Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci,...

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Page 1: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Pavithra. G. Palan.Pavithra. G. Palan.

Page 2: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Staphylococci are gram positive cocci,Staphylococci are gram positive cocci, Occur in grape like clusters,Occur in grape like clusters, In Greek; In Greek; staphyle staphyle -- Bunch of grapesBunch of grapes

KokkusKokkus - Berry - Berry

Page 3: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

CLASSIFICATION:CLASSIFICATION:

A) Based on coagulase production:A) Based on coagulase production:

1. Coagulase positive: Eg- S. aureus1. Coagulase positive: Eg- S. aureus

2. Coagulase negative: Eg- S. epidermidis2. Coagulase negative: Eg- S. epidermidis

S. saprophyticusS. saprophyticus

B) Based on pathogenicity:B) Based on pathogenicity:

1. 1. Common pathogen: Eg- S. aureusCommon pathogen: Eg- S. aureus

2. Opportunistic pathogens: Eg- S. epidermidis2. Opportunistic pathogens: Eg- S. epidermidis

S. saprophyticusS. saprophyticus

3. Non pathogen: Eg- S. homonis3. Non pathogen: Eg- S. homonis

Page 4: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

STAPHYLOCOCCUSSTAPHYLOCOCCUS AUREUSAUREUS

MORPHOLOGY:MORPHOLOGY: These are spherical These are spherical

cocci.cocci. Approximately 1Approximately 1μμm in m in

diameter.diameter. Arranged Arranged

characteristically in characteristically in grape like clusters.grape like clusters.

They are non motile They are non motile and non sporing.and non sporing.

A few strains possess A few strains possess capsules.capsules.

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CULTURECULTURE::

Media usedMedia used :- :-

i) Non selective media: Nutrient agar,i) Non selective media: Nutrient agar,

Blood agar, Blood agar,

MacConkey’s agar.MacConkey’s agar.

ii) Selective media: Salt-milk agar, ii) Selective media: Salt-milk agar,

Ludlam’s mediumLudlam’s medium

Page 6: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Cultural Characteristics:Cultural Characteristics:i) On nutrient agar-i) On nutrient agar- The colonies are large, circular, The colonies are large, circular,

convex, smooth, shiny, opaque and easilyconvex, smooth, shiny, opaque and easily

emulsifiable. Most strains produce emulsifiable. Most strains produce golden yellowgolden yellow

pigments.pigments.

Page 7: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

ii) On MacConkey’s agar-ii) On MacConkey’s agar- The colonies are small The colonies are small & pink in colour.& pink in colour.

iii) On blood agar-iii) On blood agar- Most strains produce Most strains produce ββ- - haemolytic colonies.haemolytic colonies.

Page 8: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Biochemical reactions:Biochemical reactions:

1) Catalase test- Positive.1) Catalase test- Positive.

Page 9: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

2) Coagulase test-2) Coagulase test-

i) Slide coagulase test- Positive.i) Slide coagulase test- Positive.

ii) Tube coagulase test- Positive.ii) Tube coagulase test- Positive.

SLIDE COAGULASE TEST TUBE COAGULASE TEST

Page 10: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

3) Reduces nitrate to nitrite.3) Reduces nitrate to nitrite.

4) Ferments mannitol anaerobically with acid only.4) Ferments mannitol anaerobically with acid only.

5) Urea hydrolysis test- Positive.5) Urea hydrolysis test- Positive.

6) Gelatin liquefaction test- Positive.6) Gelatin liquefaction test- Positive.

7) Produces Lipase.7) Produces Lipase.

8) Produces Phosphatase.8) Produces Phosphatase.

9) Produces Thermostable nuclease.9) Produces Thermostable nuclease.

Page 11: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

PATHOGENICITYPATHOGENICITY::

Source of infection:Source of infection:

A) Exogenous: patients or carriersA) Exogenous: patients or carriers

B) Endogenous: From colonized siteB) Endogenous: From colonized site

Mode of transmission:Mode of transmission:

A) Contact: direct or indirect( through fomites)A) Contact: direct or indirect( through fomites)

B) Inhalation of air borne droplets B) Inhalation of air borne droplets

Page 12: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Virulence factorsVirulence factors::

These includeThese include

A) Cell associated factorsA) Cell associated factors

B) Extracellular factorsB) Extracellular factors

Page 13: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

A) A) CELL ASSOCIATED FACTORSCELL ASSOCIATED FACTORS::

a) Cell associated polymersa) Cell associated polymersb) Cell surface proteinsb) Cell surface proteins

a) CELL ASSOCIATED POLYMERSa) CELL ASSOCIATED POLYMERS 1. Cell wall polysaccharide1. Cell wall polysaccharide 2. Teichoic acid2. Teichoic acid 3. Capsular polysaccharide3. Capsular polysaccharide

b) CELL SURFACE PROTEINS:b) CELL SURFACE PROTEINS:

