Introduction to Microbiology 1 ➢ Selman Waksman, Ukraine

(1944) –
Microbiology discovered streptomycin
• Study of living microorganisms including viruses - Problems
(infectious particles, acellular) ➢ Toxicity of drugs => Selective toxicity
➢ Resistance of bacteria to drugs.
• Broad discipline. Includes Bacteriology,
- Recombinant DNA Technology
Virology, Mycology, Parasitology etc.
➢ Recombinant DNA
• Micro – small, bios – life, logos – study
• Microbiology can be classified into the pure ➢ Genetic engineering/ biotechnology
science and the applied sciences. ➢ Microbial genetics – mechanism by
• Pure Science – Bacteriology, Virology, Mycology, which microbes inherit genes
Parasitology ➢ Molecular biology – structure and
function (expression) of genes
Importance of Microbiology ➢ Molecular epidemiology/ diagnostics
• Microbes play a major role in recycling essential - Branches of Applied Microbiology
elements. (Microbial Ecology - balance of ➢ Agricultural Microbiology - ecology
environment) – nitrogen fixation ➢ Environmental Microbiology
• Benefit society by their production of food, ➢ Medical and Clinical Microbiology
beverages, antibiotics and vitamins ➢ Genetic Engineering
• Causative agents of some important diseases ➢ Paleomicrobiology – ancient microbes
➢ Sanitary Microbiology – waste water sys
• Infectious diseases are a leading health-related
➢ Veterinary Microbiology - animals
issue, especially in a society where the elderly
➢ Biotechnology – recombinant DNA
population is increasing.
technology
• New infectious diseases continue to emerge and
be identified all the time.
Microbial World
• Microbiology impacts every facet of daily life.
• Microbe - general term for all living. small
• Bioremediation: use of microbes to remove
microorganisms
toxins (oil spills)
- Viruses
• Use of microbes to control crop pests (i.e
- Bacteria (Eubacteria) and (Archaebacteia)
Bacillus thuringiensis; gram positive)
- Fungi (Yeast and Molds)
• Normal microbiota (i.e skin: - Protozoa
Staphylococcus epidermidis; gram - Microscopic Algae
positive)
Viruses
Branches of Microbiology
• Smallest known infectious agents
• Bacteriology – study of bacteria
• Subcellular microorganism
• Mycology – study of fungi - Contains only nucleic acid (DNA or RNA,
• Immunology – study of immunity never both) surrounded by a protein
- Edward Jenner, UK – developed coat
vaccination (1978) - Corona Virus (RNA), Poxvirus (DNA)
- Metchnikoff, RU – discovered phagocytes - Called particles outside the body since
(1884) they must live and grow in living cells of
- Paul Ehrlich, DE – theory of immunity other organisms
(1890) - Lifeless particles that become living
• Virology – study of viruses when gained access to a cell inside the
- Beijerinck, ME – discovered intracellular body
reproduction of TMV or Tobacco Mosaic - Agents of colds, influenza, hepatitis,
Virus; coined the term “virus” (1899) warts, AIDS, herpes, MMR (measles,
• Parasitology – study of protozoa and parasitic mumps, rubella)
worms - Vaccines are mostly available to common
• Applied Branches – Chemotheraphy, Molecular viruses
Biology, etc.
- Chemotherapy Protozoans
➢ Treatment of disease by using chemical • Single-celled eukaryotic organisms,
means larger than bacteria
➢ Antibiotics produced naturally • Can be classified as amoeba, flagellates,
➢ Synthetic drugs and complex science
➢ Paul Ehrlich (1878) – used • Found in soil and water (i.e T. vaginalis;
arsenic compounds to fight disease- flagellate: zoomastigophora)
‘magic bullet’ • Illnesses
➢ Alexander Fleming, Scotland (1928) - Malaria
– discovered penicillin - Amoebic dysentery
- T. vaginalis
• Leading cause of death in developing countries
Fungi ➢ Aerobes – live with the presence of air
• Eukaryotic organisms with rigid cell wall; chitin ➢ Anaerobes – incapable to live with the
• Fungi are the causative agent of superficial presence of air
infections or skin infections. Like, ring worms, ➢ Anaerobic gram positive cocci – color
rushes, buni, an-an brought about by the violet, circle and nabubuhay ng walang
dermatophyte’s w/c are basically fungi. oxygen
➢ Aerobic gram positive cocci – color
• Yeasts violet, cirle, at kailangan may oxygen
- Single-celled ➢ Facultative – can live with oxygen or
- Reproduce by budding none
• Molds ➢ Obligate anaerobes – strictly no
- Large, fuzzy, multicellular organisms oxygen/air present
- Produce spores ➢ Obligate aerobes – strictly requires air
• Superficial Infections to grow.
- Athlete’s foot ➢ Microaerophilic – it requires oxygen to
- Ringworm (Buni) survive, but requires environments
containing lower levels of oxygen (5 –
- Thrush 10% ONLY).
• Can cause systemic infections such as - Biochemical reactions
Histoplasma capsulatum ➢ Lactose – if bacteria is able to ferment
the sugar it contains Lactose Fermenting
Bacteria if not, it is called Non-Lactose
Multicellular Parasites
Ferment
• Organisms that live on or in another organism
➢ Use of chemicals to pertain their ability
and use it for nourishment
on how they would respond when
• Parasitic worms
exposed to certain chemicals
- Usually due to poor sanitation
- Roundworms
- Flatworms
- Tapeworms
• Parasitic insects
- Bite or burrow under the skin
- Mosquitoes
- Ticks
- Lice
- Mites

Bacteria
• Single-celled prokaryotic organisms
• Reproduce rapidly (in a matter of 8 hours)
• Classification
- Shape
➢ Cocci – circular
➢ Bacilli – rod, elongated
➢ Spiral – spiral
2 types of spiral:
• Spirochetes
o has 3 genera: Treponema,
Borrelia, Leptospira
o have flexible bodies
o they have endoflagella aka axial
filament
• Spyrillum
o genus itself
o have rigid bodies
o they have exoflagella – sa labas

- Ability to retain dyes

➢ Bacteria are colorless Steps in Gram Staining
➢ Gram Staining – Gram positive (violet 1. Crystal violet (purple) – gram-positive
or blue), Gram negative (red)
o Safranin (red) 2. Grams Iodine (Mordant) – an agent that fixes the
o Crystal Violet (deep purple & crystal violet to the bacterial cell wall
bluish)
- Ability to grow with/without air
3. Decolorization – rinse the sample/slide with ➢ Prokaryotic
acetone 50% & alcohol 50% for 3 seconds. The alcohol ➢ Single, circular
will decolorize the sample if it is Gram negative, ➢ 70s but structure is similar to 80s
removing the crystal violet. ➢ Has ribosomal RNA
➢ Number of sequences shared with
4. Counter stain – to give decolorized gram-negative eukarya (3)
bacteria pink/red color (Safranin) for easier - Eukarya
identification ➢ All other living organisms (plants,
animals, and humans); have a
Classification of Microorganisms nucleus & organelles
➢ Eukaryotic
Classification of Microorganisms ➢ Several, linear
• Taxonomy ➢ 80s
- Organizing, classifying and naming of living ➢ Has ribosomal RNA
things ➢ Number of sequences
- Formal system originated by Carl von Linne shared with eukarya(all)
1701- 1778) - Example:
- Identifying and classifying organisms ➢ Kingdom – Prokaryotae
according to specific criteria ➢ Phylum – Gracilicutes
- Each organisms placed into a classification ➢ Class – Scotobacteria
system ➢ Order – Eubacteria
- Science of classification, identification, ➢ Family – Enterobacteriaceae
nomenclature, and making a system ➢ Genus – Escherichia
Classification – arrangement of ➢ Species – coli
bacteria into groups (the same
organisms can be classified
differently according to the view:
serotype classification,
antimicrobial resistance
classification, chemical reaction,
etc.)
o Placing organisms in a group of
related species
o Lists of characteristics of known
organisms
➢ Nomenclature – naming, means of
communicating; it is binomial (two
names – genus, species)
➢ Taxonomy - science of
classification, identification,
nomenclature and making a system
➢ Identification – practical use of
classification criteria to distinguish
certain organism from others
o Matching characteristics of an
“unknown” Eukaryotes
to lists of known organisms • 4 main Kingdoms:
- Protista (amoeba)
Taxonomy ➢ Complex single cell, some multicellular
• 3 Domains ➢ Absorb, photosynthesize, or ingest food
- Ribosomal RNA basis ➢ Paramecium, euglenoid, slime
- Eubacteria mold, dinoflagellate
➢ True bacteria; presence of ➢ Protozoans, algae, water molds, slime
peptidoglycan in the cell wall molds
➢ Causes a lot of diseases ➢ Grouped into clades based on rRNA
➢ Prokaryotic - Fungi
➢ Single, or few circular ➢ Some unicellular, most multicellular
➢ 70s filamentous forms with specialized
➢ Has ribosomal RNA complex cells
➢ Number of sequences shared with ➢ Absorb food
eukarya (1) ➢ Black bread mold, yeast, mushroom,
- Archaea bracket fungus
➢ Ancient (odd) bacteria; ➢ Molds, yeast and mushrooms
Extremophiles (extreme ➢ Chemoheterotrophic, cell walls of
environments, high salt, heat, etc.) chitin, develop from spores or hyphal
 fragments ribosomal RNA + + +
- Plantae signature
➢ Multicellular form with specialized sequences
complex cells Number of
sequences One Three (All)
➢ Photosynthesize food shared with
➢ Moss, fern, pine tree, nonwoody Eukarya
flowering plant Protein synthesis
➢ Mosses, ferns, nonwoody and similar - +
woody flowering plants to
➢ Cellulose cell walls Eukarya
➢ Photoautotrophic Presence of
- Algae peptidoglycan + - -
➢ Multicellular form with specialized in
cell wall
complex cells Long-chain,
➢ Ingest food Fatty branched Fatty
➢ Sea star, earthworm, finch, raccoon Cell membrane
acids hydrocarbons acids
➢ Invertebrates, fishes, reptiles, lipids
with with ether with
amphibians, birds, and mammals ester linkages ester
➢ No cell walls linkages linkages
➢ Chemoheterotrophic Sterols in - (some
- +
membrane exception)
Classification Systems in a Procaryotae
IMPORTANT DIFFERENCES
BETWEEN EUCARYOTIC AND
Prokaryotic
PROCARYOTIC CELLS
CHARACTE • Genetic material - Situated in the nucleoid
EUCARYOTIC PROCARYOTIC
RISTICS origin called nucleoid bridge
Example Animals and Plants Bacteria and Archaea
Eukaryotic
Cell Size 10-100 um in diameter 0.2-2 um in diameter
Nucleus Present Absent • Genetic material – confined in a place called
Present: Lysosomes, Golgi nucleus
Membran complex, Endoplasmic
Absent
e Reticulum, Mitochondria, Being a prokaryotic, nanggaling ang bacteria or lahat
Organelle and Chloroplasts
s
ng living organisms ay nanggaling sa mga DOMAINS.
Larger (80S) in cell; 70S
Ribosomes
in
Smaller (70S) Ang basis ng domains is because of the ribosomal RNA
organelles na meron content lahat ng content ng living
Multiple linear organism. But, they are classified into 3 domains on
DNA Single circular
chromosomes (histones)
chromosome that similarity as EUBACTERIA, ARCHAEANS and the
Cell
Division
Mitosis Binary fission EUCHARIANS.
Only in plant cells and Usually present;
Cell Wall Bacterial Morphology, Structure and
fungi (chemically simpler) Chemically
complex Classification
Cytoskelet
Present Present
on • Morphology
Plasma - Bacterial structure:
membran
Yes Usually No ➢ Capsule
e with ➢ Cytoplasm
steroid
➢ Ribosomes
Complex; composed of Composed of two ➢ Cell Wall
Flagella
multiple microtubules protein building
blocks ➢ Plasma Membrane
➢ Nuclear area (Nucleoid) containing DNA
➢ Plasmid
COMPARISON OF THREE CELLULAR DOMAINS ➢ Flagella
CHARACTERISTI BACTERIA ARCHAEA EUKARYA ➢ Inclusion
C
➢ Fimbriae
Cell Type Procaryoti Procaryotic Eucaryotic
c - Size of Bacteria
Single, or ➢ Unit of measurement: micron or
Single Several micrometer (um)
Chromosomes few,
, ,
circula
circula linear
➢ Cocci – sphere, 1um, non-motile
r ➢ Bacilli – rod-shaped, 0.5-1um in
r
70S but width; up to 3 um in length
Types of ➢ Spiral bacteria – 1-3 um in length
70S structure is 80S
Ribosomes
similar to and 0.3-0.6 um in width
80S • Cocci (circular)
Contains unique - Diplococci
 ➢ Two coccus • Many are surrounded by a sticky, protective
➢ Cocci that remain in pairs after dividing coating of sugars called the capsule or
➢ Neisseria gonnorhoeae (gram -) glycocalyx
➢ Streptococcus pneumoniae (gram +) • One circular chromosome and some small DNA
- Streptococci called plasmids
➢ Cocci that remain in chains after dividing • May have short, hairlike projections called pili on
➢ Streptococcus pyogenes (gram +) cell wall to attach to host or another bacteria
- Tetrads when transferring genetic material
➢ Cocci that divide in two planes and • Some can move by flagella, gliding over slime
remain in groups of four they secrete (e.g. Myxobacteria)
➢ Staphylococcus spp. • Some can form protective endospores around
- Sarcinae the DNA when conditions become
➢ Cocci that divide in three planes and unfavorable
remain in groups/ in cube-like groups
- Staphylococci Structures of Bacteria
➢ Cocci that divide in multiple planes and • Essential
form grape-like clusters - Cell Wall
➢ Staphylococcus aureus (gram +) ➢ Gram positive bacteria have more
prominent yet simple cell wall
• Bacilli (elongated) ➢ Gram negative bacteria have less
- Bacillus prominent yet complex components
➢ Single rod in their cell wall
➢ Klebsiella pneumoniae (gram -; - Cell Membrane
sometimes a diplobacilli) - Cytoplasm
➢ Corynebacterium diphtheriae (gram +; - Nuclear Material
club- shaped) • Particular
➢ Clostridium botolinum (gram +) - Glycocalyx – avoidance of phagocytosis
- Diplobacilli ➢ Capsule – sugar coating is firmly
➢ Appear in pairs after division attached to the bacterium
➢ Attached to each other ➢ Slimy layer – loosely attached
- Streptobacilli - Flagella – used for movement/propulsion
➢ Appear in chains after division - Pili
➢ Bacillus cereus (gram +) - Endospore – located inside the cell;
- Coccobacillus once the cell dies, the endospores
➢ Fat and short remain
• Spiral bacteria ➢ This spore can regenerate to
- Vibrio become a new bacteria again
➢ Curved spirals (coma-shaped) (sporulation)
➢ Vibrio cholerae (gram -) ➢ Survival mechanism
- Spirillum
➢ Helical shape and fairly rigid bodies
- Spirochete STRUCTURE FUNCTION
➢ Have flexible bodies Cell Wall Protects the cell and
➢ Has flagella/endoflagella (axial gives shape
filament)
Outer Membrane Protects the cell
➢ Treponema pallidum (gram -)
against some
• Other Bacterial Shapes antibiotics
- Star-shaped bacteria (only
➢ Stella present in Gram – cells)
➢ Most are Archaean Cell Membrane Regulates the movement
- Rectangular bacteria of materials into and out
➢ Haloarcula of the cell; enzymes of
➢ Halophylic bacteria respiration
➢ Most are Archaean Cytoplasm Contains DNA,
ribosomes, and organic
compounds
Structural View of Bacteria
Chromosome Carries genetic
• Microscopic prokaryotes (no nucleus nor
information inherited
membrane- bound organelles)
from past generations
• Contain ribosomes
Plasmid Contains some genes
• Enfolding of the cell membrane carry
obtain through
on photosynthesis & respiration
gene
• Surrounded by protective cell wall tic recombination;
containing peptidoglycan (protein- bacteria
carbohydrate)
 can live without this
structure

Capsule and slime layer Protects the cell

(immune
attack) and assist i
n
attaching the cell to
other surfaces
Endospore Protects the cell
against harsh
environment
al
conditions (heat
or drought)
Pilus (Pili) Attaching to other
surfaces (for genetic
recombination)
Flagellum Moves the cell

Gram positive Gram negative

Outer cell layer Peptidoglycan Outer membrane
layer is thicker; (endotoxin –
no outer lipid A), cell
membrane wall, cell
membrane
# of layers 1 2
Cell wall Membrane Porin
structures lipoteichoic (‘siev
aci e’ protein), lipo-
d, cell wall polysaccharide,
teichoic periplasmic space
acid
Flagella Basal body have 3 Basal body have 4
rings rings
 - May mga bacteria na magiging colorless in
the process because naalis yung crystal violet
and some ma rretain.
- Critical step in gram staining and will divide
largely organism into two.
- Some will be violet and some will be
colorless.
4. Counter-Stain
- Second dye aka safranin (red dye)
- Pag nilagay ang safranin sa colorless, sila
ngayon ay magkukulay pula.
- Wash it with water, then dry. Under the
microscope, you will now distinguish two
colors of bacteria: violet and red.
- Gram Positive = violet
- Gram negative = red
- Pwede naten iclassify yung bacteria using
gram staining reaction.

BACTERIAL MORPHOLOGY, STRUCTURE AND

CLASSIFICATION

Sugar coat that is firmly attached capsule in bacteria–

glycocalyx
THE 4 STEPS
Organ for adhesion – fimbriae
There are 2 dyes ang ginagamit sa gram staining
Storage area - inclusion bodies (example:
1. Crystal Violet metachromatic granules)
- Ang bacteria is colourless. Ginagamit ang
crystal violet para makulayan ang bacteria. Plasmid – circular bodies in bacteria that merong extra
genes that give the bacteria advantage to resist
- First stain na inapply ni Hans Christian Gram
2. Mordant or aka Grams Iodine antibiotics.
- Second stain
- Eto yung mag aattach sa crystal violet firmly
doon sa bacterial cell.
- Parang glue
- Chemical Notation: CV + I

Development

3. Decolorize yung slide mo using ACETONE

ALCOHOL
- 50% acetone, 50% alcohol
- May mga bacteria na maalis ang crystal
violet, may mga bacteria naman na
marretain ang crystal violet kahit na
dinecolorized mo na.
SIZE OF BACTERIA:
Essential structures of bacteria function is to protect.
They are aka THE NEEDS of the bacteria.
Particular structures of Bacteria are aka THE WANTS of grown in the presence of excess
the bacteria. nutrients and they are often observed
under laboratory conditions.
Wall-less Forms of Bacteria (Spheroplast)
• When bacteria are treated with: Structures External to the Cell Wall
- Enzymes that are lytic for the cell wall e.g. • Capsules and Slime Layers
lysozyme - Attachment
- Antibiotics that interfere with biosynthesis - Protection from phagocytic engulfment
of peptidoglycan, wall-less bacteria are - Resistance to drying
often produced. - Depot for waste products
➢ e.g. Penicillin – inhibits the cell wall - Reservoir for certain nutrients
synthesis (production of peptidoglycan) - Protection
• Periplasmic space
- Separates the peptidoglycan layer and
the cell membrane
• Capsules
− There are 2 types of capsule: glycocalyx
capsule (firmly attached sugar coat) and
the slime layer (loosely attached sugar
Structures Internal to the Cell Wall coat)
• Cell Membrane - They usually consist of polysaccharide;
- Site of biosynthesis of DNA, cell wall polymers however, in certain bacilli they are
and membrane lipids. Selective composed of a polypeptide
permeability and transport of solutes into (polyglutamic acid).
cells. - They are not essential to cell viability
- Electron transport and oxidative and some strains within a species will
phosphorylation produce a capsule, while others do not.
- Excretion of hydrolytic enzymes - Capsules are often lost during in vitro culture
• Cytoplasm • Pili
- Composed of largely water, together with - Pili are hair-like projections of the cell.
proteins, nucleic acid, lipids and small They are known to be receptors for
amount of sugars and salts. certain bacterial viruses. Chemical
- Ribosomes – numerous, 15-20nm in nature is pilin.
diameter with 70S; distributed throughout - Classification and function:
the cytoplasm; sensitive to streptomycin and ➢ Common pili or fimbriae – fine,
erythromycin site of protein synthesis. rigid, numerous, related to bacterial
- Plasmids – extrachromosomal genetic adhesion.
elements ➢ Sex pili – longer and coarser, only 1-
- Inclusions – sources of stored energy, e.g. 4, related to bacterial conjugation.
volutin • Flagella
• Mesosomes - The diameter is thin, 20 nm, and long
- Mesosomes are specialized structures with some having a length 10 times the
formed by convoluted invaginations of diameter of cell.
cytoplasmic membrane, and divided into - Due to their small diameter, flagella
septal and lateral mesosome. cannot be seen in the light microscope
- Produced by the folding of plasma membrane unless a special stain is applied.
• Nucleus - Bacteria can have one or more flagella
- Lacking nuclear membrane, absence of arranged in clumps or spread all over the
nucleoli, hence known as nucleic material or cell.
nucleoid, one to several per bacterium. - Types of Flagella:
• Plasmids ➢ Monotrichous (polar) – flagellum
- Plasmids are small, (singular)
circular/line, extrachromosomal, double- ➢ Lophotrichous – flagella on one
stranded DNA molecules. They are capable of pole of the bacteria
self-replication and contain genes that confer ➢ Peritrichous – flagella surrounding
some properties such as antibiotic the bacteria
resistance, virulence factors. ➢ Amphitrichous – flagella on both poles
- Plasmids are not essential for cellular
survival.
• Inclusions of bacteria
- Inclusions are aggregate of various
compounds that are normally involved
in storing energy reserves or building
blocks for the cell.
- Inclusions accumulate when a cell is
 Spores- circular body meant for survival.

Endospores: only Bacillus and Clostridium.

• Axial Filaments
- The rotation of these filaments causes a
corkscrewing motion by a spirochete
which helps propel it through thick
environments.
- Certain spiral-shaped bacteria, such as
spirochete Treponema pallidum, which
causes syphilis, have bundles of fibrils
that spiral the cell.
• Fimbriae
- Shorter, straighter, and thinner than flagella
- Not used for motility, but rather for
attachment to surfaces (i.e. gonorrheal
bacteria and mucous membranes).
• Endospores (Spores)
- Endospores are resting structures formed by
some bacteria for survival during adverse
environmental conditions,
- The process of endospore formation is
called sporulation; the return of an
endospore to its vegetative state is called
germination.
- Endospore provides resistance against
drying, low nutrient conditions, radiation,
high temp., organic solvents and other
chemical disinfectants.
- Built to last

DNA, RNA, Proteins,

Core
SASPs, DPA, Ca2+
Inner Membrane Lipid/ Protein
Germ cell wall Peptidoglycan
Cortex Modified peptidoglycan
Outer Membrane Lipid/ Protein
Inner space coats Proteins
Outer space coats Proteins
Exosporium Proteins
Bacteria can create either a run (counter clockwise
motion, forward motility) or a stumble (clockwise
motion, backward motility)

Amoeba – sluggish, forward movement.

