulinum toxin, B. cereus, S. aureus that
• Signs: swelling, redness, burning sensation, pain produces toxin which can disrupt the normal
in the affected areas. functioning of intestinal cell.
• Rubor, tumour, calor, dolor, function laesa PRODUCTION OF TOXINS
• During inflammation, phagocytes, neutrophils, • Exotoxin
macrophages, complement system, classical, • Endotoxin
alternative, lectin pathway, coagulation system,
primary and secondary hemostasis, cytokines TWO CLASSIFICATIONS OF TOXINS
that attracts different cells to help fight the • Exotoxins
infection. o One of the most lethal substances
IMMUNO RESPONSE o Gram Positive bacteria
o Do not require bacterial death to be
• Provides the human host with the ability to create release into circulation
a specific protective response against o Example: cytotoxins, neurotoxins and
microorganisms. enterotoxins
• It has the ability to memorize and recognize the o Bacteria: Clostridium botulinum,
structure of microorganisms. Corynebacterium diphtheriae,
• This becomes defective if a person is Staphylococcus aureus and
immunocompromised, during radiation and Streptococcus pyogenes
chemotherapy. • Endotoxins
TWO TYPES OF IMMUNITY o Composed of the lipopolysaccharide
• Natural/Innate Immunity o Present only in Gram Negative bacteria
o This includes the physical barriers e.g., o Release when bacteria die and their cell
skin, cough reflex, flushing action of wall undergo lysis which consequently
urine, pH of vagina, acidic pH of the liberates the endotoxin.
stomach. o Toxicity is due to the LIPID A portion of
LPS
• Adaptive/Acquired Immunity
o LPS may elicit fever, chills, hypotension,
o Antibodies, complement, memory B and
granulocytosis, thrombocytopenia, and
T cell.
disseminated intravascular coagulation.
TWO TYPES OF ADAPTIVE/ACQUIRED o NOTE: Endotoxic shock result of the
IMMUNITY Gram-negative septicemia
• Humoral (Antibody-Mediated) Immunity INVASION
o action of soluble proteins called • Process of penetrating and growing in tissue
antibodies occur on body fluid and • In some organism the invasion only involves a
surface of B-lymphocyte few layers of the cell while others have deep
• Cellular (Cell-Mediated) Immunity tissue penetration.
o Action of specific kind of T-lymphocyte DISSEMINATION
that directly attack the cells
• Spread of microorganisms to distant body sites.
INFECTIOUS AGENT FACTORS • Certain organisms that survive in phagocytosis
ADHERENCE are disseminated rapidly to many body sites.
• Organisms must penetrate the mucus layer and ROUTE OF TRANSMISSION
attach to the epithelium • Airborne transmission
• Main adhesins in bacteria: common pili and • Transmission by food and water
surface polysaccharide. • Close contact
PROLIFERATION • Cuts and bites

TUAZON A. 24
 • Arthropods (tick, lice) APPLICATION OF HEAT
• Zoonoses (anthropology vectors that can cause • Moist Heat
plaque, secretion of animals that causes • Dry heat
brucellosis)
EPIDEMIOLOGY MOIST HEAT
• Study of occurrence, distribution and causes of • Destroys microorganisms through coagulation of
disease and injury. enzymes and structural proteins and degradation
of Nucleic acids.
FACTORS CONTRIBUTING TO EPIDEMIOLOGY
CARRIER BOILING
• Destroys vegetative bacteria (non-sporulating).
• Person or animal that harbor and spread
organisms but does not become ill himself. • Water bath
• TEMP: 100C.
LIKLIHOOD OF BECOMING ENDEMIC
• TIME EXPOSURE: 10 to 15 minutes
• Organisms is constantly present in a population
FOUR TYPES OF CARRIERS:
• Casual/Acute/Transient Carrier
o harbors microorganisms temporarily for a
few days or weeks
• Chronic Carrier
o remain infected for a relatively long time,
sometimes its entire life.
• Convalescent Carrier
o individual recovered from infection but
continues to harbor large number of
AUTOCLAVING
pathogens
• Active Carrier • Fastest and simplest, all organisms (except for
o individual who has an overt clinical case prions) including those that contains spores
of a disease. • Only the laboratory technician can use this.
LIKELIHOOD OF BECOMING EPIDEMIC Before opening, make sure that the pressure or
PSI is completely 0.
• epidemic if affects significant large number of
• PRINCIPLE: Steam under pressure.
people in a short period of time. Ex. Influenza
• BIOLOGICAL INDICATOR:
LIKELIHOOD OF BECOMING PANDEMIC o Bacillus stearothermophilus
• affects huge population across large regions like o New Name: Geobacillus
countries and continent. Ex. COVID 19 stearothermophilus.
INCIDENCE RATE o 121C, 15 psi for 15 mins (media, liquids,
• Number of times a new event occurs in a given pipettes, utensils, etc.
period of time o 132C, 15 psi for 30-60 mins
INCUBATION PERIOD decontaminating medical wastes
o If the masking tape turns black, the
• time between exposure and onset of symptoms
Geobacillus/Bacillus stearothermophilus
MORBIDITY RATE is exposed to extreme convention.
• number of cases of a disease in a specified o Black stripes = sterilized
population during defined time interval
• Measures th infectiousness of an organisms
MORTALITY RATE
• number of death due to a disease in a population
RESERVOIR
• source of an infection
LESSON 4.1: STERILIZATION AND DISINFECTION TYNDALLIZATION
STERILIZATION • Destroys vegetative cells and
• Refers to the removal or destruction of all forms spores after 3 consecutive
of life, including bacteria spores. days of sterilization
PHYSICAL METHOD • Fractional or Intermittent
1. Application of Heat Sterilization.
2. Filtration • TEMP: 100C
3. Low/Cold Temperature • TIME: 30 minutes for 3 consecutive days
4. Desiccation and Lyophilization • Arnold’s sterilizer (free-flowing steam)
5. Osmotic Pressure INSPISSATION
6. Radiation

TUAZON A. 25
 • Sterilize protein-rich medium FLAMING
• Principle: thickening of media • Direct heating
through evaporation
• TEMP: 70C to 80C
• TIME: 2 hours for 3
consecutive days
• Ex. Lowenstein-Jensen
medium (whole egg – high protein content
medium)
PASTEURIZATION OVEN HEATING
• Partial Sterilization • For glassware, oil or
• Sterilize milk, dairy products and alcohol powders
• Eliminates food borne pathogens and organisms • TEMP: 160C to 170C
responsible for spoilage. • TIME: 1.5 to 2 hours
• Cannot eliminate bacterial endospores INCINERATION
• Most common method of treating infectious waste
and infected animals
• Widely used around the world.
• Hazardous material:
o TEMP: 870C to 980C
• Principle: Burning materials into ashes at 300C to
400C
CREMATION
• Used to control spread of communicable disease.
FILTRATION
• Method of choice for sterilization of antibiotic
solution, toxic chemicals, radioisotopes, vaccines
and carbohydrates
• Both for liquid and air substance
DEPTH FILTERS
• Made up of fibrous or granular materials
• Ex. Berkefield filter, Chamberland filter and
asbestos
TYPES OF PASTEURIZATION
Low-temperature High- Ultra-high
holding (LDH) temperature temperature
short-time (UHT)
holding (HTST)
• Batch Method • Flash Method • Milk can be
• Destroy milk- • Destroy milk- stored at room
borne borne temperature for
pathogens pathogens. two months
• Reduces • Quick healing without
spoilage of then immediate affecting its
food without flavor.
cooling.
affecting taste
• 72C for 15 • Process also
• 63C for 30
minutes seconds applicable to MEMBRANE FILTERS
the coffee • Porous membranes (0.1 um
creamer. thick)
• 140C for 3 • Composed of cellulose
seconds
acetate or polycarbonate
• Used to sterilize
pharmaceuticals, ophthalmic solution, culture
DRY HEAT media, antibiotics and oil products
• Sterilization method does not require water • Filter paper.
• Kills microorganisms by denaturating proteins
• Biological indicator: Bacillus subtilis var. niger
• New name: Bacillus atrophaeus

TUAZON A. 26
 Filtration Of • X-ray, gamma ray, infrared, ultra violet ray.
Liquid Air Bacteria, Critical IONIZING RADIATION
Filtration Filtration Yeast And Sterilizing
Molds • Cold Sterilization
Uses Uses high- Uses Uses • Causes mutation in the DNA and produce
cellulose efficiency 0.45um 0.22um peroxidase
acetate or particulate pores of membrane • Destroy vegetative cells and endospores of both
cellulose air (HEPA) membrane filters for prokaryotes and eukaryotes
nitrate filter filter parenteral • Gamma rays (1500 to 2500 radiation) & x-ray
membrane Removes Range: 0.2- solutions
with a organisms 0.45um and alcohol. • Biological indicator: Bacillus pumilus
vacuum. larger than Remove Removes NON-IONIZING RADIATION
0.3um most vegetation • Damage to cellular DNA by producing thymine
Biosafety bacteria and cells and dimers
cabinet. fungi but not spores but
• Used on exposed surface, operating rooms
virus not virus.
• Microorganisms in water are destroyed under UV
LOW/COLD TEMPERATURE lamps
• Considered bacteriostatic (bacteria slowly
• Ultraviolet rays (10um to 400um) in which 260um
multiplies)
is the most lethal.
• Reduces the rate of metabolism
• Important in food microbiology
• Blood products
• 2C to 8C for 72 hours – kills syphilis
DESSICATION AND LYOPHILIZATION
DESSICATION
• -Destroys bacteria through disruption of
metabolism involves removing of water from
microbes (bacteriostatic).
LYOPHILIZATION
• Destroys bacteria through changes in proteins
and decrease in chemical reaction. CHEMICAL METHOD
• Powdered form 1. Acid and Alkaline solution
2. Phenol
3. Alcohol
4. Halogen
5. Salt of Heavy Metals
6. Quaternary ammonium
7. Aldehydes
8. Gas sterilants
DISINFECTION
• Refers to the removal, inhibition or killing of
microorganisms usually on inanimate objects.
• Does not remove bacterial spores.
OSMOTIC PRESSURE ANTISEPTIC
• High concentration of salts and sugars to create • Applied topically on the skin
a hypertonic environment. • Inhibit sepsis formation
• Burong manga, jam • Alcohol and phenol are both antiseptic and
disinfectant
DISINFECTANT
• Applied to inanimate objects
• Lysol, Chlorine and Sodium hypochlorite (1:10)
BACTERICIDAL
• Precipitates bacterial protein and kills all bacteria
RADIATION in the specimen
• PRINCIPLES: Radiation passes through the • Ex. Strong acids.
cell’s free hydrogen and hydroxyl radical and
BACTERIOSTATIC
some peroxidase are created.
• Inhibit s the growth of organisms
• Free radicals – cause intracellular damage

TUAZON A. 27
 ACID AND ALKALINE SOLUTION • Ex. Zephiran (benzalkonium chloride and
• Hydrolyzes and coagulates proteins ceepryn) and cetylpyridinium chloride]
• DO NOT INGEST. This can damage and burn our • Pseudomonas aeruginosa in ammonium citrate
mucus membrane. medium is resistant to quats
PHENOL PHENOLICS
• Firs widely used antiseptic and disinfectant • Non sporicidal
• Destroys plasma membrane and denatures • Molecules of phenols that have been substituted
protein. by halogens, alkyl, phenyl or benzyl
• 5% Phenol; 10 to 30 mins contact time • Found in germicidal soaps Use in hospital floors
• Effective against mycobacteria • Antibactericidal effect is cell wall disruption
• Ex. Xylenols, lysols, cresols ALDEHYDES
ALCOHOL FORMALDEHYDE (HCHO)
• Non-sporicidal, denatures protein and dissolution • Generally, as formalin consist of 37% aqueous
of lipid membrane solution
• does not kill spore forming bacteria (Bacillus and • For mycobacteria, 3% to 8% HCHC. Contact time
Clostridium) 30 minutes
• Both an antiseptic and disinfectant • Commonly used in sterilizing HEPA filters
• 60% to 90% concentration (70% commonly used) Irritability factor and known carcinogen.
• Should be allowed to evaporate to achieve GLUTARALDEHYDE
complete antisepsis
• Effective against HIV and Hepatitis B for 10 mins
• Ex. Isopropanol (rubbing alcohol) and ethanol
• For medical instruments (heat-labile) that are
HALOGEN made of plastics and rubber material
• Destroys microorganisms through oxidation • 2% glutaraldehyde is germicidal in 10 minutes
• Ex. Chlorine, iodine (betadine), fluorine (zonrox) Sporicidal in 3 to 10 hours
and bromine • Rapid killing action but does not penetrate
• Tincture of iodine and iodophor are effective organic material well.
antisepsis • Pseudomonacidal, tuberculocidal, fungicidal and
• A 1:10 dilution of sodium hypochlorite is an virucidal
effective bleach GAS STERILANT
IODOPHOR ETHYLENE OXIDE (ETO)
• More stable than iodine in its pure form.
• Most commonly used gas for sterilization
• Combination of iodine and detergent.
• Antibacterial effect is the alkylation of nucleic
• Antibacterial effect is the oxidative effect of acids in spores and vegetative cells
molecular iodine and hypoiodic acid
• Concentration: 450 mg/L to 700 mg/L of chamber
• Improperly diluted iodophors may not kill space at 55C to 60C for two hours
microorganisms.
• Used to sterilize plastic petri dishes, sutures,
• 30 to 60 seconds onto the skin. catheters and heart-lung machines
CHLORINE • Biological indicator: Bacillus globigii
• Used in the form of hypochlorite PERIACETIC ACID
• CDC recommends a 1:10 dilution of 5.25% • Active against all vegetative microorganisms and
solution of sodium hypochlorite. fungal spores
• Antibacterial activity through oxidative and • Sterilize pieces of medical equipment
disinfecting effects of hypochlorous acids
DISINFECTANT SCREENING TEST
• Concentrated solution should not be used for
disinfection PHENOL COEFFICIENT (PC)
• 3 minutes (mycobacteria 10 to 30 mins) • Highest dilution that kills the bacteria after a 10-
SALTS OF HEAVY METALS minute exposure
• Destroys microorganisms by inactivating and • Potency of disinfectant is compared with phenol
precipitating cell proteins. • Test organisms: Staphylococcus aureus and
• Ex. Copper, arsenic, mercury, silver and zinc Salmonella typhi (20C or 27C)
AgNO3 eye drop for Neisseria gonorrhoeae • PC of >1 indicates disinfectant is more effective
• Mercuric chloride as antiseptic than phenol
QUATERNARY AMMONIUM COMPOUNDS
DETERGENTS
• Non-tuberculocidal and non sporicidal
• Most widely used surface active agents
• Disruption of the cell membrane leads to leakage
of cell content

TUAZON A. 28
LESSON 5: ANTIMICROBIALS (ANTIBIOTICS) NARROW- SPECTRUM
ANTIBIOTICS • Antibiotics act against a limited group of bacteria.
• Chemical substance produced by BROAD- SPECTRUM
microorganisms with the capacity to inhibit • Antibiotics act against a larger group of bacteria.
(bacteriostatic) or kill (bactericidal) other NARROW SPECTRUM BROAD SPECTRUM
microorganisms Bacitracin
• They can also be synthesized by means of Clindamycin
Ampicillin
chemical procedures that are independent from Cephalosporins
Dapsone
microbial activity. Chloramphenicol
Erythromycin
• Chemical substance produced by Ciprofloxacin
Gentamicin
microorganisms with the capacity to inhibit Rifampicin
Isonizid
(bacteriostatic) or kill (bactericidal) other Sulfonamides
Penicillin
microorganisms. Trimethoprim
Polymyxin B
• They can also be synthesized by means of Tetracyclin
Vancomycin
chemical procedures that are independent from CLASSIFICATION OF ANTIMICROBIAL AGENTS
microbial activity. • No antibiotics bacteria multiply.
CLASSIFICATION OF ANTIBACTERIALS • Bacteriostatic (static = steady) antibiotics prevent
DRUGS bacteria multiplying.
• Natural drugs • Bactericidal (cidal = wreck or kill) l antibiotics kill
o Produced by microorganisms such as the bacteria.
bacteria and fungi BACTERICIDAL
• Semi-synthetic drugs
• Aminoglycosides
o Modified natural drugs with added
chemical groups • Beta-lactams
• Vancomycin
• Synthetic drugs
o Chemically-produced drugs • Quinolones
SEMI- • Rifampin
NATURAL
SYNTHETIC
SYNTHETIC • Metronidazole
DRUGS DRUGS BACTERIOSTATIC
DRUGS
Amphotericin B Ampicillin Sulfonamides • Chloramphenicol
Erythromycin Carbenicillin Trimethoprim • Erythromycin
Kanamycin Methicillin Chloramphenicol • Clindamycin
Neomycin Ciprofloxacin • Sulfonamides
Nystatin Isoniazid • Trimethoprim
Rifampicin Dapsone • Tetracyclines
Streptomycin
Tetracycline MINIMAL-INHIBITORY CONCENTRATION (MIC)
Vancomycin • Lowest concentration of a drug that can still
Bacitracin inhibit bacterial growth
Gentamicin
Polymyxin
MINIMAL LETHAL CONCENTRATION (MLC)
Griseofulvin • Lowest concentration of a drug that can still kill
Penicillin bacteria
Cephalosporin PENICILLIN-BINDING PROTEINS
• Enzymes that mediate peptidoglycan cross-
NATURAL DRUGS linking with reduced affinity for B lactam
SOURCE ANTIBIOTICS antibiotics
(MICROORGANISMS) PRODUCED • This is used to make cell wall.
Bacillus subtills Bacitracin • B lactam antibiotics can destroy our penicillin
Bacillus polymyxa Polymyxin binding proteins.
Cephalosprium Cephalosporins
Micromonospora THERAPEUTIC INDEX
Gentamicin • The ratio of the toxic dose to the therapeutic dose
purpurea
Penicillium notatum Penicillin • The higher the therapeutic index the more
Streptomyces effective the chemotherapeutic agent
Erythromycin CLASSIFICATION OF SOME ANTIMICROBIAL
erythroceus / erythreus
Streptomyces fradiae Neomycin AGENTS BY THEIR SITES OF ACTION
Streptomyces nodosus Amphotericin B • THFA – tetrahydrofolic acid
Streptomyces noursei Nystatin • PABA – p-aminobenzoic acid
Streptomyces venezuelae Chloramphenicol

TUAZON A. 29
INHIBITORS OF METABOLISM
• Sulfonamides
• Trimethoprim BETA-LACTAM CEPHALOSP
PENICILLINASERES
INHIBITORS OF CELL WALL SYNTHESIS ISTANT PENICILLIN
ANTIBIOTICS ORINS
DRUGS
• B – Lactase Penicillin Cephalothin Methicillin
• Vancomycin Cephalosporins Cefoxitin Nafcillin
• Daptomycin Monobactam Ceftriaxone Oxacillin
• Telavancin Carbapenems Cephalexine
• Fosfomycin Cefixime Ex.
Ex. Escherichia Cefoperazone Staphylococcus
INHIBITORS OF PROTEIN SYNTHESIS coli
• Tetracyclines • Beta-Lactam ring destroys penicillin binding
• Aminoglycosides protein. PBS is seen in the cell wall.
• Macrolides • The purpose of why PBS is being broken down is
• Clindamycin to not let the microorganisms invade or multiply.
• Chloramphenicol • Escherichia coli & Staphylococcus release the
• Linezolid enzyme Beta-Lactamase enzyme.
INHIBITORS OF NUCLEIC ACID FUNCTION OR • Beta-Lactamase is used to destroy Beta-Lactam
SYNTHESIS ring.
• Fluoroquinolones
• Rifampin
INHIBITORS OF CELL MEMBRANE FUNCTION
• Isoniazid
• Amphotericin B
• Polymyxins
CELL WALL INHIBITOR
• Most selective antibiotics with high therapeutic
index
• Gram positive can resists the cell wall inhibitor.
PENICILLIN-BINDING PROTEINS
• bacterial enzymes involved in the synthesis of the
cell wall and in the maintenance of the
morphologic features of the bacterium B-LACTAM ANTIBIOTICS
INHIBITION OF TRANSPEPTIDASE
PENICILLINS
• PBPs catalyze formation of the cross-linkages
• PenicillinG
between peptidoglycan chains
• PenicillinV
PRODUCTION OF AUTOLYSINS
• Methicillin
• Many bacteria, particularly the gram-positive • Nafcillin
cocci, produce degradative enzymes (autolysins) • Oxacillin
that participate in the normal remodeling of the
• Cloxacillin
bacterial cell wall
• Dicloxacillin
• Amoxicillin
• Carbenicillin
• Ticarcillin
• Piperacillin

TUAZON A. 30
 • Mezlocillin PROTEIN SYNTHESIS INHIBITOR
• Cefoxitin • Antibiotics bind with a 30S subunits that results
• Azlocillin in the misreading of mRNA and thus interferes
with aminoacyl-tRNA binding
CEPHALOSPORINS • Also binds with a 50S subunit that results the
1ST GENERATION inhibition of peptidyl transferase and peptide
• Ampicillin chain elongation
• Cefadroxil • 30S + 50S = ribosome
• Cephalexin • Ribosome has 70S subunits (after being
• Cephalothin centrifuged)
• Cephapirin
TETRACYCLINES
• Cephradine
• Demeclocycline (Declomycin)
2ND GENERATION
• Doxycycline (Vibramycin)
• Cefazolin o Leptospirosis
• Cefamandole • Minocycline (Minocin)
• Cefonicid • Tetracycline
• Cefmetazole
• Cefotetan GLYCYLCYCLINES
• Cefuroxime • Tigecycline (Tygacil)
3RD GENERATION AMINOGLYCOSIDES
• Cefaclor • Amikacin
• Cefoperazone • Gentamicin (Garamycin)
• Ceftizoxime • Neomycin (Neo-fradin)
• Ceftazidime • Streptomycin
• Ceftriaxone • Tobramycin (Tobrex)
• Cefixime MACROLIDES/KETOLIDES
• Moxalactam • Azithromycin (Zithromax)
4TH & 5TH GENERATION o covid
• Cefepime • Clarithromycin (Biaxin)
• Cefozopran • Erythromycin (Various)
• Cefpirome • Telithromycin (Ketek)
• Cefquinome MACROCYCLIC
• Ceftobiprole • Fidaxomicin (Deficid)
• Ceftaroline LINCOSAMIDES
• Fosamil
• Clindamycin (Cleocin)
CARBAPENEMS o pimples
• Blapenem OXAZOLIDINONES
• Ertapenem • Linezolid (Zyvox)
• Doripenem OTHERS
• Imipenem • Chloramphenicol (Chloromycetin)
• Panipenem • Quinupristin / Dalfopristin (Synercid)
MONOBACTAMS DRUG THAT INHIBITS 30S SUBUNITS
• Aztreonam • Tetracyclines
• Tigemonam o Target the 30S subunit and prevent
• Carumonam binding of tRNA to mRA.
• Nocardicin A • Aminoglycosides
VANCOMYCIN o Binds to 30S subunit, distorting its
structure and causing misreading of the
• Vancomycin is a tricyclic glycopeptide that has
mRNA.
become increasingly important in the treatment of
life-threatening Methicillin-resistant
Staphylococcus aureus (MRSA) and methicillin-
resistant Staphylococcus epidermidis (MRSE)
infections, as well as enterococcal infections.
• The infection or microorganism that is in the
patient’s circulation is already resistant.
• High intensity drug.

TUAZON A. 31
 DRUG THAT INHIBITS 50S SUBUNITS
• Erythromycin
o Erythromycin and Clindamycin bind to
the 50s subunit, thus inhibiting
translocation.
• Chloramphenicol
o Inhibits peptidyl transferase.
• Linezolid
o Binds the 23S ribosomal RNA preventing
formation of the 70S initiation complex.
o Inhibits binding of 30S and 50S.
o Formation and initiation complex will not
happen because of linezolid.

NUCLEIC ACID SYNTHESIS INHIBITORS

• Inhibits the production of DNA of bacteria.
RIFAMPIN
• Rifampin inhibits RNA synthesis by binding
selectively to the β-subunit of bacterial DNA- • It is vital for the microorganism if you’re not able
dependent RNA polymerase. to produce RNA, without RNA, it won’t be
FLUOROQUINOLONES possible to produce protein.
• Inhibit bacterial DNA gyrase (topoisomerase II), • RNA polymerase is the main enzyme involved in
and therefore, DNA supercooling in GRAM- transcription.
NEGATIVE BACTERIA. They also inhibit • It reads one of the DNA strands and adds
topoisomerase IV in GRAM-POSITIVE complementary nucleotide bases to make an
organisms and thereby interfere with separation mRNA transcript. The mRNA will later be
of replicated chromosomal DNA. They are translated into protein.
bactericidal. CELL MEMBRANE INHIBITORS
• DNA gyrase is used to remove tension of DNA • Example of inhibitor is the POLYMYXIN B and E
strands. that are used for GRAM-NEGATIVE bacteria, like
• Topoisomerase IV is used to separate DNA Pseudomonas aeruginosa and also as a topical
strands. antibiotic.
• Polymyxin is a cell membrane inhibitor which
destroys the liquid portion of the gram-negative
bacteria’s outer membrane.

METRONIDAZOLE
• bound to DNA and electron-transport proteins,
thus inhibiting nucleic acid synthesis.
• Usually used in parasitic infection.

TUAZON A. 32
 • Example:
o Resistance to the glycopeptide antibiotic
(Gram-positive bacteria)
o Presence of porins and LPS (Gram-
negative bacteria
o Secretion of Beta-lactamase by bacteria
B-LACTAMASES
• Inactivates B-lactam drugs by disrupting and
opening the B-lactam ring component
• Hydrolyzes the ring to form INACTIVE
PENICILLOIC ACID
• This serves as substrate to the B-lactam ring
structure which the enzyme selectively
hydrolyzes to form an inactive penicilloic acid.
GRAM POSITIVE GRAM NEGATIVE
Secreted as exoenzymes Found in the periplasmic
space
Less protection to the
bacteria
EXAMPLE:
• Staphylococci, Haemophilus influenza, Neisseria
gonorroeae, enterobacteriaceae, Pseudomons,
Moraxella catarrhalis and Bacteroides
B-LACTAMASE INHIBITORS
ESSENTIAL METABOLITE INHIBITORS • Structural similarity with B-lactam antibiotics and
• Function of these drugs is to inhibit those function, as substrate for B-lactamase, thus
enzymes necessary for the production of DNA. reducing their harmful effects on the B-lactam
To not be able to produce folic acid, vitamin B9 antibiotics
which are necessary for the production of DNA. • Example:
• Sulfonamides – dapsone used for o Clavulanic acid
mycobacterium leprae to patients infected with o Sulbactam
leprosy. This inhibits Dihydropteroate synthase. o Tazobactam
• Trimethoprim inhibits Dihydrofolate reductase. B-LACTAM ANTIBIOTICS:
• If both enzymes are inhibited, DNA will not be • Penicillin
produced. • Cephalosporins
• Isoniazid – drug for mycobacterium tuberculosis. • Monobactam
This is used for inhibition of Mycolic acid. • Carbapenems
LESSON 5.1: ANTIBIOTIC RESISTANCE

ANTIBIOTIC RESISTANCE
• Result of both the use and overuse of
antimicrobials agents
• Arise within antibiotic-producing microorganisms
as mechanism to protect them against the action
of their own antibiotic (autotoxicity)
• Two types:
o Intrinsic resistance
o Acquired resistance
INTRINSIC RESISTANCE
• Result of the biochemical makeup of wild-type
organisms • Nature of the R group determines the drug’s
stability to enzymatic or acidic hydrolysis and
• Depends on the hydrophobic or hydrophilic
affects its antibacterial spectrum.
nature of antibiotics and on impermeability of the
cell wall to the antibiotics LACTAMASES:
• Passed vertically into a new cell • Staphylococci, Haemophilus influenza, Neisseria
• All gram-negative bacteria can mediate this type gonorroeae, enterobacteriaceae, Pseudomons,
of resistant through enzymatic inactivation of Moraxella catarrhalis and Bacteroides
Penicillin.

TUAZON A. 33
 • The PEP side chains are cross-linked as the final
step in the synthesis of peptidoglycan. This
process is blocked by penicillin.
B-LACTAMASE INHIBITORS:
• Clavulanic Acid
• Sulbactam
• Tazobactam
EXTENDED SPECTRUM B-LACTAMASES
• Inactivate extended-spectrum cephalosporins,
penicillin and aztreonam
• Indicator drugs for ESBL: aztreonam, ceftriaxone
and cefotaxime
ADDTL NOTES:
EXTENDED SPECTRUM B-LACTAMASES
IMMUNOLOGY
INHIBITORS EDWARD JENNER
• Carbapenems (imipenem, meropenem and
ertapenem) • Concept of vaccination
• Clavulanic acid (B-lactamase inhibitor) LOUIS PASTEUR AND PIERRE PAUL EMIL
ACQUIRED RESISTANCE ROUX
• Present only on certain isolates that are different • Pasteur: used the term vaccine
from parental strain • Pasteur and Roux: Anthrax vaccine
• Expressed either as a modification of target sites EMIL VON BEHRING
or enzymatic modification of antibiotics • Antitoxin: diphtheria and tetanus
• Example: MODERN THERAPY
o Chromosomal mutations such as ALEXANDER FLEMING
transformation and recombination
• Antibiotic penicillin
WAYS THAT BACTERIA ACQUIRE HOWARD FLOREY AND ERNST CHAIN
RESISTANCE • Purification of penicillin
MUTATION PAUL EHRLICH
• Through the process of cell replication, some • Salvarsan (arsphenamine) for syphilis
bacteria develop mutations that make them ABELARDO AGUILAR
resistant to antibiotics.
• Erythromycin
• Bacteria with resistant mutation have a better
chance of survival against antibiotics.
THEORY OF ANTISEPSIS
• Resistant bacteria continue to multiple, even JOSEPH LISTER
when exposed to antibiotics. • System of Antiseptic in surgery
HORIZONTAL GENE TRANSFER GERM THEORY OF DISEASE
• Antibiotic-resistant genetic material is transferred ROBERT KOCH
between different bacteria cells. This can happen • Discover
in three different ways: transformation, o Bacillus anthracis and Mycobacterium
transduction or conjugation. tuberculosis.
o Cultivate bacteria on boiled potatoes,
gelatin, meat extracts and protein
• Collaboration
o Fenny Hesse: Agar as solidifying agent

TUAZON A. 34
 o Julius Richard Petri: Petri dish
o Martinus Beijerinck and Sergei
Winogradsky: Enrichment culture and
selective media
BIOGENESIS
• Living cells arise only from pre-existing living cells
RUDOLF VIRCHOW
• Concept of biogenesis
THEODOR SCHWANN
• No growth: flask with nutrient solution after
allowing the air to pass through heated tube
HEINRICH SCHRODER AND THEODORE VON
DUSCH
• No growth: after allowing air pass through sterile
cotton wool placed on flask of heated-sterilized
medium
LOUIS PASTEUR
• Disproved the doctrine of spontaneous
generation Proposed: Aseptic technique
JOHN TYNDALL
• Dust carries germs that could contaminate sterile
broth
• Tyndallization - moist heat for 3 consecutive days
FERDINAND COHN
• Endospores
ANTOINE LAURENT LAVOISIER
• Importance of oxygen to life
SPONTANEOUS GENERATION
• life arises from non-living matters
ARISTOTLE
• Simple invertebrates arise from spontaneous
generation
FRANCESCO REDI
• Maggots could NOT arise spontaneously from
decaying meat
JOHN NEEDHAM
• Boiled mutton broth -> cloudy after pouring into
flask then sealed tightly
LAZZARO SPALLANZANI
• Heating the broth placed in sealed jar No growth
took place as long as the flask remained sealed
DISCOVERY OF MICROORGANISMS
LUCRETIUS AND GIROLAMO FRACASTRORO
• Disease caused by "invisible living creatures"
FRANCESCO STELLUTI
• Observes on bees and weevils under microscope
ANTON VAN LEEUWENHOEK
• First true microbiologist
• Father of Bacteriology and Protozoology Self-
made single lens microscope (50 to 300x)
• Used the term animalcules

TUAZON A. 35
Clinical Bacteriology (Lecture)
SECOND TERM

DASH
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science TWO
TWO
MIDTERMS
UNIT OUTLINE • If Gardnerella vaginalis is overpowered
I. Normal Flora and Micrococcaceae Lactobacillus acidophilus, it causes a disease
II. Streptococcaceae and Gram-Negative vaginosis.
Cocci • Major normal flora of the skin is Staphylococcus
III. Enterobacteriaceae epidermidis.
IV. Non-Enteric and Non-Fermentative Gram- • Normal flora that can be found on your skin are
Negative Bacilli S. aureus & S. epidermidis.
Medically important members of the normal flora
Skin
LESS
IMPORTANT
LOCATION IMPORTANT
ORGANISM
LESSON 1: NORMAL FLORA AND ORGANISM
MICROCOCCACEAE Staphylococcus
aureus,
NORMAL FLORA
Corynebacterium
• Term used to describe the various bacteria and
(diphtheroids,
fungi that are permanent residents of certain body
various
sites, especially the skin, oropharynx, colon
Staphylococcus streptococci
(produce vitamin A), and vagina (Lactobacillus Skin
epidermidis Pseudomonas
acidophilus).
aeruginosa,
• The normal flora organisms are often referred to anaerobes leg,
as commensals. Commensals (E. coli in colon, Propionibacterium),
Entamoeba coli) are organisms that derive yeasts (eg.
benefit from another host but do not damage that Candida albicans)
host.
S. epidermidis,
• The term human microbiome (microbiota) is Corynebacterium
often used to describe the normal flora. Nose S. aureus (diphtheroids),
• Seen on different sites of the body specifically various
and majority in the skin. streptococci
MEMBERS OF NORMAL FLORA AND THEIR Various
ANATOMIC LOCATIONS Viridans streptococci,
Mouth
streptococci Eikenalla
MEMBERS OF THE corrodens
ANATOMIC LOCATION
NORMAL FLORA Prevotella
Colon (majority), throat, Streptococcus
Bacteroides species intermedia,
vagina Dental plaque mutans
Porphyromonas
Candida albicans Mouth, colon, vagina gingivalis
Clostridium species Colon Various
Corynebacterium species Nasopharynx, skin, anaerobes (eg.
Gingival
(diphtheroids) vagina Bacteroides,
crevices
Enterococcus faecalis Colon Fusobacterium,
Escherichia coli and other Colon, vagina, outer streptococci,
coliforms urethra Actinomyces)
Gardnerella vaginalis Vagina Various
Haemophilus species Nasopharynx streptococci
Lactobacillus species Mouth, colon, vagina (including
Neisseria species Mouth, nasopharynx Streptococcus
Propionibacterium acnes Skin pyogenes and
Pseudomonas Viridans Streptococcus
Colon, skin Throat
aeruginosa streptococci pneumoniae),
Nose (major), skin (small Neisseria species,
Staphylococcus aureus Haemophilus
amount)
Staphylococcus Skin (major), nose, influenzae S
epidermidis mouth, vagina, urethra epidermidis
Viridans streptococci Mouth, nasopharynx

TUAZON A. 1
 Bifidobacterium, • Gram negative rods
Eubacterium, • Bacteroides and Prevotella are important
Fusobacterium, members
Lactobacillus, • Once you’re immunocompromised, Bacteroides
Bacteroides
various aerobic will invade your intestine especially if you have
Colon fragilis,
gram-negative appendicitis and if it ruptured. It can cause
Escherichia coli
rods, Enterococcus peritonitis and later on can lead to severe
faecalis and other infection or death.
streptococci, • Remainder:
Clostridium o Proteobacteria (gram negative rods)
Various ▪ Escherichia
streptococci, ▪ Salmonella
various gram- o Actinobacteria (gram positive rods)
Lactobacillus E ▪ Actinomyces
negative rods. 8.
Vagina coli group B
fragile,
streptococci MICROBIOTA PLAYS AN IMPORTANT ROLE IN
Corynebacterium
(diphtheroids), C. SEVERAL BODY FUNCTIONS:
• Weight control (obesity)
albicans
• Inflammatory bowel disease
S. epidermidis,
• Immune response
Corynebacterium
• Resistance to infectious disease
(diphtheroids),
various NORMAL FLORA OF THE SKIN
Urethra STAPHYLOCOCCUS EPIDERMIDIS
streptococci,
various gram- • Predominantly; superficially
negative rods (eg. • Non-pathogen of skin but can cause disease,
E. coli) such as Artificial Heart Valves and Prosthetic
NORMAL FLORA Joints.
• There is a distinction between the presence of • Usually causes infection to medical equipment or
these organisms and the carrier state. instruments.
• Carrier implies that an individual harbors a ANAEROBIC BACTERIA
potential pathogen and therefore can be a source • Propionibacterium and Peptococcus
of infection of others. • Deeper follicles
• There is also a distinction to be made between • Causes pimples
members of the normal flora, which are the CANDIDA ALBICANS
permanent residents, and the colonization of the • Normal flora of the skin but can cause systemic
individual with a new organism. infections once it is in the blood circulation.
• Colonization typically refers to the acquisition of • It can cause severe fungal infection.
a new organism.
NORMAL FLORA OF THE RESPIRATORY
o That particular organism should not be
found in that particular area but it’s there.
TRACT
• Colonize the nose, throat and mouth,
HUMAN MICROBIOME
• Few found in lower bronchi and alveoli
• Also known as Microbiota
• Found on the skin, mucosal surface and within
NOSE: STREPTOCOCCAL AND
the lumen of the gastrointestinal tract. STAPYLOCOCCAL SP.
• Majority of these microbes are bacteria, but yeast • Most significant: Staphylococcus aureus
(Candida albicans) and protozoa (Entamoeba THROAT: VIRIDANS STREPTOCOCCI AND
coli, found inside the intestine) are also found in NEISSERIA
the large numbers. • Streptococcus mutans
o Bacteria helps in digestion, helps in o Dental plaque (dental caries)
converting conjugated bilirubin to • Streptococcus sanguis
stercobilin (which makes the stool o Leading cause of subacute bacterial
brown). endocarditis
• Two Largest Phyla of Bacteria in the colon • Eikenella corrodens
o Fermicutes o Part of skin and soft tissue normal flora
o Bacteroides o Associated with human bites and
FERMICUTES clenched fist injuries.
• Gram positive rods o skin and soft tissue infection
• Member of genera Clostridium and ADDTL INFO: Viridans streptococci has a caramel odor.
Faecalibacterium are important members • Anaerobic bacteria
BACTEROIDES o Bacteroides, Prevotella, Fusobacterium,
Clostridium and Peptostreptococcus

