MICROBIOLOGY

Lei Anne M. Baguio, RMT

Microbiology (20%)
• Bacteriology
• Virology
• Mycology
Microbiology

• derived from the words micro, bio, and logos which means the
study of small forms of life. Small life forms include bacteria,
fungi, viruses, protozoa, slime molds, algae. They are all
member of the microbial kingdom.
CHARACTERISTICS OF
PROKARYOTIC & EUKARYOTIC
CELLS
MEDICALLY IMPORTANT
ORGANISMS
• PARASITES
Eukaryotic; possess both DNA & RNA
Possess mitochondria and ribosomes and other features of a eukaryotic
cell
Have larger ribosomes (80 Svedberg units)
Unicellular (protozoa) to multicellular (helminthes)
• FUNGI
Heterotrophic eukaryotes → obtain nutrients through absorption
Unicellular (yeasts) to multicellular (hyphae)
Monomorphic (usually opportunistic) or dimorphic (true pathogenic)
Have larger ribosomes (80 Svedberg units)
MEDICALLY IMPORTANT
ORGANISMS
• VIRUSES
Acellular, surrounded by a protein coat; smallest infectious particles
Consist of DNA or RNA but not both
Obligate intracellular parasites
Can infect all cell including bacteria (bacteriophages) and other viruses
(virophages)
Mostly host and/or host cell specific
• BACTERIA
Prokaryotic; unicellular
Possess both DNA and RNA
No true nucleus (nucleoid); no mitochondria, golgi bodies or endoplasmic
reticulum
Have smaller ribosomes (70 Svedberg units)
COMPARISON OF MEDICALLY
IMPORTANT
MICROORGANISMS
FUNDAMENTAL SHAPES OF
BACTERIA
• COCCUS
Round or Spherical
Morphologic variations: coffee bean shaped, lancet-shaped
Average diameter: 1 μm
Examples: Staphylococcus, Gonococcus, Meningococcus
• BACILLUS
• Rod-shaped
• Morphologic variations: Club-shaped-comma shaped, filamentous
• Average size: 2 x 0.5 μm
• Examples: Clostridium, Corynebacterium, Mycobacterium
FUNDAMENTAL SHAPES OF
BACTERIA
• SPIRILLUM
Spiral or coiled organisms
 3 forms:
Treponema- with fine regular coils
Borrelia- coarse, irregular coils
Leptospira- very fine regular coils with hook at one or both ends
Average size: may be as long as 40 μm
ARRANGEMENT OF BACTERIA
• Arrangement is
dependent on plane of
division of bacteria
and position taken
after cell division has
occurred.
STRUCTURES OF AN IDEALIZED
BACTERIUM
I. ENVELOPE STRUCTURES
o Glycocalyx
o Outer membrane
o Cell wall
o Periplasmic membrane
o Plasma membrane
A. Glycocalyx
• Located outside the cell that contains polysaccharide
• Also called capsule or slime layer
o Capsule -when strongly adherent to the cell wall
o Slime layer- when loosely attached
• Functions for adherence to cell surfaces
• Most bacteria potentially can have a capsule but is not essential to
the growth of the bacteria but important in survival inside the host
• Readily apparent as in S. pneumonia, B. anthracis or in C. perfringens
A. Glycocalyx
• Demonstrable by use of Hiss capsule stain, appearing as clear space
around the organism
• Size may be 2-3 X the diameter of the cell
• Significance of capsule:
o Antigenic → stimulates antibody formation
o Associated with virulence of the bacteria → delays ingestion of bacteria by
phagocytes (anti-phagocytic)
o Transformable characteristic- genes for capsule formation can be transferred
to other bacteria
A. Glycocalyx
• Composition of capsule:
o Complex polysaccharide – Streptococcus, Enterobacter arogenes
o Polymers of D(-) glutamic acid- B. anthracis
o Hyaluronic acid – Group A Streptococcus
• Can be lost if grown repeatedly in culture
B. Cell Wall
• Often referred to as murein sacculus
• Composed of peptidoglycan (murein or mucopeptide)
• Functions:
1. Gives form and rigidity to bacteria
2. Provides protection to the bacterial cell
3. Target of beta-lactum antibiotics
4. Lipoteichoic acid component- for attachement and adherence
5. Mycolic acid in acidfast bacteria – responsible for acidfastness
B. Cell Wall
• PEPTIDOGLYCAN:
• 2 amino acids: glucosamine and muramic acid.
• Chains may vary from less than 10 to more than 170 disaccharide units.
• Consists of polymeric backbones of repeating N-actylglucosamine and
N-acetylmuramic acid joined together by a tetrapeptide side chains and
cross bridges
• Diaminopimelic acid – an important element of bacterial cell wall
which an immediate precursor of lysine in GRAM NEGATIVE bacteria, it
occupies position 3.
B. Cell Wall
• LYTIC ENZYMES:
• 3 Major Groups:
1. Endo-β-1,4 N-acetylhexosamidases
2. Endopeptidases
3. Amidases
B. Cell Wall
• NON-PEPTIDOGLYCAN COMPONENTS:
• Envelope proteins: e.g. M. proteins
• Capsular polysaccharides
o Diverse capsule and surface polymers
o Soluble specific substances
o Hyaluronic acid
o Does not affect viability of the cell
B. Cell Wall
Acid Fast Bacteria: •  More than 50% of lipid components
• Mycobacteria and Nocardia are esterified mycolic acid
• Stain red with carbol fucshin and resist • Glycolipids
decolorization by acid alcohol not cell wall bound
o Trehalose, mycolates, sulfolipid,
• Mycolic Acid lipooligosaccharides, mycosides
α substituted, β hydroxyl fatty acids that and lipopolysaccharides
occur in Myobacteria as esters bound to
cell wall polysaccharides
• Contains equal amounts of
peptidoglycan, arabinans and lipids
B. Cell Wall
• Cell Wall Defective Bacteria:
1. Protoplasts-
with external surfaces free of cell wall constituents; osmotically
fragile; usually derived from gram positive bacteria.
2. Spheroplasts
have some remnants of the cell membrane, usually derived from
gram negative bacteria.
3. L forms (L phase variants)
able to grow and divide and can revert to parent bacterial form .
C. Outer Membrane
• Part of the cell wall in gram negative bacteria
• Located external to the peptidoglycan layer
• Composition:
o Lipid bilayer- attached to peptidoglycan by lipoproteins that cross the
periplasmic space
o Proteins- include porins which are involved in the transport of ions and
hydrophilic compounds
o LPS- composed of lipid A, a polysaccharide-rich core, and a polysaccharide
side chain
 Polysaccharide: designated as O antigen
  Lipid portion: responsible for biologic effects of endotoxin
C. Outer Membrane
• Functions:
1. Maintains the structure of the bacteria
2. Barrier to noxious environmental compounds
3. Molecular sieve for small water soluble molecules
D. Periplasmic Space
• Fluid-filled space located between the outer membrane and the plasma
membrane
• Contains hydrolytic enzymes and components of various transport systems
 E. Plasma Membrane (Cytoplasmic
Membrane)
• Made up of 2 layers of lipids
• The inside layer consists of actin-like filaments which is responsible for
the spiral appearance of treponemes.
• Functions:
1. Physical and metabolic barrier between interior and exterior of bacterial cell
2. Exhibits selective permeability
3. Location of bacterial electron transport system
4. Excretion of hydrolytic exoenzymes
5. Contains enzymes and receptors important in various systems of the cell
• Demonstrable by electron microscopy
 II. INTERNAL or CYTOPLASMIC
STRUCTURES
• Bacterial Nucleus (Nucleoid)
• Mesosome
• Ribosomes
• Storage Granules
 III. SURFACE APPENDAGES
• Flagella
• Pili (Fimbriae)
• Spores
A. Flagella
• Organ of locomotion; seen usually in bacilli
• Originates from cytoplasmic membrane
• Slender whip like structures → exhibit lashing, forwards and rotatory
movements
• Made up of protein called flagellin
• Parts: basal body, hook, filament
A. Flagella
• Classification:
o Monotrichous- single polar flagellum
o Lophotrichous- a tuft of flagella at one end or at both ends
o Amphitrichous- single flagella at both ends
o Peritrichous- flagella all around the bacillus
o Atrichous- without flagella
• Demonstrable by electron microscopy or silver impregnation
techniques (Fisher & Conn flagellar or Leifson stain)
• Highly antigenic (H antigen)
o Not vital to survival of bacteria
o Mutation or use of Waning blender can mean loss of flagella
B. Pili (Fimbriae)
• Pili and Fimbriae are regarded synonymous (common pili)
• Thread-like or hair-like structures found in gram negative bacteria
• Composed of structural protein (pilins) & adhesins
• Fimbriae can be distinguished from flagella by its smaller diameter
and it is not coiled
• Twitching motility
• Demonstrable by electron microscopy
B. Pili (Fimbriae)
• Types:
• 1. Common pili – thousands of pili around bacteria
• 2. Sex (F) pili – one or 2 in a bacterium
• Functions:
1. Adhesion- adherence to glycoproteins of GUT; common pili
2. Used in transfer of genetic material by the process of conjugation
(sex pili)
3. Virulence – ability to infect or damage a host
4. Antigenic – can induce antibody production
5. Antiphagocytic – prevents action of phagocytes/ prevents
occurrence of phagocytosis
C. Spores (Endospores)
• Resistant structure which enable bacteria to withstand
adverse environmental conditions; convert to vegetative
forms under favorable conditions
o Spore of C. tetani- can remain alive in soil for as long as 20 years
and as long as 5 years in chopped meat medium
• Found only in few gram positive bacteria:
1. Clostridium
2. Bacillus
3. A species of Rickettsia (Coxiella)
C. Spores (Endospores)
• Round, oval, or elliptical structures; located terminally, sub-
terminally or centrally in bacilli
• Demonstrated by Dorner spore stain, Schaeffer & Fulton stain,
heat and acetic acid method
• Components:
1. Complete copy of chromosome
2. Minimum amount of proteins and ribosomes
3. High amount of calcium bound to dipicolinic acid→ calcium
dipicolinate
4. Keratin-like protein coat
Simple or Direct Stain
• Makes use of aniline dyes (e.g. malachite green, Bismarck brown,
crystal violet, methylene blue, safranin, carbol fuchsin)
• Stained with the dye for 1 minute and organism retains the color of
the dye
• Not very informative and only demonstrates the shape and basic
structure of the organism
Differential Stain (most important)
• GRAM STAIN
• Differentiates gram
positive from gram
negative bacteria.
Gram Positive Bacteria are
stained VIOLET
Gram Negative Bacteria are
stained RED or PINK
GRAM STAIN
• Crystal Violet -is the primary stain, next step is the
• Grams Iodine -which acts as a mordant, meaning it is an agent that
fixes the primary stain to the cell wall of the organism.
• Decolorizer (95% alcohol) -which can be an acetone or ethanol or a
combination of both (remove the color of the primary stain)
• Safranin -the Counterstain (if the counterstain’s color sticks to the
organism = a GRAM NEGATIVE bacteria.)

GRAM POSTIVE = has thicker peptidoglycan layer allowing them to

absorb the primary stain.
GRAM NEGATIVE = has thinner peptidoglycan layer, a reason why
decolorizer (alcohol) removes their primary stain. They can take up the
color of the counterstain.
Differential Stain (most important)
• ACID FAST STAIN
• Differentiates acid fast from non-acid fast
bacteria GENERAL RULE for Acid Fast
• Types: staining:
Ziehl Neelsen stain (HOT method), All bacteria are NON ACID FAST
Kinyoun stain (COLD method)
with the exception of:
• Bacteria stains RED against blue or green 1. Mycobacterium
background depending on the type of
stain used
2. Nocardia (partially acid fast)
• Useful in the diagnosis of Mycobacterium
(M. tuberculosis and M. leprae)
Special (Selective) Stain
• Emphasizes certain structures or features of the bacteria
• Includes the following:
Indirect (Relief) Stain
• Used for bacteria or structures difficult to stain
• Organism or structure will be unstained while background
will appear dark
• Includes the following:
o Burri’s method: utilized Dorner or Nigrosin
o India ink method or Nigrosin to determine capsule of C.
neoformans
BACTERIAL METABOLISM
• 2 Components of Metabolism:

1. Catabolism- process of substrate breakdown and conversion to

usable energy
2. Anabolism (biosynthesis)- synthesis of cellular constituents
1. Fermentation
• Characterized by phosphorylation of substrate (intermediate) → a
pyrophosphate bond is donated to ADP
• Utilized by anaerobic organisms.
• End products: lactate, butyrate, ethanol, acetoin → accumulate in
the medium →useful for identification of bacteria, esp.
Enterobacteriaceae
• Pyruvic acid is a key metabolic intermediate
 2. Oxidation (Respiration)
• Molecular oxygen is the final electron acceptor
• Efficient energy-generating process
• Utilized by obligate aerobes and facultative anaerobes in the formation
of ATP
• Most important pathway is the Krebs or TCA (Tricarboxylic Acid) cycle.

