Staphylococcus.pptx

STAPHYLOCOCCUS
5/2/2021 Dr. Smriti Bajpai Department of Microbiology, BVC Hyderabad. 1

Introduction
Recklinghausen
(1871): first
observed in human
pyogenic
infections.
Pasteur (1880):
obtained liquid
cultures of cocci
from pus and
produced
abscesses by
inoculating them
into rabbits
Sir Alexander
Ogston: Given
name
Staphylococcus
due to typical
occurrence of cocci
in grape like
clusters in pus and
in cultures
Rosenbach (1884):
Given names of
strains S. aureus to
golden yellow
colonies and S.
albus to white
colonies on solid
media from
pyogenic
infections.
Passet (1885):
Described S.
citreus to lemon
yellow colonies.

 Family Micrococcaceae. The genus
Staphylococcus consists of 32 species
 Ubiquitous
 Gram-positive cocci
 Arranged in tetrads or grape like clusters.
 Staphylococcus is the only genus of medical
importance.
 Most common cause of localized suppurative
lesions like folliculitis to deep-seated abscess
and various pyogenic infections like
endocarditis, osteomyelitis, etc.
 Their ability to develop resistance to
Penicillin and other antibiotics enhance their
importance as human pathogen (especially in
hospital environment).

MORPHOLOGICAL PROPERTIES
 Gram-positive cocci, measuring around 1 micro
mt in diameter.
 Arranged in irregular grape-like clusters. This
appearance is due to incomplete separation of
daughter cells during successive divisions of
bacteria, which takes place in perpendicular
planes.
 The grape-like clustering is seen when the
bacteria are grown in solid media, but usually
short chains are seen when grown in liquid
media.
 Nonmotile, non sporing.
 Capsulated (microcapsule in young culture)

CLASSIFICATION
A) Based on coagulase production:
1. Coagulase positive: Eg- S. aureus
2. Coagulase negative: Eg- S. epidermidis
S. saprophyticus
B) Based on pathogenicity:
1. Common pathogen: Eg- S. aureus
2. Opportunistic pathogens: Eg- S. epidermidis
S. saprophyticus
3. Non pathogen: Eg- S. homonis

CULTURAL CHARACTERISTICS
Temperature range of 10–42°C (optimum temperature 37°C) pH range of 7.4–7.6 (optimum pH 7).
1. Nutrient agar:
 Large
 Round (measuring 2–4 mm in
diameter)
 Convex
 Well- defined colonies, smooth
shining/glistening surface
 Golden-yellow colonies due to
production of a non diffusible
golden-yellow pigment
(lipoprotein allied to carotene,
enhanced by incubation at
22°C in the presence of
oxygen, Milk agar and 1%
glycerol monoacetate agar)
 On nutrient agar slopes, the
growth gives a characteristic
“oil paint” appearance.
2. Blood agar:
 a clear zone of hemolysis
(beta-hemolysis) surrounding
the colonies
 Hemolysis is well marked on
sheep or rabbit blood agar,
especially when incubated in
an atmosphere of 20–25% CO
2. Sheep blood agar is used
for primary isolation.
 Hemolysis is weak on horse
blood agar. Human blood is
not used, as it may contain
antibiotics or other inhibitors.
 Other species of
Staphylococcus do not
produce hemolysis.

3. MacConkey agar:
 Small pink colonies due to fermentation of
lactose.
4. Selective media:
a. Mannitol salt agar (1% mannitol, 7.5%
sodium chloride, and 0.0025% phenol red
indicator, ferment mannitol with acid
production, which gives rise to yellow zone
formation around the colonies)
b. Milk agar
c. Glycerol monoacetate agar
4. Culture in liquid media:
 Produces turbidity in liquid media
 No production of pigment.

BIOCHEMICAL CHARACTERISTICS
Catalase positive. It produces enzyme catalase (which degrades H 2 O 2 into nascent oxygen and water).
Coagulase positive (test to differentiate S. aureus from S. epidermidis and other CONS)
Oxidase negative
Phosphatase positive
Ferments mannitol, sucrose, maltose aerobic conditions, acid but no gas
Liquefies gelatin
Hydrolyzes urea
Reduces nitrate to nitrite
Voges-Proskauer (VP)
Methyl red (MR)” positive
Indole negative.