1. Protein A1. Protein A

2. Clumping factor (bound coagulase)2. Clumping factor (bound coagulase)

Page 14: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Structure of Staphylococcal cell wall

Page 15: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

B) B) EXTRACELLULAR FACTORSEXTRACELLULAR FACTORS

a) Enzymesa) Enzymes

b) Toxinsb) Toxins

Page 16: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

a) a) EnzymesEnzymes::

1. Free coagulase1. Free coagulase

2. Catalase2. Catalase

3. Lipase3. Lipase

4. Hyaluronidase4. Hyaluronidase

5. DNAase5. DNAase

6. Thermonuclease6. Thermonuclease

7. Staphylokinase (Fibrinolysin)7. Staphylokinase (Fibrinolysin)

8. Phosphatase8. Phosphatase

Page 17: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

b) b) ToxinsToxins: : 1. Cytolytic toxins1. Cytolytic toxins i) Haemolysinsi) Haemolysins Alpha haemolysinAlpha haemolysin Beta haemolysin Beta haemolysin Gamma haemolysinGamma haemolysin Delta haemolysinDelta haemolysin

ii) Leucocidin (Panton-Valentine toxin) ii) Leucocidin (Panton-Valentine toxin)

2. Enterotoxin2. Enterotoxin

3.3. Toxic shock syndrome toxin (TSST)Toxic shock syndrome toxin (TSST)

44. . . . Exfoliative (epidermolytic toxin)Exfoliative (epidermolytic toxin)

Page 18: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Disease:Disease:

Diseases produced by Staphylococcus aureusDiseases produced by Staphylococcus aureus

is studied under 2 groups:is studied under 2 groups:

A) InfectionsA) Infections

B) IntoxicationsB) Intoxications

Page 19: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

A) A) INFECTIONS:INFECTIONS:

Mechanism of pathogenesisMechanism of pathogenesis::

Cocci gain access to damaged skin, mucosal orCocci gain access to damaged skin, mucosal or

tissue sitetissue site

Colonize by adhering to cells or extracellular matrix Colonize by adhering to cells or extracellular matrix

Evade the host defense mechanisms and multiply Evade the host defense mechanisms and multiply

Cause tissue damageCause tissue damage

Page 20: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Common Staphylococcal infections areCommon Staphylococcal infections are::

1) 1) Skin and soft tissueSkin and soft tissue: Folliculitis, furuncle (boil), : Folliculitis, furuncle (boil), carbuncle, styes, abscess, wound infections, carbuncle, styes, abscess, wound infections, impetigo, paronychia and less often cellulitis.impetigo, paronychia and less often cellulitis.

Folliculitis

Page 21: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Furuncle (boil)

Carbuncle

Page 22: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Styes Abscess

Page 23: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Impetigo Paronychia

CellulitisWound infection

Page 24: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

2) 2) MusculoskeletalMusculoskeletal: Osteomyelitis, arthritis, bursitis, : Osteomyelitis, arthritis, bursitis, pyomyositis.pyomyositis.

osteomyelitisosteomyelitis

3) 3) RespiratoryRespiratory: Tonsillitis, pharyngitis, sinusitis, : Tonsillitis, pharyngitis, sinusitis, otitis, bronchopneumonia, lung abscess, otitis, bronchopneumonia, lung abscess, empyema, rarely pneumonia.empyema, rarely pneumonia.

Page 25: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

4) 4) Central nervous systemCentral nervous system: Abscess, meningitis, intracranial : Abscess, meningitis, intracranial thrombophlebitis.thrombophlebitis.

55) Endovascular) Endovascular: Bacteremia, septicemia, pyemia, : Bacteremia, septicemia, pyemia, endocarditis.endocarditis.

EndocarditisEndocarditis

6) 6) UrinaryUrinary: Urinary tract infection: Urinary tract infection. .

Page 26: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

B) B) INTOXICATIOINS:INTOXICATIOINS:

The disease is caused by the bacterial exotoxins,The disease is caused by the bacterial exotoxins,

which are produced either in the infected hostwhich are produced either in the infected host

or preformed in vitro.or preformed in vitro.

