TOPIC: (ENTER TOPIC HERE) STAPHYLOCOCCUS SPECIES

INSTRUCTOR: MATTHEW TUBOLA, RMT ● Two species are commonly associated with
staphylococcal diseases in humans:
1. Staphylococcus aureus
OUTLINE OF THE LESSON ➔ More virulent strain
➔ Some people are “carriers” in nose
I. INTRODUCTION and on skin.
A. (SUB TOPICS) 2. Staphylococcus epidermidis
- (SUPER SUB TOPIC) ➔ Normal microbiota of human skin
II. TITLE #2 ➔ Can cause opportunistic infections

HIGHLIGHTING BULLET PLACEMENT PATHOGENICITY

OF TOPICS ● ‘“Staph infections result when
staphylococci break through the body’s
Aaaaa - Main topic ● (Main definition) physical barriers
Aaaaa - Sub-topics ➔ (Sub-definition / ● Low “infectious dose”
AAAA - Title of Topic Enumerate) ➔ Entry of only a few hundred bacteria can
❖ Additional detail result in disease
● Pathogenicity results from the three
NOTE: (for additional
virulence factors
information during
discussions)
➔ Evades phagocytosis by capsule
production
➔ Produces enzymes
GRAM POSITIVE COCCI ➔ Produces exotoxins

STAPHYLOCOCCUS DEFENSE AGAINST PHAGOCYTOSIS

● Normal flora of every human ● Protein A coats the cell surface
● Gram-positive cocci in clusters ➔ Binds to class G antibodies
● Catalase positive Inhibits the complement cascade
● Facultative anaerobe ● Slime layer
● Tolerant of ➔ Inhibits chemotaxis of leukocytes
➔ Salt ➔ Helps Staphylococcus attach to artificial
➔ Desiccation surfaces

ENZYMES
COAGULASE
● Converts fibrinogen into fibrin to form blood
clots
● Fibrin clots hide the bacteria from
phagocytic cells

HYALURONIDASE
● Breaks down hyaluronic acid, enabling the
bacteria to spread between cells.

STAPHYLOKINASE
● Dissolves fibrin threads in blood clots,
allowing S. aureus to free itself from clots.

LIPASE
● Digest lipids, allowing staphylococcus to
grow on the skin’s surface and in oil glands

BETA-LACTAMASE
● Breakdown penicillin

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● Allows the bacteria to survive treatment with
B-lactam antimicrobial drugs
➔ Methicillin - derivative of penicillin that
is resistant to beta-lactamase
➔ MRSA - Methicillin resistant
Staphylococcus aureus to grow on the
skin’s surface and in oil glands
TOXINS
CYTOLYTIC TOXINS
● Disrupts host’s cell membrane
● Leukocidins lyse leukocytes
EXFOLIATIVE TOXINS
● Causes skin cells to separate and slough off
TOXIC-SHOCK-SYNDROME TOXIN
ENTEROTOXINS
● Stimulate intestinal muscle contractions,
nausea, and intense vomiting associated
with Staphylococcal food poisoning
● Toxin is heat-stable; resistant to boiling
NOTE: The bacteria may die but the spore is
still present
COMPARISON OF VIRULENCE FACTORS
STAPHYLOCOCCAL DISEASES
CUTANEOUS DISEASE
● Folliculitis
➔ Hair follicle infection
● Furuncle
➔ Boil
● Carbuncle
➔ Involves connective tissue
● Impetigo
➔ Red patches; pus-filled crust
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SYSTEMIC DISEASE
● Bacteremia
➔ Presence of bacteria in the blood from
wound infections
➔ 50% are nosocomial infections
● Endocarditis
➔ Occurs when bacteria attack the lining
of the heart; 50% mortality rate
◆ Not that common
● Pneumonia
➔ Inflammation of the lungs in which the
alveoli and bronchioles become filled
with fluid
● Osteomyelitis
➔ Inflammation of the bone marrow and
the surrounding bone; from wound or
bacteremia
DIAGNOSIS, TREATMENT, & PREVENTION
DIAGNOSIS
● Detection of Gram-positive bacteria in
grape like arrangements isolated from pus,
blood or other fluids
TREATMENT
● Methicillin is the drug of choice
➔ Is a semisynthetic form of penicillin and
is not inactivated by B-lactamase
● MRSA - Methicillin-resistant Staph. Areus
➔ Vancomycin - drug of choice for
moderate to severe infections
TREATMENT FOR MRSA
● Ceftaroline (Brand name Teflaro)
➔ The first commercially available
cephalosporin with activity against:
❖ Methicillin-resistant Staphylococcus aureus
(MRSA)
❖ Penicillin-resistant Staphylococcus
pneumoniae
➔ In addition, ceftaroline is active against
most non-ESBL Enterobacteriaceae
➔ NOTE: a broad-spectrum antibiotic
PREVENTION OF MRSA SPREAD IN ICU
● All patients in ICU bathed daily with a 2%
chlorhexidine containing cloth, and had an
 antibiotic ointment of mupirocin applied ● An illness with the following clinical
twice daily inside their nose (the body site manifestations:
most commonly colonized with S. aureus) 1. Fever :greater than or equal to 102.0°F
for 5 days 2. Rash: diffuse macular erythroderma
● 44% reduction in all septicemias including 3. Desquamation: 1-2 weeks after onset
those caused by MRSA. The incidence of of illness, particularly on the palms and
MRSA colonization on these patients was soles
reduced by 37%. 4. Hypotension: systolic blood pressure
● There are about 80,000 cases of invasive less than or equal to 90 mm Hg for
MRSA infections per year, resulting in adults
11,000 deaths annually. MRSA and 5. Multisystem involvement (3 or more):
Staphylococcus in general, account for examples -
approximately one quarter of the 80,000 ➔ Gastrointestinal: vomiting or
deaths from hospital-acquired infections in diarrhea at onset
the U.S. ➔ Muscular: severe myalgia (muscle
pain)
STAPHYLOCOCCUS TOXINS ➔ Hematologic: platelets less than
TOXIC SHOCK SYNDROME 100,000/mm3
● TSS toxin produced by Staphylococcus ➔ Renal, Hepatic, Central Nervous
aureus is absorbed into the blood and System
causes shock
● Linked to many initiating Staph infections STAPHYLOCOCCAL SCALDED
SKIN SYNDROME
● Caused by exfoliative toxins (ETA & ETB) of
Staph. aureus
● Acute exfoliation of the skin typically after an
erythematous cellulitis.
● Severity varies:
➔ Few blisters localized to the site of
infection
➔ Severe exfoliation affecting almost the
entire body