TUAZON A. 2
 o Found in gingival crevices where oxygen o There are no microorganisms in the
concentration is very low. bladder. More or less contaminants that
• Actinomyces israelii can cause UTI.
o Anaerobic actinomycetes that can cause o If a man has a UTI, it is already in a
abscesses of the jaw, lungs and severe form.
abdomen. • Mycobacterium smegmatis
NORMAL FLORA OF THE INTESTINAL TRACT o Acid-fast organisms
• Stomach o Found area around the urethra of women
o contains few organisms, primarily and uncircumcised men.
because of its low pH
• Small intestine
o Small numbers Streptococci, Lactobacilli
and yeasts particularly Candida albicans.
o Larger number of organisms found in
terminal ileum.
• Colon
o Major location of bacteria in the body
o 20% of feces consists of bacteria
approximately 1011 (1011) organisms/g.
• Escherichia coli
o Leading cause of UTI (Urinary Tract
Infection)
• Bacteroides fragilis
o Important cause of peritonitis associated
w/ perforation of the intestinal wall
following trauma, appendicitis or
diverticulitis.
• Others:
o Fusobacterium and
Peptostreptococcus, other facultative
bacteria include Enterococcus faecalis.
▪ Cause UTI and endocarditis
• Pseudomonas aeruginosa
o Various infection in hospitalized patient.
o Present in 10% of normal stools
o As well soil and water
o Carriers are mostly health care workers.
NORMAL FLORA OF THE GENITOURINARY
TRACT
• Lactobacillus species - vaginal flora
o Responsible for producing the acid that
keeps the pH of the adult woman’s
DIFFERENTIATION OF EXOTOXIN AND ENDOTOXIN
vagina low
• Candida albicans
EXOTOXIN ENDOTOXIN
o Candida vaginitis
Parent Mostly Gram- Gram negative
o itchy
organisms positive bacteria bacteria
• Escherichia coli and Enterobacter
Chemical
o Cause UTI Simple protein LPS-lipid A
structure
• Group B Streptococcus (Streptococcus
Outside the Part of the outer
agalactiae) Location
living cell membrane
o UTI to sexually active women
Heat stability Heat labile Heat stable
o Newborn: sepsis and meningitis
Toxicity High Low
• Bladder
Gas gangrene,
o Sterile in healthy person Typhoid fever,
tetanus,
o Often contaminated during passage of Representative UTI and
botulism,
urine through the outermost portions of disease Meningococcal
diphtheria and
the urethra. meningitis
scarlet fever
o Most common contaminant:
Staphylococcus epidermidis, coliforms, Produce fever
Usually does not
diphtheriods and non hemolytic Fever by releasing
produce fever
streptococci. interleukin 1

TUAZON A. 3
 • Exotoxin FURAZOLIDONESUSEPTIBILITY TEST
o can also be found in some gram-negative • Uses 100 ug furazolidone and is performed on
bacteria BAP
o Exotoxin: Heat labile, except for • Result:
staphylococcal enterotoxin o Micrococci = R
• Vaccine: o Staphylococci = S
o Endotoxin: uses Toxoid vaccine
o Endotoxin: No vaccine available
• All gram-positive bacteria have NO ENDOTOXIN,
except: Listeria monocytogenes (with endotoxin-
like substance)
LESSON 1.2: MICROCOCCACEAE
OUTLINE:
FAMILY:
• Micrococcaceae
GENUS:
• Planococcus
• Micrococcus ★
• Stomatococcus
MICRODASE TEST / MODIFIED OXIDASE
• Staphylococcus ★
• Rapid test
SPECIE: (MEDICALLY IMPORTANT) • Test that detects oxidase enzyme
• Staphylococcus aureus • Paper reagent is: Tetramethyl-p-
• Staphylococcus epidermidis phenylenediamine in dimethyl sulfoxide
• Staphylococcus saprophyticus • Positive: blue to purple blue
• Staphylococcus lugdunensis • Negative: No color change
TEST TO DIFFERENTIATE MICROCOCCUS AND • Result:
STAPHYLOCOCCUS o Micrococci = Positive
• Bacitracin/Taxo A Disk Test o Staphylococci = Negative
• Furazolidone Susceptibility Test
• Lysostaphin Sensitivity Test
• Modified Oxidase/Microdase Test
• Growth on Furoxone-Tween 80-Oil Red O Agar
• Acid Production from Glycerol
• Oxidation-Fermentation (of) Reaction
BACITRACIN
• Uses 0.04 units of bacitracin and performed on
BAP (Blood Agar Plate) or MHA (Mueller Hinton
Agar)
• Result:
o Micrococci = S (susceptible)
o Staphylococci = R (resistant/resistance)
• Taxo A Disk Test
OXIDATION-FERMENTATION REACTION
• Conduct under and anaerobic condition,
staphylococci ferment the glucose while
micrococci fail to produce acid.
• Yellow = Acid formation
• Nonfermenter / Nonutilizer = color remains red

TUAZON A. 4
 DIFFERENTIATION BETWEEN STAPHYLOCOCCI • Glucose fermenter
AND MICROCOCCI IN THE ROUTINE • They are normally inhabitants of the skin, mucous
LABORATORY membranes and intestine.
TEST Staphylococci Micrococci • Staphylococci associated with human infection
Modified oxidase - + are colonizer of various skin and mucosal
Anaerobic acid surfaces.
production from + -* • Under the microscope they spherical cell that
glucose. appears in clusters or sometimes singly.
Growth on
Furoxome-Tween 80- - + CATALASE TEST
oil red O agar. • Mediates the breakdown of hydrogen peroxide
Anaerobic acid (H2O2) into oxygen and water.
production from • Positive = cupious bubble formation
glycerol in the + - (staphylococcus)
presence of • Negative = No bubble formation
erythromycin. • Note: should not take specimen from BAP
Resistance to because of peroxidase.
R+ S • i.e. agua oxinada in wound
bacitracin (0.04 units)
Lysosome (50-mg • Hydrogen peroxide end product is oxygen (O2)
R S and water (H2O).
disk)
Lysostaphin test S+ R

GRAM POSITIVE BACTERIA

BAP CULTURE
• Colonies: 4mm to 8mm
• Appear creamy, white or light gold or “butter-
looking” while other species may have gray
colonies (under BAP)
• Beta haemolytic (S. aureus)

STAPHYLOCOCCI
• Staphylococcus aureus
• Staphylococcus epidermidis
• Staphylococcus saprophyticus
• Staphylococcus lugdunensis STAPHYLOCOCCUS AUREUS
• Coagulase positive and most virulent specie of
staphylococci
• Principal virulence factor: Coagulase
• Culture: BAP golden yellow and B-haemolytic
• Cultivated by adding 7.5% to 10% NaCl which is
why they are called Halophilic microorganism
STAPHYLOCOCCI • Can cause skin, wound and deep tissue infection.
• Genus name derived from the Greek words • These are organisms that can cause infection
staphute, means “bunches of grapes” and once they enter a normally clean site during
kokkos, means “berries” trauma, abrasion of the skin, or mucosal
• Principal virulence factor is the Coagulase surfaces.
• Catalase- producing microorganism • They usually serve as normal flora in the skin, but
• Facultative anaerobe, except S. saccharolyticus if a person got hurt, trauma, or abrasion, this can
(obligate anaerobe). cause infection or abscess to the site of abrasion.
• Non-Motile, Non-spore forming

TUAZON A. 5
 INFECTION AND DISEASES (S. AUREUS) o mild inflammation of the hair follicle or
• Toxin-induced cases sebaceous gland
• Bacteremia and sepsis o single hair follicles
• Urinary tract infection • Furuncles
• Acute bacterial endocarditis o large, raised, superficial abscess can be
• Cutaneous infections extension of folliculitis
o Pigsa / Tigsa or boils • Carbuncles
o Folliculitis, furuncle, carbuncle, impetigo o develop from multiple furuncle (big lump)
and purulent abscess. o can cause fever or chills (need
• Osteomyelitis antibiotics)
• Septic arthritis (children) • Impetigo
• Food poisoning o superficial cutaneous infection usually
seen in children and characterized by
TOXIN-INDUCED CASES
crusty lesion in sides or surface of the
• Scalded skin syndrome (SSS) mouth.
o Extensive exfoliative dermatitis that • Purulent abscess
occurs primarily in newborns and o drainage needed, aseptic technique of
previously healthy children draining. Sterile or surgical.
o SSS has the enzyme exfoliation
produced by S. aureus that cleaves the
demoglein in the desmosome. There will
be a separation of skin at Stratum
granulosum.

• Toxic shock syndrome (TSS)

o Rare but potentially fatal, multi-system
disease.
o Sudden onset of fever, chills, vomiting,
diarrhea and rashes w/c rapidly progress
to hypotension and shock
o Strawberry tongue – sloughing of filiform
papillae.
o Desquamating rash
o Multi-organ involvement
o No pyogenic inflammation, blood CS
(culture and sensitivity) is negative.
o Headache, fever (102°-105°F),
confusion, sore throat, non-purulent
conjunctivitis, lethargy, vomiting,
sunburn-like rash, profuse watery
diarrhea, associated with menstruation &
tampon use, 3rd to 7th day epidermal ENZYME AND TOXIN PRODUCED BY
sloughing of palms & soles, and rapid
STAPHYLOCOCCUS AUREUS
progression (48 hours) hypotension –
• Coagulase
syncope – shock.
• Hyaluronidase (spreading factor enzyme)
• Staphylokinase
• Lipase (fat-splitting enzyme)
• Dnase enzyme (Deoxyribonuclease)
• B-lactamase
• Enterotoxin (heat-stable)
• Leukocidin (Panton-valentine leukocidin)
• Hemolysin
CUTANEOUS INFECTIONS • Exfoliatin A and B/ Epidermolytic toxin A and B
• Folliculitis • Toxic shock syndrome-1

TUAZON A. 6
 • Protein A • 90% of the isolated staphylococci are penicillin
COAGULASE resistant. They utilize semi-synthetic drugs
• It coagulates the fibrinogen in the plasma derived from penicillin.
• It promotes the formation of a fibrin layer around LEUKOCIDIN / PHANTON-VALENTINE
the staphylococcal abscess thereby protecting • Cytolytic toxin
the bacteria from phagocytosis • Attacks and kills white blood cells such as
• Two types: polymorphonuclear cells, neutrophils,
o Cell bound coagulase or clumping eosinophils, and basophils. It also attacks the
factor macrophages and monocytes.
▪ Bound to the cell wall and clots • Responsible for necrotizing skin and soft tissue
human, rabbit, or pig plasma by infection
directly converting fibrinogen into • It is a pore-forming exotoxin that suppresses
fibrin. phagocytosis.
▪ Clumping immediately. PROTEIN A
o Unbound or free coagulase • anti-phagocytic since it competes with neutrophils
▪ These are extracellular enzyme for the Fc portion of specific opsonins therefore s.
that is not bound to the cell wall aureus can resist phagocytosis.
of the microorganism but can
• An immunologically active substance that is
cause clot formation when
found in the cell wall of S. aureus.
bacterial cells are incubated with
plasma.
ENTEROTOXIN (HEAT-STABLE)
▪ Clumping takes time. Plasma • Toxin appears to act as neurotoxin that stimulate
needs to be incubated for clot vomiting through the vagus nerve.
formation. • Stable to heating by 100C for 30 mins
HYALURONIDASE • Resistant to hydrolysis by gastric and jejunal
enzyme
• Enhance invasion and survival in the tissue
• Spreading factor enzyme • Example: A, B, C1, C2, D, E and G to J
o A, B, D – responsible for food poisoning.
• Breaks down hyaluronic acid. Hyaluronic acid is
o Most common cause of food poisoning is
present in the intracellular ground substance of
enterotoxin A.
the connective tissue.
o Enterotoxin B is most commonly
STAPHYLOKINASE associated with pseudomembranous
• it causes fibrinolytic activities by dissolving fibrin enterocolitis and it is found in
clots contaminated milk products.
• Fibrinolysin HEMOLYSIN (CYTOTOXIN)
LIPASE • Enzyme that hemolyzes RBC that causes anemia
• Essential for bacterial in sebaceous areas of the and make the pigment hemoglobin which is the
body. iron available for microbial growth.
• Fat splitting enzyme. • Heme – contains iron available for microbial
• Lipase is usually produced by both coagulase growth.
positive and negative staphylococci. It is essential • Four types:
for bacterial survival in the sebaceous area. o Alpha-hemolysin
• Lipase is important in the formation of furuncle, ▪ It is a predominant hemolysin
carbuncle and boils. produced by S. aureus.
• Enzyme that causes pigsa or furuncle, carbuncle, ▪ Destroys RBS, platelets, and
and boils is lipase produced by the macrophages.
Staphylococcus aureus. o Beta-hemolysin
• Lipase helps S. aureus to survive in the ▪ Destroys sphingomyelin portion
sebaceous gland or area. of the RBC.
DEOXYRIBONUCLEASE (DNASE) o Gamma-hemolysin
• destroys DNA ▪ Less toxic than the alpha and
beta-hemolysin.
• It lowers the viscosity of exudates, giving the
▪ It is produced by all the S. aureus
pathogens more mobility.
strain that causes RBC injury in
B-LACTAMASE the culture and produce
• breaks down penicillin and other B-lactam drugs. edematous lesion.
• B-lactam ring is responsible for the destruction of o Delta- hemolysin
penicillin and penicillin binding protein. ▪ Destroys RBC and is associated
with the Phanton-Valentine
leucocidin.

TUAZON A. 7
 • Two methods:
o Slide Method
o Tube Method
RESULT
SLIDE METHOD
• Positive: Coagulum formation within 30 seconds
o Staphylococcus aureus
o Staphylococcus lugdunensis
o Staphylococcus schleiferi
TUBE METHOD
• Positive: Clot or coagulum formation after 1 to 4
hrs of incubation
• If no clot, left at room temp for additional 20 hours
• All in all 24 hours.
• Sensitive but definitive method
• This detects extracellular and free coagulase.
• Plasma and the microorganism is incubated at
35°C.
o Staphylococcus hyicus
o Staphylococcus intermedius
o Staphylococcus lutrae
o Staphylococcus delphini
o Staphylococcus schleiferi
MANNITOL FERMENTATION TEST
• Differentiate the pathogenic staphylococci from
EXFOLIATIN SEROTYPES A AND B non - pathogenic.
(SUPERANTIGENS) • Mannitol Salt Agar (MSA)
• Aka: Epidermolytic toxins A and B • pH indicator: phenol red
• A serine protease that divides the intracellular • Positive result: Yellow S. aureus colonies
bridges of the epidermis and causes extensive (surrounded by yellow halo)
sloughing of the epidermis to produce burn-like • 7.5% salt is in MSA
effect on the patient.
• Causes: Scalded-skin syndrome (SSS) or Ritter’s
disease.
TOXIC SHOCK SYNDROME TOXIN 1 (TSST-1)
• Aka: Enterotoxin F or Pyogenic exotoxin
• Chromosomal-mediated toxin
• Causes menstruation-associated TSS (using
tampon)
• It is absorbed through a vaginal mucosa thus
allowing the systemic effect seen TSS.
• Stimulates production of cytokines
DIFFERENTIAL TEST FOR STAPHYLOCOCCUS
AUREUS
• Coagulase Test
• Mannitol Fermentation Test
• Tellurite glycine Agar
• Polymyxin sensitivity Test
• Voges-Proskauer Test OTHER TEST
• Deoxyribonuclease (DNase) Test • Tellurite glycine agar
COAGULASE TEST o Jet black colonies of S. aureus
• Best criterion of Pathogenicity of Staphylococcus • Polymyxin sensitivity Test
aureus o S. aureus is resistant
• Reagent: Rabbit plasma • Voges-Proskauer (VP) Test
• Anticoagulant: EDTA or Ethylene Diamine Tetra- o Differentiate S. aureus from S.
Acetic Acid (Do not use citrate tube (blue) intermedius
because it can yield false positive result.)

TUAZON A. 8
 o Positive: Pink (acetoin) (S. aureus • Associated with community acquired UTI in
produce acetoin or acetyl methyl young sexually active females.
carbinol) • They adhere to the epithelial cells of urogenital
• Deoxyribonuclease (DNAse) Test tract
o Identify pathogenic species of • BAP: Colonies white, opaque, slightly larger than
staphylococci that produces Dnase. pin head and non haemolytic
o Culture media/medium: DNA-methyl • Urine culture: 10,000 CFU/mL (significant
green agar findings)
o Positive: Clear/colorless zone around • Biochemical test: Coagulase negative; (-) MSA
organism • Antimicrobial: Resistant to 5 ug novobiocin
o Positive: S. aureus STAPHYLOCOCCUS LUGDUNENSIS
METHICILLIN-RESISTANT STAPHYLOCOCCUS • Coagulase negative by tube method
AUREUS • Confused with S. aureus in slide method (it
• Resistant to antibiotic: methicillin, nafcillin and becomes positive)
oxacillin • More aggressive than other CoNS (coagulase
• Vancomycin is used for methicillin resistant S. negative S. aureus) in terms of infectivity
aureus. • It contains mecA gene that codes or responsible
• Acquired after prolonged stay in hospital (ICU for oxacillin resistant.
and burn patients). • Infections: endocarditis, meningitis, septicemia,
• Three types: UTI and skin and soft tissue infections.
o Hospital-acquired MRSA (HA-MRSA) OTHER TEST FOR DIAGNOSIS OF
o Community-acquired MRSA (CA-MRSA)
STAPHYLOCOCCI
o Health care-associated community onset
• Culture
(HACO-MRSA)
• PYR Test
• Chromogenic test:
o Positive, changes color of MRSA within • Double disk diffusion
24 to 48 hours using CHROM agar • Molecular Test
o If the color changes, it is methicillin • Latex agglutination
resistant. CULTURE
o Negative: colorless • BAP, MSA, PEA, CNA, CAP, BHI, Thioglycollate
OTHER MEDICALLY IMPORTANT and CHROM agar
STAPHYLOCOCCUS SPECIES Colistin-nalidixic acid
Purulent exudates
Agar
Selective for Gram
Phenylethyl alcohol positive bacteria
(PEA) Enriched with 5% sheep
blood.
Mannitol Salt Agar and Heavily contaminated
PEA specimens
Selective and differential
for MRSA
CHROM Agar Color change = MRSA
No color change = Non
MRSA
PYRROLIDONYL ARYLAMIDASE (PYR) TEST
• Differentiate coagulase-positive staphylococci by
slide method
• Detects staphylococci-producing arylamidase
enzyme
STAPHYLOCOCCUS EPIDERMIDIS • Substrate: Pyroglutamyl-B-naphthylamide (L-
pyrrolidonyl- B-naphthylamide)
• Indigenous microbiota of the skin
• Final reagent: p-demethylaminocinnamaldehyde
• Contaminant of medical instrument, catheters,
CSF shunts and Prosthetic heart valve implants • End products: L-pyrrolidone and B-
naphthylamine
• BAP: colonies white, opaque, small to medium
sized pin head and non haemolytic • Positive: Cherry red (S. lugdunensis and S.
schleiferi)
• Biochemical Test: Coagulase negative; (-) MSA
• Negative: S. aureus
• Antimicrobial Test: Susceptible with 5 ug
novobiocin
STAPHYLOCOCCUS SAPROPHYTICUS

TUAZON A. 9
 • If you don’t want to proceed with the 20 hours at
room temp to know if it is S. aureus or
S.lugdunensis, this test is what you do instead.

DOUBLE DISK DIFFUSION TEST (D-TEST) MRSA – MECHANISM – I

• Detect inducible clindamycin resistance in • Horizontally transferred DNA element – SCCmec.
staphylococci • Site specific recombination.
• Uses 15 mcg of erythromycin disk and 2 mcg of • mecA gene encodes PBP2a.
clindamycin disk and then placed into MHA or • PBP2a = 78 KDa PBP – capable of cell wall
BAP. Will then incubate overnight at 35°C. synthesis.
• Positive: flattening on one side of clindamycin • PBP2a has low affinity for all B-lactams.
zone of inhibition (near the erythromycin disk) LESSON 2: STREPTOCOCCACEAE AND
which gives the appearance of D zone GRAM-NEGATIVE COCCI
• Negative: The absence of blunting indicates
erythromycin resistance. STREPTOCOCCACEAE
• Family
o Streptococcaeae
• Genus
o Streptococcus
o Enterococcus
o Aerococcus
o Lactococcus
o Pediococcus
o Leuconostoc
o Gamella
• Specie
o Group A streptococci
o Group B streptococci
o Group C and G streptococci
o Viridans streptococci
o Streptococcus pneumonia
o Enterococci
o Abiotrophia and Granulicatella
o Streptococcus-like organisms
STREPTOCOCCI
LATEX AGGLUTINATION • Academic/Bergey’s Classification
• Detect clumping factor and Protein A • Smith and Brown Classification
• Useful for detecting altered penicillin-binding • Lancefield Classification
proteins and oxacillin resistant strains • Facultative anaerobes except for
• Utilized for both S. aureus and CoNS (coagulase peptostreptococci, which are obligate anaerobes
negative S. aureus) • Some species are capnophilic
MOLECULAR TEST – NUCLEIC ACID PROBES • Their growth enhanced by blood serum, or
AND PCR AMPLIFICATION glucose
• Utilize for the identification of mecA gene • All streptococci except the viridans group and
• Gold standard for MRSA detection Streptococcus pneumonieae are included in
• Specimen: anterior nares swab Lancefield classification.
• Microscope: Gram positive spherical cells,
arranged in chains or pairs.

TUAZON A. 10
 • Streptococcus belongs to the family of o Nonhemolytic and usually contain a
Streptococcaeae. Some specie are part of the Lancefield classification which is the N
indigenous human microbiota that can cause life antigen.
threatening infections if access to normal o It is often found in dairy products.
spherocytes is gained. They are facultative 4. Enterococcus group (formerly streptococci):
anaerobes except for peptostreptococci which grow at 10C, 45C and 37C
are obligate anaerobe. While other specie grows o Enterococcus faecalis
in the presence of oxygen, but are unable to use o Also part of the indigenous microbiota of
it for respiration like the aerotolerant anaerobes. the human intestine.
• Some specie are capnophilic and does require an SMITH AND BROWN CLASSIFICATION
increase consumption of carbon dioxide for 1. Alpha-haemolytic streptococci
growth and some, their growth is enhanced by o Partial/incomplete hemolysis of red cell
blood serum or glucose that’s incorporated in o Greenish or incomplete hemolysis
your agar plate. o Ex. Streptococcus pneumoniae
o Usually known as green streptococci
2. Beta-haemolytic streptococci
o Complete lysis of red cell
o Clear zone of hemolysis
o Ex. Streptococcus pyogenes and
Streptococcus agalactiae
3. Gamma-haemolytic/Non-haemolytic
streptococci
o Do not exhibit the lysis of red cells
o Ex. Streptococcus bovis
LANCEFIELD CLASSIFICATION
• BAP: grayish, pinpoint and translucent to slightly • Based on extraction of C carbohydrate for the
opaque while some mucoid. streptococcal cell wall.
• Biochemical test: • It is devised by Rebecca Lancefield, a
o Catalase negative microbiologist who developed the classification
o Oxidase negative method. They found out that the C carbohydrate
o No gas production could be extracted from the streptococcus cell
o Non-motile wall by placing the organisms in a dilute acid and
o Ferments carbohydrates by application of heating the suspension for
• Notorious pathogen: 10mins.
o Streptococcus pyogenes HEMOLYTIC LANCEFIELD
SPECIES
o Streptococcus pneumoniae REACTION GROUP
A S. pyogenes
B S. agalactiae
S. Dysgalactiae
Beta Hemolytic
subsp.
C
Equisimilis S.
equi
a or y
CLASSIFICATION OF STREPTOCOCCI D S. Bovis group
Hemolytic
ACADEMIC/BERGEY’S CLASSIFICATION a, B or y
D Enterococci
1. Pyogenic group: grow only at 37C Hemolytic
o Ex. Streptococcus pyogenes and group None but some
C and G streptococci a, B or y Viridans
may have A, C,
2. Viridans group: grow at both 45C and 37C Hemolytic streptococci
F, G or N
o Ex. Streptococcus mutans, a Hemolytic None S. pneumoniae
Streptococcus mitis and Streptococcus GROUP A STREPTOCOCCI
salivarius • Streptococcus pyogenes
o Not part of Lancefield classification. They • Pathogenic to humans (not considered as part of
may be alpha hemolytic or nonhemolytic. indigenous microbiota)
o It is also a indigenous microbiota of the
• Acquired through contaminated droplets
upper respiratory tract. Some may have
• Resistant to drying and can recovered from
A C G or N Lancefield antigen.
swabs several hours after collection
3. Lactic group: will grow at 10C and 37C
o Ex. Streptococcus lactis (cause normal • Species: Streptococcus pyogenes “fever
coagulation or souring of milk) producing” and flesh-eating bacterium. Usually
affects deeper tissue and organs.

TUAZON A. 11
• Principal virulence factor: M protein (triggers the • Streptokinase
production of antibody that sometimes may lead o Fibrin clot lysis
to complication, attached to the peptidoglycan o Binds to plasminogen and activates the
layer, anti-phagocytic, and usually for adherence production of plasmin
to the mucosal cell) o Activates the host’s blood factor that
• Culture BAP: colonies are small, translucent, dissolve fibrin clot.
smooth and exhibit well-defined B-hemolysis. o Responsible for the activation of
• Other virulence factors: plasminogen to produce plasmin and
o Protein F (mediates epithelial cell plasmin is responsible for degrading
attachment) fibrin clot. To remove the formation of
o Lipoteichoic acid (adherence to the thrombosis in the circulation.
respiratory epithelium) • Hyaluronidase
o Hyaluronic acid capsule (weakly o Solubilizes the ground substance
immunogenic that prevents phagocytosis hyaluronic acid.
and make its antigen) RELATED INFECTIONS AND DISEASE
o Hemolysins, toxins and enzymes. • b (Strep throat)
• Group A Streptococci contains super antigens o Spread by air droplets or close contact.
that are associated with necrotizing fasciitis or o The highly virulence strain of
toxic shock syndrome. Streptococcus pyogenes can cause
• The resistance to infection with Streptococcus outbreak of sore throat, scarlet fever. Its
pyogenes may be due to the type specific diagnoses rely on the culture of
antibodies to M protein. specimen through throat swab or direct
ENZYMES AND TOXINS PRODUCED BY antigen detection.
STREPTOCOCCUS PYOGENES • Scarlet Fever
• Hemolysin • Skin Infections
• Deoxyribonuclease (Dnase) • Rheumatic fever
• Streptokinase • Acute glomerulonephritis or Bright’s disease
• Hyaluronidase (Spreading-factor enzyme) • Streptococcal toxic shock syndrome
• Pyogenic toxins – Serotypes A, B, C and F (super SCARLET FEVER
antigens)
• Punctate exanthema overlying a diffuse erythema
o Formerly known as erythrogenic toxins
appears initially on the neck and upper chest, one
o Exotoxin B (cysteine protease): degrades
to two days following a strep throat.
protein and mediates the rash that are
• Can be spread through inhalation of respiratory
caused by Scarlet Fever
droplets.
HEMOLYSIN • Caused by: Pyrogenic exotoxins
• Streptolysin O • Some cardinal signs for this illness include
o Oxygen-labile hemolysin diffused red rashes on the upper chest, trunk, and
o Responsible for subsurface hemolysis on extremities. Also, the appearance of strawberry
BAP (anaerobically) colored tongue
o Highly antigenic and induces an antibody • Test:
response o Dick’s Test (erythrogenic toxin test)
o Serological test to be used to detect the ▪ Positive: Erythema or Redness
recent infection with Streptococcus of the skin
pyogenes is Anti-streptolysin O (ASO) o Schultz-Charlton test (anti-erythrogenic
titer test. toxin test)
o Usually, it is best to stab the agar to ▪ Positive: Blanching phenomenon
create anaerobiosis and observe the or rash fade.
subsurface lysis. ▪ Inject 0.1 ml of antitoxin
• Streptolysin S subcutaneously
o Oxygen stable hemolysin and non- ▪ Rash fades after 6-8 hours
antigenic (possible delay 14 h)
o Responsible for surface hemolysis ▪ Differentiates from other similar
(incubated aerobically). tests.
OTHER IMPORTANT ENZYME
• DNase
o Lowers the viscosity of exudates giving
the pathogen more mobility
o Four types: A, B, C and D (antigenic
enzymes)

TUAZON A. 12
 SKIN INFECTIONS • Fever > 38°C because of the presence of Group
• Cellulitis A Streptococcus which initially started as throat
o Contagious infection of infection or pharyngitis.
subcutaneous skin tissue • Streptococcus pyogenes contains M proteins and
o Characterized by redness it forms antibodies against that M protein. Without
(erythema) and knowing that this M protein has a similar structure
accumulation of fluid like the different parts of the body. Such as joints
(edema) causing joint pain, heart causing O carditis, hand
causing nodules, erythema marginatum, and
• Erysipelas Sydenham chorea.
o Acute infection of the dermal layer of the • JONES: Joint pain, O carditis, Nodules,
skin with painful, swollen, reddish spots Erythema marginatum, Sydenham’s chorea.
ACUTE GLOMERULONEPHRITIS
• Inflammatory disease of the renal glomeruli.
• Results from deposition of antigen-antibody
complexes to the kidney.
• Complication of the pharyngitis.
GLOMERULONEPHRITIS
• Impetigo (Nonbullous impetigo) • There is a formation of antigen-antibody complex
o Necrotizing fasciitis which is also known from recent streptococcus infection.
as “galloping gangrene” or “flesh-eating
• Antigen-antibody complexes are deposited in the
bacteria syndrome”
glomerulus that is present in the kidney.
o Begins as papule then develops into
• It can result to increase production of the
pustule (nana) and then will breaks down
epithelial cell lining, therefore there will be a
to formation of thick adherent crusty
scarring and loss of glomerular filtration
lesion which is also called golden or
membrane leading to decrease glomerular
honey colored crust.
filtration rate.
• Causing: Inflammation, DECREASE of
glomerular filtration rate.
• Symptoms/Manifestation: headache, increased
BP, facial/periorbital edema, lethargic, low-grade
fever, weight gain (edema), Urine (proteinuria,
hematuria, oliguria, and dysuria).

o Necrotizing fasciitis or flesh-eating STREPTOCOCCAL TOXIC SHOCK SYNDROME

bacteria syndrome: (STREP TSS)
• Condition in which the whole organ system shut
down and lead to death
• Initial strep infections leading to this condition
include pharyngitis, cellulitis and wound
infections
• Spec A or Pyrogenic exotoxin plays a major role
in the pathogenesis of this disease.
• Similar or a milder form of the Streptococcus
aureus toxic shock syndrome due to the presence
of pyrogenic exotoxin or the pyrogenic toxin A.
• It is recognizable with a pyogenic inflammation
wherein blood culture is always positive.
RHEUMATIC FEVER
• Staphylococcal toxic shock syndrome has a
• Characterized by fever, inflammation of the heart,
negative blood culture.
joints and blood vessels
• It is a complication of pharyngitis. LABORATORY TEST FOR GROUP A
STREPTOCOCCUS
• Bacitracin disk test/Taxo A (0.04 units)
o Differentiate S. pyogenes from other
Beta haemolytic groups
o Group C and G are also susceptible to
bacitracin
o (+) positive susceptibility or zone of
inhibition

TUAZON A. 13
 o Helpful in identifying group A streptococci o It is recommended that all pregnant
in the throat culture. women be screened for GBS at 35 to 37
• Sulfamethoxazole and trimethoprim (SXT) weeks’ gestation.
Test o Postpartum infections, such as
o Group B streptococci are also resistant to endometritis, which can lead to pelvic
SXT abscesses and septic shock.
o Group C streptococci are sensitive o Abortion fever.
o (+) positive Exhibits Resistance o Endometritis and wound infection.
• Pyrrolidonyl arylamidase (PYR) Test o Tricuspid valve endocarditis.
o More specific than bacitracin • Treatment:
o (+) positive Cherry Red Color o The drug of choice for treating GBS
o Detects the presence of PYR enzyme in infection in penicillin.
streptococci because the Streptococcus o S. agalactiae has remained sensitive to
pyogenes is the only beta-hemolytic penicillin and cephalosporins; however,
streptococcus specie that is positive in increasing minimal inhibitory
PYR test. Enterococci also result to concentrations (MICs) have been
positive. reported.
GROUP B STREPTOCOCCI • Laboratory diagnosis:
STREPTOCOCCUS AGALACTIAE o The GBS grow on SBA as grayish white
• Group b streptococci mucoid colonies surrounded by a small
• B-hemolytic and produce zones of hemolysis that zone of B-hemolysis.
are only slightly larger than the colonies (1-2 mm o The most useful tests are positive
in diameter) Hippurate hydrolysis and CAMP tests.
• Hydrolyze sodium Hippurate and give a positive o The definitive identification can be made
response in the so-called CAMP test. (Christie, by extracting the group antigen and
Atkins, Munch-Peterson) demonstrating agglutination with specific
anti-group B antisera.
• Part of the normal vaginal flora in 5-25% of
o Reagent used in CAMP test is the beta-
women.
lysin strip. Positive result in CAMP test is
• Part of the indigenous microbiota of the female
an arrow head or a bow tie shape beta-
genital tract and lower gastrointestinal tract.
hemolysis near the S. aureus growth.
These are nosocomial transmitted by unwashed
o In Hippurate hydrolysis test, the reagent
hands of a mother or health care personnel to a
used is, sodium Hippurate and ninhydrin.
newborn or infant. It causes the infection of the
Positive result is purple color after adding
fetus during passage through the colonized birth
ninhydrin reagent that indicates
canal and premature rupture of the mother’s
Hippurate hydrolysis. Negative = no
placenta or the membrane.
color.
• The specie included under GBS is the S.
o Serotyping can be useful for
agalactiae.
coagglutination or latex agglutination.
• Virulence factors:
GROUP C AND G STREPTOCOCCI
o Capsule
• Streptococcus dysgalactiae subsp. equisimilis
▪ Prevents phagocytes but is
ineffective after opsonization. • Streptococcus equi subsp. zooepidemicus
o Sialic acid GROUP C AND G STREPTOCOCCI
▪ A critical virulence determinant. • Organisms recovered from the upper respiratory
▪ Loss of capsular sialic acid was tract. Usually in a female reproductive organ and
associated with loss of virulence. the skin.
o Hemolysin, CAMP factor, • They may also possess the M protein similar to S.
neuraminidase, DNAae, pyogenes.
hyaluronidase, and protease. • Group C streptococci are animal pathogen and
• Infections: serves as the main source of streptokinase.
o Group B streptococcal infection during • Nasopharynx and may cause pharyngitis,
the first month of life may present as sinusitis, bacteremia, and endocarditis.
fulminant sepsis, meningitis, or • S. equi subsp. zooepidemicus is primarily an
respiratory distress syndrome. animal pathogen rarely isolated from humans.
o Intravenous ampicillin given to mothers, • Group G streptococci occur in patients with
who carry Group B streptococci and are underlying malignancies.
in labor, prevents colonization of their • Group C have been associated with acute
infants and Group B streptococcal pharyngitis.
disease.

TUAZON A. 14
• group G streptococci have hemolysins and may are important for the healthy state of the mucous
have M proteins analogous to those of group A S. membranes.
pyogenes • Principal cause of endocarditis on abnormal heart
• Clinical infections by S. dysgalactiae subsp. valves.
Equisimilis: spectrum of infections resembles S. • Some viridans streptococci (eg, S mutans)
pyogenes and includes upper respiratory tract synthesize large polysaccharides such as
infections, skin infections, soft tissue infections. dextrans or levans from sucrose and contribute
• Lancefield groups C and G are subdivided into importantly to the genesis of dental caries.
large colony and small-colony forms • Most viridans streptococci infections are treated
o The large-colony-forming isolates with with penicillin, although some resistant strains
group C and G and sometimes A and L have been reported.
antigens are classified with the pyogenic • The current classification assigns
streptococci. streptococci species in the viridans group to
o The large-colony-forming B-hemolytic one of five groups:
isolates with group C and G antigens o S. mitis group (including S. mitis, S.
belong to the subspecies S. dysgalactiae pneumoniae, S. sanguis, S. oralis)
subsp. equisimilis. ▪ Most common cause of subacute
o The small-colony-forming B- bacterial endocarditis.
hemolytic isolates with group C and G o S. mutans group (including S. mutans
antigens belong to the S. anginosus and S. sobrinus)
group, which are included in the viridans ▪ Most commonly isolated specie
streptococci. of viridans streptococci.
GROUP N STREPTOCOCCI ▪ Frequently isolated in dental
• They are rarely found in human disease states caries.
but produce normal coagulation ("souring") of o S. salivarius group (including S.
milk. salivarius and S. vestibularis)
GROUPS E, F, G, H, AND K-U STREPTOCOCCI o S. bovis group (including S. equinus, S.
gallolyticus, S. infantarius, and S.
• These streptococci occur primarily in animals.
alactolyticus)
• S. canis, can cause skin infections of dogs but.
▪ S. bovis group and the
uncommonly infects humans; other species of
enterococci possess the group
group G streptococci infect humans
antigen.D in their cell wall.
VIRIDANS STREPTOCOCCI ▪ S. bovis is no longer a valid
• Streptococcus mitis group species name. DNA studies
• Streptococcus mutans group found that S. bovis and S.
• Streptococcus salivarius group equinus were the same species,
• Streptococcus bovis group and the earlier species name, S.
• Streptococcus anginosus group equinus, was adopted.
VIRIDANS STREPTOCOCCI o S. anginosus group (including S.
• Also known as Alphaprime streptococci. They anginosus, S constellatus, and S.
basically lack the Lancefield group antigens. intermedius)
• These are indigenous microbiota of the upper ▪ Lancefield group A, C, F, G, or N
respiratory tract, the female genital tract and the antigen
gastrointestinal tract. • Infections:
• They are usually alpha hemolytic but they may o S. anginosus group:
also be nonhemolytic and beta-hemolytic. ▪ normal oral and gastrointestinal
• S. mitis, S. mutans, S. salivarius, S sanguis, and microbiota
others a-hemolytic, but they may be nonhemolytic ▪ associated with abscess
and B-hemolytic. formation in the oropharynx,
• Optochin resistant colonies are not soluble in bile brain, and peritoneal cavity
(deoxycholate) ▪ associated with invasive
• Fastidious pyogenic infections with the
• Virulence factors of Viridans streptococci tendency to form abscesses.
includes the presence of capsule, cytolysin, o S. constellatus subsp. pharyngis:
extracellular dextran, and adhesins. ▪ Pharyngitis
• Related infections include subacute bacterial ▪ normally found in the oral cavity,
endocarditis (abnormal heart valves), gingivitis, gastrointestinal tract, and female
dental caries, meningitis, osteomyelitis, and genital tract
empyema. ▪ transient normal microbiota of
• Normal flora of the upper respiratory tract, female the skin
genital tract, and the gastrointestinal tract. They

TUAZON A. 15
 ▪ most common isolates
associated with bacterial
endocarditis in native valves
and, less frequently, in prosthetic
valve infections.
o S. bovis group
▪ bacteremia, septicemia, and
endocarditis
o S. gallolyticus subsp. gallolyticus
▪ in blood cultures has a high
correlation with gastrointestinal
carcinoma.
o S. mutans group:
▪ the most commonly isolated
among the viridans streptococci.
▪ primary contributor to dental
caries
▪ bacteremia
LABORATORY DIAGNOSIS FOR VIRIDANS STREPTOCOCCUS PNEUMONIAE
STREPTOCOCCI • Also known as diplococcus or pneumococcus
• Leucine aminopeptidase • S. pneumoniae is a member of the S. mitis group
o Substrate: Leucine-B-naphthylamide • An asymptomatic member or normal respiratory
o Reagent: tract. This includes the S. pneumoniae which is a
p-dimethylaminocinnamaldehyde member of the streptococcus mitis group. It is the
o End product: B-napthylamine causative agent of lobar pneumonia.
o (+) Red color • The cell wall of S. pneumoniae contains an
o (+) LAP: Viridans streptococci, antigen, referred to as C substance - A B-globulin
enterococci, S. pyogenes, S. agalactiae in human serum, called C-reactive protein (CRP),
and S. pneumoniae reacts with C substance to form a precipitate. The
o (-) LAP: Aerococcus and Leuconostoc amount of CRP increases during inflammation
• Voges-Proskauer (VP) Test and infection.
o Culture medium: VP Test (broth) • Their main principal virulence factor is the
o Reagents: a-naphthol and KOH capsular polysaccharide
(potassium hydroxide) • The capsule is antigenic and can be identified
o End product: Acetoin with appropriate antisera in the Neufield test. In
o End color: Deep pink or Red the presence of specific anticapsular serum, the
o (+) VP: S. anginosus, S. bovis and S. capsule swells (quellung reaction).
mutans
• This reaction not only allows for identification of
• B-D-Glucuronidase (BGUR) Test S. pneumoniae but also serves to serotype
o Enzyme that is found in isolates of large, specifically the isolate.
colony-forming, B-haemolytic groups C
• Virulence Factors
and G streptococci
o capsular polysaccharide
• Antimicrobial susceptibility test o hemolysin
o Group C streptococci: susceptible to o immunoglobulin
bacitracin and SXT (sulfamethoxazole o A protease, neuraminidase, and
and trimethoprim) hyaluronidase
o Group G streptococci: resistant or
• Clinical Infections
susceptible to bacitracin
o pneumonia, sinusitis, otitis media,
• Diagnostic test for S. bovis bacteremia, and meningitis.
o Growth in Bile esculin medium o recurrent otitis media
▪ Black color o pneumococcal pneumonia cases.
o 6.5% NaCl test
• Laboratory Diagnosis
▪ Negative
o brain-heart infusion agar, trypticase soy
o PYR Test
agar with 5% sheep RBCs, or chocolate
▪ Negative
agar are necessary for good growth
o Penicillin Test
o increased CO2 for growth.
▪ Susceptible
o large zone of a-hemolysis on SBA.
o colonies: dome-shaped appearance.