A. Embden-Meyerhof-Parnas Pathway (Glycolysis)

• Major pathway of glucose catabolism → conversion of glucose to pyruvate
• Rate limiting enzyme: fructose-6-phosphate dehydrogenase
• Does not require oxygen (anaerobic)
• Used by many bacteria, including all members of Enterobacteriaceae
B. Entner-Doudoroff Pathway
• Major pathway for glucose by obligate anaerobes (lack
phosphofructokinase)
• Converts glucose-6-phosphate rather than glucose to pyruvate &
glyceraldehyde phosphate
• Generates one NADPH per molecule of glucose; uses one ATP
C. Pentose Phosphate Shunt (Phosphogluconate Pathway, HMP Shunt)
• Converts glucose to ribulose-5-phosphate → rearranged into other
carbon sugars
• Provide pentoses for nucleotide synthesis
• Produce glyceraldehyde-3-phosphate → converted to pyruvate.
A. Embden-Meyerhof-Parnas Pathway (Glycolysis)
• Major pathway of glucose catabolism → conversion of glucose to pyruvate
• Rate limiting enzyme: fructose-6-phosphate dehydrogenase
• Does not require oxygen (anaerobic)
• Used by many bacteria, including all members of Enterobacteriaceae
B. B. Entner-Doudoroff Pathway
• Major pathway for glucose by obligate anaerobes (lack
phosphofructokinase)
• Converts glucose-6-phosphate rather than glucose to pyruvate &
glyceraldehyde phosphate
• Generates one NADPH per molecule of glucose; uses one ATP
C. C. Pentose Phosphate Shunt (Phosphogluconate Pathway, HMP
Shunt)
BACTERIAL PHYSIOLOGY AND
NUTRITION
Growth
• An orderly increase in the sum of all components of an organism
• A process that entails the replication of all cellular structures,
organelles & components
• Needs a source of energy and suitable environmental conditions
• Measurement of microbial concentrations:
1. Cell concentration
2. Biomass density
REQUIREMENTS FOR GROWTH
NUTRITIONAL TYPES
• 1) Inorganic Carbon dioxide • 2) Organic compounds
• Autotrophs: making of own food • Heterotrophs: using ready-made
by reducing CO2 organic molecules for food
• Photoautotrophs (energy source: • Photoheterotrophs (energy
light): photosynthetic bacteria, source: light): purple nonsulfur
algae, cyanobacteria, bacteria, green nonsulfur
• Chemoautotrophs (energy bacteria
source: inorganic compounds): • Chemoheterotrophs (energy
iron, sulfur, hydrogen, and source: organic compounds):
nitrifying bacteria protozoans, fungi
pH- most bacteria grow at pH 6.0-8.0
a) Alkalophiles- grow best at pH 8.4-9.0
b) Neutrohphiles- grow best at pH 7.5-8.0
c) Acidophiles- grow best at pH 6.5-7.0
GRAM POSITIVE COCCI
Staphylococcus
Family Micrococcaceae
The term “Staphylococcus”
derived from Greek:
Staphyle = bunch of grapes
Kokkos = berry
meaning bacteria occurring in
grapelike clusters or berry
General characteristics:
• Gram (+) cocci, spherical, 1 micra in diameter
• Arranged in grapelike clusters
• Non-motile, non-spore former
• All are catalase positive
• Staphylococcus aureus is coagulase positive
• Grow best in aerobic conditions but may behave as facultative
anaerobes (EXCEPT S. saprophyticus which is obligate anaerobe)
• Mesophilic & chemoheterophic
• Capsulated-slime layer or biofilm - S. aureus, S. epidermidis
Culture:
• Grow readily on most bacteriologic culture media
• Aerobic or Microaerophilic environment
• Rapid growth at 37oC; pigment production best at 20oC- 25oC
• Colonies on solid media- round, smooth, raised, glistening
• S. aureus- gray to deep golden yellow colonies; (+) varying degrees of
hemolysis on BAP (Blood Agar Plate)
• S. epidermidis- gray to white on primary isolation → (+) pigments on
prolonged incubation
Growth Characteristics:
• (+) Catalase Production → differentiate from Streptococci
All staphylococcus are catalase positive
S. aureus is coagulase positive
• Slowly ferment carbohydrates → (+) lactic acid production, (-) gas
• Relative resistance to:
1. Drying
2. Heat (withstand 50oC for 30 minutes)
3. 9% NaCl
• Inhibited by certain chemicals (e.g. 3% hexachlorophene)
S. aureus
• Natural habitat: nostril (anterior nares) and skin
** S. aureus is part of our normal flora **
• Morphology:
• Gram-positive cocci, 0.5-1.5um in diameter; occur characteristically in group,
also singly and in pairs
• Form irregular grapelike clusters (since divide in 3 planes)
• Non-motile, non-sporing and few strains are encapsulated
ANTIGENIC STRUCTURES OF
STAPHYLOCOCCI
1. Cell Wall Components
a) Teichoic Acids
o Important for the attachment of the staphylococci on the mucosal surface
b) Peptidoglycan
o Polysaccharide polymer; destroyed by strong acid or exposure to lysozyme
o Effects/Function:
i. Provides rigid exoskeleton of the cell wall
ii. Elicits production of IL-1 (endogenous pyrogen) and opsonizing
antibodies
iii. Chemoattractant for PMNs
iv. With endotoxin-like activity
v. Activate complement
ANTIGENIC STRUCTURES OF
STAPHYLOCOCCI
1. Cell Wall Components
c) Protein A
• Characterized among a group of adhesins called microbial surface
components recognizing adhesive matrix molecules (MSCRAMMS) -
mediate bacterial attachment to host cell
• Other actions: bind to Fc portion of IgG (EXCEPT IgG3) → can combine
with a specific antigen. Prevents antibodies from binding to the
bacteria, hindering opsonization and acid complement
• activation
ANTIGENIC STRUCTURES OF
STAPHYLOCOCCI
2. Polysaccharide Capsule
• At least 11 serotypes → types 5 & 8 responsible for majority of infections
• Action: inhibit phagocytosis by PMNs leukocytes
VIRULENCE FACTORS: ENZYMES &
TOXINS
1. ENZYMES
a) Catalase- all Staphylococci are catalase (+) [Strep are catalase (-)]
• Used for conversion of H2O2 (hydrogen peroxide) to water (H2O) and
oxygen (O)
• Catalase test is used to differentiate Staphylococci from Streptococci
• Catalase test is positive if there is bubble formation, indicating the
presence of oxygen.
b) Coagulase- mainly produced by S. aureus
•  Binds prothrombin → (+) fibrin polymerization → fibrin deposited on the
surface of the organism → impair engulfment by phagocytic cells
•  Associated with invasive potential
•  Also called “Clumping Factor”
VIRULENCE FACTORS: ENZYMES &
TOXINS
1. ENZYMES
c) Other enzymes:
• Hyaluronidase- spreading factor
• Staphylokinase- cause fibrinolysis
• Proteinases- degrade proteins
• Lipases- degrade lipids
• ß-Lactamases- most of Staphylococci are resistant to penicillin
VIRULENCE FACTORS: ENZYMES &
TOXINS
2. TOXINS
a. Cytolysins (Hemolysins)
• Target: Eukaryotic cell membranes
• It can lead to tissue destruction and abscess formation
• Four types:
i. Alpha-hemolysin – lyse erythrocytes; damage platelets and macrophages;
severe tissue damage
ii. Beta-hemolysin (Sphingomyelinase C) – acts on sphingomyelin in the
plasma membrane of erythrocytes
iii. Delta-hemolysin- disrupts biologic membranes; possible role in S. aureus
diarrheal diseases
iv. Gamma-hemolysin- a leucocidin
VIRULENCE FACTORS: ENZYMES &
TOXINS
2. TOXINS
b. Exfoliative toxin A (epidermolytic toxin)
• Encoded by eta gene; superantigen
• Plasmid-mediated toxin
• Cause dissolution of mucopolysaccharide matrix of epidermis → cause
Staphylococcal Scalded Skin Syndrome (SSSS) or Ritter disease
• Epidermolytic toxin A – a chromosomal gene; heat stable
• Epidermolytic toxin B – produced by plasmid; heat labile
VIRULENCE FACTORS: ENZYMES &
TOXINS
2. TOXINS
c. Toxic Shock Syndrome Toxin 1 (enterotoxin F)
• Chromosomal-mediated toxin; gene found in 20% of S. aureus isolates
• Superantigen
• Bind to MHC class II molecules → (+) T cell activation
d. Enterotoxins
• A-D, G-J, K-R, U and V; superantigens; 50% of isolates Heat-stable &
resistant to GIT enzymes
• Produced when S. aureus grows in carbohydrate and protein foods →
important cause of food poisoning
• Enterotoxin → causes vomiting and diarrhea
CLINICAL SYNDROMES
1. Staphylococcal Scalded Skin Syndrome (SSSS)
• Bullous exfoliative dermatitis
• Blister- clear fluid without organisms
• 7-10 days
• Due to exfoliative toxin- a protease that can cleave the desmoglein (desmoglein
is the one holding the granulosum and spinosum layer of the skin)
2. Toxic Shock Syndrome (TSS)
• Abrupt onset of high fever, diarrhea with vomiting,myalgias, scarlatiniform rash
• Severe cases: hypotension with cardiac and renal failure
• Onset within 5 days after onset of menses in young women using high-
absorbency tampons
CLINICAL SYNDROMES
3. Food Poisoning
• Intoxication
• Incubation: 1-8 hours
• Due to enterotoxin
• Nausea, vomiting, diarrhea without fever (or with low-grade fever [38oC])
4. Staphylococci of low invasiveness
• Staphylococci of low invasiveness
5. Wound infection or infection after trauma
• Due to direct contamination
• Examples: meningitis after skull fracture, open fracture (chronic osteomyelitis)
6. Bacteremia
DIFFERENTIAL TEST FOR
STAPHYLOCOCCI
1. COAGULASE TEST
• Reagent: rabbit plasma
a. Slide method
-Detects cell bound coagulase/clumping factor on the surface of the cell
wall which reacts with the fibrinogen in the plasma
-(+) result: clot/coagulum formation within 30 seconds.
b. Tube method
-Considered a sensitive method; definitive test
-detects extracellular/unbound/free coagulase
-(+) result: clot/coagulum formation after 1-4 hours of incubation.
DIFFERENTIAL TEST FOR
STAPHYLOCOCCI
2. MANNITOL FERMENTATION TEST
• Used to differentiate pathogenic from nonpathogenic staphylococci.
• MSA (1% mannitol + 7.5% NaCl) is both a selective and differential
medium.
• pH indicator: phenol red (will turn to yellow color)
• Result: (+) yellow color – S. aureus (colonies surrounded by a yellow halo)
• S. saprophyticus – some strains also ferments mannitol (resemble S.
aureus on MSA)
Methicillin Resistant Staphylococcus
aureus (MRSA)
• S trains that are resistant to methcillin, nafcillin, and other
penicillinase-resistant penicillins due to the secretion or evolution of a
more advance penicillinase.
• Its resistance is acquired from multi-antibiotic therapy using broad-
spectrum antibiotics that favors the development of multi-drug
resistance (usually acquired by plasmid exchange).
• Another factor for developing methicillin resistance is when patient
doesn’t finish the antibiotic cycle (7 days).
• Effective treatment for MRSA is Vancomycin.
Staphylococcus epidermidis
• Part of the normal flora of the skin and may consider an opportunistic
pathogen during ectopic invasion and immunosuppression of patients.
• Coagulase negative
• Novobiocin susceptible
• Common contaminant in blood cultures.
• May cause bacteremia, infection in prosthetic heart valves, prosthetic
joints (most common infection from prosthetic operations), and
peritoneal dialysis catheters.
• Has polysaccharide capsule that allows adherence to these prosthetic
devices.
Staphylococcus saprophyticus
• Leading cause of UTI (second to E. coli) in
sexually active women and on children.
• Coagulase Negative
• Novobiocin Resistant
 Gram Positive Cocci

Catalase Test
Catalase Positive Catalase Negative
Genus Staphylococcus Genus Streptococcus

Coagulase Test

Coagulase Positive Coagulase Negative

Staphylococcus aureus
Novobiocin Test

Novobiocin Susceptible Novobiocin Resistant

Staphylococcus epidermidisStaphylococcus saprophyticus
Streptococcus
• Gram (+) in pairs or chains (when
grown in broth cultures)
• Grow in the presence of oxygen but
do not use oxygen for respiration →
Aerotolerant anaerobes
• Complex nutritional requirements
(blood or serum enriched medium);
colonies small and somewhat
transparent
• Catalase (-)
Streptococcus Classification
• Lancefield Classification
o based on the Lancefield antigen present on the surface of cocci.
o the antigen is composed of a amino sugar called C carbohydrate.
 Group A – rhamnose-N-acetylglucosamine
 Group B – rhamnose-glucosamine
 Group C – rhamnose-N-acetylgalactosamine
 Group D – glycerol teichoic acid containing d-alanine and glucose
 Group F – glucopyranosyl-N-acetylgalactosamine
Group A Streptococci
• Contains the A antigen, an amino sugar is composed of rhamnose-N-
acetylglucosamine.
• The most important specie under this group are the serotypes of
Streptococcus pyogenes which represents the group.
Group B Streptococci
• Contains the B antigen, an amino sugar is composed of rhamnose-
glucosamine.
• The most important specie under this group are the serotypes of
Streptococcus agalactiae which represents the group.
Group C Streptococci
• Contains the C antigen, an amino sugar is composed of rhamnose-N-
acetylgalactosamine.
• The most important specie under this group are the serotypes of
Streptococcus dysagalactiae spp. equimilis which represents the group.
Group D Streptococci
• Contains the D antigen, an amino sugar is composed of glycerol teichoic acid
containing D-alanine and glucose.
• The species under these group belongs to the genus Enterococcus such as
Enterococcus faecalis and Enterococcus faeceum and also a non-
enterococcus specie, the Streptococcus bovis.
Group F Streptococci
• Contains the F antigen, an amino sugar is composed of rhamnose-
glucosamine.
• the most important specie under this group are the serotypes of
Streptococcus anginosus, which represents the group.
• Under this group are: S. anginosus, S. intermedius, S. constellatus, and S. milleri
.
Hemolysis in BAM
• Alpha-hemolysis (α) – incomplete lysis of RBC → reduction of Hgb +
green pigment formation
o Characteristics of Group D, Pneumococci, and Viridans Streptococci.

• Beta-hemolysis (ß) – complete hemolysis of RBC → clearing of blood

around bacterial growth
o Characteristics of Groups A and B Streptococci.