TOXIC SHOCK SYNDROME INFORMATION

● TSS was first described in children in
1978
● Later TSS was associated with menstruating Ritter von Ritterschein disease (in newborns)
women using highly absorbent tampons. Staphylococcus toxins
● Cases of menstrual TSS (1 case per FOOD POISONING
100,000) steadily declined after withdrawing ● From the ingestion of
these tampons from the market. enterotoxin-contaminated food
● 50% of cases of TSS are not associated ● Often this type of food poisoning occurs
with menstruation. when cooked food is allowed to cool slowly
● TSS usually are complications of surgical and/or sit at room temperature for some.
and postpartum wound infections, burns, ● Distinguish staphylococcal from other types
cutaneous lesions, osteomyelitis, and of food poisoning:
arthritis. ➔ short incubation period
● Although most cases of TSS occur in ➔ brevity of illness
women, about 25% of cases occur in men. ➔ usual lack of fever

TOXIC SHOCK SYNDROME - CDC CASE SEQUENCE OF EVENTS

IDENTIFICATION

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 saprophyticus either a bright yellow or white
pigment.
● S. aureus is usually beta-hemolytic, S.
epidermidis and S. saprophyticus are almost
always nonhemolytic.

STAPHYLOCOCCUS AUREUS
● Individual colonies on agar are round,
convex and 1-4 mm in diameter with a sharp
border.
● On blood agar plates, colonies of
DIAGNOSIS, TREATMENT, & PREVENTION
staphylococcus aureus are frequently
PREVENTION OF “STAPH” INFECTIONS
surrounded by zones of clear
● Prevention of “Staph” infections
beta-hemolysis.
➔ Hand antisepsis is the most important
● The golden appearance of some strains is
measure in preventing nosocomial
the etymological root of the bacteria’s name;
infections
aureus meaning “golden” in Latin.
➔ Proper cleansing of wounds and surgical
openings
➔ Aseptic use of catheters or indwelling
needles
➔ Refrigerate food
➔ Good hygiene

LABORATORY IDENTIFICATION
● Staphylococcus is a genus of bacteria that is
characterized by a round shape (coccus or
spheroid shaped), Gram-positive (purple),
and found as either single cells, in pairs, or
more frequently, in clusters that resemble a
bunch of grapes.
● The genus name Staphylococcus is derived
from Greek terms (staphyle and kokkos) that
mean "a bunch of grapes"
NOTE:
● The left picture is of a MANNITOL SALT
AGAR showing the yellow colonies of S.
aureus. Originally, the MSA is colored
pink/red but in the case of S. aureus since it
is a mannitol fermenter, its color becomes
yellow in that agar.
STAPHYLOCOCCUS EPIDERMIDIS
● Showing y-hemolytic, porcelain-white
colonies as compared to S. aureus on BAP
● This clear distinction in colony color is seen
at all times.

STAPHYLOCOCCUS - BLOOD AGAR CULTURE

● Blood agar is both differential and enriched
medium.
● On blood agar, S. aureus usually displays a
light to golden yellow pigment, whereas S. CATALASE TEST
epidermidis has a white pigment and S.

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 ● The Catalase test is primarily used to
distinguish among Gram-positive cocci
● Members of the genus Staphylococcus are
catalase positive, and members of the
genera Streptococcus and Enterococcus are
catalase-negative.
NOTE:
● Hydrogen peroxide (H2O2), the reagent, is
dropped on a colony placed on a glass slide.
● If catalase is present, the enzyme will break
down H2O2 into oxygen and water. The
oxygen product can be seen physically or
macroscopically as BUBBLE FORMATION.
➔ BUBBLES (+) = Staphylococcus
➔ NO BUBBLES (-) = Streptococcus
or Enterococcus
COAGULASE TEST
● The only significant disease causing
bacteria of humans that produce Coagulase
enzyme are staphylococcus aureus.
● The action of the coagulase enzyme
produces clotting of the plasma by
converting fibrinogen to fibrin in the
immediate vicinity of the bacteria as a
means of protection by itself
● Used to distinguish between pathogenic and
non pathogenic members of the genus
Staphylococcus. All pathogenic strains of S.
aureus are coagulase positive whereas the
nonpathogenic species (S. epidermidis) are
coagulase negative
TUBE COAGULASE TEST
● Free coagulase secreted by S.aureus but
not CNS
● Clots rabbit plasma
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COAGULASE SLIDE TEST (CLUMPING FACTOR)
● Use BSL-2 precautions when performing
this test with an unknown organism or S.
aureus
● Detects BOUND coagulase (difference from
Tube Coagulase test)
NOTE:
● The procedure for both the TUBE and
SLIDE coagulase tests are the same.
➔ Plasma is added to a colony/growth
of bacteria, and the occurrence of
clotting is noted.
❖ CLOTTING: (+)
❖ NO clotting: (-)
MANNITOL SALT AGAR
● Mannitol salt agar (MSA) is both a selective
and differential media used for the isolation
of staphylococci from mixed cultures
● On MSA, only pathogenic staphylococcus
aureus produces small colonies surrounded
by yellow zones
● The reason for this color change is that
S.aureus have the ability to ferment
mannitol, producing an acid, which changes
the indicator color from red to yellow
● MSA components:
➔ 7.5% NaCl - selects for species of
Staphylococcus. This concentration
of salt is too high for most other
bacteria to withstand and, therefore,
inhibits their growth
 ❖ Other bacteria can only
withstand up to 6.5%.
➔ Mannitol - alcohol of the
carbohydrate mannose. Mannitol
fermentation produces acid end
products which turn the medium
yellow. Yellow indicates mannitol
positive and no color change
indicates mannitol negative
➔ Phenol red pH indicator - yellow in
acid pH (the same indicator that is
used in phenol red carbohydrate
fermentation broths)
❖ Phenol red is color red in
neutral pH.