TUAZON A. 16
 o colonies: autolytic changes result in a LESSON 2.1: GRAM NEGATIVE COCCI (NEISSERIA
collapse of each colony's center, giving it & MORAXELLA)
the appearance of a coin with a raised rim
o optochin susceptibility and bile solubility. GRAM STAIN-GENERAL RULE
S. pneumoniae is susceptible to • All cocci are gram (+) except Neisseria,
optochin, whereas other a hemolytic Veilonella, Moraxella
species are resistant. • All bacilli are gram (-) except Mycobacteria,
o The bile solubility test determines the Corynebacteria, Clostridia, Bacillus,
lysis of S. pneumoniae in the presence of Lactobacillus, Listeria, Erysiphilothrix, Nocardia,
bile salts. It correlates with optochin Actinomyces
susceptibility, that is, S. pneumoniae
• All spiral organism are reported as gram (-)
isolates are optochin susceptible and bile
• Yeasts or fungal organism are gram (+)
soluble.
o Because most isolates are susceptible, • Gram positive • Micrococcus,
penicillin remains the drug of choice for cocci Staphylococcus,
treating pneumococcal infections. (aerobes) Streptococcus
o However, S. pneumoniae has become
increasingly resistant to penicillin. • Gram positive • Peptococcus,
LABORATORY DIAGNOSIS FOR cocci Peptostreptoccocus,
(anaerobes) Sarcina
STREPTOCOCCUS PNEUMONIAE
• Gram negative • Branhamella,
• Gram stain
cocci Neisseria
o Oval or lancet-shaped
(aerobes)
o Pairs or in short chain
• Culture
• Gram negative • Veilonella
o Culture media: CAP, BHI and TSA with
cocci
5% sheep’s blood
(anaerobes)
o Young colonies: “dome-shaped”,
glistening, wet and mucoids • Gram positive • Bacillus,
o Old colonies: “coin with raised rim” bacilli Corynebacterium,
▪ Dimple-shaped (aerobes) Erysipelothrix,
• Susceptibility Test Listeria,
o Optochin disk test/Taxo P Mycobacterium,
▪ Sensitive Nocardia
▪ Ethylhydrocupreine
hydrochloride • Gram positive • Actinomyces,
• Bile Solubility Test bacilli Clostridium,
o This test will evaluate the ability of the (anaerobes) Propionobacterium
streptococcus pneumoniae to lies in the • Gram negative • Acinetobacter,
presence of bile salt. bacilli Aeromonas,
• Neufeld-Quellung Reaction (aerobes) Alcaligenes,
o Capsular swelling test Bordetella, Brucella
o Capsule will swell Enterics,
Francisella,
• Francis Test
o Detect presence of antibody against Legionella,
Pasteurella.
pneumococci
Pseudomonas,
DIFFERENTIATION OF ALPHA AND NON- Vibrio
HEMOLYTIC STREPTOCOCCI
• Gram negative • Fusobacterium
VIRIDANS STREPTOCOCCUS
CATEGORY bacilli Bacteroides
STREPTOCOCCI PNEUMONIAE
(anaerobes)
Solubility in
bile
Insoluble Soluble • The family Neisseriaceae contains the genera
Fermentation Fermenter with Neisseria, Moraxella, Acinobacter, Kingella,
Not a fermenter Eikenella, Simonsiella and Alysiella.
of inulin acid production
Sensitivity to • Simonsiella muelleri
Not sensitive Sensitive
optochin
Pathogenicity
Non pathogenic Pathogenic
to mice
Quellung test Negative Positive

TUAZON A. 17
 • Alysiella filiformis • Catalase –
• Oxidase +
SPECIES
• Kinglella denitrificans
• Kinglella kingae
• Kingella kingae • Kinglella oralis
EIKENELLA
• Bacilli
• Catalase –
• Oxidase +
SPECIES
• Eikenella corrodens • Eikenella corrodens
SIMONSIELLA
• Bacilli
• Gliding motility
• Normal human oral biota
• Large, flat multicellular filaments
• Neisseria • Catalase –
• Oxidase +
ADDTL: Normal oral biota of dogs, wounds from dog bites
may cause opportunistic infections in human hosts.
NEISSERIA
• Are obligate aerobic; non-motile and non-
hemolytic.
NEISSERIACEAE • Microscopy: Gram negative diplococci with coffee
• Plump cocci, coccobacilli or rods or kidney bean shaped, except N. elongate & N.
• Gram negative weaver (rod shaped)
• Nonmotile • Capsule: Pili
• Strictly or preferentially aerobic • Culture: small, gray-white opaque, convex and
• Optimum temperature is at 32°-36°C glistering colonies growth is best observed or
enriched medium (fastidious) – media containing
NEISSERIA
blood, serum, cholesterol, oleic acid.
• Cocci
• Blochemical tests: Oxidase (+), Catalase (+)
• Adjacent sides flattened
except N. elongata
• Cell division – two plates
• Natural habitat: Mucous membrane of the
• Catalase + respiratory and urogenital tracts.
• Oxidase – • Major pathogens: N. gonorrohoeae, N.
SPECIES meningitidis
HUMAN ISOLATES: • Are capnophilic (2-8% Carbon dioxide)
• Pathogens • Microaerophilic
o Neisseria gonorrhoeae • Has an optimal growth in a moist temperature
o Neisseria meningitidis • Are carbohydrate fermenters - primarily glucose
o Neisseria weaver (bacillus) and maltose
• Nonpathogens • Are sensitive to drying and extremes of
o Neisseria cinerea temperature - direct inoculation of specimens is
o Neisseria elongata (bacillus) required for these bacteria (N. gonorrohoeae & N.
o Neisseria flavescens meningitidis) requires immediate incubation after
o Neisseria lactamica collection and plating.
o Neisseria mucosa NEISSERIA GONORRHOEAE
o Neisseria polysaccharea • Also known as gonococcus. It is not part of a
o Neisseria sicca human microbiota.
o Neisseria subflava (biovars, flava, • Transmitted through sexual contact, infected
perflava, subflava) mother to newborn (during birth)
KINGELLA • Leading cause of sexually transmitted diseases-
• Bacilli localized to the mucosal surfaces in the initial
• Pairs and chains exposure to the organism (endocervix,
• Cell division – one plane conjunctiva, pharyngeal) surface, anorectal area
or urethra.

TUAZON A. 18
• Microscopy: gram negative intracellular o A gonococcal eye infection during
diplococci vaginal delivery through an infected birth
• Culture (CAP): Small, tan, translucent and raised canal
after 24-48 hrs of incubation 7. Septicemia
• Principal Virulence Factor. Common pili GONORRHEA IN NEWBORNS
• Other virulence factors. Receptors for • Infected as they pass
transferring- capsule; surface molecule-IgA through birth canal
protease; Cellular membrane proteins (PorB); • Eye inflammation,
and lipopolysaccharide (LOS) endotoxin-Lipid blindness
Amolety and core LOS (peptidoglycan) • Prevented by prophylaxis
• Colonial types: Ti-T5: T1 and T2 (virulent), T3 to after birth/ credes's
T5 (avirulent) prophylaxis
• In the Chocolate Agar Plate, N. gonorrhoeae is • Gonococcal ophthalmia neonatorum
characterized as molten, translucent and raised
after 24-48 hours of incubation.

SPECIMEN COLLECTION AND HANDLING

• Specimens: pus and
secretions from urethra,
CLINICAL INFECTIONS cervix, prostate, rectal
1. Gonorrhea mucosa, throat and joint
o Came from the Greek word “gonos” fluid
which means seed, and “rhoia” which • Dacron or Rayon swabs-for specimen collection
means flux. • JEMBEC, Amles', Bio bag, Gono-Pak; Transgrow
o "Flux or Flow of seed" • Cottton swabs are not used in here because of
o An acute pyogenic infecton of nonciliated the presence of toxic fatty acids in a cotton fiber.
columnar and transitional epithelium with • N. gonorrhoeae will not be recovered in a routine
short incubation period of 2 to 7 days, blood culture because of the presence of sodium
patients may be asymptomatic. polyanethol sulfonate or the SPS.
o The common site of infection includes the NOTES TO REMEMBER
endocervix, conjunctiva, pharyngeal • Swabs should be placed in a transport system
surface, anorectal area or urethra. (Amies medium with charcoal) and plated within
o Symptoms includes purulent discharge, 6 hours.
lower abdominal pain for men and • Cotton swabs should be avoided due to the
vaginal bleeding for women. presence of toxic fatty acid...
2. Purulent urethritis (men) and cervicitis
• Blood borne dissemination of N. gonorrhoeae
(female)
occurs in < 1% of all infections resulting in
o Pelvic inflammatory disease
purulent arthritis and rarely septicemia
o Untreated gonococcal cervicitis may
• If blood is first collected in vacutainer tubes, the
cause sterility and perihepatitis (Fitz-
blood must be transferred to the broth culture
Hugh Curtis Syndrome)
system within one hour of collection
3. Pharyngitis
o The chief complaint in symptomatic • Body fluids (joint fluid or CSF) should be kept at
oropharyngeal infections Room temp or placed at 37°C before plating (cold
4. Anorectal Infections sensitive organism)
o Rectal pain and bloody stool LABORATORY DIAGNOSIS
5. Purulent arthritis GRAM STAIN
o This happens when the N. gonorrhoeae • The appearance of intracellular gram-negative
penetrates the circulation. diplococci, kidney or coffee bean shaped is
o It may inhabit the joints. diagnostic.
6. Conjunctivitis (Ophthalmia neonatorum) • Avirulent forms are seen as extracellular gram-
negative diplococci.

TUAZON A. 19
 • The direct gram stain of body fluids is best • Amies, it contains
accomplished using a cytocentrifuge (Can charcoal it serves
concentrate small numbers of organisms 100 as transport
fold). media for
CULTURE (FOR CONFIRMATION) Neisseria
• Culture media: BAP and CAP - for sterile gonorrhoeae
specimens. • Amies medium
• Direct Inoculation at the bedside is optimal should be plate
• Specimens on swab should be inoculated or within 6 hours
rolled onto media in a Z pattern and a cross
streaked with a loop.
• The best method for culture and transport is to
inoculate the agar immediately after specimen
collection and place the medium in an
atmosphere of increased carbon dioxide for
transport such as candle jar, JEMBEC system or
medium with carbon dioxide atmosphere.
CULTURE (FOR CONFIRMATION)
• Is a chocolate
agar with an
enrichment
supplement
(IsoVitaleX) and
Thayer Martin Agar
antibiotics • Growth on modified New York City (MYNC) agar:
(TMA)
• Antimicrobial Small, raised, translucent colonies
Agents:
vancomycin,
colistin and
nystatin
• All TMA
Modified Thayer Martin components +
Agar (MTM) Trimethoprim
lactate
• All MTM
components
except nystatin
which is
Martin Lewis Medium
substituted by
(ML)
anisomycin
• Vancomycin
conc. Is
increased
• A transparent
medium with • Thayer-Martin Medium Agar
lysed horse • Chocolate Medium Agar
blood, horse • Biochemical Test- To differentiate the specie of
plasma and yeast our Neisseria
dialysate
New York Medium
• ANTIMICROBIAL
AGENTS:
Vancomycin,
Colistin,
Trimethoprim and
Amphotericin B
• Same NYC
GC-LECT Medium antibiotics +
Lincomycin
• Transgrow, Cary
Transport Medium Blair, Amies and
JEMBEC

TUAZON A. 20
 • Candle jar – we can utilize as a transport medium SUPEROXOL TEST
• Reagent: 20-30% H₂O₂
• (+) result: Vigorous bubbling
• Catalase test aerobic 3% H₂O₂
• Catalase test anaerobic 15% H₂O₂
BETA-LACTAMASE TEST
PROCEDU
TEST REAGENT RESULT
RE
Cephalospo A loopful of (+) deep
rin disk or organisms pink/red
Cefinase is applied color within
Cephalosporin
nitrocefin directly onto 10 mins (60
ase Test
disk a pre- mins for
moistened staphyloco
• Note: Neisseria gonorrhoeae will not grow in your disk cci)
Blood Agar plate we utilize Chocolate agar plate Citrate (+) color
in a sterile specimen buffered change (red
Acidimetric penicillin to yellow)
Method and phenol
red as an
indicator
Phosphate (+)
buffered colorless
penicillin solution –
and starch- penicilloic
iodine acid
lodometric complex reduces
method iodine and
prevents it
from
combining
with starch
CHO UTILIZATION TEST (-) purple
• It is the standard method of identifying N. DNASE TEST
gonorrhoeae • (+) Moraxella catarrhalis, streptococcus,
• Medium: cystine trypticase agar - 1% Staphylococcus aureus, Serratia
carbohydrate + phenol red • Culture Media – DNase agar with methylene
• It detects acid production from glucose, maltose, green
lactose, fructose and sucrose • Positive result- clear halo to the colonies with 18
• (+) result yellow color within 24-72 hours at 35- to 24 hours of incubation
37°C • Moraxella catarrhalis it turns to positive because
• Almost all the results for this test are positive in it secretes DNase enzyme
Neisseria gonorrhoeae and Neisseria • (-) N. gonorrhoeae
meningitidis for the utilization for glucose plus IMMUNOLOGIC TESTS
maltose.
• It employs monoclonal antibodies for the
OXIDASE TEST identification
• Reagent: 1% tetramethyl-p-phenylenediamine • It do not require pure or viable organisms and can
dihydrochloride be performed from the primary plates.
• Other oxidase-positive bacteria: N.cinereal, N. Highly specific and
meningitidis, M. catarrhalis sensitive
• (+) result: Purple color within 10s/pink colonies
then black It uses monoclonal
• Can direct in our chocolate agar or we can use a antibodies that recognizes
slide FLUORESCENT epitopes on the principal
ANTIBODY TEST (FAT) outer membrane protein
of N. gonorrhoeae.

It also confirms the

morphologic appearance
of the bacteria.
Confirms the biochemical
COAGGULATION
identification

TUAZON A. 21
 • Usually inhabits the nasopharynx and
It uses monoclonal oropharynx, but once it reaches to the blood and
antibodies directed CSF it can cause infection your meningococci
against N. gonorrhoeae
attached to killed S.
aureus cells.

(+) result: agglutination

MOLECULAR ASSAYS- NUCLEIC ACID
AMPLIFICATION
• It detects gonococcal antigen or nucleic acid
directly in specimens
• It is suitable for screening many patients
simultaneously
• Specimens: Endocervical or urethral swabs; urine
• Advantage: The ability to test for Chlamydia
trachomatis at the same time; less sensitive to CLINICAL INFECTIONS
transport and storage conditions
CHEM ILUMINESCENT NUCLEIC ACID PROBE
MENINGOCOCCEMIA
• It refers to the presence of N. meningitidis in the
• It is used for culture confirmation or direct block and can occur as an acute or chronic form
detection in endocervical or urethral swab
• It occurs with or without meningitis
specimen
• Source of epidemics: oral secretions and
• It is a rapid, direct detection of gonococcal respiratory droplets (person-to-person spread)
ribosomal RNA (rRNA) in genital and conjunctival
• Signs and symptoms: frontal headache, stiff
specimens (1-2 hours)
neck, fever (epidemic meningitis in adults)
• Disadvantages: not approved for pharyngeal or
rectal specimens; cannot identify ß-lactamase-
NOTES TO REMEMBER
producing strain; not applicable for susceptibility • Petechial skin lesions may develop during
testing. bacteremic spread due to release of endotoxin
NEISSERIA MENINGITIDS after bacterial cell lysis.
• It is the causative agent of epidemic • The Lipooligosaccharide -endotoxin complex
meningococcal meningitis activates the clotting cascade, depositing fibrin in
/meningococcemia/cerebrospinal fever/spotted small vessels, producing hermorrhage in the
fever adrenals (Waterhouse-Friderichsen syndrome),
• It is the leading cause of fatal bacterial meningitis altering peripheral vascular resistance and
leading to shock and death.
• It may be found as a commensal inhabitant of the
upper respiratory tracts of the carriers - it colonies • Individuals with a deficiency in complement C5-
the mucous membranes of nasopharynx and C8 are at risk of meningococcemia
oropharynx. • Complement C5-C8 responsible for membrane
• Human carriage Upper respiratory tract (URT) of attack complex in order to undergo obstination to
the organism without symptoms is common. easily kill the microorganism
• It has the tendency to invade serous and joint • TREATMENT: Pen G. (Penicillin)
fluids - causing pleuritis and arthritis. NEISSERIA MENINGITIDIS SPECIMEN
• Both utilization of glucose and maltose fermenter; COLLECTION AND HANDLING
requires iron for growth • Specimens: CSF, blood, nasopharyngeal swabs,
• Beta lactamase test positive petechial skin lesions
• Microscopy: intracellular or extracellular gram- • Nasopharyngeal swabs should be plated
negative diplococci encapsulated strains can immediately to the JEMBEC system or submitted
have a halo around organism on swabs placed in charcoal transport media
• Culture: encapsulated strains are mucoid in (Amies medium)
appearance bluish-gray colonies (BAP); small, • N. meningitidis is sensitive to (sodium
tan, mucoid colonies (CAP) polystyrene sulfonate) SPS, so the content in
• Serogroups: A, B, C, Y and W-135 blood culture broths should not exceed 0.025%.
• Virulence factors: CAPSULE, Pili, Polysaccharide or else we can’t isolate Neisseria meningitidis
capsule, IgA1, cellular membrane proteins (Por A • If blood is first collected in vacutainer tubes, the
and Por B and LOS endotoxin. blood must be transferred to the broth culture
• Spread of bacteria from a nasopharyngea system within one hour of collection.
infection to blood and CSF • Body fluids (joint fluid or CSF) should be kept at
RT or placed at 37°C before culture (cold
sensitive organism)

TUAZON A. 22
 NEISSERIA MENINGITIDIS LABORATORY • Pneumonia
DIAGNOSIS • Empyema
GRAM STAIN • Bacteruria
• The direct gram stain of body fluids is best • Osteomyelitis
accomplished using a cytocentrifuge (can • Ocular infection
concentrate small numbers of organisms 100- • Dog bite infection
fold)
• The highest yield of positive CSF gram stains is
obtained when specimens are concentrated
CULTURE
• Culture media: BAP, CAP and Thayer Martin Aga
(TMA)
• CLSI recommend that either a broth microdilution
or an agar dilution MIC test with cation
supplement Mueller Hinton broth (with 2-5%
laked horse blood) or Mueller Hinton Agar (with
5% sheep blood) be used.
• Neisseria lactamica resembles N. meningitidis on
gonococcal selective media MORAXELLACEAE
• Can proceed to carbohydrates utilization test • Moraxella
because Neisseria lactamica can utilize glucose, • Acinobacter
maltose, and lactose • Psychrobacter
• Neisseria gonorrhoeae – Glucose • Commensal of the upper respiratory tract
• Neisseria meningitidis - Glucose and maltose MORAXELLA CATARRHALIS (BRANHAMELLA
OXIDASE TEST CATARRHALIS)
• Reagent: 1% tetramethyl-p-phenylenediamine • "gram-negative diplococcal”
dihydrochloride • The most commonly isolated member of the
• Other oxidase-positive bacteria: N.cinereal, N. genus Moraxella
meningitidis, M. catarrhalis • Normal flora of Upper respiratory tract
• (+) result: Purple color within 10s/pink colonies (oropharynx)
then black • It is only isolated from humans - opportunistic
BETA LACTAMASE pathogen
TEST/CEPHALOSPORINASE TEST • It causes upper respiratory tract infection in
otherwise healthy children and the elderly
• Reagent: Cephalosporin disk or cefinase
nitrocefin disk • It is the 3rd most common cause of Otitis media
and Sinusitis in children; Lower respiratory tract
• (+) Result: Deep pink/red color within 10 mins (60
infection: bronchopulmonary infection
mins for Staphylococci)
• Microscopy: Small Gram-negative cocci that
IMMUNOLOGIC TEST
tend to grow in pair end-to-end; with adjacent
• Identification can be confirmed by slide sides flattened.
agglutination using pooled polyvalent grouping • Culture: Smooth, opaque, gray to white coloies
antisera with "hockey puck” appearance - colonies remain
o Latex Agglutination direct identification of intact when pushed across the plate with a loop.
antigens (CSF, serum and urine) • Biochemical tests: Oxidase (+), Catalase (+);
o Counterimmunoelectrophoresis – it reduces NO3NO2; DNAse positive
accelerates the antibody and the antigen
LABORATORY DIAGNOSIS
through a buffer diffusion medium
GRAM STAIN
CHO UTILIZATION TEST
• Intracellular gram-negative diplococci
• (+) N. meningitidis
CULTURE - BAP AND CAP
GAMMA-GLUTAMYL AMINOPEPTIDASE TEST
• Growth on BAP at 22°C and nutrient agar at 35°C
• (+) N. meningitidis it produces this kind of
• It is inhibited on gonococcal media by colistin
enzyme.
• 48 hours colony may have elevated center,
INFECTIONS REPORTED TO BE CAUSED BY
thinner wavelike periphery - "wagon wheel"
NEISSERIA SPECIES: appearance
• Meningitis
CATALASTE AND OXIDASE TESTS (+).
• Endocarditis
• Prosthetic valve infection (cause by
Staphylococcus epidermidis) CHO UTILIZATION TEST
• Bacteremia • Does not utilized any sugar in CTA medium

TUAZON A. 23
Clinical Bacteriology (Lecture)
SECOND TERM

DASH
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science TWO
TWO
• A saccharolytic in CHO degradation test - lack oxidative metabolism
DNASE TEST (+)
• The only member of family Neisseriaceae that hydrolyze DNAse.
• Definitive Test for Moraxella
• Positive result: Exhibits a clear or colorless halo around the colonies after 18-24 hours of incubation.
BUTYRATE ESTERASE TEST
• (+) blue color - M. catarrhalis
• Specimen: eye or ear culture
• Substrate used: Bromochloroindolyl butyrate
TEST N. GONORRHOEAE M. MENINGITIDIS M. CATARRHALIS
Superoxol + + -
Growth on MTM, ML,
+ + v
NYC
Growth on NA at 25 C - - v
Growth on NA at 35 C - - +
Acid production
a. Glucose + + -
b. Maltose - + -
c. Sucrose - - -
d. Lactose - - -
Dnase - - +
Reduction of Nitrate - - +
Tributyrin
- - +
Hydrolysis
B-galactosidase - - -

LESSON 3: ENTEROBACTERIACEAE • Enterobacter

• Entero = gastrointestinal tract • Escherichia
• Usually lives in the colon • Ewingella
• Hafnia
• Klebsiella
• Kluyvera
• Obesumbacterium
• Xenorhabdus
• Pantoea
• Photorhabdus
• Proteus
• Providencia
• Rahnella
• Salmonella
• Serratia
• Shigella
• Tatumella
• Leclercia
FAMILY OF ENTEROBACTERIACEAE • Leminorella
GENUS: • Morganella
• Budvicia • Trabulsiella
• Buttiauxella • Xenorhabdus
• Cedecea • Yersinia
• Citrobacter • Yokenella
• Edwardsiella

TUAZON A. 24
 GENERAL CHARACTERISTICS BACTERIAL SPECIES AND INFECTIONS THEY
• Common term: Enterobacteria COMMONLY PRODUCE
• Facultatively anaerobic, non-sporeforming, Gram BACTERIAL SPECIES DISEASE
negative bacilli Bacteriuria, septicemia,
• All members are motile at 35C with peritrichous Escherichia coll
neonatal sepsis,
flagella, except: Klebsiella, Shigella and Yersinia. meningitis, diarrheal
• All are non-encapsulated except for Klebsiella syndrome
and Enterobacter (they are + capsule) Diarrhea, dysentery
Shigella spp.
• All members are glucose fermenter and reduce usually with blood
nitrate to nitrite Diarrhea, wound
• Most of them are present in the intestinal tract as Edwardsiella spp. infection, septicemia,
commensal microbiota except for Plesiomonas, meningitis, enteric fever
Salmonella, Shigella and Yersinia (they are Septicemia, enteric
Salmonella spp.
pathogenic) fever, diarrhea
• Some organisms Serratia and Yersinia may grow Opportunistic and
at low temp 1C to 5C. hospital-acquired
Citrobacter spp
infections (wound,
urinary)
Bacteriuria, pneumonia,
Klebsiella spp.
septicemia
Opportunistic and
hospital-acquired
Enterobacter spp. infection, wound
infections, septicemia,
bacteriuria
Opportunistic and
hospital-acquired
• Microscopy: Straight Gram-negative rods or Serratia spp. infection, wound
coccobacilli with rounded ends. infections, septicemia,
• Culture on BAP: Colonies appear large, smooth bacteriuria
and gray except for Klebsiella and Enterobacter Bacteriuria, wound
with mucoid (presence of capsule) colonies and Proteus spp.
infection, septicemia
are non-haemolytic except for some strain of Opportunistic and
Escherichia coli hospital-acquired
• Biochemical tests: Providencia spp. infection, wound
o Catalase Test: Positive infections, septicemia,
o Oxidase Test: Positive; except for bacteriuria
Plesiomonas shigelloides. Opportunistic and
ANTIGEN-DETERMINANTS FOR Morganella spp. hospital-acquired
IDENTIFICATION infections
• Somatic O Antigen: heat-stable; located in the Yersinia
cell wall; used for E. coli and Shigella serotyping. • Y. pestis • Plague (black
• Flagellar H Antigen: heat-labile; found in the • Y. death)
flagellum; used for Salmonella serotyping pseudotuberculosis • Mesenteric
• Capsular K Antigen: heat-labile polysaccharide; • Y. enterocolitica adenitis,
found as K1 antigen of E. coli and Vi antigen of S. diarrhea
enterica subsp. enterica serotype Typhi (S. typhi) • Mesenteric
NOTE TO REMEMBER adenitis,
• E. coli, K. pneumoniae and K. oxytoca: ESBL diarrhea
(Extended Spectrum Beta-lactamase producer)-
producing enterobacteria Wounds contaminated
Erwinia spp.
• E. coli, P. mirabilis and K. pneumoniae are with soil or vegetation
isolated from urinary tract and can cause Wounds contaminated
Pectobacterium spp.
bacteremia. with soil or vegetation
• Citrobacter, Enterobacter and Serratia are LABORATORY DIAGNOSIS
antibiotic resistant genera. • Gram stain
• Shigella, Salmonella, E. coli and Yersinia are • Culture
associated with diarrhea. • Biochemical Test
• Molecular Test
o Triple Sugar Iron Agar Test

TUAZON A. 25
 o Sulfide Indole Motility Test • Transport Media: Amies, Cary-Blair and Stuart
o Citrate utilization Test transport media
o Lysine Iron Agar Test • Colony morphology: BAP and CAP – Large,
o Decarboxylase Test gray and smooth
o Ortho-nitrophenyl-B-Galactopyranoside • Fecal pathogen: Generally non-lactose
o Methyl red/Voges-Proskauer (MRVP) fermenter
Test • Salmonella: produce colonies with black centers
o Nitrate reduction Test in media with H2S as indicator: HEA, BSA and
o Phenylalanine deaminase XLD.
o Urea hydrolysis Test • Optimal temperature: 35C to 37C; except:
o Gelatin liquefaction Test o Serratia and Yersinia (1C to 5C)
o Malonate Test o E. coli which can also grow at 45C to 50C
GRAM STAIN
• Straight Gram-negative rods or coccobacilli with MACCONKEY-SORBITOL AGAR (MAC-
rounded ends. SOR/SMAC)
• Direct smears of stool specimen are not routinely • Used to differentiate E. coli o157:H7 (sorbitol
performed negative) from other strains of E. coli (sorbitol
• Nice to know: positive)
o Wayson stain: used to observe the • pH indicator: Neutral Red
bipolar bodies of Yersinia pestis • Result: E. coli o157:H7 exhibits clear or colorless
(identification of Yersinia pestis) colonies while other strains are PINK (o =
• VIAS – Crystal violet, Iodine, Acetone, Safranin somatic, H = flagellar)
(retained gram-negative rods)
• Right picture is the Wayson stain of Yersinia
pestis (safety pin appearance)

HEKTOEN ENTERIC AGAR (HEA)

LACTOSE FERMENTATION • Content: Bile salt, bromthymol
RAPID blue dye, salicin, lactose and
LATE LACTOSE NON-LACTOSE
LACTOSE sucrose
FERMENTER FERMENTER
FERMENTER • Bile salt and dye: inhibit the gram-
Enterobacter Citrobacter freundii Citrobacter koseri negative bacilli in GIT and
Escherichia coli Serratia Proteus
promote the isolation of
Klebsiella Hafnia Providencia
Shigella sonnei Morganella Salmonella and Shigella.
Salmonella arizoniae Shigella • pH indicator: Bromthymol blue
Yersinia Salmonella • H2S indicator: Ferric ammonium citrate
enterocolitica Yersinia • Original color is blue. Change of color = growth.
Edwardsiella tarda
• H2S = black color (Salmonella)
CULTURE • E. coli – orange-yellow color with bile precipitates
• Culture Media: BAP, CAP, MAC, HEA, XLD, around the colonies.
CIN, SSA, EMB, Bismuth sulphite Agar (BSA), • Salmonella – green colonies with black colored
Selenite F and GN Broth. center.
o BAP – Blood Agar Plate HEA RESULTS
o CAP – Chocolate Agar Plate • Lactose Fermenter: Yellow color
o MAC – MacConkey Agar o Except: Citrobacter freundii (with black
o HEA – Hektoen Enteric Agar (heavily center)
contaminated)
o XLD - Xylose Lysine Deoxycholate Agar
(Salmonella & Shigella)
o CIN – Cefsulodin Irgasan Novobiocin
Agar (Yersinia)
o SSA – Salmonella and Shigella Agar
o EMB – Eosin Methylene Blue Agar (E.
coli)

TUAZON A. 26
 • Non-Lactose Fermenter: Blue-green color
o Except: Proteus and Salmonella (green
with black center)

SALMONELLA-SHIGELLA AGAR (SSA)

• It differentiates Salmonella and Shigella from
• Non-Enteric pathogens: Orange to pinkish- other enteric bacteria.
orange • Bile salts and Brilliant green dye: Inhibits gram
positive bacteria and some lactose fermenter that
are found in the stool specimens.
• Carbohydrates: Lactose
• pH indicator: Neutral Red
• H2S indicator: Sodium thiosulfate and ferric
ammonium citrate
Salmonella Pink or colorless with black center
Shigella Pink or colorless without black center
XYLOSE-LYSINE DESOXYCHOLATE (XLD)
AGAR
• Useful in isolation of Salmonella and Shigella
species from heavily contaminated specimen
such as stool.
• Differentiate Shigella to Salmonella
• Contain lactose, sucrose, xylose, lysine and
sodium desoxycholate
• pH indicator: Phenol Red
• H2S indicator: Sodium thiosulfate and ferric
ammonium sulphate. SEROTYPING (AGGLUTINATION TEST)
Shigella Pink to red colonies • Identifies strains (serovars or serotypes) of
Salmonella Pink to red with black centers colonies microorganisms that differ in their antigenic
Other Yellow to red colonies composition.
• Commonly tested organisms: Salmonella,
Shigella and E. coli O157:H7
• Medium: BAP with 5% sheep’s blood.
• Salmonella and Shigella serotyping
o Salmonella serotyping: Heat-stable
somatic O antigen and Heat-labile
flagellar H antigen
o Shigella serotyping: based on the
somatic O antigen
• E. coli serotyping
EOSIN-METHYLENE BLUE (EMB) OR LEVINE’S o Sorbitol-negative E. coli can be
MEDIUM serotyped to identify whether somatic O
• It contains eosin Y, methylene blue, lactose and antigen and flagellar H antigen
sucrose. • E. coli O55, O111, and O127: infantile diarrhea
• pH indicator: Eosin and methylene blue • Result: (+) Agglutination
• Result: • Salmonella serotype Typhi: Produces heat-
o Lactose fermenter – Colonies of E. coli labile capsular polysaccharide Vi antigen and
exhibit a greenish metallic sheen while carries the D serogroup
those of the other LF’s exhibit purple BIOCHEMICAL TEST
color.
o Non-lactose fermenter – colonies are FERMENTATION OF SUGAR – TRIPLE SUGAR
colorless IRON AGAR
o Other – Yellow to red colonies • Fermentation of Sugar is usually detected by acid
production
• Sugars – glucose, lactose, sucrose
• Original color of TSI is orange or red

TUAZON A. 27
 • Gram negative rod utilizes glucose and lactose or • Result: Alkaline slant/Alkaline butt (K/K)
sucrose or not. • Not Enterobacteriaceae
• Identify bacteria producing gas and H2S NO LACTOSE AND SUCROSE FERMENTATION
• Two necessary enzymes for an enterobacterium (GLUCOSE FERMENTER)
to take up lactose: • Result: Alkaline slant/Acid butt (K/A)
o B-galactoside permease: facilitates the
• Glucose concentration depleted
entry of the lactose molecule.
• After 18-24hrs of incubation, organism will utilize
o B-galactosidase: hydrolyzes lactose
PEPTONE in the slant. Lead to alkaline products
into glucose and galactose
(RED)
LACTOSE, SUCROSE AND GLUCOSE
FERMENTATION
• Result: Acid slant/Acid butt
• Enterobacteriaceae attack glucose first and then
disaccharide
HYDROGEN SULFIDE (H2S) PRODUCTION
• H2S indicator: Sodium thiosulfate and ferrous
sulphate
• Result: (+) Formation of black ppt.
GAS PRODUCTION
• Result: Formation of bubble (CO2 and H2),
• If you are able to ferment the carbohydrate splitting of media in butt or complete
glucose, lactose and sucrose, acid will be displacement at the bottom.
produced.
• If you produce acid, color yellow will be emitted.
• Butt portion – glucose, Slant portion – sucrose
and lactose
• Glucose fermenter – produce acid – emit yellow
color
• A, B – glucose fermenter
• C & D – non-glucose fermenter
• A – lactose fermenter
• B, C, D – non-lactose fermenter • H2S Producer:
• Gas producer– presence of bubbles, breakages o Proteus mirabilis
or splitting of agar o Proteus vulgaris
• H2S producer – presence of black precipitate o Citrobacter freundii
CHARACTERISTICS OF TSIA: o Salmonella serotype Typhi
• Red color at pH 7.4 o Edwardsiella tarda
• Ratio of sugars: 10:10:1 (lactose, sucrose and • Gas Producer:
glucose) o Escherichia coli
• pH indicator: Phenol red o Enterobacter aerogenes
• H2S indicator: Ferrous sulphate and Sodium o Enterobacter cloacae
thiosulfate o Klebsiella pneumonia
• Should not read beyond 24 hours of incubation. o Klebsiella oxytoca
• True enteric pathogens only ferment glucose. o Serratia marcescens
INTERPRETATION OF TSIA: o Proteus mirabilis
o Morganella morganii
• Bacterial specie that are incapable of fermenting o Citrobacter freundii
glucose cannot utilize lactose o Salmonella serotype Typhi
• Lactose fermenter possess both B-galactoside o Edwardsiella tarda
permease and -galactosidase • Bold font = both gas and H2S producer
• Non-lactose fermenters (NLF) do not possess -
galactoside permease and B-galactosidase SULFIDE INDOLE MOTILITY (SIM) TEST
• Late Lactose fermenter: only B-galactosidase • (+) Sulfide: Black color formation in the butt
• Glucose and lactose fermenters are mostly • (+) Indole: Pink to “wine-colored” ring formation
opportunistic enteric. after the addition of Kovac’s reagent
• (+) Motility: Movement away from stab line that
TSIA REACTION: produces a hazy appearance.
NO FERMENTATION OF SUGARS:

TUAZON A. 28
• Note: Kovac’s reagent should be added into the
SIM medium after 18 to 24 hours of incubation.
• Semi solid media which is poked in the middle to
know if it is motile or non-motile.