• Gamma-hemolysis (γ) – no hemolysis

o Characteristics of Groups C and F Streptococci
Group A Streptococci
• Streptococcus pyogenes
 β-hemolytic streptococci
 Greek word “Streptus” meaning “chain” and the term “pyo” which means pus
producer.
 Causative agent of “strep throat” or soar throat, scarlet fever, rheumatic fever
and post-streptococcal acute glomerulonephritis.
Streptococcus pyogenes Virulence Factor
1. C carbohydrate - used by Rebecca Lancefield to divide streptococci to
groups.
2. M Protein - a major virulence factor, it inhibits the function of
complement and protects the organism from phagocytosis. The opsonin
(binding antibody for phagocytosis) produced binds the organism to aid
phagocytosis to macrophages and neutrophils (opsonization).
3. T Substance- an acid labile and heat labile protein that destroys the M
protein of the organism
4. R Protein- has the same function with T substance.
5. P Substance- a protein that are found on plasmids (nucleoprotein).
Streptococcus pyogenes Virulence Factor
1. Streptolysin O (Hemolysin) - an oxygen labile enzyme (easily
inactivated by oxygen) that destroy RBC and WBC and the reason
for beta-hemolysis in BAM. It is highly antigenic, can cause host to
produce the Anti-streptolysin O (ASO).
2. Streptolysin S (Hemolysin) - an oxygen stable enzyme, same
mechanism to ASO but not antigenic.
3. Pyrogenic Exotoxin- also called the erythrogenic toxin which is a
superantigen that is responsible for acquiring scarlet fever. It can
stimulate T lymphocytes response to pour excessive cytokines
causing Streptococcal Toxic Shock Syndrome.
Streptococcus pyogenes Diseases by Local
Invasion or Exotoxin Release
1. Streptococcal Pharygitis
-also known as “strep throat” or “sore throat” characterized by red swollen
tonsils and pharynx, and a purulent exudate on the tonsilar area with
swelling lymph nodes and high fever that last 5 days. Penicillin.
2. Skin Infections:
a) Folliculitis- infection of the hair follicle.
b) Streptococcal Cellulitis- acute, rapid spreading infection of the skin and
subcutaneous tissues characterized by pain, pain, tenderness, and erythema.
c) Impetigo (Streptococcal Pyoderma)- a vesicular, blistered eruptions becomes
crusty and flaky frequently on the mouth area on infected children.
Streptococcus pyogenes Diseases by Local
Invasion or Exotoxin Release
3. Erysepelas - resulting to the entrance of the organism in the skin
characterized by brawny edema and rapidly advancing margin of infection with
large blisters (bull
4. Necrotizing Fascitis (Streptococcal gangrene/flesh eating bacteria)- infection
of the subcuaneous tissues and fascia characterized by massive and rapid
spreading necrosis of the skin, subcutaneous tissues and muscles (myositis).
5. Scarlet Fever- derived from the development of untreated streptococcal
pharyngitis, the organism produce the enzyme pyrogenic/erythrogenic toxin
that produce fever with scarlet-red rashes that begins in the trunk of the
neck up to the extremities and face. During recrudescence the skin may
desquamate.
**Strawberry Tongue- red tongue with white spots.
Streptococcus pyogenes Delayed Antibody-
Mediated Diseases
1. Rheumatic Fever- resulted from untreated streptococcal pharyngitis
(a non-skin infection unlike scarlet fever), this disease occurs on
children of age 5 – 15.
• Rheumatic Heart Disease- an antibody-mediated disease
characterized by the excessive production of Anti-M protein
antibodies.
2. Acute Post-Streptococcal Glomerulonephritis- another antibody-
mediated inflammation caused by untreated streptococcal pyoderma
and/or streptococcal pharygitis caused by nephritogenic strains of S.
pyogenes.
Group B Streptococci
• Streptococcus agalactiae
• β-hemolytic streptococci.
• Part of vaginal normal flora of women.
• Infects neonates during delivery causing sepsis, meningitis, osteomyelitis, and
pneumonia.
Viridans Streptococci
• From the latin word “virdis” that means green, the do not belong to
the Lancefield classification, and produce α-hemolysis in BAM
(characterized by green coloration of the media surrounding the
colonies indicating incomplete hemolysis).
• Normal flora of the gastrointestinal tract frequently found in
nasopharynx and gingival crevices.
• The most important species are Streptococcus mutans, S. mitis, S.
salivarus and S. sanguis.
Viridans Streptococci
• Diseases Caused by the Viridans Streptococci:
1. Dental Carries/Plaque or Tooth Decay- caused by S. mutans, the organism
can ferment sucrose to lenvans and dextrans, an insoluble polysaccharide,
that accumulates at the surface of the enamel that actually coat a certain
part of the surface of the teeth.
2. Subacute Bacterial Endocarditis (SBE)- secondary infection from abnormal
valves (caused by congenital deformities and rheumatic or atherosclerotic
lesions and/or mitral valve prolapse).
3. Abcess- caused by the Streptococcus intermedius subgroup (S. intermedius,
S. constellatus, and S. anginosus) which are microaerophilic and part of the
GI tract flora.
Group D Streptococci
• Enterococci:
o Enterococcus faecalis
o Enterococcus faecium
• Non-Enterococci
o Streptococcus bovis
o Streptococcus equi
This group is α-hemolytic, and part of the normal flora of the human gut.
Group D Non-Enterococci
• Streptococcus bovis and S. equinus
o has same disease caused by enterococci.
o S. bovis- can cause colon malignancy in patient with bacteremia.
The Pneumococci
• Streptococcus pneumoniae
o also known as the pneumococci, common cause of pneumonia in adults,
meningitis to adults (characterized by nuchal rigidity/stiff neck), and otitis
media (middle ear infection) in children.
o do not belong to Lancefield Classification and produce α-hemolysis in BAM.
• Gram Staining: Gram-positive diplococci
• Detailed Morphology: Lancet shaped cocci
GRAM POSITIVE SPORE FORMING
RODS
Gram Positive bacilli
Bacillus anthracis
Morphology:
• Large gram positive bacilli.
• Non-motile.
• Found singly, in pairs or in long
chains.
• Capsule could be demonstrated
during growth in infected animals.
• Spores are formed in culture, dead
animal's tissue but not in the blood
of infected animals.
• Spores are oval and centrally
located.
 Bacillus anthracis
Survival in Soil
• Spores remain viable in soil for decades.
• In World War II in Scotland spores were exploded.
• Survived for >40 years and were eradicated in 1987
• Changing environmental conditions (temp. rain etc.) help in survival and multiplication.
PATHOLOGY
There are different clinical forms of anthrax:
• CUTANEOUS ANTHRAX: 95-98% of anthrax cases are of this type. Infection occur
through wounds, burns, which may progress to toxemia and septicemia. The site of
entry often produces a painless blister referred to as Malignant pustule.
• ENTERIC "INTESTINAL" ANTHRAX: Caused by the ingestion of infected meat. This
form of the disease is severe and fatal.
• PULMONARY ANTHRAX: Caused by the inhalation of large number of B. anthracis
spores. It is usually fetal. This clinical form is commonly known as "wool sorter
disease".
 Bacillus anthracis
ANTIGENIC STRUCTURE AND PATHOGENIC DETERMINANTS:
1. The Capsular Polypeptide: Composed of poly peptide of a high molecular weight
consisting of D-glutamic acid.
2. Polysaccharide Somatic Antigen: Composed of N acetulglucos-eamine and
Dgalactose.
3. Complex Protein Toxin: This toxin appear to be responsible for signs and
symptoms characteristic of anthrax. Accumulation of the toxin in tissue and its
effect on the central nervous system results in death by respiratory failure and
anoxia.
TREATMENT:
• Penicillin is the drug of choice.
• For penicillin-sensitive patients, tetracycline, erythromycin, chloramphenicol and
streptomycin may be given as alternative drugs.
Bacillus cereus
• Gram-positive, motile, facultative, aerobic sporeformer.
• Dimensions of vegetative cells are typically 1.0- 1.2 μm by 3.0-5.0 μm.
• The ellipsoidal spores are formed in a central or paracentral position
without swelling the sporangium.
• The organism does not ferment mannitol and has a very active
phospholipase (lecithinase) system. B. cereus is keyed as citrate(+),
arabinose (-), Gram (+), aerobic sporeformer.
Bacillus cereus
• Gram-positive spore forming bacilli
• Produce β-hemolysis on blood agar
• Pathogenesis & clinical features
• Spores are found on most raw foods like rice
• Spores are heat-resistant & survive rapid frying
• Produce enterotoxin – ingested → food poisoning.
• Short IP – 4-6 hours – similar to Staphylococcal food poisoning (vomiting &
diarrhoea)
• TREATMENT
• Symptomatic -fluid replacement
• Penicillin
Clostridium Species
• The clostridia are opportunistic pathogens. Nonetheless, they are
responsible for some of the deadliest diseases including gas gangrene,
tetanus and botulism. Less life-threatening diseases include
pseudomembranous colitis (PC) and food poisoning.
• Cause disease primarily through the production of numerous
exotoxins.
• perfringens, tetani, botulinum, difficile
C. tetani
• Found worldwide. Ubiquitous in soil, it is occasionally found in
intestinal flora of humans and animals.
• The cause of tetanus or lockjaw.
o Risus Sardonicus- spasm of facial muscles
o Trismus- Spasm of jaw muscles.
o Opisthotonus- spasm of vertebral muscles.
• When spores are introduced into wounds by contaminated soil or
foreign objects such as nails or glass splinters
 BIOCHEMICAL CHARACTERISTICS
• Morphology: long and slender; peritrichous
flagella,no capsule, terminal located round
spore(drum-stick apperance), its diameter greater
than vegetative cell.
• Culture:obligate anaerobic; Gram(+); swarming
occures on blood agar, faint hemolysis.
• Biochemical activities:does not ferment any
carbohydrate and proteins.
• Resistance: tolerate boiling for 60 min.alive several
ten years in soil.
• Classification and Antigenic Types: C tetani is the
only species. There are no serotypes
Clostridium tetani -Tetanospasmin
• Disseminates systemically
• Binds to ganglioside receptors
+ inhibitory neurones in CNS
• Glycine
+ neurotransmitter
• Stops nerve impulse to muscles
• Spastic paralysis
• Severe muscle contractions and spasms
• Can be fatal
C. perfringens
• Soil, fecal contamination
• Gas gangrene
o Swelling of tissues
o Gas release
o fermentation products
• Wound contamination
Toxins
toxin Biological Feature Types of Toxins
A B C D E
 lecithinase; increase the vascular
permeability; hemolytic; produces + + + + +
necrotizing activity

 Necrotizing activity, － + + － －
induces hypertension by
causing release of
catecholamines.

 increase the permeability － － － + －

of gastrointestinal wall

Necrotizing activity; increase

 the vascular permeability
－ － － － +
Toxins
• Many of these toxins have lethal, necrotizing, and hemolytic
properties;
• The alpha toxin produced by all types of C. perfringens, is a lecithinase
that lyses erythrocytes, platelets, leukocytes, and endothelial cells.
And its lethal action is proportionate to the rate at which it splits
lecithin to phosphorylcholine and diglyceride.
• The theta toxin has similar hemolytic and necrotizing effects.
• DNAase, hyaluronidase, a collagenase are also produced
Enterotoxin
• Many strains of type A produce enterotoxin, which is a heat-labile
protein and destroyed immediately at 100 ℃.
• Trypsin treatment enhances the toxin activity threefold.
• The toxin is produced primarily by type A strains but also by a few
type C and D strains.
• It disrupts ion transport in the ileum(primarily) and jejunum by
inserting into the cell membrane and altering membrane
permeability.
• As superantigen.
Gas gangrene
• Gas gangrene is a life-threatening disease with a poor
prognosis and often fatal outcome.
• Initial trauma to host tissue damages muscle and impairs
blood supply---lack of oxygenation
• Initial symptoms : fever and pain in the infected tissue.;
more local tissue necrosis and systemic toxemia. Infected
muscle is discolored (purple mottling) and edematous and
produces a foul-smelling exudate; gas bubbles form from
the products of anaerobic fermentation
Food poisoning
• Enterotoxin producing strains.
• These bacteria are found in mammalian feces and soil.
• Small numbers of the bacteria may also be found in foods and they
may propagate rapidly to dangerous concentrations if the food is
improperly stored and handled.
• The action of C. perfringens enterotoxin involves marked
hypersecretion in the jejunum and ileum, with loss of fluids and
electrolytes in diarrhea.
Cellulitis, Fasciitis
• Cellulitis, Fasciitis
• Fasciitis : a rapidly progressive, destructive process in which the
organisms spread through fascial plan es.
• Fasciitis causes suppuration and the formation of gas
• Absense of muscle involvement
• Rapidity
C. botulinum
• Anaerobic
• Gram-positive
• Rod-shaped
• Spore former
• Produces a protein neurotoxic.
• Soil, sediments of lakes, ponds, decaying
vegetation.
• Intestinal tracts of birds, mammals and
fish.
Division Transmission
• A, B, C1, D, E, F, and G. • Spores heat resistant.
• type A. 62% • canning.
• anaerobic environment
• Not all produce toxin.
• Botulism
• C and D not • eating uncooked foods
• G plasmid encoded. • spores
• GI, duodenum, blood stream,
neuromuscular synapses.
Virulence factors Botulinum toxin
• Bacterial protease • Bioterrorism
• Light chain,A,50 kDa; o not an infection
heavy chain,100kDa. o resembles a chemical attack
o 10 ng can kill a normal adult
• Disulfide bond.
• A potent toxin
Diagnosis
• By clinical symptoms alone
• Differentiation difficult.
• Most direct and effective: serum or feces.
• Most sensitive and widely used: mouse neutralization test. 48h.
Culturing of specimens 5-7d.
Treatment
• Individuals known to have ingested food with botulism should be
treated immediately with antiserum
• Antibiotic therapy (if infection)
• Vaccination will not protect hosts from botulism, however passive
immunisation with antibody is the treatment of choice for cases of botulism.
C. difficile
• After antibiotic use
• Intestinal normal flora - greatly
decreased
• Colonization occurs
• Enterotoxin secreted
• Pseudomembranous colitis
Pseudomembranous Colitis
• Pseudomembranous colitis (PC) results predominantly as a
consequence of the elimination of normal intestinal flora through
antibiotic therapy.
• Symptoms include abdominal pain with a watery diarrhea and
leukocytosis. "Pseudomembranes" consisting of fibrin, mucus and
leukocytes can be observed by colonoscopy.
• Untreated pseudomembranous colitis can be fatal in about 27-44%.
Virulence Factors
1. Anti-phagocytic capsule
• Also promote abscess formation
2. Tissue destructive enzymes
• B. fragilis produces variety of enzymes (lipases, proteases, collagenases)
that destroy tissue  Abscess Formation
3. Beta-lactamase production
• B. fragilis – protect themselves and other species in mixed infections
4. Superoxide dismutase production
• Protects bacteria from toxic O2 radicals as they move out of usual niche
GRAM POSITIVE NON-
SPOREFORMING RODS
Corynebacterium diphtheriae
• Disease caused: Diphtheria, Pseudomembrane
• Member of the normal flora of the skin.
• The pathogen release a powerful exotoxin into the bloodstream,
which specifically damages heart and neural cells by interfering
protein synthesis.

