CLOSTRIDIUM and its laboratory diagnosis.pptx

Gram-positive anaerobic bacteria which form heat-resistant spores and also the causative organisms in cases of gas gangrene, tetanus, botulism, food poisoning and pseudomembranous colitis.

1. CLOSTRIDIUM dr. nagendra kumar department of microbiology Gmc , jammu

2. CLOSTRIDIA Gram-positive anaerobic bacteria which form heat-resistant spores Commensal flora in gut of humans and animal Cause gas gangrene, tetanus, botulism, food poisoning and pseudomembranous colitis Pathogenicity Exotoxin: Pathogenic clostridia produce powerful exotoxins which are responsible for the pathogenesis and disease, e.g., tetanus, botulism and gas gangrene Invasive toxin: C. perfringens , besides being toxigenic, is also invasive and can spread along the tissues and even cause septicemia

3. Gram-positive, rods, highly pleomorphic, 3–8 micron, spore-forming, shape and position vary Spherical or oval; terminal, subterminal, central Motile, stately motility MORPHOLOGY Universities Press Pvt Ltd

4. CULTURE Anaerobic Aerotolerant Low redox potential by adding reducing substances – unsaturated fatty acids, ascorbic acid, glutathione, cysteine

5. GROWTH REQUIREMENTS pH 7–7.4 Temperature 37°C Some are thermophillic , psychrophilic BA hemolytic Robertson’s cooked meat – turbidity, gas Saccharolytic and proteolytic Litmus milk medium, production of acid, gas, clot detected

6. SPORE Central – spindle – C. bifermentans Subterminal – club – C. perfringens Oval terminal – tennis racquet – C. tertium Spherical terminal – drumstick – C. tetani Exhibit variable resistance to heat, drying and disinfectants

7. SPORE ARRANGEMENTs

8. Clinical case 1 A 50-year-old man met with a road traffic accident in which he sustained multiple fractures with open wounds and a crush injury of the leg. He was taken to the nearest hospital two days later, at which time, he was found to be in shock. He was started on supportive therapy and antibiotics. There was edema and pain at the site of injury with increased discolouration and a serous discharge. The area around the wound had crepitus on palpation. Microscopic examination of the wound discharge showed the presence of thick, brick-shaped, gram-positive bacilli along with gram-positive cocci. Based on a provisional diagnosis of gas gangrene, immediate surgical treatment with extensive excision of the local area (to prevent further spread) and intravenous penicillin with clindamycin were given. The exudate was also inoculated into Robertson’s cooked meat medium. Clostridium perfringens and peptostreptococci grew in the culture.

9. CLOSTRIDIUM PERFRINGENS Large rectangular, stout, gram-positive, capulated , non-motile bacillus, 4–6 x l µm. Pleomorphic, single, chains Spores: Subterminal, Not produced in artificial media

10. CULTURE Microphilic – aerophilic anaerobes pH – 5–8, temperature 20–50 °C (45°C) RCM (pink) – 4 – 6 hrs Media : NA, BA, thioglycolate broth, RCM BA : Polymyxin, neomycin, iron, citrate (black colonies) BA : Rabbit, sheep, human colonies show target hemolysis Litmus milk – fermentation of lactose, change of litmus – blue to red Acid coagulates casein – clotted milk disrupted – stormy fermentation

11. VIRULENCE FACTORS Strains – five types A – E Toxins – most prolific of toxin producing bacteria Four major toxins – alpha, beta, epsilon and iota Alpha toxin – most important biologically, lethal, dermonecrotic and hemolytic Table 28.6 Toxins produced by C. perfringens types

12. CLOSTRIDIUM PERFRINGENS Enzymes Neuraminidase destroys myxovirus receptors Hemagglutinin – active against RBCs Fibrinolysin Bursting factor – muscle lesions in gas gangrene Circulating factor – increase in adrenal sensitivity

