1. The Anaerobes ClostridiumBacteriodaceae

2. Anaerobes of Clinical Importance Gram(+) Spore-Forming Bacilli –Clostridium Gram(-) Bacilli: Bacteriodaceae: –Bacteroides –Fusobacterium –Porphyromonas –Prevotella

3. Clostridium Strict anaerobes, some aerotolerant Widely distributed soil, water, sewage NF in GI tract animals, humans Most are saprophytes Disease-causing species: –Survive adverse conditions by spore formation –Rapid growth in nutrition rich, decrease oxygen site –Most not invasive but produce powerful exotoxins (cytotoxin, enterotoxin, neurotoxin)

4. Clostridium: Genera C. perfringens –Food poisoning - intoxication –Myonecrosis - gas gangarene –Soft-tissue infection C. botulinum –Botulism - food poisoning (intoxication, infection) C. tetani –Tetanus - lockjaw C. difficile –Pseudomembranous colitis - antibiotic-associated disease

5. Clostridium: Staining G(+) large bacilli All motile - except C. perfringens Form endospore – oval, subterminal C. tetani - terminal spore

6. Clostridium: Lab Culture Blood Agar - Enriched –Supplemented anaerobic BA –C. perfringens produces classic double zone hemolysis Egg Yolk Agar - Differential –Lecithinase production (white precipitate) –Lipase production (sheen around surface of colonies)

7. Clostridium: Lab Culture CCFA (Cycloserine-cefoxitin- fructose agar) –Selective by antibiotics –Differential by fructose fermentation –C. difficile (yellow, ground-glass colony) Thioglycollate broth –Reducing agents eliminate oxygen –Chopped meat for nutrients Special isolation procedures: –Usually mixed culture specimens –Use heat or alcohol to kill NF before plating for Clostridium

8. Clostridium perfringens: Virulence Factors At least 12 exotoxins and enzymes Alpha Toxin - phosphopipase C) –Vascular permeability –Massive hemolysis & bleeding, tissue destruction (myonecrosis) –Hepatic toxicity, myocardial dysfunction Enzymes - gelatinase, collagenase, protease, hyaluronidase, DNase, neuraminidase Enterotoxin - food poisoning –Meats, poultry, gravy –Action resembles cholera toxin

9. C. perfringens: Infection and Disease Exogenous infection – from external source (soil, food, trauma) Endogenous infection – GI tract to sterile areas (tissues, blood) At risk: –Surgical patients –Skin trauma with soil contamination –Ingest contaminated meat products, without proper refrigeration or reheating (enterotoxin heat labile)

10. C. perfringens: Food Poisoning Relatively common Meat products infected large number MO; multiply, produce enterotoxin Ingestion of toxin contaminated food = Intoxication Short incubation, 8-24 hours before symptoms Abdominal cramps, watery diarrhea, nausea and vomiting; no fever Short, self-limiting MO and toxin may be detected in feces but not usually tested

11. C. perfringens: Myonecrosis (Gas Gangrene) Life-threatening disease Virulence of cytotoxins Intense pain ~1 week after introduction into tissue Severe systemic toxicity Painful, edematous wound, sweet or foul smelling discharge Muscle necrosis, shock, renal failure Untreated may result in death

12. C. perfringens: Soft Tissue Infection Simple contaminant of wound, heal normally with treatment Cellulitis - invasion necrotic wound –Gas accumulation –Discoloration of skin –Malodorous brown, purulent discharge Fasciitis – infection of muscle Possible rapid spread and death MO easily Gram-stained and cultured from infected tissue

13. C. perfringens: Treatment and Prevention Myonecrosis, tissue infection –Require aggressive treatment –Surgical debridement –High dose penicillin Food poisoning –Supportive treatment –Antibiotics not necessary, intoxication not infection

14. Clostridium botulinum: “ sausage ” – insufficiently smoked sausage Found in soil and water Botulinum exotoxin –Most powerful biological poison known –Works at neuromuscular junction –Prevent release neurotransmitter acetylcholine –Stops signal for muscle stimulation –Leads to flaccid paralysis

15. Food Botulism In U.S. uncommon disease; usually occurs following ingestion of inadequately processed home-canned food Contaminated with C. botulinum spores Composition and nutritive properties allow germination and toxin production i.e. pH (≥7), warm temperature Ingest inadequately heated or processed food (toxin heat labile) = Intoxication Food does not appear spoiled by smell or taste