BACTE LEC

• Lucretius, a Roman Philosopher (98-55

B.C.) and Girolamo Fracastoro, a
physician (1478-1553) believed invisible
creatures were responsible for disease.
• Francesco Stelluti observed bees and
weevils using a microscope in the early
1600s.
SPONTANEOUS GENERATION DEBATE (historical
perspectives) THEORY OF SG DISPROVED BY:

Supported by: • Francesco Redi (1626-1697) – maggot

unable to grow on meat if meat was
• Aristotle (384-322 BC) covered with gauze.
• John Needham (1713-1781) • Schwann, Friedrich Schroder and Von
• Lazaro Spallanzi (1729-1799) Dusch (1830’s) – air allowed to enter flask
• Felix Pouchet (1859) but only after passing through a heated
tube or sterile wool.
ARISTOTLE • John Tyndall (1820-1893) omission of dust
- Spontaneous generation debate – no growth. Demonstrated heat resistant
introduced by Aristotle who lived around forms of bacteria (endospores)
350BC FRANCESCO REDI (1626-1697)
- According to Aristotle “readily observable
that aphids arise from the dew which - First to formally challenge the accepted
falls on plants, fleas from putrid matter, belief of spontaneous generation.
mice from dirty hay” - Italian physician, a naturalist and poet
- This belief remained unchallenged for - Experimented on flies
more than 2000 years, UNTIL… - Redi put meat into 3 separate jars
ROBERT HOOKE (1635-1703)

- Observed living plant tissues as “little

boxes” or cells (1665)
- Used simple magnifying lens
- Suggested that all living things are made
of cells
- Cell theory

JOHN T. NEEDHAM & LAZZARO SPALLANZANI ANTON VAN LEEUWENHOEK (1632-1723)

Question: What causes tiny living things to appear in - A haberdasher/cloth’s man that uses
decaying broth? lenses to examine cloth
- He assembled hundred of microscopes
- Needham’s hypothesis: Theory of - “animalcules”
spontaneous generation - See first protozoans and called it as
- Spallanzani’s hypothesis: Microbes comes animalcules
from the air. Boiling will kill them. - First protozoologist

LOUIS PASTEUR (1822-1895)

 - “When I approach child, he inspires me in JOHN TYNDALL (1820-1893)
two sentiments; tenderness for what he
is and respect for what he may become.” - Discovered that some bacteria existed in
- French chemist two forms:
- His design of experiment settled the • Heat stable form (endospore)
argument on spontaneous generation • Heat sensitive form (vegetative
- Proved that microorganisms are present cell)
in non-living matter - Need prolonged or intermittent heating
- Microbes can be destroyed by heat to destroy the heat stable endospores
- Fermentation mediated by yeast, not air - His research resulted in a method of
- Pasteurization should be done to prevent sterilizing liquid by heating it to boiling
wine and beer spoilage by bacteria point on successive days
- Pasteur proposed that wine spoiling in an ROBERT KOCH (1843-1910)
analogy for disease (bacterial growth
made the wine “sick”) - Experimented with medium to grow on
- He hypothesized in 1857 the “Germ bacteria
theory of Disease” that microorganisms - Confirmed the Germ Theory of Disease
are responsible for infectious diseases - Originated use of a two part dish for
- Father of microbiology growing bacteria (PETRI-dish) and a
- Theory of biogenesis (golden age of technique for isolating pure bacterial
microbiology) colonies
- Developed pure culture techniques
- Work with anthrax
- KOCH’S postulate
- Cultivate petri dishes
- Gelatin una nyang ginamit, develop by
the suggestion of Walter to use agar
- Discovery of TB bacilli
VIRUS CAN EASILY MUTATE THAN BACTERIA

BY NATURE BACTERIA IS COLORLESS

GERM THEORY OF DISEASE

• Oliver Wendell Holmes Sr. (1809-1894)

believed death following childbirth often
caused by the material on hands of
midwives or attending physicians
• Ignaz Semmelweis (1818-1865) noticed
death rates higher in maternity wards
staffed by medical students than in those
attended by midwives. Death rates
decreased in summer.
JOHN SNOW (1813-1858)

- Played key role in setting standards for

good public hygiene and preventing
spread of infectious disease.
- Considered one of the fathers of
epidemiology because of his work in
tracing the source of cholera outbreak in
London. (sa well, yung pinagkukuhanan
ng tubig noon)
 PAUL EHRLICH (1854-1915)

- Hospital dermatologist
- German doctor wanted to find a “magic
bullet” an agent that would kill the
disease agent without hurting the patient
- Chemotherapy treatment using chemical
substances
- Developed Salvarsan (arsenic derivative)
Preparation 606 for syphilis “salvation
from syphilis”
WALTER HESSE (1846-1911)

- Used agar as a solidifying agent to harden

media. Agar is extracted from seaweeds
red algae.
ROCHARD PETRI (1852-1921)

- Used agar dish to provide a large area to

grow
HANS CHRISTIAN GRAM (1853-1935)
JOSEPH LISTER (1827-1912)
- Staining method that demonstrate
- Father of modern antisepsis bacteria and distinguish between gram
- Sterile surgeon positive (violet) and gram negative (red)
- English surgeon that applied germ theory bacteria.
to surgery - Use dyes (crystal violet)
- Lister experimented on carbolic acid - Another dye is safranin
spraying instruments, surgical incisions,
RAYMOND SABOURAUD (1890-1910)
wounds and dressings. This markedly
reduce the incidence of gangrene. - Developed culture media to study yeast
- Understood infection is best avoided by and molds.
preventing bacteria from getting into the - Yeast and molds are fungi
wounds in the first place. This led to the
rise of sterile surgery. DIMITRI IVANOSKI (1892)

EDWARD JENNER (1749-1823) - Tobacco mosaic virus could pass through

filters used to remove bacteria
- Credited the first vaccine
- Jenner, in the late 1700s made small SELMAN WAKSMAN (1940)
incisions or punctures with cowpox
material in arms of human subjects in - Discovered a number of antibiotic such as
order to prevent smallpox. tetracycline and streptomycin
- At first his peers doubted the safety and GERHARD DOMAGK (1895-1964)
efficacy of his treatment but eventually
the value of the cowpox inoculum was - A German chemist who discovered that
recognized. dye prontosil was effective against a
- Jenner’s works are said to have saved wide range of bacteria
more lives that efforts of any other
ALEXANDER FLEMING (1881-1955)
person in history
- Smallpox and cowpax - A Scottish biologist and pharmacologist
- Ininoculate sa 8 y/o child yung vaccine observed bacterial staphylococcus
colonies disappearing on plates
contaminated with mold
- Penicillin was discovered by accident
 GRAM STAINING:

1. Crystal violet
2. Mordant (gram’s iodine) CV + iodine
3. Decolorize slide using acetone (15%) and
alcohol (50%) ; some will be violet and
colorless
4. Apply counter stain (safranin-red) para
yung colorless maging red
SALK – inactivated polio vaccine

SABIN – oral polio vaccine

RUSKA (1938)

- First electron microscope

- The electron microscope is capable of
magnifying biological specimens up to
one million times. These computer
enhanced images of:
1. Smallpox
2. Herpes simplex
3. Mumps are magnified, respectively
150,000 and 90,000 times
- To study details structures of viruses

WATSON AND CRICK

- DNA (1953)
JACOB AND MONOD (1965)

- Did research on RNA and protein

synthesis in bacteria – last necessary step
in understanding how genetics works on a
cellular level (Replication, Transcription,
Translation – protein synthesis –
expression of traits)
- Modern science thrives today only on the
laid foundation of thousands of men and
women who did mundane routine and
often boring lab science.
-
TOPIC 2: CELL PHYSIOLOGY, METABOLISM AND
GENETICS
BACTERIOLOGY LEC

One way to be able to transport the energy inside the

cell.
2 Types:
Metabolism
NAD – it will become NADH in reduction reaction
is the process where in the cells wll derive their (reduce form)
energy and that fuel that will run a cell is called ATP.
FADH – it will become FADH2 in reduction reaction
o Energy when it’s metabolized will be in the form
of ATP. = carriers that transports energy
o It is an energy balancing act – a balance between
catabolism and anabolism
o Sum of all chemical reactions within the living
organism.

Anabolism – creating, producing, synthesizing

o Turn into simple molecules to complex ones
o When you synthesize, it most often involved
dehydration reaction, reactions that will release
water.
o You CONSUME more energy
ATP o Example : proteins to amino acids
o Mother of all rechargeable batteries Catabolism – breaking down or degrading
o ATP is consist of adenine, ribose and 3
phosphate groups o Break down of complex molecules to simpler
o So the energy that we stores in the ATP will be ones, and when you do catabolize there is a
released primarily due to drive anabolic corresponding release of energy.
reaction. o You PRODUCE more energy
o In catabolism, we will be able to generate ATP,
the ATP will be released after catabolism.
o Anabolize = synthesize

Respiration is not breathing. It is the generation of

ATP. It is an ATP generating process in which
molecules are being oxidized and there is a final
electron acceptor in the process. Respiration is the
Oxidation – removal of electrons process of converting the nutrients into usable energy
in the form of ATP.
Reduction – addition rather than loss of electrons. In
metabolism, they’re known as the basic reactions The bacteria have to undergo cellular respiration and
cellular respiration is how they can generate ATP.
Aerobic – a lot of ATP will be produce. The final Embden-Meyerhof-Parnas (EMP) pathway allows the
electron acceptor will be oxygen. metabolic use of glucose to generate ATP, NADH, and
several biosynthetic precursors such as 3-
Anaerobic – little ATP. The final electron acceptor will
phosphoglycerate or pyruvate.
be an inorganic molecule.
ALTERNATIVES:

Breakdown of glucose to CO2, water and a lot of ATP. They can produce a lot of NADPH.
Glycolysis – the glucose will be oxidized to produce The yield of ATP will only be one.
ATP and energy carrying molecules and that is NADH.
Example: E. coli

EX: Pseudomonas (gram-negative), agrobacterium and

The oxidation of glucose to pyruvic acids. rhizobium
Glycolysis has 2 steps:
1. Energy investing stage – you are going to
invest ATP in order to break the bonds in the
6th carbon molecule in such that, the product
will be fructose one, six bi-phosphate. The
product will be fructose one, six bP.
2. Energy conserving stage – will produce GAP
and DHAP. The ultimate product will be the
two molecules of pyruvic acid or pyruvates.
The end products of Glycolysis: 4 ATP, 2 NADH, and 2
pyruvic acids
Pyruvic acid to acetyl COA such that it can be accepted
in the next subpathway which is the Kreb’s Cycle.
In the process of conversion, there will be
CARBOXYLATION REACTION in such that CO2 will be
released and one NADH will be released for one
pyruvic acid.
Glycolysis will happen in CYTOPLASM. The end products: 2 NADH, 2 CO2
Not all bacteria dadaan ng glycolysis, it is called the
Embden-Meyerhof-Parnas (EMP).
 3 Main Events of ETC:
1. Redox reduction
2. Creation of proton gradient
3. Oxidative- phosphorylation of ATP
The main goal of the ETC is to release the energy in
the electron carriers to another form of energy.
Carrier molecules in the ETC (the one who will
undergo in redox reduction to pump out hydrogen):
FMN, Q, cytochromes

Oxaloacetate – Citrate - Isocitrate (release of CO2 Hydrogen ma pump out sa labas ng plasma
and NADH) – Alpha Ketoglutarate (release of CO2 membrane. Then, magkakaron ng proton gradient.
and NADH) – Succinyl CoA (GDP-an energy Tapos mapupunta sa ATP synthase, will now accept
molecule) – Succinate (FADH) - Fumarate -Malate the hydrogen that were pumped out to the energy
(NADH) that can receive now in such that it could post
correlate the ADP into ATP. ADP to ATP.
Per one acetyl CoA: 2 CO2, 3 NADH, 1 FADH, 1GDP (x2
because of 2 acteyl CoA) For every NADH na papasok sa ETC, mag ggenerate ng
3 ATP
The end products: 4 CO2, 6 NADH, 2 FADH, 2 GDP
For every FADH na papasok sa ETC, mag ggenerate ng
2 ATP
Glycolysis and Kreb’s Cycle – cytoplasm of prokaryotes
ETC is very diverse.
Eukaryotes – matrix of mitochondria

ETC will occur in the cell membrane of prokaryotes.

Eukaryotes sa mitochondria.

TOTAL: 38 MOLECULES
Maximum of 38 molecules. Theoretically, it can be 38
or less.
Aerobes can produce hydrogen peroxide.
When aerobic microbes are able to use oxygen, they
could very well undergo catabolism carbohydrates
through aerobic cellular respiration.

Obligate anaerobes would benefit in this process.

No ETC in anaerobic cellular respiration.
 Mutant type gene = aer-

Phenotype = capital letter then followed with small

letter, not italicized, starts with capital letter

The genome is the complete sets of genes in an

organism.
Bacteria size: average size 1-2mm
Chromosome of bacteria is 500x length of the
bacillum

Plasmids
o Supplementary genes that made up of double
stranded DNA that you can find inside of the
cell
o They are accessory genes
10 Mb = 1 million base pairs
o It might change some abilities or
Ex: E coli. Has 5 mega base on its genome. characteristics, but it will not definitely result
to bacterial cell death.
o Conjugation – able to transfer some of its
characteristics to other bacteria to a narrow
tube which is a sex pillus

Genotype will dictate the phenotype because the

genotype will dictate the observable characteristics.
Genes are often mnemonics of description of their
function.
Example: aerotaxis – so you can write genes as er and
should be italized
Genotypes - three lower case letters, italicized,
Bacteria reproduce by asexual reproduction and it is
aerA
through binary fission.
aerB
Binary fission – prokaryotes will reproduce asexually in
the beginning. It will double up its chromosome and
Wild type gene = aer+ extend its cytoplasmic component and elongate. It will
grow continuously, it will create in the middle of the
body. A point of fission where it could divide the single Kung gutso niya i-trasnfer ang capability, to be able to
cell that has grown into two and later on split. create sex pillus to another bacteria. That’s the last
gene na dadaan sa sex pillus. It has the cell to cell
Split into two, so that one bacteria, the mother cell will
adhesion. Some scientist consider this sexual
produce a clone of itself. 2 can become 4, 4 can become
reproduction because may attachment.
8, 8 can become 16 etc….
Kung in the middle of the docking, naalis ang sex pillus
Generation time - is the time it takes for a population
or natanggal sa pagkaka-attach, so yung bacteria na to
of bacteria to double in number. For many common
hindi siya makakapagtrasnfer ng genes because it
bacteria, the generation time is quite short, 20-60
doesn’t have the capability to create a sex pillus.
minutes under optimum conditions.
In summary: the transfer of genes to another without
cell division taking place, the genes could be
transferred.
So pagnakuha na yung genes, it will recombine it with
its own genes that would have a certain advantage,
that will be called conjugation.
Additional Notes: It could be done to transfer not only
the genes, but it could also be done in order to
transfer part of its chromosome. Pwede don ren
Vertical Gene Transfer – from mother to daughter cell padaanin ng bacteria yung part ng kanyang
chromosome to transfer the genes that is encoded in
Why are bacterial cells different?
its chromosome. So hindi lang plasmid genes ang
Because of the HORIZONTAL GENE TRANSFER. This pwedeng ibato sa kabilang bacterial cell.
now will create diversity in the population of bacteria.
So hindi magiging magkakamukha ang bacteria. It is
also the transfer of one DNA of bacteria to another
bacteria, maybe different kinds of bacteria, maybe
similar.
3 Ways How to Transfer the Genes Horizontally
1. Conjugation
2. Transformation
3. Transduction

TRANSFORMATION

CONJUGATION

Baket magkakaron ng DNA sa environment? Because

some bacterial cells when they lyses nasisira sila
because of antibiotics, harsh environmental
A picture of conjugating bacteria. Hindi lahat ng
conditions or their inability to reproduce and
bacteria have the capability to create a sex pillus.
metabolize. So it will die and the chances are the DNA
would also be freely floating in the environment. The
bacterial cells are able to take up this freely floating
DNA in the environment, and it cannot be done by all
bacterial cells. The only ones who are capable of doing
transformation or picking up the floating in the
environment are called the competent cells.
The competent cells are the bacterial cells that have TRANSDUCTION
the ability to take up freely floating DNA.
Bacteria are not always competent. Sometimes when
growth condition are optimal, like busog siya or happy,
they are not competent for the take up of the DNA.
Pero kapag ang bacteria ay gutom or stress, they could
turn on their genes that will allow them to be
competent.
Once na nakuha na yung floating DNA in the
environment, pwede niya i-recombine ito with its own
DNA transfer using bacteriophages (viruses)
genes. I-incorporate niya tong genes na ito sa existing
niyang genes. This is the process called genetic Bacteriophages or phages – are viruses, infecting
recombination, where genetic material is mixed and bacteria.
match resulting in new sequences. Phage (micrometer) is bigger than virus (nanometer)
Bacteria can end up to recombine or it can be
degraded.
Usually, only a few genes are able to be incorporated
or recombine.
Degrading - If a similar gene is already found in the
chromosome of the bacteria, yung dalawang DNA
sequence na iyon will line up beside one another and
then swap. Aalisin yung isa i-ccut then a-attach ang
bago. Virulent – breakdown of cell components to create
Example, merong 40 bacterial species within the progeny phages.
population and only 1% could carry out the full process Temperate – can also become virulent, but generally it
of transformation. And they’re only able to take a DNA will just integrate its chromosome into the bacterial
that is very similar in their own. chromosome as a prophage. Yung kanilang genes
Most of gram positive bacteria are competent. pwede nila i-insert sa bacterial chromosome, so
magkakaron ng genetic recombination in the process.
What would be the advantage of being competent for The temperate is temporary because it can choose to
transformation? The cell can express that gene and become virulent in the process.
out compete its neighbor because of that advantage
that they have taken from the expression of the gene.
This can result in the evolution of the organisms such
that iba iba na uli yung itsura and capability ng bawat
organism.

The transduction and transformation does not require

direct contact or attachment like conjugation.
The transduction, unlike the transformation, it needs
a third party to help transfer the gene.
Inject ng DNA sa bacterial cell.

Attitude of the temperate DNA.

Mix of viral DNA and bacterial DNA.
Certain part lang ng bacterial DNA ang ma ttransfer
niya.
Generalized (random genes can be incorporated)
Specialized Transduction (specific portion of genes
ang kinukuha)
MICROBIAL GROWTH REQUIREMENTS Osmotic Pressure

• Physical - Required water for growth

o Temperature - The growth of the cell is inhibited as the plasma
o pH membrane pulls away from the cell (addition of salts
o Osmotic pressure = hypertonic)
• Chemical - Hypertonic or hypotonic environment
o Sources of C, N, S, P, trace elements, O2 o Hypertonic – cellular water passes out
and organic growth factors through the plasma membrane where there
is high solute concentration, leading to cell
Temperature shrinkage and death
- Most bacteria grow only within a limited range of o Hypotonic – such as distilled water, the
temperatures water enters the cell from low to high
- Each bacterial species grows at a particular concentration, leading to swelling of the
minimum (lowest), optimum (best), and maximum cell, and burst, and death
(highest) temperatures - Halophile = salt loving
- Bacteria are very diverse when it comes to - Obligate or extreme halophiles (30% salt)
temperature o Strictly needed for hypertonic environment
- On the basis of preferred range of temperature: - Facultative halophiles (2-5% salt concentration)
bacteria can be classified as: Carbon
o Psychrophiles
▪ Psychotrophs – 0 to 15 degrees C - C is the structural backbone of living matter, all
▪ Moderate psychrophiles – 20 to 30 organisms have it
degrees C - Half of the dry weight of typical bacterial cell is C
o Mesophiles - Can be a basis for classifying organisms in basis for
▪ Involved in infectious diseases source of energy
processes o Chemotrophs – chemical source
▪ Moderate temperature (25 to 40 o Phototrophs – light source
degrees C - Energy source and carbon source mixed together
▪ Optimum: 35 – 37 degrees C o Chemoheterotrophs – carbon source is
(body temperature) organic compounds and energy source is
o Thermophiles chemicals
▪ 50 to 60 degrees C o Chemoautotrophs – energy source is
o Hyperthermophiles chemical compounds and carbon source is
▪ 80 degrees or higher from carbon dioxide
▪ Some can tolerate 121 degrees C o Photoheterotrophs - energy source is light
such as in autoclave and carbon source is organic compounds
o Photoautotrophs - energy source is light
pH and carbon source is carbon dioxide
- Most bacteria grow best in a narrow pH range near - Most organisms are classified under
neutrality between 6.5 – 7.5 chemoheterotrophs
- Very few bacteria grow below pH 4 Nitrogen, Sulfur, and Phosphorus
- Lowest recorded is pH 1, tolerated by the
chemoautotrophic bacteria (cyanobacteria) - Nitrogen has 14% in dry weight of a bacteria cell,
- Molds and yeasts, they grow at pH 5 to 6 sulphur and phosphorus about 4%
- When cultured, they produce acids, interfering with - For protein synthesis (N and S)
own growth, so it is important to make sure culture - For DNA and RNA synthesis
has chemical buffers to avoid shifting of the pH - K, Mg, and Ca are also needed as co-factors for
enzymes
Trace Elements
- Serves as co-factors for enzymes, but they are
naturally present in tap water and other media
components
• Fe
• Cu
• Mo
• Zn

Oxygen
- Metabolic systems require oxygen for aerobic
(a) Obligate aerobes require oxygen, so they will go to
respiration
the place in the tube where there is increased
- Microbes that use molecular oxygen produce more
oxygen concentration.
energy from nutrients than microbes that do not use
(b) Turbidity concentrated at bottom of the tube may be
oxygen
obligate anaerobes because clear solution is
- Oxygen can also be poisonous, we produce its toxic
observed at the top area. The bacteria fear high
form, it instigates chemical reactions in the body,
oxygen concentration so they are located at the
producing free radicals, superactive molecules that
portion with low oxygen concentration.
destroy our cells
(c) Facultative anaerobes can grow anywhere in the
- Our bodies are designed to use oxygen to its
tube, but most of them will be located at high oxygen
advantage
concentration.
• Obligate aerobes
(d) Aerotolerant anaerobes can also grow anywhere in
- strictly aerobes, oxygen is a the tube because it can tolerate oxygen.
requirement
• Facultative anaerobes Why some bacteria can and cannot tolerate oxygen?
- Part-time anaerobes, basically
aerobes
- Energy is decreased with no
oxygen
- Can use oxygen to advantage
• Obligate anaerobes
- Strict anaerobes, should live
without oxygen
• Aerotolerant anaerobes
- Anaerobes that can tolerate
oxygen
• Microaerophiles 1st tube: growth occurs only in high concentration of oxygen,
- Bacteria that can only tolerate meaning they can tolerate oxygen as much as they can,
small amount of oxygen (5-10%) because oxygen is not poison to them, they have enzymes
catalase and superoxide dismustase (SOD) to allow
- Oxygen in the air is normally
neutralization of toxic forms of oxygen that are produced in
around 21%
normal metabolism. (Obligate aerobes)
• Capnophiles
- Need carbon dioxide when they 2nd tube: grow anywhere in the tube, but most grow at the
metabolize surface. Tolerance of high concentration of oxygen means
they have the same enzymes to neutralize oxygen, catalase
and SOD. Oxygen can be used. (facultative anaerobes)
3rd tube: growth is located at the bottom of the tube. The
bacteria lack catalase and SOD to neutralize toxic forms of
oxygen (strict anaerobes), so no tolerance of oxygen.
4th tube: similar to second tube, possess catalase and SOD to bicarbondate and sodium borohydride. Cut off the
tolerate oxygen and partially neutralize its toxic forms edges and put in water. Hydrogen and carbon
(aerotolerant) dioxide are then produced.
5th tube: growth is located in the middle of the tube, meaning - Palladium catalase is also placed at the bottom of the
the bacteria are microaerophiles, can only tolerate lower lid, where there is a small container with catalase.
amount of oxygen (5-10%). The top portion has too high - The catalase will expedite and allow oxygen in the
oxygen concentration, while the bottom portion has too low jar to combine with the hydrogen produced so water
oxygen concentration, so they stay at the middle. They can is formed. Droplets of water can be seen, indicating
only produce little SOD and catalase to neutralize toxic forms the reaction is taking place, and oxygen is removed.
of oxygen. (oxygen free environment)
- When oxygen is combined with hydrogen producing
Toxic Forms of Oxygen
water, it leaves carbon dioxide in the jar.
- Can destroy cell membranes - Traditionally, it uses candle jar extinction method. A
- Singlet oxygen (1O2) is normal O2 boosted into a candle is placed in a tightly capped jar, so no more
higher energy state is extremely reactive oxygen inside.
- Superoxide free radicals (O2-) formed in small - As long as the candle is burning, it will consume all
amounts during the normal respiration of organisms. oxygen in the jar for the fire until it goes out,
Neutralized by SOD to convert into simpler indicating complete removal of oxygen.
chemicals. - It releases approximately 3% of carbon dioxide.
- In CO2 generating packet, the plate and tube to be
O2 + O2 + 2H+ → 2H2O2 + O2 filled is very limited, only for processing small
- Peroxide anion (O22) toxic form which is neutralized volumes.
by catalase converts it to water and oxygen. - It generates about 5-10% concentration, providing
only carbon dioxide.
2H2O2 → 2H2O + O2
Anaerobic Chamber
Peroxidase also can convert it to water but no
oxygen - A sophisticated way in producing anaerobic
environment
H2O2 + 2H+ → 2H2O - There is containment for mixture of oxygen provided
- Hydroxyl radical (OH-) intermediate form of oxygen through gas cylinders of nitrogen, oxygen, CO2, etc.
and probably the most reactive. It is formed in the - You can only facilitate your cultures if it tightly closed
cellular cytoplasm by ionizing radiation. and sealed when you utilize the two portions of the
chamber.
Organic Growth Factors - Greater mobility of culturing an anaerobe
- Vitamins which functions co-enzymes
- Other organic growth factors: amino acids, purines,
pyrimidines
- May be synthesized by bacteria themselves or
should be obtained from the environment
- Some bacteria would lack the co-enzymes

Anaerobic Growth Media and Methods

- Reduced media is used such as sodium
thioglycollate that chemically combine with
dissolved oxygen deplete the oxygen in the medium.
A layer should appear by the surface of the liquid.

