Tetanus and Botulism

The Genus Clostridium

The clostridia are relatively large, Gram-positive, rod-shaped bacteria. All species form endospores and have a strictly fermentative mode of metabolism. Most clostridia will not grow under aerobic conditions and vegetative cells are killed by exposure to O2, but their spores are able to survive long periods of exposure to air.

The clostridia are ancient organisms that live in virtually all of the anaerobic habitats of nature where organic compounds are present, including soils, aquatic sediments and the intestinal tracts of animals.

Clostridia are able to ferment a wide variety of organic compounds. They produce end products such as butyric acid, acetic acid, butanol and acetone, and large amounts of gas (CO2 and H2) during fermentation of sugars. A variety of foul smelling compounds are formed during the fermentation of amino acids and fatty acids. The clostridia also produce a wide variety of extracellular enzymes to degrade large biological molecules in the environment into fermentable components. Hence, the clostridia play an important role in nature in biodegradation and the carbon cycle. In anaerobic clostridial infections, these enzymes play a role in invasion and pathology.

Most of the clostridia are saprophytes but a few are pathogenic for humans. Those that are pathogens have primarily a saprophytic existence in nature and, in a sense, are opportunistic pathogens. Clostridium tetani and Clostridium botulinum produce the most potent biological toxins known to affect humans. As pathogens of tetanus and food-borne botulism, they owe their virulence almost entirely to their toxigenicity. Other clostridia, however, are highly invasive under certain circumstances.


C. perfringens

Clostridium perfringens, which produces a huge array of invasins and exotoxins, causes wound and surgical infections that lead to gas gangrene, in addition to severe uterine infections. Clostridial hemolysins and extracellular enzymes such as proteases, lipases, collagenase and hyaluronidase, contribute to the invasive process. Clostridium perfringens also produces an enterotoxin and is an important cause of food poisoning. Usually the organism is encountered in improperly sterilized (canned) foods in which endospores have germinated.

Pseudomembranous colitis in humans is caused by overgrowth ofClostridium difficile in the colon, usually after the normal flora has been distrurbed by antimicrobial chemotherapy. C. difficile produces two toxins: Toxin A is referred to as an enterotoxin because it causes fluid accumulation in the bowel. Toxin B is an extremely lethal (cytopathic) toxin.

A large amount of material, including references, on Clostridium Infections is located at the Oregon Health Science University web site.

Clostridium tetani

Clostridium tetani is the causative agent of tetanus. The organism is found in soil, especially heavily-manured soils, and in the intestinal tracts and feces of various animals. Carrier rates in humans vary from 0 to 25%, and the organism is thought to be a transient member of the flora whose presence depends upon ingestion. The organism produces terminal spores within a swollen sporangium giving it a distinctive drumstick appearance. Although the bacterium has a typical Gram-positive cell wall, it may stain Gram-negative or Gram-variable, especially in older cells.

Tetanus is a highly fatal disease of humans. Mortality rates reported vary from 40% to 78%. The disease stems not from invasive infection but from a potent neurotoxin (tetanus toxin or tetanospasmin) produced when spores germinate and vegetative cells grow after gaining access to wounds. The organism multiplies locally and symptoms appear remote from the infection site.

Because of the widespread use of the tetanus toxoid for prophylactic immunization, fewer than 150 cases occur annually in the U.S., but the disease is a significant problem world-wide where there are > more than 300,000 cases annually. Most cases in the U.S occur in individuals over age 60, which probably means that waning immunity is a significant risk factor.

Pathogenesis

Most cases of tetanus result from small puncture wounds or lacerations which become contaminated with C. tetani spores that germinate and produce toxin. The infection remains localized often with only minimal inflammatory damage. The toxin is produced during cell growth, sporulation and lysis. It migrates along neural paths from a local wound to sites of action in the central nervous system. The clinical pattern of generalized tetanus consists of severe painful spasms and rigidity of the voluntary muscles. The characteristic symptom of "lockjaw" involves spasms of the masseter muscle. It is an early symptom which is followed by progressive rigidity and violent spasms of the trunk and limb muscles. Spasms of the pharyngeal muscles cause difficulty in swallowing. Death usually results from interference with the mechanics of respiration.