16. Food Botulism Following ingestion, toxin absorbed from intestine, transported via blood and lymph to PNS Incubation - 8 hours to 8 days, 18-36 hours most common Symptoms - nausea, vomiting and diarrhea; symmetric, descending paralysis (eyes, throat, neck, trunk, then limbs) Death by paralysis of respiratory muscles Lab diagnosis by detecting toxin in food and patient (serum, feces, gastric fluid)

17. Infant Botulism Follows ingestion of spores which germinate in intestine = Infection Illness may range from subclinical to sudden infant death syndrome Honey implicated as source of spores Doesn’t occur in adults due to competing NF of GI tract

18. C. botulinum: Treatment and Prevention Respiratory, ventilatory support to patient Eliminate MO from GI tact – gastric lavage, antibiotics (metronidazole, penicillin) Administer botulinum antitoxin – antibody binds and neutralizes toxin circulating in blood Prevention –Not practical to destroy spores in food –Prevent spore germination (acid pH, high sugar content, store food at 4°C) –Destroy preformed toxin by adequate cooking of food (20 minutes, 80°C) –Infants (<1 year) not fed honey

19. Clostridium tetani Spores found in soil Transient NF GI tract of animals, humans In USA, exposure common, but disease uncommon due to DTaP vaccine Developing countries, poor access to vaccine, medical care –~1 M cases/year –20-50% mortality –Many neonatal infections Diagnosis by clinical disease presentation as lab tests (stain, culture) usually unsuccessful as MO extremely oxygen sensitive, low number; tests for tetanus toxin insenstive

20. C. tetani: Exotoxins Tetanolysin – hemolysin Tetanospasmin – neurotoxin –Travel to CNS through blood, lymph, tissue spaces, peripheral nerves –Stops release inhibitory Glycine from synapse (no signal to stop muscle contraction) –Continued excitement at synapse, spastic paralysis –“lockjaw” - muscles of jaw affected –May result in respiratory failure, death

21. C. tetani : Tetanus Due to tetanospasmin toxin Minor trauma, skin break (i.e. splinter) Infection requires relatively few MO Spores enter through wound, germinate into vegetative cells; produce toxin when sufficiently low O/R infected tissue (usually deep wound) Incubation 1-54 days, average 6-15 days Longer incubation, better prognosis

22. Tetanus Symptoms - cramps, twitching of muscles around wound; headache, neck stiffness Followed by - trismus (lockjaw), generalized symptoms (drooling, sweating, irritability, back spasms) Severe disease involves CNS – cardiac arrhythnia, fluctuation blood pressure, sweating, dehydration) Death, if occurs, from respiratory failure Neonatal tetanus –Developing countries –Umbilical stump infection by septic midwifery –>90% death of infants non-immune mothers (no DTaP vaccine)

23. Tetanus: Treatment and Prevention Debride wound, aerate well Maintain open airway Administer antitoxin – human tetanus IgG neutralizes toxin (but not in CNS) Metronidazole - to kill vegetative cells If no serious CNS symptoms and toxic effects controlled, prognosis for recovery is good Prevent disease by vaccination with tetanus toxoid – part of DTaP trivalent vaccine

24. Clostridium difficile Part of GI tract NF (in small number) In past, rarely associated human disease Today, antibiotic-associated GI disease Produces two exotoxins: –Enterotoxin A - stimulates fluid and electrolyte losses, hemorrhagic necrosis –Cytotoxin B – depolymerize actin, loss of cell cytoskeleton, cell death Antibiotic therapy can result in diarrhea, permit overgrowth of resistant MO

25. C. difficile: Pseudomembranous Colitis Often after taking ampicillin, clindamycin, cephalosporin Endogenous infection - C. difficile NF in G.I. tract Exogenous infection - person- to-person in hospital Multiplies in colon, produces toxin Colonic plaques – coalesce, form pseudomembrane; mucin, fibrin, epithelial, inflammatory cells Complications - dehydration, electrolyte loss, colonic perforation Toxin detection in stool confirms diagnosis

26. C. difficile: Treatment and Prevention Mild disease – allevate by discontinue antibiotics Serious disease – require antibiotics (metronidazole, vancomycin) Relapse ~20-30% patients due to resistant spores; allow time for spores to germinate, retreat with same antibiotics Supportive – give fluid and electrolyte replacement

