G +ve cocciG +ve cocci

StaphylococciStaphylococci

ClassificationClassification

G+ve Bacteria

Cocci (Spherical) Bacilli (Rod Shape)

Catalase +ve Catalase -ve

Aerobic

Anaerobic

Streptococci

Peptococcus Peptostreptococcus

Oxidase +ve Oxidase -ve

StaphylococciMicrococci

General properties of G +ve General properties of G +ve cocci:cocci:

G +ve Spherical cocci with a 0.5-1.5 u G +ve Spherical cocci with a 0.5-1.5 u in diameter.in diameter.

Most are Facultative anaerobes and Most are Facultative anaerobes and some are aerobic and anaerobic.some are aerobic and anaerobic.

Differ in distribution forming Diplococci Differ in distribution forming Diplococci (Pneumococci), Cluster (Staphylococci), (Pneumococci), Cluster (Staphylococci), Chain (Streptococci), and Group of four Chain (Streptococci), and Group of four (Micrococci).(Micrococci).

Non spore forming and Non motile Non spore forming and Non motile some are capsulated. some are capsulated.

Staphylococci:Staphylococci: Definition:Definition:

G+ve cocci in Grape like cluster, Non spore G+ve cocci in Grape like cluster, Non spore forming, non motile, facultative anaerobes, forming, non motile, facultative anaerobes, catalase +ve. Halophilic.catalase +ve. Halophilic.

Species:Species: Staphylococcus aureus or Staph Pyogenes----Staphylococcus aureus or Staph Pyogenes----

Golden colonies (Most pathogenic species).Golden colonies (Most pathogenic species). Staphylococcus Albus or Staph epidermidis---- Staphylococcus Albus or Staph epidermidis----

White or creamy white colonies.White or creamy white colonies. Staphylococcus citreus or Staph saprophyticus---- Staphylococcus citreus or Staph saprophyticus----

Yellow lemon colonies.Yellow lemon colonies. Normal Habitat:Normal Habitat:

Skin.Skin. - Female genital tract.- Female genital tract. Gastrointestinal tract.Gastrointestinal tract. - Upper respiratory - Upper respiratory

tract.tract. In the Nose Staphylococcus aureus found in In the Nose Staphylococcus aureus found in

50% of carrier patient.50% of carrier patient.

Virulence Factor, Protein, Enzymes and Virulence Factor, Protein, Enzymes and Toxins:Toxins:

Enzymes Produced by S. aureus:Enzymes Produced by S. aureus:CollagenaseCollagenase Break collagen.Break collagen.PhosphatasePhosphatase Break Phosphate.Break Phosphate.LipaseLipase Break Lipids.Break Lipids.HyaluronidaseHyaluronidase Break down Hyaluronic acid Break down Hyaluronic acid

(the cement (the cement between the cells)between the cells)

CatalaseCatalase Break down 3% HBreak down 3% H22OO22..CoagulaseCoagulase Convert fibrinogen into Convert fibrinogen into

fibrin fibrin clot.clot.StaphylokinaseStaphylokinaseBreak down fibrin clotBreak down fibrin clotDNaseDNase Break down DNABreak down DNAββ lactamase lactamase Destroy PenicillinDestroy Penicillin

Proteins:Proteins: Protein AProtein A Bind with FC portion of Antibodies Bind with FC portion of Antibodies

lead to prevention of lead to prevention of Opsonization.Opsonization.

Toxins:Toxins: Haemolysin (Staphylolysin): Destroy RBCs, Haemolysin (Staphylolysin): Destroy RBCs,

Macrophage and Platelets. 4 types Macrophage and Platelets. 4 types αα,,ββ,,γγ and and δδ.. Leukocidin: Destroy WBCs.Leukocidin: Destroy WBCs. Exfolative toxin: Exotoxins affect dermatocyte cause Exfolative toxin: Exotoxins affect dermatocyte cause

Staphylococcal Scalded Skin Syndrome (SSSS).Staphylococcal Scalded Skin Syndrome (SSSS). Enterotoxins: acts on intestine causing diarrhoea, vomiting Enterotoxins: acts on intestine causing diarrhoea, vomiting

and abdominal pain. and abdominal pain. Toxic Shock Syndrome Toxin-1 cause Toxic Shock Toxic Shock Syndrome Toxin-1 cause Toxic Shock

Syndrome (TSS).Syndrome (TSS). Pyrogenic toxin cause Fever.Pyrogenic toxin cause Fever.

Disease caused by S. aureus:Disease caused by S. aureus: Pyogenic Infection (wound infection).Pyogenic Infection (wound infection). U.T.I.U.T.I. Neonatal Meningitis.Neonatal Meningitis. Osteomylitis.Osteomylitis. Acute bacterial endocarditis.Acute bacterial endocarditis. Septicemia. Septicemia. Pneumonia.Pneumonia. Sinusitis.Sinusitis. Otitis Media.Otitis Media. Conjuctivitis.Conjuctivitis. Food Poisoning. (Toxic type food poisoning Take 1-6 Food Poisoning. (Toxic type food poisoning Take 1-6

hour).hour). Skin infection:Skin infection:

Carbuncles.Carbuncles. Foliculitis.Foliculitis. Impetigo.Impetigo.

Staphylococcal Scalded Skin Syndrome.Staphylococcal Scalded Skin Syndrome. Toxic Shock SyndromeToxic Shock Syndrome

Laboratory Diagnosis:Laboratory Diagnosis: Specimens:Specimens:

According to the site of infection.According to the site of infection. Culture:Culture:

Non fastideous halophilic Microorganism.Non fastideous halophilic Microorganism. Grow on Blood agar, Chocolate agar, MacConkey, CLED, Grow on Blood agar, Chocolate agar, MacConkey, CLED,

Nutrient agar, Milk agar.Nutrient agar, Milk agar. Selective media Mannitol Salt Agar MSA.Selective media Mannitol Salt Agar MSA.