Anaerobic System
- Uses gaspaks and anaerobic jars
- Culture bacteria on a plate or tube with sodium
thioglycollate, then put broth inside the jar, followed
by a gaspak, a packet of chemicals, sodium
INFECTION • Focal infection
- condition where due to infection at localized
Definition of Terms
sites like appendix and tonsil, general effects
Pathogenesis are produced
“Path” = disease • Cross infection
- when a patient suffering from a disease and new
• Pathogenesis is the steps of mechanisms involved in infection it set up from other host or external
the development of the disease. source
• Pathogenicity is the ability to cause disease. - one patient with disease transmits infection to
• Pathogen is a microorganism capable of causing another host
disease. • Nosocomial infection
• Pathology is the study of the structural and functional - cross infection occurring in hospital
manifestations of the disease. - hospital acquired infection
• Subclinical infection
Infection - where clinical effects are not apparent
- Injurious contamination of body or parts of the body - the infection is in the patient, but no signs or
by bacteria, viruses, fungi, protozoa and rickettsia or symptoms
by the toxin that they may produce • Localized infection
- Infection may be local or generalized and spread - infection that remains localized at one site
throughout the body - ex. Pimples, boils
- Once the infectious agent enters the host, it begins • Systemic/generalized infection
to proliferate and reacts with the defense - an infection that has spread throughout the body
mechanisms of the body producing infection - ex. tuberculosis
symptoms and signs
- Infectious disease – disease caused by a microbe Infection vs Infectious Disease
and the microbes that cause infectious disease are ➢ Infectious disease is a disease caused by a microbe
collectively referred to as pathogens and the microbes that cause infectious disease are
- Colonization by a pathogen collectively referred to as pathogen.
➢ Infection according to many microbiologists means
Classification
colonization only by a pathogen.
• Primary infection ➢ A person can be infected by a pathogen, but might
- initial infection with organism in host not have infectious disease.
- the first time that the host encounters a o Pathogens may colonize a certain area but
microbe it does not actually involve the disease yet,
• Reinfection no illness or signs or symptoms.
- subsequent infection by same organism in
Classification of Diseases
a host
- the same organism can attack after • Acute disease
recovery - has rapid onset usually by a relatively rapid
• Superinfection recovery
- infection by same organism in a host before - ex. Measles, mumps, influenza
recovery • Chronic disease
• Secondary infection - has a slow onset and lasts a long time to recover
- when in a host whose resistance is lowered - ex. Tuberculosis, necrosis, syphilis
by pre-existing infectious disease, a new
organism may set up an infection • Sub-acute
- the host could have a bacterial infection - disease that come on more suddenly than
from the start because of being chronic diseases but less suddenly than acute
immunocompromised, a secondary diseases
infection may be a virus
 • Latent infection ➢ The person may be immune to that particular
- diseases that may go from being symptomatic to pathogen and is the result of prior infection or
asymptomatic and then sometime later, go back vaccination.
to being symptomatic ➢ Phagocytic white blood cells may engulf and destroy
- the causative agent remains inactive for a period before it has an opportunity to multiply, invade, and
of time, then produces symptoms again cause disease.
- ex. Syphilis ➢ The indigenous microbiota at the site may produce
antibacterial factors (bacteriocins) that destroy the
Symptoms vs Signs of a Disease newly arrived pathogen.
• Symptoms – some evidence of a disease that is o Normal microbiota are very important
experienced or perceived by the patient (ex. Aches,
(Keep on improving lifestyle!!!)
nausea, itchiness)
o Symptomatic Disease (clinical disease) – a Four Periods in the Course of an Infectious Disease
disease in which the patient is experiencing 1. Incubation Period – the time that elapses between
symptoms the arrival of the pathogen and the onset of
o Asymptomatic Disease (subclinical
symptoms. It depends on nature of the microbe. No
disease) – a disease that the patient is symptoms are experienced yet.
unaware of because there are no
symptoms experienced Factors influencing the Incubation Period
• Signs – some type of objective evidence of a disease
➢ Overall health and nutritional status of the
(ex. Fever, pulse rate, blood pressure, test results,
host. Pathogen might have a difficult time to
respiratory rate)
invade if the body is healthy.
Pathogenesis of Infectious Disease ➢ Virulence of the pathogen. Involves the
mechanisms and factors of the pathogen to
Why infection does not always occur? make it able to cause disease.
➢ The microbe may land at an anatomical site where it ➢ Numbers of the pathogen that enters the
is unable to multiply. body. Some diseases take several numbers
o It may land on the skin where there are of the pathogen before initiating disease.
normal microbiota that made the bacteria
not succumb to infection because the 2. Prodromal Period – the time during which the patient
defenders counteract the microbes that starts to feel something is wrong but do not
land on the skin. experience the actual symptoms of the disease yet.
o There are factors that might affect the Signs and symptoms are present, but not specific.
survival of the microbe on that site.
➢ Many pathogens must attach to a specific receptor 3. Period of Illness – the time when the person
site before they are able to multiply and cause experiences the symptoms of the disease.
damage. Communicable diseases are most easily transmitted
o Reason why viruses in animals cannot during this stage. Identification of illness is truly
transfer to humans because they do not observed.
have specific receptor sites in human cells. 4. Convalescent period – the time when the person
➢ Antibacterial factors that destroy or inhibit the growth recovers. Person may recover from the illness but
of the microbes may be present at the site where the there may be permanent damage like disability or
pathogens are. damage of the tissues of the affected area. Death
o Some antibiotics may be present would not may also occur in this period.
promote survival of pathogen in the site.
➢ The individual’s nutritional and overall health status
often influences the outcome of the pathogen/host
encounter.
o If the resistance is high enough and
immune to a certain pathogen, the disease
may not occur, especially if the individual is
overall healthy.
 which are often glycoprotein molecules.
(host)
➢ Adhesions and Ligand – used to describe
the molecule on the surface of the pathogen
that is able to recognize and bind to a
particular receptor. (pathogen)
▪ Ex. Streptococcus pyogenes –
have adhesins called Protein F
that will adhere to the protein on
the host cell called fibronectin.
▪ Ex. HIV – have GP120, an
adhesion that must attach to CD4
➢ As the period of infectious disease continues, the or T-helper cells of the host cell.
number of microorganisms will peak at a certain ➢ Bacterial fimbriae (Pili) – long, thin, hair-
point at the period of illness. like, flexible projections composed primarily
➢ After some time, it declines as the period progresses, of an array of proteins called pilin which
and the number of microorganisms will also decline. enable the bacteria to attach to surfaces
➢ The number of microorganisms is at the lowest and cause infection.
during the period of convalescence. ▪ Ex. Neisseria gonorrhoea –
anchors itself to the urethra,
Steps in the Pathogenesis of Infectious Diseases
causing urethritis. Those that are
1. ENTRY of the pathogen into the body through portals not fimbriated can be flushed out
of entries. when the host leaks.
2. ATTACHMENT of the pathogen to some tissues • Obligate intracellular pathogen – must live within the
within the body once it has gained entrance. host cells in order to survive and multiply (e.g.
3. MULTIPLICATION of the pathogen after attaching Rickettsia, Chlamydia, Erlichia). Once pathogen is
and lodging into the tissues and cells of the body. inside the cell, the antibodies will not work.
4. INVASION/SPREAD of the pathogen to different • Facultative intracellular pathogens – able to survive
parts and tissues after multiplying. It creates signs intracellularly and extracellularly. These
and symptoms. microorganisms are able to survive within
5. EVASION of host defences by the pathogen. phagocytes. Once bacteria are ingested by WBCs,
6. DAMAGE to host, leaving the host defenceless. the phagosome will infuse with lysosomes, causing
destruction. The pathogen has the option to stay or
Virulence leave the cell, and migrate to the external
- Measure of the degree of pathogenicity surroundings to avoid bombarding of lysosome
- Vary in ability to cause disease enzymes.
- Phenotypic characteristics that enable it to • Capsule – can evade phagocytosis because they are
cause disease sticky/slimy
o Virulent strains – capable of causing • Flagella – enabled to invade aqueous regions of the
disease body that non-flagellated bacteria can’t do
o Avirulent strains – not capable of • Exoenzymes – enzymes liberated by the bacteria
causing disease outside
- Factors that are associated with the structure ➢ Necrotizing enzymes – bacteria produce
include toxins enzymes like poteases and lipases which
cause destruction of tissues (e.g. S.
Virulence Factors pyogenes)
• Attachment – pathogen must be able to anchor itself ➢ Coagulase – enable S. aureus to clot
after gaining entry. plasma and thereby form a sticky coat of
➢ Receptors and Integrins – molecules on the fibrin around themselves for protection
surface of the host cell that a particular against phagocytes, antibodies, etc. so they
pathogen is able to recognize and attach to will not be engulfed or recognized.
 ➢ Kinases or Fibrinolysins – substance that o Neurotoxin – affects nerves, most potent
will dissolve the fibrin clot that the host will exotoxin produced by Clostridium tetani
attempt to form. (tetanus) and Clostridium botulinum,
▪ Ex. Streptococcus can produce blocking nerve impulses
streptokinases to dissolve the o Enterotoxins – affects intestine, cause
fibrin. diarrhea and sometimes vomiting (Toxin B
➢ Hyaluronidase – called spreading factor produced by C. difficile damages the
because it enables the pathogen to spread surfaces of the colon leading to
throughout the connective tissue by pseudomembranous colitis)
breaking down hyaluronic acid, which holds o Toxic shock syndrome toxin (TSST) –
tissues together. Usually found in produced by strains of S. aureus and S.
Staphylococcus, Streptococcus, and pyogenes which primarily affects the
Clostridium spp. integrity of capillary walls
➢ Collagenase – breaks down collagen which o Exfoliative toxin – epidermolytic toxin
is the supportive protein found in tendons, causing sloughing of the epidermal layers
enabling the pathogen to invade tissues. of the skin leading to Staphylococcal
➢ Hemolysins – enzymes that cause scalded skin syndrome (SSSS).
destruction of RBCs. Provides iron to the o Leukocidin – toxin that destroys leukocytes,
pathogen. Can be alpha, beta, or gamma produced by Staphylococcus and
hemolysis. Streptococcus
➢ Lecithinase – enzyme produced by o Diphtheria toxin – toxin produced by
Clostridium perfringens which breaks down Corynebacterium which inhibits protein
phospholipids collectively referred to as synthesis killing mucosal epithelial cells and
lecithin. This enzyme is destructive to cell polymorphonuclears. If the bacteria cannot
membrane of RBC and other tissues. secrete the toxin, it is non-pathogenic. It is
Cause of gas gangrene. why the next step after isolated and
identified is the toxigenicity test.
Other Virulence Factors

• Endotoxins – integral part of the cell walls of Gram Characteristics of Exotoxins

negative bacteria producing septic shock, chills,
➢ Heat labile protein
fever, prostration and septicaemia. The cell wall
➢ Diffuse readily into the surrounding medium
contains LPS called Lipid A, an endotoxin. Produced
➢ Highly potent (e.g. 3kg botulinum can kill all
within bacteria cells.
the inhabitants of the world)
Characteristics: ➢ Generally formed by gram positive bacteria
and also by gram negative organisms like
➢ Proteins polysaccharide lipid complex heat
Shigella, V. cholera, and E.coli.
stable
➢ Specifically neutralized antitoxin
➢ Forms parts of cell wall (don’t diffuse into
➢ Can be separated from culture by filtration
the medium)
➢ Action is enzymatic and has specific tissue
➢ Obtained only by cell lysis once the cell wall
affinity
is destroyed, releasing Lipid A
➢ Specific pharmacological effects for each
➢ They have no enzymatic action
exotoxin
➢ Effect is non-specific action
➢ Cannot cause pyrexia (high fever) in a host
➢ No specific tissue affinity
➢ Can be toxoided (non-toxic)
➢ Active only in large doses (5-25 mg)
➢ Weakly antigenic Mechanisms by which Pathogens Escape Immune
➢ Neutralization by antibody ineffective Responses
➢ Cannot be toxoided
• Antigenic variation – pathogens are able to
➢ Produced in gram negative bacteria
periodically change their surface antigens (e.g.
Influenza). Ways of pathogens to escape defences
• Exotoxins – also poisonous and released outside.
by burying surface antigens.
Usually named after the target cell.
 o Influence of viruses: The antigens change,
and the antibodies will not work anymore.
Once they change antigens, they are
considered new strains. A small change
can make it non-specific.
• Camouflage and mimicry – hiding themselves with
host proteins (camouflage) or coat themselves
resembling the host’s antigens (mimicry), avoiding
recognition of the immune system.
• Destruction of antibodies – produces enzyme that
can destroy antibodies
o Haemophilus influenza and Neiserria
Gonorrhea can produce enzyme IgA
protease that counteracts antibody IgA that
humans produce
IMMUNITY (within host) If the second line of defense does not work, the third line will
be activated.
Definition of Terms
Non-Specific Host Defense Mechanisms
• Resistance – ability to ward off disease
o Non-specific resistance – defenses that First Line of Defense
protect against all pathogens a. Physical or Mechanical barriers: intact skin and intact
o Specific resistance – protection against mucous membranes
specific pathogens
o Skin has layers of epidermis and dermis
• Susceptibility – vulnerability or lack of resistance. If - A very effective barrier to most
susceptible, you are prone to contract disease, pathogens with few exceptions.
getting infections brought by different pathogens. - The epidermis is the thin outer layer
Host Defense Mechanisms containing Langerhans cells, dead
cells, and water proof mechanism
• Non-specific host defense mechanism provided by the keratinization.
- serve to protect the body from a variety of - The dermis is the thicker part of the
foreign substances or pathogens skin, containing connective tissue that
- cater to all types of pathogens, they will not supports the overall skin and its
recognize if it is bacteria or virus capability, so it is not easily penetrated
- serves to all substances including toxins by pathogens.
- involves two lines of defenses: - Some pathogens can still penetrate
- First line of defense – nonspecific intact skin such as hookworms and
natural barriers that restrict entry of the fungi
pathogen o Mucous membranes: line gastrointestinal,
▪ Skin genitourinary and respiratory tracts
▪ Mucous membranes - Mucus is the thick secretion that traps
▪ Secretions of skin and many microbes
mucous membranes - Two layers: outer epithelial and inner
- Second line of defense – innate, connective layer
nonspecific defense that provide rapid - Epithelial layer secretes mucus which
response against invading pathogens maintains moist surfaces
once it has breached the first line of - Although they inhibit microbial entry,
defense. they offer less protection than skin
▪ Phagocytic WBCs - Some pathogens are still capable of
(macrophages and penetrating these membranes like the
neutrophils) Trypanoma pallidum, Papilloma virus,
▪ Antimicrobial proteins against E.coli, and E. histolytica
different pathogens b. Chemical factors - digestive enzymes, acidity of
▪ Inflammatory response stomach (pH 1.5) and alkalinity of the intestine,
• Specific host defense mechanism (immune system) acidity of the vagina, lacrimal apparatus
- directed against a very particular foreign o Sebum
substance or pathogen that has entered the - Oily substances produced by
body sebaceous glands that form a
- Antigen specific immune responses, specifically protective layer over skin.
targeting and attacking invaders after bypassing - Contains unsaturated fatty acids which
the two non-specific lines of defenses. inhibit growth of certain pathogenic
- only has the third line of defense bacteria and fungi
- Third line of defense mechanism o pH
▪ Lymphocytes - low, skin pH usually between 3-5,
▪ Antibodies caused by lactic acid and fatty acids
- inhibits growth of pathogenic bateria
If the first line of defense does not work, the second line will
be activated.
 ➢ Elevated body temperature also slow down the
rate of growth of certain pathogens and can
even kill some especially fastidious pathogens.
The setting will inhibit, kill, or promote growth.
c. Interferons
➢ small, antiviral proteins that prevent viral
multiplication in virus-infected cells and
serve to limit viral infections
➢ ar
➢ Has no effect on infected cells
➢ Host-specific but not virus-specific
➢ They interfere with viral infection
➢ They are unable to see the virus infected
o Perspiration cell and when they do, they attach to the
- Produced by sweat glands. membranes of the surrounding cells and
- Contains lysozyme and acids. prevent viral replication.
o Lysozyme ➢ Effective against variety of viruses
- Enzyme that breaks down gram-
positive cell walls. Types of Interferon
- Found in nasal secretions, saliva, and • Interferon alpha – produced by B- lymphocytes,
tears. monocytes and macrophages
o Gastric juice • Interferon beta – fibroblasts and other virus infected
- Mixture of HCl, enzymes, and mucus cells
from the parietal cells. • Interferon gamma – activated by T- lymphocytes and
- pH between 1.2-3 kills many microbes NK cells
and destroys most toxins.
- Many enteric bacteria are protected by d. Inflammation
food particles ➢ localizes an infection and prevent the
➢ Helicobacter pylori spread of microbial invaders, neutralizes
neutralizes stomach acid and toxins, and aid in repair of damaged tissue
can grow in the stomach, ➢ Triggered by tissue damage due to
causing gastritis and ulcers infection, heat, wound, etc.
c. Normal microbiota ➢ Five cardinal signs:
- Microbial antagonism by indigenous microbiota • Redness (rubor)
and overall nutritional status and state of health • Pain (dolor)
- Microbiota are bacteria that are inherently • Heat (calor)
present in our body, providing protection
• Swelling (tumor)
invading pathogens, especially bacteria
• May also observe; loss of function
pathogens
(function laessa)
- They are part of health and immune system.
Purpose of Inflammation
Second Line of Defense
➢ Destroy and remove pathogens
a. Transferrin and lactoferrin
➢ If destruction is not possible, to limit effects
➢ Iron-binding proteins in the blood, preventing
by confining or localizing the pathogen and
pathogens access to this essential mineral
its products
b. Fever
➢ Repair and replace tissue damaged by
➢ Augments host mechanism by stimulating
pathogen and its products
leukocytes and destroy invaders, reducing
available free plasma iron, and inducing the Process of Inflammation
production of IL-1, which causes proliferation,
maturation, and activation of lymphocytes in the 1. Tissue damage
immunologic response. • Chemicals such as histamine, kinins,
prostaglandins, and leukotrienes are
 released by damaged cells. It calls out ▪ WBCs wander guided by the
inflammatory cells to help in the process. signals by chemotaxis
• Blood clot forms ▪ The WBCs engulfs the
• Abscess starts to form bacteria by phagocytosis
2. Vasodilation and increased permeability of ▪ The products form exudates,
blood vessels. Blood vessel diameter is which accumulates serous
increased, triggered by the chemicals released fluid, RBCs, fibrinogen, tissue
earlier. debris and WBCs breakdown
3. Phagocyte migration and phagocytosis to the products, causing swelling
site of injury. and pain
• Margination – phagocytes stick to ▪ Chemical mediators, derived
endothelium from the plasma, release
• Emigration – phagocytes squeeze between bioactive agents that act to
endothelial cells (diapedesis), going to the mediate the inflammatory
surrounding tissue response
• Phagocytosis of invading bacteria, • Histamine and serotonin – released to
destroying them and removing the respond to stimuli and causes dilation and
damaged cells. increased capillary permeability
4. Tissue repair • Plasma derived mediators – present in
plasma in precursor forms that need to be
Acute Inflammation (VIDEO NI MA’M) activated by proteolytic enzymes
➢ Early, immediate reaction of tissue to injury • Kinins – increase capillary permeability and
➢ First phase of wound healing stimulate pain receptors (bradykinin)
➢ Complex protective responses to injury that lay • Fibrin strands – part of the clotting system
the groundwork to the next stage of the body’s that traps exudates, microorganisms, and
recovery: immune response and tissue repair foreign bodies
➢ Triggered by cell or tissue damage, or presence • Complement (C3a) – promotes
of dead cells vasodilation, leukocyte chemotaxis, and
➢ Occurs before immune response is established phagocytosis
➢ Primarily involves removing causative agent and
limiting tissue damage e. Phagocytosis
➢ Involves two stages: ➢ Primarily neutrophils and macrophages
o Vascular Stage ➢ microbe is being engulfed to be contained
▪ Arterioles and venules ➢ Derived from Greek words that meant “eat
constrict briefly then dilate. and cell”
▪ Dilation promotes congestion, ➢ Carried out by WBCs: macrophages,
increasing capillary neutrophils, and occasionally, eosinophils
permeability leading to ➢ During early stages, neutrophils
movement of fluid in affected predominate in infection before
tissue, resulting to the 5 signs macrophages are involved.
of inflammation. ➢ Wandering macrophages – originate from
▪ As fluid leaves, the blood monocytes that leave blood and enter
remaining becomes viscous infected tissue, and develop into
and flows slowly for clotting to phagocytic cells
occur ➢ Fixed macrophages (histiocytes) – located
o Cellular in liver, nervous system, lungs, lumph
▪ Initiated by the WBCs in the nodes, bone marrow, and several other
site of injury tissues
▪ WBCs adhere to the vessel ➢ Phagocytes are ttracted to site of infection
wall and squeeze through the by Chemotaxis
wall to move into the inflamed
tissue
 1. Tissue injury, release of chemical signals to go and be deployed to site of
by the cells nearby to call out phagocytes invasion.
to site of injury. ➢ Cytolysis - lysis of bacteria and
2. Dilation and increased permeability of other foreign cells due to the
capillary, so the phagocytes can squeeze membrane attack complex (MAC)
in the site of injury. which produces lesions in
3. Phagocytosis of pathogens microbial membranes.
▪ Increased phagocytosis
Process of Phagocytosis by phagocytic cells.
1. Chemotaxis and adherence of microbe to They label pathogens by
phagocyte. It will depend on the virulence opsonization or by
factors of the pathogens. coating them with
2. Ingestion of microbe by phagocyte. It antibodies.
creates an extension to enclose the ➢ Inflammation – complement
microbe. components (C3a, C4a, C5a, aka
3. Formation of a phagosome, the vesicle that mast cells) trigger the release of
contains the microbe. histamine, which increases
4. Fusion of the phagosome with a lysosome vascular permeability. They also
to form a phagolysosome attract macrophages and
5. Digestion of ingested microbe by enzymes neutrophils.
6. Formation of residual body containing ➢ Opsonization – complement properties
indigestible material (C3b) bind to microbial surface and
7. Discharge of waste materials (microbial promote phagocytosis once the microbe is
fragments) out of the cell. coated. The macrophages and neutrophils
have receptors for C3b.

Specific Host Defense Mechanisms

Third Line of Defense
- Actual study of immunity
- Immunology - scientific study of the immune
system and immune responses
- The immune system is the third line of defense
against pathogens

Types of Acquired Immunity

• Active acquired immunity – body has something to

do to make it, obtained from the body’s activity and
f. Complement system
reaction towards the material injected or the
➢ involves approximately 30 different blood
pathogen itself, stimulating immunity.
proteins that interact in a step-wise manner
o Natural active acquired immunity
known as the complement cascade.
- acquired in response to the entry
➢ It is activated directly by pathogens or
of a live pathogen into the body
indirectly by antigen-antibody reactions
(e.g. in response to an actual
➢ Generates active components to fight
infection)
invading pathogens
- it has long duration
➢ Consequence of activation of the
o Artificial active acquired immunity
complement system: initiation and
- Immunity that is acquired in
amplification of inflammation
response to vaccines.
➢ Attraction and activation of
- Its duration is for many years but
leukocytes. They call out help to
must be reinforced by boosters
rescue from invading pathogens
by means of the activated proteins
 • Passive acquired immunity – the body does not do - Antigens can also be small molecules (haptens)
anything, just simply receives immunity from the that act as antigens only if they are coupled with
material. large carrier molecules such as protein but they
o Natural passive acquired immunity are less immunogenic.
- Immunity acquired by a fetus - Antigen is always complex macromolecules
when it receives maternal (mostly glycoproteins)
antibodies in utero or by an infant
when it receives maternal Classes of Immunoglobulins
antibodies contained in colostrum ▪ IgG – greatest percentage of the antibody molecules
from breast milk. in the blood, these globulins combine to small
- Its duration is from 6mos-1yr. antigen, and combine and neutralize toxins
o Artificial passive acquired immunity (antitoxins); long lived and crosses the placenta.
- Immunity that is acquired when a ▪ IgM – a pentamer, has 10 antigen binding sites, first
person receives antibodies antibodies formed in primary response to antigens;
contained in anti-sera or gamma does not cross the placenta because they are big.
globulin. Its duration is from 2-3 ▪ IgA – secretory antibodies; found in breast milk,
weeks respiratory and intestinal mucin, saliva, tears, and
vaginal secretions protect these parts of the body
Types of Immunity
from infectious agents. Total serum immunoglobulin
• Humoral immunity: Antibodies and antigens play a is IgA.
major role in the cell-mediated immunity (antibody ▪ IgD – found on the surfaces of the B- lymphocytes
mediated immunity) where it acts as specific antigen receptor.
- Mostly extracellular ▪ IgE – activities involved in both the resistance to
- An antibody is a protein produced in response parasites infections and hypersensitivity (allergy).
to foreign substance (antigen) that will react
Cell-Mediated Immunity (CMI)
specifically with that substance. The reaction
between the antibody and the antigen will lead - Does not involve production of antibodies
to destroy the antigen or the pathogen that carry because they might be useless at this point.
the antigen or inhibit it, if not kill. - Controls intracellular pathogens
- Found in blood, lymph, saliva, colostrum - Cells that participate are macrophages, T-
- The type produced depends on the antigen, helper cells, cytotoxic T-cells, NK cells, Killer
stimulus, and exposure time. cells and granulocytes.
- Antibodies are glycoproteins produced by the B - Big players are the B and T cells.
lymphocytes, transformed by plasma cells, and - Cells with intracellular pathogens, NK and Killer
binds to the specific antigen in the antigenic part cells are subpopulation of lymphocytes that
(antigenic determinant), and once it binds, they destroy infected cells where pathogens reside in
can destroy or inhibit it. - Once the cells are destroyed, the pathogens are
- Antibodies are produced by WBCs (plasma also destroyed.
cells) and may present in the blood and the body
fluids or attached to surfaces of cells. They are Lymphocytes – key players in CMI
also called immunoglobulins (Ig). a. B-lymphocytes
- Antibodies are very specific. - 10-15% of lymphocytes in peripheral blood
- Antigens are substances that stimulate the - B cells migrate to lymphoid tissues where they
animal body to produce antibodies that will produce antibodies that circulate through lymph
specifically react with the antigens and blood
- Antigens are antibody generating molecules, - They live about 1-2 weeks
antigenic, or immunogenic.
- The best antigens are the foreign proteins b. T-lymphocytes
because of its complexity.
- T cells are phagocytotic cells, it engulfs the
o Bacteria cell is a mosaic of antigen
antigen or the pathogen that carries that
determinants called the epitopes
antigen, it destroys the infected body cells, and
(antigenic sites)
it rejects the foreign tissue
 - About 70-80% in the peripheral blood
- Several types: T-helper, cytotoxic T-toxic, etc.

