Biology 216 - Lect 29 - Clostridium 5 diseases: Bot, Tet, Myonecrosis, food poisoning, AAE. I. Clostridium - general Gm+ anaerobic sporeforming rods; Ubiquitous; Powerful exotoxins and hydrolytic enzymes ;Most exogenous II. C. botulinum - botulism (botulus latin for sausage) A. Products 1. Spore - extremely heat resistant; subterminal; found everywhere in the world; Problem when survives cooking or canning. Problem with homecanning, not canning industry. 2. Botulism toxin - neurotoxin responsible for disease; Some phage encoded (can infect C. butyricum-> botulism toxin production) Most potent toxin known - 15 mg lethal to 150,000 (Butte County) 8 serotypes (A,B, E, man) - gut absorption is species specific (shown all act in vitro on all species) Subunit toxin - A (50K) and B (100K) Growth and toxin produced at neutral pH (not associated with acidic foods) Very strict range of growth and toxin production. Toxin inactivated by boiling 1 min or 80 C for 10 min (recommended boil for 10 min). MOA - Blocks release of acetylcholine at nerve-muscle junction by block Ca++ induced exocytosis. Endopeptidase which cleaves synaptobrevins of transmitter vesicle. Once thought to block Ca++ influx, since reversible by Ca++ or Ca++ ionophores. Acetylcholine necessary for muscle activation -> flaccid paralysis. 3. C2 and C3 toxin - ADP-ribosylating toxins not important in botulism. B. Diseases - 4 syndromes due to toxin. 1. Foodborne adult botulism - classic botulism (20/yr in US) More common in animals - fowl botulism(flies, inverts) - McNulty work - type C (vultures immune). a. Spores contaminate food (ubiquitous); Germinate and grow in neutral pH and anaerobic. Homecanning - green beans, mushrooms. (Cooking drives off O2) b. Toxin produced in food - little evidence of spoilage; toxin destroyed if cooked. c. Intoxication - A,B,E. Absorbed in stomach & small intestine -> lymphatics -> blood - > peripheral nerves. d. Incubation 12 - 96 hours - determined by amount of toxin ingested. e. All symptoms from toxin induced paralysis - weakness, dizziness, constipation (GI peristalsis) nausea, vomit, headache. dry painful mucus membranes in mouth, double vision, slurred speech, pharyngeal paralysis -> aspiration pneumonia, death by diaphragm paralysis or cardiac arrest (1-7 days), 20% mortality w/treatment, recovery slow, but complete. No mental CNS effects (alert), symmetric manifestations, no fever, no sensory disturbance (no CNS) Confused w/ Guillain Barre (polyneuritis, weakness following infection); Myasthenia gravis (autoimmune) - lack of acetylcholine or excess cholinesterase. Often some permanent nerve damage. 2. Wound botulism - very rare, but same syndrome as adult - don't germinate well in tissue. 3. Infant botulism - infection, not intoxication; First recognized in 1976 (50/yr in US). a. Age 2 - 26 weeks of age develop syndrome similar to botulism - Flaccid paralysis, no muscle tone, floppy baby syndrome. Found C. bot and toxin in feces; State Health Dept Infant Bot division. b. Pathos - Infants ingest C. bot Honey on pacifiers or formula supplement (>10 % honey has spores, 7/25 gm honey) AMA recommends no honey supplement to that age group. C. bot colonize large intestine - why infant and not adult? Different flora; Switch to solid foods correlate with resistance. Change in peristalsis. Conventional mice + 10(6) spore -> no toxin in feces. Gnotobiotic mice + 10 spores -> toxin in feces Hypotheses: Infant less susceptible to toxin? Less toxin produced in large intestine? Toxin absorbed poorly in large intestine (ie. lower mortality)? Expts: >2000 LD50 toxin accumulates in mouse large intestine. Feces ingested by another mouse -> toxic ie Toxin produced, but not absorbed well in large intestine. 4. Sudden Infant Death Syndrome (SIDS) - crib death; 10K/yr in US; 2 - 26 month age distribution. C. bot or toxin found in 5 % SIDS feces at autopsy. ie. C. bot may be one cause of SIDS. C. Diagnosis - difficult because rare and unexpected. 1. Clinical - History (home canned) Bilateral neuromuscular paralysis; Mental processes clear; No fever; No anaesthesia. 