1. Anaerobic Bacteria

2. Category Spore-forming: Spore-forming: rod, Gram (+)--- Clostridium rod, Gram (+)--- Clostridium Nonspore-forming: Nonspore-forming: see next slides see next slides

3. Spore-forming: rod, Gram (+)--- Clostridium rod, Gram (+)--- Clostridium Nonspore-forming: Rod, Gram (+) Propionibacterium Propionibacterium 丙酸菌属 Bifidobacterium Lactobacillus Eubacterium Actinomyces Actinomyces Rod, Gram (-) Bacteroides Fusobacterium Campylobacter Bacteroides Fusobacterium 梭菌属 Campylobacter Cocci, Gram (+) Peptococcus Peptostreptococcus Cocci, Gram (-) Veillonella Category

4. Clostridium Species The clostridia are opportunistic pathogens. Nonetheless, they are responsible for some of the deadliest diseases including gas gangrene, tetanus and botulism. Less life- threatening diseases include pseudomembranous colitis (PC) and food poisoning. The clostridia are opportunistic pathogens. Nonetheless, they are responsible for some of the deadliest diseases including gas gangrene, tetanus and botulism. Less life- threatening diseases include pseudomembranous colitis (PC) and food poisoning. cause disease primarily through the production of numerous exotoxins. cause disease primarily through the production of numerous exotoxins. perfringens, tetani, botulinum, difficile perfringens, tetani, botulinum, difficile

5. Clostridium Tetani Pathogenesis of tetanus caused by C tetani

6. General introduction C tetani is found worldwide. Ubiquitous in soil, it is occasionally found in intestinal flora of humans and animals C tetani is found worldwide. Ubiquitous in soil, it is occasionally found in intestinal flora of humans and animals C.tetani is the cause of tetanus,or lockjaw. When spores are introduced into wounds by contaminated soil or foreign objects such as nails or glass splinters C.tetani is the cause of tetanus,or lockjaw. When spores are introduced into wounds by contaminated soil or foreign objects such as nails or glass splinters

7. BIOCHEMICAL CHARACTERISTICS Morphology: long and slender; peritrichous flagella,no capsule, terminal located round spore(drum-stick apperance), its diameter greater than vegetative cell. Morphology: long and slender; peritrichous flagella,no capsule, terminal located round spore(drum-stick apperance), its diameter greater than vegetative cell. Culture:obligate anaerobic; Gram(+); swarming occures on blood agar, faint hemolysis. Culture:obligate anaerobic; Gram(+); swarming occures on blood agar, faint hemolysis. Biochemical activities:does not ferment any carbohydrate and proteins. Biochemical activities:does not ferment any carbohydrate and proteins. Resistance: tolerate boiling for 60 min.alive several ten years in soil. Resistance: tolerate boiling for 60 min.alive several ten years in soil. Classification and Antigenic Types: C tetani is the only species. There are no serotypes Classification and Antigenic Types: C tetani is the only species. There are no serotypes 2-5 x 0.3-0.5um

8. Pathogenicity No invasiveness; toxemia (exogenous infection ） No invasiveness; toxemia (exogenous infection ） produces two exotoxins: tetanolysin, and tetanospasmin(a kind of neurotoxin, toxicity strong) produces two exotoxins: tetanolysin, and tetanospasmin(a kind of neurotoxin, toxicity strong) The actions of tetanospasmin are complex and involve three components of the nervous system: central motor control, autonomic function, and the neuromuscular junction. The actions of tetanospasmin are complex and involve three components of the nervous system: central motor control, autonomic function, and the neuromuscular junction.  retrograde transport to (CNS)  delitescence ： a few days to several weeks  The two animal species most susceptible to this toxemia are horses and humans.

