Clostridium Botulinum – An Overview

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The History of Discovery


A causative agent of botulism (L. botulus – sausage, botulism – poisoning by sausage toxin) was firstly discovered and studied by E. van Ermengem in 1896. He isolated microbial pathogen both from the intestine and spleen of patients, who died from intoxication, and at the same time from the food they had ingested (ham remnants).


Classification


Botulism causative agent belongs to the order Clostridiales, family Clostridiaceae, genus Clostridium, and species C. botulinum.


Structure and Properties of C. botulinum


Morphology

Clostridium botulinum is a large gram-positive rod up to 8 μm in length. It is a motile peritrichous bacterium with oval terminal or subterminal spore. Sporeforming cell looks like tennis racket.

Clostridium botulinum

Cultivation

The optimal temperature for microbial growth is within the range 30-40°C. These clostridia are readily cultivated in anaerobic conditions at pH 7.3-7.6. Culturing on sugar-blood agar in anaerobic jar reveals filamentous irregular hemolytic colonies. The growing anaerobic culture has the smell of rancid butter. Cultivation in Kitt-Tarozzi medium results in homogenous turbidity followed by microbial precipitation.

Biochemical properties

  • Causative agents of botulism are obligate anaerobes.
  • They ferment carbohydrates (glucose, maltose, glycerol and some others) with acid and gas end products. Mixed type of fermentation results in acetic, butyric, and lactic acid.
  • Botulism clostridia express marked proteolytic activity. They produce hydrogen sulfide, ammonia, and volatile amines. Also they are able to reduce nitrates to nitrites, liquefy gelatin and coagulate milk.

Antigenic structure

  • C. botulinum is divided into 8 serovars (A, B, C1α, C2β, D, E, F and G) according to antigenic variations of microbial exotoxin. A, B, E, and F variants are found to be extremely toxic for humans.
  • Also bacteria possess flagellar H-antigen and somatic O-antigen similar in all botulism clostridia.

Virulence factors

  • C. botulinum produces the most poisonous neurotoxin known to date. One human lethal dose of dry botulinum toxin is about 0.1 ng/1 kg of body weight.
  • In anaerobic conditions clostridia start to secrete exotoxin especially after propagation in various foodstuffs (meat, fish, canned mushrooms and vegetables, etc.) Toxin production is inhibited in presence of 6-8% NaCl and in acidic conditions. Its activity is also neutralized by specific antibodies.
  • C. botulinum exotoxin is composed of A and B subunits. Subunit A is responsible for toxic activity, while B portion preserves the molecule from acid inactivation in stomach. It is also resistant to digestive enzymes of gastrointestinal tract.
  • Once ingested, the toxin is absorbed in gut. It reaches the nervous system and inhibits the release of acetylcholine at cholinergic synapses, resulting in muscular paralysis.
  • Botulinum neurotoxin is a Zn-containing metal protease that destroys synaptic proteins (e.g., vesicle-associated protein, synaptobrevin, cellubrevin and others) in cholinergic synapses of motor neurons.

Resistance

  • Heating at 90°С for 40 minutes or boiling for about 10 minutes irreversibly inactivates botulinum toxin. Heating at 80°С kills vegetative forms of clostridia within 30 minutes.
  • The spores have strong resistance and remain viable in soil and dust for years. They can withstand boiling for up to 6 hours and even keep their viability in large pieces of meat after autoclaving for 15 minutes at 120°C.
  • Standard disinfectants, such us 5% phenol, inactivate the spores of botulism clostridia after exposure for 18-24 h.

Pathogenesis and Clinical Findings in Botulism

  • Spores of C. botulinum can be found in the intestine of animals, birds and fishes. They permanently discharge spores into surrounding environment with feces. The spores retain viability in the soil for a long time and can appear on the surface of vegetables and fruits with the soil dust.
  • Infected animals and fishes are regarded as the major sources of infection.
  • Botulism is transmitted predominantly by fecal-oral route after ingestion of contaminated meat products, canned mushrooms, poultry, sausages, or vegetables, smoked and canned fish and many other products. These foodstuffs may contain germinated spores and various amounts of exotoxin, produced by viable microbial cells. Also botulinum toxin may enter the body through the wound surface.
  • Incubation period of the disease varies from several hours to 10 days and even more that depends mostly on amount of absorbed exotoxin.
  • After ingestion and intestinal absorption of exotoxin it appears in blood and invades central nervous system, muscular and other tissues. Toxin affects the neuronal nuclei of spinal cord and brain, neuromuscular junctions, cardiovascular system. Toxin binding is irreversible.
  • Anticholinergic action of toxin cause deep CNS disorders that result in dysphagia, vomiting, dry mouth, swallowing troubles, aphonia, dizziness, headache, diplopia, and eventual muscular weakness and paralysis. Diaphragm paralysis can cause the lethal outcome. Mortality rate is very high (about 20-40%).
  • Rare but severe clinical condition is infant botulism, where the ingested spores germinate directly in baby’s colon because of its poor colonization resistance, and the nascent clostridia begin to produce exotoxin.
  • Natural anti-toxic immunity is almost not created being of very low grade.
  • Recovery from botulism is followed by gradual restoration of activity of cholinergic synapses.

Laboratory Diagnosis of Botulism


  • In most cases the clinical findings of the disease are evident enough to make the right diagnosis. For laboratory diagnosis of botulism the samples of food remnants, vomit, blood and patient’s stool are examined. Stomach contents and various corpse tissues (small and large intestine, brain, spinal cord) are used for post-mortem examination.
  • The presence of botulinum toxin in the specimens is confirmed by neutralization reaction in mice or guinea pigs, by ELISA, or by indirect hemagglutination test with erythrocyte antitoxin diagnosticum.
  • For culture isolation the samples should be previously heated at 80°С for 20 min to inactivate non-sporeforming bacteria. They are next inoculated into Kitt-Tarozzi broth or other equivalent media and incubated in anaerobic conditions.
  • The isolated culture is further tested for biochemical and toxigenic properties. Culture toxin secretion is revealed by experimental mice infection. Toxin serotype identification is performed by neutralization reaction with antitoxin type-specific antibodies.
  • Toxigenicity of culture can be also confirmed by molecular genetic tests (e.g., PCR).

Treatment and Prophylaxis of Botulism


  • Non-specific measures of patient detoxication (stomach lavage, adsorbent treatment, infusion therapy) can decrease the amount of absorbed toxin.
  • Urgent passive immunotherapy includes the repeat injections of high doses of horse-derived polyvalent botulinum antitoxic sera against A, B, C, and E serovars.
  • Botulism toxoid is sometimes used to elicit specific antitoxic immunity in affected patients. The persons, suspected to use foodstuffs with botulinum toxin, are treated with polyvalent antitoxic sera in lower doses to prevent severe intoxication.
  • Non-specific prophylaxis includes the prevention of food contamination and the maintenance of established industrial sanitary conditions of meat, fish, caviar, or vegetable canning, and their proper storage. Home preservation, canning and storage of similar products can’t provide their complete decontamination, thus it should be excluded from practical use.