GROUP A STREPTOCOCCUS =
STREPTOCOCCUS PYOGENES
● Beta-hemolytic
● Infects the pharynx or skin
● Often causes disease when normal
microbiota are depleted
● Spreads through respiratory droplets
.

STREPTOCOCCUS
● Gram-positive cocci, arranged in pairs or
chains
● Catalase negative
● Facultative anaerobes
➔ Aerobes that can grow without
oxygen (anaerobic condition) BETA HEMOLYSIS
● Categorized based on: ● Streptococcus pyogenes, or group A
➔ Hemolysis beta-hemolytic streptococci (GAS), and
❖ Brown’s classification Streptococcus agalactiae, or group B
(discovered by Brown) beta-hemolytic Streptococci (GBS) blood
❖ Ability to lyse RBCs agar cultures display beta hemolysis
❖ Beta, Alpha, Gamma ● Beta hemolysis, sometimes called
➔ Lancefield Grouping complete hemolysis, is a complete lysis of
❖ Divides the streptococci into red loom cells in the media around and
serotype groups based on under the colonies: the area appears
the bacteria’s cell wall lightened (yellow) and transparent
antigens
❖ Group A and B are
pathogens

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GROUP A STREP - VIRULENCE FACTORS
1. M protein of fimbriae
● adheres to pharyngeal tissue
● resists phagocytosis
● 80 serotypes
NOTE:
● The picture on the right shows the
FIMBRIAE (color gray).
● The black dots on the picture on the
left are the M protein of the fimbriae.
2. Toxins
● Streptolysin O and S - hemolysis
➔ O: oxygen-LABILE
➔ S: oxygen-STABLE
● Erythrogenic toxin - rash
● Pyrogenic toxin - fever
3. Enzymes
● Deoxynuclease
● Hyaluronidase
● Streptokinase – lyse platelets, WBC
● Help spread bacteria through tissue
GROUP A STREPTOCOCCAL DISEASES
1. Pharyngitis (“strep throat”)
● inflammation of the pharynx
● Most common disease caused by S.
pyogenes
2. Scarlet fever
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● rash that begins on the chest and
spreads across the body
➔ The origin of the rash can
be used to differentiate from
other viral diseases (e.g.
mumps, measles).
3. Pyoderma/Impetigo
● confined, pus-producing lesion that
usually occurs on the face, arms, or
legs
4. Necrotizing fasciitis
● toxin production destroys tissues
and eventually muscle and fat tissue
➔ “Flesh-eating disease”
STREPTOCOCCUS PYOGENES - STREP
THROAT
● Symptoms:
➔ sore throat, cough
➔ high fever
➔ swollen lymph nodes
➔ “beefy” red throat
● Treatment:
➔ penicillin
● Autoimmune Complications:
➔ Rheumatic fever – inflammation
that leads to damage of heart valves
muscle
➔ Glomerulonephritis – inflammation
of the glomeruli and nephrons
obstruct blood flow through the
kidneys
❖ Can lead to renal failure
NOTE:
● The autoimmune complications are due to
immune complex deposition either in the
heart (rheumatic fever) or kidneys
(glomerulonephritis) as a result of
repeated/recurrent strep throat infections.
GROUP A STREPTOCOCCAL DISEASES
SCARLET FEVER
● Accompanies strep throat if strain
releases erythrogenic toxins
● Symptoms:
➔ strep throat
➔ “strawberry” tongue
➔ skin rash due to erythrogenic toxins
● Treatment:
 ➔ Penicillin

GROUP A STREPTOCOCCUS SKIN DISEASES NOTE: Letter A in the picture is GAS, as evidenced
● Erysipelas by the zone of inhibition.

GROUP B STREPTOCOCCUS =
STREPTOCOCCUS AGALACTIAE
● Normally colonizes the lower
gastrointestinal, genital, and urinary tracts
● Diseases
● Pyoderma (Impetigo) ➔ Most often associated with neonatal
bacteremia, meningitis, and
pneumonia
➔ Immunocompromised older patients
are at risk
● Pathogenicity
➔ Often infects newborns who have
● Necrotizing fasciitis “flesh-eating” bacteria not yet antibodies and whose
➔ Generalized as “bacteria” because mothers do not provide passive
S. pyogenes is not the only bacteria immunity
that can cause necrotizing fasciitis. ● Prevention
➔ Culture the vaginal tract (swab) at
37 weeks (of gestation) to check for
colonization of Group B
Streptococcus. If positive,
prophylactic administration of
penicillin
➔ Prophylactic administration of
BACITRACIN SENSITIVITY TEST (BETA penicillin at birth to children whose
HEMOLYTIC STREPTOCOCCI GROUP A, GAS) mothers’ urinary tracts are colonized
● In a clinical laboratory, bacitracin is useful in with group B streptococci
helping identify streptococci and other gram
positive bacteria CAMP TEST (B-HEMOLYTIC STREPTOCOCCI
● Principle: bacitracin test is used to GROUP B, GBS)
determine the effect of a small amount of ● S. agalactiae is the only species that has the
bacitracin (0.04 UL) on an organism. group B antigen
Streptococcus pyogenes (GAS) is inhibited ● CAMP - it is an acronym for “Christie, Atkins,
by the small amount of bacitracin in the disk Munch-Petersen” for the three researchers
(visible zone if inhibition of growth); other who discovered the phenomenon
beta-hemolytic streptococci usually are not. ● The CAMP test is a test to identify Group B
B-hemolytic streptococci based on their
formation of a substance called CAMP factor
that enlarges the area of hemolysis formed
by B-hemolysin from Staphylococcus aureus