Neonatal tetanus accounts for about half of the tetanus deaths in developing countries. In a study of neonatal mortality in Bangladesh, 112 of 330 infant deaths were due to tetanus. Neonatal tetanus follows infection of the umbilical stump in infants born to nonimmune mothers (therefore, the infant has not acquired passive immunity). It usually results from a failure of aseptic technique during the birthing, but certain cultural practices may contribute to infection.

Tetanus Toxin

There have been 11 strains of C. tetani distinguished primarily on the basis of flagellar antigens. They differ in their ability to produce tetanus toxin (tetanospasmin), but all strains produce a toxin which is identical in its immunological and pharmacological properties. Tetanospasmin is encoded on a plasmid which is present in all toxigenic strains.

Tetanus toxin is one of the three most poisonous substances known, the other two being the toxins of botulism and diphtheria. The toxin is produced by growing cells and released only on cell lysis. Cells lyse naturally during germination the outgrowth of spores, as well as during vegetative growth. After inoculation of a wound with C. tetani spores, only a minimal amount of spore germination and vegetative cell growth are required until the toxin is produced.

The bacterium synthesizes the tetanus toxin as a single 150kDa polypeptide chain (called the progenitor toxin), that is cleaved extracellularly by a bacterial protease into a 100 kDa heavy chain (fragment B) and a 50kDa light chain (fragment A) which remain connected by a disulfide bridge. The specific protease that cleaves the progenitor toxin can be found in culture filtrates of C. tetani. Cleavage of the progenitor toxin into A and B fragments can also be induced artificially with trypsin.

Tetanus toxin is produced in vitro in amounts up to 5 to 10% of the bacterial weight. Because the toxin has a specific affinity for nervous tissue, it is referred to as a neurotoxin. The toxin has no known useful function to C. tetani. Why the toxin has a specific action on nervous tissue, to which the organism naturally has no access, may be an anomaly of nature. The toxin is heat labile, being destroyed at 56 degrees C in 5 minutes, and is O2 labile. The purified toxin rapidly converts to toxoid at 0 degrees C in the presence of formalin.

Toxin Action

Tetanospasmin initially binds to peripheral nerve terminals. It is transported within the axon and across synaptic junctions until it reaches the central nervous system. There it becomes rapidly fixed to gangliosides at the presynaptic inhibitory motor nerve endings, and is taken up into the axon by endocytosis. The effect of the toxin is to block the release of inhibitory neurotransmitters (glycine and gamma-amino butyric acid) across the synaptic cleft, which is required to check the nervous impulse. If nervous impulses cannot be checked by normal inhibitory mechanisms, it produces the generalized muscular spasms characteristic of tetanus. Tetanospasmin appears to act by selective cleavage of a protein component of synaptic vesicles, synaptobrevin II, and this prevents the release of neurotransmitters by the cells.

The receptor to which tetanospasmin binds has been reported as ganglioside GT and/or GD1b, but its exact identity is still in question. Binding appears to depend on the number and position of sialic acid residues on the ganglioside. Isolated B fragments, but not A fragments will bind to the ganglioside. The A fragment has toxic (enzymatic) activity after the B fragment secures its entry. Binding appears to be an irreversible event. Recovery depends on sprouting a new axon terminal.

Immunity

Unlike other diseases, such as diphtheria, recovery from the natural disease usually does not confer immunity, since even a lethal dose of tetanospasmin is insufficient to provoke an immune response.

Prophylactic immunization is accomplished with tetanus toxoid, as part of the DPT vaccine or the DT (Td) vaccine. Three injections are given in the first year of life, and a booster is given about a year later, and again on the entrance into elementary school.

