ANAEROBES

1. ANAEROBES II MBBS Dr Ekta Chourasia Microbiology, GMCA

2. Anaerobes Oxidation-reduction (redox) potential CatalaseSuperoxide dismutase Polymicrobic (mixed) infection Spore formers Clostridium tetaniTetanospasmin C. perfringensLecithinase (phospholipase, alpha toxin)C. perfringens enterotoxin C. botulinumBotulinum toxin C. difficileC. dfficile enterotoxin KEY WORDS Dr Ekta, Microbiology

3. Anaerobes • Definition: Can grow in the absence of OXYGEN. • Strict anaerobes: killed by only a few minutes of exposure to O2. • Aerotolerant anaerobes: can tolerate little exposure to O2 *Anaerobes do not have enzymes for protection against the toxic effects of O2. ** most important than absence of O2 is the presence of a sufficiently low redox potential in the medium Dr Ekta, Microbiology

4. Dr Ekta, Microbiology

5. Anaerobes • Normal flora: gingival crevices, colon • Infections in all anatomic sites: abscesses, tissue necrosis, bacteremia Dr Ekta, Microbiology

6. Anaerobes: Classification • Spore forming anaerobes : Only one genus- Clostridia • Non-spore forming anaerobes – Many genus : both gram positive & gram negative bacteria. Dr Ekta, Microbiology

7. CLOSTRIDIUM • Gram positive bacilli with round or oval spores which can be Terminal : Cl. tetani Sub-terminal : Cl.perfringens Central : Cl. bifermentans • Diameter of the spore is more than that of the vegetative cell – bulging spore Cl. perfringens Cl. tetani Dr Ekta, Microbiology

8. CLOSTRIDIUM: classification based on the type of disease produced A . Tetanus Cl. tetani - Present in soil B. Gas gangrene • Established Cl. perfringens ‘gut’ organism Cl. septicum Cl. novyi • Less pathogenic Cl. histolyticum Cl. fallax • Doubtful Cl. bifermentans Cl. sporogenes Dr Ekta, Microbiology

9. CLOSTRIDIUM: classification based on the type of disease produced C. Food poisoning 1. Gastroenteritis - Cl perfringens Type A 2. Botulism - Cl. botulinum/ Soil 3. Pig-bel - Cl. perfringens type C D. Acute colitis - Cl. difficile / gut organism (pseudomembranous colitis) • Commonest cause of ‘nosocomial’ diarrhoea Dr Ekta, Microbiology

10. Cl. perfringens Dr Ekta, Microbiology

11. Cl. perfringens: Classification • Five strains, A to E • Based on the toxins they produce • Toxins: many, 4 major toxins – alpha, beta, epsilon & iota • Enzymes – collagenase, hyaluronidase Dr Ekta, Microbiology

12. Cl. perfringens : toxins • Alpha toxin – lecithinase C, a phospholipase • Damages cell membranes • Increase capillary permeability • Extravasation & increased tension in affected muscles • Lysis of erythrocytes: hemolytic anemia & hemoglobinuria • Beta, epsilon & iota - necrosis Dr Ekta, Microbiology

13. Cl. perfringens: Pathogenicity • Soft tissue infection: Gas gangrene (Type A) • Enteric infections: • Food poisoning (some strains of type A) • Gangrenous appendicitis (A + D) • Necrotising enteritis (C) • Biliary tract infections • Other diseases: • Brain abscess & meningitis • Thoracic infections Dr Ekta, Microbiology

14. GAS GANGRENE • Life threatening condition characterised by: • Muscle necrosis with edema • Sepsis • Gas production - usually a mixture of hydrogen, carbon dioxide, nitrogen and oxygen • Also called Malignant edema/ Anaerobic myositis/ Clostridial myonecrosis. • Can rapidly lead to septicemia, shock and death. • Mostly follows • Trauma e.g. burns, crush injuries, battle wounds, open fractures, large muscle involvement e.g. thigh • Features relating to thewounde.g. contamination with dirt or shrapnel • Surgeries, Abortion (especially criminal abortion) and Caesarian section • Intramuscular injections Dr Ekta, Microbiology

15. Gas gangrene: causative agents • Clostridium species • Anaerobic streptococci • Bacteroides species • Facultative anaerobes e.g. E.coli, Proteus, staphylococci. Dr Ekta, Microbiology

16. Pathogenesis of Gas Gangrene • Entry of clostridia (spores) into the wounds along with implanted foreign particles such as soil, road dust, bits of clothing or sharpnel • Anaerobic/ low oxygen tension environment • Germination of spores • Release of exotoxins (lecithinases, collagenases & hyaluronidases) • Extensive tissue damage – edema, necrosis • Gas production (crepitus) Dr Ekta, Microbiology

