1. Clostridial diseases Anthrax

2. Clostridial diseases Clostridium spp. Clostridium spp. Gram positive rods Gram positive rods Obligate anaerobes Obligate anaerobes Endospore forming Endospore forming

3. Clostridium spp. Endospores Endospores Resistant to: Resistant to: Heat Heat Disinfectants Disinfectants

4. Clostridium spp. Soil (found on just about all environmental surfaces) Soil (found on just about all environmental surfaces) Fecal matter of humans and animals Fecal matter of humans and animals Aquatic sediments Aquatic sediments

5. Clostridium spp. (obligate anaerobes) Rapidly killed by highly reactive oxygen radicals Rapidly killed by highly reactive oxygen radicals Singlet oxygen Singlet oxygen Superoxide free radicals Superoxide free radicals

6. The Clostridia genus The clostridia produce a wide variety of extracellular enzymes to degrade large biological molecules in the environment into fermentable components The clostridia produce a wide variety of extracellular enzymes to degrade large biological molecules in the environment into fermentable components Hence, the clostridia play an important role in nature in biodegradation and the carbon cycle Hence, the clostridia play an important role in nature in biodegradation and the carbon cycle In anaerobic clostridial infections, these enzymes play a role in invasion and pathology In anaerobic clostridial infections, these enzymes play a role in invasion and pathology

7. Grown in the laboratory under anaerobic conditions

8. Who are the Clostridia’s? Most of the clostridia are saprophytes but a few are pathogenic for humans Most of the clostridia are saprophytes but a few are pathogenic for humans Those that are pathogens have primarily a saprophytic existence in nature and, in a sense, are opportunistic pathogens Those that are pathogens have primarily a saprophytic existence in nature and, in a sense, are opportunistic pathogens Clostridium tetani and Clostridium botulinum produce the most potent biological toxins known to affect humans Clostridium tetani and Clostridium botulinum produce the most potent biological toxins known to affect humans

9. Clostridium perfringens C. perfringens produces a huge array of invasins and exotoxins, causes wound and surgical infections that lead to gas gangrene, in addition to severe uterine infections. C. perfringens produces a huge array of invasins and exotoxins, causes wound and surgical infections that lead to gas gangrene, in addition to severe uterine infections. Clostridial hemolysins and extracellular enzymes such as proteases, lipases, collagenase and hyaluronidase, contribute to the invasive process Clostridial hemolysins and extracellular enzymes such as proteases, lipases, collagenase and hyaluronidase, contribute to the invasive process C. perfringens also produces an enterotoxin and is an important cause of food poisoning. C. perfringens also produces an enterotoxin and is an important cause of food poisoning. Usually C. perfringens and C. botulinum is encountered in improperly sterilized (canned) foods in which endospores have germinated Usually C. perfringens and C. botulinum is encountered in improperly sterilized (canned) foods in which endospores have germinated

10. Clostridium difficile Pseudomembranous colitis in humans is caused by overgrowth ofClostridium difficile in the colon, usually after the normal flora has been disturbed by antimicrobial chemotherapy. Pseudomembranous colitis in humans is caused by overgrowth ofClostridium difficile in the colon, usually after the normal flora has been disturbed by antimicrobial chemotherapy. C. difficile produces two toxins: Toxin A is referred to as an enterotoxin because it causes fluid accumulation in the bowel C. difficile produces two toxins: Toxin A is referred to as an enterotoxin because it causes fluid accumulation in the bowel Toxin B is an extremely lethal (cytopathic) toxin. Toxin B is an extremely lethal (cytopathic) toxin.

