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1 5/9/2015 Clostridium: Anaerobic Endospore formers Filename: Clostridium.ppt.

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Presentation on theme: "1 5/9/2015 Clostridium: Anaerobic Endospore formers Filename: Clostridium.ppt."— Presentation transcript:

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2 1 5/9/2015 Clostridium: Anaerobic Endospore formers Filename: Clostridium.ppt

3 2 5/9/2015 Clostridial Diseases ä Botulism C. botulinum ä Tetanus C. tetani ä Gas gangrene C. perfringens ä Food poisoning Clostridium spp ä Pseudomembranous colitis C. difficile

4 3 5/9/2015 Clostridium ä Gram positive ä Rods ä Endospore formers ä asporogenous ä Obligate Anaerobes!!! ä Aerotolerant spp C. perfringens, C. tetani, C. botulinum, C. difficile ä Soil organisms

5 4 5/9/2015 Botulism ä Intoxication ä Foods ä Meats ä Fish ä low-medium acid canned foods ä Wild birds (limberneck) ä ducks, fish, Inuit -- whale blubber, seal fins

6 5 5/9/2015 Botulism: symptoms ä Adults ä Nerve paralysis ä shorter nerves first ä Blurred vision ä Cardiac failure ä Respiratory failure ä Intoxication ä Infants ä Failure to Thrive ä Dehydration ä Polyneuropathy ä Infection Cl. Botulinum ä umbilical cord

7 6 5/9/2015 Botulism: Symptoms 1st symptoms: weakness and dizziness soon after: blurred vision (double vision), difficulty swallowing, throat pain, constipation, abdominal pain Flaccid paralysis: Bilateral, descending, weakness of the peripheral muscles. Death: respiratory paralysis

8 7 5/9/2015 Botulism Intoxication Entry Ingestion Spread absorbed through intestine spread via blood stream moves up nerves Disease Incubation: 1-2 days Flaccid paralysis, cardiac failure, respiratory failure

9 8 5/9/2015 Botulism: Treatment Disease is progressive may not respond to treatment. -ventilatory support -gastric lavage -penicillin -antitoxin (polyvalent A,B,E) Heat food to 80 C to kill toxin and kill spores.

10 9 5/9/2015 Infant Botulism ä Infant botulism:Colonizes the GI tract of young infants. Appears as non-specific weakness. ä Flaccid paralysis: respiratory arrest. ä Mortality = 1-2% ä Some cases of sudden infant death syndrome have proven to be botulism. Eating honey.

11 10 5/9/2015 Wound botulism: ä rare -- organisms multiply in the wound. ä Can occur through umbilical cord

12 11 5/9/2015 Botulism: Lab Diagnosis Culture: culture organisms from feces, food. Heat to 80 C. Food, stool and patient’s serum. Toxin Assay: mix one portion of each specimen with antitoxin. Keep one portion antitoxin free. Keep one portion antitoxin free.

13 12 5/9/2015 Botulism Toxin Potent neurotoxin regulated by bacteriophage. Toxins: A -- E, C alpha, C beta, F, G Humans: A, B, E 150 Kd protein -- cleaved

14 13 5/9/2015 Botulism Outbreaks by Type

15 14 5/9/2015 Botulism Intoxication in USA YEAR INCIDENCE

16 15 5/9/2015 Botulism in USA; neonates YEAR INCIDENCE

17 16 5/9/2015 Botulinum Toxin ä The heavy chain attaches to the ganglioside receptors in nerves

18 17 5/9/2015 C. botulinum toxin Synaptic activity at cholinergic synapses is mediated by acetylcholine. Acetylcholine is rapidly hydrolysed by acetylcholine esterase. The result is an electrical stimulus.

19 18 5/9/2015 Sequence of Events A. Nerve stimulus -- calcium is stimulated B. Acetylcholine release into the synaptic space -- moves into post synaptic membrane and acts on specific receptors. moves into post synaptic membrane and acts on specific receptors. C. Botulinum toxin interferes with the release of acetylcholine from the synaptic vesicles.

20 19 5/9/2015 Tetanus

21 20 5/9/2015 C. tetani C.tetani looks like a tennis racket. Found in soil, carried by horses. Toxin: heat labile, 150,000 d peptide Neurotoxin: splits carboxy terminal to gangliosides on neuronal membranes. Moves to CNS by retrograde axonal transport.

22 21 5/9/2015 Tetanus Intoxication Entry wound Spread blood stream moves up nerves Disease Incubation: 1-2 days Rigid Paralysis, cardiac failure, respiratory failure

23 22 5/9/2015 Lockjaw

24 23 5/9/2015 Tetanus trismus, risus sardonicus, opisthotonos, trismus, risus sardonicus, opisthotonos, Cephalic -- poor prognosis Localized -- favourable prognosis Prevention: toxoid, 3% formaldehyde

25 24 5/9/2015 Risus sardonicus

26 25 5/9/2015 Opisthotonos

27 26 5/9/2015 Tetanus Neonatorum

28 27 5/9/2015 Tetanospasmin

29 28 5/9/2015 Toxin similarity ä Clostridium botulinum and C. tetani are Zn requiring Endopeptidases that cleave a set of proteins Synaptobrevins found in synaptic vesicles of neurons Interfere with release of neurotransmitters and the normal inhibitory function.

30 29 5/9/2015 Binding regions of tetanus toxin and botulinum toxin are different in terms of cell specificity.

31 30 5/9/2015 Tetanus Distribution

32 31 5/9/2015 Incidence of Tetanus in USA YEAR

33 32 5/9/2015 C.perfringens: Diseases 4 bacteremia 4 myonecrosis 4 gas gangrene 4 cellulitis 4 fascitis 4 food poisoning: enteritis necroticans

34 33 5/9/2015 C. perfringens ä large rectangular, hemolytic, very distinctive spreading colonies. ä target hemolysis ä found in soil and intestines, man and animals

35 34 5/9/2015 C. perfringens toxins ä Alpha toxin is a lecithinase (phospholipase C) ä lyses erythrocytes, platelets, leucocytes, endothelial cells ä Massive hemolysis and tissue destruction ä Theta Toxin- Beta hemolysis- increases permeability-- necrotizing enterocolitis ä Delta -- hemolysis ä Kappa -- collagenase

36 35 5/9/2015 C. perfringens toxins ä Mu -- hyaluronidase ä Nu -- DNAase Lambda toxin -- protease Neuraminidase -- hydrolyses serum glycoproteins Enterotoxin -- reverses water, sodium and chloride transport in the intestine (like V. cholerae) Produced by Group A.

37 36 5/9/2015 Nagler Reaction ä Presumptive identification of C. perfringens ä alpha toxin (lecithinase) hydrolyses phospholipids ä egg yolk agar becomes turbid ä specifically blocked by antitoxin

38 37 5/9/2015 Nagler Reaction blocked by Antibodies

39 38 5/9/2015

40 39 5/9/2015 Clinical Syndromes: Bacteremia - usually transient, only diagnostic with other clinical symptoms clinical symptoms Myonecrosis - gas gangrene - trauma or surgical contaminant - trauma or surgical contaminant

41 40 5/9/2015 Gas Gangrene Entry PenetratingWound Multiplication in dead anaerobic tissue Toxin production hemolysin, proteases, lipase, collagenase Disease necrosis, edema, gas

42 41 5/9/2015

43 42 5/9/2015 C. perfringens Gas gangrene Incubation: <1 week pain severe muscle necrosis shock renal failure death -crepitant -cellulitis & fascitis (no muscle)

44 43 5/9/2015

45 44 5/9/2015 C perfringens Food poisoning incubation 8-24hrs. Abdominal cramps, watery diarrhea lasts less than 24hrs. lasts less than 24hrs. Contaminated meat (left overs)

46 45 5/9/2015 C. difficile Gram + anaerobic rod. -found in normal flora -ultimate opportunistic pathogen -difficult to determine cause as the organism is ubiquitous -not difficult to culture

47 46 5/9/2015 C. difficile Diagnosis: cytotoxin - stool culture C.difficile antigen -- latex agglutination

48 47 5/9/2015 Cytotoxin slurry of stool centrifuged filter through 0.45 u filter 0.1ml - supernatant to buffer at pH 7.2 WI-38 tissue cells human diploid lung fibroblasts Add supernatant to tissue culture. Add supernatant to tissue culture. Observe for cytotoxicity 24 hrs.

49 48 5/9/2015 Mechanism of pathogenicity: Toxin A enterotoxin hypersecretion of fluid Toxin B cytotoxin cytopathic to tissue monolayers

50 49 5/9/2015 C. difficileTreatment: stop antibiotic causing disease metronidazole, vancomycin Relapses due to resistant spores. retreatment with same antibiotic neutralization with specific antitoxin obtained commercially amount of toxin present can be determined by a dilution series of the stool sample.

51 50 5/9/2015 C. difficile Culture: standard test for Clostridia include: indole, sugars, lecithinase, catalase (usually neg.)

52 51 5/9/2015 C. difficile: Latex agglutination stool buffered and centrifuged drop on slide of stool supernatant add 1 drop latex detection reagent. Latex particles coated with rabbit antibody to C.difficile antigen. In presence of C.difficile clumps can be seen by eye.

53 52 5/9/2015 Culture: Inoculate Inoculate anaerobe blood agar days egg yolk medium days Incubation temp. = 30 C except C.perfringens Inoculate cooked meat medium - (broth with meat particles)

54 53 5/9/2015 C. difficile: Culture a/ heat to destroy vegetative cells b/ alcohol spore selection for heat labile spores. Clinical syndromes: most serious is (PMC) Pseudomembranous colitis brought about by destruction of the other indigenous intestinal flora. Ranges from mild to serious. PMC self-limiting.

55 54 5/9/2015 Differential diagnosis: S.aureus, E.coli, shigellosis, acute ulcerative colitis, Campylobacter jejuni

56 55 5/9/2015 The End

57 56 5/9/2015 Key Terms ä Obligate anaerobe ä endospore ä asporogenous ä botulism ä tetanus ä gas gangrene ä pseudomembranous colitis ä myonecrosis ä Botulism toxin ä tetanus toxin ä trismus ä opisthotonus ä risus sardonicus ä C. perfringens enterotoxin ä aerotolerant ä Nagler reaction

58 57 5/9/2015 Key Terms ä Enteritis necroticans ä tetanospasmin

59 58 5/9/2015 Key Organisms ä Clostridium ä C tetani ä C botulinum ä C. perfringens ä C. difficile

60 59 5/9/2015 Key Concepts

61 60 5/9/2015 Epidemiology of Botulism ä Disease/bacterial factors ä Transmission ä who is at risk ä geography/ season ä modes of control

62 61 5/9/2015 Epidemiology of C. difficile infections ä Disease/bacterial factors ä Transmission ä who is at risk ä geography/ season ä modes of control

63 62 5/9/2015 Epidemiology of C. tetani infections ä Disease/bacterial factors ä Transmission ä who is at risk ä geography/ season ä modes of control

64 63 5/9/2015 Epidemiology of C. perfringens infections ä Disease/bacterial factors ä Transmission ä who is at risk ä geography/ season ä modes of control

65 64 5/9/2015 Short Answers ä Construct a table of the virulence factors associated with C. tetani and the biological activity of each ä Use a series of no more than four diagrams to describe the mechanism of tetanospasmin activity ä Describe the clinical manifestions of generalized, cephalic and localized tetanus

66 65 5/9/2015 Short Answers ä Construct a table listing the common clostridial species and the associated human diseases. ä Construct a table listing 5 virulence factors associated with C. difficile and the biological activity of each ä Construct a table of the virulence factors associated with C. perfringens and the biological activity of each


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