The document discusses several Clostridium species that are important anaerobic pathogens. It describes Clostridium perfringens, the main cause of gas gangrene, and its virulence factors including alpha toxin. It also discusses Clostridium botulinum, which causes botulism through its powerful neurotoxin, and Clostridium tetani, which causes tetanus through its potent exotoxin. Clostridium difficile is noted as the cause of antibiotic-associated colitis.

1. Agents of anaerobe infections Clostridia

2. General gropes of bacteria with respect to oxygen requirements Obligate (strict) aerobes Obligate (strict) anaerobes Facultative anaerobes Microaerophiles Aerotolerant microorganisms Capnophiles

3. Obligate anaerobes There are microorganisms that cannot multiply is any oxygen is present. Some members are actually killed by traces of oxygen because they cannot modify the toxic forms of oxygen produced in metabolism. Among the more important anaerobic pathogens are some species of Clostridium, Bacteroides

4. Enzyme content of bacteria with different requirement for oxygen Neither catalase nor superoxide dismutase Strict anaerobe Superoxide dismutase Aerotolerant Small amount of catalase and superoxide dismutase Microaerophile Catalase Superoxide dismutase Facultative anaerobe Catalase – H 2 O 2  H 2 O + O 2 Superoxide dismutase O 2 - +2H+  O 2 + H 2 O 2  H 2 O + O 2 Strict aerobe Enzyme content for O 2 detoxification Name

5. Cultivation of anaerobes

6. Anaerobe microorganisms that are medically important Spore-forming : Gram-positive bacteria Clostridium Nonspore-forming : Gram-positive bacteria : Actinomyces Bifidobacterium Lactobacillus Propionobacterium Gram-positive cocci : Peptococcus Peptostreptococcus Gram-negative bacteria and curved forms Anaerovibrio Bacteroides (>40 species) Fusobacterium Prevatella Campylobacter Leptotrichia Porphyromonas Treponema Borellia Gram-negative cocci Veilonella

7. General properties of Clostridia Gram-positive spore-forming rods Oval or spherical spores often have diameter more than the vegetative cell (therefore is named Clostridium) Anaerobes Catalase-negative Widely distributed in soil, vegetation, and commensals inhabit the bodies of humans and other animals There are over than 120 species General virulence factor of pathogenic clostridia is very powerful exotoxin

8. Clostridia that are implicated in serious human disease Cl.tetani causes tetanus Cl.botulinum causes botulism Cl.perfringens Cl.novyi Cl.septicum Cl.histolyticum Cl.sporogenes Cl.difficile cause causes pseudomembranous colitis (or antibiotic-associated colitis) cause gas gangrene first three – in monoculture last two – only in association with one of first

9. Clostridium perfringens The main causative agent of gas gangrene (or anaerobe infection of wound). Gram-positive rod with central or terminal endospore Encapsulated Nonmotile Anaerobes Commonly founded in human and animal intestines as well in soil Besides gas gangrene causes food poisoning with diarrhea, abdominal cramps, nausea and vomiting ( symptoms are connected with spore germination in the intestine and enterotoxin production)

10. Smear of Cl.perfringens

11. Cl.perfringens in the smear from wound exudate

12. Sporulation of Cl. perfringens

13. Five different types of Cl.perfringens are recognized, depend on which toxins they produce – A, B, C, D, E

14. Cl.perfringens virulence factors  exotoxin - lecithinase (the most potent one) kills leukocytes, causes red blood cell rupture, edema and tissue destruction by degrading the lecithin component of their membranes. On blood agar causes  -hemolysis  exotoxin causes  -hemolysis on the blood agar Enzymes: Collagenase Hyaluronidase DNase

15. Lecithinase and hemolytic activities of Cl. perfringens Absence of lecithinase effect when antitoxin is used Hemolysis Lecithinase effect Absence of hemolysis when antitoxin is used

16. Pathogenesis of gas gangrene Agent – Cl.perfringens: other Clostridia less frequently Sours of infection. The natural habitat of C.perfringens included both the soil and the human intestine. Transmission. Ednospores commonly contaminate wounds (especially puncture and gunshot), crushing injuries (if anaerobic condition) Incubation period – 1 to 5 days Spores germinate, vegetative bacilli multiply and release enzymes for invasion and exotoxin

17. Gas gangrene therefore the gas forms in tissues due to fermentation of muscle carbohydrates, amino acids and glycogen.

18. Changes in the infected tissue The gas formed in the tissue can destroy muscle structure

19. Pathogenesis of gas gangrene Symptoms: pain, edema, and a bloody exudate in the lesion, fever, tachycardia, and blackened necrotic tissue filled with bubbles of gas.

20. Treatment Surgical removal of all dead and infected tissues Hyperbaric oxygen treatment (it inhibits growth of the clostridia, thereby stopping release of toxin, and it also improves oxygenation of injured tissue) Antibiotics administration (penicillin)

21. Laboratory diagnosis Bacterioscopy. Digested muscle, Gram-positive rods, and few of no leukocytes Bacteriological method . Cultivation bacilli in anaerobic condition. Identification according morphology, biochemical properties; toxin production.

22. Botulism is the most feared type of food poisoning because it can result in paralysis and death Gram-positive anaerobe rod with subterminal spore Widely distributed in soils around the world Has very powerful exotoxin

23. Cl. botulinum morphology

24. Cl.botulinum sporulation process

25. Virulence factor Exotoxin. It is neurotoxin, meaning that it acts against the nervous system, and is one of the most powerful poisons known. The toxin acts by blocking the transmission of nerve signals to the muscles, producing paralysis. 100 milligrams of the toxin would be sufficient to kill all population of the Earth. 1 gram of purified crystallized botulinum toxin contains 10 12 fatal doses for a human

26. There are clinical forms of botulism: Botulinum food poisoning is intoxication associated with eating poorly preserved food, contaminated by Cl.botulinum spores. These spores later germinate (in anaerobic condition) and growth of the bacteria results in the release of exotoxin into the food. The neurotoxin survives stomach acid and pepsin, is absorbed into the blood stream, and is carried to nerves. Incubation period 12-36 hours. Wound botulism. The spores enter a wound or puncture, germinate, and produce toxin Infant botulism, in which the bacilli grow in the gut and produce toxin (in contrast the infant, the adult intestinal tract normally inhibits this sort of infection) Wound and infant botulism are generally milder than first form

27. Symptoms Diplopia, dysphagia, weakness, nausea, vomiting and diarrhea. Nerve involvement leads to generalized paralysis and respiratory insufficiency Respiratory paralysis is the most common cause of death. Despite treatment, about one fourth of the victims of botulism die.

28. Serotypes of Cl.botulinum and its connection with toxigenicity Bacterial chromosome - G Bacterial chromosome + F Temperate bacteriophage + E Temperate bacteriophage - D Temperate bacteriophage - С2 Temperate bacteriophage - С1 Bacterial chromosome + В Bacterial chromosome + А Location of tox-gen Human sensitivity Serotype

29. Treatment and prevention Botulism is treated by administering the antitoxin specific for the causative serotype of Cl.botulinum. Infectious botulism is treated with antibiotics – penicillin (addition to antitoxin) Immunization with toxoid is not generally available

30. Laboratory diagnosis Biological method. Botulinum toxin is demonstrable in uneaten food and the patient’s serum by neutralization test in mouse (protection test) – mice are inoculated with a sample of the clinical specimen and will die unless protected by antitoxin.

31. Susceptibility of animals to botulinum toxin Susceptible : Mouse Guinea pig Cat Hors Duck Unsusceptible : Rat Dog Chicken

32. Guinea pig, effected by botulinum toxin

33. Clostridium tetani Cause tetanus or lockjaw Gram-positive motile anaerobic rod with spherical terminal spore Does not ferment carbohydrates but ferment proteins Is a common resident of cultivated soil and the gastrointestinal tract of animals Has potent exotoxin Ziehl-Neelsen staining Red acid-fast spore at the end of bacillus

34. Cl.tetani morphology Spherical terminal spores

35. Cultural properties of Cl.tetani Swarming growth on the solid media

36. Cl.tetani sporulation process

37. Virulence factor Extremely potent exotoxin with neurotoxic activity It consist of 2 components: tetanospasmin and tetanolysin (causes lysis of erythrocytes) Tetanospasmin attaches to motor nerves where they join the muscles. The toxin is taken into the nerve by endocytosis and is carried intra-axonally to the central nervous system, where it binds to ganglioside receptors and blocks release of inhibitory madiators at spinal synapses. So tetanospasmin alters the usual regulation mechanisms for muscle contraction. As result, the muscle are released from normal inhibition and begin to contract uncontrollably.

38. Clinical symptoms The first symptoms are clenching of the jaw, followed in succession by extreme arching of the back, flexion of the arms, and extension of the legs.

39. Death is most often due to paralysis of the respiratory muscles and respiratory collapse. The fatality rate ranging from 10% to 70%.

40. Pathogenesis Spores usually enter the body through accidental puncture wounds, burns, frostbite, and crushed body parts. Spores germinate and release exotoxin. Neonatal tetanus occurs when spores enter the newborn through umbilical wound after delivery. Baby with neonatal tetanus

41. Laboratory diagnosis has limited value Bacteriological method. Biological method. Infecting of mouse causes tetanus and death. Neutralization test in mice can be used – mice protected by antitoxin (antibodies to botulinum exotoxin) remain live.

42. Tetanus in animals

43. Treatment and prevention Tetanus is treated by administering tetanus antitoxin to neutralize any toxin not yet attached to motor nerve cells An antibiotics such as penicillin For prevention use toxoid (formaldehyde-treated exotoxin)

44. C lostridium difficile Cl.defficile causes pseudomembranous or antibiotic-associated colitis. Antibiotics suppress drug-sensitive normal flora, allowing Cl.defficile to multiply and produce exotoxins A and B: toxin A is an enterotoxin that causes watery diarrhea toxin B is a cytotoxin that causes damage to the colonic mucosa, leading to pseudomembrane formation Clindamycin and ampicillin are 2 of many antibiotics that cause this colitis

45. Action of B cytotoxin to colonic mucosa Yellow-white plaques formation on the colonic mucosa.

46. Diagnosis, treatment, prevention Diagnosis. Exotoxin B is detected in filtrates of stool samples by its cytotoxin effect on cultured cells. It is identified by inhibition of cytotoxicity by specific antibody. An ELISA (immuno-enzyme test) is available for detection both exotoxins A and B. Treatment. The causative antibiotics should be withdrawn. Oral metronidazole should be given and fluids replaced. Prevention. There are no vaccines