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NOTE:
● In the pictures, the line in the middle is the
S. aureus colony. Perpendicular to that line,
colonies of the test organism are streaked.
➔ If the organism is S. agalactiae,
there will be an
ARROWHEAD/BOW TIE hemolysis
pattern in the juncture or the point
where the organism is in contact
with the S. aureus colony.
ALPHA-HEMOLYTIC STREPTOCOCCI:
STREPTOCOCCUS PNEUMONIAE
● Gram-positive diplococcus
➔ Lancet-shaped appearance
● Alpha-hemolytic
● Normally colonizes the mouth and pharynx
● Can cause disease if travels to the lungs
● Disease is highest in children and the elderly
ALPHA-HEMOLYSIS
● Streptococcus pneumoniae, Streptococcus
salivarius, viridans (including Streptococcus
mutans) are referred to collectively as
viridans streptococci, a name derived from
viridis (Latin for “green”), referring to the
green pigment formed by the partial, alpha
hemolysis of blood agar
● Encapsulated, virulent strains of S.
pneumoniae often forming highly mucoid,
glistening colonies (production of capsular
polysaccharide) surrounded by a zone of
alpha-hemolysis.
● This is sometimes called green hemolysis
because of the color change in the agar.
● Other synonymous terms are incomplete
hemolysis and partial hemolysis.
ALPHA-HEMOLYTIC STREPTOCOCCI:
STREPTOCOCCUS PNEUMONIAE
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● Alpha hemolysis is caused by hydrogen
peroxide produced by the bacterium,
oxidizing hemoglobin to green
methemoglobin.
➔ Does not destroy/lyse RBCs directly
(like in beta hemolysis)
➔ In alpha hemolysis, the destruction
of hemoglobin (which is responsible
for the red color of blood) is the
reason for the green color produced.
● Alpha hemolytic colonies with depressions in
their centers are characteristics of
pneumococci
● Some strains produce high amounts of
capsular polysaccharide which gives a
glistening appearance.
VIRULENCE FACTORS
1. Phosphorylcholine – stimulates cells to
phagocytize the bacteria
2. Polysaccharide capsule - protects the
bacteria from digestion after endocytosis
3. Protein adhesin – binds the cells to pharynx
epithelial cells
4. Secretory IgA protease – destroys IgA
(antibody found in mucous membranes)
5. Pneumolysin – lyses epithelial cells and
suppresses the digestion of the endocytized
bacteria
DISEASES CAUSED BY S. PNEUMONIAE
● Pneumococcal pneumonia
➔ Bacteria multiply in the lower lung;
cause damage to the alveolar lining;
produce an inflammatory response
➔ High fever; chest pain; SOB; sputum
production
➔ 85% occur after viral disease (i.e. flu)
● Sinusitis and otitis media
➔ Bacteria invade the sinuses or middle
ear, often following a viral infection
(usually the immune system is
weakened)
● Bacteremia and endocarditis
➔ Bacteria in the bloodstream or in the
lining of the heart
● Pneumococcal meningitis
➔ Bacteria that have spread to the
meninges
STREPTOCOCCUS PNEUMONIAE
● S. pneumoniae accounts for 25-35% of
cases of community-acquired bacterial
pneumonia leading to 40,000 deaths/year in
the US (Merck)
● Treatment – Penicillin
➔ Up to 35% of strains are resistant to
penicillin (as well as erythromycin,
Bactrim, cephalosporins)
● Prevention - Pneumovax
➔ Vaccine from purified capsular
polysaccharides
➔ Provides long lasting immunity in normal
adults; (not as effective in children, the
elderly, or AIDS patients)
ALPHA-HEMOLYTIC STREPTOCOCCI: THE
VIRIDANS GROUP
● Alpha-hemolytic (“viridans = green”)
➔ Greenish discoloration in blood agar
● No Lancefield group
➔ Lack group-specific carbohydrates
● Normal microbiota
➔ mouth, pharynx, GI tract, GU tract
● Opportunistic Disease:
➔ One of the causes of dental caries and
dental plaques; produces dextran; leads
to biofilm formation
➔ Can cause meningitis and endocarditis
OPTOCHIN TEST
● Optochin (or ethylhydrocupreine) is a
chemical used in cell culture techniques for
the of Streptococcus pneumoniae, which is
optochin-sensitive (positive result), from
other alpha- hemolytic streptococci such as
Streptococcus viridans which are resistant.
BILE SOLUBILITY TEST
● The bile (sodium deoxycholate) solubility
test distinguishes Streptococcus
pneumoniae from all other alpha-hemolytic
(viridans) streptococci
● Streptococcus pneumoniae is bile soluble
whereas all other alpha-hemolytic
streptococci are bile resistant.
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● Sodium deoxycholate (2% in water) will lyse
the pneumococcal cell wall.
● A clearing of the turbidity in the bile tube
indicates a positive test.
ENTEROCOCCUS
● E. faecalis and E. faecium
➔ Previously classified as group D
streptococci but reclassified as a
separate genus (enteroccocus and
non-enterococcus)
● Normal microbiota of the human colon
● Opportunistic disease:
➔ Urinary Tract Infection
➔ Endocarditis
● Common cause of nosocomial infections
● Treatment: Difficult to treat due to resistance
➔ Ampicillin and ceftriaxone
➔ VRE - Vancomycin Resistant
Enterococcus
ENTEROCOCCUS FAECALIS- GAMMA
HEMOLYSIS
● If an organism does not induce hemolysis,
the agar under and around the colony is
unchanged, and the organism is called
non-hemolytic or said to display gamma
hemolysis.
● Enterococcus faecalis (formerly called
Group D Streptococcus) displays gamma
hemolysis
● E. faecalis typically exhibits
gamma-hemolysis on blood agar, but some
strains are alpha hemolytic or even
beta-hemolytic (a plasmid-encoding
hemolysin called cytolysin).
EPIDEMIOLOGY OF VRE: RISK FACTORS FOR
ACQUISITION
● Prior broad spectrum antibiotics (especially
cephalosporins and vancomycin)
● Prolonged hospitalization
● Immunocompromised host
● Neutropenia
● Admission to an intensive care unit
● Renal failure requiring dialysis
 BIOCHEMICAL CHARACTERISTICS OF S.
AUREUS