• Pseudomembrane- a grayish membrane-like lesion at the pharynx of

patients composed of fibrin, leukocytes, necrotic epithelial cells, and
the bacteria.
Diphtheria
Clinical Manifestation:
1. Sore throat
2. Fever
3. Dark inflammatory exudate on pharynx (darker and thicker
than strep throat)
4. Bleeding
Diphtheria
• Treatment and Prevention:
1. Antitoxin- inactivates the powerful circulating toxin that may
prevent damage to the heart and nervous tissues.
2. Penicillin or Erythromycin- effective in killing the bacteria,
preventing further exotoxin release.
3. DPT vaccine- cause immunity during infancy. Contains formalin
inactivated diphtheria toxin.
The Diphtheria Toxin
• Heat-labile polypeptide (MW: 16,000), has a lethat dose of 0.1 μg/kg.
• Composed of two subunits:
A Subunit- blocks the protein synthesis through inactivation of the
elongation factor in protein translation. “human antibiotic”
B Subunit- a large MW protein that binds the target cell and inject
inside the A subunit.
Listeria monocytogenes
• Motile gram positive rods that produce both exotoxin and endotoxin.
• Third most common cause of neonatal meningitis (following group B
streptococci and Escherichia coli).
• Common cause of meningitis to immunocompromised patients and
pregnant women.
• Can survive inside a phagocyte, it is called facultative intracellular
organism.
Pathogenesis
• Internalin- cell wall surface protein that interacts with E-cadherins
(surface receptors of epithelial cells), promoting phagocytosis by the
epthelial cells.
• Listeriolysin O- produced by the bacteria during formation of
phagolysosome responsible for the survival of the organism inside a
phagocyte by destroying the membrane of a phagolysosome and
escape to the cytoplasm.
• ActA- another surface protein that induce host’s actin polymerization,
which propels them to cell membrane.
Pathogenesis
• Filopods- formation on the body of the bacteria after escape to a
phagocyte that chemotactize other phagocytes, epithelial cells,
hepatocytes to ingest the organism (which tend to repeat the cycle).
• Iron- important in the virulence of the organism, since it require iron
for metabolism thus invading siderophore-rich cells and from
transferrin.
Listeriosis
• Clinical Manefestation:
1. Granulomatosis infantiseptica- early onset clinical picture on
neonates results from uterine circulation and disseminate as neonatal
sepsis, pustular lesions and granulomas containing the orgnism.
2. Still-birth- the newborn is delivered dead.
3. Listeria meningoencephalitis- disease on adults especially from
immunocompromised patients caused by adjacent bacteremia from L.
monocytogenes.
Erysipelothrix rhusiopathiae
• Also known as E. insidiosa.
• Cause the disease “erysipeloid” oftenly from fishermen, fish
hanndlers, abattoir workers, butchers.
**acquire from direct inoculation at a site of cut on fingers or skin (“seal finger”
or “whale finger”). No production of pus (as erysipelas of group A streptococci
produce pus)
• Bacteremia to this organism may cause endocarditis.
Norcardia asteroides
• Causative agent for norcardiosis, characterized by acute to chronic
pulmonary infection (lobar pneumonia), impaired cell-mediated
immunity.
• Most norcardiosis are opportunistic infections.
• A coccobacilli found in soil and water and not transmitted person to
person.
GRAM NEGATIVE COCCI
General Charateristics
• Aerobic, gram-negative diplococci • Primary pathogens:
o N. gonorrhoeae
o Nonmotile
o N. meningitidis
o Oxidase positive
o Catalase positive  They are cultured on "chocolate"
agar
o Fastidious, capnophilic
 N. meningitidis is maltose fermenter
• Habitat
 N. gonorrhoeae is maltose non
o Upper respiratory tract
fermenter
o Genitourinary tract
 N. meningititidis produces no beta
o Alimentary(Digestive) tract
lactamases.
 Some of N. gonorrhoeae produce
beta lactamases.
Neisseria gonorrheae
• Never considered part of normal flora –
man is the only known host.
• Found in urogenital tract, anorectal area,
oropharynx or conjunctiva
• Transmitted by sexual contact (person to
person); infected mother to newborn
during birth.
• Leading cause of sexually transmitted
disease
VIRULENCE FACTORS
• Pili: adhesion; can turn pili expression on/off
• PII outer membrane protein (OMP) - adhesin: adhesion
• Iron uptake mechanisms: transferrin, lactoferrin binding proteins
• IgA protease: only found in pathogenic
• Neisseria important in mucosal substances
• Lipooligosaccharide (LOS): tissue destruction, generation of cytokine
storm in severe cases
 Clinical Syndromes
1. Urethritis – (+) purulent discharge and painful urination in men → may
extend to epididymis; may lead to urethral strictures
2. Cervicitis (endocervix most commonly involved)
• Primary infection in women
• May extend to urethra and vagina → (+) mucopurulent discharge
• If untreated, lead to Pelvic Inflammatory Diseases
o May cause sterility, ectopic pregnancy or perihepatitis (Fitz-High-Curtis syndrome)
3. Bacteremia- usually in patient with complement deficiency
4. Eye infections: Conjunctivitis; Ophthalmia neonatorum – passage
through birth canal; may lead to blindness
5. Fitz-Hugh-Curtis syndrome
o Present as perihepatitis, anorectal gonorrhea, & pharyngitis
Laboratory Diagnosis: Neisseria
gonorrhoeae
1.In the male, the finding of numerous neutrophils containing gram
negative diplococci in a smear of urethral exudate provides a diagnosis of
gonococcal infection.
2.In the female a positive culture is also needed.
3.Culture:
N. gonorrhoeae grows best under aerobic conditions, and most
strains require CO2 also. Gonococci are very sensitive to heating or drying.
Cultures must be plated rapidly. N. gonorrhoeae grows rapidly producing
small, raised, grey or translucent colonies after overnight incubation.
4. Oxidase test. Positive.
Treatment
• One curative dose
• Sensitivity Testing
• Blind treatment: ceftriaxone, ciprofloxacin
• Spectinomycin
• Penicillin: resistance common.
Neisseria meningitidis
• Commensal of carriers in the nasopharynx
• Cross the epithelium and enter the circulatory
system
• Primarily affects the immunocompromised, young
children, trauma victims
• Leads to septicemia and localization to the
meninges causing inflammation of the brain
• Causative agent of Meningitis (Meningococcemia)
• Highly fatal (25% even if treated)
• Encapsulated strains A, B, C, Y, W-135
Pathogenicity: Neisseria meningitidis
The virulence factors are.
1. Polysaccharide capsule. It is antiphagocytic in nature.
2. Endotoxin. It induces septic shock by causing release of cytokines.
3. IgA protease. It cleaves the IgA antibodies present in respiratory
mucosa
Clinical Conditions: Neisseria meningitidis
1. Meningitis
• The symptoms are fever, headache, stiff neck, and an increased level of
Neutrophils in spinal fluid.
2. 2.Meningococcemia.
• It occurs due to multiplication of bacteria in the blood stream.
• The severe form of it is life-threatening Waterhouse- Friderichsen
syndrome.
Laboratory Diagnosis: Neisseria
gonorrhoeae
A. Specimens include. D. Oxidase test. Positive
1. Blood for culture and smears E. Manitol fermentation
2. Spinal fluid for smear, culture, The difference between N.
chemical analysis. meningitidis and N. gonorrhoeae
B. Blood smears on gram staining show is made on the basis of manitol
gram negative bean shaped diplococci. fermentation. Meningococci
C. Culture. ferment maltose, whereas
The organism grows best on gonococci do not
chocolate agar incubated at 37°C in a F. Latex agglutination test, which
5% CO2 atmosphere. Colonies are detects capsular polysaccharide in
transparent or opaque. the spinal fluid.
Treatment
• Parenteral antimicrobial
• Start blind treatment after collection of specimens by:
Ceftriaxone or cefotaxime
Change later according to sens. Test.
Contacts: rifampicin
Prevention: vaccination (polyvalent)
The non-pathogenic Neisseria
• Normal flora of upper respiratory tract
Some members
Neisseria cinera
Neisseria lactamica
Neisseria mucosa
Neisseria sicca
Neisseria subflava
Neisseria flavescens
Nesseria polysaccharea
Moraxella catarrhalis
• Previously known as Branhamella catarrhalis
• Normal commensal of the respiratory tract
• Has become an important opportunistic
pathogen
• Advanced age, Immunodeficiency,
Neutropenia, Other debilitating diseases
• Clinical infections:
o Pneumonia
o Sinusitis
o Otitis media (3rd most common cause)
Pathogenicity: Moraxella Catarrhalis
• Endotoxin
• Pili
• Beta-lactamase

1. Oropharyngeal endogenous strains spread into normally sterile

regions of the tracheobronchial tree, the middle ear, and sinuses
2. Acute purulent exacerbation of chronic bronchitis
3. Causes 10-15% of episodes of otitis media and sinusitis
4. Rarely associated with systemic infection (endocarditis, meningitidis)
Point of Differentiation
Species Growth Acid production
BAP R.T T/M Gluc Mal Lac Suc

N. gonorrhoeae =/+ = + + = = =

N. meningitidis + = + + + = =

N. lactamica + v + + + + =

N. sicca + + = + + = +

M. catarrhalis + + = = = = =
Selected Biochemical Reactions for Identification of
Neisseria and Moraxella catarrhalis
Glu Mal Lac Suc DNa BE
N. gon + – – – – –
N. men + + – – – –
N. lac2 + + + – – –
M. cat – – – – + +

• Glu=glucose, Mal=maltose, Lac=lactose, Suc=sucrose,

DNa=DNase, BE=butyrate esterase (indoxyl butyrate substrate),
• N. gon=N. gonorrhoeae, N. men=N. meningitidis, N. lac=N.
lactamica, M. cat=Moraxella catarrhalis
• Colistin-resistant saprophytic species of Neisseria
GRAM NEGATIVE RODS
The Gram Negative Bacilli
• Family Enterobacteriaceae
• The Pseudomonads and Uncommon Gram-Negative Rods
• Vibrios, Campylobacters, Helicobacters, and Associated Bacteria
• Haemophilus, Bordetella, Brucella and Francisella
• Yersinia and Pasteurella
The Family Enterobacteriaceae
• The enteric gram-negative rods.
• Comprise the normal flora of the gut of humans, and are found in
human excreta that can cause disease to immunocompromised
patients and extra-colon colonization.
• Few are pathogens (eg. Salmonella, Shigella, Yersinia, and some
strains of E. coli)
• Categorize as Coliforms and Non-coliforms.
• Can ferment glucose rather than oxidizing it, and some are lactose
fermenters (fast and slow) producing acid products and CO2.
• LPS: Consists of three components:

1. the outermost somatic O

polysaccharide (O antigen)
2. a core polysaccharide common to all
Enterobacteriaceae (enterobacterial
common antigen)
3. lipid A
• Serologic classification
 O polysaccharides
 capsular K antigens (type-specific
polysaccharides)
 the flagellar H proteins
• Common virulence factors
o Endotoxin (Lipid A of LPS)
o Capsule
o Antigenic phase variation
o Acquisition of growth factors (e.g. Fe)
o Resistance to serum killing
o Antimicrobial resistance
The Family Enterobacteriaceae
Major Genera:
Escherichia
Shigella
Salmonella
Edwardsiella
Citrobacter
Yersinia
Klebsiella
Enterobacter
Serratia
Proteus
Morganella
Providencia
Escherichia coli
• Compose the majority of coliform normal flora of the
intestines.
• Comprise the bacteria seen in feces.
• Some strains are non-pathogenic, the pathogenic strains
have genes or plasmids for virulence factor.
• Most strains are lactose fermenters and some are slow
lactose fermenters, dextrose are easily fermented to gas.
Escherichia coli Virulence Factor
1. Mucosal Interaction
a. Mucosal adherence with pili (colonization factor)
b. Ability to invade intestinal epithelial cells.
2. Exotoxin Production
a. Heat labile and stable toxin
b. Shiga-like toxin
3. Endotoxin
-Lipid A portion of lipopolysaccharide (LPS)
4. Iron-Binding Sideropores
-Obtain iron from human transferrin or lactoferrin.
Escherichia coli Diseases
1. Diarrhea
2. Urinary Tract Infection
-the most common cause of UTI, travelling of E. coli through the use of pile
from the anus to the urethra infecting the bladder (cystitis), and sometimes the
kidney (pyelonephritis).
3. Naonatal Meningitis
-together with the group B streptococcus, the common cause of meningitis.
4. Gram-negative sepsis (Bacteremia), occurring commonly on debilitated hospitalized
patients.
-most common cause of bacteremia.
5. E. coli pneumonia
-hospital-acquired disease
Escherichia coli Virulent Strains
Enteropathogenic E.coli (EPEC)
-most important cause of self-limiting chronic diarrhea in
infants invading the small intestines. Its pathology include:
1. Loss of microvilli (effacement)
2. Formation of a cup-like filamentous actin pedestals
3. Entry to the mucosal cells
-produce watery, non-mucoid diarrhea.
Escherichia coli Virulent Strains
Enterotoxigenic E. coli (ETEC)
-common cause of “traveler’s diarrhea” or “Montezuma’s Revenge”.
Another important cause of diarrhea on infants.
-produce heat-labile exotoxins (LT) that promotes the formation of the
disease and entry of the organism to the mucosal lining.
-produce also the heat-stable exotoxins (ST) that aids in the production
of more severe diarrhea.
-invades the small intestines
-produce watery, non-mucoid diarrhea.
Escherichia coli Virulent Strains
Enterohemorrhagic E. coli (EHEC)
-produce the verotoxin (named after its cytotoxicity to Vero cells) that
acts as shiga-like toxin similar to the toxin produced by Shigella
dysenteriae.
-invades the large intestines
-associated with hemorrhagic colitis (severe form of diarrhea) with
hemolytic uremic syndrome (HUS), a disease resulting in acute renal
failure, microangiopathic hemolytic anemia, and thrombocytopenia.
-bloody, watery diarrhea.
Escherichia coli Virulent Strains
Enteroinvasive E. coli (EIEC)
-mostly copared to Shigella dysenteriae, this strains are late or non-
lactose fermenters, non-motile, and invades intestinal mucosal
epithelium of the large intestines.
-bloody, pus-filled, watery mucoid diarrhea.
Escherichia coli Virulent Strains
Enteroaggregative E. coli
-cause acute and chronic diarrhea for >14 days, secretes ST-like toxin
and hemolysin.
-invades the small intestines.
-watery diarrhea
Klebsiella spp.
Two important species under this genus:
• Klebsiella pneumoniae
o Causes pneumonia and meningitis to children and UTI to hospitalized
patients with Foley catheters.
o Pneumonia is characterized by bloody sputum (red currant jelly),
violent and frequently destroyed lungs producing cavities
(extensive hemorrhagic necrotizing consolidation of the lungs).
• Klebsiella oxytoca
-causes UTI to women.
Klebsiella spp.
-displays great amount of O antigen because of its capsule. (displayed
by mucoid colonies on agar)
-non-motile.
Enterobacter aerogenes
-this organism has a small capsule, causes UTI and bacteremia.
-highly motile.
Serratia marcescence
-a common oppurtunistic pathogen of hospitalized patients.
-causes pneumonia, bacteremia, endocarditis (narcotic adicts)
-produce prodigosin (a bright red pigment)
Proteus spp.
-highly motile (produce swarming motility on plated agar), reduce
urea to ammonia rapidly (production of urease).
-causes UTI producing alkaline (ammoniacal) urine, pneumonia, and
focal lesions (to hospitalized patients receiving intravenous infusions)
-surface antigens cross-reacts with some Rickettsia spp. OX-19 and
OX-2 of P. vulgaris, and OX-K of P. mirabilis.
Two important species
Proteus vulgaris
Proteus mirabilis
Provedencia rettgeri
-causes UTI, another member of the normal flora of the human gut.
Citrobacter freundi
-cause UTI and sepsis
Shigella spp.
-obligate pathogen of the family enterobacteriaceae.
-”inert” organism due to their tendency to show negative results in
biochemical tests.
-some strains are late-lactose fermenters, some are non-lactose
fermenters.
-all species under this genus are non-motile and are encapsulated.
-do not produce H2S.
-transmission is through oral-fecal route, and not member of the normal
flora of the gut.
-do not invade blood and lymph.
Shigella spp.
Important species:
Shigella dyseteriae- common cause of dysentery worldwide especially
to the western country.
Shigella flexneri- cause of dysentery originated in Japan and other
oriental countries.
Shigella sonnei- cause dysentery originated in the Philippines and
spread through countries of south-east Asia.
Shigella boydii- cause dysentery originated in central america.
Shigella spp. Pathogenesis
-pathogenesis is similar to EIEC, both invades the intestnal epithelium and
release Shiga toxin (shiga-like toxin for EIEC) that causes cell destruction
producing shallow ulcers and microabscesses in the large intestines and ileum
leading to tissue necrosis of the mucous membrane, bleeding, formation of
pseudomembrane on ulcerated area (contains WBCs, fibrin, cell debris,
necrotic mucous membrane).
-bloody mucoid diarrhea.
-the B subunit adheres the organism to the microvilli.
-the A subunit of the Shiga toxin targets and inactivate the 60s ribosomes
inhibiting protein synthesis of the human cells.
Salmonella typhi
-also known as Salmonella typhimurium.
-another obligate pathogen under the family enterobacteriaceae.
-non-lactose fermenter, motile with lopotrichous flagella, and are H2S
producers.
-causes the disease Typhoid Fever (Enteric Fever), and other disease
such as bacteremia with focal lesions, and enterocolitis.
-has a polysaccharide capsule the Vi antigen which surrounds the O
antigen, thus added line of protection from phagoctosis.
Typhoid Fever
-also known as “enteric fever”, invades the intestinal epithelium and
lymph nodes to systemic blood circulation and even extra-intestinal
tissue invasion.
-the organism is phagocytized by monocytes and PMNs and survive
intracellularly (facultative intracellular parasite.
-characterized by fever and malaise, abdominal pain mimicking
apendicitis, headache.
-splenomegaly may occur in tissue invasion, small-pink rash on white
people.
Salmonella Sepsis
-this has the tendency for further tissue invasion other than the
spleen and intestines (brain, lungs, bones). The organism may survive
in the bloodstreams due to the Vi antigen that prevents opsonization.
-patients with Sickle-cell anemia are prone to Salmonella
Osteomyelitis.
Genus Yersinia and Pasteurella
-these are pleomorphic gram-negative rods that exhibits bipolar
staining to grams staining and other special staining procedures
(Wright-Giemsa).
-catalase positive, oxidase negative, microaerophilic, facultative
anaerobic.
-most have animal hosts and produce serious disease to humans.
Yersinia pestis
-causative agent of the pandemic plague (black death, bubonic plague).
-nonmotile.
-Mice are usually the host, flea are infected during blood meal to the mice.
-survive and multiply inside a phagocyte monocyte when phagocytized but killed
by polymorphonucleocyte.
Type III Secretion Factor- compose of membrane spanning complex that allows
the bacteriat to inject proteins directly to cytoplasm of the host cells.
V and W antigens- capsular proteins that requires calcium prevents the organism
to opsonization.
Yersinia enterocolytica and Yersinia
pseudotuberculosis
-cause acute gastroenteritis with intermittent diarrhea, transmitted
through oral-fecal route.
-grow best at 250C, motile at 250C but nonmotile at 370C.
Pasteurella spp.
Pasteurella multocida- part of human gut flora causes respiratory and
gastrointestinal diseases by zoonosis (bites of infected animals such as
cats and/or dogs) and hemorrhagic septicemia.
Pasteurella haemolytica- causes upper respiratory infection by
zoonotic route by infected animals (epizoonotic pneumonia).
Pasteurella pneumotropica- cause pneumonia and sepsis to
immunocompromised patients through animal bites.
Pasteurella ureae- causes respiratory disease and suppurative
infections.
Pseudomonads and Uncommon Gram-
Negative Rods
-widely distributed in water and soil, most are part of the normal flora
of the human gut.
-most organism belongs to this group causes hospital-acquired
infections and UTI.
Pseudomonas aeruginosa
-saphrophytes, motile, obligate aerobic, and produce water-soluble
pigments on solid colorless media such as:
Pyocyanin- fluorescent blue pigment
Pyoverdin- fluorescent greenpigment
Pyorubin- dark-red pigment
Pyomelanin- black pigment
-causes hospital-acquired infection especially for
immunocompromised patients.
Pseudomonas aeruginosa
-contains pili as its main virulence factor that promote attachment to host’s cells.
-produce exopolysaccharide capsule from cystic fibrosis patients.
-colonize mucous membranes of the hosts causing variety of infection such as
tissue damage, fever, shock, leukocytosis and leukopenia, oliguria, DIC, and adult
respiratory distress syndome in adult.
-resistant to many antibiotics.
-produce blue-green pus, meningitis (infected lumbar puncture, UTI (infected
catheters), necrotizing pneumona.
-causes invasive (malignant) otitis externa on diabetic patients, fatal sepsis, on
leukemia and lymphoma patients, eye infections on debilitated patients.
Pseudomonas aeruginosa
Verdoglobin- a breakdown product of hemoglobin or fluorescent
pigment can be detected in wounds, burns, or urine by UV
fluorescence.
Ecthyma Gangrenosum- hemorrhagic skin lessions product of sepsis
which lession is surrounded by erythema and pus.
Classification of Some of the Medcally
Important Pseudomonads
rRNA Homology Group Genus and Specie
I.
Fluorescent Group Pseudomonas aeruginosa
Pseudomonas fluorescens
Pseudomonas putida
Nonfluorescence Group Pseudomonas stutzeri
Pseudomonas mendocina

II Burkholderia pseudomallei
Burkholderia mallei
Burkholderia cepacia
Ralstonia picketti

III Comamonas spp.

Acidovorax spp.
IV Brevundimonas spp
V Stenotrophomonas maltophilia
Burkholderia pseudomallei
-causative agent of maleioidosis (a localized supurative infection of
the skin on the inoculation site of the organism that has a big
tendency of septicemia infecting several organs)
-”safety pin” appearance in specialized staining method
Primary pneumonitis- caused by inoculation of the organism through
airways or nasopharynx.
-infection is zoonotic
Burkholderia mallei
-causative agent of Glander’s disease, a zoonotic disease
characterized by ulceration in the skin or mucous membranes
followed by lymphangitis and sepsis and primary pseumonia.
Burkholderia cepacia
-a saphrophytic organism found in soil, water, and on decaying matter
causing hospital-acquired infections.
Stenotrophomonas maltophilia
-a free-ling bacteria that gives lavender-green or gray colored colonies
on BAM.
-cause hospital-acquired infections on patients taking antimicrobial
therapy and immunosuppresed hosts.
Uncommon Gram-Negative Rods
Acinetobacter baumannii
Actinobacillus actinomycetemcomitans
Capnocytophaga ochracea (gliding motility)
Cardiobacterium hominis
Chromobacterium violaceum (violet pigment colonies)
Eikinella corodens (fastidious capnophilic organism)
Chryseobacterium meningosepticum
Kingella kingae
Moraxella catarrhalis
Vibrios, Campylobacters, Helicobacter, &
Associated Bacteria
-widely distributed in nature:
Vibrios- found in marine and surface waters.
Aeromonas- found predominantly in fresh water
Plesiomonas- exist in both cold-blooded and warm-blooded animal
gut
Campilobacters- found in may animals especially on domesticated
animals
Vibrios
-most common bacteria in surface waters worldwide.
-motile by polar flagellum, curved aerobic rods.
Medically Important Vibrios:
Organism Human Disease
V. cholerae serogroups O1 and O139 Epidemic and Pandemic cholera
V. cholerae serogroups non-O1/O139 Cholera-like diarrhea; rarely extraintestinal
infection
V. Parahaemolyticus Gastroenteritis; Extraintestinal infection
Others:
V. mimicus, V. vulnificus, V. hollisae, V. fluvalis, Ear, wound, soft tissue, and other
V. damsela, V. anginolyticus, V. metschnicovii extraintestinal infections, all common
Vibrio cholerae
-transmitted in water and development of sanitary water systems.
-causative cholera (produces profuse watery diarrhea that rapidly cause
dehydration and death) or “rice water diarrhea” containing mucous and
the organism.
-”comma-shaped” bacilli.
-actively motile by polar flagellum (heat-labile H antigen).
-has O lipopolyssacharide that confer serologic specificity and has at
least 139 O antigen groups.
-the O1 antigen consists of further subserotypes: Ogawa, Inaba, and
Hikojima.
Vibrio cholerae
-there are two types of organism that causes epidemic cholera: the
classic cholera and the “El Tor” (produce hemolysin making it positive
to Voges-Proskauer test and resistant to polymyxin B)
***O1 El Tor is similar to O139
***O139 strains produce capsule like the non-O1 strains, O1 strains
don’t have capsule.
-
Vibrio parahaemolyticus
-halophilic rods that causes acute gastroenteritis following ingestion
of contaminated sea foods and raw fish or shellfish.
-produce watery to bloody diarrhea and fecal leukocytosis.
Other Vibrios
Vibrio vulnificus- cause severe wound infection and bacteremia, and
gastroenteritis and acquired on infected oysters during warm months.
Vibrio mimicus- causes diarrhea after ingestion of uncooked sea foods
particularly oysters.
Vibrio hollisae- diarrhea
Vibrio fluvalis- diarrhea
Vibrio anginolyticus- causes eye, ear, or wound infection after exposure
to sea water.
Vibrio damsela- wound infection
Aeromonas hydrophilia
-produce UTI and wound infection to humans, rarely reported
diarrhea and produce tissue invasion due to the production of
hemolysins and cytotoxins.
Plesiomonas shigelloides
-produce diarrhea, has same antigenic structure with Shigella sonnei
but they are oxidase and DNase positive.
Campylobacter
-species under this genus cause diarrhea and systemic diseases.
Campylobacter jejuni
-the most common pathogen causing enteritis and rarely systemic
diseases same as Campylobacter coli.
-”S” or “Seagull-wing” shapes in stained slides, motile with single
polar flagellum.
-acquired through fecal-oral route, from food, drink or contact with
infected animals or animal products.
-produce bloody, pus-filled mucoid diarrhea.
Other Campylobacters
Campylobacter fetus spp. fetus- an opportunistic pathogen that causes
systemic infections to IC/IS patients.
Campylobacter lari- found in seagulls and occasionally cause diarrhea
to humans.
Campylobacter upsaliensis- from dogs and occasionally cause diarrhea
to humans.
Helicobacter pylori
-causative agent of antral gastroenteritis, (peptic) duodenal ulcers,
gastric ulcers, and gastric carcinoma.
-spiral-shaped rods, motile with multiple polar flagella, which are
catalase and urease.
-infection is characterized by ammoniacal breath. (positive to breath
test).
-sensitive to gastric acids, the organism survive on the mucosal
epithelium surrounded by neutralizing mucous.
-produce proteases that digest the mucous of the epithelial lining.
-produce gastritis and hypochorhydria with duodenal ulcers.
Haemophilus, Bordetella, Brucella
Francisella
Genus Haemophilus
-group of small gram-negative, pleomorphic bacteria (coccobacilli)
that require enriched media usually containing blood or its derivatives
for isolation.
-other Haemophilus spp. other than H. influenzae and H. ducrei are
part of the normal flora of mucous membranes and occasionally
cause disease.
Haemophilus, Bordetella, Brucella
Francisella
Haemophilus influenzae
-its serotype B is the most important pathogen under this specie.
-found in the mucous membrane of the URT, it is an important cause of
meningitis in children and occasionally cause respiratory tract of children and
adults.
-contains capsular polysaccharides of one of the six type (a – f). Serotype B is a
polyribose-robitol phosphate (PRP).
-causes meningitis, pneumonia and emphyema, fulminating obstructive
laryngotracheitis with swollen, cherry-red epiglotittis, cellulitis, septic arthritis,
otitis media and acute sinusitis, and other invasive form.
-vaccine: Haemophilus B conjugate vaccine
Haemophilus, Bordetella, Brucella
Francisella
Haemophilus aegyptius
-also known as Koch-Weeks bacilli, H. influenzae type III, H. influenzae
spp. aegyptius.
-cause a highly communicable type of conjunctivitis, Brazilian Purpuric
Fever (disease chaacterized by fever, purpura, shock and death)
Haemophilus aphrophilus
-cause infective endocarditis and pneumonia.
-member of oral and respiratory tract flora.
Haemophilus, Bordetella, Brucella
Francisella
Haemophilus ducreyi
-causes chancroid (soft chancre), which is a STD.
Chancroid/Soft Chancre- consists of ragged ulcer on the genitalia,
with marked swelling and tenderness.

