CLINICAL BACTERIOLOGY

INTRODUCTION TO MICROBIOLOGY
OUTLINE Branches of Microbiology
I. Microbiology B. Classification
A. Importance of According to
Microbiology Oxygen Major Branches
B. Branches of Requirement
• Bacteriology: Study of bacteria
Microbiology C. Structural View of
i. Major Branches Bacteria • Mycology: Study of fungi
ii. Applied i. Essential • Immunology: Study of immunity
Branches Structures o Edward Jenner, UK: Developed vaccination (1798)
C. Species and Sub ii. Particular o Metchnikoff, RU: Developed phagocytes (1884)
Species Structures
D. Gram Staining iii. Gram positive
o Paul Ehrlich, DE: Theory of immunity (1890)
II. Microbial world and Gram • Virology: Study of viruses
III. Classification/Taxonomy Negative o Beijerinck, ME: Discovered intracellular reproduction
A. Classification Bacteria of Tobacco Mosaic Virus (TMV); coined the term “virus:
B. Identification iv. Internal
(1899)
C. Nomeclature Structures of
D. Taxonomy Bactetia • Parasitology: Study of protozoa and parasitic worms
IV. Eukaryotes and v. External
Prokaryotes Structures of Applied Branches
V. Bacterial Morphology, Bacteria
Structure, and • Chemotherapy:
Classification o Treatment of disease by using chemical means
A. Morphology o Antibiotic produced naturally
o Synthetic drugs
o Paul Ehrlich (1878) – Used arsenic compounds to fight
disease – ‘magic bullet’
MICROBIOLOGY
o Alexander Fleming, Scottland (1929) – Discovered
• Study of living microorganisms including viruses penicillin
• Viruses are not living, only infectious particles o Selman Waksman, Ukraine (1944) – Discovered
• Viruses are only composed of nucleic acids and protein; streptomycin
acellular • Problems
• Microbiology is a broad discipline o Toxicity of drugs →Selective toxicity
• Includes Bacteriology, Virology, Mycology, Parasitology, o Resistance of bacteria to drugs
and many other -ologies • Recombinant DNA Technology
o Recombinant DNA
Importance of Microbiology
o Genetic engineering/biotechnology
• It plays a role in recycling essential elements (such as in o Microbial genetics – mechanism by which microbes
food chain) inherit genes
• Benefit society by their production of food, beverages o Molecular biology – structure and function (expression)
antibiotics, and vitamins of genes
• Causative agents of some important diseases o Molecular epidemiology/diagnostics
• Infectious diseases are a leading health-related issue, • Agricultural Microbiology – roles of microbes in soil
especially in a society where the elderly population is • Environmental Microbiology – ecology/water and sewage
increasing treatment
• New infectious diseases continue to emerge and be • Medical and Clinical Microbiology
identifies all the time • Genetic Engineering – genes of different microbes
• Microbiology impacts every facet of daily life • Paleomicrobiology – study of ancient microbes
• Bioremediation: use of microbes to remove toxins (oil spills) • Sanitary Microbiology – processing and disposal of garbage
• Use of microbes to control crop pests and waste; purification of water supply
• Normal microbiota (live within our bodies; Staphylococcus • Veterinary Microbiology – microbes and animals
epidermidis) • Biotechnology
o GMOSs for industrial, pharmaceutical and agricultural
applications
o Improvements of agriculture (plants and animals)
o Gene therapy: inserting a missing gene or replacing a
defective one in human cells

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 o Causative agent of superficial infections such as
Species and Subspecies
athlete’s foot, ringworm, and thrush
• Species o Can cause systemic infections
o Collection of bacterial cells which share an overall • Protozoa
similar pattern of traits in contrast to other bacteria o Single-celled eukaryotic organisms, larger than
whose pattern differs significantly bacteria
• Strain or Variety o Some of the diseases it causes are malaria, amebic
o Culture derives from a single parent that differs in dysentery, and Trichomoniasis vaginitis
structure and metabolism from other cultures of that o Found in soil and water
species (biovars, morphovars) o Leading cause of death in developing countries
• Type • Multicellular Parasites
o Subspecies that can show differences in antigenic o Organisms that live on or in other organisms and use it
makeup (serotype or serovar), susceptibility to for nourishment
bacterial viruses (phage type), and in pathogenicity o Parasitic worms
(pathotype) - Usually due to poor sanitation
- Roundworms
Gram Staining Process
- Flatworms
• Gram negative – red; Gram positive – violet/blue - Tapeworms
1. Crystal Violet o Parasitic Insects:
- To stain colorless bacteria into violet - Bite or burrow under the skin
2. Mordant (Gram’s iodine) - Mosquitoes
- To firmly attach crystal violet dye to the bacterial - Ticks
cell - Lice
3. Acetone alcohol - Mites
- 50% acetone 50% alcohol • Microscopic Algae
- Decolorizes other bacteria in the slide while there
are some bacteria that will retain the cv dye CLASSIFICATION/TAXONOMY
4. Applying counterstain (Saffranine-red) • Classification
- Colorless bacteria will be dyed red o Arrangement of bacteria into groups the same
organisms can be classified differently according to the
MICROBIAL WORLD
view: serotype classification, antimicrobial resistance
• Viruses classification, etc.
o Smallest known microorganism o Placing organisms in group of related species. Lists of
o Subcellular microorganism characteristics of known organisms
- Have only one nucleic acid surrounded by a • Identification
protein coat (Either DNA or RNA; never both at the o Practical use of classification criteria to distinguish
same time) certain organism from others
- Lifeless particles outside the body and must live o Matching characteristics of an “unknown” to lists of
and grow in living cells of other organisms known organisms
o Illnesses caused by viruses:
- Colds, influenza, hepatitis, warts, AIDS, mumps,
rubella, measles, herpes
o Vaccines are available for many viruses
• Bacteria (Eubacteria) and Archaebacteria
o Single celled prokaryotic organisms
o Reproduce rapidly
o Classification
- Shape
- Ability to retain dyes (gram staining) (red for gram
negative and violet for gram positive)
- Ability to grow with/without air (aerobes or
anaerobes)
- Biochemical reactions
• Fungi (Yeasts and Molds)
o Eukaryotic organisms with rigid cell wall because of the
presence of chitin • Nomenclature
o Yeasts o Naming; the means of communicating—it is binomial
- Single-celled (genus and specie ex. Escherichia(genus) coli(specie))
- Reproduce by budding o Must be italicized and underlined if unable to italicize
o Molds • Taxonomy
- Large, fuzzy, and multicellular organisms o Organizing, classifying, and naming living things
- Produce spores o Formal system originated by Carolus Linnaeus/Carl
von Linné (1791-1778; Father of modern taxonomy)

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 o Identifying and classifying organisms according to - Species – ex. Coli
specific criteria
o Each organism placed into a classification system
EUKARYOTES AND PROKARYOTES
o Classifications of organisms may change due to • Eukaryotes
evolution o Domain - Eukarya
o New species of microbes originate from pre-existing
Kingdom Organization Type of Representative
living species Nutrition Organisms
o Evolution usually progresses toward greater Protista A catch-all kingdom Absorb, Protozoans,
for eukaryotic photosynthesize, algae, water
complexity organisms that do not or ingest food molds, and slime
o Science of classification, identification, nomenclature, fit other kingdoms; molds
and making a system (Dumb Kings Play Chess On Grouped into clades
based on RNA
Funny Green Squares) Fungi Chemoheterotrophic; Absorb food Molds, yeasts,
- Domain – based on ribosomal RNA unicellular or and mushrooms
▪ Eubacteria (Prokaryotes) – true bacteria; has multicellular; cell
walls of chitin;
peptidoglycan develop from spores
▪ Archaea (Prokaryotes) – odd bacteria that or hyphal fragments
Plantae Multicellular; Photosynthesize Mosses, ferns,
live in extreme environments, high salt, heat, cellulose cell walls; food nonwoody and
(usually called extremophiles); ancient usually woody flowering
bacteria photoautotrophic plants
Animalia Multicellular; no cell Ingest food Invertebrates,
▪ Eukarya (Eukaryotes) – have nucleus and walls; fishes, reptiles,
organelles; plants, animals, and humans chemoheterotrophic amphibians,
birds, and
mammals
• Protozoan Classification
o Difficult because of diversity
o Simple grouping is based on motility, reproduction and
life cycle
a. Mastigophora – primarily flagellar motility, some
flagellar and amoeboid; sexual reproduction; cyst
and trophozoite
b. Sarcodina – primarily ameba; asexual by binary
fission; most are free-living
c. Ciliophora – cilia; trophozoites and cysts; most are
Characteristic Bacteria Archaea Eukarya
Cell Type Prokaryotic Prokaryotic Eukaryotic free-living; harmless
Chromosomes Single, or few, Single, Several, linear d. Apicomplexa – motility is absent except for male
circular circular gametes; sexual and asexual reproduction;
Type of 70S 70S but 80S complex life cycle—all parasitic
ribosomes structure is
• Fungal classification
similar to 80S
Contains + + + o Subkingdom: Amastigomycota
unique - Terrestrial inhabitants including those of medical
ribosomal importance
RNA signature 1. Zygomycota – zygospores; sporangiospores
sequences
and some conidia
Number of One Three Alll
sequences 2. Ascomycota – ascospores; conidia
shared with 3. Basidiomycota – basidiospores; conidia
Eukarya 4. Deuteromycota – majority are yeast and
Protein - + molds; no sexual spores known; conidia
synthesis
• Prokaryotes
similar to
Eukarya o Classification systems
Presence of + - - 1. Microscopic morphology
peptidoglycan 2. Macroscopic morphology – colony appearance
in cell wall 3. Physiological/biochemical characteristics
Cell Fatty acids Long-chain, Fatty acids
4. Chemical analysis
membrane with ester branched with ester
lipids linkages hydrocarbons linkages 5. Serological analysis
with ester 6. Genetic and molecular analysis
linkages - G+C base composition
Sterols in -(some - + - DNA analysis using genetic probes
membrane exceptions)
- Nucleic acid sequencing and rRNA analysis
- Kingdom – ex. Prokaryotae
- Phylum – ex. Graclicutes
- Class – ex. Scotobacteria
- Order – ex. Eubacteria
- Family – ex. Enterobacteriaceae
- Genus – ex. Escherichia

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• Prokaryotes vs. Eukaryotes b. Diplobacillus
- Appear in pairs after division
Prokaryotes Eukaryotes c. Streptobacillus
Cell size 0.2-2 um in dm 10-100 um in dm
- Appear in chains after division
Nucleus Absent Present
Membranous Absent Present d. Coccobacillus
Organelles - Fat and short
Cell Wall Chemically complex When present, simple - Look like cocci and bacillus
Ribosomes Smaller (70S) Larger (80S) in cell 70S • Spiral bacteria
in organelles
a. Vibrio
DNA Single circular Multiple linear
chromosome chromosomes - Curved spirals
Cell Division Binary fission Mitosis - Comma-shaped
Cytoskeleton Absent Present - Ex. Vibrio cholerae (gram -)
NOTE: b. Spirillum
• There are recent findings that bacteria now have - Helical-shaped and fairly rigid bodies
cytoskeletons - Flagella are externally located
• “70S’ – S meaning svedberg unit or kung gaano c. Spirochete
kabigat yung ribosomes - Have flexible bodies
- Are flexible because of axial filaments/endo
BACTERIAL MORPHOLOGY, STRUCTURE, AND flagella (wound around their bodies)
CLASSIFICATION - Move in a corkscrew manner
• Other Bacterial Shapes (most are Archaean)
Morphology a. Star-shaped bacteria
- Ex. Stella
b. Rectangular bacteria
- Ex. Haloarcula

Classification of Bacteria According to Oxygen

Requirement
• Aerobes need oxygen while Anaerobes do not
1. Obligate Aerobes – requires oxygen
2. Facultative – grow in the presence or absence of
oxygen
3. Microaerophilic – grow best at low levels of oxygen
4. Aerotolerant Anaerobes – oxygen not required for
growth but not harmful if present
5. Obligate Anaerobes – grow only in the absence of
oxygen; dies in its presence
• Cocci (spherical, round, ovoid)
a. Diplococci Structural View of Bacteria
- Composed of two cocci (remain in pairs after
• Microscopic prokaryotes (no nucleus nor membrane-bound
dividing)
organelles)
- Ex. Neisseria gonorrhea
• Cell wall may be a slime layer or a capsule
b. Streptococci
• Contain ribosomes
- Cocci that are in chain after dividing
• Enfolding of the cell membrane carry on photosynthesis and
- Ex. Streptococcus pneumoniae (gram +) and
respiration
Streptococcus pyogenes (gram +)
• Surrounded by protective cell wall containing peptidoglycan
c. Tetrad
(protein-carbohydrate)
- Divide into two planes and remain in groups of 4
• Many are surrounded by a sticky protective coating of
- Sometimes, Staphylococcus spp. also form in
sugars called the capsule or glycocalyx
tetrads
• One circular chromosome and some small DNA called
d. Sarcinae
plasmids
- Cocci that divide in three planes and remain in
• May have short, hairlike projections called pili on cell wall to
groups / in cube-like groups
attach to host or other bacteria when transferring genetic
e. Staphylococci
material
- Divide in multiple planes and form grape-like
• Some can move by flagella, gliding over slime they secrete
clusters
(e.g., Myxobacteria)
- Ex. Staphylococcus aureus (gram +)
• Some can form protective endospores (survival structure)
• Bacilli (rod-shaped)
around the DNA when conditions become unfavorable
a. Single bacillus
- Single rod
- Most bacilli form as single rods
- Ex. Bacillus cereus (gram +)

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 Structure Function
Cell Wall Protects the cell and gives shape
Outer Membrane Protects the cell against some antibiotics (only
present in gram negative cells)
Cell Membrane Regulates movement of materials into and out
of the cell; Enzymes of respiration
Cytoplasm Contains DNS, ribosomes, and organic
compounds
Chromosome Carries genetic information inherited from
past generations
Plasmid Contains specific genes obtain through
genetic recombination that will make them
unique; circular structures
Capsule and Slime Protects the cell (immune attack) and assist in
Layer attaching the cell to other surfaces
Endospore Protects the cell against harsh environmental
conditions, (heat or drought)
Pili Attaching to other surfaces (for genetic
recombination)
Flagella For propulsion/movement

Essential Structures
• Cell wall
o The cell wall of gram + is more prominent
• Cell membrane
• Cytoplasm
• Nuclear material

Particular Structures
• Capsule
o On type of glycocalyx (sugar coat; capsule or slime
layer
o Capsule – sugar is firmly attached to the bacterium;
Slime layer – loosely attached
• Flagella
o For propulsion/movement
• Pili
o To avoid phagocytosis
o Common pili or sex pili for conjugation
• Endospore • Structure of Peptidoglycan
o Can help damaged for survival mechanism
Gram Positive and Gram Negative Bacteria
Gram Positive Gram Negative
Outer Cell Peptidoglycan layer is Presence of outer
Layer thicker; No outer membrane (has
membrane endotoxin (causes
fever, shock sepsis,
etc.): lipid A), cell wall,
cell membrane;
consist of
lipopolysacc-haride
that is washed out o Gram Positive
during staining - Has alternating acetylglucosamine (NAG) and
No. of Layers 1 2 acetylmuramic acid (NAM)
Cell Wall Cell membrane has Porin (‘sieve’ protein), - Carbohydrate backbones are bridged by
Structures lipoteichoic acid (LTA), lipopolysaccharide,
cell wall has teichoic acid periplasmic space polypeptides with side chains of amino acids
(imparts antigenicity and
stabilized strength in cell
wall)
Flagella Basal body have 3 rings Basal body have 4
rings

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 o Gram Negative properties such as antibiotic resistance, virulence
- Peptide cross-bridges are absent factors.
o Not essential for cellular survival
• Inclusions of Bacteria
o Aggregates of various compounds that are normally
involved in storing energy reserves or building blocks
of cell. Inclusions accumulate when a cell is grown in
the presence of excess nutrients and they are often
observed under laboratory conditions e.g.,
metachromatic granules

External Structures of Bacterial Cell

• Capsule and Slime Layers
o Attachment
o Protection from phagocytic engulfment
• Bacteria without cell wall is called Mycoplasma (smallest o Resistance to drying
and cell-wall-less bacteria o Depot for waste products
• Wall-less Forms of Bacteria o Reservoir for certain nutrients
o Spheroplast (outer membrane is present) Protoplast o Protection
(outer membrane is absent); Cannot replicate • Capsules
o Bacteria becomes wall-less when treated with: o Usually consist of polysaccharide
a. Enzymes that are lytic for the cell wall e.g., o In certain bacteria, they are composed of a polypeptide
lysozyme (polyglutamic acid)
b. Antibiotics that interfere with biosynthesis of o Not essential to cell viability and some strains within a
peptidoglycan, wall-less bacteria are often specie will produce a capsule while other could not
produced e.g., penicillin o Are often lost during in vitro culture
• Pili
Internal Structures of Bacterial Cell
o Hair-like projections of the cell
• Cell Membrane o Known to be receptors for certain bacterial viruses
o Responsible for the “in and out” of substances as well o Chemical nature is pilin
as the transport of solutes of the bacterial cell o Common pili/fimbriae: fine, rigid, numerous, related to
o Site of the electron transport chain or the oxidated bacterial adhesion
phosphorylation o Sex pili: longer and coarser, only 1-4, related to
o Site where the waste/ hydrolytic exoenzymes could be bacterial conjugation
moved out of the cell • Flagella
• Cytoplasm o For movement/locomotion
o Composed largely of water, together with proteins, o The diameter is thin, 20nm, and long with some having
nucleic acids, lipids, and small amounts of sugars and a length 10 times the diameter of cell
salts o Due to their small diameter, flagella cannot be seen in
o Ribosomes – numerous; 15-20nm in diameter with light microscope unless a special stain is applied
70S; distributed throughout the cytoplasm; sensitive to o Bacteria can have one or more flagella arranged in
streptomycin and erythromycin; site of protein clumps or spread all over the cell
synthesis; a. Gram positive flagellum - Has 2 rings embedded
o Plasmids – extrachromosomal genetic elements into the basal body
o Inclusions – sources of stored energy, e.g., volutin
• Mesosomes
o Specialized structures formed by convoluted
invaginations of cytoplasmic membrane, and divided
into septal and lateral mesosomes
NOTE:
• Scientists found out that it is only produced by the
folding of the plasma membrane due to chemical
fixation techniques (artifacts) and is not actually a part
of the bacteria
• Nucleus
o Lacking nuclear membrane; absence of nucleoli, hence
known as nucleic material or nucleoid; one to several
per bacterium
o Bacteria has no true nucleus
• Plasmid
o Small, circular/line, extrachromosomal, double-
stranded DNA molecules. They are capable of self-
replication and contain genes that confer some

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 b. Gram negative flagellum - Has 4 rings embedded
into the basal body

o Sporulation

▪ Types of Flagella

a. Monotrichous – flagellum located on a

single pole
b. Lophotrichous – flagella located on one
pole
c. Peritrichous – flagella surround the
bacteria
d. Amphitrichous – flagella on both poles
of the bacteria
• Fimbriae
o Shorter, straighter, and thinner than flagella
o Not used for motility, but rather for attachment to
surfaces (i.e., gonorrheal bacteria and mucous
membranes)
• Axial Filaments
o Rotation of these filaments causes a corkscrewing
motion by the spirochete which helps propel it through
thick environments
o Certain spiral shaped bacteria such as the spirochete
Treponema pallidum which causes syphilis, have
bundles of fibrils that spiral around the cell
• Endospores (Spores)
o Resting structures formed by some bacteria for survival
during adverse environmental conditions
o The process of endospore formations is call
sporulation; The return of an endospore to its
vegetative state is called germination
o Endospore provides resistance against drying, low
nutrient conditions, radiation, high temperatures,
organic solvents, and other chemical disinfectants

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 CLINICAL BACTERIOLOGY

CELL METABOLISM AND GENETICS

OUTLINE • The actual energy that would drive the cell to be in motion.
I. Microbial Metabolism VI. Bacterial Genetics • It can synthesize or propel the bacterial cell to move around.
A. ATP A. Bacterial Genome
B. Redox Reaction B. Genome Size Oxidation-Reduction (Redox) Reaction
C. NADH and FADH C. Genotype vs.
II. Basic Metabolic Reactions Phenotype • Or Redox Reaction = chemical reactions in which electrons
III. Anabolism D. Plasmids are gained, lost or shared in a chemical reaction
IV. Catabolism VII. Bacterial Asexual • Oxidation - the loss of electrons by a molecule, atom or ion.
A. Cellular Respiration Reproduction
• Reduction – the gain of electrons by a molecule, atom or an
B. Aerobic Respiration VIII. Bacterial Sexual
Reproduction ion
i. Glygolysis
ii. PPP IX. Horizontal Gene Transfer
iii. EDP A. Conjugation
iv. Synthesis of acetyl- i. Plasmid Transfer
COA ii. Chromosome
v. Krebs Cycle Transfer
vi. ETC B. Transformation
C. Anaerobic Respiration C. Transduction
i. Fermentation i. Bacteriophage
V. Metabolic Pathways ii. Generalized
A. Polysaccharide Transduction
Biosynthesis iii. Specialized
B. Lipid Biosynthesis Transduction

MICROBIAL METABOLISM
• The sum of all chemical reations within a living organism. It NADH and FADH
is called an energy balancing act because it is a
combination of Catabolism and Anabolism • Special molecules that cells use to carry electrons (often in
• Transformation of food into energy; needs ATP. H atoms)
• Consists of the biochemical reactions bacteria use to break • This is potential energy, another way to transport energy.
down organic compounds and the reactions they use to • Two important carriers (energy):
synthesize new bacterial molecules from the resulting o Nicotinamide adenine dinucleotide 𝑁𝐴𝐷+ > ass
carbon skeletons. electrons and hydrogen > NADH
• Energy for the new constructions is generated during the o Flavine adenine dinucleotide (FAD) > add
metabolic breakdown of a substrate. electrons and hydrogen > 𝐹𝐴𝐷𝐻2
• Can be regulated in the cell either by regulation of the BASIC METABOLIC REACTIONS
production of an enzyme itself or by regulation of the activity
• Exergonic – produce more energy than they can consume
of the enzyme.
• Endergonic – consume more energy than they can
ATP – Adenosine Triphosphate produce

• Energy storing nucleotide

ANABOLISM
• Consists of an adenine, a ribose and 3 phosphate groups
• ATP = ADP + Pi, energy is released to drive anabolic • Anabolic – synthesis
reactions • Phase where the simple substances are synthesized to
complex materials which makes up the living tissue
• Involves dehydration synthesis

CATABOLISM
• Catabolic – breakdown
• Phase where there is a breakdown of complex molecules
into simple material, with the resulting occurrence of
release of energy
• Hydrolytic reactions occur
• Organisms catabolize carbohydrates as the primary energy
source for anabolic reactions via cellular respiration

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 Cellular Respiration Pentose Phosphate Pathway (option)
• The process of converting nutrients into ATP. • Pentose Phosphate Pathway (PP) or Hexose
• Molecules are being oxidize and the final electron acceptor monophosphate shunt - provides a mans for the breakdown
will be in the form of inorganic molecule or oxygen. of 5-carbon sugars as well as glucose.
• There are two types of respiration depending on the type of • It produces important intermediate pentoses used in the
bacteria: aerobes (uses oxygen) and anaerobes (doesn’t synthesis of nucleic acids, glucose from carbon dioxide in
use oxygen) photosynthesis and certain amino acids.
Aerobic Cellular Respiration • Important producer of the reduced coenzyme NADPH (from
NADP)
• Results in complete breakdown of glucose to carbon
• Yields a net gain of only 1 ATP plus 12 NADPH for each
dioxide, water and a lot of ATP
molecule of glucose oxidized.
• Final electron acceptor is oxygen
• It should undergo to the 4 sub pathways of cellular Entner-Doudoroff Pathway
respiration to convert the carbohydrates into a usable • Produces 2 NADPH and 1 ATP for each molecule of glucose
energy in the form of ATP; 4 steps are involved: • Bacteria that have enzymes for this pathway can metabolize
o Glycolysis glucose without either glycolysis or the PPP
o Synthesis of acetyl-COA
• Found in some gram-negative bacteria and none in gram
o Krebs cycle
positive bacteria
o Electron transport chain

Glycolysis Synthesis of Acetyl-CoA

• Based on Embden-Meyerhof-Parnas Pathway
• Breakdown of glucose into pyruvic acid
• Composed of 2 stages:
o Energy investing stage
▪ Needs 2 ATP to breakdown the glucose
into Glu-6-P down to Fru-1,6-bP
o Energy conserving stage
▪ From Fru-1,6-bP, it will produce DHAP
and GAP, and they will produce 2 Pyruvic
acid (1 each)
• Produces 1 𝐶𝑂2 , 1 NADH, 1 CoA per 1 Pyruvic Acid (so bale
▪ It will produce 4 ATP and 2 NADH
x2 mo sila since 2 Pyr yung naproduce during glycolysis)
• Net Gain: 2 NADH and 2 ATP (4-2 kase 2 yung ginamit na
ATP sa 1st stage) Krebs Cycle

• 1 CoA produces 3 NADH, 1 FADH, 1 GDP and 2 𝐶𝑂2 (x2 ulit

kase 2 CoA yung naproduce natin dun sa synthesis of CoA)
• FADH and NADH are electron carrier
• Occurs in the cytoplasm of most cells
• Involves splitting of a 6-carbon glucose into two 3-carbon Electron Transport Chain (ETC)
sugars.
• The sugars are then oxidized releasing and their atoms are • Most of the ATP made inn cellular respiration come from the
rearranged to form 2 molecules of pyruvic acid stepwise release of energy through a series of redox
• Results in a net gain of 2ATP and 2NADH reactions between molecules known as the electron
transport chain

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• Most occur in a membrane. The ETC is located in cristae of o Superoxide dismutase (SOD): oxygen radical >
mitochondria in eukaryotes. 𝐻2 𝑂 and 𝑂2
• In prokaryotes, ETC happens in the plasma membrane. ▪ Microbes that don’t make these enzymes cannot exists in
• 1 NADH = 3ATP, 1 FADH = 2ATP the presence of oxygen

Anaerobic Cellular Respiration

▪ Only partially breakdown glucose, into pyruvic acid and
organic waste products and a little ATP
▪ Final electron acceptor is an inorganic molecule or rarely an
organic molecule.
▪ Cells that don’t have access to oxygen, or that are obligate
anaerobes can make ATP by using something other than
oxygen as an electron acceptor (nitrate, sulfate and carbon
dioxide)
▪ Not all the ETC is used, so less ATP us produced
▪ Without catalase and superoxide dismutase, performing
aerobic respiration cannot happen

• Carrier Molecules Fermentation

o Flavoproteins: Proteins containing flavin derived ▪ An alternative system that allows glycolysis to continue
from Vit B2 (riboflavin) without the other steps of respiration
o Cytochromes: Proteins with an iron containing ▪ Releases energy from sugars or other organic molecules
group (heme) such as amino acids, organic acids, purines and pyrimidines
o Ubiquinone or CoQ: Small non-protein carriers ▪ Uses an organic molecule as the final electron acceptor
▪ Carrier molecules will process the Redox Reaction so that ▪ Not as energetically efficient as respiration
the hydrogen will be pump to the other side of the plasma ▪ Produces only 2 ATP
membrane ▪ Some fermentation pathways used by the microbes that
inhabit the human body are as follows:
Aerobic Cellular Respiration o Alcoholic fermentation
▪ Utilizes glycolysis, synthesis of acetyl-CoA, Krebs cycle, and ▪ The major end product is ethanol. This is
electron transport chain; results in complete breakdown of the pathway used by yeasts when they
glucose to carbon dioxide, water and energy ferment glucose to produce ethanol.
▪ The ultimate objective is to make molecules to do cellular o Homolactic fermentation
work ▪ The end product is almost exclusively
▪ A total of 38 molecules of ATP (approx.) are formed from 1 lactic acid. Members of the genus
molecule of glucose Streptococcus and many members of the
genus Lactobacillus ferment pyruvate
using this pathway.
o Heterolactic fermentation
▪ Some lactobacilli use a mixed
fermentation pathway, of which, in
addition to lactic acid, the end products
include carbon dioxide, alcohols, formic
acid, and acetic acid.
o Propionic acid fermentation
▪ Propionic acid is the major end product
of fermentation carried out by
Propionibacterium acnes and some
anaerobic non–spore-forming, gram-
positive bacilli.
o Mixed acid fermentation
▪ Members of the genera Escherichia,
Salmonella, and Shigella within the family
Enterobacteriaceae use this pathway for
carbohydrate fermentation and produce
a number of acids as end products: lactic,
▪ Using oxygen (1/2 Oxygen) in metabolism creates toxic acetic, succinic, and formic. The strong
waste acid produced is the basis for the positive
▪ Microbes that are able to use aerobic respiration produce reaction on the methyl red test exhibited
enzymes to detoxify oxygen: by these organisms.
o Catalase: 𝐻2 𝑂2 > 𝑂2 o Butanediol fermentation

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 ▪ Members of the genera Klebsiella,
Enterobacter, and Serratia within the Lipid Biosynthesis
family Enterobacteriaceae use this • Cells synthesize fats by joining glycerol and fatty acids
pathway for carbohydrate fermentation. • They glycerol portion is derived from dihydroxyacetone
The end products are acetoin (acetyl phosphate (glycolysis intermediate)
methyl carbinol) and 2,3-butanediol.
Detection of acetoin is the basis for the BACTERIAL GENETICS
positive VP reaction characteristic of
these microorganisms. Little acid is Bacterial Genome
produced by this pathway. Thus • Generally composed of a single, circular chromosome
organisms that have a positive VP • Haploid chromosome
reaction usually have a negative reaction • Chromosome is not enclosed by a membrane but located in
on the methyl red test, and vice versa. the nucleoid that is about 1/3 of the interior volume
o Butyric acid fermentation • Proteins aid in holding the chromosome in this nucleoid
▪ Certain obligate anaerobes, including space, which is filled with genetic material devoid of
many Clostridium spp., Fusobacterium, chromosome
and Eubacterium, produce butyric acid
as their primary end product along with
Genome Size
acetic acid, carbon dioxide, and • Complete set of genes
hydrogen. • An average bacterial chromosome is between 0.16 Mb and
10 Mb
COMPARISONS BETWEEN AEROBIC AND ANAEROBIC • In general, free-living bacteria have longer genomes and
RESPIRATION more genes than bacteria that are obligate parasites
AEROBIC RESPIRATION ANAEROBIC RESPIRATION • Bacteria thriving inside a human has a shorter genome
Requires oxygen Does not require oxygen
Complete Oxidation of Incomplete oxidation of Genotype vs. Phenotype
respiratory material respiratory material
• Genotype is the genetic composition of the organism
Produce maximum of 38 ATP Produce only 2 ATP
o Dictates the characteristic of the phenotype of the
molecules per glucose molecules per glucose
organism
Consists of glycolysis, Krebs Consists of glycolysis and
Cycle and Oxidative incomplete breakdown of • Specific genes are designated usually by 3 lower case
phosphorylation pyruvate letters that are italicized if several genes are involved
Respiratory materials are Respiratory materials are not • Usually distinguished by capital letters following the
completely oxidated completely oxidized genotype
Water is formed Normally, water is not formed o Ex: Gene: Aerotaxis = aerB, aerC, aerD (different
types)
• Some mutation sites can be seen in a gene.
o Wild type (natural setting): + superscript (allele)
▪ Ex. aer+
o Mutated: - superscript
▪ Ex. aer-
• Phenotype of an organism is an observable property
o If the phenotype is known to be controlled by a
particular gene, the property is designated by the
gene name
• Phenotype is designated starting with a capital letter
followed by 2 lowercase letters with + or – sign superscript
to indicate the wildtype or natural allele or mutant

Plasmids
METABOLIC PATHWAYS OF ENERGY USED • Small, circular and extrachromosomal DNA strands found
in some bacteria
• Microbes used ATP to provide energy for the transport of • Plasmids do not carry any genes essential for normal
substances across plasma membranes (active transport) bacterial growth or function
• Microbes also use some of their energy for flagellar motion • Some plasmids carry genes that provide a selective
• Most ATP is used in the production of new cellular advantage in the environment
components • Some plasmids carry genes that allow for bacterial
Polysaccharide biosynthesis conjugation, antibiotic resistance, or even antibiotic
production
• The carbon atoms required to synthesize glucose are
derived from the intermediated produced during glycolysis BACTERIAL ASEXUAL REPRODUCTION
and the Krebs’s cycle and from lipids or amino acids
• Binary fission: how bacteria reproduce; asexually
• Glucose units are phosphorylated and linked

4
 o A bacterial cell will duplicate its chromosome and • The final result is a copy of the plasmid in both the donor
will grow until it can divide into 2 (splitting point) and the recipient cell.
o 1 mother cell = 2 daughter cell = 4 = 8 = 16 (by • Most interesting fact that the plasmid, in addition to other
two’s) genes, carries genes that allow the recipient to become a
• Bacterial cells reproduce by making clones of themselves conjugation donor itself. Now, the recipient cell can also
• Mother cell copies its DNA chromosome the splits her cell spread the plasmid to new cells it encounters in its
in half keeping one chromosome and giving one to the new environment.
daughter cell Chromosome Transfer
• The mother and daughter cells are actually clones
• In a bacterial population, this process continues, one cell • Only a portion of the chromosome is transferred before the
dividing into two again and again and again, resulting in conjugation pilus is detached
huge populations of cells that are all clones of one another • The genes that make chromosome transfer possible are
• It is considered vertical gene transfer when DNA is passed very last genes to be sent to the recipient. So, if the
from mother to offspring cell, this is what happened in conjugation pilus becomes detached partway through the
nature the vast majority of the time. transfer, the recipient cell does not become a new donor
cell.
BACTERIAL SEXUAL REPRODUCTION (?)
• Refers to the ability of some bacterial cells to acquire new Transformation
genes from neighboring cells in their environment • Is the ability of some cells (competent cells; more on gram
• Instead of being a clone daughter cell, the cell has new positive) to take up free floating DNA found in the
genetic diversity, a mix between the vertically transferred environment (DNA contents from the destroyed bacteria)
mother cell DNA and the horizontally transferred neighbor • The DNA either has to be incorporated into the host cell
cell DNA DNA chromosome (via genetic recombination: mix and
• Hindi kino-consider ni Ms. F as sexual repro. match to the genes of bacteria) or it ends up being
HORIZONTAL GENE TRANSFER degraded
• Usually only a few genes are able to be incorporated
• Responsible in creating diversity among bacterial • If a gene that is taken up is similar to a gene already found
population in the chromosome, the two DNA sequence will line up
• The reason why not all bacteria are the same; they have the beside one another and the swap, replacing the old gene
ability to transfer genes, horizontally from 1 bacteria to with the new one.
another, different or similar types of bacteria.
• There are 3 ways that bacteria are able to carry out Transduction
horizontal gene transfer:
o Transduction: Bacteriophage
▪ Uses bacterial viruses called • Bacteriophage – virus infecting bacteria
bacteriophages, to transfer from 1 • When the Phage particle infects another bacteria, DNA
infected cell to another transfer is effected and the recipient cell acquires new
o Transformation: characters coded by donor DNA
▪ It is the ability of some cells to take up • Types of Bacteriophages:
free floating DNA found in the o Virulent bacteriophage
environment, ▪ Uses the bacterium as a viral synthesis
o Conjugation: factory
▪ Allows for the transfer of DNA through a ▪ They break down the bacterial cell
structure called a pilus from one cell to machinery to produce more phages
another ▪ Progeny phages are released from the
Conjugation bacterium as the cell lyses
o Temperate bacteriophage
• For conjugation to take place, two live bacterial cells must
▪ Can be virulent
come into direct contact with one another.
▪ Phage genome may integrate into the
• Contact between the cells is accomplished using a
bacterial chromosome as a prophage
conjugation pilus
▪ Bacteria that contain a prophage are said
• The special conjugation pilus is used specifically to attach
to be lysogenic for that phage.
to other cells and facilitate DNA transfer
• The cell that will transfer DNA is called the donor cell and Generalized Transduction
the conjugation pilus attached to its cell. • During this process, the bacteriophage chops up the host
• The conjugation pilus is a hollow, pipe-like structure that chromosome into many small fragments.
connects the cytoplasm of the donor cell to the cytoplasm • When the baby bacteriophage are being assembled, some
of the recipient cell. of these host DNA chunks can accidentally be packed into
Plasmid Transfer the new viral particles
• They are then carried to a new host cell, where they are
• This is done by copying the plasmid and sending the strand
injected and can cause genetic recombination.
of copied DNA to the recipient through the conjugation
pilus.