RESISTANCE Types
 More resistant.
 Remain viable on dried threads for 3-6 months.
 Withstand moist heat at 60°C for 30 minutes
 Grow in 10% NaCl.
 Killed rapidly by disinfectants, such as phenol,
chlorhexidine, and hexachlorophene. Resist phenol
for 15 minutes.
 1% Mercuric Chloride solution kills in 10 minutes.
 The cocci are very sensitive to aniline dyes, such
as crystal violet. The dye at a concentration of
1:500,000 and dye Brilliant green at1 in 10,000,000
inhibits the growth of the cocci.
 Uniformly resistant to lysozyme.
 Uniformly sensitive to Penicillin in pre-antibiotic era.
Soon after penicillin resistant strains began to
emerge, first in hospital then in the community at
large.

STAPHYLOCOCCAL PATHOGENICITY AND VIRULENCE
Types of disease
1. Infections
1.Cocci gain access to damaged skin, mucosal
or tissue sites
2.Colonize
3.Multiply
4.Cause tissue damage
2. Intoxication
Bacterial toxins produced in host or
preformed in vitro.
1. Cell associated polymers
2. Cell surface proteins
3. Extracellular
enzymes
4.Toxins
Source of infection:
A) Exogenous: patients or carriers
B) Endogenous: From colonized site
Mode of transmission:
A) Contact: direct or indirect( through fomites)
B) Inhalation of air borne droplets

1. Cell associated polymers
Cell wall polysaccharide
peptidoglycan
• provides rigidity and
structural integrity to the
bacterial cell.
• It has the characteristic
pentaglycine bridges that
link tetrapeptides to the
muramic acid residues.
• It activates complement
and induces release of
inflammatory cytokines.
Teichoic acid
• Antigenic component of
the cell wall. It is a polymer
of ribitol phosphate.
• Facilitates adhesion of the
cocci to the host cell
surface and protects them
from complement
mediated opsonization.
• Antibodies to teichoic acids
develop in endocarditis and
in certain other
staphylococcal infections.
The Capsular polysaccharide
• Capsulated strains are
more virulent than the
noncapsulated ones.
• Surrounds the cell wall and
protects the bacteria from
phagocytosis.
• The capsule also facilitates
adherence of the cocci to
host cells and to prosthetic
implants.

2. Cell surface proteins
Protein A
• Major protein in the cell wall, molecular weight of 13,000 Da.
• A group specific antigen, present in more than 90% strains of S.
aureus.
• Absent in both the coagulase-negative staphylococci (CONS)
and micrococci.
• Chemotactic, anticomplementary, and antiphagocytic.
• Induces platelet damage and hypersensitivity.
• Binds with Fc terminal of the immunoglobulin G (IgG), leaving
the Fab portions free to combine with its specific antigen.
• Co-agglutination
Clumping factor
• Surface antigen
• Bound coagulase responsible for coagulase test.

3. Extracellular enzymes
a. Coagulase:
•Brings about Clotting of plasma.
•The enzyme coagulase is of two types:
• (a) free coagulase (heat-labile and filterable enzyme. It has eight antigenic types (A, B, C, D, E, F, G, and H). Antigenic type A is produced by most human S. aureus
strains. The enzyme coagulase in association with coagulase- reacting factor (CRF) present in plasma converts fibrinogen to fibrin. This fibrin coats the bacterial cells,
rendering them resistant to opsonization and phagocytosis and hence making bacteria more virulent)and
• (b) bound coagulase(known as clumping factor. It is a heat-stable protein and is present in the cell wall. This enzyme brings about clumping of the staphylococci when
mixed with plasma by directly acting on fibrinogen. Lysis of the cell releases the enzyme. Unlike free coagulase, clumping factor does not need CRF for its action).
b. Catalase
•The enzyme catalase reduces H2O2 to nascent oxygen and water. This nascent oxygen causes oxidative damage of host tissue.
•This enzyme is produced after phagocytosis or during metabolism of the bacteria.
• All strains of staphylococci produce catalase unlike streptococci.
c. Hyaluronidase
•Hydrolyzes the acidic mucopolysaccharides present in the matrix of the connective issues, thereby facilitating the initiation and spread of bacteria in
tissues (infection).
d. Penicillinase and
other enzymes
•More than 90% of S. aureus produce enzyme penicillinase.
•The enzyme inactivates penicillin group of antibiotics, hence is responsible for widespread occurrence of penicillin-resistant staphylococci.
•The gene for this enzyme is acquired through plasmids.
•Other enzymes include phosphatase, deoxyribonucleases, nucleases, proteases, phospholipase, and lipases.