11. Clostridium tetani Clostridium tetani is the causative agent of tetanus. Clostridium tetani is the causative agent of tetanus. The organism is found in soil, especially heavily- manured soils, and in the intestinal tracts and feces of various animals. The organism is found in soil, especially heavily- manured soils, and in the intestinal tracts and feces of various animals. Carrier rates in humans vary from 0 to 25%, and the organism is thought to be a transient member of the flora whose presence depends upon ingestion. Carrier rates in humans vary from 0 to 25%, and the organism is thought to be a transient member of the flora whose presence depends upon ingestion. The organism produces terminal spores within a swollen sporangium giving it a distinctive drumstick appearance. The organism produces terminal spores within a swollen sporangium giving it a distinctive drumstick appearance. Although the bacterium has a typical Gram-positive cell wall, it may stain Gram-negative or Gram- variable, especially in older cells. Although the bacterium has a typical Gram-positive cell wall, it may stain Gram-negative or Gram- variable, especially in older cells.

12. Neutralization of oxygen radicals Superoxide Dismutase (SOD) Superoxide Dismutase (SOD) Catalase Catalase Clostridium spp. lacks these enzymes Clostridium spp. lacks these enzymes

13. Clostridium spp. C. tetani C. tetani C. botulinum C. botulinum C. perfringens C. perfringens C. difficile C. difficile

14. Clostridium tetani “Drumstick” appearance

15. Clostridial diseases Tetanus Tetanus Botulism Botulism Gas gangrene Gas gangrene Foodborne gastroenteritis Foodborne gastroenteritis Pseudomembrane colitis Pseudomembrane colitis

16. TETANUS Localized infection, caused by a C.tetani toxin Localized infection, caused by a C.tetani toxin General muscle spasms-due to neurotoxin General muscle spasms-due to neurotoxin

17. Trauma (often trivial) Trauma (often trivial) Chronic skin ulcers Chronic skin ulcers Umbilical cord (clay matter ritual in Africa) Umbilical cord (clay matter ritual in Africa) Abortion (unsanitary abortions) Abortion (unsanitary abortions) TETANUS (cont.)

18. 300,000/year world wide 300,000/year world wide 100 cases/year in the USA (24 fatal) 100 cases/year in the USA (24 fatal) 70% unvaccinated, or incomplete vaccination regime 70% unvaccinated, or incomplete vaccination regime Mostly 60 years and older Mostly 60 years and older

19. TETANUS (cont.) Spores in wound Spores in wound Infection and/or tissue necrosis Infection and/or tissue necrosis Low O 2 tension Low O 2 tension Spore germination Spore germination

20. TETANOSPASMIN Blocks the muscle relaxation pathway Blocks the muscle relaxation pathway Death results from spasms of respiratory muscles Death results from spasms of respiratory muscles Released from dead bacterial cells Released from dead bacterial cells

21. TETANOSPASMIN (cont.) Once it attaches to nerves, therapy is usually ineffective Once it attaches to nerves, therapy is usually ineffective

22. Advanced case of tetanus Greek tetanos, to stretch

23. TREATMENT OF TETANUS Removal of necrotized (death) tissue Removal of necrotized (death) tissue Antibiotics Antibiotics Human immunoglobulins Human immunoglobulins

24. TETANUS VACCINE -S-S- ToxinToxoid ActiveInactive Enzyme

25. TETANUS VACCINATION SCHEDULE (CDC) DTaP vaccine (Diptheria, Tetanus & acellular pertussis) DTaP vaccine (Diptheria, Tetanus & acellular pertussis) 2, 4, 6 and 12-18 months 2, 4, 6 and 12-18 months 4-6 years 4-6 years

26. FOODBORNE BOTULISM A non-infectious food poisoning A non-infectious food poisoning C. botulinum neurotoxin in contaminated food C. botulinum neurotoxin in contaminated food 34 cases in 1994 (USA) 34 cases in 1994 (USA)

27. FOODBORNE BOTULISM (cont.)

28. BOTULIN TOXIN Most potent of all natural toxins Most potent of all natural toxins Approx. 0.001 mg human lethal dose Approx. 0.001 mg human lethal dose Causes flaccid paralysis Causes flaccid paralysis Death follows cardiac failure Death follows cardiac failure