5
 Specialized Transduction
• The viral DNA goes dormant by incorporating itself into the
bacterial DNA chromosome
• The host cell survives and continues to grow and divide,
passing on the incorporated viral DNA to the clone off spring
cells
• Eventually, the bacteriophage reactivates itself and cuts
itself free from the host cell genome
• It then hijacks the cell to produce new bacteriophage that
lyse the host cell and go on to infect additional cells

Discovery of Transduction
• First, they ruled out transformation by verifying that the
culture was free contaminating DNA. Since there was no
contamination, there was no way for bacteria to take up
freely floating DNA from the environment
• Then they ruled out conjugation by keeping cells isolated
from one another by a thin membrane, If the cells couldn’t
touch, there was no way for the bacteria to conjugate
• When they still detected genetic recombination, it was time
to do some sleuthing. Eventually they discovered the tiny
bacteriophage particles in the culture and uncovered the
details of transduction.

6
 CLINICAL BACTERIOLOGY

MICROBIAL GROWTH AND CONTROL

OUTLINE ▪ Psychotrophs- grows in
I. Requirements for Growth J. Steps in the temperature within 0˚C to 15˚C
A. Physical Pathogenesis of (Alaska)
i. Temperature Infectious Disease ▪ Moderate psychophiles- able to
ii.pH K. Virulence
grow between 20˚C to 30 ˚C
iii.Osmotic pressure L. Virulence Factors
B. Chemical i.Attachment (Canada)
i.Carbon ii.Exoenzymes o Mesophiles
ii.Nitrogen, Sulfur, and iii.Endotoxin ▪ involved in infectious disease
Phophorus iv.Exotoxin process
iv.Trace Elements M. Mechanisms by Which
v.Oxygen Pathogens Escape ▪ live in moderate temperature
C. Organic Growth Factors Immune Responses (between 25˚C to 40˚C)
D. Anaerobic Growth Media III. Host Defense Mechanism ▪ the optimum temperature is between
………and Methods A. Definition of Terms 35˚C to 37˚C (body temperature)
E. Microbial Control Methods B. Host Defense
▪ When culturing bacteria, set the
II. Infection Mechanism
A. Definition of Terms C. Three Lines of incubator at ±35˚C to cater the
B. Classification of Defense Against optimum temperature that the
Infections Infection mesophiles would need.
C. Local vs. Systemic D. Non-specific Host o Thermophiles
Infections Defense Mechanism
▪ live between 50˚C to 60˚C
D. Infection vs. Infectious i.First Line of Defense
Disease ii.Second Line of o Hyperthermophiles
E. Acute, Subacute, and Defense ▪ need 80˚C or higher temperature
Chronic Disease E. Specific Host Defense requirement
F. Latent infection Mechanism ▪ some can tolerate as much as 121˚C
G. Symptoms vs. Signs of F. Two Types of
Disease Immunity (same in autoclave)
H. Pathogenesis of G. Classes of
Infectious Disease Immunoglobulin
I. 4 Periods in the Course H. CMI
of an Infectious Disease I. Lymphocytes pH
• Most bacteria grow best in a narrow pH range neutrality
between 6.5-7.5
• Each bacterial species has its own preferential growth
REQUIREMENTS FOR GROWTH at certain pH
• Lowest pH (acidic) recorded bacteria where it is able to
grow is pH 1 is Chemoautotrophic bacteria
Physical
(cyanobacteria).
• Very few bacteria grow below pH 4
Temperature • Molds and yeasts grow at pH 5 to 6, they can tolerate
• The first requirement needed by the bacteria is the temperature and low pH
correct temperature. • When bacteria are cultured in the laboratory, they often
• Most bacteria grow only within a limited range of produce acids that interfere with their own growth. That
temperatures. is why we need to ensure that the culture medium
• Each bacterial species grows at a particular minimum contains chemical buffers (in growth medium), so when
(lowest temperature which bacterial species will grow), acids are produced by the bacteria it will not lower the
optimum (temperature which bacteria will grow best) pH in the medium, they would die in too much presence
and maximum temperatures (highest temperature on of acidity.
which growth is possible for the bacteria). • Chemical buffers should be included in culture
• Bacteria is very diverse and each of it has its own mediums that we have to provide for them (peptones,
preference for temperature for them to grow. amino acids, and salts)
• On the basis of preferred range of temperature,
bacteria can be classified as:
o Psychrophiles

1
 Osmotic Pressure
• Organisms require water for growth
• The growth of the cell is inhibited as the plasma membrane
pulls away from the cell (addition of salts)
• Hypertonic or hypotonic environment
o Hypertonic environment
- Adding salt
- Will cause the cellular water (the water
from the bacterial cell inside) to pass out
through the plasma membrane whether it
is high salt concentration, from rigid low
concentration to higher region of
concentration (high solute
concentration).
- If the bacterial cellular water passes out
to the plasma membrane the bacterial Nitrogen, Sulfur, and Phosphorus
cell will shrink and will die • Nitrogen makes up the 14% of the dry weight of the bacterial
o Hypotonic environment cell. Sulfur and Phosphorous are about 4%
- Exposed to distilled water, the water • These chemicals are needed by the bacteria for synthesis
tends to enter the cell (from a region of of protein
lower concentration to a higher • For protein synthesis (Nitrogen and Sulfur)
concentration). It will enter the bacterial • For DNA and RNA synthesis
cell and the bacterial cell will swell, when
• K, Mg, and Ca are also needed as co-factors for bacterial
it cannot hold the water coming inside, it enzymes
will burst.
- Bursting means lysing of the cell, the Trace Elements
bacterial cell will die.
• Some microbes have relatively weak cell wall maybe by the
• (Fe) Iron
increase of osmotic pressure, it can also inhibit bacterial
• (Cu) Copper
growth. It is very important to provide the correct amount of
• (Mo) Molybdenum
water, hypotonicity, and hypertonicity of the environment
• (Zn) Zinc
should also be carefully considered.
• They are naturally present in tap water
• Obligate or extreme halophiles (30% salt)
• Halophiles - salt loving bacteria that can tolerate 30% salt Oxygen
• Obligate halophiles - strictly needed for a hypertonic • Metabolic systems require oxygen for aerobic respiration
environment for them to be able to live (metabolic pathways, final electron acceptor is oxygen)
• Facultative halophiles - 2-5% salt concentration is what • Microbes that use molecular oxygen produce more energy
they need in order to grow from nutrient than microbes that do not use oxygen (more
• Bacteria have diverse requirement ATP, more energy is produced when you use molecular
oxygen than those do not use oxygen)
• Oxygen is important for living organisms. Oxygen is also
Chemical
poisonous, a toxic gas. We normally produce the toxic
• Sources of C, N, S, P, trace elements, O2, and organic forms of oxygen during respiration in small amounts.
growth factors. Therefore, we also produce toxic gas as well, this is the way
how oxygen is toxic. It will instigate the chemical reactions
Carbon inside the body that can produce free radical, super active
• All organic compounds are made up of Carbon (even molecules that can destroy our cells. But our bodies are
humans) designed to capture and be able to use nutrients like oxygen
• C is the structural backbone of living matter to our advantage.
• Half of the dry weight of typical bacterial cell is carbon • Obligate aerobes – strict aerobes, oxygen is a requirement
• Chemoheterotrophs, chemoautotrophs, photoheterotrophs, • Facultative anaerobes – part time anaerobes, basically
and photoautotrophs aerobes their energy will decrease in the absence of
• Chemotrophs (chemical energy source), Phototrophs oxygen, they can use oxygen to their advantage
(light energy source) • Obligate anaerobes - strict anaerobes, they can live
• Largely, bacteria are classified under chemoheterotrophs. without the presence of oxygen
• Aerotolerant anaerobes - anaerobes that can tolerate
oxygen, they cannot use oxygen to their advantage

2
• Microaerophiles - type of bacteria that can only tolerate o Most aerobic respiration produces traces of
small amount of oxygen, around 5-10% of oxygen. Oxygen hydroxyl radicals but they are transient (go
in the air is around 21%. away).
• Capnophiles (capnophilic)- needs carbon dioxide when o In phagolysosome, once we ingest
they metabolize and live pathogens, they are killed by exposure to
these toxic forms of oxygen. These are
contained in the lysosomes.

Organic Growth Factors

• Vitamins which functions as co-enzymes
• Other organic growth factors: amino acids, purines,
pyrimidines
• May be synthesized by bacteria themselves or should be
obtained from the environment

Anaerobic Growth Media and Methods

• Reduced media is used such as sodium thioglycollate that
chemically combine with dissolved oxygen and deplete the
oxygen in the medium

Anaerobic System

• Toxic Forms of Oxygen- can destroy cell membrane.

• Singlet oxygen (1O2-)
o is normal O2 boosted into a higher energy
“Anaerobic Jar System”
state and is extremely active
• Culture the bacteria, put those culture plate or broth
• Superoxide free radicals (O2-)
inside and then put a gas pack, pack of chemicals
o formed in small amounts during the normal
(sodium bicarbonate & sodium borohydride)
respiration of organisms. SOD neutralizes
• Cut off the edge of packet, put water. Once it is filled
this.
with water, hydrogen and carbon dioxide will be
o Oxygen combined with hydrogen the SOD is
produce by this pack of chemicals. The palladium
the one that will neutralize the superoxide free
catalyst will allow the oxygen in the jar combined with
radicals on order to convert them into simpler
the hydrogen produced by this packet of chemicals.
chemicals, it will be converted by the SOD into
Oxygen and hydrogen, water is formed. When they
hydrogen peroxide and oxygen. But still,
react, you will see droplets of water inside the jar. It
peroxide is still a toxic form
means the reaction is taking place and the oxygen is
o This can start several chemical reactions; this
being removed. Carbon dioxide is the only element
is the form we normally respire
remains inside
O2 + O2 + 2H+ → H2O2 + O2
• Peroxide anion (O22-)
o Toxic from which is neutralize by catalase
converts it to water and oxygen;
2H2O2 → 2H2O + O2
o Peroxidase also convert it to water but no
oxygen, thus (in case catalase is absent)
H2O2 + 2H+ → 2H2O
• Hydroxyl radical (OH-)
o Intermediate form of oxygen and probably the
most reactive. It is formed in the cellular
“Candle Jar Extinction”
cytoplasm by ionizing radiation.

3
 INFECTION

Definition of terms

• Pathogenesis is the step of mechanisms involved in the

development of the disease
o Path- refers to disease
• Pathogenicity – the ability to cause disease
• Pathogen – is a microorganism capable of causing
“Anaerobic Chamber” disease (may be bacteria, virus, fungi, etc.)
• It has a containment for the mixture of oxygen provided • Pathology is the study of the structural and functional
in cylinders of gases (nitrogen, oxygen, etc.). It is tightly manifestation of the disease
closed and sealed. • Infection
• For greater mobility for culturing anaerobes, it is the o Injurious contamination of body or parts of the
best system for anaerobic environment setting. body by bacteria, viruses, fungi, protozoa, and
rickettsia or by the toxin that they may
produce.
Capnophiles
o May be local or generalized and spread
throughout the body
o Once the infectious agent enters the host, it
begins to proliferate and reacts with the
defense mechanisms of the body producing
infection symptoms and signs (pain, swelling,
redness, functional disorder, rise in
temperature, pulse rate, and increase in
WBC)

Classification of Infection
• Primary infection
o Initial infection with organism in host
o First time that the host will encounter a
microbe, virus, or any microbe is the primary
infection
Microbial Control Methods
• Reinfection
o Subsequent infection by same organism in a
host (after recovery)
o After recovery, the same organism will attack
the host
• Superinfection
o Infection by same organism in a host before
recovery
o Infection by the same organism before the
host could hardly recover
• Secondary infection
o When in a host whose resistance is lowered
by preexisting infectious disease, a new
organism may set up infection
o The host could have a bacterial infection in the
beginning, because of the
immunocompromised situation of the patient,
another infection set up by maybe a virus is
now on its way
• Focal infection
o It is the condition where due to infection at
localized sites like appendix and tonsil,
general effects are produced.
o Only in a certain area of the body

4
 • Cross infection
Chronic Disease
o When a patient suffering from a disease and
new infection it set up from another host or • Has a slow onset and last a long time.
external source • Examples: Tuberculosis, leprosy, syphilis
o One patient with the disease, the infection is • It will take a long time for a patient to recover from these
transmitted to another host set of illness.
• Nosocomial infection Subacute
o Cross infection occurring in hospital
• Diseases that come on more suddenly than chronic
o Hospital acquire infections
diseases but less suddenly than acute diseases.
▪ Examples: facilities, contaminated
• Somewhere in between acute and chronic illness
surfaces, contaminated materials
• Example: Bacterial endocarditis
• Subclinical infection
o It is one where clinical effect are not apparent
o The infection is in the patients but there are Latent Infections
no signs and symptoms that would tell you
that the patient is infected with the certain • A disease that may go from being symptomatic to
microbe asymptomatic and then back to being symptomatic.
• The causative agent or pathogen remains inactive for a
Local vs. Systemic Infection period of time and then becomes active again when it
changes its mind and produce symptoms again
• Example: Syphilis – for three weeks it will present signs and
Localized
symptoms, and after 2 to 6 months will have the primary
• An infection that remains localized at one site chancre, and then develop rashes, lesions, fever within 2 to
• Remains confined to a certain site 6 months’ period. Later on, after five years or few years, no
• Examples: Pimples, boils abscess symptoms will be present. Only to find out that after other
Systemic/Generalized years again, symptoms will reappear and the body is
already destructed (spinal cord and organs are really
• An infection that has spread throughout the body
destroyed)
• Example: Tuberculosis – it may spread to different
organs of the body
Symptoms vs. Signs of Disease
Primary vs. Secondary Infection
Symptoms
• Primary infections- the first disease
• some evidence of a disease that is experienced or
• Secondary Infections- the second disease
perceived by the patient only
• This is only known by the patients
Infection vs. Infectious Disease • Examples: Aches, pains nausea, itchiness
o Symptomatic disease (clinical disease) – a
• Infectious disease is a disease cause by a microbe disease in which the patient is experiencing
and the microbes that cause infectious disease are symptoms
collectively referred to as pathogens. o Asymptomatic disease (subclinical disease) – a
• Infection, according to many microbiologists, means disease that the patient is unaware of because
colonization by a pathogen. he/she is not experiencing any symptoms
• A person can be infected with a certain pathogen but
not necessarily have the infectious disease. Signs
• Infection means there are pathogens colonizing a • some type of objective evidence of a disease
certain area, but it does not actually involve in the • Examples: Fever, pulse rate, blood pressure, test result,
disease yet. A person may be infected but do not have respiratory rate, bradycardia, tachycardia
the illness yet. Meaning a person is infected, maybe not
presenting any signs and symptoms. Pathogenesis of Infectious Disease

Acute, Subacute, and Chronic Disease Questions:

• Why would infection would not necessarily result to
infectious disease?
Acute Disease
• Why would a certain infection would not lead to infectious
• Has rapid onset usually by a relatively rapid recovery
disease?
• Examples: Measles, mumps, influenza
• What are the possible reasons why a person maybe infected
• These are acute diseases you would recover from 1 to
with a certain pathogen but not necessarily have the
2 weeks
infectious disease?

5
 factors that destroy the pathogen) that destroy the
newly arrived pathogen
Why infection does not always occur?
o Importance of presence of microbiota in the
• The microbe may land at an anatomical site where
body
it is unable to multiply
o Pathogen might have landed on the skin but
there is the normal microbiota that made the 4 Periods in the Course of an Infectious Disease
bacteria not succumb to the infection because
it counterattacks those invading microbes that Incubation Period
might land on the skin. • The time that elapses between the arrival of the
o Some other reasons are the factors in the skin pathogen and the onset of symptoms
that would not promote the multiplication of • Incubation period will depend on the nature of the
the bacteria (presence of oils, chemicals) that
microbe that arrived in the body/system. (short or long)
would not allow the microbe to survive those
• This the time when you are not yet experiencing
chemicals on those conditions.
symptoms
• This time the pathogen had just arrived
• Many pathogens must attach to specific receptor
• Factors influencing the incubation period:
site before they are able to multiply and cause
o Over-all health and nutritional status of the
damage
host- the healthier you are, the lesser chance
o This is the reason why viruses that are in
or the longer it will be for the incubation
animals cannot transfer to humans because
period. The pathogens will have a difficult time
they don’t have specific receptor sites on
creating or trying to find the chance to invade
human cells.
the body
o Same with the animals to animals, the
infection cannot transfer because of the
o Virulence of the pathogen- mechanism or
unavailability of receptor sites.
factors in the pathogen will make it able to
cause disease.
• Antibacterial factors that destroy or inhibit the
growth of the microbes maybe present at the site ▪ Examples: Fungi (low virulence),
where the pathogens are Corona virus (highly virulent)
o Possibility of antibiotics, antimicrobials and
the likes are present that would not promote o Numbers of the pathogens that enters the
survival of the pathogen in that area or site. body- for some disease, it will take a several
numbers of the pathogen before they could
• The individuals’ nutritional and overall health status initiate a disease.
often influences the outcome of the pathogen/host ▪ Ex. Tuberculosis – only one live
encounter tuberculosis bacilli can cause
o If the resistance is high and immune to a tuberculosis in an
certain pathogen, there are chances on not immunocompromised patient
getting the disease. If you are overall healthy, ▪ For some disease, it will take a 106 of
(getting enough sleep, exercising) have good bacteria to be able to cause diarrhea
immunity, the body will not be succumb in the for example.
pathogen. ▪ The number of pathogens will
depend as it enters the body
• The person maybe immune to that particular
pathogen and is the result of prior infection or
Prodromal Period
vaccination
• the time during which the patient starts to feel
o Possibility of being immunized or vaccinated
something is wrong but do not experience yet the
with that particular pathogen.
actual symptoms of the disease
• Signs and symptoms are present but not specific
• Phagocytic white blood cells may engulf and
(nonspecific signs and symptoms)
destroy before it has an opportunity to multiply,
• Ex. Fever, feeling heavy
invade and cause disease
• The period wherein you know something is wrong in
o Presence of phagocytes that had a chance to
your body but don’t know the cause.
engulf the pathogen before it could create
havoc to the host

• The indigenous microbiota at the site may produce

antibacterial factors (bacteriocins – antibacterial

6
 • INVASION/SPREAD of the pathogen
Period of Illness o The spread of the pathogen will create the signs
and symptoms and at this time the host defenses
• The time when the person experiences the symptoms will be evaded by the pathogen
of the disease. Communicable diseases are mostly
easily transmitted during this stage • EVASION of host defenses
• Specific signs and symptoms are present that actually o It will create damage to the host, leaving the host
identify having illness defenseless

• DAMAGE to host
Convalescent Period
o The host defenses have been evaded by the
• The time when the person recovers. Person may pathogen so it will now create damage to the host
recover from the illness but there may be permanent
damage of tissues of the affected area Virulence
• This 4th period may be either convalescent period or • It is a measure of the degree of pathogenicity; different
death/disability (depending on the outcome) species or different strains of microbe vary in durability to
• This is the time when the person recovers cause disease.
(convalescent) • Virulence factors are the phenotypic characteristics of
• The person may recover from the illness but there may microorganisms that enable it to cause disease
be permanent damage of the affected areas (disability) • Virulent Strains – are capable of causing disease
or death • Avirulent strains – not capable of causing disease

Virulence Factors

Attachment

• To cause the disease, the microorganism must be able to

anchor themselves to cells after gaining entrance into the
body. This is where attachment factors are very important
• Receptors and Integrins
o Molecules on the surface of the host cell that a
particular pathogen is able to recognize and attach
to which are often glycoprotein molecules
TAKE NOTE: As the period of infectious disease continue, the o Surface of the host cell is the particular site where
number of the microorganisms initially in the incubation period the pathogen will be able to attach.
are below. It will peak at a certain point (including the period of o Receptors is where the pathogen will attach
illness), and then decline (the number of microorganisms). As • Adhesins and Ligand
the period progresses at the point of period of decline, the o Used to describe the molecule on the surface of
number of microorganisms will also decline, up to the period of the pathogen that is able to recognize and bind to
recovery or convalescents, wherein the number of a particular receptor
microorganisms will be at the lowest. o Therefore, the receptors and integrins are in the
host. While the adhesins and ligands are on the
pathogens.
Steps in the Pathogenesis of Infectious Disease
o Ex. Streptococcus pyogenes – they have adhesins
• ENTRY of the pathogen into the body
called protein F, it is on their surface that will
o We have several portals of entry in the body.
adhere to the protein on the host cell called
Examples: Skin and Mucous membrane
fibronectin (lock and key). Adhesion on the
pathogen and then surface/receptor protein on the
• ATTACHMENT of the pathogen to some tissues
host cell.
o Once it has gain entrance to the body it will attach
o Ex. HIV – they have the GP120 (glycoprotein 120)
to some tissues
it is an adhesin, it must attach to the CD4+ cells or
T helper cells of the host cell.
• MULTIPLICATION of the pathogen
• Bacterial Fimbriae (Pili) / Common pili
o After attaching, it will lodge into the tissues and
o Long thin, hair-like, flexible projections composed
cells of the body and multiply. The multiplication of
of primarily of an array of proteins called pilin (by
the pathogen will lead to the spread and invasion
nature it is protein) which enable the bacteria to
of the different body parts or tissues of the body
attach to the surfaces and cause infection

7
 o Bacteria like Neisseria gonorrhea (gram negative o Ex. Streptococcus can produce streptokinase
diplococci) can anchor to the inner walls of the that will be able to dissolve blood clots
urethra causing urethritis. But that Neisseria • Hyaluronidase
gonorrhea that are not fimbriated can be flushed o Called spreading factor because it enables
out when the host urinate. The fimbriated strains the pathogen to spread throughout the
are therefore more virulent than the non-fimbriated connective tissue by breaking down
strains. They are more likely to cause disease is hyaluronic acid
the virulent factor for them. o Hyaluronic acid – this is a polysaccharide
• Obligated Intracellular Pathogens cement that holds the tissues together
o Rickettsia, Chlamydia, Ehrlichia must live o Once the bacteria have this enzyme the tissue
within the host cells in order to survive and will fall apart. When the tissue is destroyed,
multiply the pathogen can spread throughout the
o When the pathogen is inside cell, antibodies connective tissue.
will not work for them because they are inside o Hyaluronidase is secreted by
the host cell Staphylococcus, Streptococcus, and
• Facultative Intracellular Pathogens Clostridium spp.
o Pathogens that are able to survive • Collagenase
intracellularly and extracellularly. These o Breaks down collagen which is the supportive
microorganisms are able to survive within protein found in tendons, cartilage and bones
phagocytes. enabling the pathogen to invade tissues
o Once the bacteria are ingested by WBCs o If the bacteria can excrete collagenase, it can
(macrophages or neutrophils), the invade tissues. This is one virulence factor of
phagosome will fuse with the lysosomes Clostridium perfringens
(contains superoxide hydrolytic enzyme • Hemolysins
peroxide) which will cause destruction of o Enzymes that cause destruction of RBCs.
bacteria. o Provides iron to the pathogen
o Once ingested, facultative in the cellular o The pathogen lyses the RBCs so that it can
pathogens could opt to leave the cell and go capture the iron
outside the host cell. o The Hemolysins could be alpha-beta or
• Capsule gamma hemolysis
o They can evade phagocytosis because they • Lecithinase
are sticky or slimy o Enzyme produced by Clostridium perfringens
• Flagella which breaks down phospholipids collectively
o They enable to invade aqueous area of the referred to as lecithin. This enzyme is
body that non-flagellated bacteria will not be destructive to cell membrane of RBC and
able to do. It is hard to be targeted by host other tissues
defense mechanism because moves around. o This is how Clostridium perfringens cause gas
gangrene.
Exoenzyme
Endotoxin
• enzymes that are liberated by the bacteria outside.
• Necrotizing Enzymes • Integral part of the cell walls of gram-negative
o Bacteria produce proteases and lipases which bacteria producing septic shock, chills, fever,
cause destruction of tissues prostration, and septicaemia during gram negative
o Ex. Proteases and Lipases – streptococcus infection. The cell wall contains lipopolysaccharide
pyogenes this strain will cause destruction of (LPS) called LIPID A, which is actually the endotoxin.
soft tissues called flesh eating necrotizing
fasciitis Characteristics of Endotoxins
• Coagulase
• Proteins polysaccharide lipid complex heat stable
o Enable S. aureus (positive for coagulase) to
• Forms part of cell wall (don’t diffuse into medium)
clot plasma and thereby form a sticky coat of
• Obtained only by cell lysis
fibrin around themselves for protection
• They have no enzymatic action
against phagocytes, antibodies, etc. so that
• Effect is non-specific action
they may not be engulfed or not be
• No specific tissue affinity
recognized by the phagocytes
• Active only in large doses 5 to 25 mg
• Kinases or Fibrinolysins
• Weakly antigenic
o Substance that will dissolve the fibrin clot that
the host will attempt to form (escape from • Neutralization by antibody infective
clots) • Cannot be toxoided
• Produce in gram negative bacteria

8
 • Highly potent, e.g. 3kg botulinum can kill all the inhabitants
Exotoxins
of the world
• They are also poisonous substances but they are • They are generally formed by Gr+ bacteria and also by some
producing within the bacterial cells and it is released Gr- organisms like Shigella, V. cholorae, and E. coli
from them. They are usually named from the target - Usually produced primarily by gram positive
organs that they affect. • Exotoxin is specifically neutralized antitoxin
• Neurotoxin • Can be separated from culture by filtration
o most potent exotoxin produced by • Action is enzymatic and it has specific tissue affinity
Clostridium tetani (causative agent of tetanus) • Cannot cause pyrexia (high fever) in a host
and Clostridium botulinum • Can be toxoided
o Toxin that will affect the nerves
o Tetanospasmin - neurotoxin of C. tetani; it Mechanisms by which Pathogens Escape Immune
will create the condition of spastic paralysis Response
o Botulinum toxin – blocks nerve impulses
producing a different kind of paralysis called • Antigenic Variation
flaccid paralysis o Pathogens are able to periodically change their
• Enterotoxins surface antigens
o Cause diarrhea and sometimes vomiting o Ex. Influenza viruses – by the time the host has
o Toxin that is produced in the intestine produced antibodies, they will come up with new
o Toxin B produced by C. difficile damages the antigens and had already shed the old ones. Even
surface of the colon leading to if the antibodies are present already, it will not be
pseudomembranous colitis effective anymore because they had changed their
• Toxic shock syndrome toxin (TSST) antigens already. Once they change their antigens,
o Produced by strains of S. aureus and S. there are new strains of influenza.
pyogenes which primarily affect the integrity o Ex. Trypanosomes – these are the blood
of capillary walls. flagellates. They can keep up their antigenic
• Exfoliative Toxin variations for 20 years. They never presented the
o Also called epidermolytic toxin, causing sloughing same appearance twice.
of the epidermal layers of the skin leading to SSSS
(staphylococcal scalded skin syndrome) cause by • Camouflage and Mimicry
Staphylococcus aureus or by some Streptococcus o They can mimic or coat and hide themselves.
pyogenes o Ex. Schistosomes - they are able to hide their
o It is usually seen in newborn babies (skin reddish foreign nature by coating themselves with host
and sloughing). The epidermal layer is removed in proteins (camouflage)
the process. o Molecular mimicry – pathogen surface would
• Leucocidin resemble the host antigens. It will not be
o Toxin that will destroy leukocytes, produced by recognized by the immune capabilities
Staphylococcus and Streptococcus
• Diphtheria toxin • Destruction of Antibodies
o Toxin produced by Corynebacterium which o Some bacteria can produce molecules or enzymes
inhibits protein synthesis killing mucosal epithelial that could destroy our antibodies produced.
cells and PMN’s (polymorphonucleus) o Ex. Heamophilus influenzae and Neisseria
o This is the one that causes diphtheria. If a gonorrhea – can produce the enzyme called IgA
Corynebacterium is not able to secrete exotoxin protease, that will counter act and destroy the
therefore it is non-pathogenic. antibody IgA that we can produce.
o This is why after identifying Corynebacterium, the
next step to do after it is isolated and identified is HOST DEFENSE MECHANISMS
to do toxigenicity test (if the Corynebacterium is
not positive in diphtheria toxin then it is not at all Definition of Terms
pathogenic) • Resistance – the ability to ward off disease
• Endotoxin is a lipid a component of the gram-negative outer o Nonspecific Resistance- Defenses that protect
membrane, and exotoxin that can be produce by the against all pathogens like virus, bacteria and fungi
bacteria and release it outside. o Specific Resistance- Protection against specific
pathogens
Characteristics of Endotoxins • Susceptibility – vulnerability or lack of resistance. If you are
• Heat labile protein susceptible, you are prone to contract disease and different
• Diffuse readily into the surrounding medium infections brought by different microbes or pathogen.

9
Host Defense Mechanism Non-specific Host Defense Mechanism

Non-specific Host Defense Mechanism First Line of Defense

• Serve to protect the body from a variety of foreign

substances or pathogens (first and second line of
defense)
• Would cater to all types of pathogens. It will not
recognize if it is a virus, microbe, or bacteria.
• Will serve for all substances (toxins or pathogens)

Specific Host Defense Mechanism

• Are directed against a particular foreign substance or

pathogen that has entered the body (third line of
defense)
• If the first line of defense did not work, the second line
of defense will be activated. And when the second
defense did not work, the third line defense will be • Physical or Mechanical Barriers: Intact Skin and Intact
activated. Mucous membranes
o Mucous Membranes: Line gastrointestinal,
Three Lines of Defense Against Infection genitourinary, and respiratory tracts.
o Two layers: Outer Epithelial and inner
connective layer.
o Epithelial layer secretes mucus which maintains
moist surfaces
▪ Although they inhibit microbial entry, they
offer less protection than skin.
o Several microorganisms are capable on
penetrating mucus membrane such as Treponema
pallidum (spirochete), papilloma virus,
Enteroinvasive e. coli, and E. histolytica.
First Line of Defense • Chemical Factors such as digestive enzymes, acidity of
stomach (pH 1.5) and alkalinity, acidity of vagina, lacrimal
• Are non-specific natural barriers which restrict entry of apparatus.
pathogens. It is naturally found in human. o Sebum: Oily substance produced by sebaceous
glands that forms a protective layer over skin.
Second Line of Defense
Contains unsaturated fatty acids which inhibit
growth of certain pathogenic bacteria and fungi.
• Macrophages and neutrophil (phagocytic leukocytes) o PH: low, skin pH usually between 3 and 5. Caused
by lactic acid and fatty acids.
• Proteins that are anti-microbial against different
o Perspiration: produced by sweat glands. Contain
pathogen
Lysozyme and acids
• Innate that provide rapid response against invading
o Lysozyme: Enzyme that breaks down gram-
pathogens once it breaches the first line of defense
positive cell walls. Found in nasal secretions, saliva
(entering the skin or mucus membrane), the second
and tears.
line of defense will be activated.
o Gastric Juice: Parietal cells secrete mixture of
Third Line of Defense hydrochloric acid enzymes, and mucus. Ph
between 1.2 to 3 kills many microbes and destroys
most toxins. Many enteric bacteria are protected
• It has antigen specific immune responses. The invaders by food particles.
that will pass in two levels of non-specific defenses will ▪ Helicobacter pylori neutralize stomach
be attack specifically. acid and can grow in the stomach,
• B and T lymphocytes causing gastritis and ulcers.
• Y shape antibodies
• It develops in the process.

10
• Microbial Antagonism: By indigenous microflora; and over • Wandering macrophages: Originate from monocytes that
all nutritional status and state of health leave blood and enter infected tissue and develop into
o More appropriate term is microbiota phagocytic cells.
o It is bacteria that inherently present in our body • Fixed macrophages (histocytes): Located in liver,
that provides protection against invading nervous system, lungs, lymph nodes, bone marrow and
pathogens (no need to activate). several other tissues.
o Part of our health immune system

Second Line of Defense

• Transferrin and lactoferrin: Are tie up iron (iron binding

protein), thereby preventing pathogens access to this
essential mineral.
o Once iron is no more available, it will not be
accessible to the bacteria (since bacteria needs
iron)
o Prevent pathogen access
• Fever: That augments host defense by stimulating
leukocytes to deploy and destroy invaders, reducing
available free plasma iron, inducing of 1L - 1, which causes
proliferation, maturation, and activation of lymphocytes in
the immunologic response.
o Elevated body temperature also slows down the
rate of growth of certain pathogens and can even
kill some especially fastidious pathogens.
o Temperature is very important factors for growth
(either kill or let them grow)
• Interferons: Are small, antiviral proteins that prevent viral
multiplications in virus infected cells and serve to limit viral
infections.
• Inflammation: Localizes an infection and prevent the
spread of microbial invaders, neutralizes toxins and aid in
• When injury happens, the cells nearby will send off chemical
the repair of damaged tissues. signals in form of chemotaxis to call out phagocytes to the
• Phagocytosis: Cell eating. Microbes are being engulfed. site of injury
• Complement system: Involves approximately 30 different • Dilation and increased permeability so that phagocytes can
blood proteins that interact in a step-wise manner known as squeeze in in the site of injury
the complement cascade.

