Species: C. botulinum
Fig 1: Gram’s staining of C. botulinum
The class Clostridia has many varieties of gram-positive bacteria distributed into three orders and eleven families. They are gram positive, obligately anaerobic, rod-shaped, often pleomorphic, nonmotile or peritrichous bacteria with cell dimensions ranging between 0.3-2.0µm x 1.5-2.0µm. The genus Clostridium is the largest and has a great impact on the food industry as they can cause food spoilage even in canned foods and are heat resistant spore formers so can survive in stressful environments in a dormant state and revive growth when exposed to normal conditions. They are killed when present in an environment containing oxygen, but however can tolerate traces of oxygen due to the presence of the enzyme superoxide dismutase (SOD) which acts as an anti oxidant and the general reaction of anti oxidation can be represented as:
M2+-SOD + O2- à M+-SOD + O2
M+-SOD + O2- + 2H+ à M2+-SOD + H2O2
C. botulinum is present in soil and marine sediments in all regions of the world. In soil, they primarily live in anoxic “pockets”, made by facultative organisms metabolizing various organic compounds. It may contaminate vegetables cultivated in the soil and may colonize the gastro intestinal tract of fish or mammals.
C. botulinum, the causative agent of botulism, produces a highly toxic, soluble exotoxin while growing in foods. These neurotoxins are synthesized as an inactive single polypeptide without a leader sequence and hence are released from the cell by bacterial lysis (Schiavo et al. 1995). The neurotoxins are of seven types A, B, C, D, E, F and G. Type C has two antigenic subtypes, C1 and C2. Of these, types A, B, E and very rarely F cause human botulism (food borne, wound and infant types), C and D affect animals and type G has been reported to not cause any type of botulism.
Four physiological groups of C. botulinum are classified according to the current system of nomenclature (groups I. II, III and IV) based on the ability of organisms to digest different protein complexes and physiological differences (optimum growth temperature, pH, water activity, and sodium chloride concentration).
Group I: strains produce neurotoxins A, B and F and are proteolytic
Group II: strains produce neurotoxins B, E and F and are non proteolytic
Group III: strains produce neurotoxins C and D and may be proteolytic or non proteolytic
Group IV: strains produce neurotoxin G and are proteolytic
Botulism in humans is categorized into four types:
Food borne botulism: caused by the ingestion of food containing the potent neurotoxin
Infant botulism: affects infants who ingest C. botulinum spores that begin to colonize in the gastro intestinal tract, reproduce and synthesize the neurotoxin that travels through the bloodstream to the central nervous system and results in flaccid paralysis. A wide range of laboratory tests show that honey could be the source of spores in infant botulism
Wound botulism: this occurs when the organism infects a wound and produces the neurotoxin that circulates in the bloodstream. Here food is not the vehicle of transmission and this is very rare in occurrence.
Unclassified: this form of botulism is very similar to the infant botulism. However, it affects the adults. This is also extremely rare.
Botulism neurotoxins (BoNTs) are the most potent toxins known and is used as a bioweapon. It causes the neuroparalytic illness by blocking synaptic transmission in peripheral cholinergic nervous system synapses. BoNTs are produced as a progenitor complex consisting of BoNT, hemagglutinin (HA), nontoxic non-HA (NTNH), and uncharacterized components like P47 and OrfX. The genes encoding the three main proteins are linked as a cluster and vary in structure among different strains. In general, the ntnh and bont genes are conjointly located to the downstream of the cluster and the upstream regions vary among different strains. BoNTs are composed of a light chain (~50kDa) and a heavy chain (~100kDa) which are linked together by a disulfide bond. BoNTs produced by different C. botulinum strains share structural and sequence homology. The light chain is a metalloprotease (has a catalytic zinc ion in the active site). The C and N terminals of the the heavy chain are associated with specific neuron binding and translocation into the nerve cell’s cytosol. Variation or deletions of amino acid sequences in the functional domains greatly alter the structure, function and antigenic properties of the complex
Fig 2: Schematic representation of the neurotoxin gene cluster in Clostridium botulinum type E, A1, and A2 strains. The gene orientation is shown by arrows. Only orfx3 in the type E gene cluster is a partial gene. Type A1 and A2 neurotoxin gene clusters are derived from C. botulinum A1 strain NCTC 2916 (GenBank accession numbers AY497357, Y14238, and X52066) and Kyoto-F (accession numbers AY497358, AB004778, X96493, X87974, and X73423).