13. GAS GANGRENE Type A Commonly seen in association with other clostridia Wound contamination Anaerobic cellulitis Muscle tissues are invaded – anaerobic myositis Fig. 28.10 Gas gangrene of the lower limb showing edema and discoloured skin

14. PATHOGENESIS OF GAS GANGRENE Exogenous: Clostridia usually enter a wound along with implanted foreign particles such as soil, road dust, bits of clothing or shrapnel. They may also be present on skin, especially that of the perineum and thighs Endogenous: Infection may also develop after surgical procedures (especially amputations for vascular disease) and even injections (especially adrenaline)

15. CLOSTRIDIUM SEPTICUM Pleomorphic bacillus Oval, central/subterminal spores Anaerobic. saccharolytic, abundant gas Four distinct toxins Alpha toxin is hemolytic, demonecrotic and lethal Gas gangrene in humans

16. CLOSTRIDIUM NOVYI Large, pleomorphic bacillus Oval, subterminal spores Strict anaerobe Type A – causes gas gangrene Large amounts of edema fluid, little or no observable gas, high mortality

17. CLOSTRIDIUM HISTOLYTICUM Oval, subterminal, bulging spores Proteolytic Gas gangrene in humans Infection – exogenous/endogenous Exogenous – implanted foreign particles Endogenous – clean surgical procedures

18. ANAEROBIC WOUND INFECTIONS Simple wound contamination – no invasion of tissue Anaerobic cellulitis – invasion of fascial planes, minimal toxin production, no invasion of muscle tissue Anaerobic myositis – gas gangrene, invasion of muscle tissue, abundant formation of exotoxins

19. CLINICAL PRESENTATION Incubation period – 7 hours to 6 weeks C. perfringens – 10–48 hours C. septicum – 2–3 days C. novyi – 5–6 days Increasing pain, tenderness and edema over the affected part Accumulation of gas – crepitus Untreated – profound toxemia and prostration

20. LABORATORY DIAGNOSIS Specimen Films – edge of affected area, tissue from necrotic area, exudate from deeper part of wound Exudate collected from depth of wound – collected by capillary pipette or swab Necrotic tissue/muscle fragments Blood cultures in C. perfringens and C. septicum infections However, C. perfringens bacteremia may occur without gas gangrene

21. MICROSCOPY C. perfringens – Gram-positive bacilli without spores C. septicum – boat- or leaf-shaped pleomorphic bacilli C. novyi – large bacilli with oval or subterminal spores Fig. 28.13 Methylene blue-stained culture specimen revealing the presence of numerous Clostridium septicum bacteria (note the absence of spores) ( Source: CDC, PHIL, Image ID 217)

22. CULTURE Robertson’s cooked meat medium: Fresh and heated blood agar Target hemolysis resulting from a narrow zone of complete hemolysis due to theta toxin and a much wider zone of incomplete hemolysis due to the alpha toxin This double zone pattern of hemolysis may fade on longer incubation

23. NAEGLER’S REACTION C. perfringens grown on media containing Fildes peptic digest of sheep blood and human serum with antitoxin on one half of the plate Colonies on the half without antitoxin will be surrounded by a zone of opacity No opacity around the colonies on the half of the plate with antitoxin Fig. 28.14 Nagler’s reaction—on the right, the presence of toxin produces opacity in the serum due to the breakdown of lecithin

24. CULTURE Fig. 28.15 Reverse CAMP test

25. TREATMENT AND PROPHYLAXIS Surgery: Most important therapeutic and prophylactic Damaged tissue removed extensively and promptly Hyperbaric oxygen Antibiotics: Metronidazole I/V – before surgery and every 8 hours Mixed aerobic and anaerobic infection: combination of metronidazole with amoxicillin and gentamicin. Passive prophylaxis – anti-gas gangrene serum given I/M 10,000 IU – C. perfringens 10,000 IU – C. novyi 5000 IU – C. septicum

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