CONSEQUENCE OF ACTIVATION OF THE COMPLEMENT

SYSTEM:
• Initiation and amplification of inflammation.
• Approximately 30 activated directly by pathogen.
• Indirectly by antigen antibody reaction.
• It generates active components in order to fight of the
invading pathogens.
o Attraction and activation of leukocytes.
▪ It sends signals to call out the leukocytes
to be deployed in site of invasion.
o Lysis of bacteria and other foreign cells.
▪ Attacking the pathogens with protein
materials PROCESS OF PHAGOCYTOSIS
o Increased phagocytosis by phagocytic cells. 1. Chemotaxis and adherence of microbe to phagocyte
▪ Labeling the pathogen by opsonization or (the adherence of the microbe to the phagocyte will
coating them by antibodies depends on the virulence factor)
PHAGOCYTOSIS 2. Ingestion of microbe by phagocyte
• Derived from the Greek words “eat and cell”. 3. Formation of phagosome
• Carried out by white blood cells: macrophages, neutrophils, 4. Fusion of the phagosome with a lysosome to form a
and occasionally eosinophils. phagolysosome
• Neutrophils: predominate early in infection 5. Digestion of ingested microbe by enzyme

11
 6. Formation of residual body containing indigestible o Interferon alpha: produced by B-
material lymphocytes, monocytes, and macrophages
7. Discharge of waste materials. o Interferon beta: fibroblasts and other virus
infected cells.
INFLAMMATION
o Interferon gamma: activated by T-
• triggered by tissue damage due to infection, heat, lymphocytes and NK cells.
wound, etc.
• Four Cardinal Major Symptoms of Inflammation Specific Host Defense Mechanism
1. Redness- Rubor
• Immunology is the scientific study of the immune system
2. Pain- Dolor and immune responses.
3. Heat- Calor • The immune system is the third line of defense against
4. Swelling- Tumor pathogens; it is a specific host defense mechanism.
but may also observe: • Two types of acquired immunity
5. Loss of function 1. Active Acquired Immunity
PURPOSES OF INFLAMMATION a. Natural active acquired immunity: that is
acquired in response to the entry of a live
1. Destroy and remove pathogens
pathogen into the body (i.e., in response to an
2. If destruction is not possible, to limit effects by actual infection). It has long duration.
confining the pathogen and its products. b. Artificial active acquired immunity: that is
3. Repair and replace tissue damaged by pathogen and acquired in response to vaccines. Its duration
its products. for many years; but must be reinforced by
boosters.
2. Passive Acquired Immunity:
a. Natural passive acquired immunity: that is
acquired by a fetus when it receives maternal
antibodies in utero or by an infant when it
receives maternal contained in colostrum. Its
duration from 6 months- 1 year.
b. Artificial passive acquired immunity: that is
acquired when a person receives antibodies
contained in anti-sera or gamma globulin. Its
duration from 2-3 weeks.

Two Types of Immunity

Humoral Immunity: Antibodies and antigens:

COMPLEMENT ACTIVATION • an antibody is a protein produced in response to

• Consequences of Complement Activation: foreign substance (antigen) that will react specifically
1. Cytolysis: Due to the formation of a membrane with that substance. The reaction between the antibody
attack complex (MAC) which produces lesions in and the antigen will lead to destroy the antigen or the
microbial membranes. pathogen that carry the antigen or inhibit it.
o The bacterial cell will lies because of the • Antibodies are produced by WBC (plasma cells) and
lesions that be created by the MAC. may present in the blood and the body fluids or
2. Inflammation: Complement components (C3a) attached to surfaces of cells. They are also called
trigger the release of histamine, which increases immunoglobins (Ig)
vascular permeability. • Antibodies are glycoproteins produced by B
o It will attract macrophages and neutrophils. lymphocytes, that once it is transformed to plasma
3. Opsonization: Complement components (C3b) cells, it become antibody secreting. It will bind to
bind to microbial surface and promote specific antigen in the antigenic determinant.
phagocytosis. • Antibodies are very particular and specific, and it reacts
INTERFERONS like lock and key. It is also called immunoglobulin.
• Antiviral proteins that interfere with viral multiplication. • Antigens are substances that stimulate the animal body
o They serve to limit the viral infection, very to produce antibodies that will specifically react with
small protein around 30,000 heat stable and the antigens.
resistant to low pH • Antigen is always complex macromolecules (mostly
• Have no effect on infected cells. glycoproteins)
• Host specific, but not virus specific • Antigen is antibody generating molecules and digenic
o Unable to save the virus infected cell but once or immunogenic
release, they attached to membranes of that • The best antigens are the foreign proteins because of
surrounding cell and prevent viral replication the complexity of the confirmation proteins
o They are effective against variety of viruses, • Bacteria are full of antigens. It is a mosaic of antigen
and they are specific only in the species of determinants (epitopes-antigenic site)
animals • Antigens can be haptens- small molecules that may act
• 3 types of Interferons as antigens only if they are coupled with a large carrier
molecule such as proteins.

12
 the peripheral blood. Several types are T-helper,
Cytotoxic T-cells, etc.
Classes of Immunoglobulin
• IgG: This is the greatest percentage of antibody molecules
in the blood, these globulins combine to small antigen and
combine and neutralize toxins (antitoxins). Long lived and
crosses the placenta.
• IgM: is a pentamer, has 10 antigen binding sites, first
antibodies formed in the primary response to antigens.
Does not cross the placenta.
• IgA: Known as secretory antibodies because they are found
in breastmilk, respiratory and intestinal mucin, saliva, tears,
and vaginal secretions protect these parts of the body from
infectious agents. It exists in three for monomer, dimere,
trimere. 10-20% of the total serum immunoglobulin is
compose of IgA
• IgD: It is found on the surfaces of the B-lymphocytes where
it acts as a specific antigen receptor.
• IgE: Its activities involved in both the resistance to parasites
infections and hypersensitivity. It is found on the surfaces of
basophils and muscles.
All antibodies are made up of proteins, globular glycoproteins. It
can be found in the blood, saliva, colostrum, lymph. The amount
in type of antibody produces during antigenic stimulation
depends on the nature of the antigen, the site of the stimulus the
amount of antigen, and the number of times of exposure.

CMI (Cell Mediated Immunity)

• Does not involved production of antibodies

• Controls intracellular pathogens
• Cells that participate are macrophages, T-helper cells,
cytotoxic T-cells, NK cells, Killer cells, and granulocyte.
• Major player in CMI are B cells and T cells.

Typical CMI Response

• Step 1: A macrophage engulfs and partially digests a

pathogen. Fragments (antigenic determinants) of the
pathogen are then displayed on the surface of the
macrophage.
• Step 2: A T-helper cell binds to one of the antigenic
determinants being displayed on the macrophage
surface. the T-helper cell produces lymphokines which
reach an effector cell of the immune system.
• Step 3: The effector cell binds to a target cell.
• Step 4: Vesicular contents of the effector cell are
discharged.
• Step 5: Toxins produced by the effector cells enter the
target cell causing disruption of DNA and organelles.
The target cell dies.

Lymphocytes

• B-lymphocytes: 10-15% of lymphocytes in Peripheral

blood. B cells migrate to lymphoid tissues where they
produce antibodies that circulate through lymph and
blood. They live about 1-2 weeks.
• T-lymphocytes: T-cells are phagocytic cells, it engulfs
the antigen or the pathogen that carries that antigen, it
destroys the infected body cells, and it rejects the
foreign tissue. About 70%-80% of the lymphocytes in

13
 CLINICAL BACTERIOLOGY

MICROCOCCACEAE, STREPTOCOCCACEAE, NEISSERIACEAE

OUTLINE • Non-motile
I. Micrococcaceae F. Virdians • Facultative anaerobes
A. Staphylococcus Streptococci • Normal inhabitants of the skin and mucous membranes
B. Micrococcus G. Treatment of (anterior nares’ main residence)
C. Stomatococcus Streptococcal and
D. S. Auereus Infection Enterococcal • Commonly cause human infections - because of the wide
i. Methods of Infections variety of virulence factors that they can produce
Identification of H. S. Pneumoniae • Species are differentiated by coagulase test, the most
Microorganisms i. Laboratory important being the coagulase positive S. aureus
ii. Laboratory identification
• Some animal species produce coagulase but are rarely
Identification of of S.
S. Aureus pnaumoniae isolated from human samples (ex. S. hyicus and S.
II. Micrococcus I. Streptococcus-like
A. Laboratory Identification organisms Micrococcus
B. Coagulase-Negative J. Laboratory
Staphylococcus Identification of intermedius)
i. Laboratory Streptococci • Opportunistic pathogens found only in
Identification of CNS K. Other Laboratory immunocompromised patients
C. Antibiotic Susceptibility of Identification of
• Of low pathogenic significance
Staphylococcal Infection Streptococci
IV. Types of Hemolysis • May be isolated as contaminant or as part of the normal
III. Streptococcus,
Enterococcus and Related V. Neisseria and Moraxella microbiota
Genera Species • M. luteus, when they grow, they give out a yellowish color
A. S. Pyogenes A. Pathogenic and they’re usually seen in tetrads
i. Laboratory Neisseria Virulence
Identification of S. Factors Stomatococcus
Pyogenes B. Neisseria
gonorrhoea
B. S. Agalactiae
i. S. Agalactiae
Infectione • Part of the normal oral microbiota
i. Laboratory • Rarely isolated from infections
Infection
Identification
ii. Laboratory
of N.
• Colonies strongly adhere to the agar surface
Identiffciation of S. • There is only one species of Stomatococcus which is now
gonorrhoeae
Agalactiae
C. Neisseria placed under Rothia genus.
C. Geoups C and G
Meningitidis o Rothia mucilaginosa is mostly associated with
D. Group D
Infections
i. Laboratory prosthetic device infections
i. Virulence
Identification of
Factors
Group D
ii. Laboratory
E. Enterococcus Staphylococcuus aureus infection
Identification
i. Laboratory
of Neisseria • Skin and wound infections – boils, furuncles and
Idenntification of
Meningitidis
Eneterococcus folliculitis
D. Moraxellla
Catarrhalis o Primarily related to skin abscesses and wound
infections because they are the primary reservoir
MICROCOCCACEAE of the anterior nares and the skin to a certain
• Members of gram-positive cocci, aerobic or facultative extent
anaerobes • Food poisoning – Stapylococcal enterotoxins A and D
• Catalase (enzyme required to neutralize the toxic forms of (heat-stable toxins pre-formed in food)
oxygen) positive (except Stomatococcus) o Rapid onset usually 2-6 hours unlike other food
• Most members are indigenous microbiota - meaning they poisoning agents which can last 18-24 hours
are part of our normal bacterial inhabitants. They are found before symptoms appear
in our skin, in the surfaces of our body. Members that are o Symptoms: Nausea, vomiting, headache,
significant among them are the: Staphylococcus, abdominal pain, and severe cramping could be
Micrococcus, Stomatococcus experienced
• Scalded skin syndrome (Ritter’s disease) – caused by
exfoliative/epidermolytic toxin and occur in newborns, and
Staphylococcus
in adults having chronic renal failure or are
• In clusters, bunch of grapes; Spherical bacteria that are immunocompromised
clustered together
• Catalse positive

1
 o Mortality rate: Low in newborn, High in adults
having chronic renal failure for Laboratory Identification of S. aureus
immunocompromised • Microscopic examination – subjecting the cultural
• Toxic shock syndrome (TSS) – caused by S. aureus that isolates of a probable S. aureus and making a smear on
produces enterotoxin F (TSST-1) the slide and stain it, you will find the characteristic
o TSST – Toxic shock syndrome toxin clustering (bunch of grape appearance) of the cocci can
o There is a strong association within the use of be observed
tampons and TSS and this can occur in both • Laboratory isolation
sexes. o First, perform a gram stain smear to identify the
• Other infections: next possible test that will lead to the final
o Staphylococcal pneumonia secondary to identification. When the gram stain smear implies
influenza can occur gram-positive cocci in clusters, it can indicate that
o Osteomyelitis can occur secondary to bacteremia it’s a Staphylococcus.
o Most outstanding association of S. aureus to o Do a catalase test (slide method) by placing a 2-3
infection: Wound, abscesses and other skin laps of 3% H2O2 on top of the slide; take the
infections colony using a loop to the H2O2. It should create
o When you culture a wound specimen, abscess strong bubbles, indicating the presence of
and exudates and other purulent discharges you catalase.
will appreciate that at least 80% of those o Culture in a mannitol salt agar (MSA) because of
conditions or samples will lead to or isolate a high concentration of salt and mannitol can
staphylococcus aureus identify S. aureus from other Staphylococcus. If
• Virulence factors isolated bacteria ferment mannitol, it is said to be
o Coagulase – a bacterial enzyme that brings S. aureus because other Staphylococcus can’t do
about the coagulation of blood or plasma it (production of yellow color).
o Protein A – a cellular component in the cell wall o Coagulase test - The clot formed at the bottom of
that helps the bacteria to avoid phagocytosis the plasma tube indicates a positive reaction
because of its ability to bind and neutralize IgG • Cultural techniques – culturing on blood agar plates,
o α, β, δ, γ Hemolysins – cytolytic toxins growing as round, smooth, white (sometimes yellowish)
▪ α – lyse RBCs, damage platelets and and beta-hemolytic (completely lyses RBCs)
even macrophages o You will see clearing at the bottom of the plate
▪ β – sphingomyelinase primarily C due to RBC lyses
▪ γ – exotoxin little to polymorphonuclear o 2 types in Blood agar plates: As white colonies
cells associated to Panton-Valentin that are medium in size or, yellow or gold
leukocydin colonies that are medium in size
▪ δ – least toxic among these cytolytic o Beta-hemolytic colonies are best appreciated in
toxins Sheep’s Blood Agar
o Exoenzymes • Biochemical identification by coagulase test or
▪ Dehyaluronidase – allows the spread of catalase test
infection, by removing the cement that o Rabbit Coagulase Plasma is use
glues connective tissues together, such o Catalase Test
as the hyaluronic acid ▪ S. aureus will be catalase positive
▪ Lipase – facilitates S. aureus to ▪ Catalase positive – Uses hydrogen
colonization of skin surfaces peroxide.
▪ Catalase is needed to convert 2𝐻2 𝑂2 →
Methods of Identification 𝑂2 + 𝐻2 𝑂 (nontoxic form). The oxygen
• Phenotypic that is present in the reaction will be
o It involves the microscopic and macroscopic observed by the bubbling reaction, when
investigations that will show the morphology of you subject the colonies to 3%
the different microorganisms. Hydrogen Peroxide (𝐻2 𝑂2 )
o It could also use the five eyes in cultural ▪ S. aureus will be catalase positive,
techniques so you will see how they will grow, converting H2O2 into its non-toxic for,
how big is their colony, what sort of color will they H2O and O2 (bubbling reaction)
be producing o Coagulase test - The clot formed at the bottom
o Phenotypic are the observable characteristics of the plasma tube indicates a positive reaction.
that you can be able to identify the different Has 2 types: Cell Bound (slide method) and Free
microbes, specifically bacteria Coagulase (tube method)
• Immunological – serological analysis employing antigen- ▪ Cell-bound coagulase (slide method)
antibody reactions that we use in lab kits and serotyping detects clumping factor in the surface of
• Genotypic – genetic techniques such as nucleic acid bacterial cells. The plasma fibrinogen in
probes, PCR, rRNA analysis, plasmid fingerprinting, etc. presence of the clumping factor will
• But in the lab, we will be just confined in just identifying S. convert fibrinogen into fibrin
aureus into mostly phenotypic means and a little of (Fibrinogen → clumping factor →
immunological means. Fibrin)

2
 ▪ Free coagulase (tube method) –
detects thrombin-like molecule called Laboratory Identification of Micrococcus
coagulase reacting factor (CRF). The • Modified oxidase test (Microdase Test) - Uses 6%
plasma contains the fibrinogen and the tetramethylphenylenediamine hydrochloride in
bacteria react with the dimethyl sulfoxide
Staphylocoagulase (extracellular o Add a drop of microdase to a suspected
molecule that will react with CRF) which micrococcus. The micrococcus is smeared into a
is now a thrombin molecule that will filter paper and then add a drop of reagent to the
convert it into fibrin (Fibrinogen → suspected isolate. Dark blue color should be
coagulase-CRF complex → Fibrin ) produced within 2 minutes when it is oxidase
▪ Place the plasma in the tube, and put positive
the inoculums of suspected o Using sterile forceps, transfer a Microdase disk
Staphylococcus, incubate to 35-37°C. from the stock bottle to a petri dish
Check for the presence of clot after four o Using a wooden applicator stick, rub a small
hours. If there is no clot, incubate the amount of several colonies of an 18-24 hour pure
tube at room temperature and read the culture grown on blood agar onto the top of
next day if there is clot formed. If a clot microdase disk
appears, it is positive for coagulase. o Incubate at room temperature for 2 minutes
▪ Read the test in four hours because it o Observe for color development. Deep blue color
produces fibrinolysis that can lyse the demonstrates positive reaction
clot within four hours, or else it would o Modified: Uses 6% instead of 1% of the same
give a false negative test. reagent
▪ Do both methods above because 5% of • Bacitracin susceptibility (Taxo A Disc)
S. aureus do not produce clumping o Mueller-Hinton agar is streaked with suspected
factor. Micrococci, and place 0.04 U of Bacitracin and
• Use of selective media from heavily contaminated incubate it overnight
specimens (MSA, PEA, CNA) o Susceptible – Zone of Inhibition
o For the selective media that you can use to • Furazolidone resistance (100µg)
culture S. aureus is basically sheep’s blood agar o Micrococci is resistant to furazolidone (antibiotic).
o You can also use selective media when you are o Growth is until the edge of plate
dealing with heavily contaminated samples or
specimens like wound exudates, etc. Coagulase-Negative Staphylococcus
o You can utilize MSA (Mannitol Salt Agar), PEA
• If the staphylococcus is not aureus, then most
(Phenylethyl Alcohol Agar) and CNA (Columbia probably it is CNS.
NaladixicAcid Agar)
• Found as normal flora in humans and animal
• Plasma coated latex particles - Serological means by the
• Often are nosocomial infections
use of this method
• Predisposing factors:
• The difference between the morphological appearance of
o Catheterization
the Staphylococcus aureus arranged in clusters vs. cocci
o prosthetic device implants
arranged in chains. Cocci that are arranged in chains is
o immunosuppressive therapy
called streptococcus. Strepto = chains: Coccus = spherical
• Most common species:
o S. epidermidis (normal skin microbiota)
o S. saprophyticus (associated with UTIs in
sexually-active, young females)
• Other coagulase-negative staphylococcus:
o S. haemolyticus and S. lugdunensis (occasionally
produce clumping factors and may yield a
positive slide test that’s why we must proceed to
the tube method) and S. schleiferi occasionally
cause wide range of infections
• Other species are not isolated frequently

Laboratory Identification of CNS

• Culture on sheep’s BAP
MICROCOCCUS o Coagulase negative urine isolates are further
• Environmental organisms tested to presumptively identify S. saprophyticus
• Normal microbiota of skin and respiratory tract using novobiocin susceptibility (urine presented a
• Common contaminants colony that is white, circular smooth without
• Coagulase-negative hemolysis, catalase-negative, coagulase negative,
gram positive cocci in clusters and resistant to
novobiocin)
o In a BAP make a lawn culture of a suspected
isolate (2) and place 0.5µg of novobiocin and

3
 place them on both sides then incubate the plate • Coagulase test will identify S. aureus being positive and
overnight in 35-37°C, the next day, observe. If negative will somehow make you think of the other two
there is no zone of inhibition the it is a S. most common coagulase negative staphylococcus which
saprophyticus, if there is a zone, then it is S. is the S. epidermidis and S. saprophyticus
epidermidis • You can distinguish S. epidermidis from S. saprophyticus
• Various commercial identification systems may be by virtue of the Novobiocin persistence tests
used • If the Novobiocin turn out to be resistant, then most
o Those that are involved automation is now being probably the isolated is S. saprophyticus.
frequently used for identification in order to • If its sensitive to Novobiocin, then it is S. epidermidis
identify coagulase-negative staphylococcus
STREPTOCOCCUS, ENTEROCOCCUS AND
Antibiotic susceptibility of Staphylococcal RELATED GENERA
• Infection
P resistance is high, especially in S. aureus isolates (85- • Members are catalase-negative
90%) • Gram-positive cocci arranged in pairs or in chains
• A common resistance is production of enzyme β- • Facultative anaerobes (aerotolerant accrdg to Mahon)
lactamase (It will neutralize the effect of β-lactam • Use of enriched medium or blood is necessary for their
antibiotics such as Penicillin and Cephalosporins) isolation
• Various β-lactamase resistant P have been developed. • Hemolysis patterns are helpful in the identification
Methicillin is the most frequently used. Oxacillin is used for • 4 ways to classify streptococcus: Nature of disease,
in-vitro susceptibility testing of methicillin resistance. physiologic characteristics, hemolysis pattern, Lancefield
• MRSA, MRSE (or ORSA/ORSE) are agents of serious classification (typing of strep by carbohydrate found in
nosocomial and community associated infections their cell wall)
o MRSA – methicilin-resistant staphylococcus
aureus Streptococcus pyogenes
o MRSE – methicilin-resistant staphylococcus
• Cell wall contains the Lancefield group A carbohydrate
epidermidis
• Lancefield group of classification is the typing or the
o ORSA – oxacillin-resistant staphylococcus aureus
classification of streptococcus by the virtue of the
o ORSE – methicilin-resistant staphylococcus
carbohydrate groups found in their cell wall.
epidermidis
• Group A strep or BHS group A
NOTE: • Virulence factors
o M-protein causes the bacterial cells to resist
• Other characteristic feature to identify S. aureus phagocytosis by enabling it to adhere to mucosal
o Mannitol fermentation – Positive cells (atleast 80 serotypes of M-protein)
o DNAse test – Positive o Streptolysin-O - causes hemolysis of RBCs
o Beta-hemolytic on blood agar (antibodies can be produced, called ASO)
o Golden yellow pigment ▪ When you do ASO titer in serological
• Mannitol Salt Agar (Selective medium) – S. aureus test, it indicates antibodies to
produces yellow color colonies due to fermentation streptolysin-O is produced. Indicating
that infection is there, in either recently
or acute infection to streptococcal
infection on going
o Streptolysin-S - lyses WBCs but not
immunogenic
▪ Antibodies are not created
o Hyaluronic acid capsule
o DNAse – all strains have this, most common is
DNAse B (antigenic)
o Spes A, B & C (Erythrogenic toxin) –
responsible for red rashes in scarlet fever
o Protein F – fibronectin binding protein, facilitates
adhesion to epithelial cells
o Streptokinase – causes lysis of fibrin clots
o Hemolysins - Beta hemolysin is very much
appreciated with it is cultured to sheep’s blood
agar
• Infections
o Pharyngitis (strep throat) & Tonsilitis
▪ These infections are frequently seen in
children ages from 5 to 15
▪ There is now a rapid strep test that can
be performed that can rapidly diagnose
strep throat

4
 o Skin infections (e.g. impetigo, necrotizing ▪ Wet the PYR test disc on the strip with
fascilitis, etc) 10 µL sterile distilled water or deionized
o Scarlet fever - you will see red rash would water (do not flood the disk)
appear on the upper chest and it will spread to ▪ Put 5-10 colonies of the tested strain
the trunk and extremities, following infection with from 18- 24 hours culture on the surface
streptococcus pyogenes of the disc with a loop and smear them
o Rheumatic fever (consequence of untreated lightly on it
pharyngitis) and glomerulonephritis ▪ Incubate the disc for 1-2 minutes at
(immunocomplexes during episodes of room temperature
pharyngitis, antigen-antibody reaction) ▪ After incubation, add 1 drop of N, N-
o Streptococcal TSS – entire organ system shuts dimethylaminocinnamaldehyde
down, happening during very severe ▪ Observe for red color development
streptococcal infections where it disseminates within 1-2 minutes
into different organ systems and eventually can ▪ PYR Test positive = S. Pyogenes (red
lead to death color)
▪ Negative – yellow color
Laboratory Identification of S. pyogenes
• Culture - collecting swab sample and culture them in
sheep’s blood agar (colonies: transparent, smooth,
sometimes beta-hemolytic)
• Gram’s stain – gram-positive cocci in chains, catalase-
negative
• Bacitracin susceptibility (Taxo A)
o Bacitracin is a drug (antibiotic) very useful for
treating superficial skin infections but it is too
toxic for systemic use; polypeptide antibiotic
produced by Bacillus subtilis
o Interferes with the peptidoglycan synthesis of
bacteria
o Create a lawn inoculum of the suspected bacteria
and incubate
o 0.4µg
o Positive – Zone of Inhibition • Gram stained S. pyogenes. Short or long chains
streptococcus cocci.
• Resistant to sulfamethoxazole (SXT)
o Inoculate (lawn culture technique) on Sheep
Blood Agar Plate using inoculating loop Streptococcus agalactiae
o Create a zigzag pattern (3 overlap) • β - hemolytic
o Put the antibiotic SXT paper disc • Cell wall contains Lancefield group B carbohydrate
o Incubate for 18-24 hrs in 35±°C • Group B Streptococcus or GBS (has sialic acid in
o Examine for inhibition. No inhibition means it is capsules, it’s loss will result to loss of virulence)
S. Pyogenes • Found as normal microbiota of the genitourinary tract
• Pyridium oral (PYR) test • Virulence factors
o Rapid method for presumptive identification of o Capsule – main component is sialic acid
bacteria based on the pyrrolidonyl arylamidase o Hemolysin – lyse rbc’s
enzyme o Christie-Atkins-Munch-Peterson (CAMP) factor
o The enzyme L-pyrrolidonyl arylamidase enlarges the area of hemolysis
hydrolyzes the L-pyrrolidonyl-β-naphthylamide o Others (DNAse, hyaluronidase) have not been
substrate to produce a β-naphthylamine shown to be factors in infection
o β-naphthylamine can be detected in the presence
of 0.1% N, N-methylaminocinnamaldehyde S. Agalactiae Infections
reagent by the production of a bright red
• Neonatal sepsis soon after birth
precipitate
• Part partum fever and sepsis
o Broth method
▪ Inoculate PYR broth with 3-5 colonies (of
suspected S. pyogenes from 18-24 Laboratory identification of S. agalactiae
hours pure culture • Culture – genitourinary tract swab, sheep’s blood agar
▪ Incubate the tube aerobically at 35-37°C • Gram’s stain – gram-positive in gram positive long chains,
for 4 hours catalase-negative. In clinical specimen: they will appear in
▪ Add 3-4 drop of PYR reagent and short chains
observe for color change • CAMP test positive – secreting a protein called CAMP
▪ Observe for red color development factor (protein B)
within 1-2 minutes o Christie-Atkins-Munch-Peterson (CAMP) – Test
o Disk method (rapid) use for presumptively identifying S. Agalactiae

5
 (because it is the only strep that has the capability
to produce CAMP/Protein B) Group C and G
o When placed perpendicularly with S. aureus, it
• Not commonly isolated
will form arrowhead hemolysis (enhanced
• Hemolytic species in Lancefield group that are
hemolysis) that synergistically reacts with the
occasionally isolated from clinical specimens
beta-lysin of sphingomyelinase of S. aureus
• Part of the normal skin microbiota
o How: in a Sheep Blood Agar Plate streak a
• Β-hemolytic
colony of S. Aureus in the middle of the agar then
• Group C – S. equi, S. zooepidemicus and S. equisimilis
perpendicularly, streak the suspected S.
• Group G – S. canis, S. anginosus, S. milleri
Agalactiae. Incubate overnight, then after 18-24
• These groups produce variety of infections including
hours observe for the arrowhead hemolysis.
pharyngitis
o Presence of arrowhead hemolysis = CAMP
• Extensive use of biochemical reagents are needed to
Positive therefore it is a S. Agalactiae
identify these groups
o It will appear as greyish white, mucoid colonies • Serological tests were developed to identify the group
carbohydrates in the cell wall for group C and G such as
Latex agglutination tests to serotype group C and G

Group D
• Include S. bovis and S. equinus
• Found as normal intestinal microbiota
• May be agents of bacterial endocarditis, UTI’s, abcesses
and wound infections
• There are associations with bacteremia caused by S. bovis
and GIT tumor. Isolation of S.bovis from the GIT indicates
presence of tumor.

Laboratory Identification of Group D Streptococcus

• α or γ/non hemolytic
• Positive bile esculin test – positive reaction is production
of black precipitate due to hydrolysis of the reagent
• Hippurate hydrolysis positive – not only it can identify S. esculin, in the presence of bile salts
agalactiae, it can also detect Campylobacter jejuni, Listeria o With an inoculating wire or loop, touch two or
monocytogenes, and Gardnerella vaginalis. It detects the three morphologically similar streptococcal
ability of the organism to hydrolyse Hippurate. colonies and inoculate the slant of the bile esculin
o Add 0.1mL of sterile water to a 12x75mm plastic medium with an S-shaped (zig-zag) motion, or
test tube streak the surface of bile esculin plate for
o Make a heavy suspension of the organism to be isolation
tested o Incubate the inoculated tube at 35-37°C for 18-24
o Using heated forceps, place a rapid hippurate hours and then observe the results
disk in the mixture o Positive – Presence of black precipitate due to
o Cap and incubate the tube for 2 hours at 35°C; the hydrolysis of the reagent esculin
the use of a water bath is preferred • No growth at 6.5% NaCl broth – if the microorganism is
o Add 0.2mL ninhydrin reagent and re-incubate for able to withstand the salt, if there is turbidity, then it is not
an additional 15 to 30 minutes a group D Strep
o Observe the solution for the development of deep o Create a 6.5% NaCl broth, put the suspected
purple color colony, incubate for 18-24 hours, 35±°C, next
day, observe for turbidity. Able to stand the salt,
NOT Group D Strep. No growth/turbidity = Group
D. Strep
• PYR negative (Same sa pyr test ng s. pyogenes pero grp
D naman ang gamit na suspected colony)
• Serotyping – using latex agglutination test

Enterococcus
• Found in the intestinal tract
• Most common is E. faecalis. Other members are E.
faecium, E. avium, E. durans
• Share characteristics of Group D, including the antigen D
• Show resistance to several of the commonly used
antibiotics
• Resistance to SXT
• PYR Negative

6
• Has similar infections to Group D, the most common being • Direct smears often reveal leukocytes and numerous
UTI gram-positive diplococci with the ends being slightly
pointed, giving a lancet-shaped appearance
Laboratory Identification of Enterococcus • Grows on sheep’s blood agar as α-hemolytic, and β-
• BE positive, 6.5% positive, PYR positive hemolytic when incubated anaerobically
• SXT resistant • Complex media such as brain-heart infusion agar (BHIA)
• Should be screened for high-level aminoglycoside- or trypricase soy agar (TSA) with 5% blood or chocolate
resistant Enterococci (HLARE) agar (CAP) are necessary for good growth
o HLARE that are used are gentamicin, amikacin • Isolates may require increased CO2 (capnophilic)
that are usually used to treat enterococcus • Colonies are α-hemolytic
• Vancomycin-resistant Enterococci (VRE) is a major • Young cultures produce a round, glistening, wet, mucoid,
concern dome-shaped appearance
o VRE are highly resistant strains to most
antibiotics. That is why resistant strains cannot
just be treated with one antibiotic alone. It is
usually combination; synergistic treatment is
being done for enterococcal infection

Viridans Streptococci
• Include those α-hemolytic streptococci (greenish
hemolysis, partial hemolysis) that lack Lancefield group
antigens and do not meet the criteria for S. pneumonia
• Part of the normal microbiota of the oropharynx and the • Gram positive diplococci, having a halo meaning it is
intestine encapsulated. The presence of numerous neutrophils or
• Fastidious and require increased CO2 polymorphonuclear cells. The presence of WBCs is an
• Frequent cause of subacute bacterial endocarditis (SBE) indication of in going infection or inflammation
• S. mutans, S. salivarus, S. anginosus, S. mitis, S. bovis – Laboratory Identification of Streptococcus pneumonia
not hemolytic
• NVS (nutritionally variant streptococcus) have been • Lanceolate shape (Football shape)
isolated from patients who have endocarditis and otitis • Αlpha-hemolytic
media • Gram positive
o Also known as pyridoxine-dependent (vitamin B6 • Susceptibility to optochin (Taxo P)
dependent) o Inhibits production of ATP in microorganisms. It
o Vitamin B6 is essential for their growth makes the cell wall of pneumonia to become
• Positive gram-stain, negative culture fragile that when the cell membrane becomes
weak, the pneumonia lyses. ≥14mm diameter
Treatment of Streptococcal and Enterococcal zone of inhibition.
infections o They appear in colonies that are greenish or
alpha-hemolytic.
• Most species are susceptible to P o Optochin is also an antibiotic that interferes with
• S. agalactiae is less susceptible than Group A and may production of adenosine triphosphate in
require a combination of Amp and an aminoglycoside microorganism
• Group D is susceptible to P o How:
• Enterococcus is usually resistant to P ▪ The media that should be used is 5%
• Enterococcus is usually treated with synergistic sheep’s blood agar
Ampaminoglycoside combination ▪ Make a lawn inoculum – overlap the
• Pneumococcal isolates are treated with Erythromycin in swabbing with the use of wire loop or a
case of P-resistant cotton swab dip ininoculum of suspected
• Linezolid is being prescribed to VRE infections isolate.
▪ Put the Taxo P in the middle of the lawn
Streptococcus pneumoniae
inoculum o Incubate to 35-37 degrees
• The cell wall doesn’t have the usual carbohydrate group with 5% CO2. After 18-24 hours, look for
by Lancefield. Rather, it has six layers composed of the zone of inhibition.
peptidoglycan and teichoic acid attached to N- ▪ You should be able to measure diameter
acetylmuramic acid, very similar to Group C Lancefield at least or more than 14 mm using a
groups 6mm disk
• Often part of the normal flora of the upper respiratory tract • Bile solubility test
(URT) o Pneumonia is lysed by bile. Bile salts will lower
• Key virulence factor is an anti-phagocytic capsule the surface tension between the bacterial cell
• There are approximately 80-90 antigenic capsule types membrane and the medium. Once the cell
• An important human pathogen-causing pneumonia, membrane is destroyed, it will accelerate the
sinusitis, otitis media, bacteremia, and meningitis organism’s autolysis.