RELATED
LIA REACTION INTERPRETATION
BACTERIA
Alkaline slant K (-) Lysine deamination Salmonella
Alkaline butt K (+) Lysine decarboxylation
Alkaline slant K (-) Lysine deamination Shigella
CITRATE UTILIZATION TEST Acid butt A (-) Lysine decarboxylation Citrobacter
• Determine the ability of an organism to utilize (+) Lysine deamination Proteus
Red slant R
o sodium citrate: carbon source Acid butt A
(-) Lysine decarboxylation Providencia
o Inorganic ammonium salts: nitrogen Morganella
source • Decarboxylation (denominator)
• (+) Result: Blue colored citrate agar slant o + purple
• pH indicator: Bromthymol blue o - yellow
• Note: If citrate is used as carbon source • Deamination (numerator)
o Green medium → Blue medium o + red
(Alkaline) o - purple
o Negative = green DECARBOXYLASE TEST (MOELLER’S
o Positive = blue METHOD)
• Citrate is the only slant agar. • Measure enzymatic ability of an organism to
• Citrate Positive: decarboxylate (or hydrolyze) an amino acid to
o Enterobacter aerogenes form an amine (putrescine or cadaverine).
o Enterobacter cloacae • Moeller decarboxylase media: Glucose, arginine,
o Klebsiella pneumonia lysine and ornithine
o Klebsiella oxytoca • pH indicator: Bromcresol purple or phenol red
o Serratia marcescens
• Promotes anaerobiosis: Mineral oil
o Proteus mirabilis (v)
• Incubation condition: Four (4) days
o Providencia rettgeri
o Providencia stuartii • Arginine = citrulline
o Citrobacter freundii • Ornithine = putrescine
o Citrobacter koseri Positive Result Alkaline product Enterobacter sp.
o Hafnia alvei (Purple or Red)
Negative Result Glucose Klebsiella sp.
LYSINE IRON AGAR (LIA) TEST fermenter
• Usually used in Proteus, Providencia and (Yellow color)
Morganella
• Interpretation:
• Organisms secretes lysine decarboxylase
(detach carboxyl groups) → cadaverine (purple
color).
• If not produce decarboxylase → butt remains
acidic (Yellow color)
• Deamination of Lysine → Form burgundy red
color on slant (proteus, providencia and
morganella)
• Deamination (removal of amine group) does not
occur → Slant remain purple
• Glucose fermenter → butt becomes acidic
(yellow)

TUAZON A. 29
 • Example:
o MR/VP: Escherichia coli (+/-)
o MR/VP: Klebsiella – Enterobacter –
Serratia – Hafnia Group (-/+)
NITRATE REDUCTION TEST
• Lysine decarboxylase • All Enterobacteriaceae can reduce nitrate to
o Lysine → cadaverine nitrite
• Arginine decarboxylase • Detects nitrate reductase production of
o Arginine → citrulline enterobacteria.
• Reagents:
ORTHO-NITROPHENYL-B- o Sulfanilic acid
GALACTOPYRANOSIDE (ONPG) o a-naphthylamine
• Identifies slow or late lactose fermenters • (+) Result: Red, water-soluble azo dye
• B-galactosidase acts on the ONPG: cleaves it • Zinc dust: confirm negative reaction
into galactose and ortho-nitrophenol
• Positive result: yellow color within 20 minutes
• If NLF: compound remain colorless

METHYL RED/VOGES-PROSKAUER TEST

• Determine the ability of an organism to produce
and maintain stable acid end products (pyruvic
acid) from glucose fermentation
• Culture media in MR test: MRVP broth/ Peptone
glucose broth
• MR reagent: Methyl Red (5-6 drops)
o Positive = red
o Negative = yellow
• VP reagents: 40% KOH and 1% a-naphthol
o Barritts test or method
o 6 drops of a-naphthol, 2 drops KOH
o Positive = red
o Negative = yellow
• Positive on MR = negative on VP (vice versa)

PHENYLALANINE DEAMINASE (PAD) TEST

• Used for differentiation of Proteus, Morganella
and Providencia, only PAD-positive genera.
• Deaminate phenylalanine to phenyl pyruvic acid
• Confirmatory reagent: 10% ferric chloride
• (+) Result: Green colored slant

TUAZON A. 30
 MALONATE TEST
• Determines if capable of utilizing sodium
malonate as it’s sole carbon source.
• (+) Result: Exhibits a blue color at pH 7.6
• Enterobacter and Salmonella are positive
• Same reaction as citrate

UREA HYDROLYSIS TEST (CHRISTENSEN’S

METHOD)
• Ability of an organism to produce the urea
hydrolyzing enzyme urease.
• Reagent: Urea disk dissolved in 1-mL distilled
water.
• End product: Ammonium carbonate (alkaline
product)
Positive Slant (orange → Magenta)
Negative Yellow MOLECULAR TESTING
Rapid urase Proteus and Morganella • Enhance recovery and identification of enteric
bacteria.
• Reclassifies genus
o Plesiomonas as enteric GI pathogens.
• Methods:
o 16S ribosomal RNA sequencing
o DNA-DNA hybridization

GELATIN LIQUE FACTION TEST

• Determine the production in the bacteria of
gelatinase (protease) which hydrolyze and
liquefies the gelatin.
• Culture media: Nutrient gelatine tube agar
• (+) Result: Liquefied gel.
Positive Serratia and Proteus
Negative Escherichia coli

TUAZON A. 31
Clinical Bacteriology (Lecture)
SECOND TERM

DASH
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science TWO
TWO

TUAZON A. 32
 • IMVic Rxn: + + - - (positive, positive, negative,
ENTEROBACTERIACEAE PART 2 negative)
• 3 rapid lactose fermenters o Rxn = reaction
o Escherichia, Enterobacter, Klebsiella o Indole, Methyl red, Voges Proskauer,
(EEK) Citrate
ESCHERICHIA COLI • TSIA: A/A (acidic slant/acidic butt)
• Most significant species in the genus Escherichia o (+) gas; (-) H2S.
• Inhabit the female genital tract, microbiota of the ESCHERICHIA HERMANII
large intestine. • Formerly called E. coli atypical or enteric group II.
• Indicator of fecal contamination in water • Isolated from CSF, wounds and blood.
purification. • Colonies: Yellow pigment
• Leading cause of nosocomial urinary tract
infection.
• Has both the sex pili and adhesive fimbriae
• Normal flora of colon and intestine.

• Culture:
o MAC: flat and dry and exhibit a pink color
(LF); some strains may be NLF.
o BAP: most strains are non-hemolytic;
some are beta-hemolytic.
o EMB: exhibit greenish metallic sheen.
• Virulence factor
o Endotoxin, common pili,
K1antigenandintimin 3
• Antigenic determinants: O, H and K antigen

TUAZON A. 33
 Virulence DIFFERENTIAL TEST FOR COMMLY ISOLATED
E. coli Strains Infection Characteristics
Factors
Enteropathogenic Infantile (+) H antigen KLEBSIELLA SPECIES
E. coli (EPEC) diarrhea and intimin K.
(stool BIOCHEMICAL PNEUMONIAE
without K. OXYTOCA
TEST SUBSP.
blood)
Enterotoxigenic E. Traveler’s Heat- Persons with PNEUMONIAE
coli (ETEC) diarrhea or stable (ST) achlorhydria are Indole - +
Montezuma’ and heat- at risk Methyl red -/+ +
s revenge labile (LT)
enterotoxi
Urease + +
n Lysine
+ +
Eneteroinvasive Dysentery- Invasin Atrichous; LF or decarboxylase
E. coli (EIEC) like or NLF Gelatin
Shigella-like (+) large plasmid - -
infection. (+) Sereny test
liquefaction
Watery Growth in
+ +
diarrhea KCN
(with WBC) Alginate
Enterohemorrhagi Hemorrhagi VerotoxinI (+) intimin + -
c E. coli (EHEC) c colitis and II or (-) MUG utilization
• EHEC Hemolytic Shiga –like Associated with
serotyp uremic toxin Thrombotic
e syndrome thrombocytopeni
O157:H Bloody c purpura
7 diarrhea
(without
WBC)
Enteroadherent E. Watery Fimbriae EAEC has a
coli (EAEC) and diarrhea “stacked-brick”
Diffusely (EAEC) appearance
Adherent E. coli UTI (EAEC
(DAEC) and DAEC)
Uropathogenic E. UTI Common Type I fimbriae
coli (UPEC) pili, adheres strongly
aerobactin to the urinary
s and tract
cytolysins
KLEBSIELLA
• Usually found in the GIT of humans and animals.
• Specie: K. pneumonia subsp. pneumoniae, K.
oxytoca, K. pneumoniae subsp. ozaenae, K.
pneumoniasubsp. rhinoscleromatis and K.
ornithinolytica.
• Culture: MAC – colonies exhibit a pink color (LF)
(+ mucoid because capsule is present)
KLEBSIELLA PNEUMONIAE
• Most isolated sp. of Klebsiella
• Causative agent of community acquired
pneumonia; cough up a “currant jelly-like”
sputum.
• Frequent cause of lower respiratory tract ENTEROBACTER
infections among hospitalized patients and in • Members resemble those of Klebsiella when
immunocompromised hosts such as newborns, grow on MAC
elderly pt and pt with on respirator. • Culture:
• VF: Polysaccharide capsule o MAC: pink color sometimes mucoid (LF)
• Differential test: (+) string test • Significant specie: E. aerogenes, E. cloacae, E.
• Neufeld-Quellung Test: Positive gergoviae and E. hormaechei
• Growth on media with Potassium: Positive • Common isolate: E. aerogenes and E. cloacae
• IMViC: - - + + • ODC Test: Positive
• TSIA rxn: A/A, (+) gas, (-) H2S • LDC Test: Positive (except for E. cloacae and E.
gergoviae)
• Growth with KCN: Positive
• Sorbitol fermentation: Positive (except E.
aerogenes and E. cloacae
• Urease test: Positive (E. cloacae)
• Malonate Test: Positive (E. cloacae)

TUAZON A. 34
 • IMViC: - - + + • S. liquefaciens: ferments arabinose and exhibits
• TSIA rxn: A/A, (+) gas, (-) H2S growth in KCN
ENTEROBACTER GERGOVIAE SERRATIA MARCESCENS
o Found in respiratory samples and rarely • Most clinically significant species of the genus
isolated from blood cultures. • Causes bacteremic outbreaks in nurseries,
ENTEROBACTER CANCEROGENUS cardiac surgery units and burn units.
(FORMERLY E. TAYLORAE) • Few strains of this species are late lactose
o Isolated with osteomyelitis following fermenter
traumatic wounds • Biochemical Test:
BIOCHEMICAL E. o Positive: Urease, gelatinase and ONPG
TEST AEROGENES
E. CLOACAE o Negative: Arabinose fermentation
LDC test + - PROTEUS
Arginine • Genus isolated from urine, wound and ear
- + infections.
dihydrolase
ODC test + + • Species can infect the proximal kidney tubules
Urease - + (w) and can cause acute glomerulonephritis (AGN),
Alginate particularly in patient with UTI and in
- - catheterization.
utilization
• Rapid urease producers; urease they produce
CRONOBACTER
splits urea in urine, raise urine pH and encourage
• Formerly: Enterobacter sakazakii renal stone formation
• Contaminant of powdered infant formula
• Species: P. mirabilis, P. vulgaris, P. penneri and
• Isolated from individual with brain abscesses and P. myxofaciens
respiratory and wound infections.
• HP: P. mirabilis and P. vulgaris
• Culture:
• Common isolate: P. mirabilis
o MAC: pink (LF)
• Culture:
o BHIA: Yellow pigment
o MAC: clear and colorless (NLF); exhibit
▪ BHIA = Brain Heart Infusion Agar
“swarming phenomenon”; and have a
• IMViC: - - + + “burnt-chocolate” or “burnt –
• TSIA rxn: A/A,(+) gas, (-) H2S gunpowder” odor.
PANTOEA • PAD: Positive
• Pantoea agglomerans • LIA: R/A (Red slant/acid butt)
o Formerly Enterobacter agglomerans • IMViC Rxn:
• Causes nosocomial outbreaks of septicemia due o P. mirabilis: - + v v
to contaminant of IV fluids o P. vulgaris: + + - v
• Triple decarboxylase: Negative • TSIA Rxn:
• Culture: o P. mirabilis: K/A, (+) gas, (+) H2S
o MAC: clear or colorless (NLF) o P. vulgaris: K/A, (+/-) gas, (+) H2S
• IMViC: - v - v BICHEMICAL
• TSIA rxn: K/A, (-) gas, (-) H2S P. MIRABILIS P. VULGARIS
TEST
SERRATIA Indole - +
• Opportunistic pathogens, associated with PAD + +
nosocomial outbreaks. LIA R/A R/A
• Resistant to a wide range antibiotic IMViC -+vv ++-v
• Culture: K/A, (+) gas, (+) K/A, (+/-) gas,
o MAC: clear and colorless (NLF); Some TSIA
H2S (+) H2S
slow or late LF
• Biochem Test:
o Positive: DNAse (+ SMS =
Staphylococcus aureus, Moraxella,
Serratia), gelatinase, lipase and ONPG
• Species: S. marcescens, S. rubidaea, S.
liquefaciens, and S. plymuthica
• IMViC rxn: - - + +
• TSIA: K/A, (+) gas, (-) H2S
• S. marcescens, S. rubidaea, S. liquifaciens and
S. plymuthica: produce pink to red colonies (due
to PRODIGIOSIN pigment) at 25C
• S. odorifera: musty and pungent odor: “rotten
potato-like” odor

TUAZON A. 35
 PROVIDENCIA • Isolated in diarrheal stool culture (extraintestinal
• Cause of nosocomial outbreaks involving burn pathogen)
units. • It produces group 1 cephalosporinase
• Species: P. alcalifaciens, P. stuartii, P. rettgeri, P. CITROBACTER KOSERI
rustigianii and P. heimbachae • Formerly C. diversus
• Culture: MAC – clear and colorless (NLF) • Outbreaks of neonatal meningitis and brain
• PAD test: Positive abscess in nursery units
• IMViC: + + - + BIOCHEMICAL
• LIA: R/A C. FREUNDII C. KOSERI
TEST
• TSIA: K/A, (-) gas, (-) H2S Indole - +
PROVIDENICA RETTGERI H2S
+ -
• Pathogen of urinary tract production
• Diarrheal disease among travelers Growth KCN + -
• Resistant to antimicrobial agents ONPG + -
PROVIDENCIA STUARTII IMViC -+-+ ++-+
A/A or K/A, (+) K/A, (+) gas, (-)
• Nosocomial outbreaks in burn units TSIA
gas, (+) H2S H2S
• Mostly resistant to antimicrobial agents
SALMONELLA
PROVIDENCIA ALCALIFACIENS
• Most pathogenic that cause enteric fever (typhoid
• Most commonly found in feces of children with fever) and acute gastroenteritis (food poisoning)
diarrhea to humans.
BIOCHEMICAL • May be transmitted by human carriers.
P. RETTGERI P. STUARTII
TEST • MOT: ingestion of contaminated animal food
Urease + - products or improperly cooked poultry, milk, eggs
Phenylalanine and direct human contact.
deaminase + + • Specie: S. enterica (type species) and S. bongori
(PAD)
• Virulence factors: Fimbriae and enterotoxin (S.
MORGANELLA enterica)
• Specie: Morganella morganii • Culture: MAC – clear and colorless (NLF)
• Culture: MAC – clear and colorless (NLF) o SSA – colorless with black center
• PAD Test: Positive • Antigenic structures:
• IMViC: + + - - o Somatic O and Flagellar H – for serologic
• LIA rxn: R/A grouping
• TSIA rxn: K/A, (+) gas, (-) H2S o Vi Antigen - antiphagocytic
• Others: (+) urease, KCN and ODC test • Main etiologic agent of enteric fever: Salmonella
serotype typhi
• Etiologic agents of paratyphoid fever: Salmonella
serotype paratyphi A, B & C and Salmonella
serotype choleraesuis
• Subspecies of S. enterica:
o I: subspecie enterica
o II: subspecie salamae
o IIIa: subspecie arizonae
o IIIb: subspecie diarizonae
o IV: subspecie houtenae
o VI: subspecie indica
• Salmonella bongori
CITROBACTER o Named after the town of Bongor in Chad,
• Genus produces colonies in MAC agar that are Africa. Isolated from a lizard in 1966
similar to those of E. coli and have a biochemical • Biochemical Test:
resemblance to those of Salmonella. o All species are motile except for
Salmonella serotype pullorum and
• Cause false-positive agglutination test with
Salmonella Salmonella serotype gallinarum
o All specie produce gas except for
• All specie grow in Simon’s citrate agar
Salmonella serotype gallinarum and
• Culture: MAC – clear and colorless (NLF) after 24
typhi.
hrs colonies exhibit a light pink color (LF) after 48
o All specie produces H2S except for
hrs.
Salmonella serotype paratyphi A.
• Specie: C. freundii and C. koseri o LDC: (+) except for Salmonella serotype
CITROBACTER FREUNDII paratyphi A.

TUAZON A. 36
 o
Urease and media w/ KCN: Negative Chronic carrier state (most
Bile, Stool,
SALMONELLA POST common complication is
BIOCHEMICAL SALMONELLA Bone marrow
SEROTYPE necrotizing cholecystitis)
TEST SP.
TYPHI • Gold standard is bone marrow.
IMViC rxn -+-+ -+-- BIOCHEMICAL DIFFERENTIATION OF
K/A, (+) gas, (+) K/A, (-) gas, (+) SALMONELLA SEROTYPES
TSIA rxn
H2S H2S Biochemical
S. S.
S. Serotype Other
Serotype Serotype
CATEGORIES OF SALMONELLA INFECTION Test
typhi paratyphi
choleraesuis Serotypes
GASTROENTERITIS Citrate
- - v* +
• One of the most common forms of food poisoning utilization
Gas
• Caused by S. enterica subsp. enterica that come production - + + +
from animal H2S
+ - v* +
• Outbreak in US in 2009 came from contaminated production
peanut butter crackers by Salmonella serotype LDC test + - + +
typhimurium ODC test - + + +
• Source of infection: Poultry products, milk and Arabinose
- + - +
fermentation
handling of pets
• MOT: contaminated kitchen utensils
• Infective dose: 106 bacteria
• Symptoms: nausea, vomiting, fever and chills,
watery diarrhea and abd. Pain
ENTERIC FEVER (TYPHOID FEVER)
• Febrile disease that develops from eating
contaminated food prepared by infected
individuals or carriers
• Causative agent: Salmonella enterica subsp.
enterica serotype typhi
• Source: human carriers, contaminated food and SHIGELLA
water • Genus closely related to Escherichia
• Cause of outbreaks: improper sewage disposal, • Non-motile, intracellular pathogens
poor sanitation and lack of clean water source • Caused Bacillary dysentery
• Symptoms: malaise, anorexia, myalgia and • Reservoir: Human
severe frontal headache • MOT: 4Fs = Flies, fingers, food and feces; water
• Hallmark of infections: “rose spots” during second from infected persons (Fecal-oral route)
week of fever • Specie: S. dysenteriae (A), S. flexneri (B), S.
BACTEREMIA boydii (C) and S. sonnei (D)
• Occurs with or without extraintestinal infection • Most virulent specie: S. dysenteriae
that is caused by non-typhidal Salmonela • S. flexneri: Gay Bowel Syndrome
species. • Antigenic structure: Somatic O
• Charac by prolonged fever and intermittent • Culture:
bacteremia o MAC: clear, fragile and colorless
• Causative agent: Salmonella serotype o SSA: colorless without black center
typhimurium, Salmonella serotype paratyphi and • IMVIC Rxn: v + - -
Salmonella serotype choleraesuis. • TSIA: K/A, (-) gas, (-) H2S
CULTURE • Biochemical Test
WEEK PRESENTATION o All specie does not produce gas from
SOURCE
Stepwise fever, anorexia, glucose except for S. felxneri
Blood o All species are mannitol fermenters
malasie, relative
1 Bone marrow except for S. dysenteriae
bradycardia and
bacteremia • All specie do not decarboxylate LYSINE
Abdominal pain, bloating, Urine • All species do not decarboxylates ORNITHINE
constipation, rose spots, Rose spots except for S. sonnei
2 • S. sonnei: Late Lactose Fermenter and ONPG
hepatosplenomegaly, Bone marrow
jaundice positive
Stool SHIGELLA DYSENTERIAE
3 Bleeding ileitis, pneumonia
Bone marrow • Most virulent and cause bacillary dysentery
4 Recovery or Death Bone marrow • Virulence factor: Shiga toxin
• Urease: Negative
• LDC test: Negative

TUAZON A. 37
 • IMViC: v + - - YERSINIA
• TSIA Rxn: K/A, (-) gas, (-) H2S • Yersinia pestis
SHIGELLA SONNEI • Known as the “plague bacillus”
• Organism self-limiting and characterized by fever • Class A bioterrorism agent
and watery diarrhea (stool w/o blood) • Only enterobacterium that is transmitted to
• It has one serotype humans through the bite of infected flea.
BACILLARY DYSENTERY • Causative agent of Bubonic plague
• Most commonly caused by S. dysenteriae type 1 • Isolated on routine culture media, grows best at
• Characterized by acute inflammatory colitis and 25C to 30C
bloody diarrhea (blood, mucus, and WBC in the • Vector: Xenopsylla cheopis (oriental rat flea)
stool) • Reservoir: Rat
• Highly communicable (<200 bacilli) • Virulence factors: Endotoxin, Coagulase and
• In young children, rectal prolapse occurs due to fibrinolysin
the excessive straining • Microscopy: Short, plump rod with a bipolar or a
• Source: Human carriers “closed safety pin appearance” (Wayson stain)
• MOT: Person to person contact, Fecal-oral route, • Culture:
4F’s, and contaminated water from infected o MAC: Clear and colorless
persons o BAP: pinpoint at 24 hours
• Symptoms: Fever, chills, abdominal cramps, o Broth: Stalactite-shaped pattern
painful bowel movement and tenesmus • IMViC: - + - -
• Complications: Ileus (obstruction of the intestine), • TSIA: K/A, (-) gas, (-) H2S
seizure and haemolytic uremic syndrome (HUS) PLAGUE
Shigella Shiga bacillus • Disease of the rodents that is casued by Yersinia
A dysenteriae type 1 pestis and is transmitted to humans through flea
Shigella Schmitz bacillus bites.
dysenteriae type 2
• Human may also acquire it by ingestion of
Shigella flexneri Flexners bacillus contaminated animal tissue and inhalation of
B Hiss and Rusell’s contaminated airborne droplets
bacillus
• Once inside human body, the bacteria multiply in
Shigella boydii Newcastle the blood and lymph
C Manchester’s
bacillus 2 FORMS OF PLAGUE
D Shigella sonnei Duval’s bacillus BUBONIC PLAGUE
• Most severe form of bacillary dysentery • Results from the bite of an infected flea
• Most common cause of epidemic dysentery • associated with high fever and painful
inflammatory swelling of the axilla and groin
(buboes)
• causes millions of death
PULMONARY PLAGUE
• Acquired by close contact with infected
individuals
• occurs secondarily to bubonic plague
• inhalation of airborne droplets
YERSINIA ENTEROCOLITICA
• Most commonly isolated specie of Yersinia
• Causative agent: enterocolitis or waterborne
gastroenteritis
• Motile in SIM at 22C but not at 35C
• Isolated from contaminated packed RBC units
and considered blood transfusion hazard
• Ability to survive in cold temperature
• MOT: ingestion of undercooked food (pork and
pork intestine and vacuum-packed meat) and
dairy products (chocolate milk) and handling of
pets.
• Reservoir: Swine, dogs, cats, rabbits and cows
• Related infections: Appendicitis-like syndrome,
arthritis and erythema nodosum

TUAZON A. 38
 • Selective medium: CIN (cefsulodin-irgasan • Inositol-brilliant green-bile salt agar: exhibit white
novobiocin) agar or green to pink color for other enterics
• Microscopy: Coccobacilli with bipolar bodies • HEA: exhibit growth
• Culture: MAC – clear and colorless; CIN – • TCBS: do not exhibit growth
exhibits “bull’s eye” appearance or dark red or • Media with NaCl: do not exhibit growth
burgundy center with transparent borders at 48 • Some strains will not grow on MAC
hours of incubation • Use of inositol-brilliant green-bile salts agar
• IMViC: v + - - enhances the recovery of plesiomonands from
• TSIA Rxn: K/A, (-) gas, (-) H2S specimens.
YERSINIA PSEUDOTUBERCULOSIS BIOCHEMICAL AND SEROLOGICAL
• Pathogens of the rodents, particularly guinea pigs • Oxidase test: Positive
• Motile in SIM at 18C to 25C but not at 35C • Decarboxylase test: Positive trio decarboxylase
• MOT: direct contact with infected animals or their test
feces and ingested of contaminated food and • Inositol fermentation: Positive
water • IMViC: + + - -
• Reservoir: farm and domestic animals (usually • TSIA rxn: K/A, (-) gas, (-) H2S
birds) • Antigenic structure: O and H antigens
• Culture: MAC – clear and colorless EDWARDIELLA
• Biochemical test: (+) Urease and rhamnose • Genus have been isolated from cold-blooded and
fermentation warm-blooded animals.
• TSIA: K/A, (+) gas, (-) H2S • Specie: Edwardsiella tarda (human pathogen)
DIFFERENTIAL TEST FOR YERSINIA SPECIES • Culture: MAC – clear and colorless
Y. Y. Y. • Urease: negative
BIOCHEMIC
AL TEST
PESTI ENTEOCOLITI PSEUDOTUBERCUL • LDC Test: Positive
S CA OSIS • IMViC Rxn: + + - -
Indole - v - • TSIA Rxn: K/A, (+) gas, (+) H2S
Methyl red + + + • E. coli is A/A, (-) H2S, greening metallic sheen
Voges-
Proskauer - - - LESSON 5: NON-FERMENTATIVE GRAM-
(25C) NEGATIVE BACILLI
Motility PSEUDOMONAS, BURKHOLDERIA,
25C - + +
37C ACINETOBACTER, STENOTROPHOMONAS AND
- - -
SIMILAR ORGANISMS
Urease - + +
Ornithine • Non-fermentative gram-negative bacilli are
decarboxyla - + - aerobic, straight, and slender gram-negative
se bacilli
Sucrose • All species except Burkholderia mallei are motile
fermentatio - + -
n and mesophilic
PLESIOMONAS • Oxidase positive, grow on MacConkey agar, and
• Plesiomonas shigelloides utilize glucose oxidatively.
• Causes secretory diarrhea • Burkholderia gladioli, Pseudomonas luteola, and
Pseudomonas oryzihabitans are oxidase-
• Cross-agglutinates with Shigella, hence the
negative
specie name shigelloides
• These organisms are able to survive on or in
• Oxidase positive
medical devices (nebulizer, dialysate fluid, saline,
• Motility: Polar flagella
catheters and hospital devices) and disinfectants.
• Associated with HIV positive with inflammatory
• Opportunistic pathogen seen in water, soil,
bowel disease
plants, vegetables, food, and hospital.
• MOT: ingestion of undercooked seafood and
• Can withstand treatment of quaternary
contaminated water
ammonium compounds and chlorhexidine.
• Microscopy: Straight bacilli w/c occur singly, in
• Enterobacteria = oxidase negative
pairs, in short chain or filamentous
• Non-fermentative = oxidase positive
• Vibriostatic test O/129: Sensitive
• They are called non-fermentative because they
• All Enterobacteria are oxidase negative except
cannot ferment lactose.
for Plesiomonas shigelloides.
CHARACTERISTIC COMMON TO GROUPS OF
CULTURE CHARACTERISTICS
NONFERMENTERS
• MAC: clear and colorless
• BAP: Shiny, opaque, smooth and non-
PIGMENTATION
haemolytic YELLOW

TUAZON A. 39
 • Chryseobacterium and Elizabethkingia spp. Brevundimonas BA Chalk white
(weak fermenters) diminuta Mac NLF
• Sphingomonas paucimobilis BA Orange pigment
Brevundimonas
• Pseudomonas (Chryseomonas) luteola NLF; only 66%
vesicularis Mac
• Pseudomonas oryzihabitans grow on Mac
• Sphingobacterium spp. Smooth and
• Pseudomonas stutzeri (light yellow) and wrinkled BA slightly raised;
colonies dirt like odor
PINK NLF; colonies
• Methylobacterium spp. become dark
Burkholderia
pink to red
• Roseomonas spp. cepacia
Mac because of
PURPLE (MACCONKEY AGAR) complex *
oxidation of
• Acinetobacter spp. lactose after 4-7
BLUE-GREEN days
• Pseudomonas aeruginosa BCSA, PC, or
Smooth
VIOLET OFPBL
• Chromobacterium violaceum (fermenter) Cream to
yellow-orange;
LAVENDER TO LAVENDER-GREEN (BLOOD
smooth and
AGAR) mucoid (24-48
• Strenotrophomonas maltophilia hours) to dry
TAN (OCCASIONALLY) BA and wrinkled (>3
• P. stutzeri days may
• Shewanella putrefaciens resemble
WRINKLED COLONIES Burkholderia Pseudomonas
• P. stutzeri pseudomallei * stutzeri); putrid
odor
• P. oryzihabitans
NLF, but appear
• Burkholderia pseudomallei
as pink colonies
ODOR Mac
(oxidizes
SWEET lactose)
• Alcalogenes faecalis NLF
• Myroides odoratus Ashdown Dry, wrinkled,
• P. aeruginosa (grapes) violet-purple
POPCORN No distinctive
Burkholderia BA
• EO-4 mallei *
appearance
• Neisseria zoodegmatis Mac NLF
NONMOTILE No distinctive
BA
• Acinetobacter spp. Pandoraea spp. appearance
• Moraxella spp. Mac NLF
• Chryseobacterium spp. and Elizabethkingia spp. Spreading and
(weak fermenters) flat, serrated
• Sphingobacterium spp. (may “glide”) edges; confluent
growth; often
• Oligella spp. (non-ureolytica)
with metallic
OXIDASE-NEGATIVE sheen; bluish
• Acinetobacter spp. green, red, or
• S. maltophilia brown pigment;
• Pseudomonas luteola and P. oryzihabitans Pseudomonas BA
often beta-
• Pseudomonas cepacian aeruginosa *
hemolytic;
H2S-POSITIVE grapelike or
• Shewanella putrefaciens corn tortilla-like
ORGANISM MEDIUM APPEARANCE odor; mucoid
No distinctive colonies seen in
BA cystic fibrosis
Acidovorax appearance
spp. (delafieldii, patients
NLF
facilis, Mac NLF
(Acidovorax
temperans) Mac Pseudomonas No distinctive
facilis does not BA
grow on Mac) fluorescens, appearance

TUAZON A. 40
montelli, disease which is a severe infection in animals
mosselii, specifically horses and donkeys.
Mac NLF
putida, and • Human infection, which can be fatal, usually
veronii begins as an ulcer of the skin or mucous
Pseudomonas BA Yellow pigment membranes followed by lymphangitis and sepsis.
fulva * Mac NLF • Inhalation of the organisms may lead to primary
Smooth, pneumonia.
nonwrinkled, • They are non-motile member of the genus and it
Pseudomonas BA
flat, brownish- has a variable growth on MAC agar plate.
mendocina *
yellow pigment • BAP culture: colonies are non-pigmented
Mac NLF • Biochemical test: oxidase variable
Dry, wrinkled, B. PSEUDOMALLEI
Pseudomonas
stutzeri and
BA adherent, buff to • Vietnamese time bomb
brown • Inhalation or direct
CDC group Vb-
Mac NLF inoculation from
3*
Mac NLF environment through
No distinctive disrupted epithelial or
appearance; mucosal surfaces
Ralstonia spp.
(insidiosa, BA
may take up to • limited to tropical and
72 hours to subtropical areas, notably
mannitolilytica,
produce visible Southeast Asia; not part of
pickettii)
colonies human flora
Mac NLF • Can survive within human macrophages
• BA 5% sheep blood agar; BCSA Burkholderia • The disease is referred to as melioidosis
cepacian selective agar; Mac MacConkey agar; (glanders-like disease); it has several forms,
NLF non-lactose fermenter; OFPBL oxidative- including formation of skin abscesses, sepsis and
fermentative base-polymyxin B-bacitracin- septic shock, abscess formation in several
lactose; PC Pseudomonas cepacian agar. internal organs, and acute pulmonary disease
BURKHOLDERIA SPP. AND RALSTONIA PICKETTII • small gram-negative bacilli; bipolar staining
• Burkholderia is an obligate aerobe, they are (safety pin appearance) is seen with Wright’s
motile because they have a polar flagella except stain or methylene blue stain
for B. mallei. It is generally non-pathogenic and is • It grows at 42 °C and oxidizes glucose, lactose,
acquired through contact of heavily contaminated and a variety of other carbohydrates
medical devices. It can grow in BAP, CAP, and • A selective medium, Ashdown medium, is
Mac. supplemented with the antibiotic colistin; colonies
B. CEPACIA on this agar are deep pink because of the
• May colonize respiratory absorption of neutral red in the medium.
tract of patients with cystic • Colonies will also exhibit an earthy odor
fibrosis • Motile with a polar flallgella.
• Intrinsic resistance • Usually isolated from muddy soil and rice
• Selective media: PC paddies.
(Pseudomonas cepacia), • MOD is inhalation or direct inoculation from the
OFPBL (oxidative- environment through a disrupted epithelial or
fermentative base, mucosal surface.
polymyxin B, bacitracin, STENOTROPHOMONAS MALTOPHILIA
lactose), and BCSA (B. • 3rd most commonly isolated, non-fermentative
cepacia selective agar). gram-negative bacilli.
• Nonwrinkled colonies produce a nonfluorescing • On blood agar, colonies have a lavender-green or
yellow or green pigment that may diffuse into the gray color with ammonia smell
media • oxidase-negative and lysine decarboxylase-
• oxidize glucose, maltose, lactose, and mannitol. positive
• lysine decarboxylase and ONPG positive • positive for catalase, DNase, esculin and gelatin
• whereas most strains are ornithine hydrolysis
decarboxylase negative and fail to reduce nitrate • hospital-acquired infections in patients who are
to nitrite receiving antimicrobial therapy and in
B. MALLEI immunocompromised patients.
• Coccobacillus • it is commonly in association with use of
• Causative agent of glanders in horses; mules, indwelling plastic intravenous catheters
and donkeys; not part of human flora or farcy

TUAZON A. 41
 • S maltophilia is usually susceptible to commonly found in clinical specimens than is P.
trimethoprimsulfamethoxazole and ticarcillin- aeruginosa.
clavulanic acid
CULTURE
• S. maltophilia also oxidizes maltose faster than
glucose (hence the species name, maltophilia, or • P aeruginosa is an obligate aerobe
“maltose loving”), and it produces a brown • sweet or grape-like or corn taco-like odor: caused
pigment on brain-heart infusion agar that contains by the presence of 2-aminoacetophenone
tyrosine. • colonies with a fluorescent greenish color
PSEUDOMONAS SPP. AND BREVUNDIMONAS • nonfluorescent bluish pigment pyocyanin
SPP. • fluorescent pigment pyoverdin, which gives a
greenish color to the agar
• Pseudomonas aeruginosa is the most commonly
encountered gram-negative species that is not a • Pseudomonas fluorescent group, which includes
member of the family Enterobacteriaceae. P. aeruginosa, P. fluorescens, P. putida, P.
veronii, P. mosselii and P. monteilii, produce
• Grows at 42°c
pyoverdin, a yellow-green or yellow-brown
• Environmental (soil, water, plants);
pigment
• Survives well in domestic environments (e.g., hot
• Some strains produce the dark red pigment
tubs, whirlpools, contact lens solutions) and
pyorubin or the black pigment pyomelanin.
hospital environments (e.g., sinks, showers,
• oxidase-positive
respiratory equipment);
• It does not ferment carbohydrates, but many
• Ingestion of contaminated food or water;
strains oxidize glucose
exposure to contaminated medical devices and
solutions; introduction by penetrating wounds; • growth at 42° C, citrate-positive, and acetamide
person-to-person transmission is assumed to utilization
occur • Mucoid phenotype of
• Pseudomonas spp. are obligate aerobe, non- Pseudomonas aeruginosa
spore forming, motile resulting from
overproduction of alginate
• TSIA = K/K, - gas, - H2S
on dialyzed tryptic soy agar.
VIRULENCE FACTORS The light blue pigment is
• Exotoxin A, which kills host cells by inhibiting caused by a low level of
protein synthesis pyocyanin production.
• Production of several proteolytic enzymes and
hemolysins (heat-labile phospholipase C and a • Pseudomonas
heat stable glycolipid) that destroy cells and aeruginosa on 6% sheep
tissue. On the bacterial cell surface, blood agar. Note the
• pili may mediate attachment to host cells. beta-hemolysis, gray,
• Some strains produce alginate, a polysaccharide gun-metal color, and
polymer that inhibits phagocytosis; provide the spreading flattened
matrix for the organisms to live in a biofilm topology.
• intrinsic resistance
• Community-acquired infections: skin
(folliculitis); external ear canal (otitis externa);
eye, following trauma; bone (osteomyelitis),
following trauma; heart (endocarditis) in IV drug
abusers; and respiratory tract (patients with cystic
fibrosis)
• Hospital acquired infections: respiratory tract,
urinary tract, wounds, bloodstream (bacteremia),
and central nervous system • Disk diffusion
• Key pathogen that infects lungs of cystic fibrosis antimicrobial susceptibility
patients test of Pseudomonas
• In some cases of bacteremia, the organism may aeruginosa on Mueller
invade and destroy walls of subcutaneous blood Hinton agar. Note the
vessels, resulting in formation of cutaneous blue-green pigment.
papules that become black and necrotic. This
condition is known as ecthyma gangrenosum.
• in swimmers or divers, a necrotizing skin rash,
referred to as Jacuzzi or hot tub syndrome
• P. fluorescens, P. putida, and P. stutzeri also are
environmental inhabitants, but they are much less

TUAZON A. 42
Clinical Bacteriology (Lecture)
SECOND TERM

DASH
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science TWO
TWO
COMMENTS REGARDING SPECIFIC ORGANISMS
• A convenient and reliable identification scheme for P. aeruginosa involves the following conventional tests and
characteristics:
o Oxidase-positive
o Triple sugar iron slant with an alkaline/no change (K/NC) reaction.
o Production of bright bluish (pyocyanin), green (pyoverdin), red (pyrorubin), or brown (pyomelanin) diffusible
pigment on Mueller-Hinton agar or trypticase soy agar.

ACINETOBACTER • oxidase negative, catalase positive, and

• plump coccobacilli that tend to resist alcohol nonmotile
decolorization, thus appearing gram positive but • purplish hue on MAC
in general, they are gram negative. • CRAB, or carbapenem-resistant Acenetobacter
• member of the family Moraxellaceae baumanii.
• A. baumannii, the glucose-oxidizing nonhemolytic • CRAB isolates are usually only susceptible to
strain colistin and tigecycline.
• A. lwoffii, the glucose-negative, nonhemolytic • A. lwoffi is susceptible to almost all antimicrobials,
strain another characteristic distinguishing it from. A.
• In the hospital environment, they have been baumanii.
associated with ventilators humidifiers, catheters,
and other devices.

TUAZON A. 43
 usually as a result of contamination of wounds
with water or soil.
• oxidase positive, motile with polar flagella
• produces a violet pigment: violacein
• Catalase positive, and indole negative
(nonpigmented strains may be indole positive).
• Isolates ferment glucose and, variably, sucrose;
they grow on MAC agar and most enteric media,
reduce nitrate, and grow at 42° C.
LESS COMMONLY ENCOUNTERED COMAMONAS AND DELFTIA
NONFERMENTATIVE, GRAM-NEGATIVE BACILLI • Comamonas spp. and Delftia spp.
ALCALIGENES AND ACHROMOBACTER • produce alkalinity in OF (oxidizing fermentation)
media, are catalase and oxidase positive, usually
• Alcaligenaceae: Alcaligenes, Achromobacter,
motile by multitrichous polar flagella, and reduce
Bordetella, Advenella, and Kertersia
nitrate to nitrite.
• oxidase-positive, grow on MAC agar, motile,
• hospital equipment and fluids
peritrichous flagella
• C. testosteroni (formerly Pseudomonas
• Alcaligenes faecalis; it is asaccharolytic,
testosteroni) and C. terrigena: nosocomial
producing alkaline reactions in carbohydrate
bacteremia
containing media.
• Delftia (Comamonas) acidovorans, can oxidize
• Achromobacter spp. can be divided into the
fructose and mannitol like the Comamonas spp.
asaccharolytic species (Achromobacter
• associated with keratitis in soft contact lens
piechaudii and Achromobacter denitrificans) and
wearers
the sacchrolytic species (Achromobacter
xylosoxidans and the unnamed Achromobacter • Delftia tsuruhatensis, a newly named species,
groups B, E, and F). has been associated with catheter related
bacteremia.
• Isolates of both Alcaligenes and Achromobacter
are found in water and are resistant to FLAVOBACTERIACEAE
disinfectants, such as chlorhexidine and • are ubiquitous in soil and water
quaternary ammonium compounds. • Nosocomial infections
BREVUNDIMONAS • Balaneatrix, Bergeyella, Chryseobacterium,
• Brevundimonas diminuta: is often considered a Elizabethkingia, Empedobacter, Myroides,
contaminant Weeksella, Wautersiella, Sphingobacterium spp.
• motile and possess a single polar flagellum, and several unnamed CDC groups belong to the
oxidize glucose, and are oxidase positive family Flavobacteriaceae.
• Esculin hydrolysis variable • All indole-negative
• B. vesicularis: meningitis, infective endocarditis • Except for Balaneatrix alpaca (motile), organisms
in the family Flavobacteriaceae are nonmotile.
• Most strains of B. vesicularis produce an orange
intracellular pigment. BALNEATRIX ALPACA
• oxidizes glucose and maltose. • pneumonia and meningitis linked to individuals
• Esculin hydrolysis positive attending a hot springs spa
• Oxidizing carbohydrate (CHO) • positive for oxidase and indole
o B. diminuta = + glucose ELIZABETHKINGIA MENINGOSEPTICA
o B. vesicularis = + glucose, + maltose • meningitis or septicemia in a newborn, especially
CDC GROUPS EO-3, EO-4, AND PARACOCCUS in conjunction with prematurity.
• EO refers to Eugonic Oxidizer. • In adults, E. meningoseptica can cause
• EO-2 exhibits a high level of homology to pneumonia, endocarditis, bacteremia, and
Paracoccus, and it has been named Paracoccus meningitis, especially in critically ill patients.
yeei. CHRYSEOBACTERIUM INDOLOGENES
• have been recovered from blood cultures • nosocomial infections including bacteremia,
• Isolates of P. yeei, EO-3, and EO-4 are oxidase- usually in immunosuppressed patients
positive, nonmotile, saccharolytic coccobacilli • distinctive yellow colonies
that grow weakly, if at all, on MAC agar. SPHINGOBACTERIUM
CHROMOBACTERIUM • Oxidase, catalase, and esculin positive but
• Chromobacterium violaceum an opportunist, generally indole negative.
attacking immunocompromised patients with
• The presence of sphingophospholipids in the cell
neutrophil deficits, including CGD (Chronic
wall is unique to the group. They are true
Granulomatous Disease) = immunologic disorder
fermenters and can also oxidize some
where they have defects in their neutrophils),
carbohydrates.

TUAZON A. 44
 • Elizabethkingia • The positive oxidase test should differentiate
meningoseptica. Note the Shewanella from the family Enterobacteriaceae
growth of yellow pigment on • S. putrefaciens produces a tan to brown pigment.
sheep blood agar (SBA) (left) • S. algae requires NaCl (halophilic) and is
and absence of growth on asaccharolytic, whereas S. putrefaciens is
MacConkey plate (right). nonhalophilic and saccharolytic.
SPHINGOMONAS
METHYLOBACTERIUM AND ROSEOMONAS • S. paucimobilis and S. parapaucimobilis.
METHYLOBACTERIUM • S. paucimobilis infections include peritonitis,
• produce a characteristic pink to coral pigment and septicemia, meningitis, leg ulcers, empyema, and
can use methanol as a sole source of carbon and splenic and brain abscesses
energy. • yellow-pigmented
• bacteremia, peritonitis, synovitis, and skin ulcers, • does not grow on MAC agar and requires more
usually in immunocompromised hosts than 48 hours for culture on SBA
ROSEOMONAS • weakly oxidase positive (some strains may be
• pink-pigmented, nonfermentative bacilli negative), motile at 18° C to 22° C but not at 37°
• unable to oxidize methanol or assimilate C
acetamide. • indole negative
• On Sabouraud dextrose agar, they produce pink, • S. parapaucimobilis are H2Spositive by the lead
mucoid, almost runny colonies acetate method, Simmon’s citrate positive, and
• nonvacuolated, coccoid bacteria, forming pairs DNase negative.
and short chains.
• catalase and urease positive
• Unlike Methylobacterium, species of
Roseomonas will usually grow on MAC agar.
RALSTONIA PICKETTII
• Environmental organism
• occasionally found in a variety of clinical
specimens, such as blood, sputa/sputum of
patients with cystic fibrosis, and urine
• The mode of transmission is uncertain but is likely
to involve exposure to contaminated materials.
• when encountered, environmental contamination
should be suspected
• slow growers, oxidase and catalase positive
• grows on MAC agar, reduces nitrate, oxidizes
glucose and xylose, and is motile by means of a
single polar flagellum.
CUPRIAVIDUS PAUCULA
• aquatic environments and has been recognized
as an opportunistic pathogen
• septicemia, peritonitis, abscesses, and
tenosynovitis, most notably in
immunocompromised patients
• C. paucula is a motile (peritrichous flagella),
oxidase-positive, catalase-positive,
asaccharolytic, gram-negative bacillus.
• Most strains can grow on MAC agar.
SHEWANELLA
• Shewanella putrefaciens and S. algae,
• Environmental sources, such as stagnant water,
natural gas (petroleum), brine, and spoiled dairy
products, may contain S. putrefaciens.
• Colonies mucoid and can cause greenish
discoloration of SBA.
• Both species are motile, ornithine decarboxylase
and nitrate reductase positive, and produce
profuse H2S in TSIA, resembling H2S producers
of the family Enterobacteriaceae.

TUAZON A. 45
Clinical Bacteriology (Lecture)
SECOND TERM

DASH
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science TWO
TWO
FINALS
UNIT OUTLINE o BAP: colonies are Alpha or Beta
I. Non-enteric Gastrointestinal Pathogens hemolytic
and Small, Pleomorphic Gram-negative • Susceptibility Test: 150ug vibriostatic O/129
Bacilli disk in MHA (susceptible)
II. Aerobic Gram-Positive Bacilli • Biochemical Test:
III. Mycobacteria (2 topics of mycobacteria) o All specie ferment glucose
IV. Anaerobic Bacteria and Rickettsia and o All specie except for V. vulnificus are
Chlamydia non-lactose fermenter
V. Cell Wall-Deficient & Spirochetes and o All species are oxidase positive and
Miscellaneous Bacteria reduce nitrates to nitrite except for V.
metschnikovii
o Motility test: Broth –polar sheated
LESSON 1: NON-ENTERIC flagella; Solid media: peritrichous,
GASTROINTESTINAL PATHOGENS unsheated flagella.
VIBRIO VIBRIO CHOLERAE
• Comma-shaped or curved bacillus • Causative agent of cholera
• Species: V. cholerae, V. parahaemolyticus, V. • Rapid darting or shooting-star motility
alginolyticus, V. metschnikovii, V. damsel and • Virulence factor: Choleragen (cholera toxin)
hollisae • Potent enterotoxins: Cholera toxin (most
commonly encountered), zottoxin and ace toxin.
• Culture:
o BAP: colonies are smooth and medium
to large sized greenish hue
• Common cause of cholera: V. cholerae O1
• Antigenic structure: Somatic O and Flagellar H
• String test: Positive
• Other tests:
o Oxidase: +
• Microscopic morphology of Vibrio using acridine o Indole: +
orange stain of Vibrio cholerae. o Lysine: +
• Usually isolated from contaminated fish, o ODC: +
shellfish, or sea foods.
SUBGROUPS AND SEROTYPES:
VIBRIO SP. • V. cholerae subgroups:
• Facultatively anaerobic and monotrichious o V. cholerae O1, V. cholerae O139 and
organisms (contains single flagella) V. cholerae non-O1
• Found in brackish water, marine water, or salt • V. cholerae O1 serotypes:
water o Ogawa (A, B), Inaba (A, C) and Hikojima
• Isolated: algae, plankton, fish and shellfish (A, B, C)
• Halophilic (salt loving): except, V. cholera and V. • Epidemic V. cholerae O1 biogroups:
mimicus o Classical and El Tor
• Mode of acquisition: raw or undercooked ▪ Classical is susceptible to
seafood. polymyxin B. Voges Proskauer
• Related infection: Cholera, wound infection, (VP) negative.
septicemia and necrotizing fasciitis ▪ El Tor are resistant to polymyxin
• Microscopy: Gram negative, short, curved and B. VP positive.
asporogenous rods.
• Common isolates of vibrio: CHOLERA
o V. cholerae O1 and non-O1, V. • Acute diarrheal infection, spread through
parahaemolyticus, V. vulnificus, V. contaminated water source
alginolyticus. • Ingestion of improperly preserved shellfish, milk
• Culture: and ice cream.
o CAP: colonies are smooth, opaque and • Hallmark: Rice watery stool (defecate usually
iridescent with greenish hue 10-30 times per day / pagdudumi)
o MAC: colonies are NLF (except: V. • V. cholerae O1: Common cause of epidemic
vulnificus, w/c is lactose fermenter)

TUAZON A. 1
 • Non-O1: Gastroenteritis.
• Choleragen
o Protein toxin,
o Produced by V. cholerae O1 strains.
Stimulates hypersecretion of water and
chloride ions and inhibits sodium ions
absorption
o 10-15 liters fluid loss and electrolytes
VIBRIO PARAHAEMOLYTICUS
• Second most common Vibrio species associated
with gastroenteritis
• Etiologic agent of “summer diarrhea” in Japan Must remember:
• MOA: Eating contaminated seafood like oysters, • Common
scallops, crabs, lobsters, shrimps and sardines o V. cholerae O1 – Cholera,
• Leading cause of pandemic: V. gastroenteritis, wound infections,
parahaemolyticus serotypes 03: K6 bacteremia
• Virulence factor: Heat stable hemolysin o V. cholerae O139 – Cholera
• Pathogenicity: Hemolysin lysis human RBC’s o Vibrio parahaemolyticus –
• Selective medium: Wagatsuma agar (high Gastroenteritis, wound infections
mannitol medium) o Vibrio vulnificus – Septicemia,
VIBRIO VULNIFICUS wound infections
• “Lactose positive” Vibrio species o Vibrio alginolyticus -
Wound infections, ear infections,
• Second to V. cholerae as cause of severe
conjunctivitis, respiratory infections,
Vibrio-associated infections
bacteremia
• Related infections: Septicemia and wound
• Rare
infection
o Vibrio metschnikovii – Septicemia,
• MOA: Eating contaminated raw oysters and fish
peritonitis
VIBRIO ALGINOLYTICUS
• Vibrio specie not commonly isolated and is CULTURE MEDIA
considered as the least pathogenic to humans. • Culture Media: Alkaline peptone water,
• Strict halophile that requires a medium with 1% thiosulfate-citrate-bile salts-sucrose agar
to 10% Nacl
(TBCS), MAC (differentiate the fermentation of
• Related infection: Eye, ear and wound
lactose) and BAP (demonstrate alpha and beta
infections (extraintestinal pathogen)
hemolysis)
LABORATORY DIAGNOSIS
• Transport Medium: Cary-Blair medium
• Gram stain
• Culture Media • Enrichment medium for V. cholerae: Alkaline
• String Test peptone water (pH 8.5)
• VibriostaticTest • Growth of vibrios requires media that contain
• Biochemical Test 0.5% Nacl except for V. cholerae and V.
• Serological Test mimicus (they are non-halophile)
• Specimen: Stool, rectal swab, pus and tissue • V. alginolyticus: tolerates up to 10% NaCl
• Gram stain: Gram neg • Pathogenic vibrio grows as non-lactose
o Straight or slightly curved fermenter on MAC
• String Test: Rgt: 0.5% sodium desoxycholate • TBCS growth:
o (+): lysis of cells releases DNA, which o TCBS inhibits: Gram-positive bacteria
can then be pulled up into a viscous o pH indicator: Thymol blue and
string. parathymol blue
• Biochemical Test: o Sucrose fermenters (Yellow colonies
o TSIA: A/A, (-) gas, (-) H2S on TCBS): V. cholerae, V. alginolyticus
o LIA: K/K
and V. metschnikovii
• V. cholerae: (+) citrate & indole o Non-sucrose fermenter (Green
• V. vulnificus: (+) indole& cellobiose
colonies on TCBS): V. mimicus, V.
• V. mimicus: (+) indole
vulnificus, V. parahaemolyticus and V.
damsel.

• Vibriostatic Test –O/129

TUAZON A. 2
 o Used to separate vibrios (susceptible) whereas neither will grow in nutrient broth with
from other oxidase-positive, glucose 6% NaCl (right) [second right pic]
fermenter like aeromonads (resistant) • Most common isolate: A. caviae
o Uses 150ug vibriostatic agent O/129 • Common isolates in GI infection: A. caviae
(2,4-diamino-6,7-diisopropylpteridine.). • Common isolates in HUS (Hemolytic Uremic
• Serological Test Syndrome): A. hydrophila and A. veronii
o V. choleraenon-O1: fail to agglutinate • Vibriostatic O/129 test: Resistant
with O1 antisera • Inositol fermentation: Negative
o V. parahaemolyticus: serotyped by O • String Test: Negative
and K antigen • Indole positive: A. caviae, A. hydrophila and A.
AEROMONAS veronii
• Comma-shaped or curved bacillus • TSIA reaction:
• Species: o A. caviae: A/A, (-) gas, (-) H2S
o Mesophilic group: A. hydrophila, A. o A. hydrophila and A. veronii: A/A, (+)
veroniicomplex, A. caviaecomplex gas, (+) H2S
o Psychrophilic group (cold temp): A.
salmonicida • Aeromonas hydrophila
exhibiting B-hemolysis on
• Gram stain of sheep blood agar.
Aeromonas

CAMPYLOBACTER
• Gram negative, small, S-shaped rods or curved
AEROMONAS SP.
• Found in fresh, estuarine and chlorinated
waters.
• Motile with single polar flagellum
• Facultatively anaerobe
• Glucose fermenter
• Grows at 4C to 42C
• Cause: Traveler’s diarrhea
• In animals: Red-leg diseases in amphibians.
• Extraintestinal human infection: Septicemia,
meningitis, keratitis and wound infections.
• Microscopy: Gram neg, straight rods
• Culture:
o BAP: large, round, raised, white and
opaque
o MAC: pink color
o CIN: “bull’s eye” appearance
• Biochemical Test: Oxidase and Catalase
Positive
• Aeromonas caviae exhibiting lactose

bacillus
• Species:
fermentation on MacConkey agar. [first left pic] o Campylobacter jejuni
• Both Aeromonas and Plesiomonas spp. will o Campylobacter fetus subsp. Fetus
grow in nutrient broth with 0% NaCl (left), o Campylobacter coli
o Camplylobacterlari

TUAZON A. 3
 o Campylobacter sputorum CAMPYLOBACTER FETUS SUBSP. FETUS
o Campylobacter concisus • Causes bacteremia and rarely associated with
o Campylobacter curvus gastrointestinal illness.
o Campylobacter rectus LABORATORY DIAGNOSIS OF
CAMPYLOBACTER SP.
• Specimen: feces, rectal swab & blood.
• Rectal swab: less-preferred specimen
• Microscopy:
o Counterstain: Carbolfuchsin
o Hanging drop prep: darting motility
o If safranin: 2-3 minutes
• Culture:
o Gray, flat, glistening and irregular with
“tailing effect” along the streak line or
“runny spreading” growth
o Transport medium: Cary-Blair medium
• Selective Media: Campy-BAP, Butzler agar,
CAMPYLOBACTER SP. Skirrow’s medium and charcoal cefoperazone
• Motile with the presence of single polar flagellum desoxycholate agar (CCDA)
and secretes oxidase
• Butzler and Skirrow’s media: both enriched
• Microaerophilic, except for C. rectus and C. and have added antibiotics.
curvus, which are both strictly anaerobes.
• CCDA contains antibiotics and is selective for
• Most recognized antecedent cause of Campylobacter species.
Guillain-Barr’e syndrome • Blood culture: two-week (2 weeks) incubation
• Also an animal pathogen (cattle and swine) that may be needed
causes sterility and abortion.
• Note: Campy can be detected effectively by a
• Enteric campylobacters: C. jejuni, C. coli and CO2 monitoring system.
C. lari SELECTIVE MEDIA FOR CULTIVATION OF
• MOA: Ingestion of contaminated water, poultry CAMPYLOBACTER SPECIES
and dairy products; handling of pets like dogs, ANTIMICROBIAL
cats and birds (occupational hazard); and sexual MEDIUM BASE
AGENT
transmission. Campy Blood Brucella agar. Vancomycin,
• Microscopy: Faintly staining, Gram-negative, Agar 10% sheep Trimethoprim,
small, curved or S-shaped rods. RBC Polymyxin B,
o Old culture: coccobacillary Amphotericin B,
o Enteric campylobacters: long spirals Cephalothin
or are seagull wing-shaped. Oxoid blood
agar base. Vancomycin,
CAMPYLOBACTER JEJUNI
Skirrow’s Lysed, Trimethoprim,
• Slow growing fastidious and asaccharolytic
defibrinated Polymyxin B
organism. horse RBC
• It has a darting motility and is unable to grow in Butzler Thioglycolate Bacitracin,
media with a high salt concentration. fluid with agar Novobiocin,
• Most common cause of bacterial gastroenteritis added. 10% Actidione,
• Can cause septic arthritis among AIDS patient sheep RBC Colistin,
• MOA: eating contaminated chicken and turkey Cefazolin
(does not multiply in foods) Nutrient agar.
• Its optimum temperature for growth is 42C Charcoal. Cefoperazone,
CCDA
Sodium Amphotericin B
• Infective dose: >10,000 organisms.
deoxycholate

TUAZON A. 4
Must remember:
• Arcobacter butzleri
o Associated with diarrheal disease and bacteremia in humans and in children with recurring
gastrointestinal illness (abdominal cramps), endocarditis, and peritonitis.
• Campylobacter jejuni
o Most common cause of bacterial diarrhea worldwide.
• Campylobacter coli
o Gastroenteritis, bacteremia in immunocompromised patients.
• Campylobacter fetus subsp. fetus
o Bacteremia in immunocompromised patients.
• Helicobacter pylori
o Common cause of duodenal ulcers and type B gastritis; possibly a risk factor in gastric carcinoma.
HELICOBACTER • Gastric tissue: best specimen for the culture of
• Gram neg, helical (S-shaped) rods, resembles H. pylori
Camphylobacter • Tissue specimen should be maintained at 4C
• Species: and processed within 2 hours of collection.
o Helicobacter pylori • Urine specimen is utilized for ammonia testing.
o Helicobacter cinaedi HELICOBACTER PYLORI
o Helicobacter fenneliae • Major cause of type B gastritis, peptic ulcer and
o Helicobacter rappini (formerly known as gastric carcinoma
Flexispira rappini) • Found in the mucous layer of the antrum and
• GIT of mammals and birds fundus of the stomach but does not penetrate
• Motile by monopolar or multi-bipolar flagella gastric epithelium
• Strong urease activity and are microaerophiles • Binds to Lewis antigen (type of blood group
• CAP: colonies are gay and translucent antigen) and the monosaccharide sialic acid.
• Biochemical Test: Oxidase and catalase • Primary habitat: Human gastric mucosa
positive • Biochemical test: Strong urease
• Route: Oral-oral and Fecal-oral • Diagnostic test: (+) urea breath (sensitive and
• Specimen: Gastric biopsy tissue, urine, feces specific).
and dental plaque HELICOBACTER CINAEDI AND
HELICOBACTER FENNELIAE

TUAZON A. 5
• Isolated from the blood of the patient with • H. pylori is urease positive (+)
bacteremia and recovered from the blood of LESSON 1.2: SMALL, PLEOMORPHIC, GRAM-
homosexual males with and without HIV. NEGATIVE BACILLI
HAEMOPHILUS
• Gram-negative, small, pleomorphic coccobacilli
or rods
• Species: H. influenza, H. ducreyi, H.
parainfluenzae, H. paraphorobilus, H.
parahaemolyticus, H. pittmaniae, H. aegypticus
and H. segnis
HAEMOPHILUS SP.
• Greek word: Haima and philos means “blood
lover”.
• Obligate parasite on the mucous membrane of
humans
• Fastidious, non-motile, caphnophilic and
facultatively anaerobic bacteria.
HELICOBACTER • Clinical specimen very susceptible to dying and
• Gram stain: extreme temperature.
o 0.1% basic fuchsin counterstain • Most specie cannot grow on pure BAP
enhances morphology • Biochemical test: (+) catalase; Oxidase (+);
o Stain for biopsy: Warthin-Starry stain, except H. segnis
silver stain or Giemsa stain. • Growth factors: X (hemin) and V (nicotinamide
• Culture: adenine dinucleotide)
o CAP, MTM, Skirrow’s agar and brucella HAEMOPHILUS INFLUENZAE
agar with 5% sheep blood • Main cause of meningitis in children
o Transport media: Stuart medium, • As early as less than 12 months, you can
cysteine brucella broth with 20% receive the vaccine for H. influenzae
glycerol and isotonic saline with 4%
• Spread: nasopharynx → lymph nodes →
glucose
meninges
o Gastric tissue biopsy specimen should
• Very fastidious and rapidly killed by phagocytes
be placed in a stuart medium
• Only member of the genus that produces IgA
o Helicobacter may require more than 5
protease
days of incubation in a capnophilic
• Does not produce endotoxin
environment.
• MOT: person to person (respiratory droplets)
• Other Test:
o Nucleic Acid amplification (PCR) is a • Culture: CAP: translucent, convex, tan-colored&
sensitive method for detecting H. pylori mucoid with “mousy” or “bleach-like odor”
o H. pylori: susceptible to metronidazole • Principal virulence factor: Polysaccharide
o Susceptibility Testing: 3 days capsule (serotypes A to F)
• Other virulence factor: IgA protease, fimbriae
and lipopolysaccharide
• Biochemical test: (-) porphyrin (necessary for
the production of heme and later on for the
production of hemoglobin)
CATEGORIES OF H. INFLUENZAE
TYPABLEFORM
• Based on capsular characteristic. of H. influenza
• Encapsulated form: types A, B, C, D, E and F
(capsular types)
• H. influenza type B (Hib): Most commonly known
type of H. influenzae. It can cause of serious
infections in human and the leading cause of
meningitis in unvaccinated children.
• Other: septicemia, arthritis, epiglottitis, tracheitis,
osteomyelitis and pneumonia

TUAZON A. 6
NON-TYPABLE FORM o Meningitis, epiglottitis, arthritis
• Does not produce capsule (non-capsulated H. AEGYPTICUS (KOCH-WEEKS BACILLUS)
strains) • Distinguishing characteristics
• Indigenous microbiota of URT and adheres to o Genetically related to H. influenzae
human EC • Growth Factor
• Second prevalent etiologic agent for otitis media. o X, V
• Other: Conjunctivitis and sinusitis • Associated Infection/disease
• Most commonly causes otitis media is o Pink eye conjunctivitis
Streptococcus pneumoniae. H. INFLUENZAE BIOGROUP AEGYPTICUS
• Distinguishing characteristics
OTHER ORGANISM o Non-typable (they don’t have capsule)
HAEMOPHILUS DUCREYI • Growth Factor
• Agent of chancroid or “soft chancre” o X, V
• This needs to be distinguished with syphilis • Associated Infection/disease
(causes hard chancre) o Brazilian purpuric fever
• Infects the mucosal epithelium, genital and non- H. HAEMOLYTICUS
genital skin, and regional lymph nodes. • Distinguishing characteristics
• Painful and tender genital lesions that advance o Beta-haemolytic
to ulcer with satellite lesion • Growth Factor
• Hallmark of chancroid: Buboes or suppurative, o X, V
enlarged, draining, inguinal lymph nodes • Associated Infection/disease
• CAP: transparent, small, non-mucoid and tan or o
yellow H. DUCREYI
HAEMOPHILUS PARAINFLUENZAE • Distinguishing characteristics
• Commonly found indigenous microbiota of the o School of fish
URT of adults. • Growth Factor
INFECTIONS CAUSED BY HAEMOPHILUS o X
INFLUENZAE • Associated Infection/disease
Non-encapsulated o Chancroid or Soft chancer
Encapsulated Strains
strains H. PARAHAEMOLYTICUS
Septicemia Otitis media with effusion • Distinguishing characteristics
Septic arthritis Conjunctivitis o Tan and dry colonies; Beta-haemolytic
Meningitis Sinusitis
• Growth Factor
Osteomyelitis Bacteremia
Cellulitis Pneumonia o V
Pencarditis • Associated Infection/disease
Pneumonia o Pharyngitis
Epiglottitis H. PARAINFLUENZAE
• Lesions of chancroid o the penis, showing a • Distinguishing characteristics
draining bubo (arrow) in the adjacent groin area. o Fructose and maltose fermentation
Chancroid is caused by Haemophilus ducreyi.. • Growth Factor
H. INFLUENZAE (PFEIFFER’S BACILLUS) o V
• Distinguishing characteristics • Associated Infection/disease
o Mousy/bleach-like odor; non-haemolytic o Endocarditis
• Growth Factor X factor – Hemin
o X, V V factor – NAD (nicotinamide adenine dinucleotide)
• Associated Infection/disease • Specimen: CSF (meningitis), sputum
(pneumonia), genital lesions or ulcer (ducreyi),
joint fluid or arthritis (caused by capsular
influenzae), vaginal swab, abscess drainage,
conjunctival swab (aegypticus) and bronchial
washing and blood.
• H. ducreyi: Cleansed with sterile gauze that is
pre-moistened with sterile phosphate-buffer
saline
o Bedside plating is preferred

TUAZON A. 7
LABORATORY DIAGNOSIS OF HAEMOPHILUS o H. aegypticus: CAP with 1%
SP. IsoVItaleXor Vitox
1. Gram-stain: Haemophilus resemble an o H. ducreyi: Nairobi biplate medium
“amorphous serous material” because of their (combination of gonococcal agar and
pleomorphic appearance. MHA with horse’s blood and
2. Porphyrin Test: (delta-aminolevulenic acid vancomycin)
Test): detects the presence of enzymes that • Serological Test
converts delta-aminolevulenic acid into o Serotypes detect thru capsular antigen
porphyrins. by Latex agglutination, capsular swelling
and immunofluorescence test
• Identifying the heme-producing species of o Neufeld quelling test: rapid direct
Haemophilus. identification of capsular antigen of H.
• Reagent influenzae
o Kovac’s reagent (other name: p- ▪ Streptococcus pneumoniae and
dimethylaminobenzaldehyde) Klebsiella pneumoniae are also
• End product: positive for Neufeld quelling
o Porphobilinogen – red color test.
o Porphyrins – reddish-orange color
(UVL 300 nm) • Haemophilus
• (+) Result influenzae satellitism
o Exhibits a red color (H. parainfluenzae, around and between
H. parahaemolyticus, H. paraphrobilus large, white, hemolytic
and H. aphrophilus) staphylococci. The
• (-) Result small, gray glistening
o H. influenza, H. haemolyticus, H. colony is H. influenzae
aegypticus and H.ducreyi. (arrow)
• Interpretation of results
o Haemophilus species that need the X
factor are unable to synthesize • This organism would be identified as
porphyrin from delta-ALA Haemophilus influenzae because it requires
(aminolevulinic acid) both X and V factors.
• Culture:
o CAP, BAP, BHI and thioglycollate
o CAP: preferred medium for • This organism
Haemophilus because it contains the X
and V factor.
o Rabbit’s or horse’s blood agar is
preferred for observing hemolysis
o All clinically significant Haemophilus,
except for H. ducreyi requires NAD
• Growth Pattern:
o Haemophilus: grows best at 35C to
37C (except H. ducreyiat 33C) and 5 to
10 CO2
o V-factor dependent Haemophilus: H.
influenzae, grows as “satellites” on BAP
and produce NAD.
o Grown anaerobically: Only NAD
required
o H. aegypticus–4 days incubation; H.
ducreyi–7 days.
o Do not grow in MAC
• Selective Media
o H. influenzae: horse’s blood-bacitracin
agar for respiratory secretion of patient
with cystic fibrosis

TUAZON A. 8
 requires V factor only and would be identified as
Haemophilus parainfluenzae.
• This organism is
positive for X factor only.
The probable species is • An Aggregatibacter aphrophilus isolate that is
Aggregatibacter not X factor dependent and is growing over the
aphrophilus because this entire surface of a trypticase soy agar plate.
species can appear to be • Aggregatibacter aphrophilus growing on sheep
hemin dependent on initial blood agar.
isolation. AGGREGATIBACTER
• Under ultraviolet ACTINOMYCETEMCOMITANS
light, the organism • Formerly known as Actinobacillus
on the bottom is actinomycetemcomitans
exhibiting a • Common cause of periodontitis
positive porphyrin • Only catalase positive in HACEK; urease
reaction. The negative
organism on the • Virulence factor: collagenase and leukotoxin
top (x factor) is porphyrin negative. • Colonies: “star-shaped appearance
HACEK (AACEK) GROUP • Serotypes: A, B, C, D, E and F
• Gram-negative, small, non-motile
• Species:
o Aggregatibacter aphrophilus (formerly
H. aphrophilus)
▪ H. aphrophilus is now under
Aggregatibacter)
o Aggregatibacter
actinomycetemcomitans • Aggregatibacter actinomycetemcomitans on
o Cardiobacterium hominis sheep blood agar. The star-shaped centers of
o Eikenellacorrodens and the colonies are not usually evident until after 48
o Kingella spp. hours of incubation and are best observed by
• Part of indigenous oral microbiota and are using x100 magnification (light microscope) or a
considered as opportunistic pathogens. stereomicroscope.
• Fastidious and most of the species grow slowly CARDIOBACTERIUM HOMINIS
(dysgonic) on BAP and CAP and require 7 to 14 • Infects the Aortic valve than other ”false-gram
days of incubation. positive” in some parts of the cells
• Slow and progressive bacterial endocarditis • The only indole-positive HACEK member;
(vegetation of fibrinous clots in heart valves). indole positive
• Utilize d-aminolevulinic acid. • BAP: capnophilic and may exhibit “pitting”
AGGREGATIBACTER APHROPHILUS • Yeast Extract: “rosette formation (flower like
• “foam-loving bacteria” appearance) and appear filamentous
• Formerly known as H. aphrophilus • Growth of colonies
• Greek: aphros and philos, “foam-loving” of Cardiobacterium hominis
• Most common species that causes over 48 hours on sheep
Endocarditis blood agar.
• Isolated from dental plaques and gingival
scrapings
• Culture: CAP, raised, convex, granular and
yellowish • Gram stain of
Cardiobacterium hominis
showing typical “rosettes”
EIKENELLA CORRODENS
“CORRODING BACILLI”
• Least common isolate
• Assacharolytic like the species of genus
Moraxella.
• Infections from human bites or clenched fist
injuries.

TUAZON A. 9
• BAP: exhibit a yellow color; pit or corrode the • Human and animal pathogens
agar; and have a “sharp bleach” odor. • Intracellular parasite
• LDC:(+) • Assacharolytic and non-encapsulated,
• Arginine dihydrolase: (-) require an increased supply of CO2 for
growth.
• Growth of Eikenella • Localized in tissue rich in erythritol
corrodens on chocolate agar. (placental tissue) and induces spontaneous
• Gram stain abortion among animals.
morphology of Eikenella • Preferred specimen:
corrodens. Blood and Bone marrow
KINGELLA SP. • BAP: small convex,
• Tendency to resist decolorization translucent, yellowish and non-
• Microscopy: Plump, square-ended, hemolytic
arranged in pairs or short chains. UNDULANT FEVER
• BAP: exhibit white to yellowish-brown • Normal temperature in the morning and then
pigmentation. followed by high temperature in the afternoon
• TMA: K. denitrificans and evening.
• K. kingae: isolated from degenerative joint and • Primary Route:
bone infections (osteoarthritis) in children o Ingestion of unpasteurized and
younger than 3 years old. contaminated milk or cheese from
• Gram stain of infected animals
Kingella kingae o Inhalation of air around animal
illustrating the plump carcasses (aerosol infection)
bacilli (square-ended) in o Penetration of ocular or oral mucosa
chains and tendency to o Direct inoculation into bloodstream
resist decolorization. BRUCELLA
• Specimen: Blood, bone marrow, skin lesions

BRUCELLA SP (BANG’S BACILLUS) and placental tissue (causes abortion)

• Small, coccobacilli arranged singly, in pairs or in • Brucella species, handled as biosafety level 3
short chain, sandy-appearance, non-motile, agent in a class III cabinet due to their aerosol
obligate aerobe mode transmission.
• Species: • Gram Stain:
o Brucella abortus o Carbolfuchsin should be substituted for
o Brucella suis safranin O to improve the gram stain
o Brucella canis • Culture:
o Brucella melitensis o BAP, trypticase soy agar (TSA), and
Castañeda’s medium
• Brucella o B. abortus: requires niacin or nicotinic
melitensis on sheep acid for growth but can be inhibited by
blood agar appear adding thionine dye.
smooth, raised, and o Isolate can be recovered after 7 days
translucent but may require prolonged incubation →
30 days
BRUCELLA • Serological Test:

TUAZON A. 10
 o Serum agglutination test (SAT): Positive ▪
Associated with vomiting and
with > 1:160 titer “whooping” or hurried, deep
o Brucella canis: not detected. respiration, last for six weeks.
Severe coughing for 15 to 25 X
in 24 hrs
o Convalescent Stage
▪ Symptoms slowly decline, last
six months after infection.
LABORATORY DIAGNOSIS
BORDATELLA SP. • Swab: Calcium Alginate or a Dacron swab
• Obligately aerobic, fastidious, Gram-negative • Gram Stain:
coccobacilli o Use of two-minute safranin or 0.2%
• Species: basic fuchsin as counterstain enhances
o Bordatella pertussis its visibility.
o Bordatella parapertussis • Serologic Test:
o Bordatella bronchiseptica o Need greater amount of bacteria
o Bordatella avium required for an agglutination test;
• Non-carbohydrates fermenters and non-motile perform subcultures from primary
except for B. bronchiseptica isolation.
• Replicate on cilated respiratory epithelial cells of o Examined using DFA (Direct fluorescent
humans antibody) stain
• Mostly inactive in biochemical test • Nucleic Acid Test:
• Culture: Bordet-Gengou agar, smooth, o PCR: rapid test, more sensitive test than
glistening and silver color. culture.
• Biochemical test: (+) catalase; (-) indole • Note:
• Growth factors: Nicotinic acid, cysteine and o Plates are incubated for 7 days at 35C
methionine without an increased CO2
o Casamino broth: transporting swab
BORDATELLA PERTUSSIS (BORDET-GENGOU
specimens.
BACILLUS)
• Etiologic agent of whooping cough
CULTURE
• Culture: Regan-Lowe agar, Bordet-Gengou
• Only infects and causes disease in humans
potato infusion agar, Modified Jones-Kendrick
• Does not survive well outside the host charcoal agar and Casamino acid broth.
• Culture: Bordet-Gengou Agar –small and shiny
and resemble “mercury drops”. Bordet-Gengou Agar: - composed of Potato
• Growth inhibitors: Fatty acid, metal ions, infusion, sheep’s blood
sulphides and peroxides and cephalexin.
• Growth protectors: Charcoal, blood and starch. - B. pertussis and B.
• Principal virulence factor: Pertussis toxin parapertussis: Hemolytic
(protein toxin) reaction
Regan-Lowe Agar - Charcoal, horse’s blood
• Other virulence factors: Filamentous
and cephalexin
hemagglutinin, tracheal toxin and pertactin
- Favorable transport and
(adherence factor) enrichment media
• Preferred specimen: Nasopharyngeal swab; Modified Jones- - Yeast extract and
other: bronchoalveolar lavage Kendrick charcoal Agar cephalexin
• Related Infection: • Do not grow on MAC, except B. bronchiseptica
o Whooping cough (IP: 7 to 14 days)
o Highly contagious, acute infection of the Species Motilit Nitrate Oxidas Ureas BAP
y Reductio e e test growt
upper respiratory tract (URT); affects n Test h
primarily children B.
• Three stages: bronchiseptic + + + + +
a
o Catarrhal Stage B.
▪ Highly communicable stage, - - - + +
parapertussis
mucous membrane B. pertussis - - + - -
inflammation and mild coughing
with runny nose. FRANCISELLA SP.
o Paroxysmal Stage • Small, obligately aerobic, non-motile
coccobacillus

TUAZON A. 11
• Species: PASTEURELLA SP.
o Francisella tularensis (subsp. tularensis • Isolated from animal bites (mainly cats) or
(type A), subsp. holarctica (type B), scratch wounds.
subsp. mediasiatica. • Virulence Factor: Endotoxin and capsule
• Category A bioterrorism, Biosafety level 3. • Culture:
• Transmitted by: vector o BAP and CAP: colonies are gray and
• Virulence factor: capsule non-hemolytic; Most specie DO NOT
• Vector: Deer flies and ticks GROW on MAC
• Reservoir: Cottontail rabbit • Biochemical Test:
o Positive: Oxidase, Catalase and Indole;
• Francisella Weakly glucose fermenter
tularensis colonies grown PASTEURELLA MULTOCIDA
on chocolate agar. After • Culture: “mushroom smell”
72 hours of incubation, • Grows only on BAP and susceptible to penicillin
gray-white, raised colonies • Biochemical Test: Positive: oxidase, OD,
with a smooth appearance indole and urease; Negative: ONPG
are visible. PASTEURELLA BETTYAE
• Glucose and fructose fermenter
• Can grow on MAC
FRANCISELLA TULARENSIS • Biochemical Test: Catalase (+); variable indole
• Microscopy: Gram negative bacilli with faint
bipolar staining
• Culture: round smooth, bluish gray to white and
slightly mucoid
• Growth Factor: Cysteine or cystine and
Thiosulfate
• Biochemical test: Weak-positive in catalase; (-)
oxidase.
• Serological Test: An agglutination titer of 1:40
is diagnostic.
• Specimen: Scrapings from infected ulcers,
lymph node biopsy and sputum
• Disease: Tularemia (deer fly or rabbit fever)
zoonotic disease can be acquired through
ingestion of, contact (arthropod bite) with or
inhalation of air around the infected tissue or production.
carcasses. LEGIONELLA SP.
• Lab Diagnosis: • Faintly staining, thin, gram-neg, bacillary or
o Gram Stain: requires acridine orange coccobacillary
o Culture: CAP, MTM, non-selective • Fastidious, Aerobic, motile and non-
buffered charcoal yeast extract (BCYE), carbohydrate fermenting
MHA and TSB. NOT MAC • Species
o Not enhanced by incubation with o Legionella pneumophila
increased CO2, 2 to 4 days for colony o Legionella micdadei (Pittsburg
growth. pneumonia Agent)
PASTEURELLA (ZOONOTIC BACTERIA) o Legionella bozemanii (WIGA Agent)
• Small, straight, Gram-negative bacilli with o Legionella dumoffii
safety-pin appearance, facultatively anaerobic • Only genus in the family Legionellaceae
and non-motile • Primarily acquired through inhalation
• Species • Culture:
o Pasteurella multocida o BCYE: appear sticky and exhibit a
o Pasteurella stomatis ”rainbow color”
o Pasteurella dagmatis • Biochemical Test
o Pasteurella bettyae o (+) catalase and gelatinase; weak-
o Pasteurella canis positive in oxidase
• Major reservoirs: Hot water system, cooling
towers and evaporative condensers.