There are 3 types-There are 3 types-

1.1. Food poisoningFood poisoning

2.2. Toxic shock syndromeToxic shock syndrome

3.3. Staphylococcal scalded skin syndromeStaphylococcal scalded skin syndrome

Page 27: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

1) 1) Food poisoning:Food poisoning:

Enterotoxin is responsible for manifestations of Enterotoxin is responsible for manifestations of staphylococcal food poisoning.staphylococcal food poisoning.

Eight types of enterotoxin are currently known, named Eight types of enterotoxin are currently known, named A, B, C1-3, D, E, and H.A, B, C1-3, D, E, and H.

It usually occurs when preformed toxin is ingested with It usually occurs when preformed toxin is ingested with contaminated food.contaminated food.

The toxin acts directly on the autonomic nervous The toxin acts directly on the autonomic nervous system to cause the illness, rather than gut mucosa.system to cause the illness, rather than gut mucosa.

Page 28: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

The common food items responsible are - milk The common food items responsible are - milk and milk products, meat, fish and ice cream.and milk products, meat, fish and ice cream.

Source of infection- food handler who is a carrier.Source of infection- food handler who is a carrier.

Incubation period- 2 to 6 hours.Incubation period- 2 to 6 hours.

Clinical symptoms- nausea, vomiting and Clinical symptoms- nausea, vomiting and diarrhoea.diarrhoea.

The illness is usually self limited, with recovery in The illness is usually self limited, with recovery in a day or so.a day or so.

Page 29: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

2) 2) Staphylococcal Toxic shock syndrome (STSS):Staphylococcal Toxic shock syndrome (STSS):

STSS is associated with infection of mucosal or STSS is associated with infection of mucosal or sequestered sites by TSST( formerly known as sequestered sites by TSST( formerly known as enterotoxin type F) producing S.aureus.enterotoxin type F) producing S.aureus.

It is fatal multisystem disease presenting with It is fatal multisystem disease presenting with fever, hypotension, myalgia, vomiting, diarrhoea, fever, hypotension, myalgia, vomiting, diarrhoea, mucosal hyperemia and erythematous rash which mucosal hyperemia and erythematous rash which desquamates subsequently.desquamates subsequently.

Page 30: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,
Page 31: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

2 types of STSS known2 types of STSS known::

i) Menstrual associated STSS: Here colonization of i) Menstrual associated STSS: Here colonization of S.aureus occurs in the vagina of menstruating S.aureus occurs in the vagina of menstruating woman who uses highly absorbent vaginal woman who uses highly absorbent vaginal tampons. tampons.

ii) Non menstrual associated STSS: Here ii) Non menstrual associated STSS: Here colonization of S.aureus occurs in other sites like colonization of S.aureus occurs in other sites like surgical wound.surgical wound.

Page 32: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

3) 3) Staphylococcal scalded skin syndrome Staphylococcal scalded skin syndrome (SSSS):(SSSS):

Exfoliative toxin produced by S.aureus is Exfoliative toxin produced by S.aureus is responsible for this.responsible for this.

It is a skin disease in which outer layer of It is a skin disease in which outer layer of epidermis gets separated from the underlying epidermis gets separated from the underlying tissues. tissues.

Page 33: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Types of SSSSTypes of SSSS::

Severe formSevere form Milder formMilder form

In new born - Ritter’s disease - Pemphigus In new born - Ritter’s disease - Pemphigus

neonatorumneonatorum

In older patients - Toxic epidermal - Bullous In older patients - Toxic epidermal - Bullous

necrolysis impetigonecrolysis impetigo

Page 34: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Toxic epidermal necrolysis

Ritter’s disease

Bullous impetigo

Pemphigus neonatorum

Page 35: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

LAB DIAGNOSIS:LAB DIAGNOSIS:

Specimens collectedSpecimens collected:: Depends on the type of infection. Depends on the type of infection.

Suppurative lesion- Pus,Suppurative lesion- Pus,

Respiratory infection- Sputum,Respiratory infection- Sputum,

Bacteremia & septicemia- Blood,Bacteremia & septicemia- Blood,

Food poisoning- Feces, vomit & the remains of suspected Food poisoning- Feces, vomit & the remains of suspected food,food,

For the detection of carriers- Nasal swab.For the detection of carriers- Nasal swab.

Page 36: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

I) I) Direct microscopyDirect microscopy: : Direct microscopy Direct microscopy

with Gram stained with Gram stained smear is useful in case smear is useful in case of pus, where cocci in of pus, where cocci in clusters are seen.clusters are seen.

This is of no value for This is of no value for

specimen like sputum specimen like sputum where mixed flora are where mixed flora are normally present.normally present.

Methods of examination:

Page 37: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

c) Gram staining: c) Gram staining: Smears are examined Smears are examined from the culture plate from the culture plate and reveals Gram and reveals Gram positive cocci(1positive cocci(1μμm in m in diameter) arranged in diameter) arranged in grape like clusters.grape like clusters.