BoNTs act in four steps:
Binding to high affinity receptors on peripheral nerve endings
Receptor mediated endocytosis
Light chain translocation across endosomal membranes to the cytosol on exposure to the endosome pH. The heavy chain acts as a channel and a transmembrane chaperone for the metalloprotease for transit from acidic endosomes to the cytosol.
Proteolytic degradation of the target
Synaptic vesicle fusion occurs when SNARE complex is formed. The SNARE proteolysis destabilizes this complex and this prevents the neurotransmitter release. This in turn inhibits muscle contraction resulting in paralysis. The heavy chain acts as a channel and a transmembrane chaperone for the metalloprotease for transit from acidic endosomes to the cytosol.
Fig 3 The botulinum neurotoxin (green and gray) docks to the neuron’s active zone (blue) by binding to two receptors in the synaptic vesicle — synaptotagmin (red) and ganglioside (yellow). The vesicle then carries the toxin-receptor complex inside the neuron, a process called endocytosis. Once inside, the vesicle releases the neurotoxin, which then blocks the release of acetylcholine, a neurotransmitter that’s responsible for muscle contraction. This results in paralysis of the muscles. (Image Credit: Axel Brunger/Stanford University,HHMI)
The canning of food products is made efficient and so the number of yearly cases has dropped to about 1000 worldwide. On an average about 110 cases are reported in the United States, of which 25% are food borne and 75% are infant botulism cases. The use of black tar heroin in California has resulted in the number of wound botulism cases as infection occurs at the injection site.
The symptoms of food borne botulism usually occur about 18-36 hours after the consumption of the contaminated food. The symptoms include double or blurred vision, drooping eyelids, dry mouth, difficulty in swallowing, slurred speech and muscle weakness initially and if untreated causes severe paralysis in other parts of the body often seen as descending paralysis of the arms, legs, trunk, and breathing muscles. Infants with botulism appear weak, lethargic and become constipated. Botulism is diagnosed by conducting a brain scan, spinal fluid examination, nerve conduction test, and a tension test for myasthenia gravis. However, the direct way to confirm botulism is identification of the toxin in the patient’s blood or stool. The patient’s serum is injected into the peritoneal cavity of mice and the serum treated with the anti-toxin is injected into the peritoneal cavity of other mice. If the mice treated with antitoxin-serum mixture live and the mice treated with just the serum die, it confirms botulism. This is called the mouse inoculation test. The bacteria can also be isolated from the serum, though its not a definitive test.
Once confirmed, the food borne and wound botulism can be treated with an anti-toxin which works by blocking the action of BoNT in the blood. A trivalent anti-toxin (effective against A, B and E) from quarantine stations by Centers for Disease Control and Prevention (CDC) or a heptavalent anti-toxin (effective against A, B, C, D, E, F and G) from the U.S army are effective. The contaminated food in the gut is removed by induction of vomiting or using enemas.
Wounds are surgically removed in patients suffering from wound botulism.
Infants are intravenously given immunoglobulins, and the treatment is called BabyBIG (Botulism Immune Globulin).
Botulism toxin is inactivated at 85 deg C for five minutes, so food borne botulism can be prevented by heating canned food before consumption. Children under a year of age should not be given honey, as it contains Clostridium spores. Wound botulism is prevented by hygiene in the affected area.
Research is being conducted for the development of vaccines against human botulism, use as a biological weapon and in cosmetic industry (to reduce wrinkles by inhibiting muscle contraction)
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