STAPHYLOCOCCUS

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STAPHYLOCOCCUS:

STAPHYLOCOCCUS By – Mr. Gunjal Prasad N. M.Sc. Medical Microbiology,

HISTORY :

HISTORY In 1871 the organism was first observed by Von Recklinghausen in human pyogenic (pus producing) lesions. Pasteur in 1880 demonstrated its pathogenicity. Sir Alexander Ougston , a Scottish surgeon, established its conclusive causative role in abscesses & suppurative (Decay in tissue producing pus) lesions. He named these as “ Staphylococcus” (In Greek - Staphyle means – bunch of grapes & kokkos – berry).

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Electronmicroscopic Picture of Staphylococci present in grape like clusters.

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Drawing by Sir Ougston of Staphylococcus he found . Gram Positive (Violet) Cocci in clusters.

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Passet in 1883 described third variety Staphylococcus citrus producing Lemon Yellow colour colonies . 1884 Rosenbach named these strains from pyogenic lesions as Staphylococcus pyogenus ( now known as Staph. aureus ) and normal skin strains as Staphylococcus albus ( now known as Staph. epidermidis ) .

INTRODUCTION:

INTRODUCTION Gram positive cocci (GPC). Arranged in grape like clusters . Ubiquitous. Commonest cause of Suppurative lesions in human beings. Able to develop resistance to Penicillin & other antibiotics. Hence important PATHOGEN in Hospital Acquired Infections or Nosocomial Infections.

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Gram stain smear showing Gram Positive Violet colour Cocci arranged in clusters

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PROPERTIES PROPOSED AS INDICATOR OF VIRULENCE ARE – Pigment production has no association with virulence . Haemolysis on Blood Agar plate. Gelatin liquefaction test POSITIVE. Lipolytic activity. Production of Urease. Production of Phosphatase. Production of Catalase. Most constant association found between virulence & production of enzyme “Coagulase”. To a lesser extent with fermentation of sugar Mannitol

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Divided into two groups on the basis of fermentation of Mannitol. Group I – Staphylococcus aureus Mannitol fermenters (Anaerobically). Coagulase production. Usually pathogenic in nature. Group II – Staphylococcus epidermidis Mannitol non fermenters. Coagulase test negative. Usually non pathogenic but cause opportunistic infections.

Staphylococcus aureus :

Staphylococcus aureus MORPHOLOGY Spherical cocci, approx. 1µm in dia. Arranged in grape like clusters. Cluster formation is due to cell division occurring in three planes with daughter cell tending to remain in close proximity. Found singly, pairs or in short chains. Non motile, nonsporing. Capsule may present, visible only microscopically. Stain readily with analyine dyes. Uniformly Gram Positive in nature.

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COLONIES ON BLOOD AGAR – Similar to those on Nutrient agar. Gives β -Haemolysis. Haemolysis best seen on sheep or rabbit blood agar plate.

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COLONIES ON Mac CONKEY’S – Very small & pink due to lactose fermentation . SELECTIVE MEDIA – BLOOD POTASSIUM TELLURITE MEDIA - Addition of 8-10% NaCl or Lethium chloride, Tellurite & Polymyxin to ordinary medium. Used for isolation of Staph. aureus from samples like faeces containing other bacteria.

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MANNITOL SALT AGAR – Selective & indicator medium contain – Nutrient agar with 1% mannitol 7.5% NaCl and Phenol red. Due to mannitol fermentation Yellow colonies formed. MILK AGAR – Contains Nutrient agar & sterilized milk. Staphylococcal strains produce various pigments for e.g. Golden Yellow, White, & Lemon Yellow.

BIOCHEMICAL REACTIONS :

BIOCHEMICAL REACTIONS BASED ON ENZYME PRODUCTION - Catalase test positive . Coagulase test positive. Production of Phosphatase. Production Deoxyribonuclase. Production of Lecithinase. BASED ON FERMENTATION – Ferments no. of sugars with the production of acid but no gas. But are of no diagnostic value. Except mannitol fermentation, which takes place anaerobically.

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TUBE CATALASE TEST Sterile Glass Rod Sterile Test tube with 3% H 2 O 2 Test isolated colony from NA Plate Break down of H 2 O 2 by enzyme Catalase into H 2 O & O 2 . Oxygen is liberated in the form of gas bubbles

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Slide Catalase Test Catalase On The Plate

Coagulase Test :

Coagulase Test Test used to identify Staph. aureus which produces the enzyme coagulase. To differentiate from Staph. epidermidis. Principle – Coagulase causes plasma to clot by converting fibrinogen (A soluble protein present in blood plasma) to fibrin (insoluble protein). Two types of coagulase are produced by most of the strains. Free Coagulase & Bound Coagulase

Toxins & Enzymes :

EXOTOXINS – 1. HAEMOLYSINS – Four antigenically distinct types called Alpha ( а ), Beta (ß), Gamma ( γ ), Delta ( δ ). All are Exotoxins. Toxins & Enzymes B. ENTEROTOXIN – Responsible for staphylococcal food poisoning. Eight antigenically distinct enterotoxins - A, B, C1, C2, C3, D, E, & H. Source of infection is usually a staphylococcal lesion on skin of fingers of food handlers.

