CLINICAL BACTERIOLOGY (LECTURE)
Toxic Shock Syndrome Toxin-1
General Characteristics
- Gram positive cocci, catalase producing,
coagulase positive
- bunches of grapes
- nonmotile, non spore-forming, and aerobic or
facultatively anaerobic except for S.
saccharolyticus
- Colonies produced after 18 to 24 hours of
incubation:
o medium sized (4 to 8 mm)
o cream-colored, white,
white or rarely light gold
o -
o -hemolytic
- Other gram-positive cocci that are occasionally
recovered with staphylococci
o Rothia mucilaginosa
o Aerococcus
o Alloiococcus otitis
General Characteristics
- catalase-producing, coagulase-negative, gram-
positive cocci
- found in the environment and as members of the
indigenous skin microbiota
- Some micrococci have a tendency to produce a
yellow pigment
Staphylococcus aureus
- most clinically significant species
- responsible for numerous infections
- An important cause of nosocomial infection
Virulence Factors
Enterotoxins
o heat-stable exotoxins
o stable at 100°C for 30 minutes
o Staphylococcal food poisoning is most
commonly caused by enterotoxins A, B,
and D.
o Enterotoxins B and C and sometimes G
and I are associated with TSS.
o Enterotoxin B has been linked to
staphylococcal pseudomembranous
enterocolitis.
o Superantigens have the ability to
interact with many T cells, activating an
aggressive, over reactive immune
response.
o Previously referred to as enterotoxin F
o At low concentrations, TSST-1 causes
leakage by endothelial cells, and it is
cytotoxic to these cells at higher
concentrations.
o TSST-1 is absorbed through the vaginal
mucosa leading to the systemic effects
seen in TSS associated with tampon use.
Exfoliative Toxin
o Also known as epidermolytic toxin
o It causes the epidermal layer of the skin
to slough off and is known to cause
staphylococcal SSS, sometimes referred
to as Ritter disease.
o This toxin has also been implicated in
bullous impetigo.
Cytolytic Toxins
o Hemolysins and leukocidins
o -Hemolysin, in addition to lysing
erythrocytes, can damage platelets and
macrophages and cause severe tissue
damage.
o -Hemolysin (sphingomyelinase C) acts
on sphingomyelin in the plasma
membrane of erythrocytes and is also
o -Hemolysin, although found in a higher
percentage of S. aureus stains and some
CoNS, is considered less toxic to cells
- -hemolysin
o Panton-Valentine leukocidin (PVL) an
exotoxin lethal to polymorphonuclear
leukocytes.
Contributing to the invasiveness
of the organism by suppressing
phagocytosis and has been
associated with severe
cutaneous infections and
necrotizing pneumonia.
Enzymes (coagulase, protease, hyaluronidase,
and lipase)
o Staphylocoagulase is produced mainly
by S. aureus.
Considered a virulence marker
o Hyaluronidase hydrolyzes hyaluronic
acid present in the intracellular ground
substance that makes up connective
tissues, permitting the spread of
bacteria during infection
o Lipases are produced by both coagulase-
positive and CoNS. Lipases act on lipids
present on the surface of the skin,
particularly fats and oil secreted by the
sebaceous glands.
Protein A
o Has the ability to bind the Fc portion of
immunoglobulin G (IgG).
o Binding IgG in this manner can block
phagocytosis and negate the protective
effect of IgG.
Infections Caused by Staphylococcus aureus
Skin and Wound Infections abscess is filled
with pus and surrounded by necrotic tissues and
damaged leukocytes
 CLINICAL BACTERIOLOGY (LECTURE)
o Folliculitis is a relatively mild
inflammation of a hair follicle or oil
gland; the infected area is raised and
red.
o Furuncles (boils) can be an extension
of folliculitis, are large, raised, superficial
abscesses.
o Carbuncles occur when larger, more
invasive lesions develop from multiple
furuncles, which can progress into
deeper tissues.
o Bullous impetigo larger and
surrounded by a small zone of erythema.
A highly contagious infection that is
easily spread by direct contact, fomites,
or autoinoculation.
Scalded Skin Syndrome.
o bullous exfoliative dermatitis that occurs
primarily in newborns and previously
healthy young children.
o This disease has also been recognized in
adults
o The severity of the disease varies from a
localized skin lesion in the form of a few
blisters, pemphigus neonatorum, to a
more extensive generalized condition
affecting 90% of the body, known as
Ritter disease.
Toxic Shock Syndrome
o rare but potentially fatal, multisystem
disease characterized by a sudden onset
of fever, chills, vomiting, diarrhea,
muscle aches, and rash, which can
quickly progress to hypotension and
shock.
o associated with use of highly absorbent
tampons
Toxic Epidermal Necrolysis
o a clinical manifestation with multiple
causes; it is most commonly drug
induced, but some cases have been
linked to infections and vaccines.
o The cause is unknown, but symptoms
appear to be due to a hypersensitivity
reaction.
o life-threatening dermatologic disorder
characterized by widespread erythema,
necrosis, and bullous detachment of the
epidermis and mucous membranes,
resulting in exfoliation and possible
sepsis and/or death.
Food Poisoning
o a type of intoxication resulting from
ingestion of a toxin formed outside the
body.
o Disease occurs when food becomes
contaminated with enterotoxin
enterotoxin-
producing strains of S. aureus by
improper handling and is then
improperly stored, which allows growth
of the bacteria and resulting toxin
production.
o Foods that are frequently incriminated
in staph food poisoning include meat
and meat products; poultry and egg