2. Detection of toxin in food, serum, gastric contents. Minute amounts (below ELISA levels) - need bioassay Mouse neutralization assay - Mouse + toxin -> death (wasp waste; diaphragm; 4 hours) Mouse + toxin + antitoxin -> no death 3. Culture feces (infant) or food. Very enriched and selective (w/antibiotics); Pre heat food or feces enhance recovery. Treat based on clinical picture. Culture for epidemiological and legal purposes. D. Treatment - 1. Immediate therapy - passive immunization; 24 hr phone # in every micro text CDC Check for horse hypersensitivity. 2. Antibiotics - infant and wound 3. Mechanical respiratory assistance - main treatment which lowers fatality. 4. Foobborne - induce vomit; purge with MgSO4 ; Remove additional toxin. 5. Guanidine HCl - enhances acetylcholine release. E. Prevention - 1. Home canning - 121 C/30 min 2. Boil home canned foods before consumption. 3. No honey for age 2 - 26 week. Toxin not formed in honey, since no water available. 4. Vaccine (toxoid of A,B,E) for researchers 5. Fowl remove carcasses. F. Uses for Botulism toxin (Botox) 1. Treat muscle spasms of face and neck, stuttering, wrinkles Parkinson's shaking, crossed eye (approved by FDA) (Previous treatment was physical nerve destruction) Requires repeat injections. III. C. tetani - tetanus; Local infection with systemic toxic effects. A. Bacteriology 1. Spore - terminal, tennis racket shape; Allows survival in soil. 2. Tetanus toxin (tetanospasmin) a. Sole virulence factor; plasmid encoded; no known function for cell (released upon lysis). LD50 130 ug/human - 1000x less toxic than botulism. Single serotype; A and B same size as botulism. Bind specific gangliosides of inhibitory neurons at spinal cord. Affect CNS reflex arc and block glycine and GABA release, block repolarization, block relaxation -> spastic paralysis. Significant sequence similarity between botulism and tetanus toxins. (Both cleave synaptobrevins of neurotransmitter vesicle, ie both have same MOA, just different target cell binding. Injection of tetanus toxin into peripheral nerves -> flaccid paralysis) Rigid paralysis - simultaneous contraction of protagonist/antagonist muscles. Facial first - lockjaw; Mouse expt in Montana b. Molecular information - bot and tet have extensive AA sequence homology. Both are endopeptidases which cleave synaptic vessicle proteins synaptobrevins. Difference lies in binding portion: Bot (peripheral nerves), Tet (CNS inhibitory neurons) B. Pathogenesis - 50/yr in US; Most over 50 years old and unvaccinated; 50K deaths/yr worldwide. 1. Exogenous - implantation of spores from soil (GI tract of many animals). Puncture wound; Rusty nail (Fe2+ reduces locally); iv drug; Tetanus neonatorum (umbilicus) Mother Ab protective passively (recommended vaccination). 2. Local colonization - remains local; may be unnoticable; Minimal growth -> LD50 toxin. 3. Toxin travels to CNS - blocks glycine release, first manifestation near infection site. 4. Clinical - 2 - 50 day incubation (usual one week) Shorter incubation and more severe disease if short route to CNS. Initially - irritable, restless, muscle stiffness, muscle spasms at site of infection. Masseter muscles first - lockjaw; sardonic grin. Muscle rigidity may last 1 min; exhaustion; hematoma (crush vessels); respiratory/cardiac arrest. Spasms precipitated by cold, noise, internal trigger (bladder or colon distention). Mortality 50% w/o treatment; 10 % w treatment. Disease does not initiate immunity (toxic dose too low) C. Diagnosis 1. Symptoms; wound; History of no vaccination (highest in unvaccinated elderly) 2. Lab diagnosis unreliable (Direct exam, tough to culture....) D. Treatment 1. Passive antitoxin (pooled human hyperimmune; horse in underdevelped countries) Toxoid given on other side of body. 2. Antibiotics to eliminate source of toxin. 3. Supportive care - barbituates, respiratory support, dark, quiet room. E. Prevention 1. Wound management - clean, remove object, disinfect, antibiotics. 