Toxic Shock Syndrome Toxin (TSST):

Toxic Shock Syndrome Toxin (TSST) This condition is characterized by-- Fever, Hypotension (abnormally low blood pressure), Vomiting, diarrhoea & An erythematous (reddening of the skin due to hyperemia of the skin) rash. Most TSST producing strains belongs to bacteriophage group-I. Type I TSST strains are most responsible for it. TSST-I producing strains colonies vagina frequently.

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Exfoliative (Epidermolytic) Toxin Two types of exfoliative toxin, A & B. Type A- Heat stable, its production is under chromosomal control. Type B- Heat labile, production is plasmid mediated. Lead to epidermal splitting resulting in blistering (formation of vesicle on or below skin layer) diseases, generalized desquamation producing (loss of bits of skin layers coming out of) Staphylococcal Scalded Skin Syndrome (SSSS). Severe form of SSSS is known as Ritter’s disease in the new born. Milder forms are pemphigus neonatorum & bullous Impetigo. ( large thin-walled blisters (bullae) arising from normal skin or mucous membrane )

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Free Coagulase – (Clumping factor) It’s a heat stable constituent of cell wall. Converts fibrinogen directly to fibrin. Causes clumping of cocci due to precipitation of fibrin on the cell surface. It also differentiate pathogenic strains from nonpathogenic. Bound Coagulase – Converts fibrinogen to fibrin by activating coagulase reacting factor present in plasma.

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Pictures showing Coagulase test with Positive & Negative test results.

Pathogenesis :

Pathogenesis Important causative agent in pyogenic lesions. Lesions are localized in nature, unlike Streptococcal spreading lesions. Coagulase enhances virulence by inhibiting phagocytosis Process in which phagocytes engulf and digest microorganisms and cellular debris; an important defense against infection. Forms a wall of fibrin clot around the lesion – Pus is thick & creamy. Disease can be classified as – Cutaneous (affecting skin – Inside or On surface), deep infections, Food poisoning, Nosocomial infections, Skin exfoliative diseases & toxic shock syndrome .

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Staph. aureus can cause skin infections. These usually remain localized; the collection of pus is called a furuncle (=boil). Cutaneous Infections – If several furuncles form & coalesce, as seen here, a larger area of infection called a carbuncle occurs. This one is at a typical site on the back of the neck. Patient with Staph. aureus infection - Impetigo

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Food poisoning – Enterotoxins is responsible for staphylococcal food poisoning. The toxin act on ANS to cause illness. Toxin produced in carbohydrate, protein foods usually cooked foods. Milk and milk products. Food may have preformed toxin but not bacteria. Source of infection is usually are food handlers with Staphylococcal lesions on hands. Ingestion of contaminated food with toxin can lead to food poisoning. Within 2-6hrs. after consumption of contaminated food it leads to nausea, vomiting, & diarrhoea. The toxin can be detected by serological tests such as latex agglutination tests & ELISA.

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Nosocomial Infections – Staphylococci are one of the important causative agent among the organisms causing nosocomial infections in hospital environment

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Skin Exfoliative diseases- SSSS is one example of exfoliative disease in which toxin spreads systematically. It is seen in small children but rarely. Toxic Shock Syndrome (TSS) – This condition is characterized by - Fever, hypotension, vomiting, diarrhoea & an erythematous rash with subsequent desquamation hyperemia of mucous membrane TSS widely known to be in association with use of tampons by menstruating women but it occurs in other conditions also.

Laboratory Diagnosis :

Laboratory Diagnosis Specimens – Pus – Suppurative lesions. Sputum – Respiratory infections. Blood – Septicemia or PUO. Urine – UTI. CSF – Meningitis. Faeces – Food Poisoning. Food OR Vomit – Food Poisoning. Nasal & Perineal Swab - Carriers.

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Collection & Transport – Always use sterile container. Follow all aseptic conditions. Follow all Universal Biosafety Precautions. Transport the specimen as early as possible to the laboratory. If delay is unavoidable, if allowed store the sample in fridge at 4 0 C.

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Laboratory Follow Up – Culture – BA/ NA/ MAC/ Mannitol Salt Agar/ Milk Agar etc. Direct Smear – Gram’s Stain. Colony morphology studies and staining. Biochemical Reactions - Enzyme detection & fermentation of sugars. Antibiotic Sensitivity testing. Bacteriophage typing. Treatment. Control Measures.

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Control Measures – Source – Isolation & Treatment. Detection, isolation & treatment of carriers among hospital staff. Sterilization & proper disinfection of instruments. Should follow all USP’s. Stop misuse of Antibiotics.

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Thank you