Clostridium

1. Clostridium perfringens Dr.T.V.Rao MD Dr.T.V.Rao MD 1

2. Genus Clostridium In Anaerobic spore bearing Gram positive bacilli Spores are wider than the body giving spindle shape The name derived from word Kolster meaning spindle Dr.T.V.Rao MD 2

3. C. perfringens : gas gangrene; food poisoning C. tetani : tetanus C. botulinum : botulism C. difficile : pseudomembranous colitis Clostridium Anaerobic. Large gram-positive rods. The spores are usually wider than the rods, and are located terminally or sub terminally. Most clostridia are motile by peritrichous flagella. Physiology and Structure Dr.T.V.Rao MD 3

4. Clostridia Large Gram positive bacilli. Straight or slightly curved rods with slightly rounded ends Anaerobic bacilli Spore bearing Spore do not germinate and growth does not normally proceed unless a suitably low redox potential Eh exists Many are Saprophytes Dr.T.V.Rao MD 4

5. SHAPES OF CLOSTIDIA The shape an position of spores varies in different species and is useful the identification of Clostridia Central and equatorial in Cl.bifermentans Sub terminal in C.perfringens Oval or terminal in Cl.tertium Spherical and terminal giving drum stick in C.tetani Dr.T.V.Rao MD 5

6. Some are Commensals But Can be Pathogenic Some are commensals of the animal & human gut which invade the blood and tissue when host die and initiate the decomposition of the corpse (dead body) Causes diseases such as gas gangrene, tetanus, botulism & pseudo-membranous colitis by producing toxins which attack the neurons pathways Dr.T.V.Rao MD 6

7. Clostridia Clostridia are biochemically active, frequently possessing both saccharolytic and proteolytic properties, although in varying degrees. Many species are highly toxigenic. The toxins produced by the organisms of tetanus and botulism attack nervous pathways and are referred to as neurotoxins. Dr.T.V.Rao MD 7

8. Clostridia of medical importance Dr.T.V.Rao MD 8

9. How they appear in Gram staining They are Gram positive, but may appear to be Gram negative. All produce spores, which enable the organisms to survive in adverse conditions, for example in soil and dust and on skin. Dr.T.V.Rao MD 9

10. Clostridium perfringens Large Gram-positive bacilli with stubby ends Capsulated Non motile Anaerobic Grown quickly on selective media Can be identified by Nagler reaction Dr.T.V.Rao MD 10

11. Some Clostridia Produce Gas gangrene The organisms associated with gas gangrene attack soft tissues by producing toxins and aggressins, and are referred to as histotoxic. C. difficile and some strains of C. perfringens produce enterotoxins. Dr.T.V.Rao MD 11

12. C. perfringens C. perfringens is a relatively large Gram-positive bacillus (about 4-6 × 1 μm) with blunt ends. It is capsulate and non-motile. It grows quickly on laboratory media, particularly at high temperatures (approximately 42°C), when the doubling time can be as short as 8 min. I Dr.T.V.Rao MD 12

13. Clostridium Causing Gas Gangrene Dr.T.V.Rao MD 13

14. Dr.T.V.Rao MD The Agent Clostridium perfringens Gram-positive bacteria Anaerobic rod 3-8 x 0.4 – 1.2 mu Found in soil, decaying matter and intestinal tract of mammals 5 types (A-E) Types B and D produce the epsilon toxin 14

15. Micro & Macroscopic C. perfringens NOTE: Large rectangular gram-positive bacilli Inner beta-hemolysis = θ toxin Outer alpha-hemolysis = α toxin NOTE: Double zone of hemolysis Dr.T.V.Rao MD 15

16. Resistance Vegetative bacteria is killed like other bacteria Cl.perfringens destroyed by boiling Cl, botulinum not killed even at 105 c 0 for less than 100 minutes All spores are killed at 121 oc in 20 minutes Halogens , Glutaraldehyde are effective on spores Metronidazole and Pencillin and Chloramphenicol are effective Dr.T.V.Rao MD 16

17. How Clostridia are Cultivated Clostridia grow well on ordinary medium under anaerobic medium Dr.T.V.Rao MD 17

18. Media used for Cultivation Liquid medium for cultivation cooked meat broth Thiglyclolate broth CMB contain unsaturated fatty acids which take up oxygen Proteolytic medium turns the medium black and Saccharolytic medium turn the meat pink Dr.T.V.Rao MD 18

19. 19 Virulence Factors Virulence factors toxins – alpha toxin – causes RBC rupture, edema and tissue destruction collagenase Hyaluronidase DNase Dr.T.V.Rao MD

20. Toxins The toxins of Cl. perfringens  toxin ( phospholipase C, lecithinase) is the most important toxin Lyses of RBCs, platelets, leucocytes and endothelial cells Increased vascular permeability with massive hemolysis and bleeding tissue destruction Hepatic toxicity and myocardial dysfunction  -toxin is responsible for necrotic lesions in necrotizing enterocolitis Enterotoxin is heat labile toxin produced in colon → food poisoning Dr.T.V.Rao MD 20