9. Clostridium tetani -Tetanospasmin disseminates systemically disseminates systemically binds to ganglioside receptors binds to ganglioside receptors inhibitory neurones in CNS inhibitory neurones in CNS glycine glycine neurotransmitterneurotransmitter stops nerve impulse to muscles stops nerve impulse to muscles spastic paralysis spastic paralysis 痉挛性麻痹 severe muscle contractions and spasms severe muscle contractions and spasms can be fatal can be fatal

10. Tetanospasmin Tetanospasmin

11. Clinical Manifestations Clinical Manifestations The initial symptom is cramping and twitching of muscles around a wound. The patient usually has no fever but sweats profusely and begins to experience pain, especially in the area of the wound and around the neck and jaw muscles (trismus). The initial symptom is cramping and twitching of muscles around a wound. The patient usually has no fever but sweats profusely and begins to experience pain, especially in the area of the wound and around the neck and jaw muscles (trismus). Portions of the body may become extremely rigid, and opisthotonos(a spasm in which the head and heels are bent backward and the body bowed forward) is common. Portions of the body may become extremely rigid, and opisthotonos 角弓反张 (a spasm in which the head and heels are bent backward and the body bowed forward) is common. Complications include fractures, bowel impaction, intramuscular hematoma, muscle ruptures, and pulmonary, renal, and cardiac problems Complications include fractures, bowel impaction, intramuscular hematoma, muscle ruptures, and pulmonary, renal, and cardiac problems

12. Clinical Manifestations Clinical Manifestations DISEASE CLINCAL MANIFESTATIONSA CLINCAL MANIFESTATIONSA Generalized Involvement of bulbar and paraspinal muscles(trismus or lockjaw, risus sardonicus, difficulty swallowing, irritability, opisthotonos);involvement of autonomic nervous system(sweating, hyper thermia, cardiac arrhythmias, fluctuations in blood pressure) Cephalic Primary infection in head,particularly ear;isolated or combined involvement of cranial nerves, particularly seventh cranial nerve; very poor prognosis Localized Involvement of muscles in area of primary injury; infection may precede generalized disease; favorable prognosis Neonatal Generalized disease in neonates; infection typically originates from umbilical stump;very poor prognosis in infants whose mothers are nonimmune Generalized disease in neonates; infection typically originates from umbilical 脐带 stump;very poor prognosis in infants whose mothers are nonimmune

14. Epidemiology Epidemiology 1 million cases of tetanus occur annually in the world,with a mortality rate ranging from20% to 50%. But rare in most developed countries. 1 million cases of tetanus occur annually in the world,with a mortality rate ranging from20% to 50%. But rare in most developed countries. In some developing countries, tetanus is still one of the ten leading causes of death, and neonatal tetanus accounts for approximately one-half of the cases worldwide. In some developing countries, tetanus is still one of the ten leading causes of death, and neonatal tetanus accounts for approximately one-half of the cases worldwide. In less developed countries, approximate mortality rates remain 85% for neonatal tetanus and 50% for nonneonatal tetanus. In less developed countries, approximate mortality rates remain 85% for neonatal tetanus and 50% for nonneonatal tetanus. In the United States, intravenous drug abusers have become another population with an increasing incidence of clinical tetanus In the United States, intravenous drug abusers have become another population with an increasing incidence of clinical tetanus In untreated tetanus, the fatality rate is 90% for the newborn and 40% for adults. In untreated tetanus, the fatality rate is 90% for the newborn and 40% for adults.

15. Immunity Immunity Humoral immunity(antitoxin) Humoral immunity(antitoxin) There is little, if any, inate immunity and the disease does not produce immunity in the patient. There is little, if any, inate immunity and the disease does not produce immunity in the patient. Active immunity follows vaccination with tetanus toxoid Active immunity follows vaccination with tetanus toxoid

16. Diagnosis Diagnosis Diagnosis is primarily by the clinical symptoms (above). The wound may not be obvious. Diagnosis is primarily by the clinical symptoms (above). The wound may not be obvious. C tetani can be recovered from the wound in only about one-third of the cases. C tetani can be recovered from the wound in only about one-third of the cases. It is important for the clinician to be aware that toxigenic strains of C tetani can grow actively in the wound of an immunized person. It is important for the clinician to be aware that toxigenic strains of C tetani can grow actively in the wound of an immunized person. Numerous syndromes, including rabies and meningitis, have symptoms similar to those of tetanus and must be considered in the differential diagnosis. Numerous syndromes, including rabies and meningitis, have symptoms similar to those of tetanus and must be considered in the differential diagnosis.