Haemophilus haemoglobinophilus
-an important dog pathogen, no cases on human yet.
Haemophilus, Bordetella, Brucella
Francisella
Haemophilus haemolyticus
-the most hemolytic organism under this genus on BAM, normal flora
of the nasopharnx and associated with URTI.
Haemophilus parainfluenzae
-normal flora of the human respiratory tract, causing endocarditis and
urethritis to ICP/ISP.
Haemophilus suis
-bacteriologically acts synergystically with swine influenza virus in
hogs.
Haemophilus, Bordetella, Brucella
Francisella
Bordetella pertusis
-causative agent of whooping cough (pertusis).
-coccobacilli showing bipolar metachromatic granules with toluidine blue
staining.
Filamentous Hemagglutinin- mediates adhesion to ciliated epithelial cells.
Pertusis Toxin- promotes lymphocytosis, sensitization to histamine, enhance
insulin secretion, and promotion of an ADP-ribosylating activity.
Adenylate cyclase toxin, Dermonecrotic toxin, Hemolysin.
Tracheal Cytotoxin- inhibit DNA synthesis in ciliated cells.
Haemophilus, Bordetella, Brucella
Francisella
Bordetella pertusis (cont)
-has pili that is important in adherence of bacteria to the ciliated
epithelial cells of the URT.
Catarrhal Stage- early stage (2 weeks) of pertusis characterized by
mild coughing and sneezing. The host is infective in this stage.
Paroxysmal Stage- cough develops on its explosive character and the
“whoop” during inhalation, and intermitent vomiting, cyanosis, and
convulsion.
Vaccine: DPT
Haemophilus, Bordetella, Brucella
Francisella
Bordetella parapertusis
-produce whooping cough-like disease.
-differentiated from B. pertusis by laboratory tests: fast grower, larger
colonies.
Bordetella bronchoseptica
-normal flora of the respiratory tract of canines, causative agent of
“Kennel Cough” and pneumonitis.
-causes snuffles in rabbits, attrophic rhinitis in swines.
 Haemophilus, Bordetella, Brucella
Francisella
Brucellae (Obligate Intracellular Parasites)
Brucella melitensis- infects goats
Brucella suis- infects swines
Brucella abortus – infects cattle
Brucella canis- infects dogs
**Human Brucelliosis/Undulant Fever/Malta Fever
-characterized by an acute bacteremic phase followed by a chronic stage that may extend
over many years with tissue involvement.
-ingestion of infected milk, mucous membrane (droplets), cutaneous (skin to skin
contact).
-Cheese from infected goat milk is the most common vehicle
Haemophilus, Bordetella, Brucella
Francisella
Francisella tularensis
-causative agent of tularemia.
-the organism may be transmitted by vector, ingestion, inhalation.
Ulceroglandular Tularemia- enlargement and necrosis of lymph nodes.
Pneumonic Tularemia- caused by inhalation of infective aerosols resulting to
peribronchial inflammation and localized pneumonitis.
Oculoglandular Tularemia- developed when infected finger touches the
conjunctiva.
Glandular Tularemia- lymphadenopathy but no ulcers.
Oropharyngeal Tularemia
Typhoidal Tularemia- - septecemia
Unusual Bacterial Pathogens
Legionella pneumophilia
-causative agent of Legionaires’ disease a pneumonia in persons
attending the American Legion Convention in Philadelphia in
contaminated aircondition.
-also the causative agent of Pontiac Fever first occur in Michigan,
characterized by fever and chills, myalgia, malaise, and headache.
-fastidious, aerobic, gram-negative rods that poorly stained by gram
staining.
Unusual Bacterial Pathogens
Legionella micdadae
-causes pneumonia in humans.
Bartonella bacilliformis- causative agent of Oroya fever and Verruga
Peruana.
Bartonella quintana- causative agent of Trench fever during WWI and
some cases of bacillary anginomatosis and Peliosis Hepatis.
Bartonella henselae- causative agent of cat-scratch disease.
Mobiluncus vaginalis- causative agent of bacterial vaginosis other than
Gardnerella vaginalis.
Unusual Bacterial Pathogens
Streptobacillus moniliformis
-aerobic, highly pleomorphic gram-negative bacilli.
-causative agent of rat-bite fever characterized by septic fever, blotchy and
petecheal rashes, and very painful polyarthritis.
Haverhill Fever-an epidemic type of rat-bite fever.
Spirillum minor- causative agent of Sodoku.
Calymmatobacterium granulomatis- causative agent of granuloma
inguinale, an uncommon STD characterized by genital ulcers.
Tropheryma whipplei- causative agent of Whipple’s disease, characterized by
abdominal pain, diarrhea, weight loss, migratory polyarthaldia.
ACID FAST BACTERIA
Two major Groups
• Mycobacterium tuberculosis complex
• Includes:
M. tuberculosis
M. bovis
M. africanum
• Nontuberculosis mycobacteria (NTMs)
MYCOBACTERIA
• Mycobacterium tuberculosis - tuberculosis
• Mycobacterium leprae - leprosy
AFB Staining
• Mycobacteria are classical acid-fast organisms. Stains used in evaluation of tissue
specimens or microbiological specimens include Fite's stain, Ziehl-Neelsen stain,
and Kinyoun stain.

• These organisms are thin rods with lipid-laden cell walls. This high lipid content
makes them acid-fast on staining.

• *Carbolfuchsin- primary stain

• *Acid alcohol (95% ethanol
• and 3% HCl)- decolorizer
• *Methylene blue – counter stain
• Only Mycobacteria and Norcardia are acid- fast.

• PRESENCE OF MYCOLIC ACIDS

Mycobacterium tuberculosis
causative agent of tuberculosis

• Mycobacterium tuberculosis. Acid-fast stain. CDC.

General Characteristics
1.) Mycobacterium tuberculosis is a fairly
large nonmotile rod-shaped bacterium distantly
related to the Actinomycetes.
2.) Mycobacterium tuberculosis is an obligate
aerobe.
3.) The bacterium is a facultative intracellular
organism, usually of macrophages.
4.) Has a slow generation time, 15-20 hours, a
physiological characteristic that may contribute to
its virulence.
5.) Catalase positive
 MYCOBACTERIAL VIRULENCE
I. Mycosides
• 1) Mycolic acid is a large fatty acid.
• 2) Mycoside is a mycolic acid bound to a carbohydrate, forming a
glycolipid.
• 3) Cord factor is a mycoside formed by the union of 2 mycolic acids
with a disaccharide (trehalose). This mycoside is only found in virulent
strains of Mycobacterium tuberculosis. Its presence results in parallel
growth of the bacteria, so they appear as cords.
• 4) Sulfatides – inhibit phagosome-lysosome
fusion.
• 5) Wax D is a complicated mycoside that acts
as an adjuvant (enhances antibody formation
to an antigen)
Cont… Virulence
• II. Iron Siderophore
• III. Facultative intracellular growth
Clinical Manifestations
• 1. PRIMARY TUBERCULOSIS
• Asymptomatic
• Overt diseases- includes the lungs or other organs
I. Asymptomatic primary infection:
• The cell mediated defenses kick in, and the foci of
bacteria become walled off in the caseous
granulomas. These granulomas then heal with
fibrosis, calcification, and scar formation.
• Organisms are still viable.
• Ghon focus – a calcified tubercle in the middle or
lower lung zone
• Ghon, or Ranke, complex - a Ghon focus
accompanied by peripheral lymph node calcified
granulomas.
II. SYMPTOMATIC PRIMARY TUBERCULOSIS
- Occurs far less frequently, more commonly in
children, the elderly, and the
immunocompromised (especially HIV infected
persons).

- *Reactivation or secondary TB
- Risk of reactivation in all persons- 10% for
lifetime
- Risk of reactivation in HIV infected- 10% per year
a.) Pulmonary tuberculosis:
This is the most common site of reactivation tuberculosis.
Infection occurs in the apical areas of the lung around the
clavicles.
b.)Pleural and pericardial infection:
Infection in these spaces results in infected fluid
collections around the lung or heart respectively.
c.) Lymph node infection:
This is the most common extrapulmonary manifestation
of tuberculosis. The cervical lymph nodes become
swollen, mat together, and drain. Lymph node
tuberculosis is called scrofula.
d.) Kidney: Patients will have red and white blood cells in
the urine, but no bacteria are seen in stain and culture.
This is called as sterile pyuria.
e.) Skeletal:
This usually involves the thoracic and lumbar
spine, destroying the intervertebral discs and
then the adjacent vertebral bodies (Pott's
disease).
f. ) Joints:
There is usually a chronic arthritis of 1 joint.
g.)Central nervous system:
Tuberculosis causes subacute meningitis and
forms granulomas in the brain.
h.)Miliary tuberculosis:
Tiny millet-seed-sized tubercles (granulomas) are
disseminated all over the body like a shotgun
blast.
DIAGNOSIS
• 1) PPD skin test: This screening test indicates an exposure sometime
in the past.
• 2) Chest X-ray: You may pick up an isolated granuloma, Ghon focus,
Ghon complex, old scarring in the upper lobes, or active tuberculous
pneumonia.
• 3) Sputum acid-fast stain and culture: When the acid-fast stain or
culture are positive, this indicates an active pulmonary infection.
ATYPICAL MYCOBACTERIA
• Mycobacterium avium-intracellulare (MAI) or Mycobacterium
avium-complex (MAC)
• - usually infects birds and other animals.
• It has now become one of the major systemic bacterial infections of
AIDS patients, usually late in the course of the disease
 Mycobacterium leprae
causative agent of leprosy (Hansen’s disease)

• A photomicrograph of Mycobacterium leprae taken from a leprosy skin lesion.

General Characteristics
• 1.) Mycobacterium leprae is an acid-fast rod.
• 2.) It is impossible to grow this bacterium on
artificial media; it has only been grown in the
footpads of mice, in armadillos, and in monkeys.
• 3.) Catalase positive
• 4.) Grows best at low temperature
• 5.) Phenolase positive- converts Dopa into a
pigmented product.
Clinical Manifestations
• Leprosy involves the cooler areas of the body. It damages the skin
(sparing warm areas such as the armpit, groin, and perineum), the
superficial nerves, eyes, nose and testes.
I. Tuberculoid leprosy (TL)
• The delayed hypersensitivity
reaction is intact, so the
lepromin skin test is usually
positive.
• The patient demonstrates
localized superficial, unilateral
skin and nerve involvement.

II. Lepromatous
leprosy (LL):

• The patient with LL cannot

mount a delayed
hypersensitivity reaction. LL
primarily involves the skin,
nerves, eyes and testes, but the
acid-fast bacilli are found
everywhere (respiratory
secretions and every body
organ).
DIAGNOSIS
• The lepromin skin test is similar to the PPD used in tuberculosis.
• It measures the ability of the host to mount a delayed hypersensitivity
reaction against antigens of Mycobacterium leprae.
• This test is more prognostic than diagnostic and is used to place
patients on the immunologic spectrum.
Multidrug Resistant Mycobacterium
tuberculosis (MDR-TB)
• Resistance to atleast isoniazid and rifampin – PRIMARY TREATMENT
for M. tuberculosis.