21. Dr.T.V.Rao MD Epsilon Toxin Produced as an inactive protoxin Activated by trypsin Removes a 13-residue N-terminal peptide Increases intestinal permeability Increases vascular permeability Vascular damage and edema in brain, heart, lung and kidneys 21

22. 22 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. Dr.T.V.Rao MD

23. C. perfringens Clinical Diseases Gas gangrene Spores germinate vegetative cells multiply, ferment carbohydrates and produce gas in the tissue. This results in distension of tissue and interference with blood supply the bacteria produce necrotizing toxin and Hyaluronidase, which favor the spread of infection tissue necrosis extends, resulting in increased bacterial growth, hemolytic anemia, then severe toxemia and death. Incubation: 1-7 days after infection. Symptoms : Crepitation in the subcutaneous tissue and muscle, foul smelling discharge, rapidly progressing necrosis, fever, hemolysis, toxemia, shock, renal failure, and death. Can be also caused by other Clostridium species. Dr.T.V.Rao MD 23

24. Mechanism of Clostridial Infections When Clostridial infection has been initiated in a focus of devitalized anaerobic tissue, the organisms multiply rapidly and produce a range of toxins and aggressins. These damage tissue by various necrotizing effects, and some have demonstrable lethal effects. They spread into adjacent viable tissue, particularly muscle, kill it, and render it anaerobic and vulnerable to further colonization, with the production of more toxins and aggressins. Dr.T.V.Rao MD 24

25. Toxic and Enzymatic Mechanisms Hyaluronidase produced by C. perfringens breaks down intercellular cement substance and promotes the spread of the infection along tissue planes. Collagenase and other proteinases break down tissues and virtually liquefy muscles. The whole of a muscle group or segment of a limb may be affected. α-Toxin , a phospholipase C (lecithinase), is generally considered to be the main cause of the toxaemia associated with gas gangrene, although other Clostridial species can produce similar manifestations. Dr.T.V.Rao MD 25

26. Clostridial Cellulitis Dr.T.V.Rao MD 26

27. Dr.T.V.Rao MD 27

28. C. perfringens Clinical Diseases Food poisoning The enterotoxin causes marked hyper secretion in jejunum and ileum. Enterotoxin: a heat-labile protein produced by some strains of C. perfringens type A. When >10 8 cells in contaminated meat are ingested and sporulate in the small intestine, enterotoxin is formed. It disrupts ion transport in the enterocytes, and induces antibodies (non-protective) in adults. Symptoms: diarrhea, usually without vomiting or fever. Necrotizing enteritis (pig-bel) : a fatal disease (acute necrosis in jejunum attributed to b -toxin) in children in New Guinea caused by type C C. perfringens . Clostridium bacteremia usually occurs in patients with tumors. Dr.T.V.Rao MD 28

29. Laboratory Diagnosis Specimen: Histological specimen or wound exudates Histological specimen transferred aseptically into a sterile screw-capped bottle & used immediately for microscopical examination & culture Specimens of exudates should be taken from the deeper areas of the wound where the infection seems to be most pronounced Microscopical examination (Gram, Spore stain etc) Gram-positive bacilli, non motile , capsulated & sporulated The spore is oval, sub-terminal & non bulging Spores are rarely observed Culture: Anaerobically at 37C On Robertson's cooked meat medium → blackening of meat will observed with the production of H2S and NH3 On blood agar → β -hemolytic colonies Dr.T.V.Rao MD 29

30. Dr.T.V.Rao MD 30

31. Specific Requirements for Anaerobic Cultivation Anaerobic Jar Candle Jar Dr.T.V.Rao MD 31

32. C. perfringens Laboratory Diagnosis Specimens: pus, necrotic tissue, feces, food, etc. Smears: large gram-positive rods with or without spores, usually in the absence of leukocytes. Culture: anaerobic culture on blood plate. Identification: “ Storming fermentation ”-- clot torn by gas in 24 hrs. Lecithinase test-- precipitate formed around colonies on egg yolk media. Biochemical tests. Dr.T.V.Rao MD 32

33. Nagler’s Reaction This test is done to detect the lecithinase activity The M.O is inoculated on the medium containing human serum or egg yolk (contains lecithin) The plate is incubated anaerobically at 37 C for 24 h Colonies of Cl. perfringens are surrounded by zones of turbidity due to lecithinase activity and the effect is specifically inhibited if Cl. perfringens antiserum containing  antitoxin is present on the medium Dr.T.V.Rao MD 33

