Clostridium: Gram-Positive Bacteria - Lesson - PDF

MODULE 3 Gram-Positive Bacteria ) Lesson No. 3.2 Gram-Positive Bacilli and Filamentous: Bacillus, Clostridium, and Erysipelothrix Lotis M. Balala, DVM, PhD College of Veterinary Medicine Visayas State University The Genus Clostridium ...

MODULE 3 Gram-Positive Bacteria ) Lesson No. 3.2 Gram-Positive Bacilli and Filamentous: Bacillus, Clostridium, and Erysipelothrix Lotis M. Balala, DVM, PhD College of Veterinary Medicine Visayas State University The Genus Clostridium  General characteristics Greek word “kloster” means spindle Largest bacilli with more than 100 species Gram-positive in young culture, old cultures are Gram variable Obligate anaerobic bacteria with unusual features: o spore formation o toxin production o normally reside in the environment or the gastrointestinal tract of humans and animals Dominant form in the human intestinal flora Clostridia spp. account for 50% or about 1013/gm stool  Arranged in pairs or short chains with rounded pointed ends Most clostridia are motile by peritrichous flagella All species form endospores and have a fermentative type of metabolism Have three important qualities: o Multiply only in the absence of oxygen o Have the ability to survive adverse conditions o Release potent toxins during process of multiplying Terminal endospore of Clostridium tetani  Cultural characteristics Slow growth on solid medium Some produce fluorescent colonies Hemolysis on blood agar Saccharolytic – ferment(CHO) into butyric acid, acetic and alcohol Proteolytic  Culture media Robertson's cooked meat broth - contain amino acids Nutrient agar Blood agar – Target or double zone hemolysis (inner-β; outer-α) Lactose egg yolk milk agar – Detection of lecithinase C Mac Conkey agar - green fluorescent colonies Thioglycolate C. botulinum on Robertson’s cooked meat broth  Clostridia produce the largest number of toxins of any bacterial class o Toxin production is the main pathogenicity mechanism Clostridium 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 C. perfringens and C. botulinum, have ovoid subterminal (OST) spores C. tetani have round terminal (RT) spores Epidemiology and Pathogenesis Four broad types of pathogenesis  Clostridium tetani Round terminal spore gives typical “drumstick” appearance Motile, grow on blood agar or cooked meat medium with swarming colonies; β-hemolysis Causative agent of tetanus Extremely sensitive to oxygen toxicity Spores highly resistant, but killed by autoclaving Found in soil, also in manure of horse, cows, sheep, dogs, chickens, rats, humans, etc. Round terminal spores give cells a “drumstick” or “tennis racket” appearance  Types of toxin produced: tetanospasmin and tetanolysin, causing the characteristic “tetanic spasm,” the clinical manifestations of tetanus an A-B toxin, released when the bacteria lyse (exotoxin) toxin binds gangliosides in synaptic membranes (synapse) Tetanospasmin – heat labile neurotoxin blocks release of inhibitory neurotransmitters resulting to continuous stimulation of excitatory transmitters (i.e., GABA, glycine) Tetanolysin – heat stable hemolysin of unknown significance Mechanism of action of tetanus toxin Clinical Forms of Tetanus  Tetanus spastic paralysis germination of the spore and production of toxin are aided by conditions that lead to low oxidation-reduction potential –necrotic tissue –calcium salts –associated pyogenic infections Risus Sardonicus Diagnosis – Depends on the clinical picture and a history of injury Prevention and Control – Active immunization with toxoid – ‘Booster shot’ for previously immunized individuals – This may be accompanied by antitoxin injected into a different area of the body – birds naturally immune Treatment – TAT:100,000 to 200,000 units single dose – Penicillin – Surgical debridement to remove the necrotic tissue – Muscle relaxants, sedation and assisted ventilation  Clostridium botulinum Fastidious growth requirements Motile, produce heat-resistant spores (ovoid, subterminal) Produce 7 distinct botulinum toxins all cause flaccid paralysis Toxin binds neuromuscular junctions and alters the mechanism for acetylcholine release Strains associated with human disease produce lipase, digest milk proteins, hydrolyze gelatin, and ferment glucose Mechanism of action of C. botulinum toxins  Disease is mostly an intoxication Causes lamziekte, limberneck effect is flaccid paralysis, due to the inability of the nerve cells to release acetylcholine swine and carnivorous animals are resistant Forms: – Foodborne, infant and wound botulism Limberneck  Diagnosis – Elisa Treatment – trivalent antitoxin (A, B, E) – ventilatory support, gastric lavage – Penicillin and metronidazole Prevention – Toxoid – Heating of home-canned food  Clostridium perfringens (C. welchii) Large, rectangular bacilli Spores rarely seen in vitro (ovoid, subterminal) Non-motile but rapidly spreading on blood agar Aerotolerant specially media supplemented with blood Five types of strains –A - E Four lethal toxins – Alpha, Beta, Epsilon and Iota  Lethal toxins Epsilon-toxin o Increases intestinal permeability causing vascular damage and edema in major organs o Liver damage o Higher blood pressure Iota-toxin o Food-borne illness Alpha-toxin and Beta-toxins o Gas gangrene – necrotizing cell membranes (humans) o Food-borne illness  Diagnosis o Direct smear and gram stain o Rapid growth culture in chopped meat media at 45oC, then isolate in blood agar four to six hours o Double zone of hemolysis o Nagler reaction – precipitation in serum or egg yolk media o Stormy fermentation (coagulation) of milk  Treatment – Removal of necrotic tissue (debridement) – Penicillin G  C. perfringens virulence factors Major Minor Exotoxins associated with C. perfringens types A-E Nagler Reaction Lecithinase (α-toxin; phospholipase) hydrolyzes phospholipids in egg-yolk agar around streak on right. Antibody against α-toxin inhibits activity around left streak.