17. infant infant DPT (diptheria, pertussis, tetanus) DPT (diptheria, pertussis, tetanus) tetanus toxoid tetanus toxoid – antigenic – no exotoxic activity Vaccination

18. Control Control The offending organism must be removed by local debridemen The offending organism must be removed by local debridemen 清创 术 toxoid toxoid TAT; Metronidazole (For more serious wounds) TAT; Metronidazole (For more serious wounds) AIDS patients may not respond to prophylactic injections of tetanus toxoid AIDS patients may not respond to prophylactic injections of tetanus toxoid

19. C. perfringens soil, fecal contaminationsoil, fecal contamination gas gangrenegas gangrene –swelling of tissues –gas release *fermentation products wound contaminationwound contamination

20. Toxinstoxin Biological Feature Types of Toxins ABCDE  lecithinase; increase the vascular permeability; hemolytic; produces necrotizing activity +++++  Necrotizing activity, induces hypertension by causing release of catecholamines. －++－－  increase the permeability of gastrointestinal wall －－－+－  Necrotizing activity; increase the vascular permeability －－－－+

21. Toxins Many of these toxins have lethal, necrotizing, and hemolytic properties; Many of these toxins have lethal, necrotizing, and hemolytic properties; The alpha toxin produced by all types of C. perfringens, is a lecithinase that lyses erythrocytes, platelets, leukocytes, and endothelial cells. And its lethal action is proportionate to the rate at which it splits lecithin to phosphorylcholine and diglyceride. The alpha toxin produced by all types of C. perfringens, is a lecithinase that lyses erythrocytes, platelets, leukocytes, and endothelial cells. And its lethal action is proportionate to the rate at which it splits lecithin to phosphorylcholine and diglyceride. The theta toxin has similar hemolytic and necrotizing effects. The theta toxin has similar hemolytic and necrotizing effects. DNAase, hyaluronidase, a collagenase are also produced DNAase, hyaluronidase, a collagenase are also produced

22. Enterotoxin Many strains of type A produce enterotoxin, which is a heat-labile protein and destroyed immediately at 100 ℃. Many strains of type A produce enterotoxin, which is a heat-labile protein and destroyed immediately at 100 ℃. Trypsin treatment enhances the toxin activity threefold. Trypsin treatment enhances the toxin activity threefold. The toxin is produced primarily by type A strains but also by a few type C and D strains. The toxin is produced primarily by type A strains but also by a few type C and D strains. It disrupts ion transport in the ileum(primarily) and jejunum by inserting into the cell membrane and altering membrane permeability. It disrupts ion transport in the ileum(primarily) and jejunum by inserting into the cell membrane and altering membrane permeability. As superantigen. As superantigen.

24. Tissue degrading enzymesTissue degrading enzymes – lecithinase [  toxin] – proteolytic enzymes – saccharolytic enzymes Destruction of blood vessels Destruction of blood vessels Tissue necrosis Tissue necrosis Anaerobic environment created Anaerobic environment created Organism spreads Organism spreads Pathogenesis

25. Without treatment death occurs within 2 days  effective antibiotic therapy  debridement  anti-toxin  amputation & death is rare

26. Gas gangrene Gas gangrene is a life-threatening disease with a poor prognosis and often fatal outcome. Gas gangrene is a life-threatening disease with a poor prognosis and often fatal outcome. Initial trauma to host tissue damages muscle and impairs blood supply----lack of oxygenation Initial trauma to host tissue damages muscle and impairs blood supply----lack of oxygenation Initial symptoms : fever and pain in the infected tissue.; more local tissue necrosis and systemic toxemia. Infected muscle is discolored (purple mottling) and edematous and produces a foul- smelling exudate; gas bubbles form from the products of anaerobic fermentation. Initial symptoms : fever and pain in the infected tissue.; more local tissue necrosis and systemic toxemia. Infected muscle is discolored (purple mottling) and edematous and produces a foul- smelling exudate; gas bubbles form from the products of anaerobic fermentation.

27. Gas gangrene As capillary permeability increases, the accumulation of fluid increases, and venous return eventually is curtailed. As capillary permeability increases, the accumulation of fluid increases, and venous return eventually is curtailed. As more tissue becomes involved, the clostridia multiply within the increasing area of dead tissue, releasing more toxins into the local tissue and the systemic circulation. As more tissue becomes involved, the clostridia multiply within the increasing area of dead tissue, releasing more toxins into the local tissue and the systemic circulation.