4. Toxins
1. Toxic shock syndrome toxin
 A protein with a molecular weight of 22,000 Da
 resembles enterotoxin F and exotoxin C.
 Production of toxin is pH dependent and occurs at pH 7–8.
 The toxin causes toxic shock syndrome (TSS) that is a potentially fatal multisystem disease presenting with fever, hypotension, myalgia, vomiting, mucosal
hyperemia and an erythematous rash which desquamates subsequently.
 TSST type 1 is most often responsible.
 .TSST is a superantigen and hence a potent stimulant of T lymphocytes, resulting in release of large amount of interleukins (IL-1 and IL-2) and tumor necrosis
factor, ultimately manifesting in TSS.
2. Enterotoxin
 A heat-stable protein can resist 100 C for 10 to 40 minutes
 Molecular weights ranging from 26,000 to 30,000 Da
 Gut-enzyme resistant
 Produced by nearly one-third of all the strains of S. aureus, and these strains belong to bacteriophage group III .
 Nine antigenic types (A, B, C 1,2,&3, D, E, G, H, I, and J) of enterotoxins formed by toxigenic strains, singly or in combination.
 Staphylococcal food poisoning; nausea, vomiting and diarrhea 2-6 hours after consuming food contaminated buy preformed toxin. by producing large amounts of
interleukins.

3. Exfoliative toxin:
 Also known as Exofoliatin
 Responsible for staphylococcal scalded skin syndrome (SSSS), an exfoliative skin disease in which the outer layer of epidermis gets
separated from the underlying tissues. Severe form of SSSS is known as Ritter’s disease in newborn and mixed epidermal necrolysis in
older patients. Milder forms are pemphigus neo notarum and bullous impetigo.
 Exfoliative toxin is of two types:
(a) toxin A (molecular weight of 30,000 Da) and (b) toxin B (molecular weight of 29,500 Da).
 Toxin A is heat stable, while toxin B is heat labile.
 Antigenic, and specific antibodies against the toxin are protective.
 The strains producing this toxin belong to bacteriophage group II.
The toxin breaks intercellular bridges in the stratum granulosum of epidermis and causes its separation from the underlying tissue,
resulting in a blistering and exfoliating disease of the skin.
4. Leukocidins
 Leukocidins include (a) alpha-lysin, (b) Panton-Valentine leukocidin (PV–leukocidin), and (c) leukolysin.
 The alpha-lysin is the most important leukocidin.
 Causes marked necrosis of the skin and hemolysis by damaging the cell membrane, leading to release of low-molecular-weight
substances from the damaged cells.
 PV–leukocidins are six in number, each consisting of two components. The molecular weight is around 32 kDa. These toxins cause
death of human leukocytes and macrophages without causing any lysis.
 Leukolysin is thermostable and causes lysis of leukocytes and necrosis of tissues in vivo.

5. Cytolytic toxins
 Membrane active substances
 Consist of 4 Hemolysins alpha, beta, gamma and delta hemolysins and 1 leucocidin
A. Alpha-hemolysin
 Lysin, protein with a molecular weight of 33 k Da
 Lethal effects on a wide variety of cell types
 Lyse RBC of rabbit but less active against sheep and human RBCs.
 Leucocidin, cytotoxic, neurotoxic, toxic to macrophages, lysosomes, muscle tissues, circulatory system.
B. Beta-hemolysin
 Sphingomyelinase
 Hemolytic for sheep cells not for human or rabbit cells
 Protein with a molecular weight of 35 k Da.
 Shows hot–cold phenomenon i.e. hemolysis initiated at 37 C, but becoming evident only after chilling.
C. Delta-hemolysin
 Protein molecular weight of 8 k Da.
 Acts primarily as a surfactant on cell membranes of erythrocytes, leucocytes, macrophages and platelets.
D. Gamma-hemolysin
• Consists of three proteins.
• The three delta-hemolysin proteins interact with one of the two PV–leukocidin proteins.

Pathogenesis of Staphylococcal Infections
Cause most common bacterial infection
Range from the trivial to the fatal
Cause characteristically localized pyogenic lesions
Adhere to the
damaged
skin, mucosa,
or tissue
surfaces
Evade
defense
mechanisms
of the host
Colonize
Multiplying in
tissues
Liberating
toxins &
cause
inflammation

Clinical features
•skin infections include impetigo, folliculitis, furuncles, carbuncles, paronychia, surgical
wound infection, blepharitis, and postpartum breast infection.
•acute bacterial endocarditis, of normal or prosthetic heart valves, which is associated
with high mortality.
•Pneumonia in postoperative patients following viral respiratory infection, leading to
empyema; it also leads to chronic sinusitisosteomyelitis in children
•The bacteria reach bone through blood stream or by direct implantation following
trauma.
•deep-seated abscesses in any organ after bacteremia.
Inflammatory
diseases
• staphylococcal food poisoning,
• (b) staphylococcal toxic shock syndrome, and
• (c) staphylococcal scalded skin syndrome.
Toxin-mediated
Staphylococcal
diseases

Inflammatory Staphylococcal diseases
Skin and soft tissue:
Folliculitis, furuncle
(boils), abscess, wound
infection, carbuncle,
impetigo
Musculoskeletal:
Osteomyelitis, arthritis,
bursitis, pyomyositis
Respiratory:Tonsilitis,
pharyngitis, sinusitis,
otitis,
bronchopneumonia, lung
abscess, empyema
Central Nervous System:
Abscess, meningitis,
intracranial
thrombophlebitis
Endovascular:
Bacteremia, septicemia,
pyemia, endocarditis
Urinary

Toxin-mediated Staphylococcal diseases
(a)Staphylococcal food
poisoning
• caused by enterotoxin.
• The enterotoxin is a preformed toxin
already present in the contaminated
food before consumption..
• The toxin acts by stimulating the
release of large amounts of
interleukins IL-1 and IL-
• heat resistant and so is not
inactivated by brief cooking.
• onset of symptoms is sudden,
appearing within 2–6 hours of
ingestion of food, self limiting
condition characterized by nausea,
vomiting, abdominal cramps, and
watery, nonbloody diarrhea
Staphylococcal toxic
shock syndrome
• Caused byTSST.
• The toxin is a superantigen, which
causes STSS by stimulating the
release of large amounts of
interleukins IL-1 and IL-2 in the body
• An acute and potentially life-
threatening condition is a multisystem
disease characterized by fever,
hypotension myalgia, vomiting,
diarrhea, mucosal hyperemia, and an
ery thematous rash followed by
desquamation of the skin, particu-
larly on palms and soles
Staphylococcal scalded skin
syndrome
• Staphylococcal scalded skin
syndrome:
• Caused by the exfoliative
toxin,exfoliatin.
• Commonly in infants and children.
• Associated with extensive exfoliation
of the skin, in which outer layer of the
epidermis is separated from the
underlying tissue and is characterized
by the appearance of extensive bullae.
These bullae when ruptured may leave
behind scalded, red, tender skin.The
lesion typically starts periorificially or
inskin folds. It usually resolves within
10 days’ time.
Complications of Staphylococcal Diseases
Complications of staphylococcal diseases include bacterial pneumonia, septicemia, arthritis, meningitis, etc. These complications are frequently
seen in persons with extreme of age, debilitated persons, and immunosuppressed hosts.
Host immunity
S. aureus infection does not cause any life-long immunity. It causes repeated infections in a susceptible host.

Epidemiology
Geographical
distribution
• Throughout the world
• One-third of the adult population is
asymptomatic carrier of staphylococci,
hospital infections worldwide
Habitat
• Primary parasites of humans and
animals, commensals on skin, in the
glands of the skin, and on mucous
membranes in the intertriginous skin
folds, perineum, axillae, and vagina
• Approximately, 35–50% of normal
adults carry S. aureus in the anterior
nares, 10% in the perineum, and 5–
10% in the vagina.
Reservoir, source, and
transmission of infection
Human cases and carriers are the
important reservoir of infection

Laboratory Diagnosis
 Specimens
 Specimens to be collected for
demonstration of staphylococci
depend on the nature of lesion.
 Microscopy
 Culture
 Identification of Bacteria:
Biochemical tests
 Antibiotic sensitivity Test
 Bacteriophage Typing

Microscopy
Gram-positive cocci arranged in
clusters and pus cells in the Gram-
stained smears of pus, wound
exudate
No value for sputum where mixed
bacterial flora is present.
Culture
On nutrient agar, large,
circular, smooth, convex, and
glistening colonies showing
golden- yellow pigments.
 On blood agar, the colonies
show a zone of beta-
hemolysis.
 Specimens from heavily
contaminated sources, such
as vomitus and feces on
selective media (e.g., mannitol
salt agar or salt milk agar).
These media inhibit growth of
Gram-negative bacteria but
allow the growth of
staphylococci and certain
other Gram-positive cocci.