7
 o This test will differentiate S. pneumonia which is
soluble by bile from alpha hemolytic which is non-
soluble or insoluble to bile
o The reagent is sodium deoxycholate; for the test
tube is 2%, for plate test 10%
• Quellung test
o Microscopic test in which capsule surrounding
the pneumococci will appear like it is swelling
o Suspension of pneumococci - Mixed on a slide
with a drop of the type-specific antiserum
o You mix a loopfull of suspected colony in equal
quantity of anti-serum. You mix the serum with
the test organism and examine it microscopically
• Pin-head colonies white colonies are indicative of
for capsule swelling.
staphylococcus
o In some test, they would recommend to put
• Pin-point translucent colonies having beta hemolysis is the
methylene blue to make it more visible
usual morphology that you can see while dealing with
o On microscopy, capsule (seen as clearing around
streptococcus
bacterial cell) becomes apparently swollen,
• Two beta hemolytic streptococcus commonly isolated in
sharply delineated and refractile
the lab
• Streptococcus pyogenes (group A streptococci) o
Streptococcus-like organisms Streptococcus agalactiae (group B streptococci)
• Aerococcus • After the preliminary test (gram stain-oxidase), the next
• Leuconostoc thing that you should do is to differentiate the two beta
• Pediococcus hemolytic (pyogenes and agalactiae) by PYR test

Laboratory Identification of Streptococci

• BAP hemolysis
o Hemolytic pattern on blood agar plate
o For s. pneumonia it will be mostly alpha hemolytic
• Bile solubility – S. pneumonia is bile-soluble
o There will be clearing in the test tube
• Optochin (ethylhydrocuprein hydrochloride/Taxo P)
susceptibility: ≥14mm with 5µg disk
• Bacitracin (Taxo A) – S. pyogenes
• Group A and B resistant to SXT
• CAMP test presumptively identifies Group B
Streptococcus – S. agalactiae • Among the beta hemolytic streptococcus that is PYR
• Esculin hydrolysis sensitive or positive is S. pyogenes
• 6.5% NaCl o If positive, the color would be violet
• PYR hydrolysis • After bacitracin sensitivity, if it is sensitive then it is
pyogenes. Once it is PYR positive, you are confirming that
Other: Laboratory Identification of Streptococci your isolate is pyogenes.
• LAP test (leucine aminopeptidase) helps differentiate • If it is resistant, possibly it is S. agalactiae. You do CAMP
Aerococcus and Leuconostoc from other Strep species test, if it is positive (arrow head hemolysis) then it is group
• Serologic testing for the detection of C-Carbohydrate of B or S. agalactiae.
the cell wall • If Hippurate hydrolysis is positive, then it is also
confirmatory test to S. agalactiae
TYPES OF HEMOLYSIS
• Beta hemolysis – there is a complete clearing of RBC,
because there is a complete lysis of RBC leaving a clear
space where the bacteria grew.
• Alpha hemolysis – greenish because of the partial
hemolysis or incomplete hemolysis of the RBC, so there
are still some unused or not completely metabolized RBCs,
not completely lysed RBCs in the process
• Gamma hemolysis or Non-hemolytic

8
 o Protein I (por) – channel or pore for nutrient and
waste diffusion
o Protein II (opa)– opacity and facilitate adherence
to phagocytic and epithelial cell
o Protein III (rmp) – reaction-modified protein
(RMP) blocks serum IgG; antibodies will not work
• Lipo-oligosaccharide (LOS) – antigenic variation
• IgA protease – render immunoglobulin A inactivated

Neisseria gonorrhoeae Infections

• Gonorrhea
o Infected males show symptoms of burning and
discharge from the urethra
o Females may be asymptomatic but can lead to
• To distinguish S. pneumonia to Viridans streptococci, you pelvic inflammatory disease (PID)
do Optochin sensitivity test o May cause sterility in males and females
• Optochin sensitive pneumococcus: To further identify • Ophthalmia neonatorum
Pneumococcus, to confirm it is S. pneumonia. You do o This is in infants from mother’s positive to
capsular swelling test, bile solubility test (clearing) or Neisseria gonorrhea, they will end up having
• Optochin sensitive pneumococcus: To further identify infection of the conjunctiva
Pneumococcus, to confirm it is S. pneumonia. You do • Gram’s stain and location (IC/EC) – gram-positive
capsular swelling test, bile solubility test (clearing) or
Laboratory Identification of N. gonorrhoeae
NEISSERIA AND MORAXELLA SPECIES o Gram-negative diplococci located intracellularly
General Characteristics or extracellularly of neutrophils
o Very important for identifying sexually disease
• Gram-negative cocci often in pairs involving male patients
• Kidney-bean shaped, capnophilic that will require 10-20% o Gram stain positive is correlated 95% strong
of carbon dioxide presumptive evidence of gonorrhea when the
• Oxidase-positive, catalase-positive gram stain is positive
• Natural habitat of Neisseria is the mucous membrane of • Culture using selective agars (Thayer Martin, MTM,
the urogenital tract Martin-Lewis and New York City) packaged in self-
• Moraxella and some Neisseria, they are inhabitants of the contained transport systems such as JEMBEC, Transgrow,
mucous membrane of the respiratory tract GonoPak – allow to have 5% CO2 so they won’t die
• Differentiation is based on acid production from • Produces acid from glucose utilization (fermentation) but
carbohydrate utilization test not for maltose, sucrose, and lactose
• Pathogenic species have fastidious growth requirements • Colonies are flat, gray, smooth and glistening
o Neisseria gonorrhea and Neisseria • Other identification Methods:
mononcytogenes, they have fastidious growth o Rapid carbohydrate degredation tests
requirement, requiring CO2 and antibiotics to o Chromogenic substrates
suppress other bacteria from growing o Immunologic assays
• Proper specimen collection is essential for successful o Nucleic acid assays
isolation - Specimens should be kept at room temperature
and plated as soon as possible
• Mucous membrane of urogenital tract (Neisseria)
o It is advisable that when you collect samples esp.
vaginal samples or urethro swab samples or
gonococcal infections. Dacron and Rayon swabs
are referred for collections because the use of
cotton swabs will allow them to ba absorb and
you will end up having negative results.
• Mucous membrane of upper respiratory tract (Moraxella
and some Neisseria)

Pathogenic Neisseria Virulence factors

• Receptors for human transferrin
• Carbohhydrate Utilization Test
• Capsule – polysaccharide capsule
o Confirmatory test that you could do to identify the
• Pili (T1-T5)
different species of Neisseria
o Piliated types (T1, T2) - Presence of pili indicate
o Using CTA (cysteine tryptic agar) containing 1%
pathogenicity.
each of these sugars (glucose, maltose, sucrose,
o Non-piliated (T3-T5)
lactose)
• Cell membrane proteins (Protein 1-3)
o The indicator is always phenol red, when they are
able to ferment the sugar. The acids that will be

9
 produced during fermentation will convert the
phenol red into yellow color

Neisseria meningitidis infections

• Primarily these infections are transmitted by droplets,
onset is abrupt
• Onset of these infections, you will experience headaches,
stiff neck and fever. Some will have pedicle lesions that
could be present.
• Meningococcal meningitis
o When this spread in the body, it will create the
condition meningococcemia (sepsis)
o The brain substance will be inflamed and it can
also progress to BIC, septic shock and therefore
• Pathogenic Neisseria are subjected to grow on a selective
later on it can also lead to hemorrhage of the
agar
adrenal gland (water house friderichsen
syndrome)
• Meningococcemia (sepsis)
o When this spread in the body, it will create the
condition meningococcemia (sepsis)
o The brain substance will be inflamed and it can
also progress to BIC, septic shock and therefore
later on it can also lead to hemorrhage of the
adrenal gland (water house friderichsen
syndrome)
• Other Neisseria are not very much implicated in clinical
disease

Virulence factors
• Pili
• Capsule - Encapsulated strains of N. meningitidis are • The antibiotics in the selective medium will not allow to
ABCYNW135 grown the N. flavescens, N. sicca and N. subflava
• POR, OPA, RMP • The N. gonorrheae and N. meningitidis can be
distinguished by the carbohydrate utilization test
• LOS endotoxin
o Glugose (+); Maltose (-) = N. gonorrhoeae
Laboratory Identification of Neisseria meningitidis o Glucose (+); Maltose (+) = N. meningitidis

• Direct microscopic count of CSF sediment revealing gram-

negative diplococci
• Culture CSF, blood and joint fluid specimens
• Nasopharyngeal swabs are cultured to detect carriers
• Growth can be seen in BAP and CAP enhanced with 5-
10% of CO2

Moraxella catarrhalis
• Part of the normal URT
• It grows on sputum sample
• It is interpreted as non-pathogenic
• It includes in the family Moraxella acinetobacter and
Moraxella citrobacter
• May cause otitis media, sinusitis, and respiratory infections
• Grows on blood, chocolate, and nutrient agars unlike
Neisseria that are very fastidious
• Able to reduce nitrate to nitrite
• Produces DNAse

10
 CLINICAL BACTERIOLOGY

ENTEROBACTERIACEAE
OUTLINE
I. Enterobacteriaceae VI . Enterobacter, Microscopic and Colony Morphology
A. Key Chracteristics Cronobacter,and • Colony morphology on non-selective media, such as SBA
B. Microscopic and Colony Pantoea and CHOC are of little value in their identification
Morphology VII. Serratia
C. Virulence and Antigenic VIII. Hafnia
• A wide variety of differential and selective media such as
Factors IX. Proteus MAC and EMB (Eosine Methylene Blue), highly selective
D. Clinical Significance X. Morganella media such as HE and XLD are available for presumptive
II. Bacterial Species and XI. Providencia identification of enteric pathogens
Infections they commonly XII. Edwardsiella o Contain one or more carbohydrates such as lactose
Produce XIII. Erwinia and and sucrose, which show the ability of the species to
III. Opportunistic Members of Pectobacterium
The Family XIV. Citrobacter
ferment specific carbohydrates
Enterobacteriaceae XV. Primary Intestinal o Fermentation is indicated by a color change in the
And Associated Infection Pathogens medium as a result of a drop in pH by a pH indicator
IV. Escheria Coli of the Family in the medium
A. Uropathogenic E. Coli Enterobacteriaceae o Species that produce H2S may be readily distinguished
B. Gastrointestinal Pathogens A. Salmonella when placed on HE or XLD agar.
i. Enterotoxigenic E. coli B. Shigella
(ETEC) C. Yersinia
o HE and XLD agars contain sodium thiosulfate and
ii. Enteroinvasive E. coli XVI. Other Genera of the ferric ammonium citrate, which produce
(EIEC) Family blackening of H2S-producing colonies
iii. Enteropathogenic E. coli Enterobacteriaceae
(EPEC) A. Budivicia Virulence and Antigenic Factors
iv. Enterohemorrhagic E. coli B. Buttiauxella
(EHEC) C. Cedecea • Controlled by several factors such as ability to adhere,
v. Enteroadherent E. coli D. Ewingella colonize, produce toxins, and invade tissue
(EAEC) E. Kluyvera • Some harbor plasmids that can provide antimicrobial
C. Extraintestinal Infections F. Leclercia resistance genes
D. Other Escherichia Species G. Leminorella
• Increasing numbers of E. coli, K. pneumoniae, and K.
V. Klebsiella H. Moellerella
A. Common Characteristics I. Obesumbacterium oxytoca clinical strains produce plasmid-mediated
i. K. pneumoniae J. Photorhabdus extended-spectrum β-lactamases (ESBLs) which can
ii. K. oxytoca K. Pragia inactivate extended-spectrum cephalosporins (e.g.,
iii. K. pneumoniae subsp. L. Rahnella cefotaxime), penicillins, and aztreonam
ozenae M. Tatumella • Antigens used in the identification of different serologic
iv. K. pneumoniae subsp. N. Trabulsiella
groups
rhinoscleromatis O. Xenorhabdus
v. K. ornithinolytica P. Yokenella o O antigen (somatic) – heat-stable, located on the cell
wall
o H antigen (flagellar) – heat-labile, surface of flagella,
ENTEROBACTERIACEAE responsible for motility
o K antigen (capsular) – heat–labile polysaccharide
Key Characteristics found only in certain encapsulated species
o K1 antigen of E. coli
• Often referred as enterics
o Vi antigen of Salmonella enterica subsp. enterica
• Gram-negative bacilli/coccobacilli serotype Typhi
• Non-spore forming, facultatively anaerobic bacilli
• Cytochrome oxidase negative, except Plesiomonas
Clinical Significance
• All are glucose-fermenting
• All reduce nitrate to nitrite, except for Photorhabdus and • Members of Enterobacteriaceae are ubiquitous
Xenorhabdus • Most enteric reside in the GI tract except for Salmonella,
• All are motile at body temperature, except for Shigella, and Yersinia
Klebsiella, Shigella, and Yersinia • Two broad categories
• None has remarkable colony morphology on supportive o Opportunistic pathogens – often a part of the usual
media, appearing large, moist, and gray on SBA, CHOC, intestinal microbiota of both human and animals
and most non-selective media; except Klebsiella, Proteus, - Outside their normal body sites, however, may
and some Enterobacter species produce serious extraintestinal,
opportunistic infections (e.g., E. coli)
o Primary pathogens – true pathogens; not present as
commensal biota in the human GI tract and produce

1
 infections resulting from ingestion of contaminated • Providencia
food or water, or from other sources (e.g. S. enterica, • Edwardsiella
Shigella spp., Yersinia spp.) • Erwinia and Pectobacterium
• Citrobacter
BACTERIAL SPECIES AND INFECTIONS THEY
COMMONLY PRODUCE ESCHERIA COLI
• Most significant species in the genus Escherichia
Bacterial species Diseases • Initially considered a harmless member of colon resident
Escherichia coli Bacteriuria, biota
septicemia, neonatal • Causes UTIs, septicemia wound infections, CNS infections,
sepsis, meningitis, diarrheal diseases
diarrheal syndrome • Primary marker of fecal contamination in water
Shigella spp. Diarrhea, dysentery purification or water quality testing
• Most strains are motile and generally possess adhesive
Edwardiella spp. Diarrhea, wound
infection, septicaemia, fimbriae and sex pili, and O, H, & K antigens
meningitis, enteric • O groups have shown remarkable cross-reactivity with O
fever antigens from other members of Enterobactriaceae, most
notably the Shigella
Salmonella spp. Septicaemia, enteric
• Serotyping for O and H antigens is often useful in
fever, diarrhea identification of strains, particularly those associated with
Citrobacter spp. Opportunistic and serious enteric disease
nosocomial infections • K antigen often masks the O antigen during bacterial
(wound, urinary) agglutination testing with specific antiserum
Klebsiella spp. Bacteriuria, • K1 antigen – identical to capsular antigen found in
pneumonia, Neisseria meningitides group B
septicemia • Appears as a lactose-positive (pink) colony with a
Enterobacter spp. Opportunistic and surrounding area of precipitated bile salts on MAC agar
nosocomial infections, • Appears with a green metallic sheen on EMB agar with the
wound infections, following properties:
septicemia, bacteriuria o Glucose, lactose, trehalose, and xylose fermentation
Serratia spp. Opportunistic and o Indole production from tryptophan
nosocomial infections, o Glucose fermentation by mixed acid pathway:
wound infections, o methyl red positive, Voges-Proskauer negative
septicemia, bacteriuria
o Does not produce H2S, DNase, Urease or
Proteus spp. Bacteriuria, wound phenylalanine deaminase
infections, septicemia o Can’t use citrase as sole carbon source
Providencia spp. Opportunistic and
nosocomial infections, Uropathogenic E. Coli
wound infections,
septicemia, bacteriuria • Most common cause of UTI in humans
• Causes acute pyelonephritis in immunocompetent hosts
Morganella spp. Opportunistic and are dominant resident in colon
nosocomial infections
• Resistant to antibacterial activity of human serum
Yersinia pestis Plague • Strains that cause UTI produce factors that allow them to
pseudotuberculosis Mesenteric adenitis, attach to the urinary mucosa (pili) and not be washed out
diarrhea by urine
enterocolitica Mesenteric adenitis, o Virulence factor:
diarrhea - Pili – adhesion to epithelial cells
Erwinia spp. Wounds contaminated - Cytolysins (hemolysins) – kill immune factor
with soil or vegetation cells and inhibit phagocytosis and chemotaxis of
Pectobacterium spp. Wounds contaminated certain WBCs
with soil or vegetation - Aerobactin – chelates iron

Gastrointestinal Pathogens
OPPORTUNISTIC MEMBERS OF THE FAMILY • Major categories of diarrheagenic E. coli based on
ENTEROBACTERIACEAE AND ASSOCIATED definitive virulence factors, clinical manifestation,
INFECTIONS epidemiology, and different O and H serotypes.
• Escherichia coli
Enterotoxigenic E. coli (ETEC)
• Klebsiella
• Enterobacter, Cronobacter, and Pantoea • Associated with diarrhea of infants and adults in tropical
and subtropical climates, especially in developing
• Serratia
countries
• Hafnia
• Major causes of infant bacterial diarrhea
• Proteus
• Traveler’s diarrhea
• Morganella

2
• Commonly spread though consumption of contaminated o Verotoxin II – biologically similar to, immunologically
food or water, poor hygiene, reduced availability of sources different from, both Stx and verotoxin I. Not neutralized
of potable water, inadequate sanitation by antibody to Stx
• High infective dose (106-1010 organisms) to initiate • Verotoxin producing E. coli may be identified by one of
disease in an immunocompetent host three methods
• Stomach acidity inhibits colonization and initiation of o Stool culture on highly different medium, with subsequent
disease serotyping
• Colonization on proximal small intestine is mediated by o Detecting verotoxin in stool filtrates
fimbriae that permits binding on intestinal microvilli o Demonstration of a fourfold or greater increase in verotoxin-
• Heat-labile toxin (LT) – similar in action and amino acid neutralizing antibody titer
sequence to cholera toxin from V. cholera • Stool culture for O157:H7 may be performed using MAC
• LT fragment A – enzymatically active portion agar containing sorbitol (SMAC) instead of lactose.
• Lt fragment B – moiety, binding portion, confers specificity, O157:H7 does not ferment sorbitol in 48 hours, and
binds to the mucosa and providing entry for A portion appears colorless
• Enzyme-labeled oligonucleotide probes detect ETEC in
Enteroadherent E. coli (EAEC)
fecal specimens
• Generally associated with two kinds of human disease:
Enteroinvasive E. coli (EIEC) diarrheal syndromes and UTIs
• Produce dysentery with direct penetration, invasion, • Two types of EAEC
and destruction of the intestinal mucosa o DAEC – associated with both UTIs and diarrheal
• Diarrheal illness is similar to that of Shigella spp. disease
although infective dose of EIEC is much higher - Uropathogenic strains are closely associated with
• Occur in adults and children alike cystitis in children and acute pyelonephritis in
pregnant women
• Fever, severe abdominal cramps, malaise, and watery
- Chronic or recurring UTI
diarrhea
o EAEC – causes diarrhea by adhering to the surface
• Strains can be non-motile and generally don’t ferment
of the intestinal mucosa
glucose
- Adheres to HEp2 cells, packed in an aggregative
• Don’t decarboxylate lysine
“stacked-brick” pattern on the cells and between
• Sereny test – ability to produce keratoconjunctivitis in the the cells by means of fimbriae
guinea pig - Watery diarrhea, vomiting, dehydration,
• It is also possible to detect invasiveness using monolayer occasional abdominal pain, mostly in children
cell cultures with HEp-2 cells (human epithelial-2 cells)
Extraintestinal Infections
Enteropathogenic E. coli (EPEC) • Newborn usually acquires infection in the birth canal just
• Causes infantile diarrhea before or during delivery, when the mother’s vagina is
• Adhesive property heavily colonized. Infection may also result if contamination
• Only certain H antigenic types within each O serogroup are of the amniotic fluid takes place
connected to intestinal infections • Septicemia and meningitis
• O serogrouping can’t differentiate this E. coli strain from • Capsular antigen K1 is the most documented virulence
strains of normal biota factor associated with neonatal meningeal infections
• Low-grade fever, malaise, vomiting, diarrhea • E. coli bacteremia in adults may result primarily from a
• Stool contains mucus, no blood present urogenital tract infection or from a GI source
• Serologic typing with pooled antisera identify EPEC
Other Escherichia Species
Enterohemorrhagic E. coli (EEC) • E. hermanii (formerly E. coli atypical/enteric group II) –
• O157:H7 strain associated with hemorrhagic diarrhea, yellow-pigmented organism isolated from CSF, wounds,
colitis, and hemolytic uremic syndrome (HUS) and blood
• Stool has NO leukocytes • E. vulneris – infected wounds, more than half the strains
• HUS is characterized by low platelet count, hemolytic also produce yellow colonies
anemia, and kidney failure • E. albertii – diarrheal disease in children
• Watery diarrhea that progresses to bloody diarrhea
with abdominal cramps, low-grade fever or absence of KLEBSIELLA
fever, no WBCs, distinguishing it from dysentery caused by • Usually found in the intestinal tract of humans and animals
Shigella spp. or EIEC infections or free-living in soil, water, and on plants
• EHEC cytotoxins • Associated with a number of opportunistic and nosocomial
o Verotoxin I – phage-encoded cytotoxin identical to infections (e.g. pneumonia, wound, UTI)
Shiga toxin (Stx) produced by S. dysenteriae type I
- Produces damage to Vero cells(African green Common Characteristics
monkey kidney cells) • Most grow on Simmons citrate and in potassium cyanide
- Reacts with and is neutralized by the antibody broth
against Stx • None produce H2S
• Few hydrolyze urea slowly
• Negative reaction with methyl red test

3
• Positive reaction with Voges-Proskauer test • Usually produce ornithine decarboxylase, unlike
• With few exceptions, indole is not produced from Klebsiella
tryptophan • Lysine decarboxylase is produced by most species but not
• Motility varies by E. gergoviae or E. cloacae
• Absence of motility distinguishes Klebsiella spp. from • E. cloacae and E. aerogenes
most of the other members of Enterobacteriaceae o Most common isolates
• Hospital acquired outbreaks of Klebsiella resistant to o Wounds, urine, blood, CSF
multiple antimicrobial agents (due to plasmid transfers)
Enterobacter cloacae and E. arogenes
K. pneumoniae
• Most common isolates
• Most commonly isolated species • Wounds, urine, blood, CSF
• Distinct feature of possessing a polysaccharide capsule,
protecting against phagocytosis and antimicrobial Pantoea agglomerans
absorption, also responsible for moist, mucoid colony
• Gained notoriety with a nationwide outbreak of
appearance
septicemia resulting from contaminated IV fluids
• Capsule is sometimes helpful in providing presumptive
• Includes species that are lysine, ornitihine, and arginine
identification
negative or “triple decarboxylases negative”
• Colonization in respiratory tracts of hospitalized patients
increases with the length of stay
Pantoea agglomerans HG XII
• Frequent cause of lower respiratory tract infections among
hospitalized patients and in immunocompromised hosts • May produce a yellow pigment
• Wound infections, UTIs, bacteremia, liver abscesses • Primarily a plant pathogen
• Antimicrobial resistance is most severe with K. pneumoniae
because of presence of K. pneumoniae carbapenemase E. gergoviae
• Found in respiratory samples and is rarely isolated from
K. oxytoca blood cultures
• Identical to K. pneumoniae except for its production of
indole and ornithine-positive isolates Cronobacter sakazaki
• Typically produces a yellow pigment
K. pneumoniae subsp. ozenae • Pathogen in neonates causing meningitis and bacteremia,
often coming from powdered infant formula
• Isolated from nasal secretions and cerebral abscesses • Isolated from cultures taken from brain abscesses and
• Causes atrophic rhinitis respiratory wound infections
• Highly associated with the presence of plasmid mediated
ESBLs, contributing to large numbers of nosocomial E. hormaechei
infections seen today
• Isolated from blood, wounds, and sputum
K. pneumoniae subsp. rhinoscleromatis
E. asburiae
• Isolates from patients with rhinoscleroma, an infection of
the nasal cavity, manifesting as an intense swelling and • Biochemically similar to E. cloacae
malformation of the entire face and neck • Isolated from blood, urine, feces, sputum, and wounds

K. ornithinolytica (Raoultella) E.dissolvens and E. nimipressuralis

• Indole positive • Newly recognized species with unknown clinical
• Ornithine decarboxylase positive significance
• Isolated from urine, respiratory tracts, and blood along with
E. cancerogenus (formerly E. taylorae)
K. planticola
• 9 Associated with osteomyelitis following traumatic wounds
K. planticola (Raoultella)
• Isolated from urine, respiratory tracts, and blood
SERRATIA
• Difficult to distinguish from K. pneumoniae • Opportunistic pathogens associated with nosocomial
outbreaks
K. variicola • Ferments lactose slowly and positive for
orthonitrophenyl galactoside (ONPG) test, except S.
• Isolated from sterile sites
fonticola
ENTEROBACTER, CRONOBACTER, PANTOEA • Differentiate from other members of the tribe by their
ability to produce extracellular DNase
• Motile • Resistance to a wide range of antimicrobials
• Enterobacter – composed of 12 species • Susceptibility tests must be performed on each isolate to
• Colony morphology resembles that of Klebsiella when determine appropriate microbial therapy
growing on MAC
• Grow on Simmons citrate medium and in potassium S. marcescens, S. rubidaea, S. plymuthica
cyanide broth • (ETEC)
Often produce pink to red pigment, prodigiosin, especially
• Methyl red = negative when isolates are incubated at room temperature
• Voges-Proskauer = positive

4
• S. marcescens is the most clinically significant
• Frequently found in nosocomial infections of the urinary P. penneri
and respiratory tract and in bacteremia outbreaks in • Can also swarm on nonselective media
nurseries and cardiac surgery and burn units
• Isolated from patients with diarrhea, although the role of
• Contamination of antiseptic solution used for joint injections
organism in disease hasn’t been proven
has resulted in an epidemic of septic arthritis
• S. plymuthica osteomyelitis was found in a motorcycle P. myxofaciens
accident
• Isolated only from gypsy moths
S. odorifera • Large amount of slime it produces
• Contains two biogroups MORGANELLA
• Emits a dirty, musty odor resembling that of potatoes
• One species: M. Morganii
o Biogroup 1 – isolated predominantly from respiratory
tract and is positive for sucrose, raffinose, and • Contain two subspecies
ornithine, may be indole positive (60%) o M. morganii subsp. morganii
o Biogroup 2 – negative for sucrose, raffinose and o M. morganii subsp. Sibonii
ornithine and has been isolated from blood and CSF, • A documented cause of UTI and has been isolated from
may also be indole positive (50%) other human body sites
• Neither species have been implicated in diarrheal illness
HAFNIA
• Isolated from a number of anatomic sites in humans and in PROVIDENCIA
the environment
P. rettgeri
• Not known to cause gastroenteritis but is occasionally
isolated from stool cultures
• Delayed positive citrate reaction is a major • Documented pathogen of the urinary tract and has
characteristic caused occasional nosocomial outbreaks
• H. alvei biotype 1 – grows in the beer wort of breweries • Implicated in diarrheal disease among travelers
and has not been isolated clinicallyERRATIA P. stuartii
PROTEUS • Implicated in nosocomial outbreaks in burn units and has
been isolated from urine cultures
• Proteus, Morganella & Providencia
• Infections caused by P. rettgeri and P. stuartii, especially
• Normal intestinal microbiota and are recognized as
in immunocompromised patients, are particularly difficult
opportunistic pathogens
to treat because of their resistance to antimicrobials
• Tribe Proteeae – distinguished from other members of
Enterobacteriaceae by virtue of the ability to deaminate P. alcalifaciens
phenylalanine
• Most commonly found in feces of children with diarrhea
• Doesn’t ferment lactose
• Role as cause of diarrhea hasn’t been proven
• Four species: P. mirabilis, P. vulgaris, P. penneri and P.
• Formerly identified as a strain of P. alcalifaciens
myxofaciens
• P. mirabilis and P. vulgaris – human pathogens
P. rustigianni
• Ascend the urinary tract, causing infections in both lower
and upper urinary tract • Rarely isolated
• Can infect proximal kidney tubules and can cause acute • Pathogenicity also remains unproven
glomerulonephritis, particularly patients with urinary tract
P. heimbachae
defects or catheterization
• Yet to be isolated from any clinical specimens
P. mirabilis and P.vulgaris
• Isolated from urine, wounds, and ear and bacteremic EDWARDSIELLA
infections • Negative for urea
• Produce “swarming” colonies on nonselective media • Positive for lysine decarboxylase, H2S, and indole
such as SBA
• Don’t grow on Simmons citrate
• Swarming is a result of regulated cycle of differentiation
from standard vegetative cells (swimmers) to E. tarda
hyperflagellated, elongated, polyploidy cells
(swarmers) capable of coordinated surface movement • Only recognized human pathogen
• Swarmer cells produce distinct odor described as “burnt • Opportunist, causing bacteremia and wound infections
chocolate” and plays a role in the ascending nature of • Pathogenic role in cases of diarrhea remains controversial
Proteus-associated UTIs
• Produces H2S and hydrolyzes urea E. hoshinae
• P. mirabilis doesn’t produce indole from tryptophan and is • Isolated from snakes, birds, and water
ornithine positive
• P. vulgaris produces indole and is ornithine negative, and E. ictalurid
ferments sucrose, therefore giving an acid/acid reaction
• Causes enteric septicemia in fish
in TSI agar (triple sugar iron agar) SERRATIA

5
 o Infections range from GI disease to mediastinal
ERWINIA AND PECTOBACTERIUM lymphadenitis and fulminant septicemia and
• Plant pathogens pneumonia
• Not significant in human infections
• Erwinia grows poorly at body temperature and fails to grow Salmonella
in selective media such as EMB and MAC • Gram-negative, facultatively anaerobic bacilli that
• Identification is more for academic interest than evaluation morphologically resemble other enteric bacteria
of their significance as causative agents of infection • On selective and differential media used primarily to isolate
enteric pathogens (e.g. MAC), salmonellae produce clear,
CITROBACTER colorless, non-lactose fermenting colonies
• Most hydrolyze urea slowly and ferment lactose, • Colonies with black centers are seen if the media (e.g. HE
producing colonies on MAC agar that resemble those of E. or XLD) contain indicators for H2S production
coli • In almost every case, they don’t ferment lactose
• All grow on Simmons citrate medium and give positive
Biochemical Features
reactions in the methyl red test
• Indole negative
• VP (Voges-Proskauer test) negative
C. freundii
• Phenylalanine deaminase negative
• Isolated in diarrheal stool cultures • Urease negative
• Pathogenic role remains unestablished • Most produce H2S, except Salmonella Paratyphi A
• Associated with infectious diseases acquired in hospital • Do not grow in medium containing potassium cyanide
settings • Virulence Factors
• UTI, pneumonias, intra-abdominal abcesses have been o Still remains uncertain
reported o Fimbriated strains appear more virulent than
• Associated with endocarditis in IV drug abusers nonfimbriated stains
• Because most (80%) C. freundii produce H2S, and some o Ability to traverse intestinal mucosa
strains (50%) fail to ferment lactose, the colony morphology o Enterotoxin produced by certain strains that cause
on primary selective media can be easily mistaken for that gastroenteritis is a significant virulence factor.
of Salmonella when isolated from stool cultures • Antigenic Structures
• Differentiation can be done through urea hydrolysis and o Somatic O antigens and flagellar H antigens are the
lysine decarboxylase primary antigenic structures used in serologic grouping
• Most (70%) hydrolyze urea, but all fail to decarboxylase of Salmonellae
lysine, whereas Salmonella fails to hydrolyze urea and most o Few strains may possess capsular K antigens,
isolates decarboxylate lysine designated Vi antigen
• Pathogen documented as the cause of nursery o Serologic identification of Vi antigen is important in
outbreaks of neonatal meningitis and brain abscesses identifying Salmonella serotype Typhi
o Heat-stable O antigen of salmonella is the
C. koseri liposaccharide (LPS) located in the outer membrane of
• Frequently found in feces, but no evidence has been found the cell wall.
that it is a causative agent of diarrhea • H antigens occur in one of two phases:
o Phase 1 flagellar antigens occur only in a small
C. amalonaticus number of serotype and determine the immunologic
identity of the particular serotype: agglutinate only with
• Isolated from sites of extraintestinal infections, such as
homologous antisera
blood and wounds
o Phase 2 flagellar antigens occur among several
strains; reacts with heterologous antisera
PRIMARY INTESTINAL PATHOGENS OF THE • Heat-labile Vi antigen is a surface polysaccharide capsular
FAMILY ENTEROBACTERIACEAE antigen found in Salmonella serotype Typhi and a few
strains of Salmonella serotype Choleraesuis
• Salmonella and Shigella produce GI illnesses in humans
• Vi antigen often blocks the O antigen during serologic
• Salmonella
typing but may be removed by heating
o Inhabit the GI tracts of the animals
• In humans, salmonellosis may occur in several forms:
o Humans acquire the infection by ingesting the
organisms in contaminated animal food products or
Clinical Infections of Salmonella
insufficiently cooked poultry, milk, eggs and dairy
products • Acute gastroenteritis or food poisoning characterized by
• Shigella vomiting and diarrhea
o Human carriers o The source of the infection has been attributed
o No animal reservoir primarily to poultry, milk, eggs, and egg products as
o Shigella dysentery usually indicates improper sanitary well as to handling pets.
o Insufficiently cooked eggs and domestic fowl, such as
conditions and poor personal hygiene
chicken, turkey, and duck, are common sources of
• Yersinia
infection.
o Transmitted by wild and domestic animals
o Salmonella gastroenteritis, also referred to as food
poisoning, occurs when a sufficient number of
organisms contaminate food that is maintained under