POSITIVE NEGATIVE OTHERS

DETERMINE WHAT ISOLATE IS THIS
● Catalase ● Oxidase ● OF test -
● Coagulase ● Indole fermentativ
Staphylococcu ● Coagulase and Catalase - presence ● Gas (-) e
s intermedius POSITIVE of free ● Hydrogen ● Beta
● Novobiocin SENSITIVE and/or sulphide hemolysis
● PYR-POSITIVE bound ● Motility on blood
coagulase ● PYR agar
Group B ● Creamy zones of Beta ● Methyl red (Pyrrolidon
Streptococcus hemolytic colonies ● VP yl
(Streptococcus ● Hippurate POSITIVE (Vogues-P Arylamidas
agalactiae) ● Bowtie type hemolysis in roskauer) e Rapid)
CAMP test ● Nitrate Test
● PYR-NEGATIVE reduction
● Gela
Staphylococcu ● Yellow Beta hemolytic ● Citrate
s aureus colonies in MSA ● Urease
● Catalase and Coagulase
POSITIVE
● Grows in 7.5% NaCl

Strep Viridans ● Alpha hemolytic

● Optochin RESISTANT
● Bile solubility test
NEGATIVE
● Bile esculin NEGATIVE

COAGULASE NEGATIVE STAPHYLOCOCCI

STAPHYLOCOCCUS
● Gram positive, non-motile cocci about 1 um
in diameter
● Form (grapelike) clusters of cells

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 ● Common inhabitants of the skin and mucous ● Selective media:
membranes ➔ Thayer Martin
● As a group, Staphylococci do not have ➔ Modified Thayer martin
spores or flagella but may form as capsules ➔ Martin Lewis
● They grow well on routine laboratory media ➔ Modified New York City medium
and are facultative anaerobes

STAPHYLOCOCCUS STREPTOCOCCUS

Clusters (grapelike) Pairs/ chains

Catalase positive Catalase negative

Coagulase positive Coagulase positive

Growth in 7.5% NaCl Growth in 6.5% NaCl

GRAM NEGATIVE COCCI

NEISSERIAE

TAXONOMIC CLASSIFICATION
● KINGDOM: Bacteria
● PHYLUM: Protobacteria
● CLASS: Betaproteobacteria
● ORDER: Neisseriales
● CLASS: Neisseriaceae
● GENUS: Neisseriae
GENERAL FEATURES
Gram negative diplococci aerobes

oxidase positive
● Catalase positive
● Aerobes and facultativeanaerobes
● Non sporing
● Non motile
● Optimum temperature: 35-36 C
● Optimum pH: 7-7.4
● 5-10 % CO2 enhances growth
● Fastidious in growth requirements
● Oxidase positive
● Catalase positive
IMPORTANT HUMAN PATHOGENS
● Superoxol test:
● Neisseria gonorrhoeae
➔ Positive in N. gonorrhoeae
● Neisseria meningitidis
➔ Negative in N. meningitidis and N.
➔ Other species normally colonize mucosal
lactamica
surfaces of oropharynx, nasopharynx and
● Carbohydrates are oxidatively utilized.
anogenital mucosal membranes.
➔ Mainly differentiated on the basis of
carbohydrate utilization tests: Glucose,
maltose, lactose and sucrose. ORGANISMS DISEASES
● Transport Media:
➔ Stuart’s media N. gonorrhoeae Urethritis, cervicitis,
➔ Amies media salpingitis, PID,

MICROBIOLOGY: BACTERIOLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 12

 proctitis, bacteremia,
arthritis, conjunctivitis,
skin lesions, pharyngitis

N. meningitidis Meningitis, bacteremia,

pneumonia, arthritis

Other neisseria Opportunistic infections

species

NEISSERIA MENINGITIDIS ● Blood Agar- weak hemolysis

● Diplococcus intrecellularis ● Liquid media- granular turbidity
● Meningitidis DIFFERENTIAL MEDIA
● Meningococcus ● Blood Agar, Chocolate Agar
● Weichselbaum first discovered and isolated ● Mueller-hinton starch, Casein hydrolysate
in 1887 from the spinal fluid. agar
MORPHOLOGY ● Modified Thayer-Martin (Vancomycin,
● Gram negative cocci Colistin, Nystatin)
● Oval/spherical
● Capsulated BIOCHEMICAL REACTIONS
● 0.6-0.8 um in size. OXIDASE TEST POSITIVE
● Arranged in pairs (adjacent sides are flat) ➔ Freshly prepared 1% oxidase reagent
● Non-motile
● Generally intracellularly or may be (Tetramethyl Para Phenylenediamine
extracellular. Dihydrochloride)
CELLULAR CHARACTERISTICS ↓
● Exacting growth requirements Culture Media
● Blood, serum, ascitic fluid ↓
● Optimal temperature: 35-36 C (25-42 C) Deep Purple Colonies
● Optimal pH: 7.0-7.4
● Growth facilitated by 5% to 10% CO2 KOVAC’S METHOD
COLONIES Growth in a loop
● Small, 1mm in diameter ↓
● Translucent
Rub on a strip of filter paper moistened with
● Round, convex
oxidase reagent
● Bluish gray
● Smooth glistening surface
↓
● Entire edges Deep purple color
● Lenticular shape
● Butyrous consistency OTHER BIOCHEMICAL REACTIONS
● Easily emulsifiable
● Smooth and rough forms
Catalase test POSITIVE

Indole and Hydrogen NOT PRODUCED

Sulphide

Nitrates NOT REDUCED

Glucose and Maltose UTILIZED with ACID

PRODUCTION only
Neisseria meningitidis in Thayer-Martin Agar (Peptone serum agar
slope, RCUT)