TUAZON A. 12
 CHARACTERISTICS OF LEGIONELLA:
• They can infect and multiply within some free-
living amoebe/amoeba (species of Hartmanella,
Acanthamoeba and Naegleria), ciliated protozoa
(Tetrahymena), and biofilms.
• Isolated from lakes, rivers, hot springs and mud
• They can tolerate up to 3 mg/L of chlorine, and
thus resist water disinfection treatments.
• Cannot grow on routine media: BAP

LEGIONELLA PNEUMOPHILA o Selective Media: BCYE with L-cysteine,

• Medium: BCYE w/ L-cysteine (pH 6.9) ferric salt and a-ketoglutatrate
• Agent of Legionnaire’s disease (1,4 and 6) and • Serological Test
Pontiac fever. o Indirect Fluorescent Antibody (IFA) –
• Invades the bronchoalveolar macrophages, most common method for Legionnaire’s
which is a facultative intracellular pathogen. disease.
• Isolated in air-conditioned units, cooling towers, o Direct Fluorescent Antibody:
humidifiers and nebulizers. detecting common Legionella species in
• Serogroups: 1 to 7 LRT. (+) yellow or green-colored bacilli.
• Culture: blue-green, glistening • Rapid methods
• Primary Clinical Manifestation: o DNA test (PCR)
o Legionnaire’s disease o Urine antigen test: detects L.
▪ Aka: legionelliosis (febrile and pneumophila as early as 3 days from
pneumonic illness) infection.
▪ MOT: Airborne spread, LESSON 2: AEROBIC GRAM-POSITIVE
inhalation of aerosol BACILLI
▪ Symptoms: High fever, non-
productive cough, headache,
neurological and severe
bronchopneumonia
o Pontiac fever
▪ Non-fatal respiratory infection
▪ Main symptom is pneumonia.
o Wound abscess and encephalitis

LEGIONELLA SP.
LABORATORY DIAGNOSIS:
• Staining
o Faint staining, undetectable in Gram
staining.
o Prolonging smear for 10 minutes
o Legionella: intracellularly and
extracellularly located in phagocytes
o L. micdadei: weakly acid-fast
• Culture

TUAZON A. 13
 • ”string of pearl appearance” – when penicillin is
used (Susceptible).
• Related disease: Anthrax
LABORATORY DIAGNOSIS
• Specimen: Malignant pustule, sputum and
blood
• B. anthracis: isolated from normally sterile site
such as blood, lung tissue and CSF.
• Staining:
o Used of old culture in Gram staining:
Gram variable
o Spore stains: Malachite green and
McFadyean stain
o Capsule stain: India ink (blood and
CSF)
• Large, ‘boxcar’ shaped, ± spores
o Fluorescent microscopy: rapid
o Bacillus, Clostridium
presumptive diagnosis
• Small, pleiomorphic, angular arrangements
• Culture Media:
o Corynebacterium, Listeria,
o Culture media: BAP, CAP, egg yolk
Cutibacterium, Lactobacillus
agar (EYA), Phenylethyl alcohol (PEA),
• Beading, filamentous Polymyxin-lysozyme-EDTA-thallous
o Nocardia, Actinomyces acetate (PLET), bicarbonate agar, and
BACILLUS nutrient broth.
• Spore-forming, aerobic or facultatively o Enrichment and Selective technique:
anaerobic, rod-shaped bacteria and isolated application of heat or alcohol shock
from the soil technique before plating on media
o Bacillus anthracis o PEA: recommended for identification of
o Bacillus cereus B. anthracis in fecal specimen.
o Bacillus thuringiensis o PEA and PLET: used in isolating
o Bacillus mycoides Bacillus from contaminated specimen
• Species only form endospores aerobically o EYA: determine B. anthracis has
• Motile with peritrichous flagella except for produced lecithinase
Bacillus anthracis and Bacillus mycoides o Gelatine Medium: Inverted pine tree
• Survive in extreme environment appearance.
• Microscopy: large, boxcar-shaped, gram-positive
rods with clear, unstained, central spores or
“empty spaces”
• Biochemical test:(+) catalase; ferments
glucose; hydrolyzes starch
BACILLUS ANTHRACIS
• Causative agent of anthrax • Medusa head colony of Bacillus anthracis. (left
• Grow aerobically or anaerobically photo)
• Not part of normal flora and not highly • Bamboo fishing rods/pole appearance of
contagious Bacillus anthracis. (right photo)
• Non-motile, halophilic organism can withstand
up to 7% NaCl
• Used as biological weapon of mass destruction
• Grows in low pH (<6.0) environment, produce
lecithinase and ferments glucose.
• Virulence factor: D-glutamic acid capsule and
protein exotoxins
• Microscopy: Gram-positive, large,
encapsulated and square-ended rod; has
“bamboo fishing rods” appearance on the
unstained central spore
• Culture: BAP –colonies have “Medusa head” • Beaten egg yolk appearance of B. anthracis
appearance with swirling projections; are non- ANTHRAX
haemolytic; and “beaten egg white” appearance • Disease primarily affects: goats and sheep, by
when inoculating loop is used. feeding on plants that are contaminated with the
• Growth Factor: Thiamine (B1) spores.

TUAZON A. 14
• MOA: inhalation of spores during exposure to o IP = Incubation period
infected animals and contaminated animal • Emetic Type
products, or cuts through skin. o Heat stable enterotoxins
• Exotoxins: cause signs and symptoms. o Ingestion of improperly cooked rice
THREE FORMS OF ANTHRAX:
• Cutaneous anthrax
o Acquired thru skin cuts and abrasions
o Small papule 2-5 days after exposure
o Appearance: “black eschar”, black,
necrotic and painless central area that
does not produce pus.
• Pulmonary anthrax/Woolsorter’s disease
o inhalation of spores into the pulmonary
parenchyma
o resembles URTI with mild fever,
dyspnea
o this is usually acquired when getting
sheep’s fur.
• Gastrointestinal anthrax
o spores are inoculated into a lesion on
the intestinal mucosa following their
ingestion.
o Signs and symptoms: Abdominal pain,
nausea, anorexia, vomiting and bloody
diarrhea.
DIAGNOSTIC TEST
• Ascoli Test (Precipitin test)
o Detects thermostable anthrax antigens o B. Cereus type I; IP: 1 to 5 hrs
o (+): formation of precipitin band after
less than 15 minutes
• Catalase Test
o Bacillus (catalase +); Clostridium
(catalase -)
• Oxidase Test
o Variable (anthrax)
• DFA Test (Direct Fluorescent Antibody Test)
o (+) cell wall polysaccharide and capsule
antigen.
BACILLUS CEREUS (“FRIED RICE” BACILLUS)
• Causes food poisoning due to the ingestion of • Frosted glass appearance of B. cereus
contaminated cooked rice dishes or other food OTHER BACILLUS
products • Bacillus subtilis (Hay Bacillus)
• Commonly encountered Bacillus in opportunistic o Most commonly encountered laboratory
infections that causes eye and ear infection. contaminant
• Exhibits motility and resistant to penicillin o Halophilic organisms, up to 7% NaCl
• BAP: large and feathery; spreading growth; o Source of Bacitracin antibiotics
have a frosted-glass appearance and b- o Cause: Eye infection among prohibited
haemolytic drug users
• Biochemical Test: Ferments salicin; (+) o BAP: large, flat, and dull with ground
lecithinase
• Virulence factors: Enterotoxins (heat-stable
and heat-labile), cerelysin, phospholipase C and
pyogenic toxin.
• Best specimen for testing: Suspected food
(>105cells/gram)
TWO TYPES OF FOOD POISONING OR
GASTROENTERITIS:
• Diarrheal Type
o Heat labile enterotoxin
o Ingestion of contaminated meat, poultry
and vegetables. IP: 8 to 16 hrs.

TUAZON A. 15
 glass appearance; maybe b-haemolytic • “club shape” or coryneform
and exhibit pigment (pink, yellow, • Cells are arranged singly, in “palisades” of
orange or brown) parallel cells or in pairs of cells connected after
o Biochemical test: Ferment mannitol, cell division to form V or L shapes: “Chinese
xylose and arabinose letters” appearance
• Bacillus pumilus • Diphtheroid, meaning “diphtheria-like”
o biological indicator: sterilization method • Specie causing human infections: C.
o BAP: large and moist; blister-like diphtheriae, C. jeikeium, C. pseudotuberculosis,
appearance and b-haemolytic C. pseudodiphthericum, C. ulcerans, and C.
• Bacillus thuringiensis urealyticum
o insect pathogens
o produces parasporal crystals that can
be utilized as pesticide.
CORYNEBACTERIUM
• Catalase positive, gram-positive rods
• Non–acid-fast, non–spore-forming, and mostly
non-branching rods
• Lipophilic Corynebacteria
o C. jeikeiumand C. urealyticum
• Non-Lipophilic Corynebacteria CORYNEBACTERIUM DIPHTHERIAE
o C. diphtheriae, C. pseudotuberculosis, • AKA: diphtheria bacillus or Kleb-Loffler
C. pseudodiphtheriticum, C. striatum, C. bacillus
• Facultative anaerobe, Inhabits the human
nasopharynx in carrier
• MOA: inhalation of contaminated respiratory
droplets or direct contact with infected
cutaneous lesions.
• Readily killed by heat and by most of the usual
disinfectants.
• Glucose and maltose fermenter
• Virulence factor: Diphtheria toxin
• Preferred medium: Enriched medium with
serum, cysteine and potassium tellurite
• Microscopy:
o Rounded ends and “club-shaped
swelling”
o highly pleomorphic cells are arranged in
ulceransand C. xerosis pairs and create X, V, Y and L formation
that closely resembles Chinese letters.
• C. diphtheriae growing on sheep blood agar.
• Biochemical Test: (-) Urease; (+) Nitrate
CORYNEBACTERIUM
reduction.
• Corynebacteria can be divided into non lipophilic
• Best specimens: Nasopharyngeal and throat
and lipophilic species
swabs
• Majority of the species are found as indigenous
THREE BIOTYPES OF CORYNEBACTERIUM
microbiota of the skin and mucous membrane of
humans and animals. Species are pathogenic to DIPHTHERIAE
animals, plants, and humans. • Intermedius
• Species are found in fresh water. o Very small, flat, dry, and grayish-black
Corynebacterium are also found in salt water, colonies; non-haemolytic
soil, and air. • Mitis
• Species are non-motile, non-encapsulated, non- o Small, black and convex that have
spore forming and highly theomorphic rods. “fried-egg appearance”
• Specie are glucose and maltose fermenters • Gravis
except for C. pseudodiphthericum and C. o Large, flat and dark gray colonies
urealyticum. “daisy-head appearance”
• Corynebacteria are slightly curved, gram-
positive rods

TUAZON A. 16
 • Albert stain used for the visualization of
metachromatic granules which is also known as
Much granules.
DIPHTHERIA TOXIN
• Heat-labile
• Produced by strains with a lysogenic b-phage
that carries the TOX gene.
• Tissue necrosis and exudates formation
(psedomembrane lining) over the tonsils, larynx
and pharynx
• Alkaline pH (7.8 to 8.0), aerobic environment
and sufficient amount of iron in the medium.
o Necrosis caused by exotoxin
RESPIRATORY DIPHTHERIA o Adherent, bleeds if scraped
• Acute, infectious disease characterized by o Severity correlates with disease severity
production of systemic toxin and false o May lead to airway obstruction
membrane lining (Pseudomembranous • Management
formation) of the throat mucous membrane → o Airway support if needed
respiratory obstruction. o Hospitalization and isolation
• S/Sx: Low grade fever, Thick mucopurulent o Equine serum diphtheria antitoxin
nasal discharge and cough (consider)
• Control: Immunization (DPT vaccine o Antibiotics (penicillin or erythromycin)
[Diphtheria, pertussis, tetanus vaccine]) o Immunize close contacts
• Cutaneous or Skin Diphtheria (Aka Veldt sore
or Barcoo rot):
o Slow healing ulcer and membrane
formations; less severe.

• Pseudomembrane (left photo)

• Veldt sore or Barcoo rot (right photo)
C. UREALYTICUM
• Most frequently isolated and most clinically
significant.
• Urinary pathogen, strict aerobe and lipophilic
• Do not ferment glucose and maltose
• Microscopy: V shaped and palisades
Corynebacterium diphtheriae • Culture: BAP –pinpoint, white smooth, non-
• Club looking bacteria haemolytic
• Causes diphtheria • Urease producer
o Infection with a characteristic tough • Most frequent isolated and most clinically
leathery membrane that forms in the significant among them all.
pharynx. C. PSEUDODIPHTHERITICUM
• Four main subspecies • Normal flora of nasopharynx
o C. diphtheriae mitis • Respiratory infection, UTI, cutaneous wound
o C. diphtheriae intermedius infection in immunocompromised.
o C. diphtheriae gravis • Microscopy: arranged in parallel rows or
o C. diphtheriae belfanti palisades and do not exhibit any other
* bold font subspecies are the most common characteristics “pleomorphism” that is similar to
other.
• Urease and nitrate positive
• Pseudomembrane C. JEIKEIUM

TUAZON A. 17
• Skin normal flora in inguinal, axillary and rectal • Microscopic Loeffler methylene blue stain of
sites. Corynebacterium spp. (right)
• Obligate aerobe and antibiotic resistant
• Immunocompromised, Common cause of
diphtheroid prosthetic valve endocarditis in
adults.
• Microscopy: Pleomorphic, club-shaped and
arranged in V-shaped
• Urease and Nitrate negative
C. ULCERANS
• Animal contact and unpasteurized dairy
products.
• Skin ulcers and exudative pharyngitis
• Associated with diphtheria-like sore throats. LABORATORY DIAGNOSIS OF
• Culture: BAP-narrow zone of b-hemolysis CORYNEBACTERIUM
• CTBA - brown halo CULTURE
• Loeffler’s serum agar: Exhibit growth • Cystine Tellurite Blood Agar
• (+): Urease and gelatinase; (-) nitrate reduction. o Preferred medium
C. PSEUDOTUBERCULOSIS for isolation and
• Animal pathogens that human can contract thru identification of
direct contact with infected animals. crynebacterium
• Dermonecrotic toxin causes death of various cell o Modification of
types the Tinsdale agar
• Diphtheria toxin (causative agent) o Selective and
Differential
• Culture: CTBA –black color and surrounded by
o Contains sheep blood, bovine serum,
brown halo
cysteine
• BAP: small and yellowish-white
o (+): Colonies of corynebacterial exhibit a
• (+) urease; (-) gelatinase black or brown color after 48 hours
LABORATORY DIAGNOSIS o (+): C. diphtheriae, C. ulcerans and C.
• Specimen: Nasopharyngeal and throat swabs, pseudotuberculosis.
urine, blood and wound discharge • Tinsdale Agar
• Calcium alginate swab: Preferred for o Sheep blood,
collection. cystine, potassium
• Staining tellurite and sodium
o Gram positive, short or slightly curved thiosulfate
rods. o (+) Black color
o Various arrangement of C. diphtheriae surrounded by halo
are due to incomplete fission during o (+) all bio types of C. diphtheriae
multiplication. • Christensen Urea Slant
o Methylene blue staining: beaded o Urea production: C. urealyticum
formation • Pai’s Slant or Loeffler’s Serum Agar
o Neisser and Albert stain: o Microscopic and
Methachromatic granules metachromatic
• Culture: granules of C.
o BAP, CAP, CTBA, Tinsdale agar and diphtheriae.
Loeffler serum agar o (+): C. diphtheriae
o Tinsdale agar and CTBA: incubated for exhibit “poached-
atleast 48 hours at 35C to 40C egg” appearance
o Metachromatic granules of C.
diphtheriaeare called Babes-Ernst
bodies
• Schick Test
o Used to determine the susceptibility of a
person to diphtheria.
o Involves the intradermal introduction of
a small amount of the diphtheria toxin
into the arm of the suspected individual.
o (+): Redness and Swelling around the
• Microscopic Gram stain of diphtheroid (left) site.

TUAZON A. 18
 o Give 0.1 ml of diluted (1/50 MLD) LISTERIOSIS
diphtheria toxin → inject intradermally → • Infection affects neonates, pregnant women and
check reaction after 2-4 days (presence immunocompromised.
of redness and swelling) • Meat products should be thoroughly cooked or
• Toxigenicity heated before consumption
o Immunodiffusion Test - Elek’s Test • After ingesting contaminated dairy products like
▪ (+): 4 mm to 5 mm fine precipitin cheese
lines at 45 angle to the streaks. • Infected pregnant women may pass the
o Guinea pig lethal Test organisms onto the fetus
▪ AKA: diphtheria antitoxin test (in
vivo test) TYPES OF LISTERIOSIS:
o Tissue Culture Test • Maternal Disease (Pregnancy)
▪ detection of diphtheria antitoxin o During 3rdtrimester of pregnancy
(in vitro) o Miscarriage or stillbirth
o Polymerase Chain Reaction o S/Sx: Flu-like illness, fever, myalgia,
▪ used for detection of toxin • Neonatal Disease
o Associated with an intrauterine infection
due to the aspiration of infected amniotic
fluid
o Infected infants are at full term and
appear healthy at birth.
o Lead to meningitis that is usually seen in
3rd week of life
genes
• Disease Of Immunocompromised
LISTERIA o Develops through the ingestion of
• Coccobacillary in form and are arranged singly contaminated dairy products and
or in short chains that resemble streptococci processed meat products.
• Culture: small, smooth, translucent, grayish- LABORATORY DIAGNOSIS
blue and are surrounded by a narrow zone of b- • Specimen: Blood, CSF, and swabs of lesion
hemolysis.
• Motility Test:
LISTERIA MONOCYTOGENES o Wet mount/hanging drop method:
• Both human and animal pathogens Exhibits a “tumbling motility” at room
• Aerobic or facultatively anaerobic and non-spore temperature
forming o SIM: Has an “umbrella-shaped” or
• Motile with peritrichous flagella and exhibits a “inverted-Christmas-tree” pattern at 25C
characteristic “tumbling motility” but not at 35C
• Grow high salt medium with up to 10% NaCl • Culture Test:
• Optimal growth: 30C to 37C but can grow at 4C o BAP, CAP, BHI, Thioglycolate medium,
(cold temp) McBride agar and Nalidixic acid medium
• Recovered from the soil, dust, water, dairy o Enrichment technique: Cold enrichment
products and processed meats (4C using broth)
• Causes miscarriage or stillbirth in humans o Colonies and haemolytic pattern may be
• Virulence factor: confused with those of group
o Listeriolysin O (hemolytic and cytotoxic) streptococci
o Catalase, o Growth: 0.5C to 45C
o Superoxide dismutase (SOD), o Requires: Slightly increased amount
o Phospholipase C and of CO2
o p60 (surface protein) • Biochemical Test:
• MOA: ingestion of contaminated food such as o Positive: Glucose fermentation,
meat, chicken, dairy products and vegetables. catalase and motility, CAMP reaction –”
• Infection: Listeriosis block-type” hemolysis, Hippurate and
Bile esculin hydrolysis, growth in 6.5%
NaCl, VP, MR
o Negative: H2S, nitrate, urease
• (Left photo) Listeria with b-hemolysis
• (Right photo) Block hemolysis using the CAMP
test.
• Umbrella motility and the inverted christmas tree

TUAZON A. 19
 appearance Listeria + + + + +
• Left tube is positive, right tube is negative. monocytog
enes
Corynebact
+ - V V V
ERYSIPELOTHRIX RHUSIOPATHIAE erium spp.
• Thin, pleomorphic rods with the tendency to Streptococ - - - + V
form long filaments that are arranged in single, cus
short chains or in a V-shaped formation. agalactiae
• Facultatively anaerobic, non-motile and non- Enterococc
-* + - V +
spore forming, Gram positive, rod-shaped us spp.
bacterium and positive H2S. • Listeria (left)
• Isolated from wild and domestic animals like • Erysipelothrix (right)
birds and fish. ARCANOBACTERIUM
• Major reservoir: Domestic swine • Classified as pleomorphic, non-motile, rod-
• Gelatin stab culture: Has a pattern of a “pipe-
cleaner” or a “test tube brush” at 22C
• Related infections and disease: Endocarditis,
septicemia and erysipeloid (red-skin infection).
• MOA: Direct contact with infected excreta, blood
and flesh of animals through skin breaks.
• Predisposed: Veterinarians and fish handlers
• Lab Diagnosis:
o Best specimen: Tissue biopsies or
aspirates from the skin.
o Localized in the deeper layer of shaped and Gram-positive
subcutaneous tissue • BAP: colonies have a narrow zone of b-
• Culture hemolysis; exhibit pitting of the agar with a black
o BAP, nutrient broth, Columbia CAN opaque dot.
agar. • Species: A. haemolyticum, A. pyogenesand A.
o BAP: pinpoint with a-haemolytic or non- bernardiae
haemolytic zone. • Biochemical: Catalase Positive
• Biochemical Test: • A. haemolyticum: lipase and lecithinase
o (+): H2S production in a TSI medium, positive and Positive reverse CAMP reaction
glucose and lactose fermentation. due to Phospholipase D.
o (-): Catalase, oxidase, esculin
hydrolysis, nitrate reduction, VP and

KURTHIA
urease. • Obligate aerobe, gram
• Erysipeloid (red-skin infection) (left photo) positive rods, Motile by
• Test tube brush or pipe-cleaner appearance peritrichous flagella
(right photo) • Culture:
SUMMARY o BAP: Large
“medusa-head” appearance
o Nutrient agar: Exhibit a Rhizoid growth
• Biochemical Test: Catalase Positive; Negative:
Gelatinase and Oxidase
GARDNERELLA VAGINALIS
DIFFERENTIATION OF LISTERIA MONOCYTOGENES • Short, pleomorphic gram-positive rod or
AND OTHER GRAM-POSITIVE BACTERIA coccobacillus that often stains gram-variable or
Gro gram-negative.
Esculi
Catal n Motil
b- wth • G. vaginalis (causative agent) → bacterial
Organism hemol in vaginosis (BV) in humans
ase Hydrol ity
ysis 6.5 • BV is characterized by a malodorous discharge
ysis
NaCl and vaginal pH greater than 4.5.

TUAZON A. 20
• BV reduction in the Lactobacillus in the vagina, • Found in the soil and organic materials
followed by an increase in vaginal pH • Can cause disease to humans and many
• The observation of “clue cells,” large squamous animals
epithelial cells with gram-positive and gram- • Culture: Cells elongate to form branching,
variable bacilli and coccobacilli clustered on the filamentous forms while some organisms form
edges, aids the diagnosis of BV hyphae on the agar surface or into the agar
• Lab Diagnosis: o Hyphae is usually present only in fungi.
o Best specimen: Vaginal discharge o Aerobic actinomycetes is considered
collected from suspected BV. before as fungi.
o G. vaginalis grows best in 5% to 7% ACID-FAST AEROBIC ACTINOMYCETES:
CO2 at a temperature of 35°C to 37C. NOCARDIA
o The medium of choice for G. vaginalis • Partially acid-fast, obligate aerobic, non-motile
is human blood bilayer Tween (HBT) and catalase positive
agar. • Microscopy: Gram negative bacilli with long,
o V (vaginalis) agar also contains human thin, beaded, branching filaments; old cultures
blood and is used for recovery of this may fragment into short, rod-shaped, coccoid
organism. forms
o When cultured on human blood, • Cell wall contains peptidoglycan, meso-
colonies are b-hemolytic, small, gray, diaminopimelic acid (DAP) and sugars such as
arabinose and galactose
• Grow on media that are used to recover fungi
• Culture: Exhibit wrinkled, chalk-like and orange-
tan pigmentation
• Resistant to lysozyme
NOCARDIA
• Related Infection:
o Acquired thru the inhalation of the
and opaque. organisms that are present in dust and
SUMMARY soil.
o Nocardia: cause invasive pulmonary
infections with hematogenous
dissemination throughout the body.
AEROBIC ACTINOMYCETES
• Actinomycetoma (Actinomycotic mycetoma)
• Gram positive bacilli, Aerobes with a branching
o Chronic, localized, painless
filamentous growth that extends along the agar
subcutaneous infection that is
due to the substrate hyphae and into the agar
characterized by the presence of sulfur
granules in the affected tissue
o Nocardia brasiliensis
• Pulmonary disease
o Confluent bronchopneumonia where the
sputum is thick and purulent although
the encapsulation of the abscess is
absent.
o Affected tissue do not have sulfur
granules.
o Nocardia cyriacigeorgica and Nocardia
farcinica.

• (left photo) Appearance of sulfur granule

collected from draining sinus tracts. These
granules contain masses of filamentous
organisms with pus materials. The arrow points
out eosinophilic projections (clubs) characteristic
due to the aerial hyphae. of sulfur granules from gram-positive bacteria.
• Filamentous rods with beaded appearance

TUAZON A. 21
• (right photo) Colony morphology of Nocardia on • Immunocompromised individuals (HIV patient)
chocolate agar. and cause slowly, progressive, granulomatous
LABORATORY DIAGNOSIS pneumonia.
• Specimen: Biopsy or drainage material • Microscopy: Coccobacilli with a ”zigzag pattern”
• Pus and tissue specimen: best samples in and a filamentous form
performing a wet mount and staining. • Culture: BAP: colonies exhibit a pale pink or
• Staining Technique: yellow color
o Modified Ziehl-Nielsen or Kinyoun stain • Differential Test:
o Gomori’s methenamine-silver stain Susceptible to lysozyme
• Culture: GORDONIA
o BAP, CAP, BHI, TMA, LJ medium, SDA • Genus varies from Gram
without Chloramphenicol, Middlebrook positive to Gram
media, Litmus milk variable rods.
o Nocardia sp. are b-haemolytic • Partially acid-fast, non-motile and catalase
o Incubation at 10% CO2 promotes the positive
growth of Nocardia species. • Culture: Smooth and slimy with irregular edges;
o May not always survive the but may appear as dry or rough; and exhibit the
decontamination procedures for presence of mycelia.
mycobacteria. • Differential Test: Susceptible to lysozyme
o Tap Water Agar: observe the TSUKAMURELLA
morphology of actinomycetes and • Genus are Gram positive and shaped like long
differentiate branching Nocardia sp. rods that fragments into three parts.
from non-branching Rhodococcus sp. • Members are Slightly acid-fast when the
Kinyoun method is used
• Culture: Colonies are circular with rhizoid
edges; has no aerial hyphae ad exhibit white or
orange pigmentation.
NON-ACID FAST, AEROBIC GRAM-POSITIVE
ACTINOMYCETES
• (left photo) Gram staining of Nocardia
demonstrating irregular staining.
STREPTOMYCES
• Gram-positive and rod-shaped with branching
• (right photo) Acid-fast staining of Nocardia
showing partially acid-fast appearance filaments
• Found in soil
RHODOCOCCUS, GORDONIA AND
• Human pathogens: Streptomyces somaliensis
TSUKAMURELLA
(Agent of Actinomycotic mycetoma)
• Aerobic, catalase-positive, branching,
• Differential Test: Susceptible
filamentous, Gram-positive bacteria that can
to lysozyme
fragment into rods and cocci
• Culture Media: BHI agar and
• Isolated in soil, fresh and marine water and
organic matter
• Primarily acquired through inhalation
• Grow on most of the non-selective media for
bacterial, mycobacterial and fungal isolation
• Infection and disease: Skin infections,
pneumonia, peritonitis, catheter-associated
sepsis
RHODOCOCCUS EQUI SDA
• Non-motile and • Culture: Colonies are dry to chalky and heaped;
partially acid-fast and some exhibit a grayish white color and “musty
is composed of basement odor”
mycolic acid with ACTINOMADURA
longer carbon chains • Microscopic and colony
• Can persist and morphology of the
replicate within species of this genus is
macrophages very similar to that of
• Acquired through respiratory route and exposure the Nocardia species.
to infected animals
• Fungal wound infection that is known as eumycetoma.
• Most common form of eumycetoma is known as mycetoma pedis → Madura foot
• Species: Actinomadura madurae and A. pelletieri

TUAZON A. 22
 • Culture: “Molar tooth” appearance
TROPHERYMA WHIPPLEI
• Gram positive actinomycete and facultative, intracellular pathogen
• Etiologic agent: Whipple’s disease
• Affects the gastrointestinal tract, joint and muscles and is characterized by abdominal pain and diarrhea.
• Isolated from human feces, saliva and gastric secretions
• Diagnostic Test: (+) Periodic Acid-Schiff Staining (PAS) macrophages.
WHIPPLE’S DISEASE
1. Encephalopathy (inflammation of brain)
2. Lymphadenopathy
3. Malabsorption and diarrhea
4. Arthritis
5. (+) PAS stain (Periodic acid–Schiff)
6. Demonstrating a tropherphyma whippli bacilli within the macrophages

LESSON 3: MYCOBACTERIA ii. Group II – scotochromogens, produces

A. Mycobacterium tuberculosis complex pigment with and without the presence
i. Mycobacterium tuberculosis of light.
ii. Mycobacterium bovis iii. Group III – non-photochromogens, does
iii. Mycobacterium africanum not produce any kind of pigment.
iv. Mycobacterium canetti ➢ Group I, II, III are slow
v. Mycobacterium microti growers.
B. Non-tuberculous mycobacteria iv. Group IV – rapid growers
i. Group I – photochromogens, produces MYCOBACTERIA
pigment when there is light, no light = no • Slender, slightly curved or straight, rod-shaped
pigment (photo – light, chromo – organisms
pigment) • Non-motile and do not form spores

TUAZON A. 23
 • The cell wall has extremely high lipid content; o Almost any organ of the body can be
thus, mycobacterial cells infected by M. tuberculosis
• Resist staining with commonly used basic • Pleurisy, an unexplained pleural effusion with
aniline dyes, such as those used in the Gram mononuclear pleurocytosis, manifests as cough,
stain, at room temperature. fever, and chest pain, resembling the
• Take up dye with increased staining time or presentation of bacterial pneumonia.
application of heat but resist decolorization with o water or pleural effusion in the lungs
acid-ethanol and upon examination through
• This characteristic is referred to as acid paracentesis (getting fluid in the lungs
fastness—hence, the term AFB—and is and observe the presence of
• Mycobacteria are strictly aerobic, but increased mononuclear pleurocytosis)
carbon dioxide (CO2) will enhance the growth of • Lymphadenitis – inflammation of the lymph
some species. nodes
• Cause pulmonary tuberculosis. • Genitourinary TB
• Studied using AFS (Acid Fast Staining) • Skeletal TB of the spine is referred to as Pott
• Important component is the Mycolic acid disease.
(targeted by the antibiotics that are used in o Positive in TB, skinny, and not able to
Mycobacteria) stand up
• Antibiotics are Isoniazid, Rifampin • Meningitis
MYCOBACTERIUM TUBERCULOSIS COMPLEX • Cerebrospinal fluid (CSF) examination usually
• Consists of M. tuberculosis, M. bovis reveals an elevated protein level, decreased
• (including the vaccination strain bacillus glucose level, and a predominance of
Calmette-Guérin), M. africanum, M. canettii, and lymphocytes.
M. microti. M. africanum • LJ medium (malachite green,
MYCOBACTERIUM TUBERCULOSIS egg white)
• Mycobacterium tuberculosis
• TB is usually a disease of the respiratory tract.
is characterized as cauliflower
• Tubercle bacilli are acquired from persons with
appearance.
active disease who are excreting viable bacilli by
sneezing or talking. • Colonies are typically raised,
with a dry, rough appearance.
• Hard tubercle or granuloma may be formed
• The colonies are
• The granuloma is an organization of
nonpigmented and classically
lymphocytes, macrophages, fibroblasts, and
described as being buff-colored
• capillaries.
• Elaboration of cord factor can result in
• With granuloma formation, healing occurs, as
characteristic cord formation.
well as fibrosis, encapsulation, and calcification,
• Optimal growth occurs at 35° C to 37° C.
with scar formation as a reminder of the past
infection. • Test results:
o Positive for niacin accumulation
o Granuloma treatment is from 9-12
o reduction of nitrate to nitrite
months
o Production of catalase
• In infected individuals, there is a potential for
o Grows on thiophene-2-carboxylic acid
reactivation of TB.
hydrazide (T2H)
TESTS USED IN PULMONARY TUBERCULOSIS
TREATMENT
• Clinical diagnosis of primary TB: Screening
• For pulmonary TB, treatment typically involves a
test or the positive PPD (purified protein
9-month course of therapy with isoniazid and
derivatives) skin test also known as Mantoux
rifampin, usually once per day the first month
skin test.
and twice a week thereafter.
• Other tests are X-ray, culture of the sputum,
o for severe/chronic TB, therapy is 12
AFS using the sputum.
months
• Diagnosis is confirmed by stained smear and o isoniazid and rifampin are the first life of
culture of sputum, gastric aspirates, or drugs for Pulmonary Tuberculosis.
bronchoscopy specimens. o Isoniazid inhibits mycolic acid.
• Extrapulmonary Tuberculosis Rifampin inhibits RNA polymerase.
o Aside from your lungs, it can also infect • Regimens also include a 2- to 8-week initial
other organs through hematogenous course of streptomycin or ethambutol
spread (spread through blood
• Pyrazinamide (PZA) may be added to the
circulation)
regimen if there is a suspicion of lowered cellular
• Miliary TB refers to the seeding of many organs immunity and a need to obtain bactericidal levels
outside the pulmonary tree with AFB through of antimycobacterial activity intracellularly in
hematogenous spread. (sesame seeds) macrophages.

TUAZON A. 24
• MDR-TB (multi drug resistant TB) is defined as MYCOBACTERIUM AVIUM SUBSP.
resistance to at least isoniazid and rifampin PARATUBERCULOSIS
o Resistance to ethambutol and • Causative agent of Johne disease, an intestinal
pyrazinamide only is not considered infection occurring as a chronic diarrhea in
MDR. cattle, sheep, goats, and other ruminants.
• Extensively drug-resistant TB (XDR-TB) is • very slow growth rate (3 to 4 months)
defined as resistance to isoniazid and • Needs mycobactin-supplemented medium for
rifampin plus resistance to any primary isolation.
fluoroquinolone and at least one of three MYCOBACTERIUM GENAVENSE
injectable second-line anti-TB drugs—the • cause of disseminated infections in patients with
aminoglycosides amikacin, kanamycin, or AIDS
capreomycin.
• enteritis and genital and soft tissue infections
o Primary drugs: RIPES (Rifampin,
• Middlebrook 7H11 agar supplemented with
Isoniazid, Pyrazinamide, Ethambutol,
mycobactin.
Streptomycin)
o Secondary drugs: Aminoglycosides • heat-stable catalase, pyrazinamidase, and
Amikacin, Kanamycin, or Capreomycin. urease
MYCOBACTERIUM BOVIS MYCOBACTERIUM HAEMOPHILUM.
• TB primarily in cattle but also in other ruminants, • Submandibular lymphadenitis, subcutaneous
as well as in dogs, cats, swine, parrots, and nodules, painful swellings, ulcers progressing to
humans. (domestic animals) abscesses, and draining fistulas are often the
clinical manifestations.
• The disease in humans closely resembles that
caused by M. tuberculosis and is treated • A unique characteristic of this organism is its
similarly. requirement for hemoglobin or hemin for growth.
• 21 days of incubation at 37° C. • Chocolate (CHOC) agar, Mueller-Hinton agar
with 5% Fildes enrichment, and Löwenstein-
• Test results:
Jensen (LJ) medium containing 2% ferric
o niacin-negative
ammonium citrate.
o do not reduce nitrate
o do not grow in the presence of (T2H) • Optimal growth temperature is 28° C to 32° C;
thiophene-2-carboxylic acid hydrazide little or no growth occurs at 37° C. cells are
strongly acid-fast, short, occasionally curved
MYCOBACTERIUM AFRICANUM
bacilli without banding or beading, and arranged
• Causes TB in Tropical Africa
in tight clusters or cords
MYCOBACTERIUM CANETTI • M. haemophilum is under group III
• Associated with AIDS patients MYCOBACTERIUM KANSASII
MYCOBACTERIUM MICROTI • M. kansasii strains have been isolated from
• Causes TB for both immunocompromised and water
competent • Infections are not normally considered
NONTUBERCULOUS MYCOBACTERIA contagious from person to person
• Slowly Growing Species • Susceptible to rifampin and ethambutol,
MYCOBACTERIUM AVIUM COMPLEX partially resistant to isoniazid and streptomycin,
• Mycobacterium avium and M. intracellulare are and resistant to pyrazinamide
part of the Mycobacterium avium complex • A multidrug regimen of isoniazid, rifampin, and
(MAC). ethambutol is currently recommended
o Also includes M. avium subsp. • Long rods with distinct crossbanding
paratuberculosis and M. avium subsp. • Colonies appear smooth to rough, with
silvaticum characteristic wavy edges and dark centers
• environmental saprophytes and have been when grown on Middlebrook 7H10 agar
recovered from soil, water, house dust, • Colonies are photochromogenic
• and other environmental sources • With prolonged exposure to light, most strains
• M. avium is a cause of disease in poultry and form dark red crystals of B-carotene on the
swine surface of and inside the colony.
• Zoonotic microorganisms • strongly catalase-positive
• the cells are short, coccobacillary, and uniformly • hydrolyze Tween 80 in 3 days
stained, without beading or banding • strong nitrate reduction
• production of a heat-stable catalase and the • Pyrazinamidase production
ability to grow on media containing 2μg/ mL of • Under Group I
T2H
MYCOBACTERIUM MALMOENSE
• Chronic pulmonary disease and cervical
lymphadenitis

TUAZON A. 25
• Resistant to isoniazid, streptomycin, p- • Young colonies grown on cornmeal agar have a
aminosalicylic acid, and rifampin and bird’s nest appearance, with characteristic
susceptible to ethambutol and cycloserine. sticklike projections.
• Short coccobacillus without cross bands on acid- • Group II
fast–stained smears. RAPIDLY GROWING SPECIES
• Colonies are smooth glistening, and opaque, MYCOBACTERIUM CHELONAE–
with dense centers MYCOBACTERIUM ABSCESSUS GROUP
MYCOBACTERIUM SCROFULACEUM • M. abscessus subsp. abscessus (formerly M.
• Cervical lymphadenitis in children abscessus), M. chelonae, and M. fortuitum.
• Group II • M. chelonae have been associated with a variety
MYCOBACTERIUM SIMIAE of infections of the skin, lungs, bone, central
• Isolated from the lymph nodes of monkeys nervous system, and prosthetic heart valves.
• Colonies on Middlebrook 7H10 agar are thin, • M. abscessus seen in patients with cystic
transparent or tiny, and filamentous fibrosis (CF)
• Group I • tap water is an important reservoir
MYCOBACTERIUM ULCERANS • positive 3-day arylsulfatase test, no reduction of
• Mycobacterium ulcerans is a rare cause of nitrate, and growth on MacConkey agar without
mycobacteriosis, also referred to as Buruli ulcer crystal violet
• Acid-fast cells are long, without beading or MYCOBACTERIUM FORTUITUM GROUP
crossbanding • M. fortuitum, M. peregrinum, and an unnamed
• Group III third species.
MYCOBACTERIUM MARINUM • Isolated from water, soil, and dust
• Mycobacterium marinum has been implicated in • Associated with localized cutaneous
diseases of fish and isolated from aquariums. infections
• Cutaneous infections in humans occur when • Middlebrook 7H11 agars after 1 to 2 days of
traumatized skin comes into contact with salt incubation colonies with branching
water or inadequately chlorinated fresh water • Filamentous extensions and rough colonies with
containing the organism. short aerial hyphae.
• Tender red or blue-red subcutaneous nodule, • Pleomorphic, ranging from long and tapered to
or swimming pool granuloma, usually occurs on short, thick rods partially acid-fast
the elbow, knee, toe, or finger • Positive 3-day arylsulfatase test and
• Susceptible to rifampin and ethambutol, reduction of nitrate
resistant to isoniazid and pyrazinamide, and MYCOBACTERIUM SMEGMATIS GROUP
partially resistant or intermediate to streptomycin • The M. smegmatis group contains two species,
• Cells of M. marinum are moderately long to long M. smegmatis and M. goodie.
rods with cross barring. • M. smegmatis has been implicated in rare cases
• M. marinum is photochromogenic; young of pulmonary, skin, soft tissue, and bone
colonies infections.
• Colonies grown in or exposed to light develop a • Cells are long and tapered or short rods with
deep yellow color. irregular acid fastness. Occasionally rods are
• Growth is optimum at incubation temperatures of curved with branching or Y-shaped forms;
28° C to 32° C. swollen, with deeper staining, beaded, or ovoid
• None reduces nitrate or produces heat-stable forms are sometimes seen.
catalase • Colonies appearing on egg medium after 2 to 4
• The organisms hydrolyze Tween 80 and days are usually rough, wrinkled, or coarsely
produce urease and pyrazinamidase. folded; smooth, glistening, butyrous colonies
• Group I may also be seen.
MYCOBACTERIUM XENOPI • Negative arylsulfatase reaction, positive iron
• Recovered from hot and cold water taps uptake, ability to reduce nitrate, and growth in
(including water storage tanks of hospitals) and the presence of 5% NaCl and on MacConkey
birds (hotspings) agar without crystal violet
• Susceptible to the quinolones (ciprofloxacin, MYCOBACTERIUM LEPRAE
ofloxacin); some isolates are susceptible to • Causative agent of Hansen disease (leprosy),
vancomycin, erythromycin, or cefuroxime an infection of the skin, mucous membranes,
• Colonies on Middlebrook 7H10 agar are small, and peripheral nerves
with dense centers and filamentous edges. • The two major forms of the disease are
• Cornmeal-glycerol agar reveals distinctive tuberculoid leprosy (localized & benign) and
round colonies with branching and filamentous lepromatous leprosy (disseminated &
extensions; aerial hyphae are usually seen in malignant)
rough colonies.