Typical CMI response

1. A macrophage engulfs and partially digests a
pathogen. Fragments (antigenic determinants)
of the pathogen are then displayed on the
surface of the macrophage.
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.
3. The effector cell binds to a target cell.
4. Vesicular contents of the effector cell are
discharged.
5. Toxins produced by the effector cells enter the
target cell, causing disruption of DNA and
organelles. The target cell dies.
Micrococcus, Streptococcus,
and Neisseria
Staphylococcus aureus infections (8)
Micrococcaceae - Most important staphylococcus
• Members are gram-positive cocci, aerobic or • Skin and wound infections
facultative anaerobes - boils, furuncles and folliculitis
• Catalase positive (except Stomatococcus) - They are the primarily related to skin
- Is an enzyme responsible for the ability to abscess, wound infection because they are
neutralize the toxic forms of oxygen, that’s the prime reservoir of the interior nares in
why they can live in the presence of the skin
oxygen) • Food poisoning
• Most members are indigenous microbiota: - Staphylococcal enterotoxins
Staphylococcus, Micrococcus, Stomatococcus o A (78%), D (38%), B (10%)
- They are part of our normal bacterial - heat-stable toxins pre-formed in food
inhabitance. They found in our skin and - rapid onset literally 6 hours and resolve
surfaces of our body. within 2-8 hrs
- nausea, vomiting, headache, stomach
Staphylococcus ache, severe cramping
o Derived from the word “staphylo” which means • Scalded skin syndrome (Ritter’s disease) caused
in clusters of bunch of grapes by exfoliative/epidermolytic toxin
o Spherical bacteria that are clustered together - can occur in newborns, and in adults
o Catalase positive having chronic renal failure or nare
o Non-motile immunocompromised
o Facultative anaerobes - immortality rate is low in newborn and high
o Normal inhabitants of the skin and mucous in adults
membranes (e.g. anterior nares) • Toxic shock syndrome (TSS) caused by S. aureus
o Commonly cause human infections that produces enterotoxin F (TSST-1)
o Species are differentiated by coagulase test, - TSST -1 – toxic shock syndrome toxic 1
the most important being the coagulase - Strong association with the use of
positive S. aureus tampons
o Some animal species produce coagulase but are - Can occur in both sexes
rarely isolated from human samples ex: hyicus - Two categories:
and inermedius o Menstruating-associated TSS
o Nonmenstruating-associated TSS
Micrococcus
Other infections
o Opportunistic pathogens found only in
immunocompromised patients • Staphylococcal pneumonia secondary to
o Of low pathogenic significance influenza can occur
o May be isolated as contaminant or as part of the • Osteomyelitis can occur secondary to
normal microbiota bacteremia – deposition of S. aureus in the
o When they grow, they give out a yellowish color diaphysis of bones
and they’re usually seen in tetrads • Staphylococcal endocarditis
• Septic arthritis
Stomatococcus
NOTES:
o Part of the normal oral microbiota
o Rarely isolated from infections • Most outstanding association of
o Colonies strongly adhere to the agar surface staphylococcus aureus would definitely be
o Rothia mucilaginosa (genus) is mostly wound, abscesses, and other skin infections.
associated with prosthetic device infections
 • When we culture wound, 80% of those o First, perform a gram stain smear to
samples would lead to staphylococcus identify the next possible test that
aureus. will lead to the final identification.
When the gram stain smear implies
gram-positive cocci in clusters, it can
Virulence factors of S. aureus (4) indicate that it’s a Staphylococcus.

• Coagulase – brings about the coagulation of o Do a catalase test (slide method) by

blood or plasma placing a 2-3 laps of 3% H2O2 on top
• Protein A – a cellular component in the cell of the slide; take the colony using a
wall that helps the bacteria to avoid loop to the H2O2. It should create
phagocytosis through neutralizing IgG strong bubbles, indicating the
• α, β, δ, γ Hemolysins – cytolytic toxins presence of catalase. Probably
- α (alpha) – lyse RBCs, damage platelets staphylococcus aureus because it is
and even macrophages positive. It can also be cultured in a
- β (beta) – sphingomyelins mannitol salt agar (MSA) because of
- γ (delta) – exotoxin little to high concentration of salt and
polymorphonuclear cells associated to mannitol can identify S. aureus from
Panton-Valentin leucocidin other Staphylococcus. If isolated
- δ (gamma) – least toxic among these bacteria ferment mannitol, it is said
cytolytic toxins to be S. aureus because other
• Exoenzymes Staphylococcus can’t produce
- Dehyaluronidase – allows the spread of yellow color. Yellow color represents
infection, removing the cement that glues the indicator of the isolated bacteria
connective tissues together, such as the in S. aureus.
hyaluronic acid • Cultural techniques
- Lipase – facilitates colonization of skin - Culturing on blood agar plates, growing as
surfaces round, smooth, white (sometimes
- Other toxins can produce by S. aureus such yellowish pigment) and beta-hemolytic
as exfoliative toxin, enterotoxins (completely lyses RBCs where it will grow)
Methods of Identification of Microorganisms (PIG) - Clearing at the bottom of the plate
• Biochemical identification by coagulase test or
• Phenotypic – microscopic and macroscopic catalase test
investigations that will show the morphology - S. aureus will be catalase positive using
of the different microorganisms; 5 I’s; any hydrogen peroxide. Catalase is needed to
observable characteristics that you can label convert hydrogen peroxide into the non-
and identify different microbes toxic form water and oxygen. The oxygen
• Immunological – serological analysis that is present in the reaction will be
employing antigen-antibody reactions that re observed by a bubbling that will produce
used in lab kits. Serotyping is also when you subject the colonies to 3%
immunological hydrogen peroxide.
• Genotypic – genetic techniques such as - Another technique is by use of serological
nucleic acid probes, PCR, rRNA analysis, means by the use of plasma coated latex
plasmid fingerprinting, etc. particles
2 Types of Coagulate Methods
Laboratory Identification of Staphylococcus aureus
- Cell-bound coagulase (slide method) –
• Microscopic examination – subjecting the detects clumping factor in the surface of
cultural isolates of a probable S. aureus and bacterial cells. The plasma fibrinogen in
making a smear, you will find the characteristic presence of the clumping factor will
clustering of the cocci can be observed convert fibrinogen into fibrin
- Laboratory isolation Fibrinogen –clumping factor -> Fibrin
- Free coagulase (tube method) – detects
thrombin-like molecule called coagulase
 reacting factor (CRF). The plasma contains • Bacitracin susceptibility (0.04 U Bacitracin)
the fibrinogen and the bacteria react with - Mueller-Hinton agar is streaked with
the Staphylocoagulase (extracellular suspected Micrococci, and place 0.04 U of
molecule that will react with CRF) which is Bacitracin and incubate it overnight.
now a thrombin molecule that will convert • Furazolidone resistance (100μg)
it into fibrin. - Micrococci is resistant to furazolidone
Fibrinogen –coagulase-CRF complex ->
Fibrin Coagulase-Negative Staphylococcus
Place the plasma in the tube, and put the • Found as normal flora in humans and animal
inoculums of suspected Staphylococcus, • If the staphylococcus is not aureus, then most
incubate to 35-37°C. Check for the presence probably its coagulase-negative
of clot after four hours. If there is no clot, staphylococcus
incubate the tube at room temperature • Often are nosocomial infections – acquired
and read the next day if there is clot during the process of healthcare
formed. If a clot appears, it is positive for • Predisposing factors include catheterization,
coagulase. prosthetic device implants and
Read the test in four hours because it immunosuppressive therapy
produces fibrinolysis that can lyse the clot
• Most common species:
within four hours, or else it would give a o S. epidermidis (normal skin
false negative test.
microbiota)
- Do both methods above because 5% of S.
o S. saprophyticus (associated with
aureus do not produce clumping factor.
UTIs in sexually-active, young
- S. aureus will be catalase positive, females)
converting H2O2 into its non-toxic form,
• S. haemolyticus, S. lugdunensis (occasionally
H2O and O2 (bubbling reaction)
produce clumping factors and may yield a
• Use of selective media from heavily
positive slide test)
contaminated specimens (MSA, PEA, CNA)
• S. schleiferi occasionally cause wide range of
Mannitol Salt Agar (MSA)
infections
Phenylethyl Alcohol Agar (PEA)
• Other species are not isolated frequently
Columbia colistin-nalidixic acid (CNA)
Laboratory Identification of CNS
Micrococcus
• Culture on sheep’s BAP
- Environmental organisms
• Coagulase negative urine isolates are further
- Normal microbiota of skin and respiratory
tested to presumptively identify S.
tract
saprophyticus using novobiocin susceptibility
- Common contaminants
• Various commercial identification systems may
- Coagulase-negative
be used.
Laboratory Identification of Micrococcus • Most commonly encountered species are
epidermidis and saprophyticus
• Modified oxidase test.
- Uses 6% tetramethyl phenylenediamine Antibiotic susceptibility
hydrochloride in dimethyl sulfoxide
- Using sterile forceps, transfer a Microdase • P resistance is high, especially in S. aureus
disk from the stock bottle to a petri dish isolates (85-90%)
- Using a wooden applicator stick, rub a • A common resistance is production of β-
small amount of several colonies of an 18– lactamase
24-hour pure culture grown on blood agar • Various β-lactamase resistant P have been
onto the top of microdase disk developed.
- Incubate at room temperature for 2 Methicillin is the most frequently used.
minutes Oxacillin is used for in-vitro susceptibility
- Observe for color development. Deep blue testing of methicillin resistance.
color demonstrates positive reaction.
 • MRSA, MRSE (or ORSA/ORSE) are agents of Streptococcus pyogenes
serious nosocomial and community associated
• Cell wall contains the Lancefield group A
infections (aureus, epidermidis)
carbohydrate
• MRSA – methicilin-resistant
• Group A strep or BHS group A
staphylococcus aureus
• M Protein
• MRSE – methicilin-resistant
o Attached to peptidoglycan
staphylococcus epidermidis
o Essential for virulence
• ORSA – oxacillin-resistant
• Colonize the throat and skin of humans
staphylococcus aureus
• ORSE – oxacillin-resistant Virulence factors of Streptococcus pyogenes
staphylococcus epidermidis
- M protein causes the bacterial cells to
resist phagocytosis by enabling it to adhere
to mucosal cells
- F protein – fibronectin binding protein,
facilitates adhesion to epithelial cells
- Lipoteichoic acid secures attachment of
cells to oral mucosal cells (w/ M proteins
and fibronectin)
- Hyaluronic acid capsule – prevents
opsonization
- Extracellular products: (6)
o Streptolysin-O causes hemolysis
of RBCs, platelets, WBCs,
(antibodies can be produced,
called ASO), highly immunogenic,
oxygen labile
o Streptolysin-S lyses WBCs but
not immunogenic (antibodies are
not created), oxygen stable
Differentiation between Staphylococci and
o DNAse – all strains have this,
Micrococci also may be important because both may
most common is DNAse B
be similar in microscopic morphology.
because they’re antigenic
TEST MICROCOCCI STAPHYLOCOCCI o Spes A, B, C & D (Erythrogenic
toxin) – immunological
OF TEST OXIDATIVE FERMENTATIVE
exotoxins, responsible for red
MODIFIED POSITIVE NEGATIVE
rashes, scarlet fever
OXIDASE TEST
o Streptokinase – causes lysis of
BACITRACIN SENSITIVE RESISTANT
SUSCEPTIBILTY fibrin clots, antibodies can be
FURAZOLIDONE RESISTANT SENSITIVE detected after infection
SUSCEPTIBILITY o Hyaluronidase spreading factor,
solubilizes ground substance in
mammalian CT
Streptococcus, Enterococcus and
Infections of Streptococcus pyogenes
Related Genera
- Pharyngitis (strep throat) & Tonsilitis –
• Members are catalase-negative, gram-positive frequently seen in children ages 5-15; if
cocci arranged in pairs or in chains ever the test is negative then culture is
recommended
• Facultative anaerobes
- Skin infections (e.g. impetigo,
• Use of enriched medium or blood is necessary necrotizingfasciitis, etc)
for their isolation - Scarlet fever – red rash appear on the
• Hemolysis patterns are helpful in the upper chest and it will spread in the trunk.
identification - Rheumatic fever (consequence of
 untreated pharyngitis) and ➢ Disk method (rapid)
glomerulonephritis o Wet the PYR test disc on the strip with
(immunocomplexes during episodes 10 µL sterile distilled water or
of pharyngitis/antigen-antibody deionized water (do not flood the
reaction; bind in the glomeruli of the disk) (YOU NEED TO MOISTEN
kidneys and damaging it resulting into MUNA)
kidney disease) o Put 5-10 colonies of the tested strain
- Streptococcal TSS – entire organ from 18- 24 hours culture on the
system shuts down that can lead to surface of the disc with a loop and
deaths. This happens during very smear them lightly on it
severe streptococcal infections where o Incubate the disc for 1-2 minutes at
it disseminates into different organ room temperature
systems o After incubation, add 1 drop of N, N-
Laboratory Identification of S. pyogenes dimethylaminocinnamaldehyde
• Culture – collecting swab sample and o Observe for red color development
within 1-2 minutes
culturing in sheep’s blood agar (small,
transparent, smooth, sometimes beta-
hemolytic) Streptococcus agalactiae
• Gram’s stain – gram-positive cocci in chains, • Cell wall contains Lancefield group B
catalase- negative carbohydrate
• Bacitracin susceptibility (Taxo A) • They have specific capsular polysaccharide -
o Bacitracin is a drug very useful for sialic acid
treating superficial skin infections. Also • Group B Streptococcus or GBS (loss of sialic
antibiotic but it is too toxic forsystemic acid in capsules, it’s loss will result to loss of
use; polypeptide antibiotic produced virulence)
by Bacillus subtilis • Found as normal microbiota of the
- Interferes with the peptidoglycan genitourinary tract
synthesis of bacteria
- Create a lawn inoculum of the suspected Virulence factors Streptococcus agalactiae
- Capsule very important virulence factor
bacteria, swab it 3 times and put Taxo A (polysaccharide which main components
and incubate. The next day read it. sialic acid)
• Resistant to sulfamethoxazole (SXT) - Other products (which do not play role in
- Just like Bacitracin susceptibility virulence)
• Pyridium oral (PYR) test o Hemolysin
- It can be test in test tube or use filter o Christie-Atkins-Munch-Peterson
papers (CAMP) factor – enlarges the area
- Rapid method for presumptive of hemolysis
identification of bacteria based on the o Others (DNAse, hyaluronidase,
pyrrolidinyl aryl amidase enzyme protease) have not beenshown to
be factors in infection
- The enzyme L-pyrrolidinyl aryl amidase
hydrolyzes the L-pyrrolidinyl-β-
naphthylamide substrate to produce a β- S. agalactiae infections
naphthylamine • Neonatal sepsis soon after birth
- β-naphthylamine can be detected in the • Part partum fever and sepsis
• Early onset – pneumonia, sepsis
presence of 0.1% N, N-methylamino
• Late onset – meningitis, sepsis
cinnamaldehyde reagent by the
production of a bright red precipitate
Laboratory identification of S. agalactiae
- WAYS TO DO THE PYR TEST
➢ Broth method • Culture – gram positive long chain; in clinical
o Inoculate PYR broth with 3-5 colonies specimens, they will appear short chains such
as in genitourinary tract swab, sheep’s blood
from18-24 hours pure culture
agar – grayish white surrounded by beta
o Incubate the tube aerobically at 35- hemolysis
37°C for 4 hours • Gram’s stain – gram-positive cocci chain,
o Add 3-4 drop of PYR reagent and catalase-negative
observe forcolor change • CAMP test positive (Christie–Atkins–Munch-
o Observe for red color development Peterson) – secreting a protein called CAMP
within 1-2 minutes factor (protein B)
 - When placed perpendicularly with S. • Group G – S. canis, S. anginosus, S. milleri
aureus, it will form arrowhead • These groups produce variety of infections
hemolysis (enhanced hemolysis) that includingpharyngitis
synergistically reacts with the beta- • Extensive use of biochemical reagents are
lysin of sphingomyelinase of S. aureus needed toidentify these groups
• Very costly to use because you need to use
biochemical reagents.
• Latex agglutination test

Group D
• Include S. bovis (1ST INDICATION OF TUMOR) and
S. equinus
• Found as normal intestinal microbiota
• May be agents of bacterial endocarditis,
- Enhanced lysis RBC (left side – UTI’s, abscesses and wound infections
positive); (right side- negative kasi
walang arrowhead eh) - There are associations with bacteremia
• Hippurate hydrolysis positive – not only caused by S. bovis and GIT tumor.
it can identify S. agalactiae, it can also - Isolation of S. bovis from the GIT indicates
detect Campylobacter jejuni, Listeria presence of tumor.
monocytogenes, and Gardnerellavaginalis.
It detects the ability of the organism to Laboratory Identification of Group D Streptococcus
hydrolyse Hippurate.
- Add 0.1mL of sterile water to a • α or γ/non hemolytic
12x75mm plastic test tube • Positive bile esculin test – positive reaction
- Make a heavy suspension of the is production of black precipitate due to
organism to be tested hydrolysis of the reagent esculin, in the
- Using heated forceps, place a rapid presence of bile salts
Hippurate disk in the mixture - BILE ESCULIN TEST - With an
- Cap and incubate the tube for 2 hours inoculating wire or loop, touch two or
at 35°C; the use of a water bath is three morphologically similar
preferred streptococcal colonies and inoculate
- Add 0.2mL ninhydrin reagent and re- the slant of the bile esculin medium
incubate for an additional 15 to 30 with an S-shaped motion, or streak the
minutes surface of bile esculin plate for
- Observe the solution for the isolation
development of deep purple color - Incubate the inoculated tube at 35-37°C for
- It detects the ability of the organism to 24 hours and then observe the results
hydrolyze Hippurate. - esculetin – form black layer
- Purple end product – positive (ATCC • No growth at 6.5% NaCl broth – if the
12386) microorganism is able to withstand the salt,
- Negative - ATCC 19615 if there is turbidity, then it is not a group D
- Must have positive and negative Strep. They should be clear
control
• PYR negative
- ATCC – American type culture
• Serotyping – using latex agglutination test
connection, collections of purely
purified bacteria
• Resistance to SXT Enterococcus
• PYR negative • Found in the intestinal tract
• Most common is E. faecalis. Other members
Groups C and G are E.faecium, E. avium, E. durans
• Hemolytic species in Lancefield group that • Share characteristics of Group D, including
are occasionally isolated from clinical the antigen D
specimens • Show resistance to several of the commonly
• Part of the normal skin microbiota usedantibiotics
• Group C – S. equi, S. zooepidemicus and S. • Has similar infections to Group D, the most
equisimilis common being UTI
 • Bile solubility test – pneumonia is lysed by
Laboratory Identification of Enterococcus bile. Bile salts will lower the surface
tension between the bacterial cell
• BE positive, 6.5% POSITIVE, PYR positive
membrane and the medium. Once the
• SXT resistant
cell membrane is destroyed, it will
• Should be screened for high-level
accelerate the organism’s autolysis.
aminoglycoside- resistant Enterococci
• Quellung test – microscopic test in which
(HLARE)
capsule surrounding the pneumococci
• Vancomycin-resistant Enterococci (VRE) is a
will appear like it is swelling
major concern
o Suspension of pneumococci
Streptococcus pneumoniae o Mixed on a slide with a drop of
• The cell wall doesn’t have the usual the type-specific antiserum
carbohydrate group by Lancefield. o Add a loopful of methylene blue
Rather, it has six layers composed of solution
peptidoglycan and teichoic acid o On microscopy, capsule (seen as
attached to N-acetylmuramic acid, very clearing around bacterial cell)
similar to Group C Lancefield groups becomes apparently swollen,
• Often part of the normal flora of the sharply delineated and refractile
upper respiratory tract (URT) Viridans Streptococci
• Key virulence factor is an anti-phagocytic • Include those α-hemolytic streptococci
capsule (greenish hemolysis, partial hemolysis) that
• There are approximately 80-90 lack Lancefield group antigens and do not
antigenic capsule types meet the criteria for S. pneumonia
• An important human pathogen- • Part of the normal microbiota of the
causing pneumonia, sinusitis, otitis oropharynx and the intestine
media, bacteremia, and meningitis • Fastidious and require increased CO2
• Direct smears often reveal leukocytes • Frequent cause of subacute bacterial
and numerous gram-positive endocarditis (SBE)
diplococci with the ends being slightly • S. mutans, S. salivarus, S. anginosus, S. mitis, S.
pointed, giving a lancet-shaped bovis – not Beta- hemolytic
appearance • NVS (nutritionally variant streptococcus) have
been isolated from patients who have
• Grows on sheep’s blood agar as α-
endocarditis and otitis media
hemolytic, and β- hemolytic when
o Also known as pyridoxine-
incubated anaerobically
dependent (vitaminB6 dependent)
• Complex media such as:
o Vitamin B6 is essential for their
o brain-heart infusion agar growth
(BHIA) • Positive gram-stain, negative culture
o trypticase soy agar (TSA) with
5% blood Laboratory Identification of Streptococci
o chocolate agar (CAP)
o necessary for good growth • BAP hemolysis
• Isolates may require increased CO2 • Bile solubility – S. pneumonia is bile-soluble
(capnophilic) • Optochin (ethylhydrocuprein hydrochloride/Taxo P)
• Colonies are α-hemolytic susceptibility: ≥14mm with 5µg disk
• Young cultures produce a round, • Bacitracin (Taxo A)
glistening, wet, mucoid, dome-shaped • Group A and B resistant to SXT
appearance • Camp test presumptively identified group B
Streptococcus
Laboratory Identification of Streptococcus • Esculin hydrolysis
pneumonia • 6. 5 NaCl
• PYR hydrol
• Susceptibility to optochin (Taxo P) –
• LAP Test (leucine aminopeptidase) helps
inhibits production of ATP in differentiate Aerococcus and Leuconostoc
microorganisms. It makes the cellwall of from other Strep species
pneumonia to become fragile that when • Serologic testing for the detection of C-
the cell membrane becomes weak, the carbohydrate of the cell wall
pneumonia lyses
- ≥14mm diameter zone of inhibition
Streptococcus-like organisms • Pneumococcal isolates are treated with
• Aerococcus Erythromycin in case of P-resistant
- Common airborne organism • Linezolid is being prescribed to VRE infections
- Associated with bacteremia, endocarditis,
UTI in immunocompromised patient
- Weak catalase/pseudocatalase
- Grows in 6.5% NaCl
- Species:
➢ Aerococcus viridans
o Bacteria and endocarditis
o Bile-esculin (+)
o PYR (+)
➢ Aerococcus urinae
o UTI, endocarditis, lymphadenitis,
peritonitis
o Bile-esculin (-)
o PYR (-)
• Leuconostoc
- Cross react with anti-group D
- Intrinsically resistant to vancomycin
- Opportunistic pathogens
- Associated with meningitis, bacteremia,
UTI, pulmonary infections
- Catalase, PYR, LAP (-)
- Bile-esculin (+)
- 6.5 NaCl (+)
- Significant species:
➢ L. citreum
➢ L. cremoris
➢ L. dextranicum
➢ L. conostoc lactis
➢ L. mesenteroides
➢ L. pseudomesenteroides
• Pediococcus
- Facultatively anaerobic
- Grow at 45 degree Celsius
- Intrinsically resistant to vancomycin
- Associated with bacteremia, abscess
formation and meningitis
- Bile-esculin, LAP (+)
- PYR (-)
- Gas from glucose (-)
- Species associated:
➢ P. acidilactici
➢ P. damnosus
➢ P. dextrinicus
➢ P. parvulus
➢ P. pentasaceus