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 ➔ 5 classes; class 5 protein (opa and
opc) is involved in attachment and
invasion epithelial cells
➔ Class 2 and 3 are major OMPs.
RESISTANCE ● LIPOOLIGOSACCHARIDES
➔ Lack repeating O-Ag (Rough LPS)
● Delicate freeze ➔ Lipid A moiety-portion that mediates
drying, induction of inflammatory cytokines
Microbanking, ● OTHER FACTORS
Liquid nitrogen ➔ IgAse
➔ Pili
HIGHLY SUSCEPTIBLE ➔ Iron is obtained from transferrin
● Heat
● Desiccation
● Disinfectants INFLAMMATORY MEDIATORS AND MOLECULES
● Penicillin INVOLVED IN MENINGOCOCCAL DISEASE
● Alterations in pH ● IL-1
CLASSIFICATION ● IL-6
● Based on capsular polysaccharide ● IL-8
● PAF
● Prostaglandins
● TNF
● H2O2
● Singlet Excited Oxygen
● Hydroxyl radical
● CD62, ELAM-1
● ICAM-1, CD 18
PATHOGENICITY
● Human pathogens- nasopharynx
● Cerebrospinal meningitis
● Meningococcal septicemia
● Rhinitis
● Pharyngitis
● Conjunctivitis

● Serotyping based on class 1 OMP and

subtyping based on class 2 and 3
● Group, type, and subtype are phenotypic
characteristics.
● Types 2, 15 and recently type 4 have been
associated with group B meningococcal
disease.
● The majority of group C meningococci
belong to type 2 (2a or 2b)

ANTIGENICITY AND VIRULENCE

● CAPSULE
➔ Antiphagocytic
➔ Opsonizing anticapsular antibodies
are protective
➔ Meningococcal vaccine
● OUTER MEMBRANE PROTEINS

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 ➔ Paralysis of cranial nerves causing
facial drooping
● Cortical venous thrombophlebitis
● Cerebral edema
● Children develop subdural effusion
● Permanent sequelae (especially in children)
include mental retardation,
deafness,hemiparesis

CLINICAL FEATURES
● Chills
● Malaise
● Prostration
● Petechial rashes
● Metastatic involvement of joints, ears, eyes,
lungs, adrenals
● 10% pneumonia

WATERHOUSE FRIDERICHSEN SYNDROME

● Fulminant meningococcemia
➔ “Fulminant” - developing or progressing
rapidly/suddenly all over the body
FEATURES ● Fatal condition causing:
● Nausea ➔ Shock
● Vomiting ➔ DIC
● Headache ➔ Multisystem failure
● Neck ➔ Deficiency of C5-C9
stiffness ➔ Pathogenic component-LPS (endotoxin)
● Lethargy ➔ Adrenal hemorrhage and shock
● Confusion ● A type of meningococcemia named after
Friderichsen

OTHER MANIFESTATIONS
● Arthritis
● Primary meningococcal pneumonia
● Meningococcal pericarditis
● Endocarditis
● Conjunctivitis
EPIDEMIOLOGY
COMPLICATIONS ● Transmission
● Cranial Nerve Palsy ➔ Airborne-droplets

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 ➔ Fomites ➔ Detection of antibodies (Chronic
● Common in children between 3 months to 5 meningococcemia)
years ● Molecular diagnosis
● Crowded conditions ➔ Detection of Meningococcal DNA-PCR.
➔ Jail ● Serological tests:
➔ Ships ➔ CFT, HA, ELISA.
➔ Army camp
CSF EXAMINATION
● MENINGITIS BELT OF AFRICA ● Sample of choice for meningitis
➔ Ethiopia- Senegal ● Obtained through spinal tap
● In 1996 there are: ● General appearance: Purulent
➔ 150,000 cases ● Biochemical parameters:
➔ 15,000 deaths ➔ Glucose <35mg/100ml
➔ Proteins 80-500mg/100ml
● Cytology: 400-20,000 PMNs/μl.
● Centrifuge the CSF sample
● Plate out the deposit on BA and CA and
incubate at 37oc in 5-10% CO2 for 24 hours.
● Add glucose broth to the remaining deposit,
incubate overnight and subculture in the
same way. Examine the colonies by Gram
stain and Oxidase test.
● Make 2 smears of the centrifuged deposit
and stain with Gram stain and methylene
blue respectively.
● FITC coupled antiserum may be examined
for the direct identification of the
INDIAN SCENARIO
meningococcal serogroup responsible for
● Meningococcal disease is endemic in Delhi
infection.
and sporadic cases of meningococcal
● Divide the supernatant into 2 aliquots
meningitis have been occurring in Delhi in
➔ One for biochemical reactions
the past.
➔ The other for meningococcal
● Isolated cases of meningococcal meningitis
polysaccharide antigen detection by
during 1985 were also reported from several
latex agglutination, CIEP or
states of India including Haryana, Uttar
coagglutination.
Pradesh, Rajasthan, Sikkim, Gujarat,
● Biochemical reactions
Jammu & Kashmir, West Bengal,
● Antibiotic sensitivity testing
Chandigarh, Kerala and Orissa.
● Serogrouping
● Serogroup A has been associated with all
the repeated outbreaks of meningitis,
although serogroup B and C have been
detected in a few sporadic cases.

LABORATORY DIAGNOSIS
●
● Blood culture
➔ Incubation for TREATMENT
4-7 days ● Penicillin G or Ceftriaxone (Confirmed
● Nasopharyngeal cases)
swab ● Empirical treatment:
● Petechial lesions ➔ Up to 3 months: ampicillin+cefotaxime
● Autopsy or ceftriaxone.
➔ Lateral ➔ 3 months to 7 years: cefotaxime or
ventricle/surface of brain & S.C. ceftriaxone.
● Retrospective evidence

MICROBIOLOGY: BACTERIOLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 16

 ➔ >7 years: ampicillin or penicillin G +
cefotaxime or ceftriaxone.
➔ Dexamethasone may be started 15
minutes before starting antibiotics.

