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  1. 1. Anaerobic Spore Forming Bacilli <ul><li>General observations </li></ul><ul><li>Definition </li></ul><ul><li>An organism that: </li></ul><ul><li>Requires reduced oxygen tension for growth </li></ul><ul><li>Fails to grow on the surface of solid media in 10% carbon dioxide in ambient air (18% oxygen) </li></ul>UMMY
  2. 2. Anaerobic Spore Forming Bacilli <ul><li>Does not use oxygen for growth and metabolism, obtain their energy from fermentation reactions </li></ul><ul><li>Grow at low or negative Eh </li></ul><ul><li>Aerobes and facultative anaerobes have the following metabolic systems which are frequently absent in anaerobes: </li></ul>MWAKIBIBI
  3. 3. Anaerobic Spore Forming Bacilli <ul><li>Cytochrome for oxygen metabolism </li></ul><ul><li>Superoxide dismutase which catalyzes: O 2 + O 2 + 2H +  H 2 O 2 + O 2 </li></ul><ul><li>Catalase which catalyzes: </li></ul><ul><li>2H 2 O 2  2H 2 O + O 2 </li></ul>MVINGE
  4. 4. Anaerobic Spore Forming Bacilli <ul><li>Approaches to culture of anaerobes </li></ul><ul><li>Absorption of O 2 </li></ul><ul><li>Alkaline pyrogallol </li></ul><ul><li>Use of aerobic organisms (Fortner’s plate) </li></ul><ul><li>Steel wool soaked in acid </li></ul><ul><li>Copper sulphate containing Tween 80 </li></ul>
  5. 5. Anaerobic Spore Forming Bacilli <ul><li>Use of reducing substances </li></ul><ul><li>Thyoglycolate –Brewer’s broth </li></ul><ul><li>Metallic iron filings </li></ul><ul><li>Cooked meat (Robertson’s cooked medium) </li></ul><ul><li>Cystein </li></ul><ul><li>Dithiothreitol </li></ul>MWAMBENE
  6. 6. Anaerobic Spore Forming Bacilli <ul><li>Exclusion of O 2 from part of medium </li></ul><ul><li>Solid medium – shake cultures </li></ul><ul><li>Deep fluid medium with 0.5% agar </li></ul><ul><li>Use of anaerobic jars </li></ul><ul><li>Mackintosh jar and fieldes cold catalyst </li></ul><ul><li>Brewer’s and Baird </li></ul><ul><li>Gas pak and gas kit system </li></ul><ul><li>Others </li></ul><ul><li>PreReduced Anaerobically Sterilized (PRAS) System </li></ul>
  7. 7. Anaerobic Spore Forming Bacilli <ul><li>Indicators of anaerobiasis </li></ul><ul><li>Chemical indicators </li></ul><ul><li>Methylene blue </li></ul><ul><ul><li>Colourless = oxidized </li></ul></ul><ul><ul><li>Blue = reduced </li></ul></ul><ul><li>Resazurin </li></ul><ul><ul><li>Colourless =  Eh </li></ul></ul><ul><ul><li>Red =  Eh </li></ul></ul>
  8. 8. Anaerobic Spore Forming Bacilli <ul><li>Bacteriological indicators </li></ul><ul><li>Strict anaerobe: C. tetani failure to grow = No anaerobiasis </li></ul><ul><li>Strict aerobe: Pseudomonas aeruginosa : If growth occurs = no anaerobiasis </li></ul>
  9. 9. Anaerobic Spore Forming Bacilli <ul><li>Pointers to anaerobic infections </li></ul><ul><li>Clinical </li></ul><ul><li>Foul smelling discharge (due to short chain fatty acids products of anaerobic metabolism) </li></ul><ul><li>Gas in tissues (due to production of carbon dioxide and hydrogen </li></ul>
  10. 10. Anaerobic Spore Forming Bacilli <ul><li>Laboratory </li></ul><ul><li>Unique morphology on Gram stain </li></ul><ul><li>Failure of organisms seen on Gram stain of original sample to grow aerobically </li></ul><ul><li>Growth in anaerobic zone of fluid media or agar deeps </li></ul><ul><li>Anaerobic growth on media containing </li></ul><ul><li>75 – 100 ul/ml of Kanamycin, neomycin or paromycin </li></ul>
  11. 11. Clostridia <ul><li>Gram-positive, spore forming, motile or non motile bacilli </li></ul><ul><li>Some species are potentially highly pathogenic to humans or animals and produce potent exotoxins </li></ul><ul><li>Found in soil (especially soil fertilized with animal excreta) and in the lower intestinal tract of humans and animals </li></ul>
  12. 12. Clostridia <ul><li>Spores </li></ul><ul><li>Terminal  Clostridium tetani </li></ul><ul><li>Subterminal  Clostridium botulinum </li></ul><ul><li>Central  C. perfringens, C. botulinum </li></ul>