Treatment of Streptococcal and Enterococcal

infections
• Most species are susceptible to Pencillin (P)
• S. agalactiae is less susceptible than Group A
and may require a combination of Amp and
an aminoglycoside
• Group D is susceptible to P
• Enterococcus is usually resistant to P
• Enterococcus is usually treated with synergistic
Amp-aminoglycoside combination
 Pathogenic Neisseria Virulence factors
• Receptors for human transferrin
• Capsule – polysaccharide capsule (N.
meningitidis)
• Pili (T1-T5) distinct colony types
o piliated types (T1, T2)
o non-piliated(T3-T5).
o Presence of pili indicate
pathogenicity.
• Cell membrane proteins
• Protein I – forms channel for nutrient to
pass and waste products to exit
• Encoded by two genes – porA & porB
(protective against host
inflammatory response)
• N. meningitidis has porA and
porB
• N. gonorrhoeae has porB only
• Protein II – opacity and facilitate
adherence to phagocytic and epithelial cell
• Protein III – reaction-modified protein
(RMP) blocks serum IgG against organism
• Lipo-oligosaccharide (LOS) – antigenic
Neisseria and Moraxella Species variation
General Characteristics • IgA protease – render immunoglobulin A
inactivated
• Gram-negative cocci often in pairs
• Kidney-bean shaped, capnophilic Neisseria gonorrheae
• Oxidase-positive, catalase-positive except N. • Humans are the only host
elongate and N. bacilliformis
• Causative agent gonorrhea
• Differentiation is based on acid
o Acute pyogenic infection of non-
production from carbohydrate
ciliated columnar and transitional
utilization test
epithelium
• Pathogenic species have fastidious gr
o Acquired by sexual contact
owth requirements
o Occur in urethra, endocervix, anal
• Proper specimen collection is essential for
canal, pharynx, conjunctiva
successful isolation o Also called “clap” from French word
• Mucous membrane of urogenital tract
clapoir meaning “brothel”
(Neisseria)
• Mucous membrane of upper respiratory
Neisseria gonorrheae Infections
tract (Moraxella and some Neisseria)
• Has short incubation period approx. 2-7 days
• Neisseria gonorrhoeae (gonococci) and
• Gonorrhea
Neisseria meningitidis (meningococci) are the
- May cause sterility in males and females
primary human pathogens of the genus - Males show symptoms of burning and
o N. gonorrhoeae – always pathogenic discharge from the urethra, strains with
o N. meningitidis – found as commensal nutritional requirement for arginine,
inhabitant of URT hypoxanthine and uracil (AHU strains) have
• Fastidious organisms, requiring enriched been isolated for asymptomatic men
media for recovery, sensitive to unfavorable - Females – endocervix most common
environment conditions site, symptoms include dysuria,
o N. gonorrhoeae and N. meningitidis cervical discharge and lower abdominal
require iron for growth pain, may be asymptomatic but can lead
▪ Compete with human host by to pelvic inflammatory disease (PID)
binding transferrin to specific • Opthalmia neonatorum
receptors o Newborn illness that is acquired during
vaginal delivery
o Gonococcal eye infection
Laboratory Identification of Neisseriae gonorrheae Laboratory Identification of Neisseria meningitidis
• Gram’s stain and location (IC/EC) – gram- • Direct microscopic count of CSF sediment
positive; gram-negative diplococci located revealing gram-negative diplococci
intracellularly or extracellularly of • Culture CSF, blood and joint fluid
neutrophils specimens
• Culture using selective agars (Thayer Martin, • Nasopharyngeal swabs are cultured to
Modified Thayer-Martin, Martin-Lewis and detect carriers
New York City) packaged in self- contained • Growth can be seen in BAP and CAP
transport systems such as JEMBEC, enhanced with 5- 10% of CO2
Transgrow, GonoPak – allow to have 5% CO2
so they won’t die Moraxella catarrhalis
• Produces acid from glucose utilization • Part of the normal URT
(fermentation) but not for maltose, sucrose, • May cause otitis media, sinusitis, and
and lactose – MLS respiratory infections
GLUCOSE-MALTOSE-SUCROSE-LACTOSE • Grows on blood, chocolate, and nutrient
agars unlike Neisseria that are very fastidious
• Able to reduce nitrate to nitrite
• Produces DNase
• Non-pathogenic upper respiratory tract

Laboratory Identification of Moraxella catarrhalis

• Specimen collection and identification
o Can be collected from middle ear
effusion, nasopharynx, sinus
aspirates, sputum aspirates or
bronchial aspirates (SSB)
o Grows in SBA and CHOC
Yellow-positive o Oxidase and catalase positive
Red-negative o Assacharolytic and positive DNase and
Butyrate esterase
• Rapid carbohydrate degradation tests
• Chromogenic substrates
• Immunologic assays
• Nucleic acid assays

Neisseria meningitidis
- Found only in humans
- Can be commensal and invasive pathogen
- Recovered from urogenital and rectal sites
- Found on mucosal surface of nasopharynx and
oropharynx
- Transmitted by respiratory droplet secretions
- Serogroups A, B, C, Y and W-135 account for
most cases of disease in the world

Neisseria meningitidis infections

• Meningococcal meningitis
• Meningococcemia (sepsis)
• Droplet transmission, onset is abrupt
• Conjunctivitis and urethritis – rare

Virulence factors of N. meningitidis

• Pili
• Capsule
• PO
• POR, OPA, RMP
• LOS endotoxin
ENTEROBACTERIACEAE On blood agars, all organisms can grow. Application of
inhibitory medium so gram positive will not grow any longer on
Key Characteristics the medium by incorporation of bile salts. They will lyse and
not grow.
 Large family of gram-negative bacilli/coccobacilli that
live in the intestine On McConkey agar, there are bile salts that would inhibit the
 Non-spore forming, facultatively anaerobic bacilli gram positive bacteria in growing. You are just confined to
 Cytochrome oxidase negative, except Plesiomonas identifying gram negative bacteria.
 All are glucose-fermenting
 All reduce nitrate to nitrite, except for Photorhabdus Example: If the organism is E. coli, it will grow in blood and
and Xenorhabdus MAC agar. In blood agar, it appears gray, moist colonies. In
o Usually in urine MAC agar, it appears as pink colonies. If differentiated with
 All are motile at body temperature, except for Staphylococcus spp, it will only grow on blood agar.
Klebsiella, Shigella, and Yersinia
EMB is used in place of MAC.
 Facultative anaerobic bacilli – can survive with or
without oxygen Should use a general medium and selective medium before
 Non-fastidious, does not need blood supplement to determining further work up.
grow
Hydrogen sulfide production:
 Most are non-encapsulated
 None has remarkable colony morphology on Virulence and Antigenic Factors
supportive media, appearing large, moist, and gray on
SBA, CHOC, and most non-selective media; except - Controlled by a number of factors such as ability to
Klebsiella, Proteus, and some Enterobacter species adhere, colonize, produce toxins, and invade tissue
- Some harbor plasmids that can provide antimicrobial
Microscopic and Colony Morphology resistance genes
- Increasing numbers of E. coli, K. pneumoniae, and K.
- Colony morphology on non-selective media, such as
oxytoca clinical strains produce plasmid-mediated
SBA and CHOC are of little value in their identification
extended-spectrum β-lactamases (ESBLs) which can
- A wide variety of differential and selective media such
inactivate extended-spectrum cephalospporins (e.g.
as MAC, highly selective media such as HE and XLD
cefotaxime), penicillins, and aztreonam
are available for presumptive identification of enteric
- Antigens used in the identification of different
pathogens
serologic groups
- Contain one or more carbohydrates such as lactose
 O antigen (somatic) – heat-stable, located
and sucrose, which show the ability of the species to
on the cell wall
ferment specific carbohydrates
o Fermentation is indicated by a color change  H antigen (flagellar) – heat-labile, surface of
in the medium as a result of a drop in pH by flagella
a pH indicator in the medium  K antigen (capsular) – heat-labile
- Species that produce H2S may be readily polysaccharide found only in certain
distinguished when placed on HE or XLD agar. encapsulated species
- HE and XLD agars contain sodium thiosulfate and  K1 antigen of E. coli
ferric ammonium citrate, which produce blackening of  Vi antigen of Salmonella enterica
H2S-producing colonies subsp. enterica serotype Typhi. It is
a special name for Salmonella
To identify: Provide a culture medium, grows on any simple capsular antigen.
medium (basal) because they are non-fastidious. Media should
have selective properties for easier identification. Clinical Significance

Lactose fermenter or non-fermenter: applying sugar to the - Most enteric reside in the GI tract except for
plate. Microorganisms should turn into red or pink. If not, they Salmonella, Shigella, and Yersinia
are colorless. - Two broad categories
  Opportunistic pathogens – often a part of the Opportunistic Members of the Family Enterobacteriaceae
usual intestinal microbiota of both human and Associated Infections
and animals. Outside their normal body
sites, however, may produce serious  Escherichia coli
extraintestinal, opportunistic infections (e.g.  Klebsiella
E. coli)  Enterobacter, Cronobacter, and Pantoea
 Primary pathogens – true pathogens; not  Serratia
present as commensal biota in the human GI  Hafnia
tract and produce infections resulting from  Proteus
ingestion of contaminated food or water, or  Morganella
from other sources (e.g. S. enterica, Shigella  Providencia
spp., Yersinia spp.)  Edwardsiella
 Erwinia and Pectobacterium
Two Major Divisions
 Citrobacter
1. Lactose Fermenters (pink) – E. coli, Kleibsiella ,
Enterobacter, etc. Escherichia coli
2. Non-lactose Fermenters (colorless) – Salmonella
- Most significant species in the genus Escherichia
Shigella, Proteus, Morganella, Yersini. This includes
- Initially considered a harmless member of colon
the pathogenic enterics.
resident biota
Bacterial Species and the Infections they Commonly - Causes UTIs, CNS infections, diarrheal diseases
Produce - Primary marker of fecal contamination in water
purification
 Escherichia coli - Bacteriuria, septicemia, neonatal - Most strains are motile and generally possess
sepsis, meningitis, diarrheal syndrome adhesive fimbriae and sex pili, and O, H, & K antigens
 Shigella spp. - Diarrhea, dysentery  O groups have shown remarkable cross-
 Edwardiella spp. - Diarrhea, wound infection, reactivity with O antigens from other
septicaemia, meningitis, enteric fever members of Enterobactriaceae, most notably
 Salmonella spp. - Septicaemia, enteric fever, diarrhea the Shigella
 Citrobacter spp. - Opportunistic and nosocomial  Serotyping for O and H antigens is often
infections (wound, urinary) useful in identification of strains, particularly
 Klebsiella spp. - Bacteriuria, pneumonia, septicaemia those associated with serious enteric
 Enterobacter spp. - Opportunistic and nosocomial disease
infections, wound infections, septicemia, bacteriuria  K1 antigen – identical to capsular antigen
 Serratia spp. - Opportunistic and nosocomial found in Neisseria meningitides group B
infections, wound infections, septicemia, bacteriuria - Appears as a lactose-positive (pink) colony with a
 Proteus spp. - Bacteriuria, wound infections, surrounding area of precipitated bile salts on MAC
septicaemia agar
 Providencia spp. - Opportunistic and nosocomial - Appears with a green metallic sheen on EMB agar
infections, wound infections, septicemia, bacteriuria with the following properties:
 Glucose, lactose, trehalose, and xylose
 Morganella spp. - Opportunistic and nosocomial
fermentation
infections
 Indole production from tryptophan
 Yersinia pestis – Plague pseudotuberculosis
- Glucose fermentation by mixed acid pathway: methyl
Mesenteric adenitis, diarrhea enterocolitica
red positive, Voges-Proskauer negative
Mesenteric adenitis, diarrhea
- Does not produce H2S, DNase, Urease or
 Erwinia spp. - Wounds contaminated with soil or
phenylalanine deaminase
vegetation
- Can’t use citrase as sole carbon source
 Pectobacterium spp. Wounds contaminated with soil
or vegetation
Uropathogenic E. coli - Non-lactose fermenter
- Diarrheal illness is similar to that of Shigella spp.
- Most common cause of UTI in humans although infective dose of EIEC is much higher
- Causes acute pyelonephritis - Occur in adults and children alike
- Strains that cause UTI produce factors that allow - Fever, severe abdominal cramps, malaise, and
them to attach to the urinary mucosa (pili) and not be watery diarrhea
washed out by urine - Strains can be non-motile and generally don’t ferment
- Cytolysins (hemolysins) – kill immune factor cells and glucose
inhibit phagocytosis and chemotaxis of certain WBCs - Does not decarboxylate lysine
- Aerobactin – chelates iron - Sereny test – ability to produce keratoconjunctivitis in
Gastrointestinal Pathogens the guinea pig
- It is also possible to detect invasiveness using
- Major categories of diarrheagenic E. coli based on monolayer cell cultures with HEp-2 cells
definitive virulence factors, clinical manifestation, - Infective dose is 106
epidemiology, and different O and H serotypes
Enteropathogenic E. coli (EPEC)
Enterotoxigenic E. coli (ETEC)
- Causes infantile diarrhea/children’s diarrhea
- Associated with diarrhea of infants and adults in - Only certain H antigenic types within each O
tropical and subtropical climates, especially in serogroup are connected to intestinal infections
developing countries - O serogrouping can’t differentiate this E. coli strain
- Major causes of infant bacterial diarrhea from strains of normal biota
- Traveler’s diarrhea - Invades the brush border containing microvilli, which
- Bacteria adheres to epithelium of small intestine with becomes lost, affecting the absorption of fluids,
adhesive factors such as fimbriae, and produces resulting into diarrhea
toxins, resulting to symptoms such as nausea, - Low-grade fever, malaise, vomiting, diarrhea
vomiting, loose stools - Stool contains mucus, no blood present
- Commonly spread though consumption of - Diagnosed by serotyping
contaminated food or water, poor hygiene, reduced
availability of sources of potable water, inadequate Enterohemorrhagic E. coli (EHEC)
sanitation - O157:H7 (most invasive) strain associated with
- High infective dose (106-1010 organisms) to initiate hemorrhagic diarrhea, colitis, and hemolytic uremic
disease in an immunocompetent host syndrome (HUS)
- Stomach acidity inhibits colonization and initiation of - Bacteria causes damage in kidneys, causing bleeding
disease  HUS is characterized by low platelet count,
- Colonization on proximal small intestine is mediated hemolytic anemia, and kidney failure
by fimbriae that permits binding on intestinal microvilli  Attaches to endothelial cells in kidneys
- Heat-labile toxin (LT) – similar in action and amino - Watery diarrhea that progresses to bloody diarrhea
acid sequence to cholera toxin from V. cholera ( with abdominal cramps, low-grade fever or absence
causes accumulation of cAMP) of fever, no WBCs, distinguishing it from dysentery
 LT fragment A (A subunit) – enzymatically caused by Shigella spp. or EIEC infections
active portion, contains the toxins - Sorbitol negative
 LT fragment B (B subunit) – moiety, binding - Causes of many deaths
portion, confers specificity, binds to the
mucosa and providing entry for A portion EHEC cytotoxins

Enteroinvasive E. coli (EIEC)  Verotoxin I – phage-encoded cytotoxin identical to

Shiga toxin (Stx) produced by S. dysenteriae type I
- Produce dysentery with direct penetration, invasion, o Produces damage to Vero cells (African
and destruction of the intestinal mucosa green monkey kidney cells)
 o Reacts with and is neutralized by the vagina is heavily colonized. Infection may also result if
antibody against Stx contamination of the amniotic fluid takes place
 Verotoxin II – biologically similar to, immunologically - Capsular antigen K1 is the most documented
different from, both Stx and verotoxin I virulence factor associated with neonatal meningeal
o Not neutralized by antibody to Stx infections
o Verotoxin producing E. coli may be identified - E. coli bacteremia in adults may result primarily from
by one of three methods a urogenital tract infection or from a GI source
 Shiga toxin-producing E. coli
o Produces Stx1 and Stx2 which are typically Other Escherichia Species
produced by Shigella dysenteriae
 E. hermanii (formerly E. coli atypical/enteric group II) -
- Stool culture on highly different medium, with
yellow-pigmented organism isolated from CSF,
subsequent serotyping
wounds, and blood
- Detecting verotoxin in stool filtrates
 E. vulneris – infected wounds, more than half the
- Demonstration of a fourfold or greater increase in
strains also produce yellow colonies
verotoxin-neutralizing antibody titer
 E. albertii – diarrheal disease in children
- Stool culture for O157:H7 may be performed using
MAC agar containing sorbitol (SMAC) instead of
Klebsiella and Raoutella
lactose. O157:H7 does not ferment sorbitol in 48
hours, and appears colorless - Klebsiella, Enterobacter, Serratia, Pantoea,
Cronobacter, Hafnia
Enteroadherent E. coli (EAEC)
- Usually found in the intestinal tract of humans and
- Generally associated with two kinds of human animals or free-living in soil, water, and on plants
disease: diarrheal syndromes and UTIs persistent - Associated with a number of opportunistic and
diarrhea, enterotoxin producer nosocomial infections (e.g. pneumonia, wound, UTI)
- Common characteristics:
Two types of EAEC  Most grow on Simmons citrate and in
potassium cyanide broth
 DAEC – associated with both UTIs and diarrheal  None produce H2S
disease  Few hydrolyze urea slowly
o Uropathogenic strains are closely associated  Negative methyl red test
with cystitis in children and acute  Positive Voges-Proskauer test
pyelonephritis in pregnant women  With few exceptions, indole is not produced
o Chronic or recurring UTI from tryptophan
 EAEC – causes diarrhea by adhering to the surface of - Motility varies
the intestinal mucosa - Absence of motility distinguishes Klebsiella spp. From
o Adheres to HEp2 cells, packed in an most of the other members of Enterobacteriaceae
aggregative “stacked-brick” pattern on the
cells and between the cells by means of K. pneumoniae
fimbriae
o Watery diarrhea, vomiting, dehydration, - Most commonly isolated species
occasional abdominal pain, mostly in - Distinct feature of possessing a polysaccharide
children capsule, protecting against phagocytosis and
antimicrobial absorption, also responsible for moist,
Extraintestinal Infections mucoid colony appearance
- Capsule is sometimes helpful in providing
- Most common cause of septicaemia and meningitis presumptive identification
among neonates - Colonization in respiratory tracts of hospitalized
 40% of the cases of Gm – meningitis patients increases with the length of stay
- Newborn usually acquires infection in the birth canal
just before or during delivery, when the mother’s
 - Frequent cause of lower respiratory tract infections P. agglomerans
among hospitalized patients and in
immunocompromised hosts - Gained notoriety with a nationwide outbreak of
- Wound infections, UTIs, bacteremia, liver abscesses septicemia resulting from contaminated IV fluids
- Includes species that are lysine, ornitihine, and
K. oxytoca arginine negative or “triple decarboxylases negative”

- Identical to K. pneumoniae except for its production of P. agglomerans HG XII

indole and ornithine-positive isolates
- Linked to antibiotic-associated hemorrhagic colitis - May produce a yellow pigment
- Similar to K. pneumoniae infections - Primarily a plant pathogen

K. pneumoniae subsp. ozenae E. gergoviae

- Isolated from nasal secretions and cerebral - Found in respiratory samples and is rarely isolated
abscesses from blood cultures
- Highly-associated with the presence of plasmid-
C. sakazaki
mediated ESBLs, contributing to large numbers of
nosocomial infections seen today - Typically produces a yellow pigment
- Causes atrophic rhinitis – tissue-destructive restricted - Pathogen in neonates causing meningitis and
to the nose bacteremia, often coming from powdered infant
formula
K. pneumoniae subsp. rhinoscleromatis - Isolated from cultures taken from brain abscesses
- Isolates from patients with rhinoscleroma, an infection and respiratory wound infections
of the nasal cavity, manifesting as an intense swelling
E. hormaechei
and malformation of the entire face and neck
- Isolated from blood, wounds, and sputum
K. ornithinolytica
E. asburiae
- Indole positive
- Ornithine decarboxylase positive - Biochemically similar to E. cloacae
- Isolated from urine, respiratory tracts, and blood along - Isolated from blood, urine, feces, sputum, and
with K. planticola wounds

Enterobacter, Cronobacter, and Pantoea E. dissolvens and E. nimipressuralis

- Motile - Newly recognized species with unknown clinical

- Colony morphology resembles that of Klebsiella when significance
growing on MAC
E. cancerogenus (formerly E. taylorae)
- Grow on Simmons citrate medium and in potassium
cyanide broth - Associated with osteomyelitis following traumatic
- Methyl red negative wounds
- Voges-Proskauer positive
- Usually produce ornithine decarboxylase, unlike Serratia
E. cloacae and E. aerogenes - Opportunistic pathogens associated with nosocomial
outbreaks
- Most common isolates
- Ferments lactose slowly and positive for ortho-
- Wounds, urine, blood, CSF
nitrophenyl galactoside (ONPG) test, except S.
fonticola
 - Differentiate from other members of the tribe by their Proteus
ability to produce extracellular DNase
- Resistance to a wide range of antimicrobials - Normal intestinal microbiota and are recognized as
- Susceptibility tests must be performed on each isolate opportunistic pathogens
to determine appropriate microbial therapy - Distinguished from other members of
- Most significant: Enterobacteriaceae by virtue of the ability to
 S. marcescens deaminate phenylalanine
- Nosocomial infections - Isolated from urine, wounds, and ear and bacteremic
- UTIs infections
- RTIs - Does not ferment lactose
- Bacteremic outbreaks - Ascend the urinary tract, causing infections in both
lower and upper urinary tract
S. marcescens, S. rubidaea, S. plymuthica - Can infect proximal kidney tubules and can cause
acute glomerulonephritis, particularly patients with
- Often produce pink to red pigment, prodigiosin,
urinary tract defects or catheterization
especially when isolates are incubated at room
- Most common isolate: Proteus mirabilis
temperature
- S. marcescens is the most clinically significant P. mirabilis and P. vulgaris
- Frequently found in nosocomial infections of the
urinary and respiratory tract and in bacteremia - Produce “swarming” colonies on nonselective media
outbreaks in nurseries andcardiac surgery and burn such as SBA
units - Swarming is a result of regulated cycle of
differentiation from standard vegetative cells
S. plymuthica (swimmers) to hyperflagellated, elongated, polyploidy
cells (swarmers) capable of coordinated surface
- S. plymuthica osteomyelitis was found in a motorcycle
movement
accident
- Swarmer cells produce distinct odor described as
S. odorifera “burnt chocolate” and plays a role in the ascending
nature of Proteus-associated UTIs
- Contains two biogroups - Produces H2S and hydrolyzes urea
- Emits a dirty, musty odor resembling that of potatoes - P. mirabilis is ornithine positive and indole negative
- Biogroup 1 – isolated predominantly from respiratory - P. vulgaris is indole positive, ornithine negative and
tract and is positive for sucrose, raffinose, and ferments sucrose
ornithine, may be indole positive (60%)
- Biogroup 2 – negative for sucrose, raffinose and P. penneri
ornithine and has been isolated from blood and CSF,
- Can also swarm on nonselective media
may also be indole positive (50%)
- Isolated from patients with diarrhea, although the role
- S. liquefaciens, S. rubidaea, S. forticola
of organism in disease hasn’t been proven
Hafnia P. myxofaciens
- Isolated from a number of anatomic sites in humans - Isolated only from gypsy moths
and in the environment - Large amount of slime it produces
- Not known to cause gastroenteritis but is occasionally
isolated from stool cultures Morganella
- Delayed positive citrate reaction is a major
characteristic M. morganii
- Composed of one specie: H. alvei
- Two subspecies
- H. alvei biotype 1 – grows in the beer wort of
o M. morganii subsp. Morganii
breweries and has not been isolated clinically
o M. morganii subsp. Sibonii
 - A documented cause of UTI and has been isolated E. hoshinae
from other human body sites
- Neither species have been implicated in diarrheal - Isolated from snakes, birds, and water
illness E. ictaluri
Providencia - Causes enteric septicemia in fish