TUAZON A. 26
 • Symptoms of tuberculoid leprosy include skin o Lepromatous lion appearance
lesions and nerve involvement that can produce • Combination of diaminodiphenylsulfone
areas with loss of sensation. (dapsone), clofazimine, and rifampin
• Patients with lepromatous leprosy if untreated, • M. leprae has not been grown on artificial
life-threatening media.
o It is characterized by skin lesions and • mouse footpad or foot pad of armadillo
progressive, symmetric nerve damage. • the length of the bacillus is at least five times the
o Lesions of the mucous membranes of width of the bacillus
the nose may lead to destruction of the • Skin test:
cartilaginous septum, resulting in nasal o Lepromin test: (+) tuberculoid
and facial deformities.
CLASSIFICATION ORGANISM
M. tuberculosis
TB complex M. africanum
M. bovis
Photochromogens (Group I) M. asiaticum
M. kansasii
M. marinum
M. simiae
M. flavescens
M. gordonae
Scotochromogens (Group II)
M. scrofulaceum
M. szulgai
Nonchromogens (Group III) M. avium complex
M. celatum
M. haemophilum
M. gastri
M. genavense
M. malmoense
M. nonchromogenicum
M. shimoidei
M. terrae
M. trivale
M. ulcerans
M. xenopi
M. abscessus
M. fortuitum group
M. chelonae group
Rapid growers (Group IV)
M. phlei
M. smegmatis
M. vaccae

SPECIES ALWAYS CONSIDERED PATHOGENS

Pulmonary and disseminated tuberculosis; millions of cases
M. tuberculosis Humans
annually in the world
M. leprae Humans Leprosy
Tuberculosis-like disease rear in North-America; M. bovis is
M. bovis Humans, cattle
closely related to M. tuberculosis
SPECIES POTENTIALLY PATHOGENIC IN HUMANS
Moderately common causes of disease
M. avium complex Soil, water, birds, fowl, Disseminated, pulmonary; very common in AIDS patients; occurs
swine, cattle, in other immunocompromised patients; uncommon in patients with
environment normal immune system
M. kansasii Water, cattle Pulmonary, other sites
Rapid growers
Cutaneous lesions most common, subcutaneous abscesses,
M. fortuitum & M. Soil, water, animals,
disseminated infection; grow in ≤ 7 days; M. fotuitum is more
chelonae marine life
susceptible to antibiotics
SAPROPHYTIC SPECIES THAT VERY RARELY CAUSE DISEASE IN HUMANS
M. gordonae Water These saprophytic Mycobacterium species are very uncommon
M. flavescens Soil, water causes of disease in humans. Positive cultures for these

TUAZON A. 27
M. fallax Soil, water mycobacteria usually represent environmental contamination of
M. gastri Gastric washings specimens and not disease. Many of the saprophytic mycobacteria
grow best at temperatures ≤ 33°C. There are many other
saprophytic Mycobacterium species not listed here that seldom if
ever appear in cultures of patients’ specimens.
UNCOMMON TO VERY RARE CAUSE OF DISEASE
M. africanum Humans, monkeys Pulmonary cultures; resembles M. tuberculosis; rare
Blood in AIDS patients; grows in liquid medium (BACTEC) and on
M. genavense Humans? pet birds?
solid medium supplemented with mycobactin j; grows in 2-8 weeks
M. haemophilum Unknown Subcutaneous nodules and ulcers primarily in AIDS patients;
requires hemoglobin or hemin; grows at 28-32°C; rare
Pulmonary tuberculosis-like (adults), lymph nodes (children); most
reported cases are from Sweden but organism may be much more
M. malmoense Unknown, environment
widespread; M. malmoense is closely related to M. avium-
intracellulare; Takes 8-12 weeks to grow
M. marinum Fish, water Subcutaneous nodules and abscesses, skin ulcers
Cervical lymphadenitis; usually cured by incision, drainage, and
M. scrofulaceum Soil, water, moist food
removal of involved lymph nodes
M. simiae Monkeys, water Pulmonary, disseminated in AIDS patients; rare
M. szulgai Unknown Pulmonary, tuberculosis-like; rare
M. ulcerans Humans, environment Subcutaneous nodules and ulcers; may be severe; M. ulcerans is
closely related to M. marinum; takes 6-12 weeks to grow; optimal
growth at 33°C suggests environmental source; rare
M. xenopi Water, birds Pulmonary, tuberculosis-like with preexisting lung disease; rare
LESSON 3: MYCOBACTERIA • Biochemical Test: (-) Niacin and Nitrate
MYCOBACTERIUM TUBERCULOSIS COMPLEX reduction
• Mycobacterium tuberculosis (Koch’s bacillus)
• Mycobacterium bovis
• Mycobacterium africanum
• Mycobacterium canetti
• Mycobacterium microti
MYCOBACTERIUM TUBERCULOSIS
• Longest replication time
• Virulence factor: Cord factor
• Culture: slow-growing; buff color; raised and
dry; “CAULIFLOWER-LIKE APPEARANCE”.
Rough colonies exhibit “cording” (Curve strands
bacilli)
• Biochemical Test: (+) Niacin and Nitrate
reduction • Vaccine given after birth (right leg) BCG, (left
• Replication time: 20 to 22 hrs leg) Hepa
MYCOBACTERIUM AFRICANUM
• Associated human cases
of tuberculosis in tropical
Africa
• Detection of organism
requires the use of
spoligotyping (spacer oligotyping)
MYCOBACTERIUM CANETTI
• Smooth strain of M.
tuberculosis
• Grows more rapidly
than M. tuberculosis (6
MYCOBACTERIUM BOVIS days)
• Tuberculosis in human and animals (cattle, • Isolated from an AIDS patient with mesenteric
dogs, cat and swine) tuberculosis
• Attenuated strain used for vaccination (Bacillus- • Biochemical Test: (+) Niacin and Nitrate
Calmette-Guerin or BCG vaccine) reduction
• Culture: Slow-growing, small, granular, rounded
and non-pigmented

TUAZON A. 28
 MYCOBACTERIUM MICROTI • Cause: Buruli ulcer (painless
• Isolated from TB patients both nodule under the skin after
immunocompromised and previous trauma)
immunocompetent individuals. • Microscopy: Moderately
long rods without cross-
banding
NON-TUBERCULOUS MYCOBACTERIA • Culture: smooth, rough and non-pigmented (6-
• Group I: Photochromogens 12 weeks incubation
• Group II: Scotochromogens • Biochemical Test: (+) Heat stable catalase
• Group III: Non-Photochromogens MYCOBACTERIUM GORDONAE (TAP WATER
• Group IV: Rapid Growers BACILLUS)
NON-TUBERCULOUS MYCOBACTERIA: SLOW • Contaminates the tap
GROWERS water used by the patients
MYCOBACTERIUM AVIUMCOMPLEX in rinsing their mouths
• Specie: M. avium, M. prior to the procedure for
intracellulare, M. avium sputum collection.
subsp. Paratuberculosis • Rarely cause infection to
and M. avium subsp. human
silvaticum (wood pigeon • Culture: Smooth and yellowish-orange colored
bacillus) • Biochemical: (+) Tween 80 hydrolysis and heat-
• Most common cause of Pulmonary infection to stable catalase, (-) nitrate reduction
human, pathogen in AIDS. MYCOBACTERIUM XENOPI
• GI tract: most common site of colonization and • Recovered from hot and cold-
dissemination in AIDS. water taps, hospital storage
• Microscopy: Pleomorphic, short coccobacilli tanks
without beading; (+) PAS • Potential pathogen of
• Biochemical: (+) Heat stable catalase pulmonary infection in adults
MYCOBACTERIUM KANSASII (YELLOW • Non-photochromogenic and
BACILLUS) scotochromogenic
• Second to M. avium • Microscopy: Long and filamentous
complex to cause NTM lung • Culture: MB7H10: small with filamentous edges
disease (Chronic cavitary • Cornmeal glycerol agar: branching filaments
pulmonary lesion) • Growth: 42°C
• Not contagious from person • Biochemical: (+) heat stable catalase,
to person pyrazinamidase
• Microscopy: Long rods with distinct • M. xenopi has been classified with non-
crossbanding photochromogenic group however, colonies are
• Culture: MB7H10: smooth to rough with dark frequently bright yellow.
centers and waxy edges • Once incubated in absence of light, they will be
• Photochromogens: Dark red crystals of 10-B- scrotochromogens with yellow pigment at 42°C
carotene MYCOBACTERIUM TERRAE COMPLEX
MYCOBACTERIUM MARINUM • Normally saprophytic and rarely causes human
• Disease of fishes and isolated from aquariums infections
• Causative agent: “Swimming pool granuloma” • Species: M. terrae, M. triviale and M.
red or bluish red nodule on the elbow, knee, toe nonchromogenicum
or finger. Occurs when an open wound comes in • Microscopy: Short to medium coccobacilli
contact with contaminated chlorinated fresh • Culture:
water or salt water o M. triviale: rough and dry
• Natural Reservoir: Fresh water and salt water o M. terrae: smooth
• Microscopy: Long rods with cross barring o M. nonchromogenicum: smooth to
• Culture: smooth to tough, wrinkled, yellow rough and white to buff
• Biochemical test: (+) Tween 80 hydrolysis and
heat stable catalase, (+) growth in 5% NaCl (M.
terrae)

MYCOBACTERIUMULCERANS
• Third most common Mycobacterium species
after M. tuberculosis and M. leprae • Left: M. triviale. Right: M. terrae

TUAZON A. 29
 DISTINGUISHING
SPECIE MICROSCOPY CULTURE BIOCHEMICAL TEST
FEATURES
Saphrophytes; rarely
Dysgonic smooth and (+) High level of heat
M. asiaticum Acid-fast coccoid cells cause human
pigmented colonies stable catalase
infections
M. genavense Distinct acid-fast cells Dysgonic colonies; (+) semi-quantitative Fastidious; Do not
requires an extended and heat stable grow on routine
incubation (6-8 catalase; media; recovered in
weeks) (+) Pyrazinamidase BACTEC culture
Rough to smooth and
non-pigmented; The
Distinct acid-fast cells;
recommended media
short or curved,
include CA, MHA, Requires hemoglobin
M. haemophilum without beading,
with 5% Fildes or hemin for growth
appears clusters or
enrichement, LJ
cords
medium with 2% ferric
ammonium citrate
M. malmoense Short coccobacilli Non-pigmented, (+) Tween 80 Growth at 22C
without crossbands smooth, glistening hydrolysis, heat- requires 7 to 12
and opaque colonies stable catalase and weeks of incubation
with dense centers pyrazinamidase

OTHER NTM-SLOW GROWERS

M. simiae Short coccobacilli Filamentous colonies; (+) Niacin and high One of the very few
yellow and smooth level of heat stable NTM that produces
colonies after catalase niacin
extended incubation

Light yellow to deep (+) High-level, heat

M. scrofulaceum Medium to long rods orange colonies with stable catalase and
dense centers urease

M. szulgai Medium to long rods Yellow to orange and (+) Heat stable
with cross barring smooth to rough catalase
colonies

NON-TUBERCULOUS MYCOBACTERIA • Exhibits greater

• Group IV: Rapid Growers / Fast growers resistance to
MYCOBACTERIUM FORTUITUM antimicrobial agents
• Most common, rapidly • Microscopy: Strongly
growing mycobacteria acid-fast with
that are associated with pleomorphism in young
localized cutaneous and cultures
soft tissue infections • Culture: Rough to smooth, non-pigmented and
• Microscopy: have no filamentous branching
Pleomhorpic–long to • MAC: growth without crystal violet
short, thick rods MYCOBACTERIUM ABSCESSUS SUBSP.
• Old cultures: partially AFB ABSCESSUS
• Cultures: • Reservoir: Tap water
o MB7H11: branching and filamentous • Related infection: Chronic lung
with aerial hyphae disease and Otitis media
o MAC: growth in media without • Culture: MAC –exhibit growth in a
CRYSTAL VIOLET medium without CRYSTAL
MYCOBACTERIUM CHELONAE VIOLET
• Associated with cutaneous infections in • Biochemical: (+) 3-day
immunocompromised persons arylsulfatase; (-) Nitrate

TUAZON A. 30
 MYCOBACTERIUM SMEGMATIS o Claw-shaped hands
• Related infections: o Pendulous ear lobes
Pulmonary, skin and bone o Saddle nose
infections • Suppressed (low resistance)
• Microscopy: Long and
tapered rods with partial
acid-fastness; maybe
beaded or ovoid in form
• CULTURE:
o MB7H10: smooth or rough
o Culture: MAC –exhibit growth in a
medium without CRYSTAL VIOLET
• Biochemical: (-) 3-day arylsulfatase; (+) Nitrate
reduction
MYCOBACTERIUM LEPRAE
• Chronic skin disease, mucous membrane and
nerve tissue
• Not considered contagious disease
• MOT: Person to person contact through
inhalation (nasal secretions), Contact with
infected skin, arthropod bites and ingestion of
breast milk and transplacental transmission for
infants
• Forms: Tuberculoid (localized & benign) and
Lepromatous Leprosy (disseminated &
malignant)
• Microscopy: Rod shaped and exhibit “cigar-
pocket” or “pocket-fence”
• Skin test: Fernandez and Mitzuda Reaction
LABORATORY DIAGNOSIS
• Acid-Fast Staining (Biopsy)
• Culture: Footpads of Mice, definitive Tests
• Serological Test:
o Fluorescent leprosy antibody absorption
test
o DNA amplification
o ELISA
• Heat stable catalase: Negative

TUBERCULOID LEPROSY ((TT)

• Well demarcated, dry patch
• Minimal disfigurement
o No leonine facies
o No claw-shaped hands
o No pendulous earlobes
• Good immune response (high resistance)

LEPROMATOUS LEPROSY (LL)

• Disfigurement is there
o Leonine facies

TUAZON A. 31
 LESSON 4: ANAEROBIC BACTERIA AND RICKETTSIA AND CHLAMYDIA
ANAEROBIC BACTERIA

ENDOGENOUS ANAEROBES COMMONLY INVOLVED IN HUMAN INFECTIONS

INFECTION ANAEROBE
Actinomycosis Actinomyces israelii, other Actinomyces spp.
Antibiotic-associated diarrhea;
Clostridioides (Clostridium) difficile
• Pseudomembranous colitis
Bacteremia Bacteroides fragilis group, fusobacteria, clostridia,
peptostreptococci
Bacteroides spp., Prevotella spp., Porphyromonas spp.,
Brain abscess Fusobacterium spp., Clostridium spp. (these infections are
often polymicrobial)
Infections of the female genitourinary tract Peptostreptococci, Bacteroides spp., Clostridium spp.,
Prevotella bivia, Prevotella disiens, Actinomyces israelii
(IUD associated)
B. fragilis group, other Bacteroides spp., Fusobacterium
Intraabdominal infections, liver abscess, peritonitis, spp., Clostridium perfringens, other
perineal and perirectal infections Clostridium spp., peptostreptococci (frequently
polymicrobial)
Myonecrosis C. perfringens, Clostridium novyi, Clostridium septicum
(80%–95% of the cases)
Peptostreptococci, Porphyromonas spp., Fusobacterium
Oral, sinus, dental infections
spp. (often polymicrobial)
Aspiration pneumonia, pleuropulmonary infections Porphyromonas spp., Fusobacterium nucleatum,
peptostreptococci, B. fragilis group, Actinomyces spp.

ENDOGENOUS ANAEROBES OF VARIOUS ANATOMIC SITES

SITE ANAEROBES
Skin Propionibacterium, peptostreptococci
Peptostreptococci, Actinomyces, Propionibacterium,
Upper respiratory tract
Campylobacter, Fusobacterium, Prevotella, Veillonella
Actinomyces, Eubacterium/Eggerthella, peptostreptococci,
Oral cavity Campylobacter, Fusobacterium, Prevotella,
Bifidobacterium, Porphyromonas, Veillonella
Intestine Bifidobacterium, Eubacterium/Eggerthella, Clostridium,

TUAZON A. 32
 peptostreptococci, Bacteroides fragilis group,
Parabacteroides, Bilophila, Campylobacter, Fusobacterium,
Porphyromonas, Prevotella, Sutterella, Veillonella
Peptostreptococci, Bifidobacterium, Fusobacterium,
Genitourinary tract
Lactobacillus, Mobiluncus, Prevotella, Veillonella

POTENTIAL VIRULENCE FACTORS OF ANAEROBIC BACTERIA

ANAEROBES KNOWN OR
POTENTIAL VIRULENCE FACTOR POSSIBLE ROLE
THOUGHT TO POSSESS
Polysaccharide capsules Promotes abscess formation; Bacteroides fragilis, Porphyromonas
antiphagocytic function gingivalis
Fimbriae, fibrils enable organisms to
Adherence factors B. fragilis, P. gingivalis
adhere to cell surfaces
CLOSTRIDIAL TOXINS, EXOENZYMES
Collagenases Catalyze the degradation of collagen Certain Clostridium spp.
Cytotoxins Toxic to specific types of cells C. difficile
DNases Destroy DNA Certain Clostridium spp.
Enterotoxin Toxic to cells of the intestinal mucosa C. difficile
Lyse red blood cells liberating
Hemolysins Certain Clostridium spp.
hemoglobin
Hyaluronidase Catalyzes the hydrolysis of hyaluronic Certain Clostridium spp.
acid, the cement substance of tissues
Catalyze the hydrolysis of ester
linkages between fatty acids and
Lipases Certain Clostridium spp.
glycerol of triglycerides and
phospholipids
Neurotoxins (e.g., botulinum toxin, Destroy or disrupt nerve tissue C. botulinum, C. tetani
tetanospasmin)
Catalyze the splitting of host
Phospholipases Certain Clostridium spp
phospholipids (lecithinase)
Proteases Split host proteins by hydrolysis of Certain Clostridium spp
peptide bonds
o Non-encapsulated; except C.
GRAM-POSITIVE ANAEROBIC SPORE-FORMING perfringens.
BACILLI o Single haemolytic reaction; except C.
• Clostridum spp. perfringens
o Specie: C. perfringens, C. novyi, C. CLOSTRIDIUM PERFRINGENS (GAS
histolyticum, C. bifermentans, C. GANGRENE BACILLUS)
sordellii, C. innocuum, C. botulinum and • Most commonly isolated member
C. tetani • Virulence factor: a-toxin and enterotoxin
o Histotoxic: C. perfringens, C. novyi, C. • Microscopy: “Boxcar-shaped” bacilli with
histolyticum, C. septicum, C. subterminal spores
bifermentans • Biochemical:
• Most commonly causes myonecrosis is C. o (+) Lecithinase – EYA (egg yolk agar)
perfringens o (+) Nagler test – Lecithovitellin
CLOSTRIDIUM o (+) Reverse CAMP Test –arrowhead-
• Obligate anaerobes, catalase negative, Gram- shaped zone of hemolysis towards test
positive, spore-forming bacilli organism
• Toxins: acquired through ingestion or open • Culture:
wounds that have been contaminated with soil o BAP: dome-shaped and grayish white
• Virulence contributors: Collagenase, with double zones of hemolysis
hyaluronidase, lecithinase (soil destruction) and ▪ Alpha and beta zones (double
phospholipase zones)
• Carbohydrate fermenter; except: C. tetani and o Litmus milk: Stormy fermentation of milk
C. histolyticum • Related Disease:
• Characteristics: o Gas gangrene (myonecrosis)
o Form endospores anaerobically ▪ Blister that has water inside,
o Motile; except: C. perfringens, C. tissue necrosis
ramosum and C. innocuum. o Clostridial necrotizing enteritis or
o Have swollen sporangia except: C. Enteritis necroticans
perfringens and C. bifermentans

TUAZON A. 33
 ▪ Ingested beta enterotoxin in a • BOTULISM: double or blurred vision, impaired
contaminated food. speech, difficulty in swallowing, weakness and
▪ Bloody diarrhea, abdominal pain paralysis.
CLOSTRIDIUM TETANI (TACK HEAD o Two types of botulism:
BACILLUS) ▪ Foodborne botulism
• Endospore is usually in dust, soil, or dirt/fecal(?) • Usually due to ingestion
of animals in the farm of the preform toxin in
• Virulence factor: Tetanospasmin (neurotoxin) preserved or meat-
• Microscopy: Drumstick or tennis-racket based food or canned
appearance (terminal spores) goods.
• Culture: • Commonly caused by
o BAP: slow, anaerobic, heavy, smooth, botulism toxin A.
and swarming growth, narrow zone of b- ▪ Infant botulism
hemolysis • Actual infection caused
• Biochemical: (+) gelatinase and indole; (-) by ingesting the
Lecithinase and lipase organism from the raw
• Infection: Tetanus, Tetanus neonatorum honey or through
• Tetanospasmin: endopeptidase cleaves the breastfeeding for
synaptic vesicle membrane protein, infants.
Synaptobrevin. CLOSTRIDIUM DIFFICILE
o Cause tension or cramping and twisting • Most Common cause of antibiotic-associated
in skeletal muscles that surrounds the diarrhea and pseudomembranous colitis (bloody
wound and tightness of jaw muscles. diarrhea with necrosis of colonic mucosa).
• Tetanus: Characterized by trismus or lock jaw • Acquired in hospitals by individuals who are
and risus sardonicus or distorted grin. IP: 3 to 21 receiving antibiotics
days. • “Infection control dilemma” among hospitalized
o Symptoms: muscle rigidity, difficulty of patients.
swallowing, rigidity of the abdomen, • Ferments fructose-producing formic acid that
chest, back & limbs changes the color of medium to pink to yellow.
• Virulence factor: Toxin A (enterotoxin) and
Toxin B (cytotoxin).
• Microscopy: Chains up to 6 cells that are
aligned from end to end with oval subterminal
endospores.
• Culture:
o Cycloserine-cefoxitin-fructose agar
• (left) Gramstained appearance of terminal (CCFA) – colonies exhibit yellow color
spores of Clostridium tetani and a “ground-glass” appearance.
• (right) Gram-stained appearance of subterminal o BAP: “Horse stable” odor; non-
spores of Clostridium sordellii haemolytic and produce fluorescent
CLOSTRIDIUM BOTULINUM (“CANNED FOOD” chartreuse. (under UV)
BACILLUS)
• Usually found in soil and aquatic sediments. LABORATORY DIAGNOSIS
• Potential bioterrorism agent COLLECTION, TRANSPORT AND STORAGE:
• Presence of subterminal spores • If cannot processed immediately: should be kept
• Virulence factor: Botulism toxin in room temp.
• Culture: BAP-b-haemolytic colonies • Transport promptly to the laboratory under
• Infection: Botulism anaerobic condition or with minimal O2
• Botulism toxin: neurotoxin that is considered exposure.
as one of the most potent natural toxins known RECOMMENDED SPECIMEN FOR ANAEROBIC
to man. CULTURE
o Cleaves the synaptic vesicle membrane • Specimen must be collected at the actual site;
protein, synaptobrevin. swabbing of mucosal surface is insufficient.
o Preventing exocytosis and the release • Needle aspiration: best specimen for anaerobic
of the neurotransmitter, Acetylcholine. culture
o Small amount to produce paralysis and • Swabs: only be used when performing
death aspiration is not possible or if a biopsy specimen
o Botulism antigens/agent: A to G (7 is not available.
antigenic types) • Swab should be placed into a 0.5 mL sterile
o Cause human disease: A, B and E thioglycolate broth.

TUAZON A. 34
 • Food and fecal specimens that are suspected of DIFFERENTIAL TEST:
C. perfringens food poisoning should be • Catalase Test:
transported at 4°C. o Catalase negative; Rgt: 15% H2O2
UNACCEPTABLE SPECIMENS FOR • Direct Nagler Test:
ANAEROBIC CULTURE: o Using EYA plate and C. perfringens type
• Swabs, Sputum, Bronchial washings, Feces and A antitoxin.
effluents from ileostomy and colostomy, and o (+): Inhibition of the lecithinase reaction
gastric and small bowel contents. that is produced by C. perfringens.
GRAM STAIN • Mouse Neutralization Test
• Spores are not observed in Gram-stained o Definitive identification test for C.
smears of clinical specimens that contain botulism
clostridia, unless incubated for many days. • Reverse CAMP Test:
• C. ramnosum and C. clostridioforme: Gm (-) o Confirm the presence of C. perfringens.
o (+) Arrowhead-shaped zone of
CULTURE:
hemolysis at the intersection of the two
• Culture media: Anaerobic blood agar, streaks towards clostridium isolates,
thioglycollate, EYA, CCFA, PYG, brucella blood • Cell Culture Cytotoxicity Test
agar, PEA and CNA. o Gold standard test for detection of C.
• Transport media: pre-reduced anaerobically difficile toxin. Requires 2-3 days to
sterilized (PRAS) medium and Amies medium. achieve positive result.
• Cycloserine and Cefoxitin in CCFA: Inhibit gram- • Lipid/Lipase And Lecithinase Test
negative coliforms. o (+) Lecithinase: opaque zone
• Neutral red: pH indicator in CCFA ▪ C. perfringens, C. bifermentans,
• EYA: detect lecithinase and lipase activity. C. novyi
o (+) Lipase “Mother of pearl” app. Or
gasoline on water appearance.
▪ C. botulinum, C. novyi
PRESUMPTIVE IDENTIFICATION OF GRAM-POSITIVE ANAEROBES
COLONY MORPHOLOGY ON BLOOD SPOT
IDENTIFICATION CELLULAR MORPHOLOGY
AGAR INDOLE
Clostridium difficile Large, flat colonies; barnyard odor, Thin rods, rare spores Negative
chartreuse fluorescence
Large, irregular-shaped, double zone of β-
C. perfringens Boxcar, large, square rods Not done
hemolysis
C. septicum Smoothly swarming Thin rods, subterminal spores Negative
C. sordellii Very large, lobate, irregular, flat Thin rods, subterminal spores Positive
C. tetani Smoothly swarming but slow growing Swollen terminal spores Positive
“Peptostreptococci” Small, peaked, circular Cocci, pairs and chains Not done
Cutibacterium acnes Small, opaque, enamel white, circular Coryneform rods Positive
(catalase-positive)

ACCEPTABLE SPECIMENS FOR ANAEROBIC BACTERIOLOGY

ANATOMIC SOURCE SPECIMENS AND RECOMMENDED METHODS OF COLLECTION
Central nervous system Cerebrospinal fluid, aspirated abscess material, tissue from biopsy or autopsy
Dental, ear-nose-throat specimens Aspirated abscess material, biopsied tissue
Localized abscesses Needle and syringe aspiration of closed abscesses
Decubitus ulcers Aspirated pus
Sinus tracts or draining wounds Aspirated material
Specimens obtained during surgery from depths of wound or underlying bone
Deep tissue or bone
lesion
Pulmonary Aspirate obtained by direct lung puncture; pleural fluid obtained by
thoracentesis; open lung biopsy; sulfur granules from draining fistula
Intraabdominal Aspirate from abscess, ascites fluid, peritoneal fluid, tissue
Urinary tract Suprapubic bladder aspiration
Female genital tract Aspirate from loculated abscess; culdocentesis specimen
Other Blood, bone marrow, synovial fluid, biopsied tissue from any normally sterile
site

TUAZON A. 35
 DIFFERENTIAL CHARACTERISTICS OF ANAEROBIC NON-SPORE FORMING BACILLI AND COCCI
DISTINGUISHING
ORGANISM GRAM STAIN REACTION
CHARACTERISTICS
Actinomyces Anaerobic, straight or slightly curved, Gram- Young colonies –spider-like or wooly
positive rods that are banded or beaded appearance
Old colonies – “molar tooth”
appearance
Pale-staining, pleomorphic, Gram-negative rods Grayish-white, circular, smooth, and
Bacteroides fragilis
with a “safety pin” appearance non-hemolytic
Bacteroides ureolyticus Pale-staining, thin, Gram-negative rods Colonies corrode (pit) the agar
Gram-positive diphtheroids; that are coccoid or
Small, white, shiny and convex
Bifidobacterium spp. pointed in shape with bifurcated (forked) ends
colonies
which resemble a shape of a “dog bone”
Clostridium septicum Gram positive rods in young culture that turn Formation of Rhizoid margins that
Gram negative with age; Have subterminal resemble “medusa head”
spores
Pleomorphic, Gram-positive rods that are
Eubacterium Fluorescent chartreuse color
seagull wing-shaped
Fusobacterium nucleatum Spindle-shaped, Gram-negative rods that The medium exhibits a green color
resemble a Capnocytophaga upon air exposure; colonies have
“breadcrumb-like” appearance
Gram variable rods or short coccobacilli that
Lactobacillus Pinpoint colonies
resemble streptococci
Leptotrichia Large-fusiform Gram-negative rods “Raspberry-like” colonies
Gram positive cocci that are paired singly, pairs
Peptococcus niger Small black and shiny colonies
or in tetrads
Peptostreptococcus Large, Gram-positive coccobacilli in chains Grayish-white colonies that emits foul
anaerobius odor.
Brown, mucoid colonies with brick red
Porphyromonas Gram negative coccobacilli
fluorescence
Propionibacterium Diphtheroids-like, Gram-positive rods; that have Small, grayish-white colonies
(anaerobic diphtheroids) a palisade arrangement
White, Shiny colonies with a brick-red
Prevotella Gram negative rods
fluorescence
Veillonella parvula Tiny Gram-negative diplococci Red fluorescence

PRESUMPTIVE IDENTIFICATION OF GRAM-NEGATIVE ANAEROBES

COLONY
COLONY MORPHOLOGY ON CELLULAR SPOT
IDENTIFICATION MORPHOLOGY ON
BLOOD AGAR OR KVLB AGAR MORPHOLOGY INDOLE
BBE AGAR
Bacteroides fragilis Large (>1 mm) Large, convex, black Regular Not done
group gray
Campylobacter Tiny rods or
Translucent, pitting (some) No growth Negative
ureolyticus coccobacilli
Bilophila Tiny, translucent Translucent, with Regular to filamentous Negative
wadsworthia black center at 72 h
Fusobacterium Fusiform, thin with
Ground glass or breadcrumb No growth Positive
nucleatum pointed ends
Porphyromonas Small, translucent or opaque, No growth Tiny coccobacilli Positive
brick red fluorescence on blood
agar, no growth on KVLB agar
Small, translucent or opaque,
Prevotella brick red
No growth Tiny coccobacilli Negative
intermedia fluorescence on blood agar and
KVLB agar
Veillonella Small, translucent or opaque, No growth Tiny diplococci Negative
brick red
fluorescence on blood agar, no
growth on
KVLB agar

TUAZON A. 36
 CHARACTERISTICS OF SOME CLINICALLY ENCOUNTERED ANAEROBIC COCCI

OTHER MICROORGANISMS PROPIONIBACTERIUM ACNE

BACTEROIDES FRAGILIS • Indigenous microbiota of the skin
• Most commonly isolated anaerobes from blood • Frequently isolated from blood culture
cultures LACTOBACILLUS
• B-Lactamase producer, non-motile and • Pleomorphic, Gram-positive rods
saccharolytic. • Non-motile, aerotolerant
• Cause: intra-abdominal abscesses. • Species: L. acidophilus, L. fermentum, L.
• Culture: Bacteroides bile esculin (BBE) agar – vaginalis and L. salivarius
exhibit a gray color and growth with 20% bile
and cause the blackening of the originally LACTOBACILLUS ACIDOPHILUS
yellow-colored agar. • Part of indigenous microbiota of the mouth, GIT
• Biochemical: (+) Esculin hydrolysis and vaginal canal.
ACTINOMYCES ISRAELLI • Protects female genital tract from urogenital
• Most common cause of infections
actinomycosis • Related infection: Bacterial vaginosis
• Indigenous microbiota of oral • Differential medium: Tomato juice agar (pH 3 to
cavity. 4)
• Molar tooth appearance • Biochemical Test: (-) catalase, H2S and esculin
hydrolysis.