28. Food poisoning Enterotoxin producing strains. Enterotoxin producing strains. These bacteria are found in mammalian faeces and soil. These bacteria are found in mammalian faeces and soil. Small numbers of the bacteria may also be found in foods and they may propagate rapidly to dangerous concentrations if the food is improperly stored and handled. Small numbers of the bacteria may also be found in foods and they may propagate rapidly to dangerous concentrations if the food is improperly stored and handled.

29. Food poisoning more than 10 8 vegetative cells are ingested and sporulate in the gut, the toxins can act rapidly in the body, causing severe diarrhea in 6-18 hours, dysentery, gangrene, muscle infections more than 10 8 vegetative cells are ingested and sporulate in the gut, the toxins can act rapidly in the body, causing severe diarrhea in 6-18 hours, dysentery, gangrene, muscle infections The action of C. perfringens enterotoxin involves marked hypersecretion in the jejunum and ileum, with loss of fluids and electrolytes in diarrhea. The action of C. perfringens enterotoxin involves marked hypersecretion in the jejunum and ileum, with loss of fluids and electrolytes in diarrhea.

30. Cellulitis, Fasciitis Cellulitis, Fasciitis Cellulitis, Fasciitis Fasciitis : a rapidly progressive, destructive process in which the organisms spread through fascial plan es. Fasciitis : a rapidly progressive, destructive process in which the organisms spread through fascial plan es. Fasciitis causes suppuration and the formation of gas Fasciitis causes suppuration and the formation of gas Absense of muscle involvement Absense of muscle involvement rapidity rapidity

31. Necrotizing Enteritis Necrotizing Enteritis Rare, acute necrotizing process in the jejunum Rare, acute necrotizing process in the jejunum Abdominal pain, bloody diarrhea, shock, and peritonitis Abdominal pain, bloody diarrhea, shock, and peritonitis Mortality: 50% Mortality: 50% Beta-toxin-producing C. perfringens type C Beta-toxin-producing C. perfringens type C Septicemia Septicemia

32. Who is at risk? Surgical patients; patient after trauma with soil contamination. Surgical patients; patient after trauma with soil contamination. People who ingest contaminated meat products (without proper refrigeration or reheating to inactivate endotoxin) People who ingest contaminated meat products (without proper refrigeration or reheating to inactivate endotoxin)

33. Epidemiology C. perfringens type A: the intestinal tract of humans and animals, soil and water contaminated with feces. forms spores under adverse environmental conditions and can survive for prolonged periods. C. perfringens type A: the intestinal tract of humans and animals, soil and water contaminated with feces. forms spores under adverse environmental conditions and can survive for prolonged periods. Type B to E strains colonize the intestinal tract of animals and occasionally humans. Type B to E strains colonize the intestinal tract of animals and occasionally humans.

34. Epidemiology Type A: gas gangrene, soft tissue infections and food poisoning Type A: gas gangrene, soft tissue infections and food poisoning Type C: enteritis; necroticans Type C: enteritis; necroticans

35. lecithinase production lecithinase production Laboratory identification Double Hemolysis Circles

36. C. botulinum

37. Biological Features Anaerobic Anaerobic Gram-positive Gram-positive rod-shaped rod-shaped sporeformer sporeformer produces a protein neurotoxic. produces a protein neurotoxic. soil, sediments of lakes, ponds, decaying vegetation. soil, sediments of lakes, ponds, decaying vegetation. intestinal tracts of birds, mammals and fish. intestinal tracts of birds, mammals and fish.

38. ---A, B, C1, D, E, F, and G. ---type A. 62% ---Not all produce toxin. ---C and D not ---G plasmid encoded. Division

39. ---spores heat resistant. canning. anaerobic environment ---Botulism eating uncooked foods spores ---GI, duodenum, blood stream, neuromuscular synapses. Transmission

40. ---bacterial protease ---light chain,A,50 kDa; heavy chain,100kDa. ---disulfide bond. ---A potent toxin Virulence factors

41. Botulinum toxin binds peripheral nerve receptors binds peripheral nerve receptors acetylcholine neurotransmitteracetylcholine neurotransmitter inhibits nerve impulses inhibits nerve impulses flaccid paralysis flaccid paralysis death death respiratoryrespiratory cardiac failurecardiac failure

42. Botulinum toxin Bioterrorism Bioterrorism not an infectionnot an infection resembles a chemical attackresembles a chemical attack 10 ng can kill a normal adult10 ng can kill a normal adult

43. ---4: foodborne, infant, wound, undetermined. ---Certain foods; wound not. ---Foodborne botulism, consumption. ---Infant botulism, 1976, under 12m. ---ingestion, colonize and produce toxin in the intestinal tract of infants. honey. ---increased. ---internationally recognized. Epidemiology

44. ---18-36 hours: ---weakness, dizziness,dryness of the mouth. ---Nausea,vomiting. ---Neurologic features: blurred vision, inability to swallow, difficulty in speech, descending weakness of skeletal muscles, respiratory paralysis. Clinical syndromes

45. Botulism( 肉毒中毒 ) food poisoning food poisoning rarerare fatalfatal germination of spore germination of spore inadequately sterilized canned food inadequately sterilized canned food homehome not an infection not an infection

46. Infection with C. botulinum Neonatal botulism Neonatal botulism uncommonuncommon the predominant form of botulismthe predominant form of botulism colonization occurscolonization occurs no normal flora to compete no normal flora to compete unlike adult unlike adult

47. Wounds extremely rareextremely rare an infectionan infection

48. Immunity ---specifically neutralized, antitoxin. ---toxoided, make good antigens. ---does not develop, amount toxic. ---Repeated occurrence. ---Once bound, unaffected by antitoxin. ---circulating toxin,neutralized, injection of antitoxin. ---treated immediately with antiserum. ---multivalent toxoid,unjustified,infrequency. experimental vaccine.

49. Diagnosis ---by clinical symptoms alone ---differentiation difficult. --- most direct and effective: serum or feces. ---most sensitive and widely used: mouse neutralization test. 48h. Culturing of specimens 5-7d.

50. Treatment Individuals known to have ingested food with botulism should be treated immediately with antiserum. Individuals known to have ingested food with botulism should be treated immediately with antiserum. antibiotic therapy (if infection) antibiotic therapy (if infection) Vaccination will not protect hosts from botulism, however passive immunisation with antibody is the treatment of choice for cases of botulism.Vaccination will not protect hosts from botulism, however passive immunisation with antibody is the treatment of choice for cases of botulism.

51. Prevention ---proper food handling and preparation. --- spores survive boiling (100 degrees at 1 atm) 1h. ---toxin heat-labile, boiling or intense heating, inactivate the toxin. ---bulge, gas, spoiled.

52. C. difficile After antibiotic useAfter antibiotic use Intestinal normal flora --greatly decreasedIntestinal normal flora --greatly decreased Colonization occursColonization occurs Enterotoxin secretedEnterotoxin secreted Pseudomembanous colitisPseudomembanous colitis

53. Pseudomembranous Colitis Pseudomembranous colitis (PC) results predominantly as a consequence of the elimination of normal intestinal flora through antibiotic therapy. Pseudomembranous colitis (PC) results predominantly as a consequence of the elimination of normal intestinal flora through antibiotic therapy. Symptoms include abdominal pain with a watery diarrhea and leukocytosis. "Pseudomembranes" consisting of fibrin, mucus and leukocytes can be observed by colonoscopy. Symptoms include abdominal pain with a watery diarrhea and leukocytosis. "Pseudomembranes" consisting of fibrin, mucus and leukocytes can be observed by colonoscopy. Untreated pseudomembranous colitis can be fatal in about 27-44%. Untreated pseudomembranous colitis can be fatal in about 27-44%.

54. Therapy Discontinuation of initial antibiotic (e.g. ampicillin) Discontinuation of initial antibiotic (e.g. ampicillin) Specific antibiotic therapy (e.g. vancomycin) Specific antibiotic therapy (e.g. vancomycin)

55. no oxidative phosphorylationno oxidative phosphorylation fermentationfermentation killed by oxygenkilled by oxygen lack certain enzymeslack certain enzymes –superoxide dismutase *O 2 - +2H + H 2 O 2 –catalase *H 2 O 2 H 2 0 + O 2 –peroxidase *H 2 O 2 H 2 0 /NAD to NADH Obligate (strict) anaerobes

56. Strict anaerobe infectious disease Sites throughout body Sites throughout body Muscle, cutaneous/sub-cutaneous necrosis Muscle, cutaneous/sub-cutaneous necrosis Abscesses Abscesses

58. Bacterial Flora of the Body SiteTotal BacteriaRatio (per/ml or gm) Anaerobes:Aerobes Upper Airway Nasal Washings10 3 -10 4 3-5:1 Saliva10 8 -10 9 1:1 Tooth Surface10 10 -10 11 1:1 Gingival Crevice10 11 -10 12 1000:1 Gastrointestinal Tract Stomach 10 2 -10 5 1:1 Small Bowel10 2 -10 4 1:1 Ileum10 4 -10 7 1:1 Colon10 11 -10 12 1000:1 Female Genital Tract Endocervix10 8 -10 9 3-5:1 Vagina10 8 -10 9 3-5:1

59. Problems in identification of anaerobic infections air in sample (sampling, transportation)air in sample (sampling, transportation) –no growth identification takes several days or longeridentification takes several days or longer –limiting usefulness often derived from normal floraoften derived from normal flora –sample contamination can confuse

60. Virulence Factors 1. Anti-phagocytic capsule Also promote abscess formationAlso promote abscess formation 2. Tissue destructive enzymes B. fragilis produces variety of enzymes (lipases, proteases, collagenases) that destroy tissue  Abscess FormationB. fragilis produces variety of enzymes (lipases, proteases, collagenases) that destroy tissue  Abscess Formation 3. Beta-lactamase production B. fragilis – protect themselves and other species in mixed infectionsB. fragilis – protect themselves and other species in mixed infections 4. Superoxide dismutase production Protects bacteria from toxic O 2 radicals as they move out of usual nicheProtects bacteria from toxic O 2 radicals as they move out of usual niche

61. Characteristics of Anaerobic Infections 1. Most pathogenic anaerobes are usually commensals Originate from our own floraOriginate from our own flora 2. Predisposing Conditions Breeches in the mucocutaneous barrierBreeches in the mucocutaneous barrier  displace normal flora  displace normal flora Compromised vascular supplyCompromised vascular supply Trauma with tissue destructionTrauma with tissue destruction Antecedent infectionAntecedent infection

62. Characteristics of Anaerobic Infections 3. Complex Flora Multiple species Multiple species Abdominal Infection  Avg of 5 speciesAbdominal Infection  Avg of 5 species 3 anaerobic 3 anaerobic 2 aerobic 2 aerobic Less complex then nl floraLess complex then nl flora Fecal flora 400 different speciesFecal flora 400 different species Those predominant in stool are not infecting species Those predominant in stool are not infecting species Veillonella, Bifidobacterium  rarely pathogenic Veillonella, Bifidobacterium  rarely pathogenic Species uniquely suited to cause infection predominateSpecies uniquely suited to cause infection predominate 4. Synergistic Mixture of Aerobes & Anaerobes E. coli  Consume O 2 E. coli  Consume O 2 Allow growth of anaerobes Anaerobes  promote growth of other bacteria by being antiphagocytic and producing B- lactamases Anaerobes  promote growth of other bacteria by being antiphagocytic and producing B- lactamases

63. Clues to Anaerobic Infection 1. Infections in continuity to mucosal surfaces 2. Infections with tissue necrosis and abscess formation 3. Putrid odor 4. Gas in tissues 5. Polymicrobial flora 6. Failure to grow in the lab BIOCHEMICAL KITS e.g. API SYSTEMe.g. API SYSTEM GAS CHROMATOGRAPHY volatile fermentation productsvolatile fermentation products

64. Bacteroides fragilis Major disease causing strict anaerobicMajor disease causing strict anaerobic after abdominal surgery after abdominal surgery non-spore-former non-spore-former Prominent capsule Prominent capsule – anti-phagocytic – abscess formation Endotoxin Endotoxin – low toxicity – structure different than other lipolysaccharide

65. Enterobacteriaceae (facultative anaerobes) – commonly cause disease – low numbers gut flora Strict anaerobes Strict anaerobes – much less commonly cause disease – high numbers gut flora – high numbers gut flora.