products; salads such as egg, tuna,
chicken, potato, and macaroni; bakery
products such as cream-filled pastries,
cream pies, and chocolate eclairs;
sandwich fillings; and milk and dairy
products.
o Foods kept at room temperature are
susceptible to higher levels of toxin
production when contaminated with
toxin producing staphylococci and they
are more commonly associated with
food poisoning
Other Infections
o Staphylococcal pneumonia has been
known to occur secondary to influenza
virus infection.
Characterized by multiple
abscesses and focal lesions in
the pulmonary parenchyma.
o Staphylococcal bacteremia leading to
secondary pneumonia and endocarditis
has been observed among intravenous
drug users.
o Staphylococcal osteomyelitis occurs as
a manifestation secondary to
bacteremia.
The infection develops when the
organism is present in a wound
or other focus of infection and
gains entrance into the blood.
Staphylococcus epidermidis
- Infections are predominantly hospital acquired.
- Some predisposing factors are instrumentation
procedures such as catheterization, medical
implantation, and immunosuppressive therapy.
- a common cause of healthcare acquired UTIs.
- Prosthetic valve endocarditis is most commonly
caused by S. epidermidis
- Considered as normal skin biota but is a common
source of hospital-acquired infection and often a
contaminant in improperly collected blood
culture specimens
 CLINICAL BACTERIOLOGY (LECTURE)
- Some predisposing factors for hospital-acquired
infections include instrumentation procedures
such as catheterization, medical implantation,
and immunosuppressive therapy.
Staphylococcus saprophyticus
- associated with UTIs in young women.
- It is the second most common cause after E. coli
- This species adheres more effectively to the
epithelial cells lining the urogenital tract than
other coagulase negative staphylococci (CoNS).
o Staphylococcus epidermidis
o Staphylococcus saprophyticus
o Staphylococcus haemolyticus
o Staphylococcus lugdunensis
- It is rarely found on other mucous membranes or
skin surfaces.
Staphylococcus lugdunensis
- can cause both community-associated
community associated and
hospital acquired infections.
- This organism can be more virulent and can
clinically mimic S. aureus infections.
- known to contain the gene mecA that encodes
oxacillin resistance.
- It is an important pathogen in infective
endocarditis, septicemia, meningitis, skin and
soft tissue infections, UTIs, and septic shock.
Other Coagulase-Negative Staphylococci
o S. warneri
o S. capitis
o S. simulans
o S. hominis
o S. schleiferi
- A wide range of infection have been associated
with these organisms, including endocarditis,
septicemia, and wound infections
- Other species of coagulase negative
staphylococci are found as normal biota in
humans and animals
o Although they are not commonly seen as
pathogens, their role in site of infection
is well established and they cannot be
automatically discarded as
contaminants
SPECIMEN COLLECTION AND HANDLING
- Clinical materials collected from infected sites
should be transported to the laboratory without
delay to prevent drying, maintain the proper
environment, and minimize the growth of
contaminating organisms
- Specimens should be taken from the site of
infection after appropriate cleansing of the
surrounding area to avoid contamination by the
skin microbiota.
MICROSCOPIC EXAMINATION
Staphylococci appears as
gram positive cocci in
clusters
ISOLATION AND IDENTIFICATION
- Commonly used agar for isolation and
identification:
o Sheep blood agar (SBA)
general culture media
o Mannitol salt agar (MSA)
selective culture media
contains at least 10% of salt,
sugar mannitol, and phenol red
as pH indicator
Staphylococcus aureus ferments
mannitol and produces a yellow
halo on mannitol salt agar media
as a result of acid production
altering the pH
Although mannitol salt agar is
not typically used in the clinical
identification, it may still be
used to purify staphylococci
from contaminating organisms
for further characterization
o Columbia colistin nalidixic acid agar
(CNA)
o Phenylethyl alcohol (PEA) agar
o CHROM agar
Selective or differential media
For identification of methicillin
resistant Staphylococcus aureus
- Staphylococci produce round, smooth, white,
creamy colonies on SBA after 18 to 24 hours of
incubation at 35°C to 37°C.
Cultural Characteristics (Summary)
S. aureus can produce hemolytic zones around
the colonies and may rarely exhibit pigment
production (yellow) with extended incubation.
S. epidermidis small to medium sized,
nonhemolytic, gray-to-white colonies. Some are
weakly hemolytic
S. saprophyticus forms slightly larger colonies,
with about 50% of the strains producing a yellow
pigment.
S. haemolyticus produces medium sized
colonies, with moderate or weak hemolysis and
variable pigment production.
S. lugdunensis often hemolytic and medium
sized, although small colony variants can occur.
Identification Methods
Catalase Test This test differentiates catalase-
positive micrococcal and staphylococcal species
from catalase-negative streptococcal species
o Principle: The enzyme, catalase, can
convert hydrogen peroxide to water and
oxygen. The presence of enzyme in
bacterial isolate causes rapid
elaboration of bubbles.
o Reagent: 3% hydrogen peroxide
o Results:
Positive: rapid bubble formation
Negative: no rapid bubble
formation
o In performing catalase test, false
positive can also occur
Caused by some organism such
as enterococci that produces
 CLINICAL BACTERIOLOGY (LECTURE)

peroxidase (slowly catalyzes the Modified oxidase test can be used to rapidly
breakdown of hydrogen differentiate staphylococci
staphylococci from micrococci.
peroxide) Most staphylococci are negative, whereas
Sample contaminated from micrococci are positive.
blood agar and use of platinum
wire

Oxidation-Fermentation Test Staphylococci

ferment glucose, whereas micrococci fail to
produce acid under anaerobic conditions.
Coagulase Test The test is used to differentiate o Staphylococci that fail to grow or
Staphylococcus aureus (positive) from coagulase produce acid anaerobically: S.
negative staphylococci (negative). saprophyticus, S. auricularis, S. hominis,
o Principle: S. xylosus, and S. cohnii

cell wall and reacts directly with

fibrinogen.
The presence of bound
coagulase correlates with free
coagulase, an extracellular
protein enzyme that causes the Pyrrolidonyl Arylamidase can be used to
formation of a clot when S. differentiate S. aureus (negative) from S.
aureus colonies are incubated lugdunensis, S. intermedius, and S. schleiferi
with plasma (positive).
o Reagent: Rabbit plasma o The substrate, pyroglutamyl- -
naphthylamide (-pyrrolidonyl- -
naphthylamide; PYR), is hydrolyzed to L-
-naphthylamine,
which combines with p-
o Another Staphylocoagulase producing dimethylaminocinnamaldehyde to form
staphylococci aside from Staphylococcus a red compound.
aureus are: Vogue-Proskauer (VP) test differentiates S.
Staphylococcus intermedius aureus (positive) from S. intermedius (negative)
Staphylococcus delphini o A positive result is the formation of
Staphylococcus lutrae acetoin from glucose or pyruvate.
Some strains of Staphylococcus o Other VP positive Staphylococci:
hyicus S. lugdunensis
o Some species or some isolates such as S. haemolyticus
Staphylococcus lugdunensis and S. schleiferi
Staphylococcus schleiferi also can
occasionally mistaken for coagulase
positive staphylococci because of the
presence of clumping factor
TEST STAPHYLOCOCCI MICROCOCCI
o 2 Types of coagulase test: Modified oxidase +
Slide coagulase test used for Anaerobic acid production from
+ *
glucose
the determination of bound
Growth on Furoxone-Tween 80-oil
coagulase or clumping factor red O agar
+
Tube coagulase test to detect Anaerobic acid production from
glycerol in the presence of +
the free coagulase erythromycin
o False positive reactions: Resistance to bacitracin (0.04 units) Resistance Sensitive
Can be done with the use of Lysosome (50-mg disk) Resistance Sensitive
Lysostaphin test Sensitive Resistance
citrate and colonies from high
* Micrococcus kristinae and Micrococcus varians are positive
cell media concentrations Some stains show opposite reaction
Some strains are able to
produce fibrinolysin, which
dissolves the clot after 4 hours
of incubation at 35°C, and it may
appear to be negative if allowed
to incubate longer than 4 hours
Since citrate utilizing organisms
may yield false positive result,
plasma containing
Ethylenediamine tetraacetic
acid (EDTA) rather than citrate
should be used