PREVENTION
● Meningococcal polysaccharide vaccines
(univalent and polyvalent vaccines).
CULTURAL CHARACTERISTICS
ANTIMICROBIAL PROPHYLAXIS ● Aerobes/ facultative anaerobes
● Rifampicin ● pH: 7.2 - 7.6
● Ciprofloxacin ● Temperature: 35C - 36C
● 5-10% CO2
VACCINES ● Chocolate agar, Mueller-Hinton agar, Thayer
● Five serogroups, A, B, C, Y and W135 are martin media, Modified TM medium,
responsible for virtually all cases of the Modified NYCM
disease in humans.
● Vaccines are currently available against four COLONIES
of the five strains, and a vaccine against the ● Small
B strain is in development. ● Round
● Menactra, Menomune of Sanofi-Aventis, ● Translucent
Mencevax of GlaxoSmithKline and ● Convex/slightly umbonate
NmVac4-A/C/Y/W-135 (has not been ● Granular surface, lobulated margins
licensed in the US) of JN-International
Medical Corporation are the commonly
used vaccines.
● Vaccines offer significant protection from
three to five years (plain polysaccharide
vaccine Menomune, Mencevax and
NmVac-4) to more than eight years
(conjugate vaccine Menactra)

NEISSERIA GONORRHOEAE
● 1879 - Albert L.S.
Neisser
➔ First described in
gonorrhoeal pus.
● 1885 - Bumm
➔ Cultured & proved it’s
pathogenicity
● 1960 - Douglas Kellogg
➔ Discovered phase
variation in gonococci.
● Fred Sparling
➔ Showed that small colony gonococci
were piliate and virulent large colony
gonococci were non piliated and BIOCHEMICAL FEATURES
avirulent. ● Utilize only glucose
● Catalase positive
MORPHOLOGY ● Oxidase positive
● Gram negative ● Superoxol test positive
● Diplococcus Adjacent sides concave
● Kidney shaped RESISTANCE
● Exclusively intracellular Pili-adhesion to ● Delicate
mucosal surface ● Killed: heat, drying, antiseptics.

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 ● Dies: 1-2 hours outside the body in
Designation Location Contribution
exudates.
● Culture dies: 3-4 days.
PilE Major fimbrial Initial binding to
● Survives: slant culture - 35 ̊c-sterile paraffin
protein epithelial cells
wax.
● Stored: (-70 ̊c) for years
P.II (Opa) Outer Contributes to
membrane invasion
ANTIBIOTIC RESISTANCE
protein
● Chromosomally resistant N. gonorrhoeae
(CMRNG)
P.I (Por) Outer May prevent
● PPNG (Penicillinase producing N.
membrane phagolysosome
gonorrhoeae)
porin formation in
➔ Mainly 3.2MDa and 4.4MDa plasmids
neutrophils
(TEM-1 β lactamase).
and/or reduce
● Tetracycline resistant N. gonorrhoeae
oxidative burst
(TTNG): 25.2MDa plasmid- tetM gene.
LOS Outer Elicits
ANTIGENIC PROPERTIES
membrane inflammatory
● Pili
lipooligosacch response,
● Hair-like structure
aride triggers release
● Composed of repeating peptides subunits
of TNF
(pilins)
➔ Pilins contain 159 amino acids. P.III (Rmp) Outer Elicits formation
● Cyanogen bromide digestion has recognized membrane of ineffective
3 regions. Region 2 is receptor binding protein antibodies that
region and 3 is type specific region. block bactericidal
antibodies
against P.I and
LOS

Tbp1 and Outer Iron acquisition

Tbp2 membrane for growth
receptors for
transferrin

Lbp Outer Iron acquisition

membrane for growth
● P1: Principal outer membrane protein. receptor for
● P2: OPA lactoferrin
● P3: Binds with P1 to form porin channels
● LOS: Damage to cilia sloughing of epithelial
cells.
NOTE: Responsible for the dead cells and for the
pus when infected with the bacteria.
● Lacks somatic O antigen. Sialation protects
from antibody mediated defense processes.
● Peptidoglycan: C6 hydroxyl group is o-
acetylated. Induces fever, ciliary damage,
epithelial damage, arthritogenic and
activates complement. Also prevents lytic
action of lysozymes.
● IgAse: Cleaves Fc portion of sIgA.
● Alpha protein or factor.

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 IN WOMEN
Note: First, there is sexual contact then the bacteria ● Urethra & cervix
will enter, it will attach to the microvilli of our mucus ➔ Affected
membrane cells (anal, vaginal, pharyngeal). It will ● Vaginal mucosa
then be endocytosed, it will either multiply inside the ➔ Not affected
host cell or it will penetrate the basement membrane ➔ Stratified squamous epithelium
and spread. It will still survive even inside the ◆ If destroyed, it can be changed to a
neutrophils and macrophages. It can go out the new set of cells.
PMNs together with the pus. ➔ Acidic pH
● Vulvo-vaginitis
➔ Prepubertal girls
● Urethra, cervix, bartholin’s glands, F.T
● P.I.D & Salpingitis-sterility
● Peritonitis
● Perihepatic inflammation (Fitz-Hugh-Curtis
Syndrome)