27. Bacteriodaceae NF of oropharynx, urogenital tract, colon Anaerobes predominant over aerobes (10-1,000x) in colon Few cause infection, opportunistic pathogen Bacteroides fragilis - most commonly isolated anaerobe pathogen

28. Bacteriodaceae: Gram Stain G(-) straight, curved, helical rods Bacteroides – pleomorhpic Fusobacterium – long, slender, pointed ends Porphyromonas – small, pigments Prevotella – small, pigments

29. Bacteriodaceae: Lab Culture Nonselective media –CBA plates plus vitamin K 1, hemin, yeast extract, L-cystine Selective media –KVLB (Kanamycin-Vancomycin Laked BA) - freezing, thawing whole blood –BBE (Bacteroides Bile Esculin agar) – selective, differential –PEA (phenylethyl alcohol agar) – growth all obligate anaerobes Incubate strict anaerobic conditions At 35-37 0 C, 48 hours before opening anaerobic jar

30. Bacteriodaceae: Lab Culture Thioglycollate broth –Liquid media –Enriched; chopped meat, glucose –Thioglycolic acid (reducing agent) remove oxygen, anaerobic atmosphere deeper in tube –Resazurin - reduction indicator; presence of O 2 = pink

31. Bacteriodaceae Lab ID Each colony - Gram stain, subculture to plates (aerobic, anaerobic) to confirm anaerobe Species ID - bile tolerance, pigment production, sensitivity to antibiotics (vancomycin, kanamycin, colistin) Gas Liquid Chromatography (GLC) – used to differentiate anaerobes by major by- products, mixed acids

32. Bacteriodaceae: Virulence Factors Capsule – adhesin, antiphagocytic Fimbriae – adhesin Endotoxin – LPS of gram(-) cell wall Protease – degrade IgA Enzymes - collagenase, phosphotase, RNAse, DNAse

33. Bacteriodaceae: Clinical Significance As human NF cause serious infections when gain access to normally sterile tissue, organ, fluid At risk: –Surgical, trauma patient –Disrupt patient normal mucosa –Patient aspirate oral secretions (with NF) into RT

34. Infection: Mixed Culture Gram(-) Anaerobes Respiratory tract – causes ~50% chronic infection of sinus, ear; may spread to blood, CNS (brain abscess) Peridontal - involved in all infections Intraabdominal – anaerobes recovered Gynecological – PID, abscess, endometritis, surgical wound infection Skin and soft tissue – colonize wound, progress to disease

35. Bacteriodes: Treatment and Prevention Manage infection – antibiotics + surgical intervention (incision, drainage, aerate) Many isolates produce β-lactamases Antibiotics: –Metronidazole (anaerobes incorporate drug into DNA; making it unstable and disrupted) –Carbapenems (imipenem) –β-lactam + β-lactamase inhibitor (piperacillin-tazabactam) Bacteroides NF, endogenous infection difficult to prevent Prophylactic antibiotics - patients with mucosa disrupted by diagnostic or surgical procedure

36. Case Study 8 - Clostridium A 61-year-old woman with left-sided face pain came to the emergency department of a local hospital. She was unable to open her mouth because of facial muscle spasms and had been unable to eat for 4 days because of severe pain in her jaw. Her attending physician had noted trismus (motor disturbance of trigeminal nerve, spasm of masticatory muscles, difficulty in opening the mouth) and risus sardonicus (spasmodic grin).

37. Case Study 8 - Clostridium The patient reported that 1 week before presentation, she had incurred a puncture wound to her toe while walking in her garden. She had cleaned the wound and removed small pieces of wood from it, but she had not sought medical attention. Although she had received tetanus immunizations as a child, she had not had a booster vaccination since she was 15 years old. The presumptive diagnosis was made.

38. Case Study 8 - Questions 1. How should this diagnosis be confirmed? 2. What is the recommended procedure for treating this patient? Should management wait until the laboratory results are available? What is the long-term prognosis for this patient? 3. Compare the mode of action of the toxins produced by C. tetani and C. botulinum. 4. C. difficile causes what diseases? Why is it difficult to manage infections caused by this organism?

39. Class Assignment Textbook Reading –Chapter 22 Anaerobes of Clinical Significance Important Concepts In Anaerobic Bacteriology Frequently Encountered Anaerobes and Their Associated Diseases Omit: Remaining last three Sections of reading Omit: Key Terms Omit: Learning Assessment Questions