P. rettgeri Erwinia and Pectobacterium

- Documented pathogen of the urinary tract and has - Plant pathogens
caused occasional nosocomial outbreaks - Not significant in human infections
- Implicated in diarrheal disease among travelers - Erwinia grows poorly at body temperature and fails to
P. stuartii grow in selective media such as EMB and MAC
- Identification is more for academic interest than
- Implicated in nosocomial outbreaks in burn units and evaluation of their significance as causative agents of
has been isolated from urine cultures infection
- Infections caused by P. rettgeri and P. stuartii,
especially in immunocompromised patients, are Citrobacter
particularly difficult to treat because of their resistance
to antimicrobials - Most hydrolyze urea slowly and ferment lactose,
producing colonies on MAC agar that resemble those
P. alcalifaciens of E. coli
- All grow on Simmons citrate medium and give positive
- Most commonly found in feces of children with reactions in the methyl red test
diarrhea
- Role as cause of diarrhea hasn’t been proven C. freundii

P. rustigianii - Isolated in diarrheal stool cultures

- Pathogenic role remains unestablished
- Formerly identified as a strain of P. alcalifaciens - Associated with infectious diseases acquired in
- Rarely isolated hospital settings
- Pathogenicity also remains unproven - UTI, pneumonias, intra-abdominal abcesses have
been reported
P. heimbachae
- Associated with endocarditis in IV drug abusers
- Yet to be isolated from any clinical specimens - Because most (80%) C. freundii produce H2S, and
some strains (50%) fail to ferment lactose, the colony
Edwardsiella morphology on primary selective media can be easily
mistaken for that of Salmonella when isolated from
- Negative for urea stool cultures
- Positive for lysine decarboxylase, H2S, and indole - Differentiation can be done through urea hydrolysis
- Don’t grow on Simmons citrate and lysine decarboxylase
- Most (70%) hydrolyze urea, but all fail to
E. tarda
decarboxylase lysine, whereas Salmonella fails to
- Only recognized human pathogen hydrolyze urea and most isolates decarboxylate lysine
- Opportunist, causing bacteremia and wound
C. koseri
infections
- Pathogenic role in cases of diarrhea remains - Pathogen documented as the cause of nursery
controversial outbreaks of neonatal meningitis and brain abscesses
C. amalonaticus Biochemical Factors of Salmonella

- Frequently found in feces, but no evidence has been - In almost every case, they don’t ferment lactose
found that it is a causative agent of diarrhea - Indole negative
- Isolated from sites of extraintestinal infections, such - VP negative
as blood and wounds - Phenylalanine deaminase negative
- Urease negative
Primary Intestinal Pathogens of the Family - Most produce H2S, except Salmonella Paratyphi A
Enterobacteriaceae - Do not grow in medium containing potassium cyanide

Salmonella Virulence Factors of Salmonella

- Salmonella enterica (pathogenic) and Salmonella - Still remains uncertain

bonggori - Fimbriated strains appear more virulent than
- Exclusively a human pathogen. nonfimbriated strains
- Motile unlike Shigella - Ability to traverse intestinal mucosa
- Produces H2S - Enterotoxin produced by certain strains that cause
- Gram-negative, facultatively anaerobic bacilli that gastroenteritis is a significant virulence factor
morphologically resemble other enteric bacteria
- On selective and differential media used primarily to Antigenic Structures of Salmonella
isolate enteric pathogens (e.g. MAC), salmonellae
 Somatic O antigens and flagellar H antigens are the
- Produce clear, colorless, non-lactose fermenting
primary antigenic structures used in serologic
colonies
grouping of Salmonellae
- Colonies with black centers are seen if the media
 Few strains may possess capsular K antigens,
(e.g. HE or XLD) contain indicators for H2S
designated Vi antigen
production
 Serologic identification of Vi antigen is important in
- S. enterica causes typhoid fever, enteroclotis and
identifying Salmonella serotype Typhi
bacteremia
 Typhoid fever is a known infection  Heat-stable O antigen of salmonella is the
(salmonella enterica serotype typhi), where liposaccharide (LPS) located in the outer membrane
the stool is bloody, and the organism is now of the cell wall.
found in the blood or urine.  H antigens occur in one of two phases
 Liver may be inflamed, such that there are o Phase 1 flagellar antigens occur only in a
pale pink rose spots on the patient’s small number of serotype and determine the
abdomen. immunologic identity of the particular
 Gall bladder may also be inflamed, and may serotype; agglutinate only with homologous
store large number of the organisms. It is the antisera
major reservoir. o Phase 2 flagellar antigens occur among
 When they get well, they shed off the several strains; reacts with heterologous
organisms in their stools. antisera
 Fever has a pattern, such as spiking, which  Heat-labile Vi antigen is a surface polysaccharide
is a problem. Needs medications capsular antigen found in Salmonella serotype Typhi
- Enterocolitis, swelling of intestine, is same to and a few strains of Salmonella serotype
gastroencolitis. Choleraesuis
 Fever is lowgrade, and can resolve on its - Vi antigen often blocks the O antigen during
own within 4 days. serologic typing but may be removed by
 The typical reaction is diarrhea, nausea, heating
vomiting, headache, after 48 hours of
Clinical Infections of Salmonella
ingestion of sufficient number of organism.
In humans, salmonellosis may occur in several forms:
  Acute gastroenteritis or food poisoning characterized - “Rose spots” (blanching, rose-colored papules around
by vomiting and diarrhea the periumbilical region) appear during the second
 Typhoid fever, most severe of enteric fever, caused week of fever
by Salmonella serotype Typhi; and enteric fevers
Bacteremia
caused by other Salmonella serotypes (e.g.
Salmonella Paratyphi and Choleraesuis) - Involvement of reticuloendothelial system, particularly
 Nontyphoidal bacteremia the liver, spleen, intestines, and mesentery
- Carrier state following Salmonella infection - Dissemination to multiple organs
- With the exception of Salmonella Typhi and - With and without extraintestinal foci of infection
Salmonella Paratyphi, salmonellae caused by nontyphoidal Salmonella, is characterized
organisms infect various animals that serve primarily by prolonged fever and intermittent
as reservoirs and sources of human bacteremia
infections - Serotypes most commonly associated are
- Salmonella Typhi and Paratyphi have no Typhimurium, Paratyphi, and Cholerasuis
known animal reservoirs; infections only
seem to occur in humans. Carriers are often Carrier state
the source of infection
- Individuals who recover from infection may harbor the
Gastroenteritis organisms in the gall bladder, which becomes the site
of chronic carriage
- “Food poisoning” ingesting contaminated food - Excretions of organisms through feces either
Salmonella strains associated with this infection are continuously or intermittently
usually found in animals (e.g. S. enterica subsp. - May be terminated by antimicrobial therapy if
enterica) gallbladder infection is not evident
- Salmonella Typhimurium was responsible for a - Cholecystectomy might be a solution
nationwide outbreak linked to peanut butter-
containing products Salmonella Infection Observations
- Occurs when a sufficient number of organisms
 Young children – experience fever and gastroenteritis
contaminate food that is maintained under inadequate
with brief episodes of bacteremia
refrigeration, thus allowing growth and multiplication
 Adults – experience transient bacteremia during
of the organisms
episodes of gastroenteritis or develop symptoms of
- Infective dose is higher than required for shigellosis
septicemia without gastroenteritis
- Nausea, vomiting, fever, chills, watery diarrhea and
abdominal pain
Shigella
- Symptoms usually appear within a few days, with few
or no complications - Serogroup A: S. dysenteriae; B: S. flexneri, C: S.
boydii, D: S. sonnei
Enteric fevers
- Most severe is S. dysenteria, because of the Shiga
- Prolonged fever toxin, an exotoxin.
- Enteric fever caused by Salmonella Typhi is known as  Highly infectious, can be transmitted to many
“typhoid fever,” a febrile disease resulting from fles, feces, fingers, foods, friends, and
ingestion of food contaminated with the organisms contaminated water.
originating from infected individuals or carriers - Not normal resident of intestinal tract of humans. Can
- Salmonella Typhi has no known reservoir; humans be found in animals but never in humans.
are the only source of infection - Does not produce H2S
- Paratyphoid fevers: Salmonella serotypes Paratyphi - Can cause bacillary dysentery
A, B, and C, and Salmonella serotype Cholerasuis
S. dysenteriae
- Paratyphoid fevers are less sever
- Cause bacillary dysentery
 - Presence of blood, mucus, and pus in stool - Bloody diarrhea that progresses to
- Non-motile dysentery
- Except for certain types of S. flexneri, they do not - Extremely painful bowel
produce gas from glucose movements, containing blood and
- Urease negative mucus
- H2S negative - Rectal prolapse may result from
- No lysine decarboxylation excessive straining
- Unlike Escherichia spp., Shigella spp. do not utilize - Ileus (obstruction of intestines) with
acetate or mucate as carbon source marked abdominal dilatation,
possible leading to toxic megacolon
S. sonnei
Yersinia
- Decarboxylates ornithine
- Slowly ferments lactose – delayed positive - Y. enterocolitica is most common and predominantly
fermentation of lactose with formation of pink colonies isolated.
only after 48 hours of incubation - Facultative anaerobes, nonmotile but some are
- ONPG positive motile.
- On differential and selective media, they appear as - Has a required temperature to be nonmoitle (37
clear, nonlactose-fermenting colonies degrees).
- Susceptible to various effects of physical and - Motile at room temperature.
chemical agents (e.g. disinfectants, high - Not found in intestinal tract.
concentration of acid and bile) - Exhibits bipolar appearance, staining at only the ends,
resembling a safety pin.
Antigenic Structures
- Specimen depends on the case and type of Yersinia
- Divided into four major O antigen groups implicated. It can be aspirates, sputum, CSF, stool,
- Several serotypes exist within each species, with the etc.
exception of S. sonnei, which only has one serotype  An example of media is blood agar with
- All possess O antigens and certain strains possess K supplements
antigens - Y. pseudotuberculosis and Y. enterocolitica causes
- Shigella K antigens, when present, interfere with sporadic cases of mesenteric lymphadenitis in
detection of O antigen during serologic grouping (can humans, especially in children, and generalized
be removed through heating) septicemic infections in immunocompromised hosts
- Non-motile and therefore lack H antigen
Yersinia pestis
Clinical Infections
- VW antigen system
- Humans are the only known reservoir  V antigen is a virulence factor responsible, a
- Transmission occurs by direct person to person cell-bound protein, attached to the Yersinia
contact, and spread takes place via fecal-oral route body.
with carriers as the source  W antigen is an excreted antigen made of
- May be transmitted by flies, fingers, food or water glycoprotein that would lead to
contaminated by infected people hemoconcentration and shock.
- Low infective dose (<200 bacilli) - Yersinia would be virulent in virtue of
- Bacillary dysentery – penetration of intestinal lipopolysaccharide, more potent, more dangerous 50
epithelial cells following attachment of the organisms times more than other enterics.
to mucosal surfaces, local inflammation, shredding of - Gram-negative, short, plump bacillus
intestinal lining, and formation of ulcers following - When stained with methylene blue or Wayson stain, it
epithelial penetration shows intense staining at each end of the bacillus
o S. dysenteriae Type 1 (bipolar staining) which gives it a “safety- pin”
appearance
 - Grows at body temperature although room - Non-fermentation produces a colorless, translucent
temperature is preferred colon
- Causative agent of plague, primarily a disease of - A slightly modified medium called CIN II can be used
rodents transmitted to humans by fleas to simultaneously isolate most Aeromonas spp.
- It creates the three infections of plague: bubonic,
septicemic, pneumonic. Y. pseudotuberculosis
 Bubonic / glandular – skin, buboes, pigsa, - Pathogen of rodents, especially guinea pigs
brought about by flea bites coming from - Birds are natural reservoirs
infected rats. - Pseudotubercles – caseous swelling
 Multiplies intracellularly of - Spreads to the mesenteric lymph nodes, producing a
phagocytes. generalized infection
 Leads to more WBCs, leading to - Septicemia accompanied by mesenteric
enlargement of the affected site. lymphadenitis, similar to appendicitis
 Painful regional lymph nodes as - Appears as a typical-looking plague bacillus
buboes (swollen lymph nodes) - Differentiated from Y. pestis by its motility by 18-
 High fever, vomiting, diarrhea, if not 22°C, urease production, and ability to ferment
treated within 3-5 days it is very
rhamnose
fatal.
 Septicemic – entrance to blood where Y. ruckeri
the organisms proliferate in the blood
stream and respiratory tract, - Causative agent of red mouth disease in fish
 Pneumonic – highly contagious, easily
Other Genera of the Family Enterobacteriaceae
spread, high mortality.

Yersinia entercolitica Budivicia

- Gram-negative coccobacilli with bipolar staining - Based on DNA hybridization, B. aquatica is a group of
- Grows on SBA and MAC closely related organisms
- Optimal growth at 25-30°C (motility noted at 25°C - Not as closely related with other members of
and not at 35°C) Enterrobacteriaceae, but qualifies to belong in the
- Produces an infection that mimics appendicitis (acute family
enteritis) - Usually found in water
- Erythema nodosum – inflammatory reaction - Occasionally occurs in clinical specimens
characterized by tender, red nodules that may be
accompanied by itching and burning
Buttiauxella
- Stools may contain blood - Consists of 7 species isolated from water
- Can be acquired from contact with household pets - Only B. agrestis and B. noackiae have been
- Pigs as natural reservoir isolated from human species
- Sepsis associated with transmission of contaminated - Biochemically similar to Citrobacter and Kluyvera
packed RBCs - 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 crystal violet as inhibitory agent - Five species:
- Inhibits normal colon microbiota better than MAC agar  C. davisae – most commonly isolated
- Fermentation of mannitol resuts in a drop in pH  C. lapagei
around the colony, causing the pH indicator, neutral  C. neteri
red, to turn red at the center of the colony and the bile  Cedecea species types 3 and 5
to precipitate - Most have been recovered from sputum, blood, and
wounds
Ewingella Photorhabdus
E. americana - Three species
 P. luminescens
- Most isolates came from human blood cultures or o subsp. Luminescens
respiratory specimens o subsp. Akhurstii
- Thought to be related to Cedecea Kluyvera o subsp. laumondii
- Three closely-related species
 P. asymbiotica
 K. ascorbata
 P. temperate
- Small zones of inhibition in
- Natural habitat is lumen of entomopathogenic
cephalothin and carbenicillin disk
nematodes but strains have occasionally been
susceptibility tests
isolated from human species
- Does not ferment glucose at 5°C
- Occur in two phases with the property of
luminescence in phase 1 only
 K. cryocrescens - Most strains produce pink, red, orange, yellow, or
- Large zones of inhibition in cephalothin green pigmented colonies on nutrient agar and
and carbenicillin disk susceptibility tests especially on nutrient-rich agar
- Ferments glucose at 5°C - Negative for nitrate reduction
 K. Georgiana
- Very similar to the two species but is Pragia
negative for gas production during
fermentation of glucose - P. frontium is the only species in this genus
- Found in respiratory, urine, and blood - Produces H2S, a Shigella-like odor on nutrient agar
cultures - “Pig sty-like” odor on Endo agar overlaid with a
- May produce a blue-violet pigment, sloping surface of the agar
usually but not exclusively on nonblood- - Utilizes citrate and oxidizes gluconate
containing media - Most strains are positive for methyl red
- One isolate from stool of healthy human
Leclercia - No indication of pathogenicity for humans or animas

- 58 isolates from human clinical specimens, including Rahnella

blood, urine, sputum, and feces
- 27 isolates from nonhuman sources R. aquatilis

L. adecarboxylata - Psychrotolerant, grows at 4°C

- Have no single characteristic that distinguishes them
- Yellow pigment on primary isolation from other Enterobacteriaceae
- Similar IMViC reactions to E. coli but is triple - Resemble E. agglomerans, but has a weak
decarboxylase negative phenylalanine deaminase reaction
- Potassium cyanide, gelatin, lysine, ornithine, and
Leminorella motility negative
- Enteric group 57 - Lack of yellow pigmentation
- Occasionally isolated from human clinical specimens,
- Two species
including wound infections, bacteremias, feces from
 L. grimontii
patients with acute gastroenteritis, and septicemia,
 L. richardii
especially from immunocompromised patients
- Produce H2S
- Weak reactions with Salmonella antisera
Tatumella
- Leminorella spp. are relatively inactive
- Clinical significance is unknown - T. ptyseos is the only species
- Isolated from urine, feces, and water
 - Unusual organism
- Stock cultures may be kept frozen in sheep RBCs or
freeze-dried, but they will die in a few weeks on agar
slants
- Show more biochemical reactions at 25°C than at
35°C
- Motile at 25°C but not at 35°C
- Large 15-36mm zones of inhibition around penicillin
disks
- Slow-growing, producing tiny colonies, relatively
nonreactive in laboratory media
- Isolated from human sources, especially sputum, and
may be a rare cause of infection

Trabulsiella
- T. guamensis is the only species in this genus, and
although it is very rarely isolated, it is biochemically
similar to Salmonella
- Isolated from vacuum-cleaner contents on the island
of Guam when environmental indoor dirt samples
were being collected
- Also isolated from human feces

Xenorhabdus
X. nematophilus

- Grows best on 25°C

- Does not reduce nitrate to nitrite
- Isolated from nematodes
- Not found in human specimens

Yokenella
Y. regensburgei

- Thought to be another species of Hafnia,

biochemically similar but differs in VP test results
(Hafnia is positive, Yokenella is negative)
- Isolated from human specimens
 - P. asymbiotica  Also isolated from human feces
- P. temperate
 Natural habitat is lumen of entomopathogenic Xenorhabdus
nematodes but strains have occasionally been isolated  X. nematophilus
from human species  Grows best on 25°C
 Occur in two phases with the property of  Does not reduce nitrate to nitrite
luminescence in phase 1 only  Isolated from nematodes
 Most strains produce pink, red, orange, yellow, or  Not found in human specimens
green pigmented colonies on nutrient agar and
especially on nutrient-rich agar Yokenella
 Negative for nitrate reduction  Y. regensburgei
 Thought to be another species of Hafnia, biochemically
Pragia similar but differs in VP test results (Hafnia is
 P. frontium is the only species in this genus positive, Yokenella is negative)
 Produces H2S, a Shigella-like odor on nutrient agar  Isolated from human specimens
 “Pig sty-like” odor on Endo agar overlaid with a
sloping surface of the agar Vibrio, Aeromonas, Plesiomonas, and Campylobacter
 Utilizes citrate and oxidizes gluconate Species
 Most strains are positive for methyl red
 One isolate from stool of healthy human Vibrio
 No indication of pathogenicity for humans or animas  They are temperature sensitive in that in temperate
climates when water temperatures exceeds 20°C, as in
Rahnella the summer months, vibrios can easily by isolated
 R. aquatilis from water, suspended particulate matter, algae,
 Psychrotolerant, grows at 4°C plankton, fish, and shellfish.
 Have no single characteristic that distinguishes
them from other Enterobacteriaceae General Characteristics of Vibrio
 Resemble E. agglomerans, but has a weak  Clinical Manifestations
phenylalanine deaminase reaction - Ranging from mild gastroenteritis to cholera and
 Potassium cyanide, gelatin, lysine, ornithine, and from simple wound infections to fatal septicemia
motility negative and necrotizing fasciitis
 Lack of yellow pigmentation - Most common isolates
 Occasionally isolated from human clinical specimens,  V. cholera (serogroups O1 and non-O1)
including wound infections, bacteremias, feces from  V. parahaemolyticus
patients with acute gastroenteritis, and septicemia,  V. vulnificus
especially from immunocompromised patients  V. alginolyticus
 Microscopic Morphology
Tatumella - Asporogenous, gram-negative rods measuring
 T. ptyseos is the only species approximately 0.5-0.8µm in diameter by 1.5-
 Unusual organism 3.0µm in length
- Stock cultures may be kept frozen in sheep RBCs - Polar, sheathed flagella when grown in broth
or freeze-dried, but they will die in a few weeks on but can produce petritrichous, unsheathed
agar slants flagella when grown on solid media
- Show more biochemical reactions at 25°C than at - Curved gram-negative rods
35°C - Can be highly pleomorphic especially under
- Motile at 25°C but not at 35°C suboptimal growth conditions
- Large 15-36mm zones of inhibition around  Physiology
penicillin disks - Facultatively anaerobic
 Slow-growing, producing tiny colonies, relatively - All clinically species are oxidase positive and
nonreactive in laboratory media able to reduce nitrate to nitrite except for V.
 Isolated from human sources, especially sputum, and metschnikovii
- Most are generally susceptible to vibriostatic
may be a rare cause of infection
compound O/129 (2,4-diamino-6,7-
Trabulsiella diisopropylpteridine), exhibiting a zone of
inhibition to a 150µg Vibriostat disk on either a
 T. guamensis is the only species in this genus, and
Mueller-Hinton or trypticase soy agar
although it is very rarely isolated, it is biochemically
- Positive string test observed as mucoid
similar to Salmonella
“stringing” reaction after emulsification of
 Isolated from vacuum-cleaner contents on the island
colonies in 0.5% sodium desoxycholate
of Guam when environmental indoor dirt samples
were being collected

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 - All species, except for V. cholera and V. mimicus, - Different phage susceptibility patters
are halophilic or salt-loving and require the  V. cholerae serogroup O141 appears to be associated
addition of Na+ for growth with sporadic cholera-like diarrhea and bloodstream
- Can be differentiated from the similar genera infections
Aeromonas and Plesiomonas by mean of key  Other non-O1 serogroup strains have been implicated
biochemical and growth requirement in a variety of extraintestinal infections including
characteristics cholecystitis, ear infections, cellulitis, and septicemia
 Antigenic structure  Some O139 strains share cross-reacting antigens with
- Subgroups of V. cholerae Aeromonas trota, a somewhat uncommon cause of
 V. cholerae O1 diarrheal disease
o Ogawa (A, B)
o Inaba (A, C) Vibrio parahaemolyicus
o Hikojima (A, B, C)  Second most common Vibrio species implicated in
o Epidemic cholera gastroenteritis
 V. cholerae O139  Cause of “summer diarrhea”
o Epidemic cholera  V. parahaemolyticus serogroup O3:K6 is implicated
 V. cholerae non-O1 – phenotypically in numerous food-borne outbreaks
resembles V. cholerae but fail to agglutinate  Found in aquatic environments but limited to coastal
in O1 antisera or estuarine areas despite a halophilic requirement of
 Shares a common flagellar (H) antigen and 1-8% NaCl
somatic (O) antigen  Gastrointestinal disease is self-limited
 V. parahaemolyticus can also be serotypes by means of  Watery diarrhea, moderate cramps or vomiting, and
its O and K (capsule) antigen little if any fever
 Occasionally isolated from extraintestinal sources
Vibrio cholerae such as wounds, ear and eye infections, even in a case
 Vibrio cholerae O1 is the causative agent of cholera, of pneumonia
also known as Asiatic cholera or epidemic cholera  Kanagawa phenomenon – most strains produce a
 Cholera is an acute diarrheal disease that is spread heat-stable hemolysin that is able to lyse human RBC
mainly through contaminated water in a special high-salt mannitol medium (Wagatsuma
 Manifests in acute cases as a severe gastroenteritis agar)
accompanied by vomiting followed by diarrhea  These strains are considered Kanagawa toxin positive,
 “Rice water” stools, contains numerous flecks of whereas other isolates are Kanagawa toxin negative
mucus
 Can result in a rapid fluid and electrolyte loss that Vibrio vulnificus
leads to dehydration, hypovolemic shock, metabolic  Commonly referred to as the “lactose-positive” Vibrio
acidosis, and death in a matter of hours  Second most serious type of Vibrio infection
 Choleragen – cholera enterotoxin  Two categories
 2 Toxic A subunits and 5 binding B subunits - Primary septicemia – surmised to occur through
 Toxin initially binds to the GM1-ganglioside receptor the gastrointestinal route after the consumption of
on the cell membrane via the B subunits shellfish, especially raw oysters
 A2 subunit facilitates entrance of A1 subunit - Wound infections
 Once inside the cell, the active A1 subunit stimulates
the production of adenylate cyclase through Vibrio alginolyticus
inactivation of the G-protein  Least pathogenic for humans and one most frequently
 Accumulation of cyclic adenosine monophosphate isolated
(cAMP) along the cell membrane, which stimulates  Common inhabitant of marine environments
hypersecretion of electrolytes (Na+, K+, HCO3-) and  Strict halophile, requiring at least 1% NaCl and can
water out of the cell and into the lumen of the intestine tolerate up to 10% NaCl
 The gastrointestinal tract’s absorptive ability is  Nearly all isolates are from extraintestinal sources
overwhelmed, resulting in the massive outpouring of such as eye and ear infections or wound and burn
watery stools infections
 Resistant to tetracycline and doxycycline  Can be an occupational hazard for most people in
 Epidemic V. cholerae O1 strains occur in two constant contact with seawater
biogroups: classic and El Tor
 El Tor has been the predominant biogroup in the last Laboratory Diagnosis of Vibrio spp.
two pandemics  Specimen Collection and Transport
- Voges-Proskauer positive - Vibrios are not fastidious, and only a few special
- Hemolyzes RBCs collection and processing procedures are
- Inhibited by polymyxin B (50µg), and is able to necessary to ensure the recovery of vibrios from
agglutinate chicken RBCs clinical material

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 - Whenever possible, body fluids, pus, or tissues - Their general susceptibility to the vibriostatic
should be submitted, but swabs are acceptable if agent O/129 (150 µg) and positive “string test”
they are transported in an appropriate holding distinguishes them from Aeromonas
medium, such as Cary-Blair, to prevent - Inability to ferment inositol (except for V.
desiccation cincinnatiensis and some strains of V.
- Buffered glycerol saline is not recommended as a metschnikovii) separates them from Plesiomonas
transport or holding medium because the - Their positive oxidase reaction (except for V.
glycerol is toxic for vibrios metschnikovii) separates them from the
- Even strips of blotting paper soaked in liquid stool Enterobacteriaceae (excluding Plesiomonas
and placed in airtight plastic bags are considered shigelloides), and a fermentative metabolism
viable specimens for up to 5 weeks separates them from the oxidative Pseudomonas
- Stool specimens should be collected as early as  Definitive identification
possible in the course of the illness, before the - It is important to note that with the halophilic or
administration of any antimicrobial agents salt-loving vibrios, it often is necessary to add at
 Culture media least 1% NaCl to most biochemical media to
- The salt concentration (0.5%) in most obtain reliable reaction results
commonly used laboratory media, such as - Rapid and semiautomated identification systems
nutrient agar or sheep blood agar (SBA), is - Serology
sufficient to support the growth of any vibrios
present SBA or chocolate (CHOC) agar, vibrios Antimicrobial Susceptibility of Vibrio spp.
produce medium to large colonies that appear  Both Mueller-Hinton agar and broth contain
smooth, opaque, and iridescent with a sufficient salt to support the growth of the Vibrio spp.
greenish hue most often isolated from clinical specimens
- The SBA plate should also be examined for the  The recommended antimicrobial susceptibility testing
presence of α- or β-hemolysis. On MacConkey methods are standardized disk diffusion
(MAC) agar, the pathogenic vibrios usually grow (KirbyBauer) or dilution susceptibility testing
as nonlactose fermenters methods
- However, lactose-fermenting species such as V.
vulnificus may be overlooked and incorrectly Aeromonas
considered to be members of the family  Consists of ubiquitous oxidase-positive, glucose-
Enterobacteriaceae, such as Escherichia coli. It fermenting, gram-negative rods that are widely
therefore is imperative to determine the oxidase distributed in freshwater, estuarine, and marine
activity of any suspicious Vibrio-like colony environments worldwide
- This can be accomplished by either directly testing  They are frequently isolated from retail produce
colonies from SBA or CHOC agar plates with sources and animal meat products
oxidase reagent or by subculturing any  Responsible for a diverse spectrum of disease
suspicious lactose-fermenting colonies on MAC syndromes among a variety of warm- and cold-
to an SBA plate for next-day testing blooded animals including fish, reptiles, amphibians,
- This is necessary because lactose-positive colonies mammals, and humans
from selective-differential media such as MAC or  Phylogenetic evidence from molecular studies
cefsulodin-irgasin-novobiocin agar may give resulted in the proposal of a separate family
false-positive oxidase reactions Aeromonadaceae from Vibrio
- If a selective medium is warranted, either because
of the clinical history (exposure to seafood or General Characteristics of Aeromonas spp.
seawater) or for geographic reasons (coastal area  Aeromonads are straight rods (1.0 to 3.5 µm long by
resident or recent foreign travel), thiosulfate 0.3 to 1.0 µm wide), and most are motile by means of
citrate bile salts sucrose (TCBS) agar is a single polar flagellum
recommended. It differentiates sucrose-  Aeromonads are classified into one of two groups: the
fermenting (yellow) species such as V. cholerae, mesophilic group or the psychrophilic group
V. alginolyticus, V. fluvialis, V. furnissii, V. - The mesophilic group consists of three different
cincinnatiensis, V. metschnikovii, and some V. complexes or groups of species
vulnificus strains from the nonsucrose-  A. hydrophila complex which includes A.
fermenting (green) vibrios: V. mimicus, V. hydrophila, A. bestiarum, and certain motile
parahaemolyticus, V. damsela, and most V. strains of A. salmonicida
vulnificus strains  A. veronii complex includes A. veronii biovar
 Presumptive identification sobria (formerly misidentified as A. sobria), A.
- Vibrios can be easily confused with other genera, veronii biovar veronii, A. jandaei, A. trota, and
including Aeromonas, Plesiomonas, A. schubertii
Enterobacteriaceae, and even Pseudomonas  A. caviae complex includes the species A.
caviae, A. media, and A. eucrenophila

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 - These organisms are considered mesophiles  A. veronii biovar sobria has also been linked
because they grow well at 37°C to cholera-like disease characterized by
- All motile abdominal pain, fever, and nausea
- The psychrophilic group consists of only one - Extraintestinal infections
species, A. salmonicida, which is a fish pathogen  Most aeromonad wound isolates are A.
with several subspecies. This organism is hydrophila, A. veronii biovar sobria, or A.
nonmotile and is considered a psychrophile schubertii. This last species has to date been
because it grows best at 22-25°C, and when such isolated almost exclusively from aquatic
strains are found to be nonmotile, they are not wound infections and bloodstream infections
considered human pathogens  A. veronii biovar veronii and surgical wound
 All aeromonads, in general, can typically grow from infections involving the use of leeches for
4-42°C medicinal therapy following plastic surgery to
 Clinical manifestations relieve venous congestion has been noted.
- Intestinal infections These patients can develop serious
 The aeromonads are recognized as enteric aeromonad wound infections. It appears that
pathogens, albeit not in the same manner as the leech Hirudo medicinalis has a symbiotic
the more common enteric pathogens like relationship with this particular aeromonad
Salmonella, Shigella, or V. cholerae species within its gut, wherein the organisms
 The level and pattern of virulence is more like aid in the enzymatic digestion of the blood
the multifactorial patterns of the various E. ingested by the leech
coli subgroups associated with enteric disease  Aeromonad sepsis appears to be the most
(e.g., enterotoxigenic E. coli, invasive type of Aeromonas infection and
enterohemorrhagic E. coli) likewise has a strong association with the
 Screening of stool specimens for the presence species A. veronii biovar sobria, A. jandaei, and
of these organisms, followed by further A. hydrophila.
identification to the species level, is
appropriate Laboratory Diagnosis of Aeromonas spp.
 Five diarrheal presentations are observed  Culture media
in patients in whom an Aeromonas has been - Aeromonads grow readily on most media used
isolated from their stools for both routine and stool cultures
o An acute, secretory diarrhea often - After 24-hour incubation at 35° C, aeromonads
accompanied by vomiting appear as large round, raised, opaque colonies
o An acute, dysenteric form of diarrhea with an entire edge and a smooth, often
(similar to shigellosis) with blood and mucoid surface
mucus - Frequently, an extremely strong odor is present,
o A chronic diarrhea usually lasting more and pigmentation ranges from translucent and
than 10 days white to buff colored
o A cholera-like disease including rice- - Hemolysis is variable on SBA, but most major
water stools clinical species, such as A. hydrophila, A. veronii
o The nebulous syndrome commonly biovar sobria, and A. jandaei, display strong β-
referred to as “traveler’s diarrhea” hemolysis
(similar to enterotoxigenic E. coli) - The most commonly isolated species, A. caviae,
 Most cases are self-limiting, but in the is nearly always nonhemolytic or at best
pediatric, geriatric, and immunocompromised weakly α-hemolytic on SBA
populations, supportive therapy and - Although aeromonads grow on nearly all enteric
antimicrobials are often indicated media, they often are overlooked on MAC agar,
 A. caviae is the species most frequently especially in stool cultures because a number of
associated with gastrointestinal infections, aeromonads ferment lactose
especially in neonate and pediatric  A. caviae - this particular species is nearly
populations, and has been associated with always pink on MAC agar because of positive
inflammatory bowel disease lactose fermentation and is therefore
 Other species associated with diarrhea generally overlooked as “normal biota” E. coli
include A. hydrophila and A. veronii (biovars - The combined use of ampicillin sheep blood
sobria and veronii) agar and a modified cefsulodin-irgasin-
 More serious complications, usually from novobiocin (CIN II) plate (with only 4 µg of
infections with A. hydrophila and A. veronii cefsulodin instead of 15 µg), might yield the
biovar sobria, include hemolytic uremic highest recovery of aeromonads
syndrome or kidney disease that may - However, the incorporation of ampicillin in the
require a kidney transplant blood agar may inhibit some A. caviae as well as all
A. trota strains because the hallmark feature of A.
trota is its unusual universal susceptibility to

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 ampicillin. Therefore SBA without ampicillin is Aeromonas from oxidase-positive nonfermenting
preferred Pseudomonas isolates
- On CIN medium (either the standard CIN  Definitive identification
formulation for enteric Yersinia or the modified - Definitive identification of the aeromonads is
CIN II), Aeromonas will form pink-centered accomplished with a small number of
colonies from the fermentation of mannitol, with conventional and readily available biochemical
an uneven, clear apron-resembling Yersinia tests and antimicrobial markers and the use of a
enterocolitica. However, an oxidase test simple dichotomous key, Aerokey II
performed on SBA colonies will easily separate the
oxidase-positive aeromonads from the oxidase- Plesiomonas
negative yersinias  Oxidase-positive, glucose fermenting, facultatively
- The use of an enrichment broth generally is not anaerobic, gram-negative bacilli that are motile by
considered necessary. However, if such a medium polar flagella
is warranted for detecting chronic cases or  Found in both soil and aquatic environments, but
asymptomatic carriers, alkaline peptone water is because of intolerance to increased NaCl and a
recommended. This can be inoculated, incubated minimum growth temperature of 8°C, they are
overnight at 35° C, and subsequently subcultured generally found only in the fresh and estuarine waters
to appropriate plate media of tropical and subtropical climates
 Presumptive identification  Clinical manifestations
- An important screening procedure for - Gastroenteritis
aeromonads is to perform an oxidase test and a  At least three major clinical types of
spot indole on suspicious colonies on SBA, gastroenteritis are caused by Plesiomonas
especially β-hemolytic colonies o The more common watery or secretory
- A positive oxidase distinguishes aeromonads diarrhea
from the family Enterobacteriaceae (except for o A subacute or chronic disease that lasts
Plesiomonas shigelloides), and most clinically between 14 days and 2 to 3 months
relevant aeromonads are indole positive o A more invasive, dysenteric form that
- The presence and type of hemolysis among resembles colitis
multiple aeromonad-colony types in a single  Most cases are self-limiting, but antimicrobial
culture often are the only clues to an infection therapy is indicated in severe and prolonged
involving more than one species of Aeromonas cases
- The best tests to distinguish the aeromonads from  Reports of P. shigelloides infection in patients
Vibrio spp. are the string test (usually negative with human immunodeficiency virus
for aeromonads and positive for vibrios) and infections are increasing, as are associations
testing for sensitivity to O/129 (usually with inflammatory bowel disease
aeromonads and plesiomonads are resistant - Extraintestinal infections
and most vibrios are susceptible)  Occupational exposure can be a source of
- An additional test to separate aeromonads and infections for veterinarians, zookeepers,
plesiomonads from most vibrios is that of aquaculturists, fish handlers, and athletes
determining the ability to grow in the participating in water-related sports
presence of NaCl  More serious infections, such as bacteremia
 Aeromonads and plesiomonads grow quite and meningitis, usually occur only in severely
well in nutrient broth with 0% NaCl, but not immunocompromised patients or neonates
in 6% NaCl. Conversely, most vibrios  Recent reports include cases of continuous
(specifically the halophilic species) cannot ambulatory peritoneal dialysis-associated
grow in 0% NaCl but thrive in 6% NaCl and peritonitis
even higher concentrations of NaCl  Furthermore, biliary tract disease has been
 However, because both V. cholerae and V. identified as a possible risk factor for
mimicus are nonhalophilic and grow quite bacteremia with this organism
well without additional salt, any salt tolerance
test must be used in conjunction with both the General Characteristics of Plesiomonas spp.
string test and the O/129 disk to distinguish  Plesiomonads are straight (0.8 to 1 µm by 3 µm),
aeromonads from this major pandemic gram-negative bacilli that occur singly, in pairs, or
cholera species and the less common sucrose- in short chains or filamentous forms
negative V. mimicus  They do not form spores or capsules and are motile
- For separation of aeromonads from plesiomonads, by monotrichous or two to five lophotrichous
one can utilize the fermentation of inositol— flagella
where aeromonads are negative and  The genera Plesiomonas and Shigella share both
plesiomonads are positive biochemical and antigenic features, and plesiomonads
- Lastly, the ability to ferment glucose, with or often cross-agglutinate with Shigella sonnei, S.
without the production of gas, distinguishes

dane.
 dysenteriae, and even S. boydii—hence the species - This is in large part because of its unique “positive
name shigelloides trio” profile of positive ornithine and lysine
 However, unlike Shigella, the organism P. shigelloides decarboxylases and arginine dihydrolase
appears to possess a much lower virulence reactions, combined with the fermentation of
potential, with a low symptomatic carriage rate inositol
among humans, and is oxidase-positive - Quantitative polymerase chain reaction assay
 Plesiomonas can be serotyped by somatic O antigens for P. shigelloides
(50 groups) and their flagellar H antigens (17
groups). Campylobacter and Campylobacter-like Species
 Campylobacters were formerly classified with the
Laboratory Diagnosis of Plesiomonas spp. vibrios because of their positive oxidase and
 Culture media characteristic microscopic morphology, but DNA
- Plesiomonas spp. grow readily on most media homology studies have shown that Campylobacter
routinely used in the clinical laboratory spp. do not belong with the vibrios
- After 18-24 hours incubation at 35° C, shiny,  Unlike the vibrios, which are fermentative, most
opaque, nonhemolytic colonies appear, with a campylobacters are asacchrolytic
slightly raised center and a smooth and entire  Based on ribosomal RNA (rRNA) sequence studies,
edge Wolinella recta and Wolinella curva have been found to
- Because most strains ferment lactose, albeit as a be similar to the campylobacters. Therefore these two
“delayed” positive reaction, the easiest screening species have been transferred to the genus
procedure is an oxidase test performed on Campylobacter as C. rectus and C. curvus
colonies from nonselective media, such as SBA or  Although they may appear to be strict anaerobes,
CHOC agar they have been grown in a microaerophilic
- Although a specialized medium is not environment
recommended for the detection of plesiomonads  Microaerophilic organisms require oxygen, but at a
from stool specimens, and because certain strains concentration less than that of room air; 5% is
are inhibited on eosin-methylene blue or MAC normally optimal
agars, the use of inositol brilliant green bile  Campylobacter spp. have been known to cause
salts agar can enhance the isolation of abortion in domestic animals such as cattle, sheep and
plesiomonads swine
 Plesiomonas colonies are white to pink on  Campylobacter jejuni – most common cause of
this medium, and most coliform colonies bacterial gastreoenteritis
are either green or pink  Some species are sexually transmitted
- Plesiomonas will not grow on TCBS agar but will  Campylobacter fetus subsp. fetus – isolated most
grow quite well on CIN, a selective agar for frequently from blood cultures and is rarely associated
Yersinia spp., as opaque (non-mannitol with gastrointestinal illness
fermenting) colonies with an opaque apron.
- However, they must be distinguished from other Clinical Significance of Campylobacter Species and
oxidase positive organisms, such as Aeromonas Campylobacter-Like Organisms
and Pseudomonas, that will also grow on CIN Campylobacter Species Clinical Significance
- Because plesiomonads are often susceptible to Arcobacter butzleri Associated with diarrheal
ampicillin, any agar used as a possible means of disease and bacteremia in
detecting plesiomonads in clinical samples should humans and in children
not contain this antibiotic with recurring
 Identification gastrointestinal illness
- Plesiomonas shigelloides can be presumptively (abdominal cramps
differentiated from similar genera with several Arcobacter cryaerophilus Isolated from cases of
key tests. The positive oxidase activity separates human bacteremia and
it from the Enterobacteriaceae, sensitivity to the diarrhea
vibriostatic agent O/129 separates it from Arcobacter nitrofigilis
Aeromonas, and its ability to ferment inositol Campylobacter fetus subsp. Rarely involved in human
separates it from all Aeromonas and nearly all venerealis infections
Vibrio spp. Campylobacter sputorum
- It can also be separated from the halophilic Vibrio biovar faecalis
spp. by its ability to grow in nutrient broth with Campylobacter concisus Involved in periodontal
0% NaCl coupled with its inability to grow in disease; has also been
nutrient broth with 6% NaCl recovered from individuals
- Most rapid identification systems include P. with gastrointestinal illness
shigelloides in their databases and appear to be Campylobacter fetus subsp. Bacteremia in
able to identify it with a fairly high degree of fetus immunocompromised
accuracy patients

dane.
Campylobacter Enteric disease in swine; - Cramps and bloody diarrhea often follow the
hyointestinalis occasionally associated in initial signs. Patients may experience fever and
human enteric illness chills and, rarely, nausea and vomiting
Campylobacter jejuni Most common cause of - In most patients, the illness is self-limited and
bacterial diarrhea usually resolves in 2 to 6 days. Untreated patients
worldwide can remain carriers for several months
Campylobacter lari Enteritis very similar to - Other enteric Campylobacter infections (i.e., those
that caused by caused by C. coli and C. lari) have similar clinical
Campylobacter jejuni manifestations
Campylobacter mucosalis - Strong evidence suggests that Campylobacter
Campylobacter sputorum infection plays a role in Guillain-Barré
biovar bubulus syndrome, an autoimmune disorder
Campylobacter rectus Associated with periodontal characterized by acute paralysis due to damage to
disease; has been recovered the peripheral nervous system. Many patients
from patients with root with GBS test positive for antibodies to
canal infections and Crohn Campylobacter
disease - It is believed that antibodies produced during a
Campylobacter upsaliensis Potential pathogen in Campylobacter infection bind to gangliosides
humans, causing found on peripheral nerves. Cross-reactivity with
gastrointestinal illness and these nerve cells in an autoimmune response may
bacteremia in both be responsible for this debilitating nerve disorder
immunocompetent and  Helicobacter pylori
immunocompromised - Helicobacter pylori has been primarily linked to
patients gastric infections. Once acquired, H. pylori
Helicobacter canadensis Isolated in stool specimens colonizes the stomach for a long time and can
from patients with diarrhea, cause a low-grade inflammatory process,
pathogenesis unknown producing a chronic superficial gastritis
Helicobacter cinaedi Recovered from blood of - Although it does not invade the gastric epithelium,
homosexual males with or the infection is recognized by the host immune
without AIDS and from system, which initiates an antibody response. The
blood and feces of children antibodies produced are not protective, however
and adult females - H. pylori is also recognized as a major cause of
Helicobacter felis type B gastritis, a chronic condition formerly
Helicobacter fennelliae Recovered from rectal associated primarily with stress and chemical
irritants
swabs and blood of
homosexual men with or - In addition, based on recent data, the strong
without AIDS presenting association between long-term H. pylori infection
and gastric cancer has raised more questions
with proctitis, proctocolitis,
and enteritis regarding the clinical significance of this organism
- There is speculation that long-term H. pylori
Helicobacter muridarum
infection resulting in chronic gastritis is an
Helicobacter mustelae
important risk factor for gastric carcinoma
Helicobacter pylori Common cause of duodenal
- Other species of helicobacters, including H. cinaedi
ulcers and type B gastritis;
and H. fennelliae, have been associated with
possibly a risk factor in
human gastroenteritis, generally in
gastric carcinoma
immunocompromised patients
- More recently gastroenteritis has also been linked
Clinical Manifestations of Campylobacter spp. and to H. canadensis, H. canis, H. pullorum, and H.
Campylobacter-like Organisms winghamensis
 Campylobacter - In addition, H. cinaedi has been isolated from
- Several Campylobacter spp. have been implicated blood of patients with bacteremia and patients
in human infection: C. fetus, C. jejuni, C. coli, C. with human immunodeficiency virus infection
sputorum, C. concisus, C. curvus, and C. rectus
- C. fetus contains two subspecies: C. fetus subsp. Laboratory Diagnosis of Campylobacter spp. and
fetus and C. fetus subsp. venerealis Campylobacter-like Organisms
- Patients infected with C. jejuni present with a  Specimen collection and transport
diarrheal disease that begins with mild abdominal - Campylobacter fetus subsp. fetus can be
pain within 2 to 10 days after ingestion of the recovered in several routine blood culture media
organisms - Campylobacter spp. that cause enteric illness are
isolated from stool samples and rectal swabs, the
less-preferred specimen

dane.
 - If a delay in processing the stool specimen is therefore to isolate this organism, media should
anticipated, it can be placed in a transport be incubated at 37°C
medium such as Cary-Blair to maintain the - Enteric Campylobacter and Helicobacter species
viability of the organisms require a microaerophilic and capnophilic
- A common stool transport medium, buffered environment. The ideal atmospheric environment
glycerol-saline, is toxic to enteric campylobacters for these organisms contains a gas mixture of 5%
and should therefore be avoided O2, 10% CO2, and 85% N2 for Campylobacter
- H. pylori can be recovered from gastric biopsy spp. and 5% to 10% O2 and 5% to 12% CO2 for
materials. Samples must be transported quickly to Helicobacter spp.
the laboratory - Except for C. rectus and C. curvus, a strict
- Stuart medium can be used to maintain the anaerobic environment does not support the
viability of the organisms if a delay in processing growth of most Campylobacter spp.
is anticipated  Presumptive identification
- Tissue samples may also be placed in cysteine- - Microscopic morphology
Brucella broth with 20% glycerol and frozen at  Campylobacter spp. are curved, non–spore-
−70°C forming, gram-negative rods that measure
 Culture media approximately 0.2 to 0.9 µm × 0.5 to
- An enriched selective agar, CAMPY-BAP (blood 5.0 µm
agar plate), is a commonly used medium to  Enteric campylobacters may appear as long
isolate C. jejuni and other enteric campylobacters spirals, S shapes, or seagull-wing shapes
- This commercially available medium contains  These organisms may appear as coccobacilli
Brucella agar base, 10% sheep red blood cells, in smears prepared from older cultures
and a combination of antimicrobials:  On Gram stained smears, these organisms
vancomycin, trimethoprim, polymyxin B, stain poorly
amphotericin B, and cephalothin  For better visualization, carbolfuchsin is
- Other selective media that have been successful in recommended as a counterstain; if safranin
recovering Campylobacter spp. are Butzler is used, counterstaining should be extended to
medium and Skirrow’s medium 2 to 3 minutes
- Medium V, a modification of the original Butzler  They exhibit a characteristic “darting”
medium, contains cefoperazone, rifampin, colistin, motility on hanging drop preparations or
and amphotericin B; it seems to inhibit normal when visualized under phase-contrast
colon microbiota better than the original microscopy
formulation  Arcobacter spp. have a microscopic
- CAMPY-CVA (cefoperazone-vancomycin- morphology similar to that of Campylobacter
amphotericin B) medium has been reported to spp.
provide better suppression of fecal biota, even  H. pylori also appears similar to
when this medium is incubated at 37° C campylobacters, but one ultra structural study
- Incubation at 37° C allows the recovery of has shown that Helicobacter has multiple
Campylobacter spp. that are inhibited at 42° C flagella at one pole, unlike the single polar
- C. fetus subsp. fetus, C. rectus, and C. curvus can be flagellum of campylobacters
isolated using routine culture media - Colony morphology
- Charcoal-based blood-free media, such as  Typical colony morphology of C. jejuni and
charcoal cefoperazone deoxycholate agar, are other enteric campylobacters is moist,
also available “runny looking,” and spreading
- To recover H. pylori, a combination of a  Colonies are usually nonhemolytic; some are
nonselective medium, such as CHOC agar or round and raised and others may be flat
Brucella agar with 5% horse red blood cells,  C. fetus subsp. fetus produces smooth,
and a selective medium, such as Skirrow’s agar, convex, translucent colonies
may be used  A tan or slightly pink coloration is observed
- It is important that the inoculated medium be in some enteric campylobacter colonies
fresh and moist and that the culture be incubated  Other Campylobacter species produce
in a microaerophilic environment with colonies similar to those of C. jejuni
increased humidity  Although most do not produce pigment, C.
 Incubation mucosalis and C. hyointestinalis can produce a
- C. jejuni and other enteric campylobacters grow dirty yellow pigment
optimally at 42°C  Definitive identification
- Growth of normal colon organisms is inhibited at - Isolates from stool specimens and rectal swabs
this higher temperature can be presumptively identified as Campylobacter
- C. fetus subsp. fetus, on the other hand, is a rare spp. by being oxidase positive, observing the
stool isolate, and growth is suppressed at 42°C; characteristic Gram-stained microscopic
morphology, and the characteristic motility

dane.
 - The microscopic morphology is very important  Some have fruity, sweet or unique odors
because it differentiates Campylobacter from  They may display unique colony morphologies and
other bacterial species (i.e., Aeromonas, pigmentation
Pseudomonas) that are oxidase positive and can  Often MDR or Multi-Drug Resistant
grow at 42°C in a microaerophilic environment
- To observe the typical motility, organisms should
be suspended in Brucella or tryptic soy broth
- Distilled water and saline seem to inhibit motility
- A positive hippurate hydrolysis is an important
characteristic for the identification of C. jejuni
- H. pylori may be presumptively identified in a
gastric biopsy specimen by testing for the
presence of a rapid urease reaction. The collected
tissue sample is placed onto Christensen’s urea
medium and incubated at 37°C for 2 hours. A
rapid color change suggests the presence of H.
pylori
- Urease activity can also be detected by the urea
breath test, which is reportedly both sensitive
and specific and is recommended for monitoring
therapy Biochemical Tests for Identification of NF(-)R
- H. pylori infection can also be diagnosed by fecal  Hugh-Leifson OF medium
antigen detection, microscopic examination of  Oxidase
stained gastric tissue, and DNA amplification tests  Decarboxylation of amino acids
(i.e., polymerase chain reaction)  Motility
 Nitrate reduction
Non-Fermenting and Miscellaneous Gram-Negative
 Acetamide
Bacilli
 Growth at 42°C
General Characteristics  Pigments
 Prefer wet environments such as sinks, respiratory  Colony morphologies
equipment, flower vases, etc.  Distinct odors
 Usually not part of the healthy human microbiota
Types of Pigments
 Considered opportunistic and can colonize and infect
immunocompromised individuals  Pyoverdin
 Often found as transient or colonizing microbiota of  Fluorescein
hospitalized individuals and can become nosocomial  Fluorescein+Pyocianin
pathogens  Pyomelanin
 Aerobic, Gram negative rods  Pyoverdin+Fluorescein
 Do NOT use carbohydrates as a source of energy or  Pyoverdin+Pyomelanin
degrade them through metabolic pathways other than  No Pigment
fermentation
 Most are obligate aerobes and grow poorly if at all Hugh-Leifson OF Medium
under anaerobic conditions 1. Obtain pure, isolated colonies from an 18-24 hour
 Oxidizers and non-fermenters culture
 Asaccharolytic, do not degrade carbohydrate at al 2. For each test organism, inoculate tubes in duplicate.
Inoculate by stabbing the agar to approximately ¼
 Usually display abundant growth on sheep BAP and
inch from the bottom
CAP within 24-48 hours
3. Apply sterile mineral oil, sterile melted paraffin, or
sterile melted petrolatum to one of each duplicate
Clues that Suggest the Isolation of a Non-Fermenter
tubes. Tighten the cap of the overlaid tube, and
(NF)
loosenthe cap of the non-overlaid tube
 The organism does not ferment carbohydrates
4. Incubate both tubes aerobically at 35°C for up to 14
 Alkaline slant/No change deep butt reaction in TSI and
days
KIA
5. Examine tubes daily for color change
 Require oxygen for the metabolism of carbohydrate if
 Positive: a positive carbohydrate utilization test is
they are able to use them at all
indicated by the development of a yellow color in
 They fail to ferment carbohydrates the medium
 NF can be oxidase positive - Oxidative: development of a yellow color in the
 May fail to grow or show poor growth on McConkey open tube only
agar

dane.
 Non-Fermenting and Miscellaneous Gram-Negative
Bacilli
General Characteristics
o Prefer wet environments such as sinks, respiratory
equipment, flower vases, etc.
o Usually not part of the healthy human microbiota
o Considered opportunistic and can colonize and
infect immunocompromised individuals
o Often found as transient or colonizing microbiota
Notes:
of hospitalized individuals and can become
nosocomial pathogens • ex of fruity odor: pseudomonas aeruginosa
o Aerobic, Gram negative rods (able to grow in the • TSI – Triple Sugar Ion agar
presence of oxygen) they are 3 types of sugars contain in this medium:
o Do NOT use carbohydrates as a source of energy glucose, fructose and sucrose
or degrade them through metabolic pathways no change in the reaction of the butt meaning
other than fermentation (also biochemically inert) they are not able to degrade the sugars that are
o Most are obligate aerobes and grow poorly if at all contained in this medium
under anaerobic conditions K/K = alkaline that there is no reaction in the
o Oxidizers and non-fermenters slant, no reaction in the butt
o Asaccharolytic, do not degrade carbohydrate at al numerator – slant / denominator - butt
o Usually display abundant growth on sheep BAP • Citrate
and CAP within 24-48 hours urea is negative, citrate is positive
Clues that Suggest the Isolation of a Non-Fermenter(NF) from green to blue color
o The organism does not ferment carbohydrates • SIM
o Note: Usually when the proteins are degraded in the 3 type of test:
surface of the agar slant, alkaline byproducts are Sulfide = negative
created that will turn the phenol red indicator from Indole = negative
red orange to pink. They will not be able to degrade
Motility = positive
almost most of the proteins, so they are generally
inert. They have flagella
o Alkaline slant/No change deep butt reaction in TSI • LIA (lysine iron agar) - negative
and KIA • Nitrate - will be positive in pseudomonas
aeruginosa
• MR – test are negative in pseudomonas
aeruginosa
• Most of them aren’t able to present positive
} butt
o
results because they are biochemically inert.
They may display unique colony morphologies

} slant o
andpigmentation
Often MDR or Multi-Drug Resistant

Biochemical Tests for Identification of NF(-)R

o Violet reaction – positive; colorless - negative o Hugh-Leifson OF medium
o Require oxygen for the metabolism of o Oxidase – most of them will be positive
carbohydrate if they are able to use them at all o Decarboxylation of amino acids (glycine, arginine)
o They fail to ferment carbohydrates o Motility
o NF can be oxidase positive o Nitrate reduction
o May fail to grow or show poor growth on o Acetamide reduction
o Growth at 42°C – pseudomonas positive
McConkey agar
o Pigments – pseudomonas aeruginosa can have various
o Some have fruity, sweet or unique odors combination of pigments; Mueller Hinton or Nutrient
agar
 o Colony morphologies the open tube only
o Distinct odors • Fermentative: development of a yellow color in
Relative Frequency of the Type of Pigments Produce by both open and closed tubes (anaerobic or
Pseudomonas Aeruginosa Strains aerobic environment)
2. Negative: a negative carbohydrate utilization test is
indicated by the absence of a yellow color (media
remains green or turns blue)
• Non-oxidizer/Non-fermenter

Types of Pigments
• Pyoverdin - 63.1% of pseudomonas will be able to
produce Pyoverdin pigment (yellow green)
• Fluorescin – a pigment that can glow (also yellow
From left to right.
green)
1. Control
• Fluorescein + Pyocianin = when these are combined Left side – has mineral oil
you will produced the bluish-greenish Right side – open and no over layer of mineral oil
• Pyomelanin – brown pigment 2. E.coli
• Pyoverdin + Pyocianin – black na Positive in both aerobic and anaerobic tubes
• Pyoverdin + Fluorescin – combination of most Carbohydrates was able to metabolize by the
frequently isolated pigment organism
• Pyoverdin + Pyomelanin Facultative anaerobe
• No pigment 3. Pseudomonas
• Pyoverdin is the most isolated pigment produced by Able to utilize the carbohydrates in the presence of
pseudomonas pigment oxygen
Hugh-Leifson of Medium Oxidizers
1. Obtain pure, isolated colonies from an 18-24 hr 4. Alcaligenes
culture. No reaction at all
2. For each test organism, inoculate tubes in
duplicate. Inoculate by stabbing the agar to Pseudomonas
approximately ¼ inch from the bottom. o Gram negative bacilli belongingto
3. Apply sterile mineral oil, sterile melted paraffin, or o Pseudomonadaceae
sterile melted petrolatum to one of each duplicate o Motile by means of a single polar flagellum
tubes. Tighten the cap of the overlaid tube, and o Non-spore forming
loosen the cap of the non-overlaid tube. o Capsulated “Polysaccharide capsule”
4. Incubate both tubes aerobically at 35 degrees o Aerobic
o Breakdown glucose by oxidation i.e. Oxidative
Celsius plus or minus two for up to 14 days.
o Oxidase and catalase positive
5. Examine tubes daily for color change. o It has very simple nutritional requirements i.e. non-
Note: fastidious
- Able to differentiate organism if it is an oxidizer or o The most important pathogenic organism is P.
fermenter of carbohydrates (glucose) aeruginosa
OF Medium Expected Results o Optimum temperature is 37°C, and is able to grow at
1. Positive: a positive carbohydrate utilization test is 42°C (thermo resistant)
indicated by the development of a yellow color in o It is resistant to high concentrations of salts, dyes,
the medium (from blue to yellow) weak antiseptics, and many antibiotics
• Oxidative: development of a yellow color in o Common inhabitants of soil, water, GIT
 o P. fluorescens, putida and stutzeri o Reduces nitrate
o MOT is still unknown because of the location which o Decarboxylates arginine
they can be found. It is an environmental o It has the ability to adhere to the surfaces by fimbriae
organism. o Requires a break in the body’s defenses for an
o Catheters, shunts, devices that can lead to infection to begin
opportunistic infections. o Has natural resistance to many antibiotics
produced by bacteria and fungi
Pseudomonas aeruginosa o It is invasive (protease enzymes) and toxigenic
o GNR that may be encapsulated o Infection consists of 3 stages:
o Most strains are motile by means of single polar ✓ Bacterial attachment and colonization
flagellum ✓ Local invasion
o Common inhabitant of the environment such as ✓ Dissemination by means of blood and
soil, water, and plants systemic disease
o Survives well in wet environments o Most often found in hospitalized individuals
o Metabolism is oxidative and requires the presence
of oxygen Virulence Factors P. aeruginosa
o Will grow in the absence of oxygen if nitrate is o The ability to invade tissue is usually due to toxins
available as a respiratory electron acceptor and enzymes that breakdown physical barriers,
(possible to see growing in anaerobic condition, damage host cells and help the organism evade
even though it is considered an obligate aerobes host defenses
just to have to supply nutrients) o Extracellular protease: elastase protease (elastin)
o Rarely part of human microbiota and alkaline protease (proteins, collagen, etc)
o Opportunistic pathogen o Produces cytotoxins and hemolysins such as
o Often beta-hemolytic, with rough spreading flat phospholipase and lecithinase
colonies with a ground glass consistency with o The blue pigment appears to impair the ability of
serrated or jagged edges (hemolyze chip red cells the upper respiratory cilia to sweep out mucous
in BSA) and debris
o Colonies often display metallic sheen and blue- o Kills the cells of lung epithelium by preventing
green pigment (combination of pyocianin and catalase activity within the cell (not be able to
pyoverdin) neutralize toxic oxygen)
o Pigmentation can also be red or brown o It causes apoptosis of WBCs
o Often beta-hemolytic with a sweet fruity odor o Other virulence factors: LPS or endotoxin,
o (grape-like or taco-shell like) Exoenzyme S, Exotoxin A
o Colonies can be of 2 types: large and smooth with
flat edges and an elevated appearance and the Clinical Significance of P. aeruginosa
other mucoid appearance o Pseudomonas is involved in respiratory infections,
o Mucoid colony consistency is attributed to prevalent in ICU patients on ventilators and
the receiving ling-term therapy with broad spectrum
o production of alginate slime (presence of slime antibiotics
layer will prevent phagocytosis and it will have the o Individuals with cystic fibrosis are often colonized
ability to colonize areas because of the slimy by a unique mucoid strain that is difficult to
production of this alginate and able to cause eradicate
disease)
o Associated with bacteremia and septicemia
o Pigments: pyoverdine or fluorescein that is yellow
o Causes endocarditis in intravenous drug users and
and seen only as fluorescence under a UV light;
those with prosthetic heart valves
pyocyanin, a blue pigment produced abundantly
o Well-known cause of swimmer’s ear
inmedia of low iron content
o Major cause of bacterial keratitis often due to
o When combined the 2 pigments produce the
extended contact wear or poor contact lens
characteristic blue-green pigment seen often in
hygiene
cultures
o Prefers cartilages and joints of the bones of the
Pseudomonas aeruginosa Identification skull and trunk causing chronic osteomyelitis
o Grows well on most lab media o UTI caused by catherization, instrumentation, or
o Oxidizes glucose, fructose, xylose but not surgery
lactose or sucrose o Produces skin and soft tissue infections (burn
o Deaminates acetamide wound,pyoderma and dermatitis, folliculitis)
o Grows at 42°C Prevention and Treatment
 o Observation of proper isolation procedures, o Non-fluorescent GNB that is widely distributed in the
aseptic techniques and careful cleaning and environment
monitoring of respirators, catheters and other o Rare opportunistic pathogen best known as a soil
instruments denitrifier
o Treatment with anti-pseudomonal beta-lactam, o Often recognized by its unusual colony BAP:
fluoroquinolones and aminoglycosides, adherent, wrinkled, or leather, hard and dry with
carbapenem (imipenem) either a yellow or brown pigment
o Current standard is combination of anti- o It is difficult to mix into suspension, colonies resemble
pseudomonal beta-lactam and aminoglycoside B. pseudomallei, NLF on Mc
(carbonicilin and gentamycin) o Can grow at wide range of temperature 4-4.5°C;
optimum 35°C
Pseudomonas fluorescens and Pseudomonas putida o Motile, oxidase(+), no pigment is produced
o Often described together
o Motile, aerobic, oxidase + GNB Colony of P. stutzeri
o Produces pyoverdine or fluorescein but pyocyanin o Arginine decarboxylate negative
o Remember that only a non-fermentative gram o Oxidizes maltose and hydrolyze starch
bacilli, only P. aeruginosa is able to produce o Has been associated with bacteremia and septicemia,
pyocyanin. bone infection, endocarditis eye infection, meningitis,
skin infections, and UTIs
Pseudomonas fluorescein o Demonstrates at least 2 antibiotic resistance
o An environmental organism found in soil, water, mechanisms:
plants, and contaminated food such as milk o Alteration of OM proteins and LPS profiles
o Rarely isolated from clinical specimens because andpresence of beta-lactamases
they grow poorly at 35°C o Treatment include the use of aminoglycosides,
o Optimum growth temperature: 25-30°C (may cause tmp-sxt, Te, fluoroquinolones and third gen.
spoilage in milk) Cephalosporin (has four generations; but only use
o Motile, oxidase (+) 3rd and 4th generation)
o Same typical colonies on BAP, wet and gray. NLF
on McConkey ANOTHER GENUS
o Can grow at 4°C and hydrolyze gelatin (food Acinetobacter
spoilage of refrigerated food; associated with o Member of the family Moraxellaceae
spoilage of chicken and processed meat) o Consists of 25 DNA homology groups, 11 species
have been officially named
Pseudomonas putida o A. baumanii, A. lwoffi, and A. haemolyticus
o Also, an environmental organism found in soil and o Ubiquitous in the environment, in soil, water and
water foodstuffs in the hospital environment
o Known for its ability for bioremediation such o Associated with ventilators, humidifiers,
as breakdown of oil and toluene catheters,and other devices
o Has been isolated from lizards, insects, and o About 25% of adults carry the organisms on the
mammals skin,7% carry the organisms in their pharynx
o Optimum temperature for growth: 25°C, NLF on Mc
o Hospitalized patient may become easily colonized
o Motile, oxidase(+), fails to grow at 42°C
o Opportunistic pathogens, second to P. aeruginosa
3 Most Common Pseudomonas in. isolation frequency
o A. baumanii have been associated with UTI
(common) , pneumonia, tracheobronchitis, or
both, endocarditis, septicemia meningitis, cellulitis,
trauma, burns, introduction of foreign body
o A. baumanii have been reported in eye infections
o A. lwoffi is much less virulent and when isolated
indicates contamination or colonization rather
than infection

Pseudomonas stutzeri Identifying Characteristics of Acinetobacter

 o Strictly aerobic, gram negative coccobacilli Stenotrophomonas maltophilia
o Oxidase negative, catalase(+), non-motile o A motile, gram negative, oxidase negative rod
o (they are exact opposite of pseudomonas) o Found in soil, plants, and water as well as a
o Can appear as gram positive cocci in smears nosocomial pathogen
made from blood cultures o Originally included in the Pseudomonas, then became
o The purplish hue produced on McConkey may Xanthomonas, now it is the only species in the genus
resemble a lactose fermenting bacterium Stenotrophomonas
o pH 5.5 to 6.0; temperature 30-35°C are preferred
o A. baumanii is saccharolytic, A. lwoffi is Identifying Characteristics of S. maltophilia
assacharolytic o Appears as non-distinct, straight or slightly curved
o Produces a K/NC reaction rod in singles or pairs
o Majority of beta-hemolytic organisms are o BAP colonies are non-hemolytic, large, smooth and
called A.haemolyticus shiny, can be gray-white but may have a slightly
yellow pigment with a distinctive ammonia-like odor
Clinical Significance when the agar plate lid is initially removed
o Able to form biofilms on inanimate objects o Colonies may exhibit a lavender-green discolorationof
o Presence of pili allows the organisms to the agar in areas of heavy growth
adhere toepithelial cells o NLF, may exhibit a brownish discoloration of
o Once it adheres, it is able to create McConkey agar
proteins thatcan cause cell death o In blood agar plate, non hemolytic, large, smooth,
o It possesses LPS as part of its cell wall shiny
o Tends to be resistant to disinfectants
o Oxidase negative
o Can survive in moist and dry surfaces
o Strong rapid oxidation of maltose
o Best known for its multi-drug resistance
o Weaker oxidation of glucose
o Has the ability to acquire resistance to many o Lysine decarboxylase (+)
major classes of antibiotics including newer o DNAse (+)
beta-lactams o TSI: K/C
o The presence of resistance plasmids is a
significant virulence feature Clinical Significance
o A. baumanii has been named “gram negative o Important nosocomial pathogen in debilitated and
MRSA” immunosuppressed individuals
o The digestive tract, skin surface, and mucous o Produces proteases and elastases, and has LPS being
membranes of patients in ICU are reservoir sites a GNB a part of its cell wall
for this organism o Difficult to distinguish between colonization and
o Transmission occurs due to contact from hands of infection
healthcare workers or from environmental o Its presence is a marker that the patient is
reservoirs deteriorating
o A. baumanii infections outside the hospital are very o Commonly present in medical devices such as IV/CV
rare and urinary catheters
o Pneumonia is the most common infection
Prevention and Treatment of Acinetobacter o Cystic fibrosis patients can be colonized with S.
o Standard and Contact precautions maltophilia in addition to other fermenters
o Careful use of antibiotics o Its presence in urine is usually due to colonization
o Treatment should be based on susceptibility test of the urinary catheter or manipulation of the
results urinarytract
o Most A. baumanii are susceptible to the o It can be isolated in wounds. Unless there is a
carbapenems (Imipenem and Meropenem) presenceof WBCs in the form of pus, it may only be
Piperacillin-Tazobactam, most third generation a colonizer
Cephalosphorins, aminoglycosides
o Colistin is one of the few antimicrobials that can be Prevention and Treatment
used on MDR strains but is potentially nephrotoxic o Standard and Contact precaution
and neurotoxic (carefully given to parents) o S. maltophilia is intrinsically resistant to many
o Tigecycline, a tetracycline is a promising agent antibiotics due to production of beta-lactamases
and carbapenemases, plasmids, alteration of OM
ANOTHER GENUS proteins and an efflux mechanism
 (efflux mechanism - capacity to pump out o Extremely pathogenic for those with cystic fibrosis
antimicrobials in your system) o Possesses few virulence factors LPS and ability to
o It is well-known for developing resistance to adhere to mucin, resistance to multiple antibiotics
new antibiotics quickly o Studies show the presence of B. cepacian prior to
o Susceptible to TMP-SXT, Colistin and Polymyxin B lung transplant often results in death after
o Therapy usually requires a rifampin plus a transplantation
fluoroquinolone or beta-lactam
o Broth dilution is the methods recommended Treatment and Prevention
for susceptibility tests o Requires isolation of patient when hospitalized
o Antibiotic therapy rarely eradicates the organism
ANOTHER GENUS fromthe CF respiratory tract
Burkholderia cepacian Complex o Resistant to aminoglycosides
o A complex 9 subspecies not usually differentiated o Multiple antibiotics are necessary for treatment like
o Very significant motile GNB often associate Minocycline, Meropenem, Ceftazidime,
with apatient population esp. patients with Fluoroquinolones, Chloramophenicol
cystic fibrosis o It is susceptible to TMP-SXT
o Formerly Pseudomonas
o Inhabitants of soil and water, not part of Burkholderia pseudomallei
normal human microbiota o Oxidase(+), motile, aerobic GNB that is straight or
o Well-known as a plant pathogen but not as slightly curved
nosocomialpathogen and does not cause harm o Grows on standard lab media at 35°C in a CO2
to healthy individuals atmosphere
o Can be found in the hospital environment, tap o Has a smooth and mucoid colony or a dry wrinkles
water, disinfectants, soaps and lotions colony similar to P. stutzeri on BAP
o A characteristic musty or earthy odor
Identifying Characteristics o Colonies appear pink on McConkey due to oxidation
o Able to grow o n sheep’s BAP, CAP, and of lactose
McConkey o Decarboxylates arginine whereas P. stutzeri, B.
o NLF but after 4-7 days, they may become cepacian and B. gladioli is negative
dark pink or red due to oxidation of lactose o Organism is resistant to colistin and Polymyxin B
o BCSA (Burkholderia cepacia Selective Agar) o Motile, GNB found in soil and water
selects and differentiates the organism based o Associated with rice paddy surface waters
on the presence of CV, polymyxin B, Gm and Va o It can be transmitted person to person via aerosols
o OFPBL(oxidative-fermentative polymyxin B- o Causes meliodosis
bacitracin-lactose) is also selective and o Known as “Vietnamese time bomb”
differential. Colonies are yellow due to o The organism can survive in phagocytes and can
oxidation of lactose reactivated decades after the initial infection when
individuals become immunocompromised
Burkholderia cepacia o Reactivation often appears as multiple abscesses
o Commercial ID system may have difficulty in throughout the body and skin
identifying B. cepacian
o Molecular methods are now recommended Alcaligenes faecalis
o Weakly oxidase (+) o Inhabits the environment and not part of human
o TSI: K/NC microbiota
o Able to decarboxylate lysine o Can be found in hospital environment such as
o Able to oxidize glucose, maltose, and lactose respiratory equipment and disinfectants
o B. cepacian is able to oxidize mannitol while o Oxidase (+), motile, GNB produces a thin spreading
S.maltophilia is DNAse positive colony with irregular edges on sheep’s BAP
o The negative arginine reaction of B. cepacian o It can produce a green discoloration of the agar
willdifferentiate it from B. pseudomallei and o It smells green apple, NLF on McConkey
o B. gladioli is unable to oxidize maltose and lactose o Assaccharolytic, tends to create a strong alkaline
reaction on OF medium which appear as a blue color
o Reduces nitrate to nitrite
o Can cause opportunistic infections in
Clinical Significance immunocompromised individuals and those with
 underlying disease
o Has the ability to colonize cystic fibrosis patient
who are intubated

Achromobacter xylosoxidans
o Aerobic, motile, oxidase(+), NLF GNB
o Found in moist environment
o It can oxidize glucose and xylose
o Opportunistic pathogen capable of
causingbacteremia, meningitis, pneumonia,
peritonitis
o Capable of colonizing the respiratory tract of
persons with CF, medical equipment and
solutions

Elizabethkingia meningoseptica
(Chryseobacteriummeningospeticum)
o Formerly Flavobacterium meningospeticum
o Founds in soil, plants, water, food, and hospital
water sources including incubators, sinks,
faucets, saline solutions and respiratory
equipment
o Not part of normal human flora
o Can survive in chlorinated water
o It is an oxidase(+), non-motile, slightly
filamentous or thread-like GNB
o BAP colonies are circular, smooth, and
glistening with a light-yellow pigment
o May not grow well on McConkey
o Positive indole using an Ehrlich’s reagent
o Often associated with meningitis and
bacteremia inpremature infants
o Also implicated in pneumonia, cellulitis, and
abscesses in immunocompromised patients