Clostridium:Anaerobic Endospore formers Filename: Clostridium.ppt
Clostridial Diseases • BotulismC. botulinum • TetanusC. tetani • Gas gangreneC. perfringens • Food poisoningClostridium spp • Pseudomembranous colitisC. difficile
Clostridium • Gram positive • Rods • Endospore formers • asporogenous • Obligate Anaerobes!!! • Aerotolerant spp C. perfringens, C. tetani, C. botulinum, C. difficile • Soil organisms
Botulism • Intoxication • Foods • Meats • Fish • low-medium acid canned foods • Wild birds (limberneck) • ducks, fish, Inuit -- whale blubber, seal fins
Adults Nerve paralysis shorter nerves first Blurred vision Cardiac failure Respiratory failure Intoxication Infants Failure to Thrive Dehydration Polyneuropathy InfectionCl. Botulinum umbilical cord Botulism: symptoms
Botulism: Symptoms 1st symptoms: weakness and dizziness soon after: blurred vision (double vision), difficulty swallowing, throat pain, constipation, abdominal pain Flaccid paralysis: Bilateral, descending, weakness of the peripheral muscles. Death: respiratory paralysis
Botulism Intoxication Entry Ingestion Spread absorbed through intestine spread via blood stream moves up nerves Disease Incubation: 1-2 days Flaccid paralysis, cardiac failure, respiratory failure
Botulism: Treatment Disease is progressive may not respond to treatment. -ventilatory support -gastric lavage -penicillin -antitoxin (polyvalent A,B,E) Heat food to 80 C to kill toxin and kill spores.
Infant Botulism • Infant botulism:Colonizes the GI tract of young infants. Appears as non-specific weakness. • Flaccid paralysis: respiratory arrest. • Mortality = 1-2% • Some cases of sudden infant death syndrome have proven to be botulism. Eating honey.
Wound botulism: • rare -- organisms multiply in the wound. • Can occur through umbilical cord
Botulism: Lab Diagnosis Culture: culture organisms from feces, food. Heat to 80 C. Food, stool and patient’s serum. Toxin Assay: mix one portion of each specimen with antitoxin. Keep one portion antitoxin free.
Botulism Toxin Potent neurotoxin regulated by bacteriophage. Toxins: A -- E, C alpha, C beta, F, G Humans: A, B, E 150 Kd protein -- cleaved
INCIDENCE YEAR Botulism Intoxication in USA
INCIDENCE YEAR Botulism in USA; neonates
Botulinum Toxin • The heavy chain attaches to the ganglioside receptors in nerves
C. botulinum toxin Synaptic activity at cholinergic synapses is mediated by acetylcholine. Acetylcholine is rapidly hydrolysed by acetylcholine esterase. The result is an electrical stimulus.
Sequence of Events A. Nerve stimulus -- calcium is stimulated B. Acetylcholine release into the synaptic space -- moves into post synaptic membrane and acts on specific receptors. C. Botulinum toxin interferes with the release of acetylcholine from the synaptic vesicles.
C. tetani C.tetani looks like a tennis racket. Found in soil, carried by horses. Toxin: heat labile, 150,000 d peptide Neurotoxin: splits carboxy terminal to gangliosides on neuronal membranes. Moves to CNS by retrograde axonal transport.
Tetanus Intoxication Entry wound Spread blood stream moves up nerves Disease Incubation: 1-2 days Rigid Paralysis, cardiac failure, respiratory failure
Tetanus trismus, risus sardonicus, opisthotonos, Cephalic -- poor prognosis Localized -- favourable prognosis Prevention: toxoid, 3% formaldehyde
Toxin similarity • Clostridium botulinum and C. tetani are Zn requiring Endopeptidases that cleave a set of proteins.......... Synaptobrevins found in synaptic vesicles of neurons Interfere with release of neurotransmitters and the normal inhibitory function.
Binding regions of tetanus toxin and botulinum toxin are different in terms of cell specificity.
C.perfringens: Diseases • bacteremia • myonecrosis • gas gangrene • cellulitis • fascitis • food poisoning: enteritis necroticans
C. perfringens • large rectangular, hemolytic, very distinctive spreading colonies. • target hemolysis • found in soil and intestines, man and animals
C. perfringens toxins • Alpha toxin is a lecithinase (phospholipase C) • lyses erythrocytes, platelets, leucocytes, endothelial cells • Massive hemolysis and tissue destruction • Theta Toxin- Beta hemolysis- increases permeability-- necrotizing enterocolitis • Delta -- hemolysis • Kappa -- collagenase
C. perfringens toxins • Mu -- hyaluronidase • Nu -- DNAase Lambda toxin -- protease Neuraminidase -- hydrolyses serum glycoproteins Enterotoxin -- reverses water, sodium and chloride transport in the intestine (like V. cholerae) Produced by Group A.
Nagler Reaction • Presumptive identification of C. perfringens • alpha toxin (lecithinase) hydrolyses phospholipids • egg yolk agar becomes turbid • specifically blocked by antitoxin
Clinical Syndromes: Bacteremia - usually transient, only diagnostic with other clinical symptoms Myonecrosis - gas gangrene - trauma or surgical contaminant
Gas Gangrene Entry PenetratingWound Multiplication in dead anaerobic tissue Toxin production hemolysin, proteases, lipase, collagenase Disease necrosis, edema, gas
C. perfringens Gas gangrene Incubation: <1 week pain severe muscle necrosis shock renal failure death -crepitant -cellulitis & fascitis (no muscle)
C perfringens Food poisoning incubation 8-24hrs. Abdominal cramps, watery diarrhea lasts less than 24hrs. Contaminated meat (left overs)
C. difficile Gram + anaerobic rod. -found in normal flora -ultimate opportunistic pathogen -difficult to determine cause as the organism is ubiquitous -not difficult to culture
C. difficile Diagnosis: cytotoxin - stool culture C.difficile antigen -- latex agglutination
Cytotoxin slurry of stool centrifuged filter through 0.45 u filter 0.1ml - supernatant to buffer at pH 7.2 WI-38 tissue cells human diploid lung fibroblasts Add supernatant to tissue culture. Observe for cytotoxicity 24 hrs.
Mechanism of pathogenicity: Toxin A enterotoxin hypersecretion of fluid Toxin B cytotoxin cytopathic to tissue monolayers
C. difficileTreatment: stop antibiotic causing disease metronidazole, vancomycin Relapses due to resistant spores. retreatment with same antibiotic neutralization with specific antitoxin obtained commercially amount of toxin present can be determined by a dilution series of the stool sample.
C. difficile Culture: standard test for Clostridia include: indole, sugars, lecithinase, catalase (usually neg.)