The document discusses several Clostridium species including C. perfringens, C. botulinum, C. tetani. It covers their characteristics, virulence factors, diseases caused like gas gangrene, food poisoning, botulism. Management includes prompt surgery, antitoxins, antibiotics for C. perfringens. C. botulinum toxin blocks acetylcholine release causing flaccid paralysis. Its toxin is neutralized by antitoxin.

1. Clostridium perfringens Clostridium botulinum Dr Kamran Afzal Classified Microbiologist

2. Clinically important Gram positive bacilli Spore forming 1. Bacillus 2. Clostridium Non spore forming 1. Corynebacterium 2. Listeria Bacilli w/ branching filaments 1. Actinomyces 2. Nocardia

3. Spores

4. Spores Clostridia form endospores under adverse environmental conditions Spores are a survival mechanism Spores are characterized on the basis of position, size and shape Most Clostridium spp., including Cl. perfringens and Cl. botulinum , have ovoid subterminal (OST) spores Cl. tetani have round terminal (RT) spores

5. 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

6. 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 (pseudomembranous colitis) Commonest cause of ‘nosocomial’ diarrhoea

7. Introductory Characteristics Obligate anaerobes Gram positive Capable of producing endospores Rod-shaped Greek word for spindle, kloster Club-shaped Endospores form club end

8. Clostridium Associated Human Diseases

9. Clostridium perfringens

10. Characteristics Gram positive Rod-shaped Non-motile Anaerobic Capsulated Double zone of haemolysis Five types of strains A - E Four lethal toxins Alpha, Beta, Epsilon and Iota

11. Epidemiology Lives in soils esp manured and cultivated lands Persists in human and animal intestinal tracts and faecal matter Can survive higher temp (45 0 C) Doubling time can be as short as 8 minutes Food poisoning strains produce spores that can resist boiling for several hours Gas gangrene strains produce spores that are inactivated by boiling for few minutes

12. Virulence Factors Major Minor Enzymes Hyaluronidase, Proteinase and Collagenase

13. Lab Diagnosis Large rectangular gram-positive bacilli Inner beta-hemolysis = θ toxin Outer alpha-hemolysis = α toxin Double zone of haemolysis Lack of inflammatory cells

14. Nagler Reaction (egg-yolk agar) NOTE: Lecithinase ( α -toxin; phospholipase) hydrolyzes phospholipids in egg-yolk agar around streak on right. Antibody against α -toxin inhibits activity around left streak Cl perfringens Cl histolyticum Cl septicum Cl novyii

15. Clostridial food poisoning Cl perfringens Heat resistant Enterotoxin Carriers for food poisoning strains Survival of heat resistant spores in bulk meals Sporulation in gut - Short IP and watery diarrhoea for 24-48 hours Usually symptoms occur within 6-24 hours of ingestion and can last ~24 hours

16. Salmonella spp Staphylococcus aureus Campylobacter spp Clostridium perfringens

17. Necrotizing enteritis (Pig-bel) New Guinea natives have pork feasts Clostridia survive in under-cooked meat Production of Beta toxin by Cl perfringens Type C Beta toxin acts on small intestine

18. Gas Gangrene

19. Pathogenesis Dead tissue, blood clots, aerobic organisms In an injury Development of ANAEROBIC CONDITION (Exogenous infection) Germination of spores Gas gangrene oedema, necrosis, gas production toxaemia, myositis crepitus

20. Management Prompt surgical intervention Sutures are removed, fascial compartments are incised to release tension, necrotic tissue is debrided Hyperbaric oxygen Special pressurized chamber Antitoxin antiserum A polyvalent antitoxin antiserum containing Cl perfringens , Cl novyii and Cl septicum antitoxin Now replaced with intensive antimicrobial therapy

21. Antimicrobial therapy

22. Clostridium botulinum

23. Epidemiology Found in soil, sediments of lakes, ponds, coastal waters, decaying vegetation Intestinal tracts of birds, mammals and fish Usually seen in canned foods Hams, sausages, fish, liver paste, honey and home preserved meat products and vegetables

24. Characteristics Anaerobic Gram-positive bacillus that forms oval sub-terminal spores Botulinum Toxin - One of the most poisonous natural substances known Seven toxigenic subtypes of the organism A, B, C, D, E, F and G The principle cause of human disease A,B and E

25. Spores Spores can withstand boiling in water for several hours Destroyed by moist heat at 120 0 C within 5 min Insufficient heating in the process of preserving foods – Botulism Canning factories should ensure ‘adequate heating’ in all parts of canned contents Resistance of some spores to irradiation

26. Action of Toxin Structure: Synthesized as a polypeptide chain that cleaves into two chains, a light and heavy linked by disulfide bonds Blocks release of Ach Failure to release neurotransmitter Zinc-dependent endopeptidase that cleaves synaptobrevins Flaccid Paralysis Permanent binding and damage

27. Mechanism of Action of Toxin Toxin is neurotoxic protein Not inactivated by stomach / intestinal enzymes Destroyed by heating at 100 0 C for 20 mins Action : Block release of Acetylecholine at synapses and NMJ of peripheral and cranial nerves - flaccid paralysis

28. Symptoms Begin 8-36 hours after ingestion Length : 2 hours to 14 days after entering circulation Preliminary symptoms: weakness, dizziness, dryness of mouth, nausea, vomiting After Neurological disturbance: blurred vision, inability to swallow, difficulty in speech, descending weakness of skeletal muscles Respiratory paralysis and cardiac failure - death

29. Clinical Syndromes Flaccid paralysis Food-borne: ingestion of foods in which spores have germinated and grown in, considered an intoxication – most common form Wound: infects a wound and then produces toxins that spread through the bloodstream – very rare Infant: infection establishes itself in the bowels of infants, colonizes and produces the toxin – common source is honey Unidentified: source is unknown, usually from intestinal colonization with in vivo production of toxin – usually from surgeries

30. Lab diagnosis Clinical samples Suspected food, faeces or vomitus Cl botulinum cultured anaerobically Toxin detection Toxin-Anti toxin neutralization by inoculation in mice

31. Treatment Priorities To remove unabsorbed toxin from stomach and small intestine To neutralize unfixed toxin by giving polyvalent antitoxin (A,B and E) To give relevant intensive care and support Penicillin or other beta-lactam antibiotics? A prophylactic dose of polyvalent antitoxin should be given to all the persons who have eaten ‘suspicious food’ containing botulism toxin

32. Alternative botulism uses Botox A-Type botulism is an active ingredient Biological Warfare Poisonous to humans World War II gelatin capsules with a lethal dose slipped into food or drink 1 gram crystalline toxin dispersed evenly and inhaled = 1 million deaths

33. What are the causative agent(s) of gas gangrene? Cl. perfringens Cl. novyii Cl. difficile Cl. septicum Cl. botulinum

34. What are the causative agent(s) of gas gangrene? Cl. perfringens Cl. novyii Cl. difficile Cl. septicum Cl. botulinum

35. Which one is not a Clostridial toxin? Neurotoxin Histotoxin Aflatoxin Enterotoxin

36. Which one is not a Clostridial toxin? Neurotoxin Histotoxin Aflatoxin Enterotoxin