6
 inadequate refrigeration, thus allowing growth and o Individuals who recover from infection may harbor the
multiplication of the organisms. organisms in the gall bladder, which becomes the site
o Most cases of Salmonella gastroenteritis are self- of chronic carriage
limiting. o Excretions of organisms through feces either
o The antimicrobials of choice include chloramphenicol, continuously or intermittently
ampicillin, and trimethoprim-sulfamethoxazole. o May be terminated by antimicrobial therapy if
• Typhoid fever, most severe of enteric fever, caused by gallbladder infection is not evident
Salmonella serotype Typhi; and enteric fevers caused by o Cholecystectomy might be a solution
other Salmonella serotypes (e.g. Salmonella Paratyphi
and Choleraesuis) Shigella
o Prolonged fever
• Not members of normal GI microbiota and can cause
o Bacteremia
bacillary dysentery
o Involvement of the reticuloendothelial system
o Dissemination to multiple organs • S. dysenteriae
o A febrile disease that results from the ingestion of food • Cause bacillary dysentery
contaminated with the organisms originating from • Presence of blood, mucus, and pus in stool
infected individuals or carriers. Characteristics of Shigella
o Typhoid fever occurs more often in tropical and
subtropical countries, where foreign travelers are more • Non-motile
likely to acquire the infection. • Except for certain types of S. flexneri, they do not
o Improper disposal of sewage, poor sanitation, and lack produce gas from glucose
of a modern water system have caused outbreaks of • Urease negative – do not hydrolyze urea
typhoid fever when the organisms reach a water • H2S negative – do not produce hydrogen sulfide
source. • No lysine decarboxylation – do not decarboxylate lysine
• With the exception of Salmonella Typhi and Salmonella • Unlike Escherichia spp., Shigella spp. do not utilize
Paratyphi, salmonellae organisms infect various animals acetate or mucate as carbon source
that serve as reservoirs and sources of human infections • Fragile organism
• Salmonella Typhi and Paratyphi have no known animal • S. sonnei
reservoirs; infections only seem to occur in humans. o Decarboxylates ornithine
Carriers are often the source of infection o Slowly ferments lactose – delayed positive
• Paratyphoid fevers: Salmonella serotypes Paratyphi A, B, fermentation of lactose with formation of pink
and C, and Salmonella serotype Cholerasuis colonies only after 48 hours of incubation
o Paratyphoid fevers are less severe o ONPG positive
o “Rose spots” (blanching, rose-colored papules around • On differential and selective media, they appear as clear,
the periumbilical region) appear during the second nonlactose-fermenting colonies
week of fever
• Susceptible to various effects of physical and chemical
• Gastroenteritis
agents (e.g. disinfectants, high concentration of acid and
o “Food poisoning” ingesting contaminated food
bile)
o Salmonella strains associated with this infection are
• Susceptible to the acid pH of stoll, feces suspected of
usually found in animals (e.g. S. enterica subsp.
containing Shigella organisms should be plated
enterica)
immediately onto laboratory media to increase recovery of
o Salmonella Typhimurium was responsible for a
nationwide outbreak linked to peanut buttercontaining the organism
products • Antigenic structures
o Occurs when a sufficient number of organisms • Divided into four major O antigen groups (that are
contaminate food that is maintained under inadequate biochemically similar)
refrigeration, thus allowing growth and multiplication of • Several serotypes exist within each species, with the
the organisms exception of S. sonnei, which only has one serotype
o Infective dose is higher than required for shigellosis • All possess O antigens and certain strains possess K
o Nausea, vomiting, fever, chills, watery diarrhea and antigens
abdominal pain • Shigella K antigens, when present, interfere with
o Symptoms usually appear within a few days, with few detection of O antigen during serologic grouping (can be
or no complications removed through heating)
• Nontyphoidal Bacteremia • K antigen is heat labile and can be removed by boiling cell
o With and without extraintestinal foci of infection caused suspension Shigellae are nonmotile and they lack H
by nontyphoidal Salmonella, is characterized primarily antigens
by prolonged fever and intermittent bacteremia • Non-motile and therefore lack H antigen
o Serotypes most commonly associated are
Typhimurium, Paratyphi, and Cholerasuis Clinical Infections of Shigella
o Salmonella infection observations • Shigella spp. can cause dysentery, species vary in
o Young children – experience fever and gastroenteritis epidemiology, mortality rate, and severity of disease
with brief episodes of bacteremia
• S. sonnei is the predominant isolate, followed by S. flexneri
o Adults – experience transient bacteremia during
• S. sonnei infection is usually a short, self -limiting disease
episodes of gastroenteritis or develop symptoms of
characterized by fever and watery diarrhea
septicemia without gastroenteritis
• Carrier state following Salmonella infection

7
• The recognition of gastroenteritis in men who have sex lymphadenitis, especially in children; and generalized
with men, in which S. flexneri has been the leading isolate septicemic infections in immunocompromised hosts
• Persons with HIV infections are also at increased risk • The DNA relatedness between Y. pestis and Y.
• In developing countries, S. dysenteriae type 1 and S. pseudotuberculosis is about 90%
boydii are the most common isolates • Y. enterocolitica produces an infection that can mimic
• S. dysenteriae type 1 remains the most virulent species, appendicitis
with significant morbidity and high mortality • Other members of the genus Yersinia are found in water,
• Humans and other primates are the only known reservoir soil, and lower animals; isolates occasionally have been
• Transmission occurs by direct person to person contact, found in wounds and the urine of humans
and spread takes place via fecal-oral route with carriers as • To Y. enterocolitica, have caused intestinal disease has not
the source been found
• May be transmitted by flies, fingers, food or water
contaminated by infected people Y. pestis
• Young children in daycare centers, particularly infants • Causative agent of plague, primarily a disease of rodents
younger than 1 year of age, are the most susceptible transmitted to humans by fleas
• Seen in people living in crowded and inadequate housing • Three forms:
and in people who participate in anal-oral sexual activity • Bubonic or glandular form
• Low infective dose (<200 bacilli) – high communicable o Most common, usually results from flea bite
• Bacillary dysentery – penetration of intestinal epithelial o Symptoms appear 2 to 5 days after infection.
cells following attachment of the organisms to mucosal o Include high fever with painful regional lymph nodes
surfaces, local inflammation, shredding of intestinal lining, known as buboes (swollen lymph nodes) begin to
and formation of ulcers following epithelial penetration appear.
• Shigellosis vary from asymptomatic to severe forms of the • Pneumonic form
disease initial symptoms o Occurs secondary to bubonic plague when organisms
o Marked by high fever, chills, abdominal cramps, and proliferate in the bloodstream and respiratory tract
pain accompanied by tenesmus, appear • Septicemic form
approximately 24 to 48 hours after ingestion of the o Occurs when the bacteria spread to the bloodstream
organisms • It is gram-negative, short, plump bacillus
o Organisms, which originally multiply in the small o When stained with methylene blue or Wayson stain,
intestine, move toward the colon, where they may be it shows intense staining at each end of the bacillus
isolated 1 to 3 days after the infection develops (bipolar staining) which gives it a “safety pin”
• Bloody stools containing mucus and numerous leukocytes appearance
follow the watery diarrhea, as the organisms invade the • Grows at body temperature 37° C and it has a preferential
colonic tissues and cause an inflammatory reaction growth temperature of 25° to 30° C
• S. dysenteriae Type 1 • It is a class A bioterrorism agent
o Bloody diarrhea that progresses to dysentery
o Extremely painful bowel movements, containing blood
Y. enterocolitica
and mucus
• Rectal prolapse may result from excessive straining • Gram-negative coccobacilli with bipolar staining
• Ileus (obstruction of intestines) with marked abdominal • Grows on SBA and MAC
dilatation, possible leading to toxic megacolon • Optimal growth at 25-30°C (motility noted at 25°C and not
o One of the most serious complications cause by at 35°C)
shigellosis • Can be acquired from contact with household pets
• Other complications of shigellosis include seizures, • Found in a wide variety of animals, including domestic
infection, and HUS, a complication among the shigellae swine, cats, and dogs
exclusively associated with S. dysenteriae type 1 • Pigs as natural reservoir
shigellosis • Sepsis associated with transmission of contaminated
• The effects of shigella toxin have been implicated as the packed RBCs
mechanism responsible for the signs of disease • Human infections most often occur after the ingestion of
• The detectable toxin levels produced by S. dysenteriae contaminated food, often pork, and vacuum-packed deli
type 1 are higher than those produced by other Shigella meat, beef, lamb, chicken, and possibly chocolate milk and
spp. water
• Potential risk of transmitting this organism is its ability to
survive in cold temperatures
Yersinia
• Y. enterocolitica sepsis associated with the transfusion of
• Yersinia currently consists of 14 named species, most are contaminated packed red blood cells
considered environmental species • Common findings in cases of Y. enterocolitica infections:
• Three species are considered human pathogens: Y. pestis, o Produces an infection that mimics appendicitis (acute
Y. pseudotuberculosis and Y. enterocolitica enteritis) – occurs un older children and adults
• Y. pestis is the causative agent of plague o Erythema nodosum – inflammatory reaction
• Y. pseudotuberculosis and Y. enterocolitica have caused characterized by tender, red nodules that may be
sporadic cases of gastroenteritis; mesenteric accompanied by itching and burning
o Stools may contain blood

8
 o Enlarged mesenteric lymph nodes and inflamed ileum
and appendix OTHER GENERA OF THE FAMILY
• Y. enterocolitica infections manifest in several forms: an ENTEROBACTERIACEAE
acute gastroenteritis, an appendicitis-like syndrome
o Less frequently: septicemia, arthritis, and erythema
nodosum Budivicia
• Systemic infection is higher among elderly adults or those • Based on DNA hybridization, B. aquatica is a group of
with underlying diseases: closely related organisms
o liver cirrhosis, diabetes, acquired immunodeficiency • Not as closely related with other members of
syndrome, leukemia, aplastic anemia, and other Enterrobacteriaceae, but qualifies to belong in the
hematologic conditions • family
• Acute enteritis, the most common form of the infection, • Usually found in water
characterized by acute gastroenteritis with fever • Occasionally occurs in clinical specimens
ccompanied by headaches, abdominal pain, nausea, and • Consists of 7 species isolated from water
diarrhea
Buttiauxella
• Cold enrichment used to increase the recovery in fecal
samples suspected of containing this organism • Only B. agrestis and B. noackiae have been isolated from
• Fecal material is inoculated into isotonic saline, kept at 4° human species
C for 1 to 3 weeks and with weekly subculturing to • Biochemically similar to Citrobacter and Kluyvera
selective agar for Yersinia • DNA hybridization differs it from other genera
• Cefsulodin-igrasan-novobiocin (CIN) agar – selective
media for Y. enterocolitica Cedecea
• Incorporates cefsulodin, irgasan, novobiocin, bile salts, and • Five species:
crystal violet as inhibitory agents o C. davisae – most commonly isolated
Inhibits normal colon microbiota better than MAC agar o C. lapagei
Have added a differential property (mannitol) to the o C. neteri
medium, named Yersinia-selective agar (YSA) base o Cedecea species types 3 and 5
• Fermentation of mannitol resuts in a drop in pH around the • Most have been recovered from sputum, blood, and
colony, causing the pH indicator, neutral red, to turn red at wounds
the center of the colony and the bile to precipitate
Ewingella
• Non-fermentation produces a colorless, translucent colony
• A slightly modified medium called CIN II can be used to • E. americana – only species of the genus Ewingella
simultaneously isolate most Aeromonas spp. • Most isolates came from human blood cultures or
respiratory specimens
Y. enterocolitica • It was first thought to be related to Cedecea
• Pathogen of rodents, especially guinea pigs • DNA hybridization confirmed the placement of these
• Farm and domestic animals and birds are natural organisms in separate genera
reservoirs
Kluvyera
• Turkeys, geese, pigeons, doves, and canaries have yielded
positive cultures for this organism • Three closely-related species: K. ascorbata, K.
• Pseudotubercles – caseous swelling cryocrescens, and K. georgiana
• Human infections: rare- close contact with infected animals • K. ascorbata
or their fecal material or ingestion of contaminated drink o Most common clinical isolate
and foodstuff o Small zones of inhibition in cephalothin and
carbenicillin disk susceptibility tests
• When ingested, they spread to the mesenteric lymph
o Don’t ferment glucose at 5°C
nodes, producing a generalized infection that is usually
• Cephalohtin and carbenicillin disk susceptibility tests
self-limiting
separate the first two species
• Spreads to the mesenteric lymph nodes, producing a
o K. cryocrescens
generalized infection
- Large zones of inhibition in cephalothin and
• The clinical manifestations include:
carbenicillin disk susceptibility tests
o Septicemia accompanied by mesenteric
- Ferments glucose at 5°C
lymphadenitis, similar to appendicitis o K. georgiana
• It ppears as a typical-looking plague bacillus - Very similar to the two species but is negative for
• Differentiated from Y. pestis by its motility by 18-22°C, gas production during fermentation of glucose
urease production, and ability to ferment rhamnose • Found in respiratory, urine, and blood cultures
• Y. ruckeri • Most strains are nonpigmented, but occasional isolates
o Causative agent of red mouth disease in fish may produce a reddish-blue or violet pigment
• May produce a blue-violet pigment, usually but not
exclusively on nonblood-containing media
• All resemble E. coli colonies on MAC agar

9
 • Negative for potassium cyanide, gelatin, lysine, ornithine,
and motility
Leclercia • Lack of yellow pigmentation
• The name Leclercia was proposed in 1986 for 58 isolates • Occasionally isolated from human clinical specimens,
from human clinical specimens, including blood, urine, including wound infections, bacteremias, feces from
sputum, and feces patients with acute gastroenteritis, and septicemia,
• 27 isolates from nonhuman sources especially from immunocompromised patients
• Isolated more recently in pure culture from a septicemia
and wounds Tatumella
• L. adecarboxylata
• T. ptyseos is the only species
o Yellow pigment on primary isolation
• Unusual organism
o Similar IMViC reactions to E. coli but negative for
• Stock cultures may be kept frozen in sheep RBCs or freeze-
lysine and ornithine decarboxylase and arginine
dried, but they will die in a few weeks on agar slants
dihydrolase
• Show more biochemical reactions at 25°C than at 35°C
Leminorella • Motile at 25°C but not at 35°C
• Enteric group 57 • Large 15-36mm zones of inhibition around penicillin disks
• Leminorella was proposed as a genus with two species: L. • Slow-growing, producing tiny colonies, relatively
grimontii and L. richardii nonreactive in laboratory media
• Produce H2S • Isolated from human sources, especially sputum, and may
be a rare cause of infection
• Show weak reactions with Salmonella antisera
• Complete biochemical testing differentiates Leminorella
Trabulsiella
from Salmonella
• Leminorella spp. are relatively inactive • T. guamensis is the only species in this genus, and although
• Clinical significance is unknown it is very rarely isolated, it is biochemically similar to
• Isolated from urine, feces, and water Salmonella
• Isolated from vacuum-cleaner contents on the island of
Guam when environmental indoor dirt samples were being
Photorhabdus collected
• Three species • Also isolated from human feces
o P. luminescens • Its role in disease is unknown
- subsp. luminescens
- subsp. akhurstii Xenorhabdus
- subsp. laumondii • X. nematophilus
o P. asymbiotica
• Grows best on 25°C
o P. temperate
• Does not reduce nitrate to nitrite
• Natural habitat is lumen of entomopathogenic nematodes,
• Isolated from nematodes
but strains have occasionally been isolated from human
• Not found in human specimens
species
• Occur in two phases with the property of luminescence in
Yokenella
phase 1 only
• Most strains produce pink, red, orange, yellow, or green • Y. regensburgei
pigmented colonies on nutrient agar and especially on • Thought to be another species of Hafnia, biochemically
nutrient-rich agar, such as trypticase soy agar and egg yolk similar but differs in VP test results (Hafnia is positive,
agar Yokenella is negative)
• Negative for nitrate reduction • Isolated from human specimens

Pragia Moellerella
• P. frontium is the only species in this genus • Genus Moellerella contains one species, M. wisconsensis
• Produces H2S, a Shigella-like odor on nutrient agar • Moellerella is positive for citrate, methyl red, lactose, and
• “Pig sty-like” odor on Endo agar overlaid with a sloping sucrose
surface of the agar • It is negative for lysine, ornithine, arginine decarboxylase,
• Utilizes citrate and oxidizes gluconate and indole
• Most strains are positive for methyl red • It resembles E. coli growing on enteric media
• One isolate from stool of healthy human • The clinical significance of this organism has not been
• No indication of pathogenicity for humans or animas established
• It has been isolated from feces in two cases of diarrhea,
Rahnella infected gallbladders, and a bronchial aspirate
• R. aquatilis
Obesumbacterium
• Psychrotolerant, grows at 4°C
• Have no single characteristic that distinguishes them from • Obesumbacterium proteus biogroup 2 is more closely
other Enterobacteriaceae related to Escherichia blattae
• These are fastidious, slow-growing organisms at 37° C
• Resemble E. agglomerans, but has a weak phenylalanine
deaminase reaction • Have not been found in human specimens.

10
 CLINICAL BACTERIOLOGY LECTURE

NON ENTERIC GIT PATHOGENS

OUTLINE • Most common isolates
I. Vibrio C. Colony Morphology o V. cholera (serogroups O1 and non-O1)
A. General Characteristics D. Clinical Significance o V. parahaemolyticus
B. Clinical Manifestations E. Clinical o V. vulnificus
C. Microscopic Manifestations
o V. alginolyticus
Morphology F. Epidemiology
D. Physiology G. Pathogenesis
E. Vibrio Species H. Laboratory Diagnosis Microscopic Morphology
i. V.cholerae IV. Helicobacter pylori • Asporogenous, gram-negative rods measuring
ii. V. parahaemolyticus A. Microscopic approximately 0.5-0.8µm in diameter by 1.5- 3.0µm in
iii. V. vulnificus Morphology length
iv. V. alginolyticus B. Clinical Infections
F. Laboratory Diagnosis C. Laboratory Diagnosis
• Polar, sheathed flagella when grown in brothbut can
II. Aeromonas V. Plesiomonas produce petritrichous, unsheathed flagella when
A. General Characteristics A. General grown on solid media
B. Clinical Manifestations Characteristics • Curved gram-negative rods
C. Laboratory Diagnosis B. Epidemiology
III. Campylobacteraceae C. Infections
A. General Characteristics D. Clinical
Physiology
B. Microsopic Morphology Manifestations • Can be highly pleomorphic especially under
E. Laboratory Diagnosis suboptimal growth conditions
• Facultatively anaerobic
VIBRIO
• All clinically species are oxidase positive and able to
• They are temperature sensitive in that in temperate reduce nitrate to nitrite except for V. metschnikovii
climates when water temperature exceeds 20°C, as in the • Most are generally susceptible to vibriostatic
summer months compound O/129 (2,4-diamino-6,7-
• Can easily be isolated from water, suspended particulate diisopropylpteridine), exhibiting a zone of inhibition to a
matter, algae, plankton, fish, and shellfish. 150µg Vibriostat disk on either a Mueller-Hinton or
trypticase soy agar
General Characteristics • Positive string test observed as mucoid “stringing”
• Motile with polar flagella reaction after emulsification of colonies in 0.5%
• Catalase + sodium desoxycholate
• Oxidase +. • All species, except for V. cholera and V. mimicus, are
• Grow best in alkaline media halophilic or salt-loving and require the addition of Na+
• Often found in brackish water for growth
• Temperature sensitive • Can be differentiated from the similar genera
• Risk of infection transmitted by eating undercooked or raw Aeromonas and Plesiomonas by mean of key
marine products biochemical and growth requirement characteristics
• 4 most common vibrio species encountered in the lab:
Antigenic Structure
o V. cholerae
o V. parahaemolyticus • Subgroups of V. cholerae
o V. vulnificus o V. cholerae O1
o V. alginolyticus - Ogawa (A, B)
- Inaba (A, C)
Clinical Manifestations - Hikojima (A, B, C)
• Ranging from mild gastroenteritis to cholera and from - Epidemic cholera
simple wound infections to fatal septicemia and o V. cholerae O139
necrotizing fasciitis - Epidemic cholera
• Most common isolates o V. cholerae non-O1 – phenotypically resembles V.
o V. cholera (serogroups O1 and non-O1) cholerae but fail to agglutinate in O1 antisera
o V. parahaemolyticus o Shares a common flagellar (H) antigen and somatic
o V. vulnificus (O) antigen
o V. alginolyticus o V. parahaemolyticus can also be serotypes by means
of its O and K (capsule) antigen
Clinical Manifestations
• Ranging from mild gastroenteritis to cholera and from
simple wound infections to fatal septicemia and
necrotizing fasciitis

1
 dehydration, hypovolemic shock, metabolic acidosis, and
Vibrio Species
death in a matter of hours
Vibrio cholerae • Choleragen – cholera enterotoxin
• 2 Toxic A subunits and 5 binding B subunits
• Based on O antigen
• Toxin initially binds to the GM1-ganglioside receptor on
o O1 – the causative agent of cholera
o O139 – share cross-reacting antigens with the cell membrane via the B subunits
Aeromonas trota • A2 subunit facilitates entrance of A1 subunit
o Non-O1 – have been implicated in a variety of • Once inside the cell, the active A1 subunit stimulates the
extraintestinal infections including cholecystitis, production of adenylate cyclase through inactivation of the
ear infections, cellulitis, and septicemia. G-protein
• Based on Biotypes • Accumulation of cyclic adenosine monophosphate (cAMP)
o Classical - along the cell membrane, which stimulates hypersecretion
o El Tor – able to agglutinate chicken red blood cells of electrolytes (Na+, K+, HCO3-) and water out of the cell
• Based on Serotypes and into the lumen of the intestine
o Ogawa • The gastrointestinal tract’s absorptive ability is
o Inaba overwhelmed, resulting in the massive outpouring of watery
o Hikojima stools
• Virulence Factors • Resistant to tetracycline and doxycycline
o Cholera • Epidemic V. cholerae O1 strains occur in two biogroups:
- Toxins classic and El Tor
▪ Mucinase • El Tor has been the predominant biogroup in the last two
▪ Cholera toxin or Choleragen: an enterotoxin, pandemics
consist of 2 toxic A subunits and 5 binding B o Voges-Proskauer positive
subunits. o Hemolyzes RBCs
▪ Once ingested, the bacteria colonize the small
o Inhibited by polymyxin B (50µg), and is able to
intestine, in which they multiply and produce
agglutinate chicken RBCs
choleragen
o Different phage susceptibility patters
▪ Coregulated pilus: adherence to mucosal cells
▪ Adhesion factor • V. cholerae serogroup O141 appears to be
▪ Hemagglutination protease: induces intestinal associated with sporadic cholera-like diarrhea and
inflammation and degradation of tight junctions bloodstream infections
▪ Siderophores : iron sequestration • Other non-O1 serogroup strains have been implicated
▪ Neuraminidase: increase toxin receptors in a variety of extraintestinal infections including
- Symptoms cholecystitis, ear infections, cellulitis, and septicemia
▪ Profuse vomiting • Some O139 strains share cross-reacting antigens with
▪ Watery diarrhea Aeromonas trota, a somewhat uncommon cause of
▪ Sunken eyes diarrheal disease
▪ Watery diarrhea and profuse vomiting can lead
Vibrio parahaemolyticus
to severe dehydration which leads to death.
- Mode of Transmission • Number 1 cause of Summer Diarrhea in Japan
▪ Not an airborne disease • Second most common Vibrio species implicated in
▪ Bacteria that causes cholera come from the gastroenteritis
feces of the infected people - It may be spread • V. parahaemolyticus serogroup O3:K6 is implicated
through flies or leakage from sewage in numerous food-borne outbreaks
contaminated with the bacteria. • Found in aquatic environments but limited to coastal or
▪ Ingestion of contaminated water and food. estuarine areas despite a halophilic requirement of 1-
- Treatment 8% NaCl
▪ Oral rehydration salts • Gastrointestinal disease is self-limited
▪ Intravenous fluids • Watery diarrhea, moderate cramps or vomiting, and
▪ Stool culture little if any fever
- Prevention • Occasionally isolated from extraintestinal sources
▪ Making sure of having clean a drinking water such as wounds, ear and eye infections, even in a case
▪ Wash hands with clean water and soap of pneumonia
▪ Wash fruits and vegetables with safe clean • Kanagawa phenomenon – most strains produce a
water – peeling if possible heat-stable hemolysin that is able to lyse human RBC
▪ Eat cooked food while hot in a special high-salt mannitol medium (Wagatsuma
▪ Cover food to protect from flies agar)
▪ Keep cook food away from raw food • These strains are considered Kanagawa toxin positive,
▪ Don’t drink unpasteurized milk whereas other isolates are Kanagawa toxin negative
• Vibrio cholerae O1 is the causative agent of cholera, also
Vibrio vulnificus
known as Asiatic cholera or epidemic cholera
• Cholera is an acute diarrheal disease that is spread mainly • Commonly referred to as the “lactose-positive” Vibrio
through contaminated water • Second most serious type of Vibrio infection
• Manifests in acute cases as a severe gastroenteritis • Found in warm salt waters
accompanied by vomiting followed by diarrhea • Causes severe skin and soft tissue infections esp. in
• “Rice water” stools, contains numerous flecks of mucus shellfish handlers
• Can result in a rapid fluid and electrolyte loss thatleads to
• Two categories

2
 o Primary septicemia – surmised to occur through o This is necessary because lactose-positive
the gastrointestinal route after the consumption of colonies from selective-differential media such as
shellfish, especially raw oysters MAC or cefsulodin-irgasin-novobiocin agar may
o Wound infections give false-positive oxidase reactions
• Virulence o If a selective medium is warranted, either because
o Resistant to complement and antibody-mediated of the clinical history (exposure to seafood or
serum killing (thus systemic infections) seawater) or for geographic reasons (coastal area
o Anti-phagocytic capsule resident or recent foreign travel), thiosulfate
o Production of hydrolytic enzymes (cytolysins, citrate bile salts sucrose (TCBS) agar is
recommended. It differentiates sucrose-
collagenase, protease)
fermenting (yellow) species such as V. cholerae,
• Treatment: Doxycycline
V. alginolyticus, V. fluvialis, V. furnissii, V.
Vibrio alginolyticus cincinnatiensis, V. metschnikovii, and some V.
vulnificus strains from the nonsucrose-
• Least pathogenic for humans and one most frequently fermenting (green) vibrios: V. mimicus, V.
isolated parahaemolyticus, V. damsela, and most V.
• Common inhabitant of marine environments vulnificus strains
• Strict halophile, requiring at least 1% NaCl and can • Presumptive identification
tolerate up to 10% NaCl o Vibrios can be easily confused with other genera,
• Nearly all isolates are from extraintestinal sources such including Aeromonas, Plesiomonas,
as eye and ear infections or wound and burn infections Enterobacteriaceae, and even Pseudomonas
• Can be an occupational hazard for most people in o Their general susceptibility to the vibriostatic agent
constant contact with seawater O/129 (150 µg) and positive “string test”
distinguishes them from Aeromonas
Laboratory Diagnosis o Inability to ferment inositol (except for V.
• Specimen Collection and Transport cincinnatiensis and some strains of V.
o Vibrios are not fastidious, and only a few special metschnikovii) separates them from Plesiomonas
collection and processing procedures are o Their positive oxidase reaction (except for V.
necessary to ensure the recovery of vibrios from metschnikovii) separates them from the
clinical material Enterobacteriaceae (excluding Plesiomonas
o Whenever possible, body fluids, pus, or tissues shigelloides), and a fermentative metabolism
should be submitted, but swabs are acceptable if separates them from the oxidative Pseudomonas
they are transported in an appropriate holding • Definitive identification
medium, such as Cary-Blair, to prevent o It is important to note that with the halophilic or
desiccation salt-loving vibrios, it often is necessary to add at
o Buffered glycerol saline is not recommended as a least 1% NaCl to most biochemical media to obtain
transport or holding medium because the glycerol reliable reaction results
is toxic for vibrios o Rapid and semiautomated identification systems
o Even strips of blotting paper soaked in liquid stool o Serology
and placed in airtight plastic bags are considered • Antimicrobial Susceptibility
viable specimens for up to 5 weeks o Both Mueller-Hinton agar and broth contain
o Stool specimens should be collected as early as sufficient salt to support the growth of the Vibrio
possible in the course of the illness, before the spp. most often isolated from clinical specimens
administration of any antimicrobial agents o The recommended antimicrobial susceptibility
• Culture media testing methods are standardized disk diffusion
o The salt concentration (0.5%) in most commonly (KirbyBauer) or dilution susceptibility testing
used laboratory media, such as nutrient agar or methods
sheep blood agar (SBA), is sufficient to support o cholerae susceptible to doxycycline or
the growth of any vibrios present SBA or ciprofloxacin
chocolate (CHOC) agar, vibrios produce medium o Most vibrios are susceptible to gentamycin,
to large colonies that appear smooth, opaque, tetracycline and chloramphenicol
and iridescent with a greenish hue AEROMONAS
o The SBA plate should also be examined for the
presence of α- or β-hemolysis. On MacConkey
General Characteristics
(MAC) agar, the pathogenic vibrios usually grow
as nonlactose fermenters • Consists of ubiquitous oxidase-positive, glucose-
o However, lactose-fermenting species such as V. fermenting, gram-negative rods that are widely
vulnificus may be overlooked and incorrectly distributed in freshwater, estuarine, and marine
considered to be members of the family environments worldwide
Enterobacteriaceae, such as Escherichia coli. It • They are frequently isolated from retail produce
therefore is imperative to determine the oxidase sources and animal meat products
activity of any suspicious Vibrio-like colony • Responsible for a diverse spectrum of disease
o This can be accomplished by either directly testing syndromes among a variety of warm- and cold-
colonies from SBA or CHOC agar plates with blooded animals including fish, reptiles, amphibians,
oxidase reagent or by subculturing any mammals, and humans
suspicious lactose-fermenting colonies on • Phylogenetic evidence from molecular studies
MAC to an SBA plate for next-day testing resulted in the proposal of a separate family
Aeromonadaceae from Vibrio

3
• Aeromonads are gram negative straight rods (1.0 to 3.5 d. A cholera-like disease including rice- water
µm long by 0.03. to 1.0 µm wide), and most are motile by stools
means of a single polar flagellum e. The nebulous syndrome commonly referred to as
• All aeromonads, in general, can typically grow from 4-42°C “traveler’s diarrhea” (similar to enterotoxigenic E.
• Oxidase and Indole positive (mostly) coli)
• Glucose fermenter o Most cases are self-limiting, but in the pediatric,
geriatric, and immunocompromised populations,
• Motile with single polar flagellum
supportive therapy and antimicrobials are often
• salmonicida are non-motile and atrichous
indicated
• Ubiquitous in fresh and salty water o A.caviae is the species most frequently associated
• Virulence factors: exotoxins (enterotoxin, hemolysin, with gastrointestinal infections, especially in neonate
cytotoxin and protease), adherence factors (S-layer, and pediatric populations, and has been associated
capsule, pili) Type III secretion system (T3SSS) with inflammatory bowel disease
• MOT: Food and water o Other species associated with diarrhea include A.
• Aeromonads are classified into: hydrophila and A. veronii (biovars sobria and veronii)
a . Mesophilic o More serious complications, usually from infections
- Grow best at 37°C; All motile by single pole flagellum with A. hydrophila and A. veronii biovar sobria, include
- These organisms are considered mesophiles hemolytic uremic syndrome or kidney disease that may
because they grow well at 37°C require a kidney transplant
- All motile o A.veronii biovar sobria has also been linked to cholera-
like disease characterized by abdominal pain, fever,
▪ Hydrophila complex which includes A.
and nausea
hydrophila, A. bestiarum, and certain
motile strains of A. salmonicida • Extraintestinal infections
▪ Veronii complex includes A. veronii biovar o Most aeromonad wound isolates are A. hydrophila, A.
sobria (formerly misidentified as A. sobria), veronii biovar sobria, or A. schubertii. This last species
A. veronii biovar veronii, A. jandaei, A. has to date been isolated almost exclusively from
trota, and A. schubertii aquatic wound infections and bloodstream
infections
▪ Caviae complex includes the species A.
caviae, A. media, and A. eucrenophila o A. veronii biovar veronii and surgical wound
b. Psychrophilic infections involving the use of leeches for medicinal
- Grow best at 22-25°C therapy following plastic surgery to relieve venous
- Non-motile congestion has been noted. These patients can
▪ Salmonicida (fish pathogen) develop serious aeromonad wound infections. It
appears that the leech Hirudo medicinalis has a
c. Motile group: A. hydrophila, A. caviae, A. sobria
- Positive for Catalase, starch hydrolysis, lecithinase, symbiotic relationship with this particular aeromonad
phosphatase, arginine dihydrolase, ONPG species within its gut, wherein the organisms aid in the
hydrolysis enzymatic digestion of the blood ingested by the leech
d. Non-motile group: A. salmonicida o Aeromonad sepsis appears to be the most invasive
- Ferments arabinose, trehalose, galactose, mannose type of Aeromonas infection and likewise has a strong
and dextrin association with the species A. veronii biovar sobria, A.
- Negative for growth in KCN broth, nutrient broth with jandaei, and A. hydrophila.
7.5% NaCl, ornithine decarboxylase, tetrathionate Laboratory Diagnosis
reductase,
- Some strains produce pigments • Culture media
o Aeromonads grow readily on most media used
Clinical Manefistations for both routine and stool cultures
• Intestinal infections o After 24-hour incubation at 35° C, aeromonads
o The aeromonads are recognized as enteric pathogens, appear as large round, raised, opaque colonies
albeit not in the same manner as the more common with an entire edge and a smooth, often
enteric pathogens like Salmonella, Shigella, or V. mucoid surface
cholerae o Frequently, an extremely strong odor is present,
o The level and pattern of virulence is more like the and pigmentation ranges from translucent and
multifactorial patterns of the various E. coli white to buff colored
subgroups associated with enteric disease (e.g., o Hemolysis is variable on SBA, but most major
enterotoxigenic E. coli, clinical species, such as A. hydrophila, A. veronii
enterohemorrhagic E. coli) biovar sobria, and A. jandaei, display strong β-
o Screening of stool specimens for the presence of hemolysis
these organisms, followed by further identification to o The most commonly isolated species, A. caviae,
the species level, is appropriate is nearly always nonhemolytic or at best weakly
o Five diarrheal presentations are observed in patients α-hemolytic on SBA
in whom an Aeromonas has been isolated from their o Although aeromonads grow on nearly all enteric
stools media, they often are overlooked on MAC agar,
a. An acute, secretory diarrhea often accompanied especially in stool cultures because a number of
by vomiting aeromonads ferment lactose
b. An acute, dysenteric form of diarrhea (similar to - A, caviae - this particular species is nearly
shigellosis) with blood and mucus always pink on MAC agar because of positive
c. A chronic diarrhea usually lasting more than 10 lactose fermentation and is therefore
days generally overlooked as “normal biota” E. coli

