The Anaerobes

1. The Anaerobes Clostridium Bacteriodaceae

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 K1, 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 35-370C, 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 O2 = 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 todifferentiate 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