Clostridium perfringens

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Clostridium perfringens:

Clostridium perfringens

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Spore-forming Bacilli Orginally cultivated by Achalme 1891 Also called C.welchii Normal Inhabitant of large intestines Iraq produced 90 gallons of C. perfringens 1945: Japan Used shrapnel bomb containing C. perfringens on ten Chinese victims

Introductory Characteristics:

Introductory Characteristics Obligate anaerobes Capable of producing endospores Rod-shaped, named after Greek word for spindle, kloster Club-shaped, as well: endospores form club end

The Basics:

The Basics Gram positive Rod-shaped Non-motile Anaerobic Five types of strains A - E Four lethal toxins Alpha, Beta, Epsilon and Iota

Morphology :

Morphology Large gram positive bacilli with straight, parallel sides and rounded or truncated ends, occurring singly or in chains or small bundles. Spores- Oval, central or subterminal and not bulging but are rarely seen in artificial culture or in in material from pathologic lesions

Ecology:

Ecology Found in guts of human, domestic pets, wild animals. Their spores exist in soil. Existance of their spores in soil indicates faecal contamination. Widespread in nature, on dust, soil, air, water, faecal.

Cultural Characteristics:

Cultural Characteristics Anareobe pH 5.5-8 Temp 37-45 Enrichment Media- Robertson Cooked broth Blood agar plates streaked post enrichment have higher growth Robertson cooked meat media- Pink but is not digested. Acid reaction and sour odor Cl. Perfringens requires 13-14 amino acids and 5-6 vitamins to grow. This requirement also indicates their prescence on foods having high proteins.

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NOTE: Large rectangular gram-positive bacilli Inner beta-hemolysis = θ toxin Outer alpha-hemolysis = α toxin NOTE: Double zone of hemolysis

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Glucose blood agar- large smooth, regular, convex, slightly opaque disks. Colonies of most strains demonstrate target hemolysis after overnight incubation (Theta toxin ) Reverse CAMP test- Synergistic b hemolysis. Other colonies include one with raised opaque center and flat radially striate transparent border

Biochemical Reactions:

Biochemical Reactions Sacchrolytic. Glucose, maltose, lactose and sucrose fermented with acid and gas IMViC- -+-- H 2 S- plenty produced Nitrates to Nitrites

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Litmus Milk medium- Fermentation of lactose leads to formation of acid. Acid clots the milk and clotted milk is disrupted due to vigorous gas production. Stormy fermentation or stormy clot reaction

Resistance:

Resistance Spores are destroyed within 5 minutes Except food poisoning strains Type A and C Autoclaving is lethal

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C. perfringens Virulence Factors Major Minor

Classification:

Classification There are four main exotoxins . They are alpha , beta, epsilon and iota . According to 4 main exotoxins , Cl . Perfringens strains are divided to 5 sub group and they are named as A,B,C,D,E. A- alpha B- alpha , beta, epsilon C- alpha , beta D- alpha , epsilon E- alpha , iota

Alpha toxin:

Alpha toxin Type A strain Lecithinase Lyse erythrocytes, platelets, leukocytes and endothelial cells. Lethal, dermonecrotic and hemolytic Lysis of hot cold variety being best seen after incubation.

Nagler’s reaction:

Nagler’s reaction Basis Lecithinase C is alpha toxin, splits lecithin into phosphoryl choline and diglyceride in presence of Ca ++ and Mg++ ions. Reaction seen as an opalescnce in serum or egg yolk media and is neutralized by antitoxin

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C. perfringens Nagler Reaction NOTE: Lecithinase ( α -toxin; phospholipase) hydrolyzes phospholipids in egg-yolk agar around streak on right. Antibody against α -toxin inhibits activity around left streak.

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Procedure: Culture plate – 6%agar, 5%peptic digest of sheep blood and 20%human serum or 5%egg yolk prepared. Neomycin s ulphate makes it more selective inhibiting coliforms and aerobic spore bearers. One half of plate 2-3 drops of antitoxin are spread and allowed to dry. Plate inoculated with test organisms

Interpretation:

Interpretation On section no antitoxin colonies show zone of opalescence. There will be no opacity around the colonies on half of plate with antitoxin due to specific neutralization. Not specific cause of other lecithinase producing bacteria.