• Risk factors:
(1) Previous treatment for TB
(2) Residence in an area endemic for drug resistance
(3) Close contact with individual infected with MDR-TB
TREATMENT: Aminoglycosides and fluoroquinolones
Extensively Drug Resistant Mycobacterium tuberculosis
(XDR-TB)
• Resistance to isoniazid and rifampin PLUS
resistance to any fluoroquinolones and atleast ONE
of THREE injectible second line anti TB drugs:
amikacin, kanamycin, or capreomycin.

• Treatment: around 30% to 50%

• Treatment will be less effective, increase side effects
and expensive.
Specimen Collection
• Pulmonary Specimen
• Gastric Lavage Specimen
• Urine Specimen
• Fecal Specimen
• Tissue and Body fluid Specimen
• Blood Specimen
• Wounds, Skin Lesions and Aspirates
Digestion and Decontamination of Specimen
• To ensure optimal recovery of mycobacteria.
• Example: Sputum- abundance of nonmycobacterial organism
Purpose:
(1) To liquefy the sample through digestion of the proteinaceous material
(2) To allow the chemical decontaminating agent to contact and kill the
nonmycobacterial organism.
*In liquefaction, mycobacteria can be concentrated through centrifugation
Digestion and decontamination: Sputum, gastric washing, brochial washing, and
transtracheal aspirate.
Decontamination: voided urine, autopsy tissue, abdominal fluid.
SPECIMENS FROM STERILE SITES: DO NOT REQUIRE decontamination
Digestion and Decontaminating Agents
• Sodium Hydroxide
• N Acetyl L cysteine
• Benzalkonium Chloride
• Oxalic Acid *
STAINING for ACID FAST
Ziehl Neelsen Stain – Hot method
Kinyoun stain- Cold method

Auramine or auramine-rhodamine fluorochrome stain

• Requires a fluorescent microscope
• Read at 250X-450X magnification
• AFB appear yellow against a black background
Ziehl Neelsen Stain
Culture Media
• Combination of different culture media is required.
• 1 solid medium + liquid medium

SOLID MEDIA
• Agar Based
• Middlebrook 7H10 and Middlebrook 7H10 selective
• Middlebrook 7H11 and Middlebrook 7H11 selective
• Middlebrook biplate (7H10/7H11S agar)

• Egg Based
• Lowestein-Jensen (L-J)
• L-J Gruft
• L-J with pyruvic acid
• L-J with iron
Solid Media ( Egg Based Media)
• Examples: Lowenstein Jensen (LJ), Petragnani,
and American Thoracic Society (ATS) media.
• Fresh whole eggs
• Potato flour
• Glycerol
• Salts, milk
• Malachite green
• Note: Selective media w/ antimicrobial agents, sometimes
used in combination with non selective media.
• Non selective media- L.S of 1 year
• Opaque
Solid Media (Agar Based Media)
• Examples: Middlebrook 7H10 and 7H11
• Defined salts
• Vitamins
• Cofactors
• Glycerols
• Malachite green
• Agar
• OADC Enrichment – Oleic acid, bovine albumin, glucose and
beef catalase.
• Middlebrook 7H11medium- 0.1% casein hydrolysate
recovery of isoniazid-resistant strains of M. tuberculosis
• Clear agar based media
• Drug susceptibility test without altering drug concentration
 • Taxonomic separation of rapidly growing
mycobacteria

• M.onfortuitum
GROWTH group
MacConkey Agar – growth
without CRYSTAL5-11 days,
VIOLET change in
medium color
• M. chelonae
• M. smegmatis- 25%

• Submit to 3 day arylsulfatase test

• M. fortuitum and M. chelonae are POSITIVE
• M. smegmatis – RARELY positive
• Tubed Media- slanted position with loose screw
caps for atleast 1 week.
• Plated Media- placed in a CO2 permeable plastic
bag or wrapped with CO2 permeable tape.
• Skin or lesions specimen suspected with M.
marinum or M. ulcerans, additional set of solid
media is required to be inoculated and incubated
at 25 to 30˚C
• Chocolate Agar plate- incubated 30 to 32˚ C for
recovery of M. haemophilum.
LIQUID MEDIA
• Examples: Middlebrook 7H9 broth and Dubos Tween albumin

• Non selective liquid media

• Subculturing stock strains
• Picking single colonies
• Preparing inoculum for in vitro testing
• Mycobacterium spp. Grow more rapidly in liquid medium. It can be used for both primary
isolation and subculturing.

• Reduces the turn around time for isolation of acid fast bacilli to approx. 10 days compared
with 17 days or more for conventional solid media.

• Growth  acid fast stain  subcultured to solid agar

• NOTE : For optimal recovery of Mycobacterium, a minimum combination of liquid medium

and solid media is recommended.
Methods in Identification
Molecular
biology

Positive
AFB culture

Morphology

Genotypic
Phenotypic Approach
Approach
Biochemical
and growth tests DNA probes
(Slow)

with amplification
Immunochromatography
(fast) w/o amplification

DNA Sequencing

251
PHENOTYPIC Methods
• Growth parameters
• Biochemical characteristics
• Analysis of cell wall lipids

• --Limitations

• * Molecular and genetic investigations

Phenotypic Method
• 1.) Growth characteristics- preliminary identification
• - Rate of growth
• - Colonial Morphology and texture
• - Pigmentation
• - Permissive incubation temperature
GROWTH RATE
• Examine cultures after 5 days and 7 days for appearance of grossly
visible colonies.

• INTERPRETATION
• Rapid growers- produce colonies within 3 to 4 days after sub culture.

• Slow growers- requires more than 7 days

Mycobacteria require pH between 6.5 to 6.8

• *M.flavescens- exhibit false-positive rapid growth
• *M. genavense- requires extended incubation (6-8) weeks
PIGMENT PRODUCTION
• Photochromogens- produce pigmented colonies after exposure to light.
• Ex: M. kansassi
• Scotochromogens- produce pigmented colonies even in the absence of light.
• Ex: M. gordonae and M. scrofulaceum
• Nonchromogens- not affected by light.
• Ex: M. tuberculosis

• *M. szulgai- scotochromogen at 35˚C

• - nonpigmented at 25 ˚C to 30 ˚C
Photoreactivity of Clinically important
Mycobacteria
• Nonchromogens • M. ulcerans
• -Slow growers
• M. tuberculosis • - Rapid growers
• M. avium- • M. chelonae • Scotochromogen
intracellulare • M. forfuitum group • -Slow growers
• M. bovis • M. gordonae
• M. celatum • Photochromagens • M. szulgai
• M. gastri • -Slow growers • M. scrofulaceum
• M. genavense • M. asiaticum • M. xenopi
• M. haemophilum • M. kansassi • -Rapid Growers
• M. malmoense • M. marinum • M. phlei
• M. terrae complex • M. simiae • M. smegmatis group
Biochemical Testing
• 1.) Niacin Accumulation
Principle: The accumulation of niacin in the medium
caused by lack of an enzyme that converts niacin to
other metabolite is the characteristic of M.
tuberculosis.
Reagent strip with Cyanogen bromide
Expected Result:
• POSITIVE- Yellow liquid (M.tuberculosis)
• NEGATIVE- Liquid is clear

• Note: Should be performed only from cultures on L-J

that are atleast 3 weeks old and with atleast 50
colonies, otherwise, insufficient niacin is accumulated
making it undetectable.
2.) Nitrate reduction test
Principle: Presence of nitrite (product of nitroreductase enzyme)
is detected upon addition of several reagents  red colored
product.
Reagent: Hydrochloric acid, sulfanilamide, and N-
napththylenediamene dihydrochloride
Expected Result:
POSITIVE- pink to red color
(M. tuberculosis, M. kansassi, M. szulgai, M. forfuitum )
NEGATIVE- no color
(Scotochromogens, MAC)
* Add powdered zinc
= red color = unreduced nitrate
= organism is nitroreductase -negative
• M. tuberculosis reduces nitrates to nitrites.
• Several species may reduce nitrates.
• Cultures tested:
• 4 weeks old
• abundant growth.

259
3.) Urease Test
Principle:Ability to hydrolyze urea (releasing ammonia)
used in identifying both scotochromogens and
nonphotochromogens.
• Used to distinguish M. scrofulaceum from M. gordonae
• Expected Result:
• Positive: Pink to Red Color
• (M. scrofulaceum, M. szulgai, M. flavescens, M. bovis,
M. tuberculosis and M. gastri)
• Negative:
• (M. avium complex, M. xenopi, M.terrae complex, and
M. gordonae)
4.) Catalase test

• Intracellular, soluble enzyme.

• Release of O2 and production of bubbles.
• Virtually all mycobacteria possess catalase
enzymes, however not all strains produce a
positive result after the culture is heated to 68C
for 20 min.

261
Catalase test at 68°C, pH 7.0
• All mycobacteria produce catalase, usually thermoresistant but
M.tuberculosis produces thermolabile catalase
• 1 tube: incubation at 68 °C for 20 minutes
• 1 tube: incubation at room temperature
for 20 minutes
• detection by H2O2

• 68 °C test at pH7 :
strains of M. Tuberculosis
lose catalase activity.
262
Catalase test at 68 °C, pH 7.0 –
results and interpretation

• Both tube forms bubbles= catalase positive- thermoresistant = (M. fortuitum or

M. gordonae)
• Unheated tube= bubbles, heated tube= no bubbles= catalase positive –
thermolabile= M. tuberculosis complex
• Both tube = no bubbles= catalase negative= rare isoniazid resistant strains of M.
tuberculosis

263
Biochemical Testing
• 6.) Tween 80 Hydrolysis Test
Principle: Tween 80 hydrolysis test is used mainly
to differentiate slow–growing Mycobacterium
(Scotochromogens) with similar colony
appearance, M. gordonae (positive) and M.
scrofulaceum (negative). This test is also useful in
identifying M. kansasii (positive results within 24
hours). Tween 80 hydrolysis is detected by a
change in color of the indicator from light orange
to pink – red due to the production of oleic acid.
Biochemical Testing
Expected Result:
• POSITIVE-Pink-red substrate (Tween 80
hydrolyzed)
• NEGATIVE-No change in colour (No hydrolysis of
Tween 80)

• Note: The reagent should be stored at 4oC in the

dark. Do not store or incubate tubes in the light.
The red color of a positive reaction is not due to
change in pH, rather is due to hydrolysis of
Tween 80, which modifies the optical rotation of
light passing through the substrate.
Biochemical Testing
• 7.) Tellurite Reduction
Principle: Reduction of colorless potassium
tellurite of black tellurium in 3 to 4 days is a
characteristic of MAC and is helpful in
distinguishing MAC from other nonchromogenic
species. All rapid growers are able to reduce
tellurite in 3 days.
Biochemical Testing
Expected Result:
• POSITIVE-Smooth, fine, black precipitate(smoke-
like action)(MAC)
• NEGATIVE-Gray clumps(No smoke-like action)(M.
tuberculosis)
Biochemical Testing
• 8.) Arylsulfatase Test
Principle: The arylsulfatase test is used for
identification of Mycobacteria, particularly M.
fortuitum and M. chelonei, which may produce
sufficient arylsulfatase to give a positive reaction
within 3 days. The enzyme arylsulfatase converts
phenolphthalein disulfate (colourless) to free
phenolphthalein (red at alkaline pH).
Biochemical Testing
Expected Result:
• POSITIVE-Pink to red(M. fortuitum)
• NEGATIVE-No color change(M. intracellulare)
Biochemical Testing
• 9.) Sodium Tolerance Test
Principle: High salt concentration(5% NaCl) in egg-
based media inhibits the growth of most
mycobacteria. M. flavescens, M. triviale, and
most rapidly growing Mycobacterium spp. are
exceptions that do grow in the presence of 5%
NaCl.
Biochemical Testing
Expected Result:
• POSITIVE-Substantial growth
• NEGATIVE-Little or no growth
Biochemical Testing
• 10.) TCH or T2H Test
Principle: T2H(Thiophene-2-Carboxylic Acid
Hydrazide) distinguishes M. bovis from M.
tuberculosis. M.bovis is susceptible to lower
concentrations of T2H than MTB. Variability in
inhibition exists, depending on the concentration
of the inhibitory agent and temperature of
incubation.
Biochemical Testing
Expected Result:
• POSITIVE-No growth(susceptible)
• NEGATIVE-Growth(resistant)
SUMM
ARY
 Acid fast bacilli

Colonies visible
10d – 8 wks
< 7 days
= tubercle bacilli
NO tubercle bacilli + MOTT
= MOTT

YES Pigmentation

+ NO
NO
= tubercle bacilli
PNB
+ MOTT
-
= tubercle bacilli
+ few MOTT

Heat stable catalase

Thermo labile = tubercle bacilli Nitrate reductase

-
+
M.tuberculosis M.bovis
M. africanum M. africanum

• MOTT- Mycobacteria other than tuberculosis

• PNB- rho nitrobenzoic acid
Tubercle bacilli identification chart with niacin
test
Acid fast bacilli

Colonies visible
10d – 8 wks
< 7 days
= tubercle bacilli
NO tubercle bacilli + MOTT
= MOTT

YES Pigmentation

+ NO
= tubercle bacilli
PNB
+ MOTT
-
= tubercle bacilli
+ few MOTT

M.tuberculosis
niacin +
M. africanum
-
M.bovis
thermolabile
M.bovis M. africanum
Catalase
M. africanum
+ few MOTT NO tubercle bacilli
thermoresistant
= MOTT 278
Special Lab Determinations
I. Antigen-Protein Detection
Detection of microbial products or components to diagnose
infections caused by M. tuberculosis.
Examples:
• Tuberculostearic acid- presence in CSF- tuberculous meningitis.
• Adenosine deaminase- increased level of production- certain
infections caused by M. tuberculosis.
II. Nucleic Acid Amplification

• PCR- used to detect M. tuberculosis directly in clinical specimens.

