GRAM POSITIVE COCCI & RODS CLASSIFICATION: (A) Cocci, divided into: Catalase positive : Staphylococcus Catalase negative : Streptococcus & anaerobic cocci

(B) Rods (bacilli), divided into: Branching, divided into: a) Aerobic : Nocardia, Streptomyces, Actinomadura. b) Anaerobic : Actinomyces.

2. Non-branching divided : a) Anaerobic : Clostridium b) Aerobic : divided into: (i) Catalase negative : Loctobacillus, Gardnerella, Erysipelothrix. (ii) Catalase positive : divided into: @ Sporing, e.g.: Bacillus @ Non-sporing : Listeria, Corynebacterium

STAPHYLOCOCCUS SPECIES: S. aurous, main pathogenic species. 2. S. epidermidis. 3. S. saprophyticus.

(40% of healthy people carry S. aureus). NORMAL HABITAT: *Skin,* intestinal tract, * upper respiratory tract, * nose (40% of healthy people carry S. aureus).

PATHOGENICITY: 1- Abscess, boils, styes, impetigo. 2- Secondary infection to: insect bites, ulcers, burns, wound . 3- Conjunctivitis (newborn). 4- Hospital cross-infection.

5- Septicaemia, endocarditis, osteomyelitis. 6- Pneumonia, empyema. 7- Mastitis. 8-Antibiotic-associated enteritis 9- Food poisoning : secretion of enterotoxin (B) in contaminated meat, milk, & milk products.

10- Scalded skin syndrome in young children (release of exfoliatin toxin). 11- Toxic shock syndrome, (colonization of vagina, other parts of body).

ENZYMES AND TOXINS 1-Coagulase enzyme: clots plasma & prevents phagocytosis 2- Leucocidin: destroy WBC . 3- Lytic exotoxin: destroys RBC and platelets. 4- Deoxyribonuclease enzyme : destroys DNA.

5- Hyaluronidase enzyme, helps spread in tissues. 6- Lipase enzyme, breaks down fats. 7- Staphylokinase enzyme, causes fibrinolysis.

8- Exfoliatin, causes scalded skin syndrome (skin peeling) 9- Enterotoxin B, causes food poisoning. 10- Beta-lactamase enzyme, destroys Beta-lactam ring, leads to penicillin resistance.

S. epidermidis : May cause @ Bacteraemia, due to : @ contamination of * cannulae, shunts, * indwelling catheters

S. SAPROPHYTICUS: Associated with : @ Cystitis. @ Pyelonephritis. @ Acute urethritis (women)

LABORATORY DIAGNOSIS SPECIMENS: Pus & sputum for microscopy and culture. 2. Blood for culture. 3. Stool, vomit, remains of food, (food poisoning) 4. Nasal swabs (carriers)

MICROSCOPY: @ Staphylococci : * non-motile, * non-capsulated, * gram positive, * 1 µm in diameter, * occur: cluster, pairs, single

@ Aerobic , anaerobic , carboxyphilic, S. aureus: use: CULTURE: @ Aerobic , anaerobic , carboxyphilic, S. aureus: use: Blood agar & chocolate agar: colonies yellow-cream, golden, white, 1-2 mm, raised, may be Beta-haemolytic. b) Mc Conkey :colonies 0.5 mm, lactose not fermented. c) Mannitol salt agar: To isolate S. aureus from stool in food poisoning.

2. S.epidermidis: 3. S. saprophyticus: @ Same media of S.aureus @ Colonies white, non-haemolytic. 3. S. saprophyticus: @ Same above media. @ Colonies white or yellow, non- haemolytic. @ Does not grow anaerobicaly, or on Mc Conkey agar.

BIOCHEMICAL REACTIONS: @ S.aureus: 1-Coagulase: identifies pathogenic strains 2-DNAse test: if coagulase is not clear. 3-Catalase test: Staph. catalase positive 4-Most Staph. strains ferment mannitol 5-S.aureus grows on 15-25% NaCl media

S.epidermidis & saprophyticus: Coagulase negative. DNAse negative. Catalase positive. Grow on mannitol salt agar. S. saprophiticus ferments mannitol + acid production . 6. S. epidermidis does not ferment mannitol.

COAGULASE TEST: Bound: detected by slide S. aureus has 2 types of coagulase: Free: detected by tube Bound: detected by slide @ Slide coagulase test is confirmed by tube test if: Slide is negative & S. aureus is isolated from a very ill patient b) Slide is not clear.

SLIDE METHOD: Put a saline drop on each end of a slide Mix a colony on each saline drop to make a thick suspension. Colonies are taken from B.A. or N.A. 3. Add a drop of plasma to one suspension. Mix, look for clumping within 10 sec. second suspension is a negative control. 4. Control Strains: a) S.aureus: Positive control. b) E.coli: Negative control.

TUBE METHOD: Plasma is diluted 1:10 in saline . Label 3 test tubes: T = Test organism Pos = Positive control Neg = Negative control (E.coli ) 2. Pipette 0.5 ml plasma into each tube. 3. Add 5 drops of test organism to tube (T) 3. Add 5 drops of positive control to tube (Pos) 4. Add 5 drops of negative control to tube (Neg). 5. Mix, & incubate all tubes at 37˚C. Look for clotting after one hour. No clotting, check tubes every ½ hr for 6 hrs.

Positive: organism hydrolysed DNA in medium DNAse Test: Culture organisms on media containing DNA. Divide the DNA plate into 3 parts: a) Test organism is streaked on part (1). b) Positive control is streaked on part (2). c) Negative control is streaked on part (3). Incubate overnight at 37˚C. Flood plate with HCl & pour off excess. Note clear zones around colonies within 5 min Results: @ Positive DNAse: Clear @ Negative DNAse: Not clear Positive: organism hydrolysed DNA in medium 7. Controls: Positive : S.aureus -Negative : E. coli

Catalase Test: Take colonies from a medium not containing blood (catalase is in RBC). Use nutrient agar Place 2ml H2O3 solution in test tube. Use a wood stick (not wire), to mix colonies with hydrogen perioxide solution. Note appearance of O2 bubbles. Controls: Positive: Staph. species. Negative: Strep. species.

BACTERIOPHAGE TYPING: @ To study: * epidemiology of hospital cross-infection * food poisoning sources of infection @ S. aureus strains producing enterotoxin belong to phage Group III.

Penicillin, Methicilin, Cephalosporin SENSITIVITY @ Sensitive to: Flucloxacillin, Cephalosporin, Fucidin, Clindamycin, Penicillin, Erythromycin, Lincomycin. Resistance to: Penicillin, Methicilin, Cephalosporin