BIOCHEMICAL CHARACTERISTICS
Catalase positive. It produces enzyme catalase (which degrades H 2 O 2 into nascent oxygen and water).
Coagulase positive (test to differentiate S. aureus from S. epidermidis and other CONS)
Oxidase negative
Phosphatase positive
Ferments mannitol, sucrose, maltose aerobic conditions, acid but no gas (mannitol characteristic feature for identification)
Liquefies gelatin
Hydrolyzes urea
Reduces nitrate to nitrite
Voges-Proskauer (VP)
Methyl red (MR)” positive
Indole negative.

Identification of bacteria
Coagulase Test
Tube CoagulaseTest
 Detect free coagulase.
 0.1 ml young overnight broth
added to 0.5 ml 1:10 dilution of
human plasma
 Incubated in water bath at 37 C
for 3-6 hrs.
Slide CoagulaseTest
 Detect bound coagulase or
clumping factor.
 Test isolate emulsified in a drop
of saline on slide.
 1 drop plasma added to emulsion
Result
Positive
(Clot formation)
Negative
(No Clot)
Result
Positive
(Clumping)
Negative
(Clumping)

Antibiotic
sensitivity test
 This test is performed as a
guide to treatment.
 Penicillin, oxacillin,
cephalothin, gentamicin,
erythromycin, clindamycin
and ampicillin-sulbactam.
 No resistance was detected
for oxacillin, cephalothin
and ampicillin-sulbactam.
5/2/2021
Dr. Smriti Bajpai Department of Microbiology, BVC Hyderabad.
26

Bacteriophage typing
 Useful in epidemiological studies
 Based on the susceptibility of cocci to bacteriophages.
 Carried out by pattern method, where a set of 23 standard
typing phages of S. aureus is used to type staphylococcal
isolates and distinguish them from one another by their
patterns of susceptibility to lysis.
 In this method, th strain of S. aureus to be typed is
inoculated on a nutrient agar plate to produce a lawn culture.
After drying the plate, various phages at their routine test
dilution (RTD) are applied over marked squares on plate.
Such plates are then incubated overnight at 30°C and
observed for the presence or absence of lysis of the colonies
by the phages.
 The phage type of a strain is known by designation of the
phages that lyse it.
 Thus, if a strain is lysed by phages 83A, 84,and 85, it is
called phage type 83A/84/85.
 By this method, most of the strains of staphylococci can be
classified and are divided into five lytic groups, while there
are a few which cannot be classified and constitute the
unclassified group.
 The national reference centre for staphylococcal phage
typing in India is located in the Department of Microbiology,
Maulana Azad Medical College, New Delhi.

Treatment
Skin and soft tissue infections are treated best with local wound care with or without topical antibiotics (e.g., neomycin).
Spontaneous or surgical drainage of pus and debridement of necrotic tissue is an effective mode for treatment of staphylococcal abscess.
Systemic antibiotics are necessary for deep-seated and systemic infections. Antibiotics should be chosen based on drog sensitivity test.
Benzyl penicillin most effective. For life threatening staphylococcal infections vancomycin.
Treatment of Carriers
Treatment of carriers by local application of mupirocin (antibiotic) and chlorhexidine (antiseptic).
In resistant cases rifampicin along with other oral antibiotic effective in long term suppression or elimination of the carrier state.

PREVENTION:
• Isolation & treatment of MRSA patients.
• Detection of carriers among hospital staff, their isolation & treatment.
• Avoid indiscriminate usage of antibiotics

Coagulase Negative Staphylococci( CoNS )
Two species of coagulase negative Staphylococci can cause human infections-
1. Staphylococcus epidermidis
 It is a common cause of stitch abscesses.
 It has predilection for growth on implanted foreign
bodies such as artificial valves, shunts,
intravascular catheters and prosthetic appliances
leading to bacteraemia.
 In persons with structural abnormalities of urinary
tract, it can cause cystitis.
 Endocarditis may be caused, particularly in drug
addicts.
2. Staphylococcus saprophyticus
 It causes urinary tract infections, mostly in sexually
active young women.
 The infection is symptomatic and may involve the
upper urinary tract also.
 Men are infected much less often.
 It is one of the few frequently isolated CoNS that is
resistant to Novobiocin.

CHARACTERS S. aureus S. epidermididis S. saprophyticus
COAGULASE + _ _
NOVOBIOCIN SENSITIVITY Sensitive Sensitive Resistant
ACID PRODUCTION FROM
MANNITOL FERMENTATION
ANAEROBICALLY
+ _ _
PHOSPHATASE + + _

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