29. Definitive Diagnosis only establish with toxin identification: only establish with toxin identification: Isolation of toxigenic cultures and identification of the involved type C or D toxin with the aid of serum neutralization in mice or guinea-pigs. Isolation of toxigenic cultures and identification of the involved type C or D toxin with the aid of serum neutralization in mice or guinea-pigs. Toxin detection in clinical samples collected for laboratory analysis (intestinal contents) Toxin detection in clinical samples collected for laboratory analysis (intestinal contents)

30. Type of tests to ID botulism tests may include a brain scan, spinal fluid examination, nerve conduction test (electromyography, or EMG), tests may include a brain scan, spinal fluid examination, nerve conduction test (electromyography, or EMG), The most direct way to confirm the diagnosis is to demonstrate the botulinum toxin in the patient's serum or stool by injecting serum or stool into mice and looking for signs of botulism The most direct way to confirm the diagnosis is to demonstrate the botulinum toxin in the patient's serum or stool by injecting serum or stool into mice and looking for signs of botulism The bacteria can also be isolated from the stool of persons with foodborne and infant botulism The bacteria can also be isolated from the stool of persons with foodborne and infant botulism These tests can be performed at some state health department laboratories and at CDC These tests can be performed at some state health department laboratories and at CDC

31. BOTULIN TOXIN (cont.) Home preserves (pH 5 and above) frequent source of botulism Home preserves (pH 5 and above) frequent source of botulism Not formed in pH below 4.7 Not formed in pH below 4.7 Molds may shift pH to above 4.7 Molds may shift pH to above 4.7 Destroyed by boiling 10 min Destroyed by boiling 10 min Treatment of Botulism  NONE Treatment of Botulism  NONE

32. BOTULISM PREVENTION Preservation of foods at pH below 4.7 Preservation of foods at pH below 4.7 Salt (brine) and sugar Salt (brine) and sugar Nitrites in cured foods (remember the nitrites/nitrates in hot dogs, cured ham, processed meats) Nitrites in cured foods (remember the nitrites/nitrates in hot dogs, cured ham, processed meats) Boiling food 10 min Boiling food 10 min

33. INFANT BOTULISM Predominant form in the USA Predominant form in the USA 75-100 cases/year in the USA 75-100 cases/year in the USA

34. INFANT BOTULISM (cont.) Less than 6 months old children Less than 6 months old children Associated with ingestion of honey Associated with ingestion of honey Honey has endospores in it naturally  bees pick up the endospores from the flowers Honey has endospores in it naturally  bees pick up the endospores from the flowers Immature intestinal microflora of infants leads to infant botulism Immature intestinal microflora of infants leads to infant botulism children may receive medical attention because of symptoms such as constipation, poor sucking action, a weak cry, and a general, progressive muscle weakness. children may receive medical attention because of symptoms such as constipation, poor sucking action, a weak cry, and a general, progressive muscle weakness.constipation

35. Infant botulism detection tests!! The diagnosis is confirmed by the detection of the organism or its toxin in the infant’s stool The diagnosis is confirmed by the detection of the organism or its toxin in the infant’s stool Toxin isolation and identification are accomplished via mouse lethality testing, with typing (type C or D toxin) confirmed by neutralization of toxin by specific sera (antibodies—immunoglobulins) Toxin isolation and identification are accomplished via mouse lethality testing, with typing (type C or D toxin) confirmed by neutralization of toxin by specific sera (antibodies—immunoglobulins)

36. Where are these endospores commonly found? More than 90% of reported cases (infant botulism) in the USA come from California, Utah, and southeast Pennsylvania; this is likely a consequence of high concentrations of C. botulinum spores in the soil of these regions More than 90% of reported cases (infant botulism) in the USA come from California, Utah, and southeast Pennsylvania; this is likely a consequence of high concentrations of C. botulinum spores in the soil of these regions

37. WOUND BOTULISM C. botulinum develops in grossly contaminated wounds C. botulinum develops in grossly contaminated wounds 19 cases in 1995 19 cases in 1995 Very common with black tar heroine users/skin popping Very common with black tar heroine users/skin popping

38. blepharospasm and strabismus BOTOX Therapeutic use of botulinum toxin

39. What is Blepharospasm? Blepharo means "eyelid". Spasm means "uncontrolled muscle contraction". The term blepharospasm can be applied to any abnormal blinking or eyelid tic or twitch resulting from any cause, ranging from dry eyes to Tourette's syndrome to tardive dyskinesia.

40. What is Strabismus? Strabismus, also known as crossed or turned eye, is the medical term used when the two eyes are not straight. It occurs in approximately 2% to 4% of the population.

41. GAS GANGRENE

42. Tissue necrosis (death) from lack of blood supply Tissue necrosis (death) from lack of blood supply Caused by C. perfringens Caused by C. perfringens Neglected wounds (anaerobic conditions) provide a suitable environment for C. perfringens growth Neglected wounds (anaerobic conditions) provide a suitable environment for C. perfringens growth

43. Clostridium perfringens Gas gangrene

44. GAS GANGRENE (cont.) Highly lethal if untreated Highly lethal if untreated C. perfringens ferments muscle proteins and carbohydrates producing H 2 and CO 2 C. perfringens ferments muscle proteins and carbohydrates producing H 2 and CO 2 Predisposing factors: Predisposing factors: Dirt in wound Dirt in wound Long delay before wound care Long delay before wound care Induced abortion Induced abortion

45. GAS GANGRENE (cont.) Predisposing factors: Predisposing factors: Arteriosclerosis Arteriosclerosis Diabetes Diabetes

46. Gas gangrene in arm

47. Bone fracture with gas in surrounding muscular tissue

48. Gas gangrene in foot

49. Gas gangrene in foot

50. Gas gangrene in buttocks

51. TREATMENT Removal of necrotized (dead) tissue Removal of necrotized (dead) tissue Amputation Amputation Hyperbaric O 2 Hyperbaric O 2 Antibiotics Antibiotics

52. Hyperbaric chamber for gas gangrene treatment

53. Anthrax

54. Anthrax Caused by Bacillus anthracis (aerobic;G+; endospore former) Caused by Bacillus anthracis (aerobic;G+; endospore former) commonly found in the soil (South and Central America, Southern and Eastern Europe, Asia, Africa, Caribbean and the Middle East) commonly found in the soil (South and Central America, Southern and Eastern Europe, Asia, Africa, Caribbean and the Middle East) primary disease of domesticated and wild animals- particularly herbivores primary disease of domesticated and wild animals- particularly herbivores humans become infected when they come into contact with diseased animals (flesh, bones, hides, hair and excrement) humans become infected when they come into contact with diseased animals (flesh, bones, hides, hair and excrement)

55. Robert Koch-1876, 1877 isolated and obtained a pure culture Robert Koch-1876, 1877 isolated and obtained a pure culture Louis Pasteur-1881, developed a vaccine Louis Pasteur-1881, developed a vaccine

56. Robert Koch Koch was a doctor and he had a detailed knowledge of the human body – something that Pasteur, as a research scientist – lacked. Koch was a doctor and he had a detailed knowledge of the human body – something that Pasteur, as a research scientist – lacked. He was also skilled in experiments, the result of his work in natural sciences He was also skilled in experiments, the result of his work in natural sciences Qualities that also proved to be important were his ability to work for long periods of time and his patience Qualities that also proved to be important were his ability to work for long periods of time and his patience However, Koch was also difficult to work with and could not tolerate anyone telling him that his theories were wrong However, Koch was also difficult to work with and could not tolerate anyone telling him that his theories were wrong

57. Robert Koch In 1872, Koch became district medical officer for a rural area near Berlin. He started to experiment with microbes in a small laboratory he had built for himself in his surgery. In 1872, Koch became district medical officer for a rural area near Berlin. He started to experiment with microbes in a small laboratory he had built for himself in his surgery.

58. Anthrax vaccine--Pasteur In France at that time many cattle suffered from anthrax, a serious disease from which many of them died. In France at that time many cattle suffered from anthrax, a serious disease from which many of them died. anthrax, after many experiments Pasteur succeeded in producing a weakened & harmless culture of anthrax bacteria after many experiments Pasteur succeeded in producing a weakened & harmless culture of anthrax bacteria He inoculated cattle & sheep with this giving them a mild form from which they recovered He inoculated cattle & sheep with this giving them a mild form from which they recovered When these animals were introduced with others who had a severe form they remained unaffected. They were immune. When these animals were introduced with others who had a severe form they remained unaffected. They were immune.

59. Gram Stain-Gram Positive Robert Koch’s Photos

60. Human cases of Anthrax are rare: 1/100,000 risk Human cases of Anthrax are rare: 1/100,000 risk Three forms: Three forms: Cutaneous-Most common form Cutaneous-Most common form acquired through a cut or abrasion of the skin, which comes into contact with spores from the soil or a contaminated animal acquired through a cut or abrasion of the skin, which comes into contact with spores from the soil or a contaminated animal Inhalation- Inhalation- acquired by the inhalation of spore-containing dust where animal hair or hides are handled acquired by the inhalation of spore-containing dust where animal hair or hides are handled Intestinal- (Speculated) Intestinal- (Speculated) consumption of contaminated meat consumption of contaminated meat

61. Cutaneous-Spores germinate, vegetative cells multiply and a lesion (black=necrotic tissue) develops at the site of infection Cutaneous-Spores germinate, vegetative cells multiply and a lesion (black=necrotic tissue) develops at the site of infection Extreme cases involve bacteria in the bloodstream which can be fatal (25%) Extreme cases involve bacteria in the bloodstream which can be fatal (25%) Inhalation-Symptoms may resemble a common cold, progressing to abrupt fever and chest pain. After several days, severe breathing problems and shock, resulting in death (hemorrhage)—leads to 100% death if left untreated Inhalation-Symptoms may resemble a common cold, progressing to abrupt fever and chest pain. After several days, severe breathing problems and shock, resulting in death (hemorrhage)—leads to 100% death if left untreated Intestinal-Inflammation of the intestinal tract, nausea, loss of appetite, vomiting, severe diarrhea and death (25-60%) Intestinal-Inflammation of the intestinal tract, nausea, loss of appetite, vomiting, severe diarrhea and death (25-60%)

62. Cutaneous Anthrax

63. Treatment: Antibiotics Antibiotics Effective if given within 24 hours, or before the bacteria enter the bloodstream Effective if given within 24 hours, or before the bacteria enter the bloodstream Penicillin, Tetracyclines, fluoroquinolones (Cipro) Penicillin, Tetracyclines, fluoroquinolones (Cipro) Vaccine Vaccine protective antigen (composed of a fraction of the toxin) protective antigen (composed of a fraction of the toxin) 95% protective 95% protective *No evidence of person-to-person transmission *No evidence of person-to-person transmission

64. Anthrax Toxin-Symptoms and disease is caused by a toxin Anthrax Toxin-Symptoms and disease is caused by a toxin made up of a protease (protein-digesting enzyme) made up of a protease (protein-digesting enzyme) B. anthracis form endospores B. anthracis form endospores Spores may survive in the soil, water and on surfaces for many years Spores may survive in the soil, water and on surfaces for many years Destroyed by autoclaving, burning, or chlorination Destroyed by autoclaving, burning, or chlorination

65. Biological Warfare Any disease-causing organism that is used as a weapon Any disease-causing organism that is used as a weapon Anthrax has particularly useful features to be used as a weapon Anthrax has particularly useful features to be used as a weapon Stable in the environment (endospores) Stable in the environment (endospores) spores that can be inhaled spores that can be inhaled once spores are inhaled, vegetative cells grow and produce lethal toxins once spores are inhaled, vegetative cells grow and produce lethal toxins