  13. 13. Clostridia <ul><li>Some decompose </li></ul><ul><li>– protein  proteolytic </li></ul><ul><li>– CHO  Sacharolytic </li></ul><ul><li>– neither </li></ul><ul><li>Some are saprophytic </li></ul>
  14. 14. Clostridia <ul><li>Medically important species </li></ul><ul><li>Gas gangrene group: </li></ul><ul><ul><li>C. perfringens ( C. welchii ) </li></ul></ul><ul><ul><li>C. septicum </li></ul></ul><ul><ul><li>C. oedematiens </li></ul></ul><ul><ul><li>C. histolyticum </li></ul></ul><ul><li>C. tetani </li></ul><ul><li>C. botulinum </li></ul><ul><li>C. difficile </li></ul>
  15. 15. Clostridia perfringens <ul><li>Morphology and growth characteristics </li></ul><ul><li>Strongly Gram +ve rod, non motile </li></ul><ul><li>Short, thick, strainght parallel sides and rounded edges </li></ul><ul><li>Spores rarely seen </li></ul>
  16. 16. Clostridia perfringens <ul><li>Sporulation favoured by alkaline environment and absence of fermentable CHO </li></ul><ul><li>If spores are produced, they are large, oval and central </li></ul><ul><li>Capsulated especially at the site of infection </li></ul><ul><li>Capsule disappears when organism is grown on culture </li></ul>
  17. 17. Clostridia perfringens <ul><li>Colonies are 2 – 4 mm in diameter, semitranslucent </li></ul><ul><li>On BA </li></ul><ul><li>Zone of hemolysis caused by θ toxin </li></ul><ul><li>Wider zone of incomplete hemaolysis caused by  toxin (lecithinase C, phospholipase) </li></ul>
  18. 18. Clostridia perfringens <ul><li>Partial hemolysis becomes complete if the plate is refrigerated and rewarmed </li></ul><ul><li>On egg yolk agar it produces diffuse opalescence due to phospholipase </li></ul><ul><li>In cooked meat it produces gas but being predominantly saccharolytic it does not digest meat </li></ul>
  19. 19. Clostridia perfringens <ul><li>Ferments lactose with acid and gas production, thus in litmus milk the organism breaks down lactose producing acid which makes the milk clot </li></ul><ul><li>The clot is ruptured and broke down into pieces (stormy clot) </li></ul>
  20. 20. Clostridia perfringens <ul><li>Antigenic structure </li></ul><ul><li>Serotypes </li></ul><ul><li>Types A – E according to combination of exotoxins produced </li></ul><ul><li>The exotoxins are antigenic </li></ul><ul><li>Toxins </li></ul>
  21. 21. Clostridia perfringens <ul><li> -toxin (phospholipase, lecithinase C) </li></ul><ul><li>hydrolyses lecithin and sphengomyelin thus disrupts the cell membranes of various host cells,including erythrocytes, leucocytes and muscle cells </li></ul><ul><li>θ -toxin oxygen labile hemolysin; alters capillary permeability and toxic for heart muscle </li></ul><ul><li>Responsible for  -hemolysis on blood agar </li></ul>
  22. 22. Clostridia perfringens <ul><li>Enterotoxin – produced by some strains of C. perfringens type A; responsible for food poisoning, released into the upper GIT when ingested vegetative forms undergo sporulation, causes diarrhoea by reversing the flow of water and electrolytes in the small intestine, precise mechanism not yet defined </li></ul>
  23. 23. Clostridia perfringens <ul><li> -toxin, an enterotoxin produced by some strains of C. perfringens type C, induces intestinal paralysis, inflammation and gangrenous necrosis – necrotizing enteritis (enteritis necroticans) </li></ul><ul><li>Enzymes </li></ul><ul><li>Collagenase </li></ul><ul><li>Deoxyribonuclease </li></ul><ul><li>Hyaluronidase </li></ul><ul><li>Proteases </li></ul>
  24. 24. Clostridia tetani <ul><li>Morphology and Growth characteristics </li></ul><ul><li>Widely distributed in nature: soil, GIT of man and other animals </li></ul><ul><li>Gram +ve rods, delicate, may appear filamentous after 48 hrs incubation at 37  C </li></ul><ul><li>Round terminal spores – drumstick appearance </li></ul><ul><li>Flagellated and motile </li></ul>