Incubation:Incubation: At 37°C aerobically for 24 hours.At 37°C aerobically for 24 hours.

Colonial Morphology:Colonial Morphology: Small, low convex, dry white or creamy white, some are Small, low convex, dry white or creamy white, some are ββ

hemolytic.hemolytic. Lactose fermenting on CLED and MacConkey.Lactose fermenting on CLED and MacConkey. Pigmented on Milk agar or Nutrient agar.Pigmented on Milk agar or Nutrient agar. S. aureus produce Yellow colonies on Mannitol Salt agar due to S. aureus produce Yellow colonies on Mannitol Salt agar due to

Mannitol fermentationMannitol fermentation

S. aureus on BAP:S. aureus on BAP:

ΒΒ-hemolysis on BAP:-hemolysis on BAP:

Colonial morphology on BAP:Colonial morphology on BAP:

Mannitol Fermentation by S. Mannitol Fermentation by S. aureus:aureus:

Gram stain:Gram stain:

Biochemical reaction:Biochemical reaction: Catalase test:Catalase test:

To differentiate between Staphylococci and To differentiate between Staphylococci and Streptococci.Streptococci.

Slide methods: Drop of 3% HSlide methods: Drop of 3% H22OO22 + colonies + colonies taken by wouden stick or glass rods. Examine taken by wouden stick or glass rods. Examine for air bubbles.for air bubbles.

Precausion:Precausion:Do not use metal loop.Do not use metal loop.Do not use culture that grow on Blood agar. Do not use culture that grow on Blood agar.

Coagulase Test:Coagulase Test:Used to differentiate between S. aureus and other Used to differentiate between S. aureus and other

Staphylococci.Staphylococci.2 Enzymes Bound Coagulase (Slide methods)2 Enzymes Bound Coagulase (Slide methods)Free coagulase (Tube methods)Free coagulase (Tube methods)

Coagulase testCoagulase test

Slide coagulase: Used for bound Slide coagulase: Used for bound coagulasecoagulase

Control Control TestTest

Colony+NaclColony+Nacl

+Undiluted +Undiluted

(Nacl + Colony) (Nacl + Colony) Plasma Plasma

Tube coagulaseTube coagulase Used for detection of free coagulase.Used for detection of free coagulase. Need:Need:

1/10 diluted plasma1/10 diluted plasma Organism under test on Nutrient broth.Organism under test on Nutrient broth. Water pathWater path

Procedure:Procedure: On 0.5 ml 1/10 diluted pooled plasma we add 5 On 0.5 ml 1/10 diluted pooled plasma we add 5

drop from the broth and incubate on the water drop from the broth and incubate on the water path at 37°C; examined for fibrin clot by tilting the path at 37°C; examined for fibrin clot by tilting the tube every 30min, 1, 2, 4, 6, and after 24 hours.tube every 30min, 1, 2, 4, 6, and after 24 hours.

Why we make the test on broth?Why we make the test on broth? Why we use diluted plasma?Why we use diluted plasma? Why we examine periodically?Why we examine periodically?

S. aureus Coagulase +ve while S. epidermidis S. aureus Coagulase +ve while S. epidermidis and S. saprophyticus Coagulase –ve.and S. saprophyticus Coagulase –ve.

DNaseDNasePrinciple:Principle:

DNase break down DNADNase break down DNADNA precipitated by 1N HCLDNA precipitated by 1N HCL

Method:Method:On DNA containing media subculture On DNA containing media subculture

tested organism on the center.tested organism on the center.Incubate at 37°C for 24 hours.Incubate at 37°C for 24 hours.Flood the plate by 1N HCL and examined Flood the plate by 1N HCL and examined

for clearance of precipitation around the for clearance of precipitation around the colonies of S. aureus. colonies of S. aureus.

DNase:DNase:

Phosphatase testPhosphatase test

S. aureus produce phosphatase while other S. aureus produce phosphatase while other staphylococci not produce the enzyme.staphylococci not produce the enzyme.

Principle:Principle: Phosphatase enzyme break down phosphate Phosphatase enzyme break down phosphate

from Phenol phathalin phosphate agar, which from Phenol phathalin phosphate agar, which give free Phenol phathalin which react with give free Phenol phathalin which react with ammonium vapor to give red colour.ammonium vapor to give red colour.

Procedure:Procedure: Sub-culture organism under test on Phenol Sub-culture organism under test on Phenol

Phthalin Phosphate agar. Incubate at 3 7°C for Phthalin Phosphate agar. Incubate at 3 7°C for 24 hours, than add ammonium vapour on the 24 hours, than add ammonium vapour on the cover of the plate and examined for red colour cover of the plate and examined for red colour formation.formation.

S. saprophyticusS. saprophyticus Disease:Disease:

U.T.I (3U.T.I (3rdrd most common cause). most common cause). Lab. Diagnosis:Lab. Diagnosis:

Specimens:Specimens: UrineUrine Culture: BAP, CLED, MacConkey.Culture: BAP, CLED, MacConkey. Catalase +ve.Catalase +ve. Coagulase –ve.Coagulase –ve. Non mannitol fermenters.Non mannitol fermenters. DNase –ve.DNase –ve. Phosphatase –ve.Phosphatase –ve.

Differentiation between Coagulase –ve spp.Differentiation between Coagulase –ve spp. By Novobiocin sensitivity:By Novobiocin sensitivity:

S. saprophyticus S. saprophyticus ResisitantResisitant S. epidermidis S. epidermidis SensitiveSensitive

Novobiocin resistant:Novobiocin resistant: