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1. Good Luck with the last chance！ 19：30-21：30 Oct. 19th, Fri

2. Anaerobic Bacteria

5. Obligate Anaerobes • In the presence of oxygen, anaerobic bacteria produce toxic products such as superoxide and hydrogen peroxide, but they lack superoxide dismutase (SOD), catalase and peroxidase that detoxify these products:

6. SOD: O2-+2H+ H2O2 Catalase: H2O2  H2O + O2 Peroxidase: H2O2 H2O/NAD to NADH

7. No oxidative phosphorylation • Infection caused by anaerobes usually occurs in the sites with low oxidation-reduction potential such as periodontal pocket, intestinal tract and vagina. • Anaerobes are co-infected with other facultative anaerobe which use up oxygen to establish a local anaerobic environment.

8. Source of anaerobic infection • Endogenous infection: caused by anaerobes of normal flora which are non-spore formers. • Exogenous infection: The pathogens are usually anaerobic spore-formers and come from the environment (e.g., soil). Clostridia is the unique genus of anaerobic spore-formers to cause human diseases.

9. Clostridia • There are at least 118 species, the clinically important species: • Clostridia tetani • Clostridia perfringens • Clostridia botulinum • Clostridia difficile

10. Clostridium tetani spore vegetative

11. Gram-positive rods that form terminal spores

12. Culture: clustering growth with hemolysis occurs on blood agar.

13. Biochemical activity: does not ferment any carbohydrates. • Resistance: spores but not its vegetative form can tolerate boiling for 60 min and stay alive for several ten years in soil.

14. Clostridia tetani is found in soil. It is occasionally found in intestinal flora of humans and animals. • Clostridia tetani is the cause of tetanus when the spores enter wounds.

15. Process to cause tetanus Spore-contaminated soil or other foreign objects • Narrow deep wounds with limited blood and oxygen supply • co-infection with facultative anaerobe can use up the local oxygen Spores germinate and then produce tetanus toxins. The organism still remains in the local wound.

16. Clostridia tetani produces two exotoxins called tetanus toxins: • Tetanolysin: its virulent role remains unknown. • Tetanospasmin: it is a neurotoxin with strong toxicity and the most important virulent factor of the bacterium.

17. Pathogenesis of tetanospasmin • one heavy chain (H chain) • C end：binds to the ganglioside receptors of inhibitory neurones • N end：helps in entrance to the cells • one light chain (L chain) • It contains a zinc endopeptidase • Blocks the release of inhibitory neuronal mediators g-GABA (g-氨基丁酸) and glycin (甘氨酸) • Stops inhibitory nerve impulse to skeletal muscles, resulting in persistent muscle contraction.

18. Typical symptoms include sardonic smile, lockjaw, neck rigidity, opisthotonos and dyspnea

19. This baby has tetanus. The infection is usually caused by exposing to Clostridia tetani when cutting umbilical cord.

20. This the neonatal tetanus patient displays sardonic smile, lockjaw and dyspnea

21. The adult  tetanus patient shows opisthotonos

22. Among all animal species, horses and humans, are most susceptible to tetanospasmin • If not treated in time, about 20% of the patients are died of suffocation and respiratory failure

23. The wound is treated by debridement (清创术) to destroy anaerobic environment.

24. Although antibiotics (streptomycin and erythromycin) are used as part of the treatment, tetanus patients must be promptly treated with tetanus antitoxin (TAT) to neutralize free tetanospasmin. • 1500 ~ 3000 U for prevention • 100.000 ~ 200.000 U for therapy

25. Tetanus toxoid is a component of DPT vaccine (diphtheria toxoid, killed whole cell pertussis, tetanus toxoid).

26. Clostridia • There are at least 118 species, the clinically important species: • Clostridia tetani • Clostridia perfringens • Clostridia botulinum • Clostridia difficile

27. Gram-positive rods, have capsule, can form terminal spores

28. Form double hemolysis circles on blood agar plates. • The α-hemolysis is caused byα-toxin while the β-hemolysis byθ-toxin.

29. Stormy fermentation • Clostridium perfringens shows “stormy fermentation” in litmus milk. Acid turns the pH indicator litmus from blue to pink. The acid and enzymes coagulate proteins to curd. The gas generated in the milk breaks the coagulated proteins.

31. Diseases • Wound contaminated by soil (main source) and mammalian feces • Gas gangrene refers to serious tissue swelling due to release of gas (fermentation product of the bacterium) and tissue necrosis

33. The death can occur within 2 days if untreated. Treatment includes debridement, antitoxin and antibiotic therapy.

34. Diseases • Common diseases: • Gas gangrene • Food poisoning • Other diseases: • Necrotizing Enteritis • Cellulitis (蜂窝织炎) • Septicemia

35. Food poisoning • Marked hypersecretion in jejunum and ileum with loss of fluids and electrolytes in diarrhea.

36. Necrotizing enteritis • an acute necrotizing process in the jejunum with symptoms of abdominal pain, bloody diarrhea and peritonitis. • The death rate of this disease is as high as approximately 50%.

37. Virulent factors • Clostridium perfringens produces over 10 types of toxins. Some toxins are hemolytic, proteolytic, saccharolytic enzymes. Some are lethal and necrotic. • alpha-toxin is the most important, it lyses erythrocytes, platelets, leukocytes and endothelial cells.

38. According to antigenic differences of 4 major toxins, the bacterial strains can be divided into A~E toxic types. • Type A is clinically the most important. Type A can also produce enterotoxin to cause food poisoning.

39. Morphology • Stormy fermentation • alpha-toxin (lecithinase)

40. Debridement (Gas gangrene) • A large dose of antibiotics (penicillin) • Antitoxin against alpha-toxin and hyperbaric oxygenation (高压氧疗法) • No vaccine is available

41. Clostridia • There are at least 118 species, the clinically important species: • Clostridia tetani • Clostridia perfringens • Clostridia botulinum • Clostridia difficile

42. Clostridium botulinum

43. Clostridium botulinum is aGram-positive, anaerobic, spore-forming bacillus. • It produces an enterotoxin (botulinum toxin) that causes food poisoning (botulism). • According to the antigenicity of botulinum toxin, The microbe can be divided into A~G types. • Among the 7 types, type A and then type B strains cause most disease.

44. The foods that are easily contaminated by the toxins or spores are sausage and canned foods abroad, and fermented-bean preparations in China. • In adult, the toxin or spore causes botulism (death rate 50-70%). • In infant (especially younger than 6 months), the toxin or spore causes infant botulism (death rate 1-2%) .

45. Pathogenesis of botulinum toxin • Botulinum toxin is a nuerotoxin. It binds to other non-toxic proteins to form a complex. It is released, when the bacteria are dead and broken. • When the complex enters intestines, the alkaline condition makes botulinum toxin released.

46. Botulinum toxin binds the cellular receptor of neuromuscular junction and then enters the cells to block the release of acetylcholine (乙酰胆碱). • Acetylcholine is a neurotransmitter, which mediates nerve impulses. Therefore, flaccid paralysis occurs.

47. The typical symptoms are double vision and squint (eye), dyscatabrosis (throat), and dyspnea. The patients are usually died of respiratory failure. • 10 ng of Botulinum toxin can kill an adult.

48. Sample collection: foods and patients’ feces. • Identification of botulinum toxin in the samples

49. The patients must be promptly treated withA, B, E multi-valent antitoxinto neutralize the free toxins. • Sometimes antibiotic therapy is needed. • Vaccination does not protect hosts from botulism.

50. Non-spore-forming anaerobesare strict anaerobesand present in large numbers in intestine (95-99% of the total bacterial mass), mouth and genitourinary tract as normal flora. • These anaerobes can be divided into anaerobic Gram-positive or negative cocci or rods belonging to 23 genera. 11 genera are associated with human diseases.