2. Vaccine - Formalin toxoid (single serotype) DPT - 2,4,6,18,5 yr; dT - 10 years (used to be 5 years, but hypersensitivity) Maternal updated vaccination recommended due to passive transfer of IgG. IV. Myonecrosis, gas gangrene - histotoxic Clostridia A. Bacteriology - usually polymicrobic - invasive Clostridia Shorter Gm+ rods; seldom sporulate in vitro. BAP - double zone of hemolysis (not always apparent) Litmus milk - acid, coagulation, gas -> stormy fermentation (historical) Products - invasive enzymes (proteases, DNases, collagenases....) Alpha toxin most important (lecithinase, phospholipase C) - phage encoded. Lecithin -> diglyceride + phosphoryl choline Lecithin is major lipid in cell membranes Capillary endothelial cells -> leakage, edema, myonecrosis Organisms - C. perfringens (80%); C. novyi (40 %), C. septicum (20 %) - all alpha + B. Pathogenesis - mild to myonecrosis (based on # organisms; toxigenicity; tissue damage) 1. Transmission - May be endogenous (fecal flora) or exogenous (soil) Deep crushing wound (devitalizing) - contaminated by objects, soil, clothing... (Ex. automobile, war, compound fracture, septic abortion) 2. Colonization - low O2; vascular damage; facultatives. 3. Grow and produce alpha toxin - several day incubation, painful, rapidly fatal Alpha toxin -> necrosis -> low O2 -> spread; bloody exudate, edema, muscle liquifaction. Gases (CO2/H2) - gas gangrene. Lack of PMN due to ischemia - advances. 4. CNS manifestation - delerium, disorientation - mechanism unknown. 5. Prognosis - w/o surgery (100 % fatal); w/ surgery (20 %) 6. Possible outcomes: a. Local growth only (non-toxigenic or low inoculation) b. Invasion into healthy tissue (tox+) C. Diagnosis 1. Clinical picture: Injury, appearance (edema, discoloration, spreading, gas), pain 2. Laboratory - specimen from deep within the wound a. Direct exam - Gm+ rods; no inflammatory cells (dissolved) b. Culture - rich medium, biochemical ID Egg yolk - halo of diglyceride from alpha toxin (Nagler - Egg yolk with anti toxin) BAP - double zone of hemolysis - due to several hemolysins (one is alpha toxin) D. Treatment 1. Debride all involved tissues (amputation) - fatal if reaches trunk Uterine infection - hysterectomy 2. Antibiotics 3. Anti-alpha toxin passive immunization has been used 4. Hyperbaric O2 - chamber w/ high O2 (whole body), 1 hour pulses/3 per day V. Food Poisoning - C. perfringens. A. Pathos - Ingestion of food heavily contaminated with C. perfringens 10(9) Toxin produced during sporulation in stomach. Sort of an intoxication. Enterotoxin - Spore associated (sporulation negative mutants do not produce enterotoxin) B. Intestinal mucosal cell desquamation -> diarrhea and abdominal pain; Self limiting. VI. Antibiotic associated enterocolitis - C. difficile (Range from diarrhea -> Pseudomembranous colitis) A. Pathogenesis - first described in 1977 1. Endogenous - 3 % human intestines; 20 % in hospital patients (Hosp heavily contaminated with spores; carriage selected for) 2. Always associated with antibiotic therapy - dissrupt normal flora; Ave follow treatment 1 week. Clindamycin (anaerobic therapy) and B-lactams. 3. C. difficile produce several enterotoxins (A & B). No evidence that C. difficile attach 4. Toxins -> A & B by most strains; internalized; Coregulated Mucosal damage - diarrhea Inflammed colonic mucosa; plaque-like lesions (2 - 5 cm) Pseudomembranous yellow coat (similar to diphtheria) - tissue below is necrotic. 5. Disease - Early diarrhea (mild) - suspect if antibiotic patient; Untreated -> Later - Pseudomembrane May lead to shock B. Diagnosis 1. Clinical history - following antibiotic therapy Frequent stools (5 loose stools/day); Sigmoidoscopy for lesions. 2. Lab diagnosis - culture and toxin detection in feces a. Culture - Selective media with antibiotics, quantify. Culture less significant than toxin detection. ID by fermentation, egg yolk, spore b. Toxin detection - Cytotoxicity of fecal filtrate -> rounding of fibroblast cell line (One treated with toxin other with toxin + anti-toxin) Companies will do this - send specimen frozen -> culture and toxin assay ELISA and Latex agglutination for simple toxin detection (toxin B, not A). (Latex detects C. difficile product that is not toxin) C. Treatment 1. Discontinue current antibiotic 2. Switch to vancomycin (effective vs C. difficile) early 3. Fluid/electrolyte and shock.