4
 o The combined use of ampicillin sheep blood • Definitive identification
agar and a modified cefsulodin-irgasin- - Definitive identification of the aeromonads is
novobiocin (CIN II) plate (with only 4 µg of accomplished with a small number of
cefsulodin instead of 15 µg), might yield the conventional and readily available biochemical
highest recovery of aeromonads tests and antimicrobial markers and the use of
o However, the incorporation of ampicillin in the a simple dichotomous key, Aerokey II
blood agar may inhibit some A. caviae as well as all
A. trota strains because the hallmark feature of A.
CAMPYLOBACTERACEAE
trota is its unusual universal susceptibility to
ampicillin. Therefore SBA without ampicillin is
General Characteristics
preferred
o On CIN medium (either the standard CIN formulation • Campylobacters were formerly classified with the vibrios
for enteric Yersinia or the modified CIN II), Aeromonas because of their positive oxidase and characteristic
will form pink-centered colonies from the microscopic morphology, but DNA homology studies
fermentation of mannitol, with an uneven, clear apron- have shown that Campylobacter spp. do not belong with the
resembling Yersinia enterocolitica. However, an vibrios
oxidase test performed on SBA colonies will easily • Unlike the vibrios, which are fermentative, most
separate the oxidase-positive aeromonads from the campylobacters are asacchrolytic
oxidase-negative yersinias • Based on ribosomal RNA (rRNA) sequence studies,
o The use of an enrichment broth generally is not Wolinella recta and Wolinella curva have been found to be
considered necessary. However, if such a medium is similar to the campylobacters. Therefore these two species
warranted for detecting chronic cases or have been transferred to the genus Campylobacter as C.
asymptomatic carriers, alkaline peptone water is rectus and C. curvus
recommended. This can be inoculated, incubated • Although they may appear to be strict anaerobes, they
overnight at 35° C, and subsequently sub cultured to have been grown in a microaerophilic environment
appropriate plate media • Microaerophilic organisms require oxygen, but at a
• Presumptive identification concentration less than that of room air; 5% is normally
o An important screening procedure for aeromonads is optimal
to perform an oxidase test and a spot indole on • Campylobacter spp. have been known to cause abortion in
suspicious colonies on SBA, especially β-hemolytic domestic animals such as cattle, sheep and swine
colonies • Campylobacter jejuni – most common cause of bacterial
o A positive oxidase distinguishes aeromonadsfrom gastroenteritis
the family Enterobacteriaceae (except for Plesiomonas • Some species are sexually transmitted
shigelloides), and most clinically relevant • Campylobacter fetus subsp. fetus – isolated most
aeromonads are indole positive frequently from blood cultures and is rarely associated with
o The presence and type of hemolysis among multiple gastrointestinal illness
aeromonad-colony types in a single culture often are • Found in the intestines of avian species
the only clues to an infection involving more than one • Thermophilic species grow better at 42 C
species of Aeromonas
o The best tests to distinguish the aeromonads from Microscopic Morphology
Vibrio spp. are the string test (usually negative for • Curved or spiral-shaped gram-negative rod
aeromonads and positive for vibrios) and testing for • Exhibits corkscrew or darting motility by polar flagella
sensitivity to O/129 (usually aeromonads and • Non-spore forming
plesiomonads are resistant and most vibrios are
• Enteric campylobacters- may appear as long spirals or S-
susceptible)
or seagull-wing shapes.
o An additional test to separate aeromonads and
plesiomonads from most vibrios is that of determining • Gram-stained smears, these organisms stain poorly
the ability to grow in the presence of NaCl • For better visualization, carbol-fuchsin is recommended as
o Aeromonads and plesiomonads grow quite well in a counterstain
nutrient broth with 0% NaCl, but not in 6% NaCl. • If safranin is used, counterstaining should be extended to 2
Conversely, most vibrios (specifically the halophilic to 3 minutes.
species) cannot grow in 0% NaCl but thrive in 6% NaCl • Campylobacter spp. exhibit a characteristic “darting”
and even higher concentrations of NaCl motility on hanging drop preparations or when visualized
- However, because both V. cholerae and V. under phase contrast microscopy.
mimicus are nonhalophilic and grow quite well
without additional salt, any salt tolerance test must Colony Morphology
be used in conjunction with both the string test and • The typical colony morphology of C. jejuni and other enteric
the O/129 disk to distinguish aeromonads from campylobacters is moist, runny looking,and spreading.
this major pandemic cholera species and the less • Colonies are usually nonhemolytic; some are round and
common sucrose- negative V. mimicus raised and others may be flat.
o For separation of aeromonads from plesiomonads, one
• C. fetus subsp. fetus produces smooth, convex, translucent
can utilize the fermentation of inositol— where
colonies. A tan or slightly pink coloration is observed in
aeromonads are negative and plesiomonads are
some enteric campylobacter colonies.
positive
• Other Campylobacter species produce colonies similar to
o Lastly, the ability to ferment glucose, with or without those of C. jejuni. Although most do not produce pigment,
the production of gas, distinguishes Aeromonas C. mucosalis and C. hyointestinalis can produce a dirty
from oxidase-positive nonfermenting Pseudomonas yellow pigment
isolates

5
 Helicobacter mustelae
Clinical Significance Helicobacter pylori Common cause of duodenal
ulcers and type B gastritis;
possibly a risk factor in gastric
Campylobacter Species Clinical Significance carcinoma
Arcobacter butzleri Associated with diarrheal disease
and bacteremia in humans and in Clinical Manefistations
children with recurring
gastrointestinal illness (abdominal
• Several Campylobacter spp. have been implicated in
cramps
human infection: C. fetus, C. jejuni, C. coli, C. sputorum, C.
concisus, C. curvus, and C. rectus
• C. fetus contains two subspecies: C. fetus subsp. fetus
Arcobacter cryaerophilus Isolated from cases of
human bacteremia and C. fetus subsp. venerealis
• Patients infected with C. jejuni present with a diarrheal
disease that begins with mild abdominal pain within 2 to 10
Arcobacter nitrofigilis
days after ingestion of the organisms
Campylobacter fetus subsp. Rarely involved in human
• Cramps and bloody diarrhea often follow the initial
venerealis infections
signs. Patients may experience fever and chills and,
Campylobacter sputorum
rarely, nausea and vomiting
biovar faecalis • In most patients, the illness is self-limited and usually
Campylobacter concisus Involved in periodontal disease; resolves in 2 to 6 days. Untreated patients can remain
has also been recovered from
carriers for several months
individuals with gastrointestinal
illness • Other enteric Campylobacter infections (i.e., those
caused by C. coli and C. lari) have similar clinical
Campylobacter fetus subsp. Bacteremia in manifestations
fetus immunocompromised • Strong evidence suggests that Campylobacter
patients infection plays a role in Guillain-Barré syndrome, an
Campylobacter Enteric disease in swine;
autoimmune disorder characterized by acute paralysis
hyointestinalis due to damage to the peripheral nervous system. Many
occasionally associated inhuman
patients with GBS test positive for antibodies to
enteric illness
Campylobacter
Campylobacter jejuni Most common cause of
• It is believed that antibodies produced during a
bacterial diarrhea Campylobacter infection bind to gangliosides found on
worldwide peripheral nerves. Cross-reactivity with these nerve
Campylobacter lari Enteritis very similar tothat
caused by Campylobacter jejuni
Epidemiology
Campylobacter mucosalis
cells in an autoimmune response may be responsible
Campylobacter sputorum Lung, axillary, groin abcesses
for this debilitating nerve disorder
biovar sputorum
• Campylobacteriosis is the most common form of acute
Campylobacter rectus Associated with periodontal
infectious diarrhea in developed countries involving mostly
disease; has been recovered from
patients with rootcanal infections
young children
and Crohn • Cause abortion in domestic animals, such as cattle,
disease sheep, and swine (primarily zoonotic organisms)
• Most common cause of bacterial gastroenteritis worldwide
Campylobacter upsaliensis Potential pathogen in humans, Campylobacter jejuni
causing gastrointestinal illness • Transmission: direct contact with animals and handling
andbacteremia in both infected pets such as dogs, cats, and birds
immunocompetent and
• Transmission: Indirectly by the consumption of
immunocompromised patients contaminated water and dairy products and improperly
cooked poultry.
• Transmission: Some person to person and sexually
transmitted
Helicobacter canadensis Isolated in stool specimens from
• Humans acquire infections primarily by ingestion of
patients with diarrhea, pathogenesis contaminated food particularly by eating raw or uncooked
unknown poultry
Helicobacter cinaedi Recovered from blood of • Common causes of foodborne gastrointestinal illness
homosexual males with or without
• C. coli and C. lari causes gastrointestinal disease
AIDS and from blood and feces of
children • C. fetus subsp fetus has been most frequently from blood
and adult females cultures and associated with gastrointestinal illness.
Helicobacter felis • Feco-oral transmission also occurs
Recovered from rectal swabs and • Infectious dose can be as low as 500 - 1,000,000 organisms
Helicobacter fennelliae
blood of homosexual men with or depending on host factors including immunity
without AIDS presenting
with proctitis, proctocolitis, and Pathogenesis
enteritis • Non-motile strains and those lacking adhesin are avirulent
Helicobacter muridarum • Damages the mucosa of the jejunum, ileum and colon

6
• Virulence factors: endotoxin, flagellum, adhesins, S-layer 5% horse red blood cells, and a selective medium,
protein, enterotoxins and cytotoxins such as Skirrow’s agar, may be used
o It is important that the inoculated medium be fresh and
moist and that the culture be incubated in a
microaerophilic environment with increased
humidity
o Susceptibility to Nalidixic acid or Cephalotin
- Antigen detection: Lior or Penner serotyping
- Antibody detection: Latex particle agglutination

Laboratory Diagnosis
• Specimen collection and transport
o Campylobacter fetus subsp. fetus can be
recovered in several routine blood culture media
o Campylobacter spp. that cause enteric illness are
isolated from stool samples and rectal swabs, the
less-preferred specimen
o If a delay in processing the stool specimen is • Incubation
anticipated, it can be placed in a transport medium o C. jejuni and other enteric campylobacters grow
such as Cary-Blair to maintain the viability of the optimally at 42°C
organisms o Growth of normal colon organisms is inhibited atthis
o A common stool transport medium, bufferedglycerol- higher temperature
saline, is toxic to enteric campylobacters and should o C. fetus subsp. fetus, on the other hand, is a rare stool
therefore be avoided isolate, and growth is suppressed at 42°C therefore
o H. pylori can be recovered from gastric biopsy to isolate this organism, media should be incubated at
materials. Samples must be transported quickly to the 37°C
laboratory o Enteric Campylobacter and Helicobacter species
o Stuart medium can be used to maintain the viability of require a microaerophilic and capnophilic
the organisms if a delay in processing is anticipated environment. The ideal atmospheric environment
o Tissue samples may also be placed in cysteine- for these organisms contains a gas mixture of 5%
Brucella broth with 20% glycerol and frozen at O2, 10% CO2, and 85% N2 for Campylobacter
−70°C spp. and 5% to 10% O2 and 5% to 12% CO2 for
• Culture media Helicobacter spp.
o An enriched selective agar, CAMPY-BAP (blood agar o Except for C. rectus and C. curvus, a strict
plate), is a commonly used medium to isolate C. jejuni anaerobic environment does not support the
and other enteric campylobacters growth of most Campylobacter spp.
o This commercially available medium contains
• Definitive identification
Brucella agar base, 10% sheep red blood cells, and
o Isolates from stool specimens and rectal swabs
a combination of antimicrobials: vancomycin,
can be presumptively identified as Campylobacter
trimethoprim, polymyxin B, amphotericin B, and
spp. by being oxidase positive, observing the
cephalothin
characteristic Gram-stained microscopic
o Other selective media that have been successful in
morphology, and the characteristic motility
recovering Campylobacter spp. are Butzler medium
and Skirrow’s medium o The microscopic morphology is very important
o Medium V, a modification of the original Butzler because it differentiates Campylobacter from other
medium, contains cefoperazone, rifampin, colistin, and bacterial species (i.e., Aeromonas, Pseudomonas) that
amphotericin B; it seems to inhibit normal colon are oxidase positive and can grow at 42°C in a
microbiota better than the original formulation microaerophilic environment
o CAMPY-CVA (cefoperazone- o To observe the typical motility, organisms should be
vancomycin- amphotericin B) medium has been suspended in Brucella or tryptic soy broth
reported to provide better suppression of fecal biota, o Distilled water and saline seem to inhibit motility
even when this medium is incubated at 37° C o A positive hippurate hydrolysis is an important
o Incubation at 37° C allows the recovery of characteristic for the identification of C. jejuni
Campylobacter spp. that are inhibited at 42° C o H. pylori may be presumptively identified in a gastric
o C. fetus subsp. fetus, C. rectus, and C. curvus can be biopsy specimen by testing for the presence of a rapid
isolated using routine culture media urease reaction. The collected tissue sample is placed
o Charcoal-based blood-free media, such ascharcoal onto Christensen’s urea medium and incubated at
cefoperazone deoxycholate agar, are also available 37°C for 2 hours. A rapid color change suggests the
o To recover H. pylori, a combination of a nonselective presence of H. pylori
medium, such as CHOC agar or Brucella agar with o Urease activity can also be detected by the urea

7
 breath test, which is reportedly both sensitive and
specific and is recommended for monitoring therapy Clinical Infections
o H. pylori infection can also be diagnosed by fecal • Primarily linked to gastric infections
antigen detection, microscopic examination of stained • H. pylori colonizes the stomach for a long time and cause a
gastric tissue, and DNA amplification tests (i.e., low-grade inflammatory process, producing a chronic
polymerase chain reaction) superficial gastritis.
• Does not invade the gastric epithelium
HELICOBACTER PYLORI • Most common cause of chronic gastritis (type B gastritis)- a
• Bacterium found in the stomach of over half of the world’s chronic condition formerly associated primarily with stress
population and chemical irritants.
• Can cause inflammation of the stomach lining • Most common cause of duodenal ulcers
• In fact, complications from H. pylori ulcers is thought to have • Second most common cause of gastric ulcer
been the cause of the death for the famous writer, James • Associated with development of gastric cancer and gastric
Joyce lymphoma
• H. pylori is a gram-negative bacterium that’s shaped like a • Long term H. pylori infection resulting in chronic gastritis is
curved rod and it has 2 to 6 flagella, kind of like multiple tails, an important risk factor for gastric carcinoma
all at one end which it uses for movement. • H. pylori being classified as carcinogen
• It’s positive for urease, oxidase and catalase • H. canaedi and H. fenelliae: human gastroenteritis generally
in immunocompromised patients.
• Microaerophile (needs oxygen to survive, but requires less
than the levels typically found in atmosphere) Laboratory Diagnosis
• 4 Regions of the Stomach
o Cardia • Collection and Transport
o Fundus o Organism is recovered from gastric biopsy specimens
o Body o Samples must be transported quickly to the laboratory
o Pylorus o Stuart medium maintains the viability of the organism if
o Made up of two main parts: the antrum and the pyloric delay is anticipated
canal, which connects to the first section of the small o Tissue samples may be placed in cysteine-brucella
intestines called the duodenum. broth with 20% glycerol and frozen at -70 C
• Mucosa o Culture and urease production (Urea breath test)
o 3 Cell Layers • Culture Media
- Epithelial Layer (innermost layer) – absorbs and o To recover H. pylori, combination of:
secretes mucus and digestive enzymes. Dips - Nonselective medium: CHOC agar or Brucella
down below the surface of the stomach lining to agar with 5% horse red blood cells
form gastric pits. - Selective medium: Skirrow’s agar
- Lamina Propria (middle layer) – has blood and • Clinical Presentation
lymph vessels o Asymptomatic
- Muscularis Mucosa (outermost layer) – layer of o Peptic ulcer disease (gastric, duodenal)
a smooth muscle that contracts and breaks down o Non-ulcer dyspepsia
food
o Gastric carcinoma
o Gastric Pits
o Gastric mucosa-associated lymphoid tissue lymphoma
- Are contiguous with gastric glands below which
- MALT lymphoma, MALToma, marginal zone B-cell
contain various epithelial cell types – each
lymphoma of MALT type
secreting a variety of substances.
• Infection
- Example: foveolar cells, or surface mucus cells,
o Spread through fecal-oral, gastro-oral, and perhaps
secrete mucus (mix of water and glycoproteins
oral-oral transmission, from one infected individual to
that coats the stomach epithelial cells.)
another.
- With all of these digestive enzymes and
- Through contamination of food and water
hydrochloric acid floating around, the stomach
and duodenal mucosa would get digested if not - Directly with fecal matter, vomitus or saliva
for this mucus which coats and protects the o Once it arrives within the stomach, H. pylori use its
epithelial flagella to propel toward the stomach lining.
- Parietal cells – secrete hydrochloric acid to help - It will migrate to regions where pH is less acidic
maintain an acidic pH in the stomach. (antrum – has fewer parietal cells)
- Chief cells – secrete pepsinogen to digest - It then uses adhesin proteins to stick to the surface
proteins. of foveolar cells where it can release a number of
- G cells – secrete gastrin which has a number of virulence factors which help it both survive and
effects, including stimulation of parietal cells thrive, and cause damage to the mucosa.
- Urease – one of the most important enzymes for
Microscopic Morphology their survival, because it converts urea in the
gastric juices to carbon dioxide and ammonia
• Helicobacter spp. have either a single polar flagellum or
- Ammonia – basic, locally neutralizes the acid
multiple flagella at one pole.
gastric environment and protects H. pylori from the
harsh, acidic environment of the stomach.

8
• Virulence Factors o Fecal antigen test
o Cause extensive damage to epithelial cells. o Blood titers
o Some strains of H. pylori produce cytotoxin-associated o Urease breath test – with labeled urea can be used to
gene A or cagA and exotoxin vacuolating cytotoxin A detect the bacteria or enzymes
or vacA o Upper gastrointestinal endoscopy – can be used to get
o Cytotoxin-associated gene A (cagA) – interferes with a gastric biopsy
the attachments between epithelial cells that normally o Culture H. pylori from a gastic biopsy
help protect the underlying mucosal layers
o No clinical symptoms, H. pylori is not typically treated.
- Induces inflammatory immune response within the But if it’s detected in a person with a family history of
gastric lining, called gastritis gastrointestinal cancers; or when clinical symptoms are
• Chronic Infections present – triple therapy is effective and consists of two
o cagA – has been linked to the development of two antibiotics. Typically, amoxicillin and clarithromycin,
gastric cancers: and proton pump inhibitor
- Mucosa-Associated Lymphoid Tissue (MALT) • Indications for Testing
Lymphoma – involves B cells o Active peptic ulcer disease or history of peptic ulcer
- Adenocarcinoma – involves cells within the disease (unless H. pylori eradicated)
gastric glands o Low-grade gastric mucosa-associated lymphoid tissue
o Exotoxin Vacuolating Cytotoxin A (vacA) – causes lymphoma or history of endoscopic resection of early
epithelial cell death and exposes the underlying gastric cancer
mucosal layers to gastric acid. o Uninvestigated dyspepsias
- As more and more cell layers die, deep holes o Long-term NSAID or aspirin use
through the mucosa form ulcers. o Unexplained iron-deficiency anemia (after evaluation
o Inflammation – also stimulates the G cells to secrete for other causes)
gastrin, which in turn stimulates parietal cells to o Immune thrombocytopenia in adults
produce even more hydrochloric acid. • Treatment
o All this excess acid can spur the formation of both o Treating H. pylori infections consists of a macrolide,
gastric ulcers and duodenal ulcers – main distinction amoxicillin, and proton pump inhibitor.
being the exact location of the gastritis o An alternative regimen consisting of metronidazole,
o Antral gastritis – leads to duodenal ulcers tetracycline, and bismuth salt can be used.
o Corpus gastritis – leads to gastric ulcers o Treatment for H. pylori infections should be
o Very deep ulcers can erode underlying blood vessels administered for 7 to 14 days to eradicate the infection.
and may even erode all the way through the wall of the o Proton-pump inhibitors, antibiotics, Bismuth salts
stomach or duodenum in particular causing a o Typically treated with combinations of antibiotics along
perforation. with a PPI. According to the Guideline from the
o Long standing duodenal ulcers in the pyloric canal, American College of Gastroenterology, an
can sometimes have so much edema or scarring that - “...initial course of eradication therapy…generally
they obstruct the normal passage of gastric contents offers the greatest likelihood of treatment success.
into the duodenum. Thus, careful attention to the selection of the most
o Majority of individuals with H. pylori don’t have any appropriate first-line eradication therapy for an
symptoms and it may play an important role in the individual patient is essential.”
natural stomach ecology. - “The main determinants of successful H. pylori
o Chronic infections, particularly with strains that have eradication are the choice of regimen, the patient’s
cagA or vacA adherence to a multi-drug regimen with frequent
- Heartburn side-effects, and the [susceptibility] of the H. pylori
strain to the combination of antibiotics
- Shortness of breath
administered.”
- Loss of appetite with or without weight loss
- Pain in the abdomen just above the stomach that PLESIOMONAS
worsens a few hours after eating. • Oxidase-positive, glucose fermenting, facultatively
o Ulcer that leads to blood vessel erosions may cause: anaerobic, gram-negative bacilli that are motile by
- Blood in vomit or feces, if a nearby artery is polar flagella
eroded, it could lead to rapid blood loss and shock. • Found in both soil and aquatic environments, but
o Perforation: because of intolerance to increased NaCl and a
- Air can collect under the diaphragm, irritating the minimum growth temperature of 8°C, they are
phrenic nerve, and sending referred pain up to generally found only in the fresh and estuarine waters
the shoulder of tropical and subtropical climates
o Iron deficiency anemia
- Bacteria sequester dietary iron – literally eating it General Characteristics
before you can get to it
• Plesiomonads are straight (0.8 to 1 µm by 3 µm),
• Diagnosis gram-negative bacilli that occur singly, in pairs, or
o Slender, curved Gram-negative bacillus in short chains or filamentous forms
o Urease-producer • They do not form spores or capsules and are motile
o Mucosa stomach by monotrichous or two to five lophotrichous
o Growth flagella
o Microaerophilic atmosphere, slow • The genera Plesiomonas and Shigella share both
o Group 1 carcinogen (gastric carcinoma) biochemical and antigenic features, and plesiomonads

9
 often cross-agglutinate with Shigella sonnei, S. o Furthermore, biliary tract disease has been
dysenteriae, and even S. boydii—hence the species identified as a possible risk factor for bacteremia
name shigelloides with this organism
• However, unlike Shigella, the organism P. shigelloides
appears to possess a much lower virulence potential, Laboratory Diagnosis
with a low symptomatic carriage rate among humans, and • Culture media
is oxidase-positive o Plesiomonas spp. grow readily on most media
Epidemiology routinely used in the clinical laboratory
o After 18-24 hours incubation at 35° C, shiny,
• Plesiomonas can be serotyped by somatic O antigens (50 opaque, nonhemolytic colonies appear, with a
groups) and their flagellar H antigens (17 groups). slightly raised center and a smooth and entire
• Found is soil and aquatic (fresh or estuarine) environments edge
• Does not tolerate increased salt (NaCl) concentration o Because most strains ferment lactose, albeit as a
• Minimum growth temperature of 8oC “delayed” positive reaction, the easiest screening
• Like the genus Aeromonas – distributed among both warm- procedure is an oxidase test performed on
and cold-blooded animals. colonies from nonselective media, such as SBA or
CHOC agar
Clinical Infections o Although a specialized medium is not
• Most infections are transmitted from contaminated seafood recommended for the detection of plesiomonads
and water. from stool specimens, and because certain strains
• A single infection results in a moderately increased risk for are inhibited on eosin-methylene blue or MAC
diarrheal disease. It may be pathogenic in the presence of agars, the use of inositol brilliant green bile salts
another pathogen especially rotavirus or pathogenic E. coli agar can enhance the isolation of plesiomonads
• Intestinal – ingestion of contaminated water or food,
- Plesiomonas colonies are white to pink on this
medium, and most coliform colonies are
particularly uncooked or undercooked seafood: oysters,
either green or pink
clams or shrimp.
o Plesiomonas will not grow on TCBS agar but will
o Self-limiting Gastroenteritis:
grow quite well on CIN, a selective agar for Yersinia
- More common watery or Secretory diarrhea spp., as opaque (non-mannitol fermenting)
- Subacute or Chronic Disease – Lasts between 14 colonies with an opaque apron.
days and 2 to 3 months. o However, they must be distinguished from other
- A more invasive, dysenteric firm that resembles oxidase positive organisms, such as Aeromonas
colitis and Pseudomonas, that will also grow on CIN
o Extra-intestinal o Because plesiomonads are often susceptible to
- Bacteremia ampicillin, any agar used as a possible means of
- Meningitis detecting plesiomonads in clinical samples should
not contain this antibiotic
NOTE: Self-limiting means the condition will resolve on its own
• Identification
and has no long-term harmful effect on a person’s health.
o Can be presumptively differentiated from similar
genera with several key tests. The positive
Clinical Manefistations
oxidase activity separates it from the
• Gastroenteritis Enterobacteriaceae, sensitivity to the vibriostatic
o At least three major clinical types of agent O/129 separates it from Aeromonas, and its
gastroenteritis are caused by Plesiomonas ability to ferment inositol separates it from all
o The more common watery or Aeromonas and nearly all Vibrio spp.
secretorydiarrhea o It can also be separated from the halophilic Vibrio
o A subacute or chronic disease that spp. by its ability to grow in nutrient broth with
lasts between 14 days and 2 to 3 0% NaCl coupled with its inability to grow in
months nutrient broth with 6% NaCl
o A more invasive, dysenteric form o Most rapid identification systems include P.
thatresembles colitis shigelloides in their databases and appear to be
o Most cases are self-limiting, but antimicrobial able to identify it with a fairly high degree of
therapy is indicated in severe and prolonged cases accuracy
o Reports of P. shigelloides infection in patients with o This is in large part because of its unique
human immunodeficiency virus infections are “positive trio” profile of positive ornithine and
increasing, as are associations with inflammatory lysine decarboxylases and arginine
bowel disease dihydrolase reactions, combined with the
• Extraintestinal infections fermentation of inositol
o Occupational exposure can be a source of o Quantitative polymerase chain reaction assay
infections for veterinarians, zookeepers, for P. shigelloides
aquaculturists, fish handlers, and athletes
• Treatment
participating in water-related sports
o Generally resistant to Penicillin
o More serious infections, such as bacteremia and
meningitis, usually occur only in severely o There are reports of resistance to more than one
immunocompromised patients or neonates aminoglycoside (e.g., gentamicin, tobramycin,
o Recent reports include cases of continuous amikacin)
ambulatory peritoneal dialysis-associated o Quinolones, Cephalosporins & Carbapenems are
peritonitis effective

10
 CLINICAL BACTERIOLOGY

NON-FERMENTING AND MISCELLANEOUS GRAM-NEGATIVE BACILLI

OUTLINE o Usually they will fail to ferment carbohydrates, usually
I. General Characteristics C. Pseudomonas there will be cytochrome oxidase positive
II. Clues that Suggest the stutzeri • They fail to ferment carbohydrates
Isolation of a Non- D. Acinetobacter • NF can be oxidase positive except for a few unlike in
Fermenter E. Stenotrophomonas
III. Biochemical test for ID maltophilia
Enterobacteriaceae wherein most of them are oxidase
IV. Types of Pigments F. Burkholderia negative except for Plesiomonas shigelloides
V. Hugh-Leifson OF medium cepacian Complex • May fail to grow or show poor growth on McConkey agar
A. OF Medium Expected G. Bukholderia • Some have fruity, sweet or unique odors
Results pseudomallei
o An example of having a fruity, sweet odors are the
B. 3 Types of Reaction H. Alcaligenes faecalis
VI. Characteristics of I. Achromobacter bacteria Pseudomonas aeruginosa
Pseudomonas xylosoxidans • They may display unique colony morphologies and
A. Pseudomonas J. Elizabethkingia pigmentation
aeruginosa meningoseptica • Often Multi drug resistant (MDR)
B. Pseudomonas (Chryseobacterium
fluorescens and P. meningosepticum)
putida

GENERAL CHARACTERISTICS
• Prefer wet environments such as sinks, respiratory
equipment (ventilators), flower vases
• Usually not part of healthy human microbiota
• Considered opportunistic and can colonize and infect
immunocompromised individuals
• Often found as transient or colonizing microbiota of
hospitalized individuals and can become nosocomial
pathogens
• Aerobic, Gram-negative rods
• Do not use carbohydrates as a source of energy or
degrade them through metabolic pathways other than
fermentation
• Most are obligate aerobes and grow poorly if at all under NOTE:
anaerobic conditions
• Pseudomonas Aeruginosa will not be able to ferment
• Oxidizers and non-fermenters
the sugars that is contained in TSI (Triple Sugar Iron
• Asaccharolytic, do no degrade carbohydrates at all
Agar means there are 3 kinds of sugar namely, lactose,
• Usually display abundant growth on sheep BAP and CAP
glucose and sucrose) so there is no change in the
within 24-48h
reaction in the slant or butt meaning they are not able to
degrade the sugars contained in a medium.
CLUES THAT SUGGEST THE ISOLATION OF A
• K/K, alkaline reaction, the numerator represents the
NON-FERMENTER
slant and the denominator the butt, if there is no change
• The organism does not ferment carbohydrates in both meaning they are not able to degrade the sugars
o Usually when the proteins are degraded on the contained in a medium. TSI - negative
surface of the agar slant, alkaline byproducts are • Urea is also negative
created that will turn the phenol red indicator from • Citrate is positive
red-orange to pink. So, they will not be able to • SIM sulfide – negative; indole - negative and motility –
degrade most of the proteins, so they are generally positive
inert. • LIA – negative
• Alkaline slant or no change deep but reaction in TSI and • Nitrate – positive
KIA • Methyl Red and VP – negative
o In TSI you will find them having an alkaline slant and a
butt with no change in the color. BIOCHEMICAL TEST FOR IDENTIFICATION
• Require oxygen for the metabolism of carbohydrates if • Hugh-Leifson OF medium
they are able to use them at all • Oxidase test (most of them will be positive)
• Decarboxylation of amino acids

1
• Motility • No. 4: Pyomelanin: Brown pigment, 4.5% only of
• Nitrate reduction pseudomonas will be able to produce this
• Acetamide • No. 5: Combination of Pyoverdin + Pyocianin: Black
• Growth at 42°C (for Pseudomonas, they should be positive) • No. 6: Most frequently isolated, will be the combination of
• Pigments (Pseudomonas aeruginosa itself can have various pyoverdine+ fluorescein
pigments and combinations of pigments that when you look • No. 7: Pyoverdin and Pyomelanin combine together
at them, they’re so colorful when you isolate them in culture • No. 8 not have pigment on this pseudomonas.
media especially Mueller-Hinton or nutrient agar)
• Colony morphologies NOTE: Pyoverdin is the most frequently isolated pigment
• Distinct odors produce by pseudomonas followed by pyoverdine combine
with flourescin

HUGH-LEIFSON OXIDATIVE FERMENTATIVE

MEDIUM
• Obtain pure, isolated colonies from an 18 to 24 hour culture.
o So the procedure is that you obtain pure isolated
colonies from an 18-24 Hour culture. When you do
culture, and you perform streak technique, you should
be able to get isolated colonies. If not, then you have to
exercise more for you to be able to isolate. There is no
use to do T-streak, quadrant streak techniques if you
are not able to isolate colonies
• For each test organism, inoculate tubes in duplicate.
Inoculate by stabbing the agar to approximately ¼ inch from
the bottom.
NOTE: In this picture you can see that Pseudomonas o Then inoculate 2 OF medium tubes by stabbing the
aeruginosa will have Pyocyanin, it is the blue water soluble agar approx. 1/4 inch from the bottom. You do not stab
pigment. Pyorubin, a reddish pigment also that can be the all throughout until the bottom.
produced by Pseudomonas aeruginosa • Apply sterile mineral oil, sterile melted paraffin, or sterile
melted petrolatum to one of each duplicate tubes. Tighten
the cap of the overlaid tube, and loosen the cap of the non-
TYPES OF PIGMENTS overlaid tube.
• Incubate both tubes aerobically at 35 degree C for up to 14
days.
• Examine tubes daily for color change.

NOTE:

• You see that we have one tube that is anaerobically

incubated because we have tightly capped the tube and
the other one is aerobic because we have loosened the
cap.
• So the growth of microorganisms in this medium will be
either by utilizing the tryptone contained in hugh-leifson
medium. It will result in an alkaline reaction, dark blue
color will be seen.
• Or it can also be that the growth of microorganism will
be because of utilizing the glucose or any other sugar
for that matter which will result in the production of
acid.
• Remember in metabolism, fermentation, the end result
will be the production of acids and acids, once it is
formed will turn the indicator found in OF medium which
is bromothymol blue from blue to yellow. Meaning when
glucose is utilized, you will see yellow color tube so that
• No. 1: Pyoverdin, most of the Pseudomonas aeruginosa is a positive carbohydrate utilization test.
will be able to produce this type of pigment. Yellow-green
pigment called Pyoverdin (number 1 pic) OF Medium Expected Results
• No. 2: Fluorescein pigment: A yellow green pigment that • Positive: A positive carbohydrate utilization test is indicated
will floure or glow. by the development of a yellow color in the medium.
• No. 3: Combination of pigments: When fluorescein and o Oxidative: Development of a yellow colour in the open
pyocianin will mix together or combine. The greenish and tube only.
bluish will become green pigment

2
 o Fermentative: Development of a yellow colour in both • It has very simple nutritional requirements i.e. non
open and closed tubes. fastidious (can grow in any type of media)
• Negative: A negative carbohydrate utilization test is • The most important pathogenic organism is P. aeruginosa
indicated by the absence of a yellow color (media remains • Optimum temp. is 37°C, and it is able to grow at 42°C
green or turns blue) (slightly thermo-resistant)
o Non-oxidizer/Non-fermenter • It is resistant to high concentrations of salts, dyes,
weak antiseptics, and many antibiotics (multi-drug
NOTE:
resistant)
• When you see a yellow color in the medium in the open • Common inhabitants of soil, water, GIT
tube only, meaning the organism is an oxidizer. In the • P. fluorescens, putida and stutzeri
presence of oxygen, it is able to ferment the sugar which • Mode of transmission to humans is still unknown
turned the indicator bromothymol blue to yellow. • Environmental organism (you can get it:
• Closed tube - anaerobic environment immunocompromised state, indwelling catheters, shunts,
• Open tube - aerobic environment devices that would lead to opportunistic infections)
hgf
f
gF
GF
fd

3 Types of Reaction on Hugh-Leifson OF Medium Pseudomonas aeruginosa

• Most important pathogenic Pseudomonas
FGD
gf

• GNR that may be encapsulated

• Most strains are motile by means of a single polar
flagellum
• Common inhabitants of the environments such as soil, water
and plants
• Survives well in wet environments
• Metabolism is oxidative and requires the presence of
oxygen
• Obligate aerobe BUT will grow in the absence of oxygen if
nitrate is available as a respiratory electron acceptor
• Possible to see pseudomonas aeruginosa growing under
anaerobic condition even though it is considered an
obligate aerobe. You just need to supply some nutrients.
• No. 1 Tube: has mineral oil, controlled
• Rarely part of human microbiota
• No. 2 Tube: open, no overlaid mineral oil, controlled
• Opportunistic pathogen
• No. 3 Tube: aerobic, positive (carbohydrate was able to be
metabolized by organism) • Often beta-hemolytic, with irregular, rough spreading
flat colonies with a ground glass consistency with
• No. 4 Tube: anaerobic, positive
serrated or jagged edges
o Facultative anaerobes can metabolize carbohydrate
o They can hemolyze sheep’s red cells on SBA
like E. coli
• Colonies often display metallic sheen and blue-green
o Most of the Enterobacteriaceae members will be able
pigment
to present with this result because they are positive to
o Blue pigment: pyocyanin
fermentation and also they can oxidize the glucose
once it is in the anaerobic environment o Green pigment: pyoverdine
• No. 5 and 6 Tubes: oxidizers, they are able to utilize the • Pigmentation can also be red or brown (or black)
carbohydrate or glucose in the presence of oxygen, but it is • Often beta-hemolytic with a sweet fruity odor (grape-like
not a fermenter because it not able to utilize the glucose in or taco-shell like)
the anaerobic environment • Colonies can be of 2 types:
o Pseudomonas is an oxidizer, not a fermenter o large and smooth with flat edges
• No. 7 Tube: anaerobic, no reaction, non-saccharolytic o elevated appearance and the other mucoid
• No. 8 Tube: aerobic, no reaction, non-saccharolytic appearance

NOTE: Hugh-Leifson OF Medium is a very important test for

Flat colony
distinguishing oxidizers and fermenters belonging to the
group non-fermenting, Gram-negative bacilli.