  25. 25. Clostridia tetani <ul><li>Strictly anaerobic </li></ul><ul><li>Swarming growth on BA; swarming prevented by firm agar (2-3%) or tetanus antitoxin </li></ul><ul><li>Tiny colonies with rhizoids (medusa head appearance) </li></ul><ul><li>On horse BA they are  -hemolytic which tends to change into  -hemolysis after prolonged incubation </li></ul>
  26. 26. Clostridia tetani <ul><li>Hemolysis is due to tetanolysin </li></ul><ul><li>No effect on egg yolk </li></ul><ul><li>Grows in cooked meat but no effect on the meat </li></ul><ul><li>Can be identified by gas chromatography </li></ul><ul><li>Isolation difficult in the lab </li></ul>
  27. 27. Clostridia tetani <ul><li>Antigenic structure </li></ul><ul><li>10 serotypes on the basis of specific flagellar antigens </li></ul><ul><li>All share a common O antigen </li></ul><ul><li>Toxins </li></ul><ul><li>Tetanospasmin (exotoxin), polypeptide, MW 160,000, released in lysis; production under control of a plasmid gene </li></ul>
  28. 28. Clostridia tetani <ul><li>Neurotoxin,inhibits release of acetylcholine, thus interfering with neuromuscular transmission </li></ul><ul><li>Inhibits post synaptic spinal neurons by blocking the release of an inhibitory mediator = generalized muscular spasms, hypereflexia and seizures </li></ul><ul><li>0.1 ug lethal to humans, treatment with formaldehyde  toxoid </li></ul><ul><li>Tetanolysis = hemolysin </li></ul>
  29. 29. Clostridium botulinum <ul><li>Morphology and growth characteristics </li></ul><ul><li>Widely distributed in nature, present in soil, vegetables and gut of man </li></ul><ul><li>Gram +ve, straight rounded edges with oval or rounded centrally placed or subterminal spores </li></ul><ul><li>Strict anaerobe </li></ul>
  30. 30. Clostridium botulinum <ul><li>Antigenic structure </li></ul><ul><li>8 types (A – H) based on serological tests of the neurotoxin </li></ul><ul><li>A, B, E and F = cause botulism in man </li></ul><ul><li>C and D = cause botulism in animals and birds </li></ul><ul><li>G = pathogenicity unknown </li></ul>
  31. 31. Clostridium botulinum <ul><li>Toxins </li></ul><ul><li>8 antigenically distinct neurotoxins; types A, B and E toxins </li></ul><ul><li>MW 150,000, highly toxic, lethal dose for humans < 1 µ g </li></ul><ul><li>Heat labile, destroyed rapidly at 100 C, resistant to GIT enzymes </li></ul><ul><li>Production under control of a temperate phage </li></ul>
  32. 32. Clostridium botulinum <ul><li>Acts on neuromuscular junctions by inhibiting release of acetylcholine, resulting in muscular paralysis </li></ul><ul><li>Voluntary and autonomic cholinergic nervous systems affcted </li></ul>
  33. 33. Clostridium difficile <ul><li>Morphology and growth characteristics </li></ul><ul><li>First isolated from stools of healthy newborns over three decades ago </li></ul><ul><li>Role as an enteric pathogen recently documented </li></ul><ul><li>Widespread in environment, occurs in GIT flora of 2-4% of healthy adults in the community </li></ul>
  34. 34. Clostridium difficile <ul><li>Many hospitals heavily contaminated by spores of this organism </li></ul><ul><li>Slow growing in culture, hence its name </li></ul><ul><li>Antigenic structure </li></ul><ul><li>Toxins </li></ul><ul><li>Medically important strains produce two distinct large polypeptide toxins, A and B </li></ul><ul><li>Toxins encoded in chromosomal genes and released during stationary or post stationary growth phases of the vegetative organisms (or during cell lysis) </li></ul>
  35. 35. Clostridium difficile <ul><li>Toxin A – primarily an enterotoxin; has cholera toxin-like activity and also causes extensive mucosal damage leading to formation of hemorrhagic fluid rich in albumin </li></ul><ul><li>Toxin B – highly potent cytotoxin, decreases cellular protein synthesis and disrupts the microfilament system of cells (like diphtheria toxin) </li></ul><ul><li>Animal studies suggest that toxin A induced mucosal damage allows absorption of toxin B from the human gut </li></ul>