FLUORESCENCE UNDER LONG-WAVE ULTRAVIOLET LIGHT

ORGANISM COLOR OF FLUORESCENCE
Prevotella (pigmented) Brick red
P. bivia, P. disiens Light orange to pink (coral)
Porphyromonas Brick red (some no fluorescence)
Fusobacterium Chartreuse (yellow/green)
Veillonella Red but fades rapidly
Clostridium ramosum Red
C. innocuum, C. difficile Chartreuse
Eggerthella lenta Red or no fluorescence

PRIMARY SETUP MEDIA RECOMMENDED FOR RECOVERY OF ANAEROBES

MEDIUM ORGANISMS COMMENTS
Anaerobic blood agar (CDC) Supports growth of almost all obligate An enriched medium containing sheep
and facultative anaerobes, best for blood for enrichment and detection of
anaerobic gram-positive cocci hemolysis, vitamin K (required by
some Porphyromonas spp.), and yeast
extract
Bacteroides bile esculin agar Supports growth of bile-tolerant A selective medium containing

TUAZON A. 37
 Bacteroides spp., some strains of gentamicin (which inhibits most
Fusobacterium mortiferum, Klebsiella aerobic organisms), 20% bile (which
pneumoniae, enterococci, and yeasts inhibits most anaerobes), and esculin;
may grow to a limited extent used primarily for rapid isolation and
presumptive identification of members
of the B. fragilis group, which grow
well on Bacteroides bile esculin agar
(because of their bile tolerance) and
turn the originally light-yellow medium
to black (because of esculin
hydrolysis)
Brucella blood agar Supports growth of almost all obligate An enriched medium containing sheep
and facultative anaerobes, best for red blood cells for enrichment and
gram-negative bacteria detection of hemolysis, casein
peptones, dextrose, yeast extract,
vitamin K, and hemin
A selective medium containing
kanamycin (which inhibits most
facultative gram-negative bacilli),
vancomycin (which inhibits most gram-
positive organisms and vancomycin-
Supports growth of Bacteroides and
sensitive strains of Porphyromonas
Kanamycin–vancomycin–laked Prevotella spp.; yeasts and
spp.), and laked blood (which
blood agar kanamycin-resistant, facultative, gram-
accelerates production of brown-black
negative bacilli will also grow
pigmented colonies by certain
Prevotella spp.); used primarily for
rapid isolation and presumptive
identification of pigmented species of
Prevotella
Phenylethyl agar Supports growth of almost all obligate A selective medium containing sheep
anaerobes (gram-positive and gram- red blood cells and phenylethyl
negative) and gram-positive, alcohol; used primarily to suppress the
facultative anaerobes growth of any facultative, gram-
negative bacilli (e.g.,
Enterobacteriaceae) that might be
present in the clinical specimen,
especially swarming Proteus spp.
A selective medium containing sheep
red blood cells and the antimicrobials
Supports growth of almost all obligate colistin and nalidixic acid; used
Colistin–nalidixic acid blood agar anaerobes (gram-positive and gram- primarily to suppress the growth of any
plate negative) and gram-positive, facultative, gram-negative bacilli (e.g.,
facultative anaerobes Enterobacteriaceae) that might be
present in the clinical specimen,
especially swarming Proteus spp.
Anaerobic broth (e.g., thioglycollate Supports growth of almost all types of Because obligate anaerobes can be
and chopped or cooked meat) bacteria; in thioglycollate broth, overgrown by more rapidly growing
obligate aerobes and microaerophiles facultative organisms present in the
grow near the top, obligate anaerobes specimen and killed by their toxic,
at the bottom, and facultative metabolic by-products, thioglycollate
anaerobes throughout the broth broth serves only as a
backup source of culture material
(e.g., in case there is no growth on
plated media because of a jar failure
or presence of antimicrobial agents in
the specimen); chopped meat
carbohydrate broth can be used in
place of thioglycollate broth; broth
cultures should never be relied on
exclusively for isolating anaerobes
from clinical material

TUAZON A. 38
Clinical Bacteriology (Lecture)
SECOND TERM

DASH
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science TWO
TWO
LESSON 4.1: RICKETSSIACEAE A ND andersoni)
RELATED ORGANISMS Dog ticks
RICKETTSIA (Dermacentor
variabilis)
• Simplest bacterial form and considered as
Brown Dog
transitional organism between bacteria and
ticks
virus.
(Rhipicephalu
• Fastidious bacteria and obligate, intracellular
s sanguineus
parasites.
and
• Gram-negative cell wall, motile, will not grow in Amblyomma
cell-free media
cajennense
• Multiply: Binary fission TRANSITIONAL GROUP
• Survive briefly outside of their host Mouse mite
• Microscopy: Small, pleomorphic, gram-negative Rickettsia akari Rickettsial pox (Liponyssoide
bacilli. They do not undergo any intracellular s sanguineus)
developmental cycle. Rickettsia felis Flea-borne spotted Flea bite or
• Culture: require living cells for growth fever feces
• MOA: Human become infected following the bite TYHPUS GROUP
of an infected arthropod vector. (lice, tick, mites) Rickettsia Epidemic Body louse
• Mode of prevention: Avoid contact with prowazekii typhus/Brill- (Pediculus
respective vector Zinsserdisease humanus
• Accidental host: Humans corporis)
• Agents of bioterrorism: R. prowazekii and R. Squirrel flea
rickettsii. (Orchopeas
• Rickettsia is divided into three groups: howardi)
o Spotted fever group Squirrel louse
o Transitional group (Neohematopi
o Typhus group nus
PATHOGENSIS OF RICKETTSIA sciuriopteri)
SPOTTED FEVER GROUP: Rat flea
Endemic murine
• Rocky mountain spotted fever (RMSF) most Rickettsia typhi (Xenopsylla
typhus
serious rickettsial infection. cheopis)
• For RMSF, humans are the accidental hosts and SCRUB TYPHUS GROUP
ticks are the main vector and reservoir. Chigger
• Rashes developed on the palms of the hands Orientia (Leptotrombid
Scrub typhus
and soles of the feet. tsutsugamuchi ium deliense)
• Boutonneuse fever: Rashes that are similar to bite
RMSF but on face. ANAPLASMATACEAE
• Tache noires (black spot): present in BF Lone star tick
Ehrlichia Human Monocytic
• Rickettsialpox: rashes face and extremities (Amblyomma
chaffeensis ehrlichiosis
americanum)
TYPHUS GROUP
Anaplasma Human Deer tick
• Endemic typhus characterized by rashes on phagocytophila granulocytoropic (Ixodes
the face, palms, and soles of feet of the sick. anaplasmosis scapularis
• Rashes are not commonly observed and Ixodes
• Inhalation of aerosol from dried infected flea pacificus)
feces is also a mode of transmission of OTHER ORGANISMS
Rickettsia typhi infection. Coxiella Q fever Inhalation of
INFECTION/DISEAS VECTOR/MO burnetti aerosol and
ORGANISM
E T infected
SPOTTED FEVER GROUP animals
Rickettsia Boutonneuse fever Ticks Feces of body
conorii or (Rhipicephalu louse
Mediterranean s sanguineus) Bartonella
Trench fever (Pediculus
spotted fever quintana
humanus
Rickettsia Rocky Mountain Wood ticks corporis)
rickettsii Spotted Fever (Dermacentor Bartonella Cat scratch disease Kitten scratch

TUAZON A. 39
henselae Bacillary or bite • MOA: inhalation of contaminated aerosols from
angiomatosis dried animal feces and ingestion of
Sandfly contaminated unpasteurized milk
Bartonella Oroya fever and
(Lutzomyia) • Animal reservoir: Cattle, goats and sheep
bacilliformis verruga peruana
bite EHRLICHIA
ORIENTIA TSUTSUGAMUSHI • Genus belongs to the family Anaplasmataceae
• Belong to family Rickettsiaceae • Species of the genus are Gram-negative
• Categorized as a separate genus due to the coccobacilli that undergo an intracellular
absence of lipopolysaccharide and development cycle following the infection of
peptidoglycan and the presence of 54 to 58 kDa circulating WBC’s (replicate ion occurs in the
major surface protein leukocytes)
• It replicates in the cytoplasm of its host cell and • Microscopy: Presence of intravacuolar
is released through a process that involves microcolony that resembles “mulberries” or a
“pinching off” the host cell morula.
• MOA: Bite of an infected arthropod vector THREE DEVELOPMENTAL STAGES
• Vector: Leptotrombidum delicense (Chigger) • Elementary body (infective form)
• Accidental host: Humans and rats • Initial bodies
ANAPLASMA PHAGOCYTOPHILA • Morulae
• It causes human ➢ Natural hosts: Human and animals (dog and
granulocytotropic deer)
anaplasmosis ➢ Primary vector: Amblyomma americanum
• Vector: Ixodes pacificus ➢ Species: E. chaffeensis and E. ewingii
(Western black-legged ➢ Infection: Human monocytic ehrlichiosis
tick) and Ixodes scapularis (deer tick) LABORATORY DIAGNOSIS FOR RICKETTSIAEAE
• Reservoir: Peromyscus leucopus (white-footed DIRECT METHODS
mouse) • Immunohistology (Immunofluorescence and
BARTONELLA immunoenzyme stains)
• The species of this genus are facultative, o Skin biopsy is utilized or usually used.
intracellular and Gram-negative bacilli. • Giemsa stain
• They live within the RBC in their natural CULTURE
mammalian hosts • Culture media: Yolk sacs of embryonated eggs
• Species can be cultivated in CAP with 5% CO2 and tissue culture
or in charcoal yeast extract agar. • Rickettsia, Ehrlichia and Anaplasma can be
• Some species exhibit a “twitching motility in wet isolated from human in an antibiotic-free,
mounts (B. bacilliformis and B. henselae). centrifugation-enhanced shell vial cell culture
INFECTIOUS AND • Columbia blood agar with 5% defibrinated blood
SPECIES
DISEASES for B. bacilliformis
Trench fever (louse-borne • Lung tissue cells are the preferred medium for
B. quintana
disease) C. burnetti
B. henselae Cat scratch disease SEROLOGICAL TEST
B. elizabethae Infective endocarditis
B. bacilliformis Oroya fever (chronic • Only test preferred for diagnosis of rickettsial
verruga peruana) and disease.
febrile acute haemolytic • It is used to confirm rickettsiosis/ricketsioses
anemia during convalescence stage.
Cat scratch disease • Antibodies to rickettsia can be detected until at
B. clarridgeiae least 2 weeks after the infection.
(secondary agent)
COXIELLA BURNETTI • Rickettsiosis are seldom diagnosed serologically
during the acute stage of the illness due to the
• Only specie in the genus Coxiella
absence of an early antibody response.
• Causative agent of Q (Query) fever which is a
systematic infection of the lungs
INDIRECT IMMUNOFLUORESCENT ANTIBODY
• Extremely contagious and can be considered as (IFA) ASSAY
a potential bioterrorism agent • Gold standard serologic test or reference
• Can survive extracellularly because of its method for rickettsioses and Q fever.
endospore-like body WEIL-FELIX REACTION
• Can infect birds and rodents, which in turn • Presumptive test for rickettsioses
excrete the organisms via their urine, feces and • Agglutination of certain strains of Proteus
birth products vulgaris by serum from patient.
• Not transmitted by arthropod vectors

TUAZON A. 40
 • Individual with Q fever, ehrlichiosis and CHLAMYDIA TRACHOMATIS
rickettsial pox do not produce Weil-Felix • Major sexually transmitted pathogens
antibody • Principal cause of: PID (pelvic inflammatory
MICROIMMUNOFLUORESCENT DOT TEST disease) and ocular trachoma
• Excellent for detecting antibodies to Rickettsia • Travel through the birth canal where infants can
• Used for early diagnosis of RMSF be infected during birth
NUCLEIC ACID AMPLIFICATION (PCR) • Associated within fertility and ectopic pregnancy
• Cat scratch disease can be performed through • Natural host: Humans
PCR testing of lymph nodes. • Unique features:10 stable plasmids
• PCR a diagnostic tool for ehrlichiosis • Biovars: Lymphogranuloma venereum (LGV),
CHLAMYDIACEAE mouse and pneumonitis trachoma
• Genus are non-motile, small (0.2to1.5um) and • Serovars of C. trachomatis based on MOMP
have Gram-negative cell wall (major outer membrane protein) antigenic
differences:
• Obligate, intracellular organisms that requires
living cells for growth • Serotypes A, B, Ba, C: endemic trachoma
• Serotypes L1, L2, L2a,
• Do not possess cytochromes and cannot
synthesize their own ATP. L2b, L3 – LGV
• They are called “energy parasites” because they • Serotypes D to K, Da,
depend on the eukaryotic cells of their host for Ia, Ja – PID, urethritis,
metabolism, growth and reproduction. cervicitis, epididymitis
and inclusion
• Replicate by binary fission in the cytoplasm of
conjunctivitis
infected cells.
TWO MORPHOLOGIC FORMS: RELATED INFECTION:
o Trachoma
RETICULATE BODY (RB) o LGV
• replicative and non-infectious form o Inclusion conjunctivitis
• Intracellular and metabolically active form of TRACHOMA
Chlamydia.
• Chronic inflammation of the
• Multiply: Binary fission conjunctiva that lead to
ELEMENTARY BODY (EB) blindness
• Infectious form. • Cause distortion of the eyelids
• Extracellular from a Chlamydia and is spherical (eyelashes become
in shape. misdirected and turned-in)
• Resembles Gram-negative bacilli with a rigid cell • MOT: Contact with contaminated objects, hand
wall to hand contact with
• Infects cells through inducing active carriers and through
phagocytosis vectors (flies)
LYMPHOGRANULOMA
VENEREUM (LGV)
• Sexually transmitted
disease which has a multi-system involvement
• Small, painless ulcer or papule appears initially
and then nodules (buboes) develop after several
weeks
INCLUSION CONJUNCTIVITIS
• Characterized by an abundant eye discharge
and swollen conjunctiva
• Affects infants who acquired it through aspiration
or ocular exposure during birth

HUMAN DISEASES CAUSED BY CHLAMYDIACEAE SPECIES

SPECIES SEROVARS DISEASE HOST
Chlamydia trachomatis A, B, Ba, C Trachoma Humans
Inclusion conjunctivitis (adult
and newborn)
D, Da, E, F, G, H, I, Ia, J, Nongonococcal urethritis
Humans
Ja, K Cervicitis
Salpingitis
Pelvic inflammatory disease

TUAZON A. 41
 Endometritis
Acute urethral syndrome
Proctitis
Epididymitis
Pneumonia of newborns
Perihepatitis (Fitz-Hugh–
Curtis syndrome)
L1, L2, L2a, L2b, L3 Lymphogranuloma
venereum
Pneumonia, bronchitis
Chlamydophila
1 Pharyngitis Humans
pneumoniae
Influenza-like febrile illness
Chlamydophila psittaci 10 Psittacosis Birds
Endocarditis
Abortion

LABORATORY DIAGNOSIS o Complement fixation: family-reactive

• Specimen: Urethra and cervical secretions, antibodies. Used to diagnosed LGV.
conjunctiva discharge, nasopharynx and rectal Genus specific antigen. (+) Titer: > 1:64
swabs and material aspirated from fallopian o Microimmunofluorescence (MICRO-
tubes and epididymis IF) Assay: Used for type-specific
• Use Dacron and rayon-tipped swabs is antibodies of C. trachomatis
preferred. ▪ Preferred method for
CULTURE: identification of C. trachomatis
• Culture media: Buffalo green monkey kidney ▪ It can be used for the diagnosis
cells, He La 299 cells, Hep-2 cells, McCoy cells of LGV, trachoma, inclusion
and Cyclohexamide-treated McCoy cells. conjunctivitis, and chlamydial
neonatal infection.
CYTOLOGIC EXAMINATION:
▪ (+): IgM titer of 1:32
• Cell scrapings from conjunctiva of newborns or
persons with ocular trachoma.
IMMUNOASSAY
• Enzyme immunoassay (EIA): Most commonly
• Direct fluorescent antibody (DFA)
used rapid antigen assay
o Fluorescein-isothiocyanate-conjugated
monoclonal antibofy • Detects the outer membrane LPS chlamydial
antigen or MOMP antigen
ANTIGEN DETECTION AND NAAT
o Major outer membrane protein
• Nucleic acid amplification is the most sensitive
method for detection of C. trachomatis.
• LPS antigen major antigen that is detected
• Specimen: Endocervical and urethral swabs.

CHLAMYDOPHILA PSITTACI
• Causative agent of psittacosis or ornithosis
• Endemic pathogen of birds’ specie such as
SERODIAGNOSIS parrots, parakeets, chicken and ducks
• Extractable LPS and elementary body with keto- • MOA: Inhalation of infected aerosols from dried
deoxyoctonate is the primary antigen that can be bird excreta or handling of infected birds
identified in genus specific test. • Commonly used test: Complement fixation
• Negative test: exclude chlamydial infection. with titer of >1:32

TUAZON A. 42
 • Sensitive method: Direct • Human pathogen that is transmitted through
microimmunofluorescence aerosol droplets.
• Precautionary measures: Only laboratories • One of the major causes of infectious respiratory
with biosafety level 3 facilities can perform the disease
isolation and cultivation of the specimens • Associated with pneumonia, bronchitis,
CHLAMYDOPHILA PNEUMONIAE pharyngitis and sinusitis.
• Formerly known as the Chlamydia pneumonia • Specimen for isolation: Nasopharyngeal
strain TWAR. aspirates, sputum and throat swabs.
o TWAR – Taiwan Acute Respiratory • Culture media: He LA cells or Hep-2 cell lines
Chlamydia • Preferred method: Microimmunofluorescent
assay.
PROPERTIES C.TRACHOMATIS C. PSITTACI C. PNEUMONIAE
Host range Humans Birds Humans
Elementary body Round Round Pear-shaped
Round, vacuolar
Inclusion morphology and Variable, dense Levinthal-
Halberstaeder-Prowazek Round, dense
inclusion body Cole-Lillie bodies
bodies
Stain used Macchiavello stain and
Lugol’s Iodine Giemsa stain
Giemsa stain
Glycogen-containing
Present Absent Absent
inclusions
Susceptibility to
Sesceptible Resistant Resistant
sulfonamides
Trachoma, LGV and
Disease Psittacosis Pneumonia, Pharyngitis
inclusion conjunctiva
Number of Serovars 20 10 1

COMPARATIVE PROPERTIES OF MICROORGANISMS

• Zoonotic disease acquired by humans through

inhalation of dried excreta of animals or infected
birds.
• Causes: mild flu, lung infection
• Also known as Ornithosis or Chlamydiosis.
LESSON 5: CELL WALL-DEFICIENT &
SPIROCHETES AND MISCELLANEOUS
BACTERIA
CELL WALL DEFICIENT BACTERIA
• Mycoplasma
• Ureaplasma
• Specie: M. pneumoniae, M. hominis, M.
fermentans, M. pirum, M. penetransand U.
PARROT DISEASE urealyticum
• Psittacosis (1929) MYCOPLASMA AND UREAPLASMA
o Ducks, pigeon, hen, sparrow, cockatiels, • Both mollicutes
macaws, budgerigars

TUAZON A. 43
 • Smallest free-living organisms that are capable • Swab: Calcium alginate and Dacron swabs.
of growing in artificial media. • Transport medium: SP4 (sucrose phosphate
• Pleomorphic organisms that lack cell wall buffer, horse serum and neural red).
• They found mainly in the oropharyngeal, upper • Mycoplasma and Ureaplasma grow slowly
respiratory and genitourinary tracts. than most of the other bacteria
• Slow growing, fastidious and facultative • M. hominisis the only species that is capable of
anaerobes that replicate by binary fission. growing on BAP and CAP
• Requires sterols (cholesterol) for membrane • M. Pneumoniae requires biphasic culture
function and growth system and incubation of up to three weeks in
• Common parasites of the genital tract and their chamber with 5% to 10% CO2
transmission is related to sexual activity. • Urea and/or arginine: incorporated into the
• Etiologic agent: Primary atypical pneumonia or media to detect the presence of U. urealyticum
walking pneumonia and M. hominis and produce alkaline reaction.
• Extremely fastidious • Mycoplasma in Shepard’s 10B arginine
• MOA: Inhalation of contaminated aerosol medium: red color. Blood culture: not
droplets recommended
• Initiation of Disease: Attachment to respiratory • Serodiagnosis
mucosal cells, evasion from phagocytosis and o ELISA: Most commonly used
modulation of the immune system. serological method for diagnosis of
• Culture: SP4 broth: Yellow color Mycoplasma and Ureaplasma.
ATYPICAL PNEUMONIA • Manganese Chloride Urea Test
o Rapid identification test for U.
• Can occur as separate incidents or as outbreaks
urealyticum
in closed population such as in school, military
o Reaction for the test is observed under
camps and within family members.
a dissecting microscope.
MYCOPLASMA HOMINIS & UREAPLASMA o (+) results: dark brown precipitate of
UREALYTICUM(GENITAL MOLLICUTES) manganese oxide around the colonies.
• Recovered from the genital tract of healthy o U. urealyticum utilizes manganese
adults. chloride in the presence of urea.
• Cause prostatitis and pelvic inflammatory
disease. • Dienes stain of
• Colonization among infants occurs during Mycoplasma spp. colonies
passage through an infected birth canal and demonstrating typical fried
results in the isolation of these organisms from egg appearance
their nose and throat.
• M. hominis: Causative agent of postabortal
fever and postpartum fever in women. • Typical mixed sizes of
• Culture: Mycoplasma spp. on
o M. hominis: “Fried-egg” appearance on primary isolation
plated medium media, Mycoplasma
o U. urealyticum: “dark-brownish lumps” salivarium.
on a A7 or A8 Agar media.
LABORATORY DIAGNOSIS
• Specimen: Throat swab, serum,
bronchoalveolar lavage, sputum and lung tissue
for M. pneumoniae
• Genital mycoplasma: urethral, vaginal or
endocervical swab, blood, urine, prostatic
secretions and semen.
• No direct method or gram staining can be
used for identification since mycoplasma
and ureaplasma lacks cell wall.
CULTURE:
• Culture media: Beef or Soybean protein with
serum, fresh yeast extract, biphasic SP4
medium, pleuropneumonia-like organism
(PPLO) broth or agar with yeast extract and
horse serum, A8 agar (usually used for
ureaplasma), Shepard’s 10B arginine broth and
modified New York City medium.

TUAZON A. 44
Clinical Bacteriology (Lecture)
SECOND TERM

DASH
Our Lady of Fatima University – Pampanga
College of Medical Laboratory Science TWO
TWO
SUMMARY OF ASSOCIATION OF GENITAL MOLLICUTES WITH UROGENITAL AND NEWBORN DISEASES
Disease, Target Mycoplasma Mycoplasma
Ureaplasma spp. Comments
Population hominis genitalium
Nongonococcal Ureaplasma spp.
urethritis cause some cases,
None Weak Strong
but the proportion is
unknown
An association with a
few cases of chronic
Prostatitis None Weak None disease has been
reported; a causal
relation is unproven
Epididymitis Mycoplasma spp. are
None None Weak not an important
cause
M. hominis is often
Vaginitis and associated with
None None None
cervicitis disease, but a causal
relation is unproven
Pelvic inflammatory M. hominis causes
disease Strong Strong None some cases, but the
proportion is unknown
Recent studies
indicate that M.
Postpartum fever Strong None Weak
hominis may be a
major cause
Urinary calculi Ureaplasma spp.
cause calculi in male
rats, but no
None None Weak convincing evidence
exists that they cause
natural human
disease
M. hominis causes
Pyelonephritis Strong None None
some cases
Involuntary infertility Ureaplasma spp. are
associated with
Weak None
altered motility of
sperm
An association exists,
Chorioamnionitis Strong None Strong but a causal relation
is unproven
Low birth weight An association exists,
None None Strong but a causal relation
is unproven
Further clarification is
Neonatal infections,
needed, but
including sepsis,
Strong Strong importance is growing
pneumonia,
in a selected prenatal
meningitis
population
Neonatal period, These findings need
particularly preterm further clarification
delivery, very low birth because most
weight; clinical signs Strong Weak Strong neonatal infections
compatible with resolve without
meningitis (CSF), therapy, but in low
pneumonia (trachea), socioeconomic

TUAZON A. 45
sepsis (blood) groups the diagnostic
workup of newborns
should include CSF
and blood cultures for
detection of
mycoplasmas. This
includes low-birth-
weight and preterm
newborns, in whom
traditional CSF cell
counts and cultures
would be negative

• Identification of Mycoplasma-infected cell culture using DNA fluorochrome

stain (Hoechst 33258 stain). A, Mycoplasma orale. B, Uninfected Vero cell culture
highlighting the DNA-rich nucleus. C, Mycoplasma salivarium. The mycoplasma
appear as small, pinpoint, fluorescent bodies throughout the background.

• Mixed isolation of Mycoplasma hominis and Ureaplasma urealyticum showing

why U. urealyticum was originally called “T” for “tiny strain”.

COMPARATIVE FEATURES OF VARIOUS LABORATORY METHODS USED TO DETECT MYCOPLASMA

PNEUMONIAE, MYCOPLASMA HOMINIS, AND UREAPLASMA UREALYTICUM
Detection Method Mycoplasma pneumoniae Mycoplasma hominis Ureaplasma urealyticum
NONSEROLOGIC
Method of choice using
Method of choice, but must
urease detection, but must
Culture Traditionally difficult differentiate infection
differentiate infection from
from colonization
colonization
Indirect Respiratory antigen for
immunofluorescence early-stage infection
Research use only but is
promising
Polymerase chain reaction Assays are being evaluated Assays are being evaluated Assays are being evaluated
SEROLOGIC
Traditional assay but <50%
seroconvert; need fourfold
rise between acute-phase
Complement fixation
and convalescent sera; >32
single titer may be
suggestive
Immunofluorescent Measures IgG and IgM Measures IgG and IgM
antibody separately separately; not
recommended
Latex agglutination IgM/IgG IgG only
Enzyme immunoassay Method of choice
Reactive IgM, IgG, and IgA,
but IgM level may remain
elevated for 1 year

TUAZON A. 46
SPIROCHETES AND MISCELLANEOUS BACTERIA fresh blood, injuries from contaminated needle
• Treponema sticks and handling specimen
• Borrelia • Symptoms: Chancre (hard chancre), fever, sore
• Leptospira throat, headache, rashes (palm and soles) and
• Miscellaneous: Streptobacillus moniliformis, gummas on skin.
Spirillum minus, Klebsiella granulomatis, o Soft chancre: H. ducreyi
Capnocytophaga and Enterococci • Stages of syphilis:
GENERAL CHARACTERISTICS OF o Primary syphilis
SPIROCHETES o Secondary syphilis
o Latent stage
• Belongs to the order
o Tertiary stage or Late stage
Spirochaetales
• Unusual morphologic
PRIMARY SYPHILIS
features • Appearance of hunterian or hard chancre,
• Facultatively anaerobic • Painless, usually seen on the genitalia
or aerobic • Develops 10 to 90 days after infection
• Various types of motility • No systemic signs and symptoms
pattern SECONDARY SYPHILIS
• Have fibrils or axial filaments which are flagella- • Develops 2 to 12 weeks after appearance of
like organelles that wrap around the bacterial chancre
cell wall and facilitate motility (exhibiting a • All lesions that are observed seen in this phase
“corkscrew like” winding) are highly infectious
• General microscopy: slender, helical and • Chancre heals but organisms are still
unicellular bacteria disseminated via blood stream
• Genera: Treponema, Borrelia and Leptospira. • Symptoms: Fever, Sore throat, headache and
TREPONEMA rashes (palms and soles)
• Greek word: Trepein means “to turn” and nema LATENT STAGE
“thread”, “turning thread” • Disease becomes subclinical but not necessarily
• Darkfield microscope dormant
• Infects only human • Occurs within more than a year of infection
• Stain poorly with Gram or Giemsa stains • In this stage, diagnosis can be made only by
• Reproduction: Transverse fission serological test
• Microscopy: Thin, spiral organisms with three TERTIARY STAGE/LATE SYPHILIS
axial filaments. Cell end are pointed and
• Tissue-destructive phase
covered with a sheath.
• Appears 10 to 25 years after initial infection
TREPONEMA PALLIDUM SUBSP. PALLIDUM
• In this stage, individuals are not usually
• Causative agent of syphilis infectious
• Microaerophiles which survives longer in the • Complications: Central nervous disease
presence of 3% to 5% oxygen, (neurosyphilis), cardiovascular abnormalities,
• Killed rapidly at 42C eye disease and granuloma-like lesions
• Remains visible in whole blood or plasma for (gummas)
atleast 24hrs, which is potential importance in LABORATORY DIAGNOSIS
blood transfusion. • Specimen: Skin lesions (cleaned with saline)
• Can cross intact mucous membrane and • Oral lesions: should not be examined because
placenta non-pathogenic spirochetes will lead to false-
• Darkfield microscopy: White against a dark positive results.
background and long with fine spirals that have MICROSCOPIC EXAM
10 to 13 coils and three fibrils/periplasmic
flagella. • Direct examination of exudates is recommended
and motility
• Generationtime:30hours
• Definitive test: Darkfield microscopy
• Diagnostic Test:
o Treponemal reagin • Stain: Levaditi’s stain and Fontana-Tribondeau
o Non-treponemal reagin stains.
SYPHILIS • Direct detection in lesions: FITC (Fluorescein
isothiocyanate-labeled antibody)
• AKA: French disease / Italian disease / The SERODIAGNOSIS (SECONDARY AND
Great Pox
TERTIARY)
• Disease of blood vessels and perivascular areas
• Known as “great imitator” • Non-treponemal Test/Non-Specific Test –
• MOA: sexual contact, congenital transmission, Screening test. Detects the presence of non-
skin contact with active lesion; transfusion of specific antibody or antibody-like protein like

TUAZON A. 47
 your regain or Wassermann antibodies. This is GENERAL CHARACTERISTICS OF BORRELIA
used to monitor syphilis. • Slow growing sphirochetes that multiply by
o RPR - Rapid Plasma Reagin binary fission
o VDRL - Venereal Disease Research • Composed of 3 to 10 loose coils and is actively
Laboratory motile
o USR - Unheated Serum Reagin • Species have 15 to 20 axial filaments and two
o TRUST - Toluidine Red Unheated insertion disks
Serum Test • They stain well with Geimsa stain and can be
o ELISA - Enzyme-Linked Immunosorbent visualized by using brightfield microscopy.
assay • Species that have been grown in vitro are
• Treponemal Test/Specific Test – Confirmatory microaerophilic.
test. Usually used to detect the presence of BORRELIA SPP.
antibody to the treponemal antigens.
o FTA-ABS - Fluorescent Treponemal
BORRELIA RECURRENTIS
Antibody Absorption • Agent of louse-borne/ epidemic/ European
o MHA-TP - Microhemagglutination Assay relapsing fever
for Treponema Pallidum • Vector: Louse (Pediculus humanus)
(Microhemagglutination Test for • Reservoir: Humans
Treponema palli- dum) BORRELIA HERMSII, B. TURICATE, B. DUTONI
o TPHA - Treponema Pallidum AND B. PARKERI
Hemagglutination • These agents are tick-borne relapsing fever/
o TPPA - Treponema Pallidum Particle endemic/ American relapsing fever.
Agglutination Assay • Vector: Soft ticks (Ornithodoros)
MOLECULAR TEST BORRELIA BURGDORFERI, B. GARINII AND B.
• PCR used for neurosyphilis detection during AFZELII
tertiary syphilis • Agents of Lyme disease (B. burgdorferi)
• Western blot detection of congenital syphilis. • Vector: Hard ticks (Ixodes) – Ixodes pacificus,
TREPONEMA SPP. Ixodes scapularis (deer tick), Ixodes
TREPONEMA PALLIDUM SUBSP. PERTENUE persulcatus, Ixodes dammini
• Causative agent of Yaws or frembesia tropica • Transmission: Bite of the Ixodes ticks
• Acquired by direct contact through skin breaks • Natural hosts of ticks: Deer and Rodents
with an infected lesion. (Peromyscus leucopus or white-footed mouse)
• Non-venereal infection, chronic ulcerative sores • All stages of ticks (larval, nymph and adult) can
TREPONEMA PALLIDUM SUBSP. ENDEMICUM harbour the organisms and transmit disease
• Causative agent of endemic non-venereal RELATED DISEASES AND LABORATORY
syphilis or Bejel DIAGNOSIS
• Non-venereal syphilis, appearance of primary • Related diseases
lesion near mouth. Pimple-like lesions on trunk, RELAPSING FEVER
arms and legs • It is an acute infectious disease with recurring
TREPONEMA CARATEUM febrile episodes (2 to 10 relapses)
• Causative agent of pinta or carate • Symptoms: Fever, headache, myalgia (2 to 15
• Acquired by contact with infected skin days after infection)
• Pinta: skin infection characterized with primary LYME DISEASE
lesions or graduall enlarging papule with • Acute, recurring inflammatory infection involving
enlargement of regional lymph nodes. (Red to the large joints, like knees.
blue macular rash) • Hallmark of infection are erythema migrans
TREPONEMA DENTICOLA & TREPONEMA (bull’s eye lesion on the skin) and swelling
SOCRANSKI LABORATORY DIAGNOSIS
• Ulcerative gingivitis and chronic periodontitis. MICROSCOPIC EXAMINATION
BORRELIA (BLOOD SPIROCHETES) • Giemsa and Wright stain
• Borrelia recurrentis, Borrelia dutoni, Borrelia o Darkfield microscopy, Blood culture after
hermsii, Borrelia turicatae, Borrelia parkeri, 2 to 3 weeks of incubation at 35C.
Borrelia afzelii, Borrelia burgdorferi (most • Relapsing Fever
commonly encountered, causes lyme disease) o Specimen: Peripheral blood
and Borrelia garinii o Spirochetes in peripheral blood, stained
• B. recurrentis (arrows) in as blue colored.
blood. • Lyme Disease
o Specimen: Blood, CSF, and Biopsy
specimen

TUAZON A. 48
 o Tissue section: Warthin-Starry stain is o Entry through breaks in the skin,
used. mucous membranes or conjunctiva
CULTURE o Direct contact with the urine of carriers
• Culture media: Barbour-Stoenner-Kelly medium like rats
or Chick embryo o Contact with bodies of water that are
• Organisms are slow-grower and requires 7 to 14 contaminated with the urine of the
days at 35C carriers
o Upon entry, Leptospira rapidly invades
SERODIAGNOSIS
the bloodstream and spread throughout
• Relapsing fever: Serological test reveals the CNS and Kidneys.
increased titers to Proteus OXK antigens (up to TYPES OF LEPTOSPIROSIS
1:80)
ICTERIC LEPTOSPIROSIS OR WEIL
• Lyme disease: Serology is the standard method
SYNDROME
for the diagnostic testing of this disease.
• IGM and IGG antibodies are detected in the • Severe form of illness that affects the liver and
serum. kidneys and causes vascular dysfunction
• Serology: ELISA and IFA (Indirect • Death up to 10% of the cases
Immunofluorescent Assay) • Icteric – jaundice/yellowish discoloration
MOLECULAR TESTS (paninilaw)
ANICTERIC LEPTOSPIROSIS
• PCR is important in diagnosis of B. burgdorferi
DNA in urine. • Symptoms: Septicemic stage of infection, high
LEPTOSPIRA fever and severe headache (3 to 7 days)
• Leptospira interrogans (pathogenic species, followed by the immune stage
looks like a question mark)) and Leptospira • Hallmark of immune stage: Aseptic meningitis.
biflexa (non-pathogenic species) • Absence of yellowish discoloration of the skin
• Leptospira interrogans LABORATORY DIAGNOSIS
serotype • Specimen: Blood, CSF and tissues for the
bacterimic phase (first week); urine for the
immune phase (second week)
• Microscopic Examination
• Darkfield microscopy, can be used for the
detection of motile leptospires in the specimens.
• Culture
GENERAL CHARACTERISTICS OF o Culture media: Fletcher’s medium,
LEPTOSPIRA Ellinghausen-McCullough-Johnson-
• Genus belongs to the family Leptospiraceae Harris (EMJH) medium, Bovine serum
under the order of Spirochaetales. albumin, Stuart’s broth and Noguchi’s
• Species of this genus are obligate aerobes medium
which can be grown in artificial media. o Fletcher’s and EMJH media are semi-
• They are tightly coiled and are highly motile with solid media.
hooked ends. (looks like a question mark) • Serodiagnosis
• They live in the lumen of the renal tubules and o Commonly used methods for antigen
shed into the urine detection: ELISA, Radio immunoassay
(RIA) and immunomagnetic capturing
• Recommended animals for cultivation:
o Antigen detection:
Hamsters and guinea pigs
Immunofluorescence and
• Microscopy: Tightly coiled, Thin, flexible
immunohistochemistry
organisms with two long axial filaments that
o Reference method: microscopic
exhibit a spinning motility.
agglutination (MA) using living cells
• Virulence factor: Hemolysin (L. interrogans)
• Molecular Test
• Growth factor: Hemoglobin and thiamine o Detects leptospiral DNA in infected
• Animal Reservoir: Rats and Dogs patients
LEPTOSPIRA SPP. o Methods: Polymerase chain reaction
LEPTOSPIROSIS OR INFECTIOUS JAUNDICE and hybridization techniques.
• Zoonotic disease in humans caused by MISCELLANEOUS BACTERIA
Leptospira interrogans • Streptobacillus moniliformis
• Acquired in home and recreational settings • Spirillum minus/minor
(swimming pools) • Klebsiella granulomatis
• Symptoms: Fever, headache, myalgia, anorexia • Capnocytophaga
and vomiting. • Enterococci
• MOA: • Gardenella

TUAZON A. 49
 STREPTOBACILLUS MONILIFORMIS arthralgia; and lymphadenitis;
• Etiologic agent: Rat-bite fever and Haverhill recurrent fever if and recurrent
fever in humans untreated fever if untreated
• Gram-negative bacillus Diagnosis Culture and Dark group
• Normally found in oropharynx of wild and serology microscopy,
laboratory rats microscopy of
• Facultatively anaerobic Giemsa-stained
• Non-motile, non-encapsulated and non- blood smear,
haemolytic and animal
• Diene’s stain: required to demonstrate the L- inoculation
form colonies Penicillin (L
• Microscopy: Yeast-like shape, Chains of bacilli forms not
Penicillin: in case
sensitive to
• Broth: fluff or breadcrumbs appearance Antibiotic of endocarditis,
penicillin). Both
• BAP: Fried-egg appearance with dark center therapy addition of an
forms sensitive
SPIRILLUM MINUS/MINO aminoglycoside
to streptomycin
• Rat-bite fever known as sodoku in humans and tetracycline
• Grow non artificial culture media Mortality Higher if Lower if
• Strictly aerobic, closely related Neisseria untreated (10%) untreated (6%)
• Direct visualization of specimen (blood, CAPNOCYTOPHAGA
exudates or lymph node tissue) using Giemsa • GNR with long fusiform shape.
stain, Wright stain or darkfield microscopy is
• Fastidious facultative anaerobe that grows
recommended.
slowly and needs enriched agar.
• Microscopy: Thick, helical, gram-negative
• C. animorsus
bacilli with 2 to 3 coils and are motile by
o Most common cause of severe disease
polytrichous polar flagella.
in humans.
KLEBSIELLA GRANULOMATIS o Normal flora in oral cavity of dogs (and
• Formerly known as Calymmatobacterium cats)
granulomatis • Classic clinical scenario:
• Etiologic agent: Granuloma inguinal or o Septic shock with fever, rash,
donovanosis: Sexually transmitted disease hypotension, renal failure → can evolve
nodule enlarged with beefy, erythematous, to purpura fulminans or gangrene.
granulomatous and painless lesion that easily • Risk factor for severe disease
bleed. o Contacts with clog (bite/scratch),
• Culture: Yolk sac or fresh egg yolk medium. immunocompromised host, asplenia,
• Microscopy: Blue rods with “safety pin” app and cirrhosis.
is surrounded by a pink capsule, presence DONOVAN BODIES
Donovan bodies in mononucleated endothelial
cells.
CAPNOCYTOPHAGA
• Indigenous microbiota of the oral cavity of
humans and animals (dog and cats)
• Resembles HACEK group in their CO2
requirements for enhanced growth
• Gliding motility on solid surface
• Facultatively anaerobic with a negative reaction
to most biochemical tests
• Microscopy: Gram-negative rods to filamentous
or spindle-shaped bacteria
• Culture: BAP or CAP: slight yellow or orange DONOVANOSIS
pigmentation.
COMPARISON OF RAT-BITE FEVER CAUSED BY
STREPTOBACILLUS AND SPIRILLUM SPECIES
Streptobacillus
Spirillum minus
moniliformis
Incubation Short (10 days) Long (15 days)
period
High fever, Fever,
headache, chills, headache, chills,
Symptoms
myalgia, rash, rash,
arthritis, lymphangitis,

TUAZON A. 50
ENTEROCOCCI (GROUP D STREPTOCOCCI)
• Specie: Enterococcus faecalis, Enterococcus
faecium, Enterococcus avium, Enterococcus
gallinarum, Enterococcus durans, Enterococcus
raffinosus
ENTEROCOCCUS SPP.
• Belong to family Streptococcaceae
• Produce D antigen
• Indigenous microbiota of human and animals’
intestinal tracts
• Not highly pathogenic but are frequent causes of
nosocomial infections
• Resistant to multiple antimicrobial agents
• Most common isolates: E. faecalis
• Virulence factor: Extracellular serine protease,
gelatinase and cytolysin
• Related infections: UTI, endocarditis,
bacteremia, wound infections
• Non-haemolytic or maybe alpha or beta
haemolytic
• Laboratory test: (+) Bile esculin and PYR; (+)
growth in 6.5% Nacl (Halophilic)
GARDENELLA VAGINALIS
• Gram-variable
• Coccobacillus
• Low numbers in normal vaginal flora
o Mostly lactobacilli
o Keep vaginal pH <4.5
• Whiff Test or KOH Test
o Specimen: Vaginal secretions
o Reagent: 10% KOH
o (+) Result: Exhibits “fishy amine
odor
o Also look for clue cells

GOODLUCK!

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TUAZON A. 51