17. Pathogenesis of Gas gangrene Dr Ekta, Microbiology

18. Clinical Presentation • Initially - no skin changes just pain • Systemic symptoms e.g. fever, dehydration • Once nerves damaged anaesthesia occurs • Paralysis • Skin changes - cellulitic progressing to dark purple; develop vesicles and bullae • Subcutaneous air on palpation (may not be present early on) • Foul smelling discharge • Oedema • Necrotic or haemorrhagic tissue • Patients may also present in septicaemic shock with tachycardia, hypotension, fever, stupor Dr Ekta, Microbiology

19. Laboratory diagnosis • Specimen – • muscles at the edge of infected area • exudates from the site of active infection/ deeper parts of wound • necrotic tissue. • Microscopy– scanty pus cells, regularly shaped (box-car shape) gram +ve bacilli with / without spores Dr Ekta, Microbiology

20. Laboratory Diagnosis • Culture Media • Robertson cooked meat medium (RCM) • Media containing reducing substances like unsaturated fatty acids, ascorbic acid, thioglycollic acid, glutathione • Culture methods • McIntosh-Fildes’ anaerobic jar • Gaspak method • Anaerobic Bio-hood • Deep butt culture Anaerobic Bio hood Dr Ekta, Microbiology

21. Gaspak McIntosh-Fildes’ anaerobic jar Dr Ekta, Microbiology

22. Laboratory Diagnosis • Cultural Characteristics • RCM Broth – meat turns pink (saccharolytic reaction) • Litmus milk test/ Stormy fermentation • Target Hemolysis Dr Ekta, Microbiology

23. Laboratory Diagnosis • Detection of Alpha toxin: “Nagler Reaction” Dr Ekta, Microbiology

24. Management of gas gangrene Dr Ekta, Microbiology

25. Management • Supportive therapy - e.g. analgesia, oxygen, intravenous fluids and good nourishment • Surgical - radical debridement of necrotic tissue (may require amputation if limb involved) • Antibiotics - these do not work alone as they are unable to penetrate the necrotic tissue. Cover gram negative, gram positive and anaerobes e.g. combination of penicillin, gentamicin and metronidazole • Hyperbaric oxygen therapy - kills anaerobic C. perfringens; but efficacy not proven Dr Ekta, Microbiology

26. Cl. difficile • Gram +ve bacilli with large, oval & terminal spores • Normally present in the gut – 3% of healthy adults, 66% of infants • Disease caused - • Pseudomembranous colitis PMC • Antibiotic associated diarrhoea AAD • Antibiotic associated colitis AAC Dr Ekta, Microbiology

27. Pathogenesis of PMC • Complication of oral antibiotic therapy – • Clindamycin • Lincomycin • Ampicillin • Tetracycline • Chloramphenicol • Occurs due to alteration in normal gut flora and overgrowth of Cl. difficile – • 4-9 days after starting antibiotic therapy • Up to 6 weeks after discontinuation • Person to person spread - spores shed in feces Dr Ekta, Microbiology

28. Pathogenesis of Antibiotic Associated Diarrhoea Dr Ekta, Microbiology

29. Risk factors • Admission to intensive care unit • Advanced age >65 years • Antibiotic therapy (overuse) • Prolonged hospital stay • Immunosuppressive therapy • Multiple and severe underlying diseases • Recent surgical procedure • Sharing a hospital room with a difficile-infected patient Dr Ekta, Microbiology

30. PSEUDOMEMBRANOUS COLITIS Virulence factors Enterotoxin - (Toxin A) Cytotoxin - (Toxin B) Diagnosis Clinical suspicion Culture of feces Detection of toxin Management Discontinue antibiotics - Ampi/Tetra/Clinda Oral metronidazole Oral vancomycin Dr Ekta, Microbiology

31. Prevention • Good personal hygiene • Bathroom, kitchens & other areas to be cleaned regularly with detergents / disinfectants • Isolation of patient with difficile diarrhoea Dr Ekta, Microbiology

32. Summary – Cl. perfringens Anaerobic jar/ Gas pak RCM - sacchrolytic Gas gangrene: Cl perfringens Stormy fermentation / litmus milk test Acquired by wound exposure to contaminated soil / dust Demonstration of toxin by Nagler’s reaction Alpha toxin (Lecithinase C): edema, crepitus Other toxins - necrosis Wound debridement, antigas gangrene serum, antibiotic, amputaion, hyperbaric oxygen Gram postive bacilli: box car appearance Food poisoning, intestinal infections Dr Ekta, Microbiology

33. Summary – Cl. difficile Gram positive bacillus, oval terminal spores Antibiotic associated colitis / Pseudomembranous colitis Enterotoxin and Cytotoxin Ampicillin, Clindamycin, Lincomycin, Tetracycline, Chloramphenicol Treatment: Metronidazole / Vancomycin Dr Ekta, Microbiology

34. Clostridium tetani • Gram +ve bacilli with terminal, spherical spore • Drumstick appearance • Motile with peritrichous flagella • Present in soil, intestines of humans & animals and inanimate objects like street & hospital dust, cotton wool, bandages, catgut, wall plaster, clothings, etc Dr Ekta, Microbiology