PowerPoint Presentation:

Epsilon-toxin Increases intestinal permeability causing vascular damage and oedema in major organs Liver damage Higher blood pressure Iota-toxin Food-borne illness

Pathogenesis:

Pathogenesis Soft tissue infections caused by it are subdivided into Cellulitis- Serious form of wound infection. Clostridial species can colonize wounds and skin. Fasciitis- Suppurative myositis, pus in muscle plane Myonecrosis/gas gangrene

Gas Gangrene:

23 Gas Gangrene Clostridium perfringens most frequent clostridia involved in soft tissue and wound infections - myonecrosis Spores found in soil, human skin, intestine, and vagina Predisposing factors – surgical incisions, compound fractures, diabetic ulcers, septic abortions, puncture wounds, gunshot wounds

Virulence Factors:

25 Virulence Factors Virulence factors toxins – alpha toxin – causes RBC rupture, edema and tissue destruction collagenase hyaluronidase DNase

Pathology:

26 Pathology Not highly invasive; requires damaged and dead tissue and anaerobic conditions Conditions stimulate spore germination, vegetative growth and release of exotoxins, and other virulence factors. Fermentation of muscle carbohydrates results in the formation of gas and further destruction of tissue.

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27

Treatment and Prevention:

28 Treatment and Prevention Immediate cleansing of dirty wounds, deep wounds, decubitus ulcers, compound fractures, and infected incisions Debridement of disease tissue Large doses of cephalosporin or penicillin Hyperbaric oxygen therapy No vaccines available

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Septicemia- Blood culture alarming. Occur in association with with malignancy and may involve localized myonecrosis Food poisoning- Common buy underappreciated. Short incubation period, no fever, abdominal cramps and watery diarrhea. Clinical course lasting less than 24 houres

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Enteritis necroticans Colitis Clostridial endometritis

Lab Diaganosis of Gas gangrene:

Lab Diaganosis of Gas gangrene Specimen- Edge of affected muscles Exudates from wound Necrotic tissue and muscle fragments.

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Microscopy- Gram stained films- Thick stubby gram positive rod C.perfringens or C.sordellii Citron bodies, boat or leaf shaped pleomorphic bacilli with irregular staining may indicate C. septicum C.tetain slender rods with round terminal end

culture:

culture Fresh and heated blood agar Serum or egg yolk agar Meat broth Ifentification from the above

Lab diagnosis of Food poisoning:

Lab diagnosis of Food poisoning Isolation in higher than normal number from feces or ingested food Cooked meat broth inoculated and heated at 100 C for 30m min and incubated for 18 hours. Identification by morphology. Isolated from feces except in large number. More specific diagnosis by ELISA

Treatment:

Treatment Surgery Antibiotics Penicillin,metronidazole and Aminoglycoside Passive immunization Hyperbaric oxygen Active immunization

Clostridium difficile:

Clostridium difficile

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1935 Difficulty in isolating Antibiotic associated colitis in 1977

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Morphology- Gram positive rod with oval subterminal spores. Spores are large, oval and terminal. Non hymolytica, saccharolytic and weakly proteolytic

C. Difficle Toxins Produced:

Cytotoxin: Toxin B Associated with Inflammation C. Difficle Toxins Produced Enterotoxin: Toxin A Protein toxin released by C. difficile in the lower Intestine Frequently cytotoxic Alters the permeability of the epithelial cells of the intestinal wall. Pore forming toxins, secreted by bacteria, form pores in cell membranes causing cells death.

Toxins:

Toxins Enterotoxin (Toxin A) and Cytotoxin ( Toxin B ) Toxin A primarily responsible for diarrhea. Capable of producing fluid accumulation Toxin B potent cytotoxin capable fo cytopathogenic effects

Pathogenesis:

Pathogenesis Antibiotic associated diarrhea and pseudomembranous colitis ( life threatening condition ) Alteration of normal flora either permitting overgrowth of relatively resistant organisms or making patient more susceptible to exogenous acquistion . Disease occurs if organisms proliferate in colon and produce toxins

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Three drugs- Clindamycin Ampicillin Cephalosporins Severity of diease varies from mild diarrhea to fulminat pmc

Lab diagnosis:

Lab diagnosis Isolation of bacilli can be isolated from feces by enrichment and selective culture procedures. Demonstration of toxins by hep 2 and human diploid cell cultures

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Treatment- discontinuing antibiotics

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