CHARACTERISTICS OF PSEUDOMONAS
• Gram-negative bacilli belonging to the
Pseudomonadaceae
• Motile by means of a single polar flagellum
• Non-spore forming
• Capsulated “Polysaccharide capsule” NOT polypeptide
• Aerobic
• Breakdown glucose by oxidation i.e. Oxidative
• Oxidase and catalase positive

3
 Mucoid colony Clinical significance
• Pseudomonas is involved in respiratory infections, prevalent
in ICU patients on ventilators and receiving long-term
therapy with broad spectrum antibiotics
• Individuals with cystic fibrosis are often colonized by unique
mucoid strain that is difficult to eradicate
• Associated with bacteremia and septicemia
• Causes endocarditis in intravenous drug users and those
with prosthetic heart valves
• Well known cause of swimmer’s ear
• Major cause of bacterial keratitis often due to extended
contact wear or poor contact lens hygiene
• Prefers cartilages and joints of the bones of the skull and
• Mucoid colony consistency is attributed to the production of trunk causing chronic osteomyelitis
alginate slime • UTI caused by catheterization, instrumentation or surgery
o It will prevent phagocytosis and have the advantage to • Can produce disease in the GIT in immunocompromised
colonize area because of the slimy production of the and necrotizing enterocolitis in infants
alginate and cause disease • Produces skin and soft tissue infections (burn wound,
• Pigments: pyoverdine or fluorescein that is yellow and pyoderma and dermatitis, folliculitis)
seen only as fluorescence under a UV light; pyocyanin, a
blue pigment produced abundantly in media of low iron Prevention and treatment
content • Observation of proper isolation procedures, aseptic
• When combined the 2 pigments produce the characteristic techniques and careful cleaning and monitoring of
blue-green pigment often seen in cultures respirators, catheters and other instruments (which are
attach to the patients)
Identifying Characteristics
• Treatment with anti-pseudomonal beta-lactam,
• Grows well on most lab media fluoroquinolones and aminoglycosides, carbapenem
• Oxidizes glucose, fructose, xylose but not lactose or (imipenem)
sucrose • Current standard is combination of anti-pseudomonal beta-
• Deaminates acetamide lactam and aminoglycoside
• Grows at 42°C
• Reduces nitrate Pseudomonas fluorescens and Pseudomonas
• Decarboxylates arginine putida
o Ability to adhere to surfaces by means of fimbria • Often described together
• Requires a break in the body’s defenses for an infection to • Motile, aerobic, oxidase positive and gram-negative
begin bacilli
• Has a natural resistance to many antibiotics produced by • Produces pyoverdine or fluorescein but not pyocyanin
bacteria and fungi
• It is invasive and toxigenic Pseudomonas fluorescens
• Infection consists of 3 stages:
• An environmental organism found in soil, water, plants and
o Bacterial attachment and colonization contaminated food such as milk
o Local invasion o May cause spoilage in milk
o Dissemination by means of blood and systemic disease
• Rarely isolated from clinical specimens because they grow
• Most often found in hospitalized individuals
poorly at 35°C
• Optimum growth temperature: 25 to 30°C
Virulence factors
• Motile
• The ability to invade tissue is usually due to toxins and • Oxidase positive
enzymes that break down physical barriers, damage host • Same typical colonies on BAP, wet and gray, Non- lactose
cells and help the organism evade host defenses fermenter on McConkey
• Extracellular protease: elastase (elastin) and alkaline • Can grow at 4°C and hydrolyze gelatin (involved in food
protease (proteins, collagens etc.) spoilage: chicken and processed meat)
• Produces cytotoxins and hemolysins such as
phospholipase and lecithinase Pseudomonas putida
• The blue pigment appears to impair the ability of the upper • Also an environmental organism found in soil and water
respiratory cilia to sweep out mucous and debris
• Known for its ability for bioremediation such as
(pyocyanin)
breakdown of oil and toluene
• Kills the cells of lung epithelium by preventing catalase o Use of microorganisms to solve environmental
activity within the cell concerns
• It causes apoptosis of WBCs • Has been isolated from lizards, insects and mammals
• Other virulence factors: LPS or endotoxin, exoenzyme S, • Optimum temperature for growth: 25°C, non-lactose
exotoxin A fermenter on McConkey

4
• Motile NOTE: Right now, the Cephalosporins are grouped into 4
• Oxidase positive different generations. 1st, 2nd, 3rd, 4th generations. You
• Fails to grow at 42°C can use the 3rd generation Cephalosporins, if you can use
the third, you can use the 4th generation of Cephalosporins
Biochemical differentiation of the fluorescent
Pseudomonad group Acinetobacter
• Member of the family Moraxellaceae
Test P. aeruginosa P. fluorescens P. putida
• Consists of 25 DNA hemology groups, 11 species have
Pyocyanin + - -
been officially named
production
• Most commonly encountered Acinetobacter in the lab are
Pyoverdin + + +
production A. baumanii, A. lwoffi (letter L yan) and A. haemolyticus
Growth at + - - • Ubiquitous in the environment, in soil, water and
42°C foodstuffs, in the hospital environment
Acetamide + - - • Associated with ventilators, humidifiers, catheters and
deamination other devices (just like Pseudomonas, associated with
Gelative + + - tubings)
hydrolysis • About 25% of adults carry the organisms on the skin, 7%
Nitrate + - - carry the organisms in the pharynx
reduction • Hospitalized patients may become easily colonized
(especially those that are immunocompromised)
Pseudomonas stutzeri • Opportunistic pathogens, second to P. aeruginosa in
• Non-fluorescent gram-negative bacilli that is widely isolation frequency
distributed in the environment • A. baumanii have been associated with UTI, pneumonia,
• Rare opportunistic pathogen best known as soil tracheobronchitis, or both, endocarditis, septicemia,
denitrifier meningitis, cellulitis, trauma, burns, introduction of foreign
o Reduce the nitrogen on the soil and return to the body
atmosphere • A. baumanii has been reported in eye infections
• Often recognized by its unusual colony on BAP; adherent, • A. lwoffi is much less virulent than A. baumanii and when
wrinkled or leathery, hard and dry with either a yellow or isolated indicates contamination or colonization rather than
brown pigment infection
• It is difficult to mix into suspension, colonies resemble B.
pseudomallei, non-lactose fermenting on McConkey Identifying Characteristics
• Can grow at temperature of 4-45oC; optimum 35oC • Strictly aerobic (just like Pseudomonas), gram negative
• Motile coccobacilli
• Oxidase positive • Oxidase negative, catalase positive, non-motile (exact
• No pigment is produced opposite of Pseudomonas)
• Can appear as gram positive cocci in smear made from
Colony of P. stutzeri blood cultures
• Arginine decarboxylase negative • The purplish hue produced on McConkey may resemble
• Oxidizes maltose and hydrolyze starch a lactose fermenting bacterium (but it is not)
• Has been associated with bacteremia and septicemia, bone • pH 5.5-6.0, temperature 30-35°C are preferred
infection, endocarditis, eye infection, meningitis, skin • A. baumanii is saccharolytic (can metabolize sugar,
infections and UTIs carbohydrate but not lactose), A. lwoffi is assaccharolytic
• Demonstrates at least 2 antibiotic resistance (will not be able to metabolize sugar and carbohydrate)
mechanisms: o So when you see a K/NC reaction it is either a
o Alteration of OM proteins Pseudomonas or Acinetobacter because these two are
o LPS profiles and presence of beta-lactamases non-fermenting, gram negative bacilli
• Treatment include the use of aminoglycosides, tmp-sxt, Te, • Majority of beta-hemolytic organisms and most likely
fluoroquinolones and third gen Ceph. hemolysing the blood are called Acinetobacter
haemolyticus
• Produces K/NC reaction

5
NOTE: This is how they will appear in McConkey, that’s why • Colonies ma exhibit a lavender-green discoloration of the
I have said “purplish hue” or a color that is mistakenly called agar in areas of heavy growth
lactose fermenter because of the pink colonies • NLF, may exhibit a brownish discoloration of McConkey
agar
Clinical Significance
• Able to form biofilms on inanimate objects
• Presence of pili allows the organisms to adhere to epithelial
cells (has fimrbia just like the Pseudomonas that can adhere
to epithelial cells)
• Once it adheres, it is able to create proteins that can cause
cell death
• It possesses LPS as part of its cell wall
• Tends to be resistant to disinfectants
• Can survive in moist and dry surfaces
• Best known for its multi-drug resistance just like
pseudomonas
• Has the ability to acquire resistance to many major classes
of antibiotics including newer beta-lactams
• The presence of resistance plasmids is a significant
• Oxidase negative
virulence factor
• Strong rapid oxidation of maltose
o Plasmids – very important for multi-drug resistance
• Weaker oxidation of glucose
• A. baumanii has been named “gram negative MRSA”
• Lysine decarboxylase positive
NOTE: Remember that MRSA is gram positive, the
• DNAse positive
version for gram negative MRSA, remember that MRSA
• TSI: K/NC (no change in the triple sugar iron agar)
is multi-drug resistant, so this is associated with A.
baumanii
• The digestive tract, skin surface and mucous membranes of
patients in ICU are reservoir sites for this organism
• Transmission occurs due to contact from hands of
healthcare workers or from environmental reservoirs
• A. baumanii infections outside the hospital are very rare

Prevention and Treatment

• Standard and Contact precautions
• Careful use of antibiotics
• Treatment should be based on antimicrobial susceptibility
test results
• Most A. baumanii is susceptible to the carbapenems
(Imipenem and Merepenem) Piperacillin-Tazobactam, most
third generation Cephalosphorins, aminoglycosides NOTE: In Blood Agar Plate, non-hemolytic, large, smooth
NOTE: If everything else is resistant, there is no and shiny colonies like this
susceptible antibiotic to treat Acinetobacter* Colistin is
one of the few antimicrobials that can be used on MDR Clinical Significance
strains but is potentially nephrotoxic and neurotoxic • Important nosocomial pathogen in debilitated and
• Tigecycline, a tetracycline is a promising agent in terms of immunosuppressed individuals
multi-drug resistance of Acinetobacter • Produces preoteases and elastases, and has LPS being a
GNB a part of its cell wall
Stenotrophomonas maltophilia
• Difficult to distinguish between colonization and infection
• Maltophilia (philia- love, therefore, love for maltose) • Its presence is a marker that the patient is deteriorating
• A motile, gram negative, oxidase negative rod • Commonly present in medical devices such as IV/CV and
• Found in soil, plants and water as well as a nosocomial urinary catheters
pathogen • Pneumonia is the most common infection
• Originally included in the Pseudomonas, then became • Cystic fibrosis patients can be colonized with S. maltophilia
Xanthomonas, now it is the only species in the genus in addition to other fermenters
Stenotrophomonas • Its presence in urine is usually due to colonization of the
urinary catheter or manipulation of the urinary tract
Identifying Characteristics
• It can be isolated in wounds. Unless there is a presence of
• Appears as non-distinct, straight or slightly curved rod WBCs in the form of a pus, it may only be a colonizer
in singles or pairs
• BAP colonies are non-hemolytic, large, smooth and Prevention and Treatment
shiny, can be gray-white but may have a slightly yellow • Standard and Contact precaution
pigment with a distinctive ammonia-like odor when the
agar plate lid is initially removed

6
• S. maltophilia is intrinsically resistant (naturally) to many • The negative arginine reaction of B. cepacian will
antibiotics due to production: differentiate it from B. pseudomallei and
o To inactivate beta lactam antibiotics • B. gladioli is unable to oxidize maltose and lactose
o Carbapenemases – will neutralize carbapenemase
imipenem antibiotics
o Able to acquire plasmid
o Alter outer membrane protein and efflux mechanisms-
they have the capacity to come out antimicrobials out
in their system. Efflux pump so allow the microorganism
regulate their internal environment by removing these
toxins substances such as antibiotics. So that the
effects of antibiotics will not be exerted.
• It is well known for developing resistance to new
antibiotics quickly
• Susceptible to TMP-SXT, Colistin and Polymyxin B
• Therapy usually requires a rifampin plus a fluoroquinolone
or a beta-lactam
o Incase of Strenotrophomonas maltophilia, you cannot
do the usual standard Kirby Bauer Disc Diffusion Test.
Because this diffusion can produce inaccurate result
when ahhh you deal with Strenotrophomonas NOTE: This picture in McConkey is very very beautiful
maltophilia it will not give reproducable result. isolation of colonies. The one created this is a machine
• Broth dilution is the method recommended for susceptibility streaker
tests
Clinical Significance
Burkholderia cepacian Complex
• Extremely pathogenic for those with cystic fibrosis
• A complex of 9 subspecies not usually differentiated • Possesses few virulence factors lipopolysaccharides and
• Very significant motile gram-negative bacilli often associate ability to adhere to mucin, resistance to multiple antibiotics
with a specific patient population esp. patients with cystic • Studies show the presence of B, cepacian prior to lung
fibrosis transplant often result in death after transplantation
• Formerly Pseudomonas NOTE: It is so important to insure recovery and accurate
o Because of advances in DNA homology, it is now place identification of B. cepacia especially with regards to
in another genus Burkholderia Cystic fribrosis patient. Because the Identification,
• Inhabitants soil and water, not part of normal human Isolation and Definitive presence of cepasia would mean
microbiota death sentence to cystic fibrosis patients even after
• Well known as a plant pathogen but not as nosocomial transplantation.
pathogen and does not cause harm to healthy individuals
o If you have existing condition and pre-existing Prevention and Treatment
condition, you might get this kind of bacteria. • Requires isolation of patient when hospitalized
• Can be found in the hospital environment, tap water, • Antibiotics theraphy rarely eradicates the organism form the
disinfectants, soaps and lotions. CF respiratory tract
• Resistant to aminoglycosides and other multiple drugs
Identifying Characteristics • Multiple antibiotics are necessary for treatment like
• Able to grow on sheep’s BAP, CAP and McConkey Minocycline, Meropenem, Ceftazidime,
• Non lactose fermenters but after 4-7 days, they may Fluoroquinolones, Chloramphenicol
become dark pink or red due to oxidation of lactose in • It is susceptible to TMP-SXT
McConkey
• BCSA (burkholderia cepacia selective agar) selects and Bukholderia pseudomallei
differentiates the organism based on the presence of CV, • Oxidase positive, motile, aerobic gram negative that is
polymyxin B, Gm and Va. straight or slightly curved
• OFPBL is also selective and differential. Colonies are yellow • Grown on standard lab media ate 35°C in a CO2
due to oxidation of lactose atmosphere
o OFB – Oxidative Fermentative Polymyxin b Bacitracin • Has a smooth and mucoid colony or a dry wrinkled
and Lactose medium colony similar to P. stutzeri on BAP
• Commercial ID system may have difficult in identifying B. • A characteristic musty or earthy odor
cepacian
• Colonies appear pink on McConkey due to oxidation on
• Molecular methods are now recommended lactose
• Weakly oxidase positive • Decarbocylates arginine whereas P. stutzeri, B. cepacian
• TSI: K/NC and B. gladioli is negative
• Able to decarboxylate lysine • Organism is resistant to colistin and Polymyxin B
• Able to oxidize glucose, maltose and lactose • Motile, gram negative found in soil and water
• B. cepacia is able to oxidize mannitol while S. maltophilia is • Associated with rice paddy surface waters
DNAse positive

7
• It can be transmitted person to person via aerosols is aminobenzaldehyde. But indole test for
• Causes melioidosis: an infection disease which aim to Elizabethkingia meningoseptica Erlich reagent should
exhibit a local infection, pulmonary infection or fatal be used.
septicemia especially to immunocompromised/suppressed • Often associated with meningitis and bacteremia in
patient premature infants
• Known as “Vietnamese time bomb”- during Vietnam war • Also implicated in pneumonia, cellulitis and abscesses in
1960-1970 soldiers inhaled this organism as they marched immunocompromised patients
through the raise paddy surface water and swaps. They
inhaled aerosols. For additional Information Read Your Book hehe
• The organism can survive in phagocytes and can reactive
decades after the initial infection when individuals become
immunocompromised
• Reactivation often appears as multiple abscesses
throughout the body and skin

Alcaligenes faecalis
• Inhabits the environment and not part of human microbiota
• Can be found in hospital environment such as respiratory
equipment and disinfectants
NOTE: Most all of members of this group they are not
part of our human microbiota. Mostly hospital,
environmental pathogens associated with ventilator,
humidifier, respiratory equipments etc.
• Oxidase positive, motile gram negative produces a thin,
spreading colony with irregular edges on sheep’s BAP
• It can be produce a green discoloration of agar
• It smells green apple, non-lactose fermenting on MCConkey
• Assaccharolytic, tends to create a strong alkaline
reaction of OF medium which appears as a blue color
o OF medium – not able to oxidize and ferment
• Reduces Nitrate to Nitrite
• Can cause opportunistic infections in immunocompromised
individuals and those with underlying disease
• Has the ability to colonize cystic fibrosis patients who are
intubated

Achromobacter xylosoxidans
• Aerobic, motile, oxidase positive, non-lactose
fermenting, gram negative bacilli
• Found in moist environments
• It can be oxidize glucose and xylose
• Opportunistic pathogen capable of causing bacteremia,
meningitis, pneumonia and peritonitis
• Capable of colonizing the respiratory tract of persons with
CF, medical equipment and solutions

Elizabethkingia meningoseptica
(Chryseobacterium meningosepticum)
• Formerly Flavobacterium meningosepticum
• Found in soil, plants, water, food and hospital water sources
including incubators, sinks, faucets, saline solutions and
respiratory equipment
• Not part of nomal human flora
• Can survive in chlorinated water
• It is an oxidase positive, non-motile, slightly filamentous
or thread-like gram negative bacilli
• BAP colonies are circular, smooth, and glistening with a
light-yellow pigment
• May not grow well on McConkey
• Positive indole using an Ehrlich’s reagent
o Erlich’s reagent uses cyline, extract the indole. The
usual confirmatory reagent with the presence of indole

8
 CLINICAL BACTERIOLOGY

FASTIDIOUS GRAM-NEGATIVE BACILLI

OUTLINE
I. Fastidious Gram-Negative V. Bordatella General Characteristics
Bacilli A. Bordatella Pertussis • Gram-negative
A. General VI. Pasteurella
Characteristics Pasteurella Bettyae • Pleomorphic
II. Haemophilus VII. HACEK GROUP • Short
A. Haemophilus A. Aggregabtibacter • Non-motile
Influenzae aphrophilus
B. Haemophilus B. Aggregatibacter • Rods, often assuming coccobacilli
Parainfluenzae Actinomycetem- • Facultative anaerobes
C. Haemophilus Comitans • Obligate parasites (in respiratory tract)
Aegyptius C. Cardiobacterium
D. Haemophilus Hominis
• Ferment carbohydrates
Influenzae Biogroup D. Eikenella Corrodens • Oxidase positive
Aegyptius E. Kingella • Catalase positive
E. Haemophilus Ducreyi i. Kingella
• Reduce nitrates to nitrites
III. Francisella Denitrificans
A. Francisella Turensis ii. Kingella Kingae
IV. Legionella F. Capnocytophaga Species
A. Legionella • H. influenzae
Pneumophilia
• H. parainfluenzae
FASTIDIOUS GRAM-NEGATIVE BACILLI • H. haemolyticus
• Fastidious, miscellaneous, pleomorphic, small, gram- • H. parahaemolyticus
negative • H. aphrophilus
• Do not grow readily on routinely used lab media (ordinary, • H. paraphrophilus
such as Mac Conkey and Blood agar) • H. paraphrohaemolyticus
• Facultative anaerobes, obligate anaerobes, others are • H. pittmaniae
aerobic or microaerophilic • H. aegyptius
• Human infections with these bacteria are relatively • H. segnis
uncommon • H. ducreyi
• Haemophilus, Franciscella, Legionella, Bordetella and
Virulence Factors
Brucella
• Polysaccharide capsule – frequently present on isolates
• HACEK (Haemophilus parainfluenza, Aggregatibacter,
from clinical specimens
Cardiobacterium, Eikenella and Kingella) causing SBE
• Serotyping of capsular antigens is important because of
(subacute bacterial endocarditis)
the clinical significance of type b capsule
HAEMOPHILUS
Laboratory Diagnosis
• Derived from the Greek word “blood-lover”
• Non-pathogenic or produce opportunistic infections Specimen Processing and Isolation
• 10% of the microbiota of the upper respiratory tract
• Common sources include blood, cerebrospinal fluid
• Require performed growth factors present in the blood:
(CSF), middle-ear exudate, joint fluids, upper and lower
o X Factor (hemin or hematin; X for unknown) – used in
respiratory tract specimens, swabs from conjunctivae,
the synthesis of catalase, peroxidase, and in the
vaginal swabs, and abscess drainage.
cytochrome electron transport system
• Bronchial washing – for culture of lower respiratory tract
o V Factor (nicotinamide-adenine dinucleotide (NAD); V
• Direct plating on selective media at the bedside
for vitamin) – NAD is a co-enzyme that transfers
ispreferred (Haemophilus spp. are fast drying)
electrons from one reaction to another
o Both are additives
Microscopic Morphology
o Both are found inside RBCs, but only X factor is
• Small, gram-negative coccobacilli to long filaments
directly available
• Coccobacillary morphology – more predominant form in
o Haemophilus species with the prefix para- only
clinical specimens
require V factor for growth
• Acridine orange or Methylene blue stain – help in
• Multiply slowly in culture
detecting Haemophilus spp.
• Capnophilic – requires 5-10% CO2

1
 • V factor dependent do not grow on SBA because the red
Colony Morphology blood cells are still intact, and the sheep red bloodcells
• SBA, CHOC, and MacConkey (MAC) agar plates contain enzymes (NADases) that hydrolyze V factor
• Gram-negative pleomorphic coccobacilli on CHOC agar • Lysing of the red blood cells by heat in the preparation of
• Haemophilus spp. will not grow on MAC agar and SBA CHOC agar releases both the X and the V factors and
• Difficult to pick up inactivates NADases
• Produce a “clumpy” nonhomogeneous appearance when • Haemophilus Quad Plate
suspended in saline o Four zones: media with X factor only, V factor only, X
and V factors, and X and V factors with horse red
Laboratory Identification
blood cells
X and V Factor Requirement Porphyrin Test
• Use of impregnated strips or disks – for dentification of • Some Haemophilus spp. are able to use delta-
Haemophilus spp. and some of the Aggregatibacter spp aminolevulinic acid (ALA) as a substrate to synthesize
• Carryover may produce erroneous or less than definitive heme factor, in the process porphyrins are created
results causing H. influenzae to be misidentified as H. • Performed in agar, in broth, or on a disk
parainfluenzae • Principle: Based on the ability of the organism to convert
• When Haemophilus spp are grown anaerobically they do the substrate ALA into porphyrins or porphobilinogen,
not require heme but still require NAD which are intermediates in the synthesis of X factor
• When H. influenzae isolate was incubated anaerobically in • Porphobilinogen
this test, it could be misidentified as H. parainfluenzae o Detected by the addition of Kovacs reagent after 35°C
• H. influenzae – requires both X and V factors for 4 hours incubation
o Red color forms in the lower aqueous phase
o Kovacs reagent (p-dimethylaminobenzaldehyde)
0.5mL for inoculation
• Porphyrins
o Detected using an ultraviolet light with a wavelength
of about 360 nm (Wood’s lamp)
o Reddish-orange or pink fluorescence form under UV
light
• H. parainfluezae – requires V factor only o Much more accurate means of determining X factor
requirement compared to X and V factor disks
o Advantage: X factor is not required (no carryover)
o Disadvantage: Primary identification is based
on a negative test result
• Porphyrin negative = ultraviolet is negative = no
fluorescence = no color change occurs in addition of
Kovacs reagent = cannot synthesize heme (require heme)
= X factor positive
• Porphyrin positive = ultraviolet is positive = fluorescence
present = color change in addition of Kovacs reagent =
• A. segnis – requires only V factor, oxidase negative can synthesize heme (do not require heme) = X factor
• Aggregatibacter aphrophilus negative
• Porphyrin negative (top); porphyrin positive (bottom)

• Procedure:
Horse Blood Agar- Satellitism
o Make a lawn inoculum in a non-nutrient agar (Mueller-
Hinton.) Making a suspension of bacteria in saline, • Hemolysis is determined on HBA since it cannot
hemolyze sheep’s blood
putting a suspected Haemophilus spp. then using a
cotton swab to dip into the inoculum, which is then • Hemolysis is subtle and best viewed with light originating
spread out to the Mueller-Hinton in 3 streaks from behind the agar plate
overlapping (60°) • Stabbing the area of inoculation enhances the hemolytic
reaction

2
• X and V factors are found in the blood that’s why Horse o It is frequently present on initial isolation but can be
blood agar is used rapidly lost on subculture
• Satellitism occurs when an organism such as o It produces 6 different antigenic types of capsule:
Staphylococcus aureus, Streptococcus pneumoniae, or A, B, C, D, E, F
Neisseria spp. produces V factor as a byproduct of - based on differences of the capsular
metabolism (they obtain V factor from the SBA) polysaccharide
• Haemophilus isolate obtains X factor from the SBA o Serotype B strains
• Based on the factors required for growth and the - Present in encapsulated H. influenzae
presence of hemolysis: - Unique polymer composed of ribose, ribitol, and
o H. haemolyticus – beta hemolytic on horse blood phosphate (polyribitol phosphate [PRP]) Leading
o H. influenzae – non hemolytic cause of meningitis in unvaccinated children
o Misidentifying H. haemolyticus as H. influenzae may - Has antiphagocytic property and
result in overtreatment anticomplementary activity
o Not all strains are encapsulated
Biochemical Test
o Latex agglutination test is a rapid test for detection
• Carbohydrate fermentations - help further differentiate of these capsular antigens (most important - serotype
the Haemophilus spp. B)
• Indole, Urease, and Ornithine - used to biotype some of • IgA Proteases
the Haemophilus spp. o Has the ability to cleave secretory IgA
Treatment o Secretory IgA is present on human mucosal surfaces
of the respiratory tract
• Chromogenic cephalosporin test and acidometric test
o H. influenzae is the only member that produces IgA
– rapid tests used to detect β-lactamase
protease
o Positive β-lactamase – bacteria is resistant to
• Adherence mechanisms by fimbriae and other
ampicillin and amoxicillin
o Chromogenic cephalosporin test structures
o Most nonencapsulated strains are adherent to
- A disk impregnated with Nitrocefin, is moistened
with a drop of water human epithelial cells, whereas most serotype b
- Red color of the area develops when the β- strains are not
o Tendency to cause systemic infections
lactam ring of Nitrocefin is broken by the β-
lactamase enzyme • Outer membrane proteins and lipopolysaccharide
- Occurs in 5 minutes (LPS)
o Acidometric test o Antibody directed against these antigens may play a
- A strip impregnated with benzylpenicillin and a significant role in human immunity
pH indicator, bromocresol purple, is moistened - LPS have a paralyzing effect on the sweeping
with one or two drops of sterile distilled water motion of ciliated respiratory epithelium.
- If the β-lactam ring of the benzylpenicillin is Clinical Significance
broken by the β-lactamase, penicilloic acid is • “Pfeiffer bacillus” named after Richard Friedrich
formed, causing a drop in pH. Johannes Pfeiffer who discovered it in the great influenza
- Color change occurs from purple (negative) to pandemic in 1892
yellow (positive) • It does not cause influenza although it was originally
- Occurs 5 to 10 minutes believed to be the cause of most of the victims of flu
HAEMOPHILUS INFLUENZAE • It is often a secondary invader in the individuals infected
by Influenza A
• Major pathogen within Haemophilus spp.
• Organism is transmitted person to person via respiratory
• Appear as coccobacilli, thread-like rods and high
droplets resulting in sinusitis and infection of the middle
pleomorphism is evident
ear
• Colonies are round, small, and convex, non-hemolytic
• The most common manifestations of illness in elderly and
• Most H. influenzae produce acid from glucose and xylose
debilitated adults are bronchitis and pneumonia
but not lactose or sucrose
• Treatment with beta-lactam antibiotics can be useful if
• Variable reaction for urease and indole
the organism does not produce beta-lactamase.
• H. aegyptius can be distinguished from H. influenzae by a
Ceftriaxone, Fluoroquinolones may be required.
negative xylose reaction
• The original vaccine consisted only of the type b
• All H. influenzae from clinical samples should be serotyped
polysaccharide. Today, 4 conjugated vaccines for type
especially for patients <15 years of age
b are available
Virulence Factor • May be carried in the URT by approximately 2-4% of
• Capsule healthy individuals
o Plays the most significant role in the pathogenesis of • The carrier rate is much higher (up to 80%) for non-
invasive disease typable forms that do not have a capsule

3
• The non-encapsulated strains are usually non-invasive.
They cause contiguous spread of localized infection. Colony Morphology
• The encapsulated strains are invasive, in which • CHOC agar: translucent, tannish, moist, smooth, and
bacteremia plays a significant role. convex, with a distinct “mousy” or bleachlike odor
• Pneumonia and bacteremia caused by serotypes a, d, • H. influenzae biogroup aegyptius resembles H. influenzae
and f in immunocompromised adults and neonatal sepsis • Encapsulated strains of H. influenzae grow larger and
caused by serotype c. more mucoid than the nonencapsulated strains
• Meningitis Antibiogram Pattern
o Serotype B
• Most strains are susceptible to ampicilin
o Bloodstream invasion and bacteremic spread follow
• 25-30% are β-lactamase positive
colonization, invasion, and replication of this organism
• Most strains are susceptible to newer cephalosphorins
in the respiratory mucous membranes.
(Cefotaxime and Ceftriaxone)
o Headache, stiff neck, and other meningeal signs are
usually preceded by mild respiratory disease. Treatment
• Epiglottis • For H. influenzae:
o Rapid onset, acute inflammation, and intense edema o Cefotaxime or ceftriaxone – drug of choice
that may cause complete airway obstruction, requiring o Trimethoprim-sulfamethoxazole, imipenem, and
an emergency tracheostomy. ciprofloxacin
o The peak incidence occurs in children between 2 and o Therapy with ampicillin and Chloramphenicol
4 years of age. • For Non–life-threatening H. influenzae infection:
• Bacterial Tracheitis o Amoxicillin-clavulanate
o A serious life-threatening disease in young children. o An oral second or third-generation cephalosporin
o It can arise after an acute, viral respiratory infection. o Trimethoprim-sulfamethoxazole
o Use of broad-spectrum anti-microbial agents during
the early stages of the disease is imperative because HAEMOPHILUS PARAINFLUENZAE
thick secretions can occlude the trachea • Resemble H. influenzae in both microscopic and colonial
morphology
INFECTIONS • In blood cultures, the organism may appear as large
Encapsulated strains Non-encapsulated strains clumps of filamentous rods
• Septicemia • Bronchitis (in older • ALA positive and require only NAD (V factor)
• Septic Arthritis patients)
• Non-hemolytic on HBA
• Meningitis • Emphysema
• • Produce acid and gas from glucose and sucrose but not
• Osteomyelitis COPD
• Arthritis • Otitis media with lactose or xylose
• Tracheitis effusion • Variable for urease and catalase
• Cellulitis • Conjunctivitis • Negative for indole
• Pericarditis • Sinusitis
• Pneumonia • Bacteremia Clinical Significance
• Epiglottis • Pneumonia (in older • Part of the normal microbiota of the human respiratory
patients) tract and oral cavity
• Meningitis
• Etiologic agent of endocarditis, pharyngitis, urethritis
• Neonatal sepsis
and URT
Specimen Processing and Isolation • Mitral valve – primary site of the infection
• They are capable of colonizing the GIT tract and have
• CHOC (chocolate) agar – commonly used medium to
been implicated in sporadic cases of peritonitis and
isolate H. influenzae
cholecystitis
o Incubation:
- 33° C and 37° C (temperature)
Colony Morphology
- 5% to 10% CO2 (atmosphere)
- 18 to 24 hours • CHOC agar: tannish and drier with a medium to large size
• Bacitracin (300 mg/L) – added for the isolation of compared to H. influenzae
Haemophilus spp. from respiratory specimens, to reduce • CHOC agar: H. parahaemolyticus resembles H.
overgrowth of normal respiratory microbiota parainfluzae
• Horse or Rabbit Blood agar: H. parainfluenzae is beta
Microscopic Morphology hemolytic wheareas H. influenzae is nonhemolytic
• Observed in Gram-stained direct smears
HAEMOPHILUS AEGYPTIUS
• Clear, nonstaining areas (halos)
• Small and pleomorphic • Koch-Weeks bacillus
• Genetically related to H. influenzae
• Stains a faint pink
• It was observed in conjunctivitis exudates from Egyptians
• Amorphous serous material (serum-like or proteinaceous
by Koch in 1883, hence the species name.
background material)

4
• H. aegyptius is associated with an acute, contagious o First half: consist of GC agar base with 2% bovine
conjunctivitis, commonly referred to as “pinkeye” hemoglobin and 5% fetal calf serum
o Second half: consist of MH agar with 5%
Specimen Processing
chocolatized horse blood
• CHOC agar supplemented with 1% IsoVitaleX or Vitox
o GC agar contains 1% hemoglobin, 5% fetal calf
• Should be held for at least 4 days
serum, 1% IsoVitaleX, and 3 mg/L of vancomycin
HAEMOPHILUS INFLUENZAE BIOGROUP o Both sides contain vancomycin (resistant)
AEGYPTIUS o Vancomycin helps reduce the growth of commensal
biota from genital specimens and improves the
• Can cause conjunctivitis, primarily in pediatric populations
detection of H. ducreyi.
• Causes a severe systemic disease known as Brazilian
• 5% to 10% CO2 atmosphere containing high humidity
purpuric fever (BPF) in hot climates
• Grows best at 33°C
• Characterized by recurrent or concurrent conjunctivitis,
• Should be held for at least 7 days
high fever, vomiting, petechial/purpural rash, septicemia,
shock, and vascular collapse Microscopic Morphology
• Mortality rate: 70% within 48 hours after onset • Pale staining gram-negative coccobacilli
• School of fish – arranged singly, or in groups (clusters)
HAEMOPHILUS DUCREYI
• Railroad tracks – loosely coiled clusters lined up in
• Small gram-negative coccobacilli that tend to grow in short parallel
chains, clumps, or whorls within lesions • Fingerprints arrangement
• Individual bacteria exhibit bipolar staining
• Organism is very difficult to culture (takes too long to grow
Colony Morphology
and easy to die)
• CHOC agar: small, flat, smooth, nonmucoid, transparent to
• Scrapings of an ulcer is best accomplished on CAP
opaque colonies, or appears tan or yellow
containing Isovitalex and Va, incubation at 33°C in 10%
CO2 Treatment
• Plates must be incubated for 10 days • Erythromycin – drug of choice
• Yellowish to gray, dome-shaped, smooth colonies • Azithromycin, ceftriaxone, or ciprofloxacin, TMP-5XT-E
• Requires heme factor (X factor) but not NAD (V factor)
FRANCISELLA
• Unable to synthesize porphyrin and does not cause
• 2 species: F. tularensis and F. philomiragia but only F.
hemolysis on HBA
tularensis is pathogenic for humans
• Most are catalase negative, urease negative, indole
• Appear as non-motile, non-spore forming, strictly
negative
aerobic, gram-negative bacilli or coccoid bacteria
• Acid is produced from lactose, sucrose and xylose
• Facultative intracellular pathogens/ Facultative
Virulence Factors anaerobes (can live inside and outside the cell)
• Hemolytic cytotoxin, a cytolethal distending toxin, pili, • Fastidious and may require supplementation with
hemoglobin binding protein and lipooligosaccharide cysteine, cystine, or thiosulfate
• CHOC agar, modified Thayer-Martin, buffered charcoal
Clinical Significance yeast extract agars, Mueller-Hinton, and Tryptic soy broths
• H. ducreyi is a strict human pathogen can be used
• Not part of the normal microbiota • MC and EMB will not support growth
• Causes chancroid
• Grow after 48 hours
o It is a highly communicable sexually transmitted
genital ulcer disease (GUD) FRANCISELLA TULARENSIS
o Commonly referred to as soft chancre
o Hard chancre – syphilis • Has four subspecies or biovars: subsp. tularensis (type
o All patients who have GUD should also be tested for A), subsp. holarctica (type B), subsp. mediasiatica, and
human immunodeficiency virus along with syphilis and subsp. novicida.
herpes virus • Does not grow on most routinely used laboratory media
o Causes suppurative (pus forming), enlarged, • Very small, transparent colonies will generally appear on
draining, inguinal lymph nodes (buboes) cysteine-supplemented agar when incubated for 3 days at
• Common sites of infection: penis or the labia or within
37°C aerobically
the vagina
• Bacteria are transmitted by direct sexual contact • Pinpoint colonies may appear after 24 hours of incubation
• Unlike syphilis chancre, the lesion is painful • Amino acid cysteine is required
• Men have symptoms related to the inguinal tenderness • Best growth is obtained on blood-cysteine-glucose agar,
and genital lesions enriched chocolate agar or Thayer Martin agar or BCYE
• Women are asymptomatic or other media that contain cysteine
• Immunity to the organism does not develop after infection • It is a Category A biological agent by CDC
• Condom use
• Organism is highly infectious and should be handled on
Specimen Processing and Isolation BSL 3 precautions
• Nairobi bioplate medium

5
• Organisms are extremely small, intracellular GNCB that • Slide agglutination
stains poorly on Gram’s stain and highly pleomorphic • Single serology test
• Satellite negative, X and V factors negative
• Oxidase, urease, indole, and ornithine negative Biochemical Identification
• Weakly positive for catalase and produces β-lactamase • Relatively inert biochemically
• Biochemical tests are not used for ID and not
Clinical Significance
recommended
• F. tularensis subsp. tularensis causes the most severe • Organisms are usually missed in smears from tissue
disease specimens because of their small size and intracellular
• F. tularensis subsp. novicida is an opportunistic pathogen, nature, high pleomorphism and pale staining with Gram’s
primarily causing disease in immunocompromised stain
individuals • The organisms have a thin capsule that consists of lipid,
• F. tularensis subsp. holarctica and mediasiatica produce a proteins, and carbohydrates
similar disease to F. tularensis subsp. tularensis, but • Direct FAT (fluorescent antibody test) is used for the
infections are rarely fatal identification of the organisms in the tissues and sputum
• As few as 50 organisms can cause infection through the specimens
cutaneous (ulceroglandular form) or inhalation • An antibody titer of 160 in a single specimen is highly
(pneumonia) routes suggestive of infection
• Present in wide variety of wild animals, birds and even • A four-fold increase in antibody titer in paired serum
some fishes and amphibians samples taken 2 weeks apart is strongly indicative of active
• Common reservoir are rabbits, muskrats, and squirrels disease
• Infection can occur by direct contact with a dog or cat that
has had a contact with an infected animal BIOTYPES
• Ticks and deerflies are the most common arthropod BIOTYPE A BIOTYPE B
vectors • Found in US and North • More widespread
• Routes of transmission: America • Found in Western and
o Bite of an arthropod • Highly virulent Eastern hemispheres
• Transmission is through and is associated with
o Direct contact with an infected animal
the bite of a tick that has water and rodents
o Ingestion of contaminated meat or water acquired the organism
• Males and children under the age of 10 years have the from infected wild
highest incidence of tularemia rabbits
• Hunters, vets, and taxidermist are at increases risk for
Treatment
infection
• Manifestations are highly dependent on the route of • Streptomycin is the drug of choice or Gentamicin over a
transmission period of 10 days.
• Fever, malaise, headache, and pain in the involved region • Ceftraixone is not effective – because of the presence of
are noted beta-lactamase
• Infection occurs most often through minute abrasions in • Vaccine provides partial immunity
the skin resulting in greatly enlarged regional lymph nodes LEGIONELLA
that sometimes drain for weeks and become necrotic
• Contains 50 known species, 20 of which have been
INFECTION isolated from humans
Tularemia • Ubiquitous gram-negative bacilli acquired by humans
• Zoonotic disease aka Lemming and Water rat trapper’s primarily through inhalation
disease
Clinical Significance
• Can be transmitted through ingestion, inhalation, or
arthropod-bite • Produce a spectrum of symptoms from mild upper
• Can occur in pneumonic, glandular, oropharyngeal, respiratory tract infections to pneumonia
oculoglandular and typhoidal forms • Associated with nosocomial infections
• The most common clinical form is ulceroglandular • Most human cases of legionellosis are caused by L.
• Agent of tularemia or glandular fever or tick fever
pneumophila
and occasionally as deer fly fever or rabbit fever
• The genus name was given in honor of Edward • Legionella spp. are transmitted to human hosts from these
Francis who studied the organism extensively environmental sources primarily via aerosolized particles,
• The species name is based on the location where such as those produced by normal tap water pressure.
the organisms were discovered (Tulare, CA) • The factors that contribute to the ability of Legionella spp.
to colonize these sources include:
Presumptive Identification o The ability to multiply over the temperature range of
• Direct fluorescent antibody 20° to 43° C and survive for varying periods at 40° to
• Immunohistologic staining with monoclonal antibody 60° C
• PCR

6
 o The capacity to adhere to pipes, rubber, plastics, and • L. micdadei is weakly acid-fast in tissue and stains best
sediment and persist in piped water systems even with the modified Kinyoun procedure
when flushed • The faint-staining, pleomorphic gram-negative bacilli may
o The ability to survive and multiply within free-living be found outside of and within macrophages and
protozoa and in the presence of commensal bacteria segmented neutrophils
and algae • Direct fluorescent antibody (DFA) test - provides a
useful method of confirming that an isolate is a Legionella
Yellow Green Blue White sp. and for identifying the more common species and
Autofluorescence Autofluorescence serogroups of the genus
• L. birminghamensis • L. bozemanii
• Acid treatment of specimens contaminated with bacteria
• L. cincinnatiensis • L. dumofii
• L. hackeliae • L. gormanii Isolation Methods
• L. jordanis • L. lytica
before inoculation enhances isolation of Legionella spp.
• L. longbeachae • L. parisiensis
• L. maceachernii • L. tusconensis o In this procedure, an aliquot of the specimen is first
• L. micdadei diluted 1: 10 with 0.2 N KCl-HCl and allowed to stand
• L. oakridgensis for 5 minutes.
• L. pneumophila† o Inoculated medium is incubated at 35° to 37° C in air
• L. sainthelensi for at least 7 days.
• L. wadworthii o Usually within 3 to 5 days, Legionella spp. colonies
Blue White or Yellow No color are visible.
Green Autofluorescence
• Fastidious, aerobic bacteria that will not grow on sheep
• L. anisa • L. feeleii
• L. lansingensis blood agar (SBA) and require L-cysteine for growth
• Tiny colonies may appear on chocolate agar (CHOC) agar
Virulence Factors that contains L-cysteine
• Organism’s ability to enter, survive, and multiply within • Buffered charcoal yeast extract (BCYE) agar with L-
the host’s cells, especially bronchoalveolar cysteine is best for Legionella isolation
macrophages, and the ability to produce proteolytic
Colony Morphology
enzymes
• BCYE: Grayish-white or blue green, convex, and
• Cause intracellular infections in humans but also survive in
glistening, measuring approximately 2 to 4 mm in diameter
an extracellular environment.
• The central portion of young colonies has a “ground-
Laboratory Diagnosis glass” appearance, light gray and granular
• The periphery of the colony has pink and/or light blue or
Specimen collection and handling bottle green bands with a furrowed appearance
• Include sputum, bronchoalveolar lavage, and bronchial
Treatment
washings
• Sputum purulence screens are not used • Treated with a macrolide such as azithromycin or a
• Transtracheal aspiration, lung tissue, blood, fluoroquinolone
• wound and abscess material, and pleural, peritoneal, and • An alternate drug is doxycycline
pericardial fluids LEGIONELLA PNEUMOPHILIA
• Respiratory secretions • Discovered after an outbreak of severe respiratory illness
• and body fluids (except blood) are submitted in sterile, at an American Legion convention that took place in 1976
leak proof containers in Philadelphia
• Small pieces of tissue may be overlaid with sterile water • Can cause Pontiac fever, an influenza like illness that
• Saline or buffer should not be used in processing or initially occurred during an outbreak in Michigan
transporting specimens because of the inhibitory effects of • Ubiquitous in the environment where warm and moist
sodium condition prevail
• Specimens should be refrigerated if more than 2 hours • Have been recovered from lakes, streams, mud, and soil
pass between collection and processing • No known animal reservoir
• Transport specimens to a reference laboratory on wet ice, • Of more than 40 species, L. pneumophila is the most
and freeze specimens at −70° C if processing will be common cause of disease in humans.
delayed for several days • There are at least 10 serogroups with serogroup 1 being
Microscopic Examination the cause of the 1976 outbreak
• Pleomorphic, weakly staining, gram-negative bacilli that • Aerobic, gram-negative rods that can be isolated on
BCYE supplemented with 1% α-ketoglutarate
are approximately 1 to 2 µm × 0.5 µm in size.
• The organism requires iron salts, cysteine, and high
• Extending the safranin counterstaining time to at least 10
humidity (moist) for growth
minutes can enhance the staining intensity of the
organisms. • Best growth is obtained at a pH of 6, 9, 37°C and 90%
humidity

7
• Antibiotics are sometimes added to the medium to prevent Legionnaire’s Disease
overgrowth by other bacteria • Febrile disease with pneumonia
• Growth in blood cultures usually requires at least 2 weeks • Typically presents in three major patterns:
incubations o Sporadic cases – most common and usually occur
in the community
• Specimens for culture include bronchial washings, lung
o Epidemic outbreaks – characterized by short
biopsies, pleural fluid, and blood duration and low attack rates
• Sputum is not an optimal choice for culture because of o Nosocomial clusters – occurring in compromised
abundance of Upper Respiratory Tract microbiota patient populations
• Blood could be as specimen choice • Pneumonia is the predominant manifestation of
• Fluoresces under long-wave fluorescent light (Wood’s legionellosis, and the organisms are among the top four
causes of community acquired bacterial pneumonia
lamp) exhibiting a pale-yellow green fluorescence
• Bacterial pneumonia – caused by Streptococcus
• Many patients are diagnosed retrospectively by an pneumoniae
indirect FAT a 4-fold rise in anti-Legionella antibody to a • Atypical pneumonia – caused by Mycoplasma
titer of 128 or greater is considered positive pneumoniae, Chlamydophila pneumoniae, and
Legionella
Culture Characteristics
• Incubation period: 2 to 10 days
• Can be distinguished from other members of the genus by • S/s: non-productive cough, fever, headache, and
its production of the oxidase enzyme and its ability to myalgia, rales, dyspnea, and shaking chills
hydrolyze hippurate • Pulmonary infiltrates develop, sputum may be bloody or
• On Grams stain, the bacteria appear relatively thin (0.5-1.0 purulent
• Infections of the kidneys, liver, heart, central nervous
um in width) rods but they do not stain well with Grams
system (CNS), lymph nodes, spleen, and bone marrow
stain as well as cutaneous abscesses
• Basic fuchsin is often used as a counterstain for 3 • Bacteremia, renal failure, liver function abnormalities,
minutes watery diarrhea, nausea, vomiting, headache,
• Visible colonies usually appear about 3-4 days of confusion, lethargy, and other CNS abnormalities
incubation on an appropriate medium • The most important test is culture of the organism and
• Colonies may be round or flat with entire edges, glistening, should always be attempted even when employing a
rapid test such as urine antigen detection
and convex
Pontiac fever
• They appear to have ground glass speckling like a
• Febrile disease without pulmonary involvement or non-
shattered windshield pneumonic form legionellosis
• Pigmentation can vary from colorless, grayish, pale green • Short incubation period of about 2 days
to indescent pink or blue • S/s: flulike symptoms of fever, headache, and myalgia
• Colonies may be translucent that last 2 to 5 days

Clinical Significance BORDETELLA

• Agent of Legionnaire’s disease most common in males • Small gram-negative bacilli or coccobacilli
over 35 years who have risk factors of smoking, • All are obligate aerobic bacteria
emphysema, and other chronic respiratory conditions • Grow best at 35° to 37° C
• Organisms are acquired through inhalation of aerosols • Do not ferment carbohydrates, oxidize amino acids, are
created by contaminated air conditioners to destruction by relatively inactive in biochemical test systems, and
PMNs produce catalase, although this is variable in B. pertussis.
• Inhibit fusion of phagosomes and lysosomes • Bordetella spp., except B. pertussis, are less fastidious and
• Disease ranges from a mild, short-term febrile illness to an will grow on MAC agar or media containing blood
acute purulent pneumonia with an intra alveolar exudate
Species
Serologic Testing • Fastidious specie:
• Indirect fluorescent antibody (IFA) o B. pertussis – oxidate positive
• Enzyme immunosorbent assay • Six nonfastidious species:
• For IFA, heat- or formalin-killed bacteria are fixed to a o B. holmesii – oxidase negative
microscope slide o B. parapertussis – oxidase negative
• The specificity of the test is enhanced when paired sera o B. trematum – oxidase negative
• A fourfold rise in IFA titer to at least 1 : 128 from the acute o B. avium – oxidase positive
serum phase (obtained within 1 week of onset of o B. bronchiseptica – oxidase positive
symptoms) to the convalescent serum phase (3 to 6 weeks o B. hinzii – oxidase positive
later) • Bordetella pertussis and B. parapertussis are primary
human pathogens of the respiratory tract, causing
whooping cough or pertussis
• B. bronchiseptica and B. avium are respiratory tract
pathogens of wild and domestic birds and mammals
• B. hinzii appears to be an avian commensal

8
• B. bronchiseptica is an opportunistic human pathogen,
causing respiratory and wound infections Colony Morphology
• B. holmesii and B. trematum are respective agents of • Charcoal–horse blood and Regan-Lowe media: young
immune-compromised bacteremia and wound or ear colonies are smooth, glistening, and silver, resembling
infection. mercury droplets
Laboratory Diagnosis • Colonies turn whitish-gray as they age

BORDETELLA PERTUSSIS
Specimen collection and handling • Strict aerobe
• Nasopharyngeal aspirates or swabs (calcium alginate or • coccobacillus- singly or in pairs
Dacron polyester with a flexible Gram negative • Transmission by aerosolized droplets
• Small wire shaft) are the specimen of choice for culture, • Non-invasive
DFA, and PCR testing • Strictly human pathogen (no animal reservoir)
• Should be plated directly onto culture media or transferred • Best culture on media containing charcoal to neutralize
to an appropriate transport system at the bedside inhibitory effects (charcoal will inhibit other commensals)
• Two swabs are collected, one through each of the • Bordet-Gengou (potato-sheep-blood-glycerol) medium
external nares that includes Pen G
• Fresh media or transport systems and swabs are needed • Regan- Lowe is often used as transport medium
• Transported at room temperature • Incubation is 3-7 days at 35°C in a moist enclosure such
• If the transit time is less than 2 hours or DFA is the as sealed plastic bag.
desired test, the swab can be expressed into a solution of
1% casein hydrolysate (casamino acids) broth Virulence Factor
• Overnight or several days transport: use half-strength • Filamentous hemagglutinin (FHA) and pertactin (a 69-
charcoal agar containing 10% horse blood and 40 mg/L kDa outer membrane protein) – facilitate attachment to
cephalexin (Regan-Lowe transport medium). ciliated epithelial cells
• Pertussis toxin (PT) – protein exotoxin that produces a
Nucleic Acid Detection wide variety of responses in vivo
• Detection of Bordetella spp. nucleic acid by PCR from o Main activity: Modification of host proteins by ADP-
nasopharyngeal swabs is a primary rapid diagnostic ribosyl (adenosine diphosphate) transferase, which
strategy interferes with signal transduction
• Should use at least two DNA targets (e.g., IS481 and • B. parapertussis and B. bronchiseptica contain the
pxtS1). structural gene for PT but do not express the complete
operon
Microscopic Examination
• Adenylate cyclase toxin – inhibits host epithelial and
• Small, fat bacilli or coccobacilli with intense peripheral immune effector cells by inducing supraphysiologic
yellow-green fluorescence and darker centers concentrations of cyclic adenosine monophosphate
• DFA test should only be used along with culture because (cAMP)
the lack of sensitivity diminishes the clinical utility of a • Tracheal cytotoxin – contributes to pathogenesis by
negative result, and false positive results can occur even in causing ciliostasis, inhibiting DNA synthesis, and
experienced hands promoting cell death.
• DFA should not be used as a replacement for culture
• Slides for DFA testing may be prepared directly from Clinical Significance
swab specimens or following expression of the material • Humans are only known source
from the swab to a solution of 1% casein hydrolysate • Cause whooping cough or pertussis
• Two slides should be prepared • Most cases occur in children under 5 years of age
• Slides are dried, heat-fixed, and stained on the same • Most death occurs in infants
day of collection or stored at −70° C and heat-fixed • Transmission occurs by inhalation of contaminated
immediately before staining. aerosols from individuals who have the early stage of
disease
Isolation Method
• Catarrhal phase – initial phase
• Charcoal agar supplemented with 10% horse blood and o Symptoms are insidious and nonspecific
40 mg/L cephalexin (Regan-Lowe transport medium) o Include sneezing, mild cough, runny nose, and
• Plates for the recovery of Bordetella spp. should be perhaps conjunctivitis, although infants can develop
incubated at 35° C in ambient air for a minimum of 7 days apnea and/or respiratory distress.
• Most isolates of B. pertussis are detected in 3 to 5 days, o Infection is highly communicable because of the
whereas B. parapertussis is detected a day or so sooner large number of organisms in the respiratory tract.
• Stereomicroscope should be used to detect the colonies o Cultures are not often performed at this stage
before they become visible to the unaided eye. because the symptoms are nonspecific
o May last 1 to 2 weeks
• Paroxysmal phase – second phase

9
 o The hallmark of this phase is the sudden onset of • First isolated by Sir David Bruce on the island of Malta
severe, repetitive coughing followed by the • Based on DNA analyses, there is only one species B.
characteristic “whoop” at the end of the coughing melitensis however bacteria are often still referred by their
spell original species or designations
o Whooping sound is caused by the rapid gasp for air • B. melitensis (goat and sheeps), B. suis (swine), B. abortus
following the prolonged bout of coughing (cattle), B. canis (dogs)
o Coughing spells may occur many times a day and • Causes Brucellosis or also known as Malta fever or
are sometimes followed by vomiting. undulant fever
o Young children may experience apnea and/or • Small gram-negative, non-motile, unencapsulated
pneumonia and require aid in maintaining a patent bacteria that do not form spores (non-spore forming) and
airway may appear as coccobacilli or bacillI
o B. parapertussis generally causes a similar disease • Strictly aerobic that have a complex growth requirement
with milder symptoms • The bacteria are facultative intracellular pathogens that
o Last from 1-6 weeks; may extend to 10 weeks can reside within phagocytic cells.
• Convalescent phase – third phase • They will grow on well-defined media containing amino
o Begins within 4 weeks of onset with a decrease in acids, vitamins, salts, and glucose
frequency and severity of the coughing spells • Grow slowly on sheep’s BAP or CAP with 5-10% CO2
o Recovery is gradual • It stains irregularly and pale with Gram’s stain
o Coughing lessens but fits of coughing may return • Inactive metabolically
o Complete recovery may require weeks or months • Do not produce acid or gas in significant amounts,
o Last about 2-3 weeks • Reduce nitrates to nitrite
o Susceptible to other respiratory infections • Oxidase positive, Catalase positive, Urease positive
Pathogenesis • Brucella spp. can vary in their requirement for CO2 and
production of a positive result for hydrogen sulfide
• Two stage process of disease:
using the lead acetate method.
o Respiratory colonization
o These tests can also differentiate brucellae from
- 7-10 days
similar organisms (e.g., Bordetella bronchiseptica,
- NO symptoms
Acinetobacter spp., and H. influenzae) when
- Positive cultures
combined with growth on SBA, colony morphology,
o Toxin-mediated disease
specimen source, and testing for X and V factor
- Composed of AB Toxin
requirements
- Exotoxins are controlled by central locus
• Infection can be laboratory acquired
• BygAS – two components signal transduction system to
sense the environment and regulate gene expression Brucella Identification
Treatment • Short, coccobacillary forms, bipolar staining sometimes
evident
• Erythromycin
• Colonies appear after 3-5 days of incubation on enriched
• Vaccine
media as small, smooth, convex, transparent, non-
o Killed bacterial cell suspension-DPT vaccine
hemolytic
o Vaccine- Induced immunity after five to ten years
• On subculture colonies tend to become rough because of
• Acellular vaccines
loss of the capsule
Biochemical Identification • They will grow on well-defined media containing AA,
• Biochemical reactions are generally not used for ID vitamins, salts, and glucose
• A saline nasal wash and nasopharyngeal swab are the
Pathogenesis
most common specimens
• Different biovars express different amount of LPS antigens
• DFA and slide agglutination tests are performed to detect
A, M and L
the bacteria in nasopharyngeal aspirate specimens as well
• Biovars and are differentiated in the ability to produce H2S
as colonies on culture
and susceptibility to certain dyes
• DFA must be used in tandem with culture due to its low
• In the animals, Brucella localize in the pregnant uterus
sensitivity
because of the presence of Erythritol in allantoic and
• Polyclonal fluorescent labeled conjugates for both B.
amniotic fluids
pertussis and B. parapertussis
• Abortion is a major manifestation of the disease in animals
• Serologic tests for antibodies are not very useful for rapid
• Bacteria remain viable in dry soil for up to 60 days
diagnosis because agglutination and precipitating
• The bacteria localize in mammary glands of animals can
properties do not appear until the 3rd week of illness.
be shed in milk or cheeses or other products.
BRUCELLA
• Intracellular bacteria that are usually found in animals with
humans being accidental hosts

10
 • P. multocida is the most frequently isolated species and
Clinical Manifestation includes three subspecies and has 3 subspecies:
• Brucellosis, infection with bacteria from the genus multocida, septica and gallcida
Brucella, is an important zoonotic disease found • P. multocida consists of 5-serogroups (A, B, C, D, E)
throughout the world. defined by capsular antigens
• Brucella spp. are considered category B select biological • Colonizes mucous membranes of the upper respiratory
agents by the Centers for Disease Control and Prevention tract and gastrointestinal tracts of mammals and birds
(CDC). • Human infections occur from bites and scratches inflicted
• Should be handled under BSL-3 conditions by animals
• The four species that are most commonly associated with • Most common isolates are Pasteurella multocida
human illness are B. melitensis, B. abortus, B. suis, and B.
Clinical Manifestation
canis.
• Two other species are B. ovis and B. neotomae; in recent • Systemic, pneumonic, cutaneous form of infections
years, additional species have been isolated from marine • Localized infection after a bite or scratch
mammals. • Respiratory tract infection
• Brucellosis has been described by both the disease • Life-threatening systemic diseases (e.g., meningitis,
course (undulant fever) and geographic locations where bacteremia)
cases have occurred (Mediterranean, Crimean, and Malta • Colonizes mucous membranes of the URT and GIT of
fevers). mammals and birds
• Brucellosis is acquired through aerosol, percutaneous, • Human infections occur from bites and scratches inflicted
and oral routes of exposure. by animals
• It can be difficult to diagnose brucellosis through direct • Most common isolated species is Pasteurella multocida
examination of a clinical sample, most often blood or bone Culture Characteristics
marrow, and the ability for direct isolation and culture can
• Growth on 5% blood or chocolate shows small, smooth,
vary between acute and chronic manifestations.
convex colonies
• 3 clinical stages of Brucellosis: acute, sub-chronic and
• “Musty” odor
chronic
• No growth on McConkey agar
o Symptoms of acute infection are non-specific (fever,
• Oxidase positive
malaise, headache, anorexia, myalgia, and back pain)
• Catalase positive
and usually occur within 8 weeks of exposure.
• Ferment glucose with weak to moderate acid
Brucellosis is a systemic, deep-seated disease
production without gas.
resulting in various long-term sequelae.
• In TSIA, a weak glucose fermentation reaction appears.
o Sub chronic or undulant form appear after a year of
exposure with undulating fevers (characterized by • All Pasteurella spp. grow on SBA and CHOC agar,
normal temperatures in the morning followed by high producing grayish colonies
temperatures in the afternoon and evening), arthritis, • Conversely, MAC agar does not support the growth of
and epididymoorchitis (inflammation of the most Pasteurella spp.
epididymis and testis) in males. • Growth on SBA in the absence of satellitism or in pure
o Chronic form presents after 1 year of exposure with culture combined with bipolar staining may differentiate
symptoms such as depression, arthritis and chronic Pasteurella from Haemophilus.
fatigue • P. multocida produces non-hemolytic colonies on SBA
• Farmers, vets and abattoir or slaughterhouse workers that may appear mucoid after 24 hours of incubation at
are at greatest risks for infection 37° C followed by the production of a narrow green-to
• Incubation period ranges from 1-6 weeks brown halo around the colony after 48 hours.
• Onset is slow and insidious and disseminated via the Microscopic Examination
lymphatics and the blood stream
• Very small gram-negative rods
• Proliferation of mononuclear cells is a major histologic
• Bipolar staining with Giemsa or Methylene blue
finding
• “Safety-pin” appearance when the poles of the cells are
• Fever may have a daily, periodicity, rising in the
more intensely stained
afternoon and falling at night, malaise, weakness and
non-specific aches and pains PASTEURELLA BETTYAE
• Immunity is good after recovery although reinfection is • Has been isolated from placenta, amniotic fluid, blood,
possible rectal sites, abscesses, and urogenital specimens
• Isolates are fastidious, capnophilic coccobacilli
PASTEURELLA
• Facultative anaerobe, non-motile
• 17 species have been identified based on DNA
• Ferment glucose and fructose
hybridization
• Catalase positive, indole positive and oxidase variable
• P. canis is associated with dogs
• They may grow on McConkey
• P. stomatis and P. dogmatis are associated with dogs and
cats and also isolated from human

11
 • Isolates are catalase positive, negative for X and V
HACEK GROUP
factors
(Haemophilus, Aggregatibacter, Cardiobacterium, • Do not grow in McConkey, oxidase variable
Eikenella, Kingella) and Capnocytophaga
• Urease negative, indole negative, esculin negative and
• HACEK is an acronym of the first initial of each genus that citrate negative, oxidase variable
belong to the group • Ferments glucose but not lactose or sucrose
• Haemophilus spp. Aggregatibacter aphrophilus and
paraphrophilus Virulence Factor
• Aggregatibacter actinomycetemcomitans formerly • Composed of 6 serotypes (A, B, C, D, E, F) based on its
Actinobacillus actinomycetemcomitans surface polysaccharides, of which A, B, and C are the
• Cardiobacterium hominis most common
• Eikenella corrodens • Include collagenase, leukotoxin that is toxic to
• Kingella spp. polymorphonuclear cells and monocytes
• Capnocytophaga spp.
Clinical Significance
o Has similar requirements as the HACEK group
• Normal oral microbiota of humans
• HACEK is an old term, but it’s now called AACEK
• Human tissue infections attributed to cattle, sheep, pig,
General Characteristics and horse bites
• Gram-negative bacilli • Has been isolated from blood, lung tissue, abscesses of
• Capnophilic – require an increased CO2 (5%-10%) the mouth and brain and sinuses
environment • Causes SBE and periodontitis
• The latter four members of the HACEK group are
Antibacterial Susceptibility
considered to be more dysgonic (slower or poorer
growing) – in contrast to eugonic • P. aminoglycosides, 3rd gen
• Include both fermentative and non-fermentative gram- • Cephalosphorins, quinolones, C and Te sensitive
negative bacilli • Resistance to ampicillin, vancomycin and erythromycin is
common
Clinical Significance • Usual treatment for endocarditis is with penicillin and an
• Significant cause of endocarditis (infective and bacterial) aminoglycoside.
o Involves the heart valves; the lesion (referred to as
CARDIOBACTERIUM HOMINIS
vegetation) is composed of fibrin, platelets,
polymorphonuclear cells, monocytes, and • A pleomorphic, non-motile, fastidious gram-negative
microorganism bacilli
o Include tooth extraction, history of endocarditis, • Gram stains of the bacilli often show false gram-positive
gingival surgery, mitral valve prolapse reactions in parts of the cell
• Usual normal flora of the oral cavity, allowing for their • On Gram stain, organisms tend to form rosette swellings,
introduction in the bloodstream and resultant infections. long filaments, or stick-like structures in yeast extract
• All organisms are opportunistic pathogen in • Grow slowly on SBA and CHOC agar but not at all on
immunocompromised hosts McConkey agar
• They can create the so-called condition as SBE (sub- • Incubation in a humid atmosphere with 5% CO2 enhances
acute bacterial endocarditis) growth
• Fermenter when serum is added
AGGREGATIBACTER APHROPHILUS • Ferments glucose, mannitol, sucrose, and maltose
• Aphros “foam loving” or needing high concentration of • Isolates are oxidase positive, catalase negative and
CO2 indole positive
• Found in dental plaque and gingival scrapings • Negative for urease, nitrate, gelatin and esculin
• Most prevalent cause of endocarditis
Clinical Significance
• Clinical features of infections: fever, heart murmur, CHF,
• Normal microbiota of the nose, mouth, and throat
and embolism
• May be present in the gastrointestinal tract
• Colonies are convex, granular and yellow with opaque
zone near the center on CAP • Usual manifestation is endocarditis often presenting with
large vegetations and no demonstrable fever
AGGREGATIBACTER • Infects the aortic valve
ACTINOMYCETEMCOMITANS • Associated with meningitis
• Formerly Actinobacillus
• 6 species have been recovered from humans Antibacterial Susceptibility
• Produce small bacilli to coccoid gram-negative bacilli • Sensitivity can be seen to β-lactams, chloramphenicol,
that are non-motile and tetracycline with variable response to
• Fermenter when serum is added to the carbohydrate aminoglycosides, erythromycin, clindamycin, and
• Growth is star-shape with 4-6 points at the center of the vancomycin.
colonies after 48 hours • Usual therapy includes Penicillin and aminoglycosides

12
 • Catalase negative, superoxol negative, urease negative,
EIKENELLA CORRODENS
indole negative, esculin negative, gelatin negative, citrate
• Fastidious, gram-negative coccobacilli
negative
• Non-motile and assacharolytic (very similar to the
Moraxella spp.) KINGELLA KINGAE
• Corrodes (pits) the surface of agar • Recognized as an important pathogen in pediatric
• Non-hemolytic but may show greening around the population
colonies on SBA • Weakly fragments glucose and sucrose but not maltose
• A bleach-like odor from the agar surface may be obvious • May produce a yellow pigment
• Isolates do not grow on grow on EMB and Mc • Has two types of colonies: spreading or corroding or a
• In broths, may adhere to the sides of the tubes and smooth, convex β-hemolytic colonies
produce granules • Gram stains showing plump rods in chains
• Oxidase positive, catalase negative and often produce a • Isolates have been obtained clinically from blood, bone,
yellow pigment joint fluid, urine and wounds
• Lysine and ornithine decarboxylase positive • Major green negative bacterium isolated from
• Arginine dihydrolase negative degenerative joint and bone infection in children <3
years
Clinical Significance • Causes endocarditis in adults and school-age children
• Normal microbiota of the oral and bowel cavities • Most isolates are susceptible to most antibiotics
• Human bites or fights infection
CAPNOCYTOPHAGA
• Associated with poor dental hygiene or oral surgery
• Belongs to the the family Flavobacteriaceae and includes
• Reported as a cause of meningitis, empyema,
dysgonic fermenter called DF-1 and DF-2
pneumonia, osteomyelitis, arthritis, and post-op
• Genus consists of 7 species
infections
• Fastidious, facultatively anaerobic, gram-negative
• Shows least predilection for attachment to heart valves
bacteria
among HACEK
• Thin, and often fusiform (pointed ends) resembling
Antibacterial Susceptibility Fusobacterium spp.
• Resistant to Clindamycin and aminoglycosides and • Spindle-shaped, coccoid, and curved filaments may be
narrow spectrum cephalosporins also seen
• In vitro, isolates demonstrate sensitivity to penicillin, • Flagella are absent but produce gliding mostly on solid
ampicillin, cefoxitin, chloramphenicol, carbenicillin, surfaces
and imipenem • Colonies are adherent and produce a yellow-orange
pigment mostly are non-hemolytic
KINGELLA
• Ferments glucose, sucrose, lactose, maltose although
• 3 species: K. kingae, K. denitrificans, and K. oralis
TSI is negative
• Coccobacillary to short bacilli with squared ends that
• Negative for most biochemical reactions
occur in pairs or short chains
• Reduce nitrates and hydrolyzes esculin
• Tend to resist decolorization in Gram stain
• Oxidase negative, catalase negative, indole negative
• May grow in MTM, resemble colonies of Neisseria when
they do not pit agars which many usually do Clinical Significance
• No growth on McConkey • 5 of its species are normal microbiota of the oral cavity
• Non-motile, oxidase positive, catalase negative, • Not commonly involved in endocarditis but is associated in
fermenters of glucose with no gas septicemia with patients with neutropenia
• Colonize the upper respiratory tract, especially the • Most are isolated from dental plague
tonsils • C. ochracea is the most common clinical isolate
• Associated with poor dental hygiene or oral surgery • C. canimorsus can cause a fulminant, life-threatening
• Very important in pediatric patients, it can create infection in humans following a dog or cat bite
diseases that will present as bone diseases, septicemia Antibacterial Susceptibility
KINGELLA DENITRIFICANS • Susceptible to imipenem, erythromycin, clindamycin,
• Has two types of colonies: smooth, convex type and a tetracycline, chloramphenicol, quinolones, and β-lactams
spreading corroding type • Resistant to the aminoglycosides
• Rarely isolated but has been associated with bacteremia • Penicillin is the drug of choice
and abscess
• Unlike Neisseria gonorrhoeae, K. denitrificans is both
catalase and superoxol negative.
• Positive for glucose fermentation and nitrate reduction
• Does not grow on MAC

13