35. Clostridium tetani • Ten serological types based on agglutination • All types produce the same toxin • Toxins : • Tetanospasmin - neurotoxin • Tetanolysin • Tetanospasmin – responsible for tetanus, antigenic, can be neutralised by antitoxin & can be toxoided Dr Ekta, Microbiology

36. Pathogenesis of Tetanus Source of Infection Contaminated soil / dust Mode of transmission Injury, puncture wounds, wound contamination Unhygienic Complication Septic abortion, Cutting umbilical stump Ear boring / circumcision / unsterile injections Virulence factor Neurotoxin (tetanospasmin) Dr Ekta, Microbiology

37. Pathogenesis of Tetanus Injury & entry of spores Transported to CNS via peripheral nerve Germination of spores Fixed to gangliosides at the presynaptic inhibitory motor nerve endings Toxin release (tetanospasmin) Blocks the release of inhibitory neurotransmitters across the synaptic cleft (Presynaptic Inhibition) Toxin absorption by motor nerve endings Abolition of spinal inhibition - results in muscle rigidity & spasms Dr Ekta, Microbiology

38. Dr Ekta, Microbiology

39. Clinical Features • Incubation period: commonly 6-12 days • I.P. is influenced by • Site & nature of wound • Dose & toxigenicity of organism • Immune status of patient * IP is of prognostic significance (short IP – grave prognosis) • Affects skeletal muscle • Begins with lock jaw (trismus) stiffness of the neck, difficulty in swallowing, and rigidity of pectoral and calf muscles (ophisthotonus) • Severe muscle spasm may end in respiratory failure. Dr Ekta, Microbiology

40. Tetanus – spastic paralysis Ophisthotonus Dr Ekta, Microbiology

41. Neonatal Tetanus • A form of generalized tetanus that occurs • in newborn infants • if the mother has never been immunized. • through infection of the unhealed umbilical stump, particularly when the stump is cut with a non-sterile instrument. Dr Ekta, Microbiology

42. Laboratory diagnosis • Specimen - excised tissue bits, necrotic depths of wounds • Microscopy - Gram +ve bacilli with spherical & terminal spore (Drum stick appearance) • Culture • BA – swarming • RCM with polymyxin B (selective) – mild proteolytic reaction Dr Ekta, Microbiology

43. Laboratory diagnosis • Demonstration of Toxin • on BA with 4% agar & incorporation of antitoxin on one half – hemolysis around colonies only on the half without antitoxin • Not reliable – demonstrates tetanolysin Dr Ekta, Microbiology

44. Treatment • Mild tetanus casescan be treated with: • Tetanus immune globulin (TIG) IV or IM • Metronidazole IV for 10 days • Diazepam • Tetanus vaccination (TT) • Wound debridement • Wound toilet Dr Ekta, Microbiology

45. Treatment • Severe tetanuscases will require admission to intensive care (special isolated units). In addition to the measures listed for mild tetanus: • Human TIG injected intrathecally (increases clinical improvement from 4% to 35%) • Tracheostomy and mechanical ventilation for 3 to 4 weeks • Magnesium, as an IV infusion, to prevent muscle spasm • Diazepam as a continuous IV infusion • Full course of active immunisation to patient recovering from tetanus Dr Ekta, Microbiology

46. Prevention • Surgical prophylaxis • Antibiotic prophylaxis • Immunoprophylaxis – • Passive - human TIG • Active – Tetanus toxoid (TT), DPT DPT - Primary: 2, 4, 6 & 18 months Booster: 5 years & only DT every 10 years • Combined – TIG + TT Dr Ekta, Microbiology

47. Clostridium botulinum • Causes Botulism, a paralytic disease • Classified into 8 types based on the toxins produced – A, B, C1, C2, D, E, F G • All are neurotoxins & identical in their activity except C2 which is a cytotoxin • Botulinum exotoxin is • Produced intracellularly • Released only on death or autolysis of cell. • Slow acting neurotoxin Dr Ekta, Microbiology

48. Botulism and its types • Presents as food poisoning • Three types – • Food borne • Wound botulism: local production & absorption of toxin, very rare • Infant botulism: ingestion of spores in food (honey), less than 12 months Dr Ekta, Microbiology

49. Foodborne botulism (flaccid paralysis) • Ingestion of preformed toxins - A, B, E & F • Source – preserved (canned) food like meat & meat products, fish & canned vegetables • Type E – associated with fish & seafoods • Cans are often inflated & show bubbles on opening • Symptoms – vomiting, constipation, difficulty in swallowing, speaking & breathing. Develops after 12-36 hrs * Flaccid paralysis (4Ds) – diplopia, dysphagia, dysarthria, dilatation of pupil • End in death due to respiratory paralysis Dr Ekta, Microbiology

50. Pathogenesis of Botulism Dr Ekta, Microbiology