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

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

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

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

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

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

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

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.

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.

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

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.

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

13 5/9/2015 Botulism Outbreaks by Type

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

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

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

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.

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.

19 5/9/2015 Tetanus

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.

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

22 5/9/2015 Lockjaw

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

24 5/9/2015 Risus sardonicus

25 5/9/2015 Opisthotonos

26 5/9/2015 Tetanus Neonatorum

27 5/9/2015 Tetanospasmin

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.

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

30 5/9/2015 Tetanus Distribution

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

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

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

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

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.

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

37 5/9/2015 Nagler Reaction blocked by Antibodies

38 5/9/2015

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

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

41 5/9/2015

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

43 5/9/2015

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)

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

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

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.

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

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.

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

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.

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)

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.

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

55 5/9/2015 The End

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

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

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

59 5/9/2015 Key Concepts

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

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

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

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

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

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