34. 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. Dr.T.V.Rao MD 34

35. Nagler Reaction Positive Nagler Reaction Procedure of Nagler Reaction Dr.T.V.Rao MD 35

36. Collecting a Infected Tissues If there are sloughs of necrotic tissue in the wound, small pieces should be transferred aseptically into a sterile screw-capped bottle and examined immediately by microscopy and culture. Specimens of exudate should be taken from the deeper areas of the wound where the infection seems to be most pronounced. Gram smears are prepared. Dr.T.V.Rao MD 36

37. Proceeding with our observations If gas gangrene exists, typical Gram-positive bacilli may predominate, often with other bacteria present in a mixed infection. However, there is usually a pronounced lack of inflammatory cells. Initiation of treatment should not await a full laboratory report and early discussion with the bacteriologist is crucial. Dr.T.V.Rao MD 37

38. Importance of Direct Smears A direct smear of a wound exudate is often of great help in providing evidence of the relative numbers of different bacteria that may be participating in a mixed infection or may merely be present as contaminants, but the distinction is not invariably easy and joint discussions are important. Dr.T.V.Rao MD 38

39. Biochemical Tests Cl. perfringnes characterized by: It ferments many carbohydrates with acid & gas It acidified litmus milk with stormy clot production Nagler reaction is positive Dr.T.V.Rao MD 39

40. C. perfringens Treatment Treatment for Suppurative myositis or myonecrosis: Prompt and extensive debridement. Antibiotics (penicillin) administration. Hyperbaric oxygen may "detoxify" patients rapidly. Efficacy of antitoxins is doubtful. C. perfringens food poisoning requires only symptomatic care. Prevention, and Control Preventive measures: surgical debridement and prophylactic antibiotics. Dr.T.V.Rao MD 40

41. 41 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 Dr.T.V.Rao MD

42. Importance of Prompt Antibiotic Treatment Antibiotic therapy is started immediately in very high doses. This must take account of the likely coexistence of coliform organisms, Gram-positive cocci and faecal anaerobes. Accordingly, penicillin, metronidazole and an aminoglycoside may be given in combination. Alternatively, clindamycin plus an aminoglycoside or a broad-spectrum antibiotic, such as meropenem or imipenem, may be considered. Much intensive supportive therapy is needed. Dr.T.V.Rao MD 42

43. Surgical attention a Urgent priority Prompt and adequate surgical attention to the wound is of the utmost importance Sutures are removed, and necrotic and devitalized tissue is excised with careful debridement. Facial compartments are incised to release tension. Any foreign body is found and removed. The wound is not resutured but is left open after thorough cleansing, and loosely packed. Dr.T.V.Rao MD 43

44. Use of Anti Gas gangrene High titre serum ? The experts have opined that use of Anti Gas Gangrene Serum injection is no longer the preferred treatment for such patients. For the stated reasons, this drug is not in use and opinions may differ Dr.T.V.Rao MD 44

45. Food poisoning strains' of C . perfringens Few strains of C. perfringens can cause food posinoing.Carrier rates for 'typical food poisoning strains' of C. perfringens range from about 2% to more than 30% in different surveys across the world. These bacteria also occur in animals; thus, meat is often contaminated with heat-resistant spores. When meat is cooked in bulk, heat penetration and subsequent cooling is slow unless special cooking precautions are taken. Dr.T.V.Rao MD 45

46. The pathogens propagate in spite of cooking During the cooling period surviving spores may germinate and multiply in the anaerobic environment produced by the cooked meat. Anyone eating this will consume the equivalent of a cooked meat broth culture of the organism. Dr.T.V.Rao MD 46

47. The organisms are protected The organisms are protected from the gastric acid by the protein in the meal and pass in large numbers into the intestine. The vehicle of infection is usually a pre-cooked meat food that has been allowed to stand at a temperature conducive to the multiplication of C. perfringens Dr.T.V.Rao MD 47

48. ENTERITIS NECROTICANS (PIGBEL) Subgroup of C. perfringens type C that produces heat-resistant spores is the cause of a disease that affects New Guinea natives when they have pork feasts. The method of cooking the pork allows the clostridia to survive. When the contaminated meat is eaten along with a sweet potato vegetable that contains a proteinase inhibitor, a toxin (the β-toxin) is able to act on the small intestine to produce a necrotizing enteritis. A successful vaccination programme has reduced the incidence of pigbel dramatically Dr.T.V.Rao MD 48

49. Diagnosis of Food poising Strains Diagnosis depends upon the isolation of similar strains of C. perfringens from the faeces of patients and from others at risk who have eaten the suspected food, and from the food itself. Numbers usually exceed 106 organisms/g faeces. The isolates can be sent to a reference laboratory for special typing to prove their relatedness. Dr.T.V.Rao MD 49

50. Care of the Food a Priority The occurrence of this type of food poisoning is an indictment of the catering practices concerned, as food has to be mishandled to allow the chain of events to take place. Nevertheless, How ever C . perfringens is among the most common causes of food poisoning. Dr.T.V.Rao MD 50

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