Note: After incubation period it will now cause

symptoms.
IN MAN

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 IN BOTH THE SEXES
● Proctitis
● Conjunctivitis
● Arthritis
● Ulcerative endocarditis
● Rarely meningitis
● Pyemia
● Non-Venereal-Gonococcal Ophthalmia
○ Occurs during normal vaginal
delivery, when it will pass in the
vaginal wall containing bacteria, it
can affect the eyes. Sometimes
babies are given prophylaxis to the
eyes.
● Gonococcal bacteremia
● Hemorrhagic papules - hands,forearm;
arthritis - knee,ankles
ANORECTAL GONORRHOEA
● Anorectal pain/pruritus
● Tenesmus
● Purulent rectal discharge
● Rectal bleeding
● Pharyngeal Gonorrhoea
● Ocular Gonorrhea
○ Can also be transmitted if you
haven’t disinfected your hand when
handling a positive specimen and
you scratch your eyes.
● Autoinoculation from infected site
EPIDEMIOLOGY
● Source: human carrier
● Mode of infection: venereal
○ Venereal-meaning through sexual
contact
● Non-venereal: Ophthalmia Neonatorum
● 1% Silver nitrate - CREDE’S METHOD
● 1970 Global incidence: 16million
● Higher in B-blood group people.
● Most common in the age group of 15-29
years.
● Risk factors include unprotected intercourse
and multiple sexual partners.
● Proctitis , throat infections , arthritis and
disseminated infections can also be seen.
MICROBIOLOGY: BACTERIOLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 20
LABORATORY DIAGNOSIS
SAMPLES TO BE COLLECTED
● MEN:
➔ Urethral discharge(acute cases),
‘morning drop’ of secretion in chronic
cases.
➔ Exudate may be obtained after prostatic
massage.
➔ Centrifuged deposits of urine may be
used.
● WOMEN:
➔ Urethral discharge, cervical swabs.
Although repeated sampling of multiple
sites is ideal, a single well taken
endocervical swab will detect
approximately 90% of gonococcal
infections in women.
➔ High vaginal swabs not to be collected
(1 in 3 infected women likely to be
missed).
● Other samples include rectal swabs, throat
swabs, blood, swabs from skin lesions, pus
aspirated from joints, conjunctival swab in
neonatal ophthalmia.
● Purulent material should be collected using
dacron or rayon swabs because calcium
alginate and cotton swabs may contain
materials toxic for Gonococci.
● Microscopy:
➔ Gram stained smear reveals Gram
negative kidney shaped diplococci.
➔ If mostly intracellular then positive, if
extracellular then equivocal and if not
found then negative
◆ If extracellular, investigate further
● CULTURE:
➔ Direct plating of the sample followed by
immediate incubation at 36-37C in a
moist atmosphere containing 5-10%
CO2.
● If direct plating and immediate incubation is
impracticable several transport and culture
systems are available.
● Amies transport media and Stuart’s media.
● Biochemical tests
 ● Serology
● Molecular methods
TREATMENT AND PROPHYLAXIS
● Ceftriaxone + doxycycline or tetracycline
or azithromycin.
○ Either of the 3 drugs
● If penicillin sensitive (NON PPNG) then
amoxicillin + probenecid and doxycycline.
● No effective vaccine.
● Health education, contact tracing,
monogamy and use of condoms are the only
preventive measures.
OTHER NEISSERIA SPECIES
● N. elongata ssp. nitroreducens
● N. weaveri ssp nov
● N.gonorrhoeae ssp kochii
● N. flavescens
● N.mucosa
Note:
● The following are considered as
commensals
MORAXELLA
● Moraxella species were formerly called
Neisseria and later Branhamella species
● Separated from Neisseriae based on DNA
base composition, fatty acid composition
and inability to produce acid from
carbohydrates.
● Moraxellas may be involved in opportunistic
infections in compromised patients.
● These occur as components of the normal
flora of the upper respiratory tract, the
conjunctiva, the skin and genital tract.
MORAXELLA CATARRHALIS
● Oval Gram negative cocci about 0.8 μm in
size
● May occur singly, but majority of these are
diplococci with adjacent sides flattened.
● Sometimes these may occur in groups of 4.-
tetrads
● May be found within polymorphonuclear
leukocytes.
○ Can be intracellular
● Aerobes
● Non-sporing, non-capsulated, and
non-motile.
● Optimum temperature is 36C but many
strains may grow at 22C .
● CO2 is not an absolute requirement.
○ Difference between Neisseria and
Moraxella
● Most strains grow on Nutrient agar.
MICROBIOLOGY: BACTERIOLOGY | MLS 24 / MT 12 ASSESSMENT | DINO | PAGE 21
● Some strains grow on selective media for
pathogenic Neisseriae.
● Colonies on Blood agar or Chocolate agar
are 1-2 mm in size, non-haemolytic,
friable, white or grayish, convex with an
entire margin later becoming irregular. After
48 hours colonies are larger, more elevated
with a raised opaque center.
● Oxidase positive
● Catalase positive
● Do not utilize carbohydrates.
● Reduce nitrates to nitrites.
● Produce DNase.
● More resistant than meningococcus and
gonococcus.
● Cultures may remain viable for several
months at 20C if prevented from drying.
May survive in sputum for 3-4 weeks.
○ Culture is easy to store
● Susceptible to a wide range of antibiotics but
many strains produce β-lactamase and are
resistant to penicillin and ampicillin.
● It is found in the respiratory tracts of
1.5-5.4% of healthy young and middle-aged
adults, 26.5% of healthy elderly people and
50.8% of healthy children.
● It is therefore recognized as a significant
potential pathogen of the respiratory
tract, including the sinuses, bronchi and
larynx.
● 20% of the cases of sinusitis are due to M.
catarrhalis.
● Chronic bronchitis among smokers is often
caused by M. catarrhalis.
● Although laryngitis is often caused by this
organism, about 90% of cases are due to
viruses.
Note:
● The only specie that is pathogenic to man
LABORATORY DIAGNOSIS
● Specimens collected are sputum and
transtracheal aspirates.
● Microscopy
● Culture (some strains on selective media
used for pathogenic Neisseriae).]
● Oxidase test, RCUT test, test for
β-lactamase production.
OTHER MORAXELLA SPECIES
● Moraxella lacunata
➔ Causes purulent conjunctivitis (angular
blepharoconjunctivitis)
● Moraxella lacunata and M. atlantae require
serum for growth
 ● Loeffler's medium supports the growth of
M. lacunata and M. liquifaciens
● Moraxella lacunata produces gelatinase.

OLIGELLA
● Recently delineated from Moraxella on the
basis of DNA:Rrna hybridization and
serological tests.
● O. urethralis is a rare cause of septic
arthritis.
● Can be misidentified as N. gonorrhoeae
as it is non-motile, Gram negative
diplococcus, both oxidase and catalase
positive which will grow on Thayer-Martin
medium. However, Oligella will grow on
MacConkey's agar,
Note:
● Oligella can grow to MacConkey agar, while
Neisseria can not

END OF TRANS

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