• 2 kits available:
• 1.) Amplicor Mycobacterium tuberculosis test
• 2.) Amplified Mycobacterium tuberculosis direct test
INTRACELLULAR
PATHOGENS
 Rickettsia: General Characteristics

Classification – Family Rickettsiaceae with 3 medically important genera

• Rickettsia
• Coxiella
• Rochlimaea
Morphology and cultural characteristics
• All are obligate intracellular parasites (except Rochlimaea) that can grow in both
phagocytic and nonphagocytic cells.
• Rochlimaea can be cultivated on artificial media containing blood
• Obligate intracellular parasites.
• Small Gram (-) coccobacilli (0.3-0.5 um)
• Cell membrane similar to Gram (-) bacteria with LPS & peptidoglycan
 Rickettsia: General Characteristics

-Others are grown in embryonated eggs or tissue culture

-Cultivation is costly and hazardous because aerosol
transmission can easily occur
-Small, pleomorphic coccobacilli
-Gram stain poorly, but appear to be G-
-Stain readily with Giemsa
-All, except Coxiella, are transmitted by arthropod vectors.
 Rickettsia: General Characteristics

The family Rickettsiaceae is taxonomically divided into three genera:

1. Rickettsia (11 species)--obligate intracellular parasites which do not
multiply within vacuoles and do not parasitize white blood cells.
2. Ehrlichia (2 species) – obligate intracellular parasites which do not multiply
within vacuoles but do parasitize white blood cells.
3. Coxiella (1 species) – obligate intracellular parasite which grows
preferentially in vacuoles of host cells.
4. Bartonella (3 species) – intracellular parasite which attacks the red blood
cell.
 Rickettsia: Transmission

-Rickettsiae are transmitted through vectors.

Transmission
-Rickettsia are usually introduced into human skin by the bite of an insect (flea or
louse) or an arachnid (tick or mite)
R. rickettsii invades the endothelial cells that line the blood vessels
Incubation period: ~1 week
Virulence factors of Rickettsial species
-changes in the host cell phagocytosis
-bacterial surface protein
 Rickettsia: Pathogenesis

During the first few days of incubation period

-local reaction caused by hypersensitivity to tick or vector
products
Bacteria multiply at the site & later disseminate via lymphatic system
Bacteria is phagocytosed by macrophages (1st barrier to rickettsial
multiplication)
After 7-10 days organisms disseminate, replicate in the nucleus or
cytoplasm of endothelial cells causing vasculitis
 Rickettsia: Pathogenesis

Infected cells show intracytoplasmic inclusions & intranuclear inclusions

Endothelial damage & vasculitis progress causing
-development of maculopapular skin rashes
-perivascular tissue necrosis
-thrombosis & ischemia
Disseminated endothelial lesion lead to increased capillary permeability, edema,
hemorrhage & hypotensive shock
Endothelial damage can lead to activation of clotting system ---> Disseminated
intravascular coagulation (DIC)
Rickettsia: Pathogenesis
 Rickettsia: Classification (Accdg. To Dease
Caused)

Spotted Fever Group

Organism Disease
R. rickettsii Rocky Mountain spotted fever
R. akari Rickettsialpox
Fievre Boutonneuse fever,
Mediterranean/Israel spotted fevers,
R. conorii
South African Tick Fever, Kenya/India Tick
Typhus, Tache Noire (Eschar)
R. sibirica Siberian (North Asian) tick typhus
R. australis Australian tick typhus
R. japonica Oriental spotted fever
 Rickettsia: Classification (Accdg. To Dease
Caused)

Typhus Group
Organism Disease
Epidemic (Louse-borne) typhus
Recrudescent typhus (Brill-Zinsser
R. prowazekii
Disease)
Sporadic typhus
Murine typhus
R. typhi
Endemic (Flea-borne) typhus
 Rickettsia: Classification (Accdg. To Dease
Caused)

Scrub Typhus Group

Organism Disease
Orienta. tsutsugamushi Scrub typhus
 Rickettsia: Classification (Accdg. To Dease
Caused)

Coxiella Group
Organism Disease
Coxiella burnetii Q fever (Quintana)
 Rickettsia: Classification (Accdg. To Dease
Caused)

Bartonella Group
Organism Disease
Oroya Fever
Bartonella bacilliformis
Veruga Peruana
Cat Scratch Fever
B. henselae
Bacillary Angiomatosis
Rochalimaea (Bartonella) quintana Trench Fever
 Rickettsia: Classification (Accdg. To Dease
Caused)

Erlichia Group
Organism Disease
Erlichia chaffaeensis Human monocyte ehrlichiosis
Neorickettsia sennetsu Human monocyte ehrlichiosis
Anaplasma phagocytophilium Human granulocyte anaplasmosis
Ehrlichia ewingii Human granulocyte ehrlichiosis
Rickettsia: Classification (Accdg. To Vector)
1. Louse-borne:
European epidemic typhus (Rickettsia prowazekii),
Brill's disease (Rickettsia prowazekii),
Trench fever (Bartonella quintana)
2. Flea-borne
Endemic murine typhus (Rickettsia typhi),
Cat scratch fever /Bacilliary angiomatosis/ (Bartonella henselae)
3. Mite-borne
Scrub typhus (Orientia /Rickettsia tsutsugamushi),
Rickettsialpox (Rickettsia akari)
Rickettsia: Classification (Accdg. To Vector)

4. Tick-borne
Rocky Mountain Spotted Fever (Rickettsia rickettsii),
North Asian tick typhus (Rickettsia siberica),
Fievre boutonneuse (Rickettsia conorii),
Queensland tick typhus (Rickettsia australis),
Q-fever (Coxiella burnetii),
Spotted fever (Rickettsia rhipicephali),
Ehrlichiosis (Ehrlichia canis, Ehrlichia chaffeensis)
5. Fly-borne
Oroyo fever / Verruga peruana (Bartonella bacilliformis)
 Rickettsia: Clinical Findings

Clinical significance – the diseases caused by Rickettsia are all characterized by fever,
headache, myalgias, and usually a rash.
Typhus fevers – incubation is 5-18 days.
Symptoms include a severe headache, chills, fever, and after a fourth day, a
maculopapular rash caused by subcutaneous hemorrhaging as Rickettsia invade the
blood vessels.
The rash begins on the upper trunk and spread to involve the whole body except
the face, palms of the hands, and the soles of the feet.
The disease lasts about 2 weeks and the patient may have a prolonged
convalescence.
 Rickettsia: Clinical Findings

Two types of typhus may occur:

Epidemic typhus – caused by R. prowazekii and transmitted by human lice
as it bites and defecates in the wound.
-This occurs in crowded areas causing epidemics. Mortality rates are high in
untreated cases. Following an initial attack, some individuals may
harbor the organism of a latent infection with occasional relapses =
Brill-Zinsser disease
Endemic typhus – caused by R. typhi and transmitted to man by rat fleas.
-The disease occurs sporadically, but is clinically the same, but less severe
than epidemic typhus.
 Rickettsia: Clinical Findings

Rocky mountain spotted fever – caused by R. rickettsii and transmitted by ticks

that must remain attached for hours in order to transmit the disease.
An incubation of 2-6 days is followed by a severe headache, chills, fever,
aching, and nausea.
After 2-6 days a maculopapular rash develops, first on the extremities,
including palms and soles, and spreading to the chest and abdomen.
If left untreated, the rash will become petechial with hemorrhages in the
skin and mucous membranes due to vascular damage as the organism
invades the blood vessels.
Death may occur during the end of the second week due to kidney or
heart failure.
 Rickettsia: Clinical Findings

Rickettsial pox – caused by R. akari and transmitted by a mouse mite.

After a 1-2 day incubation a papule develops at the entry site and within
1-2 weeks a fever, malaise, and headache develop followed by a rash.
The disease is mild and usually not fatal.
Q fever – caused by Coxiella burnetii. The infection is acquired by inhalation
of infectious material.
After an incubation of 14-26 days there is a sudden onset of fever, chills,
and headache, but no rash.
The disease is characteristically an atypical pneumonia lasting 5-14 days
with a low mortality rate
 Rickettsia: Clinical Findings

Trench fever – caused by Rochalimaea quintana and transmitted

by body lice. Was a major problem during WW I and WW II.
After an incubation of 6-22 days, the patient experiences a
headache, exhaustion, leg pains, and a high, relapsing fever.
A roseolar rash occurs and the patient usually recovers.
Rickettsia: Treatment

Chloramphenicol or tetracycline
Wear protective clothing and use insect repellents
 Rickettsia: Laboratory Diagnosis

1. Culture & isolation

-Difficult & dangerous because of the highly infectious nature of rickettsiae
2. Serologic test
A. Weil-Felix test: based on cross-reactivity between some strains of Proteus &
Rickettsia
B. Complement fixation: not very sensitive & time consuming
C. Indirect fluorescence (EIA): more sensitive & specific; allows discrimination
between IgM & IgG antibodies which helps in early diagnosis
D. Direct immunofluorescence: the only serologic test that is useful for clinical
diagnosis, 100% specific & 70% sensitive allowing diagnosis in 3-4 days into the
illness
 Rickettsia: Laboratory Diagnosis

3. Presumptive laboratory diagnosis is based on the finding of

rickettsial- like organisms in tissue or blood. Although the
organisms are gram- negative, they only weakly take the counter
stain, safranin. Therefore, special staining procedures are used.
Infected tissue may be stained with:
i. Macchiavello stain--organisms are bright red against the blue
background of the tissue.
ii. Castaneda stain--blue organisms against a red background.
iii. Giemsa stain--bluish purple organisms.
 Chlamidiae

-are obligate intracellular bacterial parasites of humans, animals

and birds with tropism for squamous epithelial cells and
macrophages of the respiratory and gastrointestinal tracts.
 Chlamidiae

Important Chlamydia and Their Serotypes:

Species Serotypes Disease
C. trachomatis A, B, Ba, C Trachoma
C. trachomatis D, E, F, G, H, I, J, K Conjunctivitis, genital
chlamidiasis, infant pneumonia
C. trachomatis L1, L2, L3 Lymphogranuloma venereum
C. psittaci Many serotypes Psitacosis
C. pneumoniae One serotype Acute respiratory disease
 Chlamidiae: Laboratory Diagnosis

-Are energy parasites that use ATP produced by the host cell
-A Giemsa stain can be used to visualize chlamydial inclusions in tissues.
Identification
Direct methods – stain tissues with Giemsa or use a direct
fluorescent antibody technique.
The most sensitive method is to culture the organisms in tissue
cultures and then stain the infected tissue culture cells
 Chlamidiae: Pathogenesis
-A complement fixation serological test is available as are DNA based tests.
Virulence factors:
-Toxicity from attachment and penetration
Clinical significance:
Chlamydia trachomatis – serotypes A-K and L1,2,3; the serotype determines
the clinical manifestation.
Genital tract infection (serotypes D-K) – is the major cause of nongonococcal
urethritis; is sexually transmitted and frequently found concomitantly with N.
gonorrhoeae
In males symptoms include urethritis, dysuria and it sometimes progresses to
epididymitis
 Chlamidiae: Pathogenesis

-In females symptoms include mucopurulent cervical inflammation which can

progress to salpingitis and PID.
Inclusion conjunctivitis – this occurs in both newborns and adults and a
genital tract infection is the source of the infection (serotypes D-K); is a
benign, self-limited conjunctivitis which heals with no scarring
Newborns – are infected during the birth process and the infection manifests
1-2 weeks after birth as a mucopurulent discharge that lasts2 weeks and then
subsides.
-Some may develop an afebrile, chronic pneumonia
 Chlamidiae: Pathogenesis

In adults – causes an acute follicular conjunctivitis with little discharge.

Trachoma (serotypes A-C) – is the single, greatest cause of blindness in
underdeveloped countries.
-Transmission is by direct contact and in poor, less developed countries children
may be infected in the first three months of life.
-Chronic infection and reinfection are common and result in conjunctival scarring
and corneal vascularization.
-The scars contract causing the upper lid to turn in so that the eyelashes cause
corneal abrasions.
-This leads to secondary bacterial infections and results in blindness.
 Chlamidiae: Pathogenesis
Lymphogranuloma venereum (serotypes L1, 2, 3) is a venereal disease that occurs in
poor, tropical areas.
-Upon infection, widespread dissemination takes place and a primary, painless
lesion (either a vesicle or an ulcer) occurs at the site of entry within a few days.
-This heals with no scarring.
-A secondary stage occurs 2-6 weeks later with symptoms of regional suppurative
lymphadenopathy (buboes) that may drain for a long time and be accompanied
by fever and chills.
-Arthritis, conjunctival, and CNS symptoms may also occur.
-A tertiary stage may occur and is called the urethrogenital perineal syndrome.
-This is characterized by structural changes such as non-destructive elephantiasis
of the genitals and rectal stenosis.
 Chlamidiae: Pathogenesis

Chlamydia psittaci – naturally infects avian species and non-

primate animals causing mild to severe illness.
-In man causes psittacosis (ornithosis) and is acquired by
contact with an infected animal.
-Infection can range from subclinical to fatal pneumonia.
-Most commonly causes an atypical pneumonia with fever,
chills, dry cough, headache, sore throat, nausea, and vomiting.
 Chlamidiae: Treatment

Treatment/antimicrobic susceptibility
C. trachomatis :
Trachoma – systemic tetracycline, erythromycin; long
term therapy is necessary
Genital tract infections and conjunctivitis –
tetracyclines and erythromycin
C. psittaci – same as above