II) II) CultureCulture::

a) Media used:a) Media used:

b) Cultural Characteristics:b) Cultural Characteristics:

Page 38: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

d) Biochemical reactions:d) Biochemical reactions:

III) Antibiotic sensitivity tests done as a guide to III) Antibiotic sensitivity tests done as a guide to treatment.treatment.

IV) Bacteriophage typing is done for IV) Bacteriophage typing is done for epidemiological purposes. epidemiological purposes.

V) Serological tests are not useful.V) Serological tests are not useful.

Page 39: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

TREATMENT:TREATMENT: Drug resistance is common.Drug resistance is common. Benzyl penicillin is the most effective antibiotic, Benzyl penicillin is the most effective antibiotic,

if the strain is sensitive.if the strain is sensitive. Cloxacillin or Methicillin is used against Cloxacillin or Methicillin is used against

beta-lactamase producing strains.beta-lactamase producing strains. MMethicillin ethicillin RResistant esistant SStaphylococcus taphylococcus aaureus ureus

((MRSAMRSA) strains have become common.) strains have become common. Vancomycin is used in treatment of infections Vancomycin is used in treatment of infections

with MRSA strains.with MRSA strains.

Page 40: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

EPIDEMIOLOGY:EPIDEMIOLOGY:

Staphylococci are primary parasites of human Staphylococci are primary parasites of human beings and animals.beings and animals.

Hospital infections caused by staphylococci Hospital infections caused by staphylococci deserve special attention because of their deserve special attention because of their frequency & they are caused by strains resistant to frequency & they are caused by strains resistant to various antibiotics.various antibiotics.

Staphylococci are the common cause of Staphylococci are the common cause of postoperative wound infection and other hospital postoperative wound infection and other hospital cross infections.cross infections.

Page 41: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

PREVENTION:PREVENTION:

Isolation & treatment of MRSA patients.Isolation & treatment of MRSA patients.

Detection of carriers among hospital staff, their Detection of carriers among hospital staff, their isolation & treatment.isolation & treatment.

Avoid indiscriminate usage of antibiotics.Avoid indiscriminate usage of antibiotics.

Page 42: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

CoCoagulase agulase NNegative egative SStaphylococci( taphylococci( CoNSCoNS ): ):

Two species of coagulase negativeTwo species of coagulase negative

Staphylococci can cause human infections-Staphylococci can cause human infections-

1. Staphylococcus epidermidis1. Staphylococcus epidermidis

2.2. Staphylococcus saprophyticus Staphylococcus saprophyticus

Page 43: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

S. EpidermidisS. Epidermidis::

It is a common cause of stitch abscesses.It is a common cause of stitch abscesses.

It has predilection for growth on implanted foreign bodies It has predilection for growth on implanted foreign bodies such as artificial valves, shunts, intravascular catheters such as artificial valves, shunts, intravascular catheters and prosthetic appliances leading to bacteraemia.and prosthetic appliances leading to bacteraemia.

In persons with structural abnormalities of urinary tract, it In persons with structural abnormalities of urinary tract, it can cause cystitis.can cause cystitis.

Endocarditis may be caused, particularly in drug addicts.Endocarditis may be caused, particularly in drug addicts.

Page 44: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

S.saprophyticusS.saprophyticus::

It causes urinary tract infections, mostly in It causes urinary tract infections, mostly in sexually active young women.sexually active young women.

The infection is symptomatic and may involve the The infection is symptomatic and may involve the upper urinary tract also.upper urinary tract also.

Men are infected much less often.Men are infected much less often.

It is one of the few frequently isolated CoNS that It is one of the few frequently isolated CoNS that is is resistantresistant to to NovobiocinNovobiocin..

Page 45: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Distinguishing features of the major species ofDistinguishing features of the major species of

staphylococcusstaphylococcus

CharactersCharacters S.aureusS.aureus S.epidermididisS.epidermididis S.saprophyticusS.saprophyticus

CoagulaseCoagulase ++ -- --

Novobiocin Novobiocin sensitivitysensitivity

SensitiveSensitive SensitiveSensitive ResistantResistant

Acid from Acid from mannitol mannitol fermentation fermentation anaerobicallyanaerobically

++ -- --

PhosphatasePhosphatase ++ ++ --

Page 46: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

Novobiocin sensitivity test

Page 47: Pavithra. G. Palan.. Staphylococci are gram positive cocci, Staphylococci are gram positive cocci, Occur in grape like clusters, Occur in grape like clusters,

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