Whenever a previously-immunized individual sustains a potentially dangerous wound, a booster of toxoid should be injected. Wherever employed, intensive programs of immunization with toxoid have led to a striking reduction in the incidence of the disease.

Clostridium botulinum

C. botulinum is a large anaerobic bacillus that forms subterminal endospores. It is widely distributed in soil, sediments of lakes and ponds, and decaying vegetation. Hence, the intestinal tracts of birds, mammals and fish may occasionally contain the organism as a transient. Seven toxigenic types of the organism exist, each producing an immunologically distinct form of botulinum toxin. The toxins are designated A, B, C1, D, E, F, and G). In the U.S. type A is the most significant cause of botulism, involved in 62% of the cases. Not all strains of C. botulinum produce the botulinum toxin. Lysogenic phages encode toxin serotypes C and D, and non lysogenized bacteria (which exist in nature) do not produce the toxin. Type G toxin is thought to be plasmid encoded.

Pathogenesis of Botulism

Food-borne Botulism

In food-borne botulism the botulinum toxin is ingested with food in which spores have germinated and the organism has grown. The toxin is absorbed by the upper part of the GI tract in the duodenum and jejunum, and passes into the blood stream by which it reaches the peripheral neuromuscular synapses. The toxin binds to the presynaptic stimulatory terminals and blocks the release of the neurotransmitter acetylcholine which is required for a nerve to simulate the muscle.

Food-borne botulism is not an infection but an intoxication since it results from the ingestion of foods that contain the preformed clostridial toxin. In this respect it resembles staphylococcal food poisoning. Botulism results from eating uncooked foods in which contaminating spores have germinated and produced the toxin. C. botulinum spores are relatively heat resistant and may survive the sterilizing process of improper canning procedures. The anaerobic environment produced by the canning process may further encourage the outgrowth of spores. The organisms grow best in neutral or "low acid" vegetables (>pH4.5).

Clinical symptoms of botulism begin 18-36 hours after toxin ingestion with weakness, dizziness and dryness of the mouth. Nausea and vomiting may occur. Neurologic features soon develop: blurred vision, inability to swallow, difficulty in speech, descending weakness of skeletal muscles and respiratory paralysis.

Botulinum toxin may be transported within nerves in a manner analogous to tetanospasmin, and can thereby gain access to the CNS. However, symptomatic CNS involvement is rare.

Infant Botulism

Infant botulism is due to infection caused by C. botulinum. The disease occurs in infants 5 - 20 weeks of age that have been exposed to solid foods, presumably the source of infection (spores). It is characterized by constipation and weak sucking ability and generalized weakness. C. botulinum can apparently establish itself in the bowel of infants at a critical age before the establishment of competing intestinal bacteria (normal flora). Production of toxin by bacteria in the GI tract induces symptoms. This "infection-intoxication" is restricted to infants. C. botulinum organisms, as well as toxin can be found in the feces of infected infants. Almost all known cases of the disease have recovered. The possible role of infant botulism in "sudden infant death syndrome-SIDS" has been suggested and is under investigation. C. botulinum, its toxin, or both have been found in the bowel contents of several infants who have died suddenly and unexpectedly.

The Botulinum Toxins

The botulinum toxins are very similar in structure and function to the tetanus toxin, but differ dramatically in their clinical effects because they target different cells in the nervous system. Botulinum neurotoxins predominantly affect the peripheral nervous system reflecting a preference of the toxin for stimulatory motor neurons at a neuromuscular junction. The primary symptom is weakness or flaccid paralysis. Tetanus toxin can affect the same system, but the tetanospasmin shows a tropism for inhibitory motor neurons of the central nervous system, and its effects are primarily rigidity and spastic paralysis.

Botulinum toxin is synthesized as a single polypeptide chain with a molecular weight around 150 kDa. In this form the toxin has a relatively low potency. The toxin is nicked by a bacterial protease (or possibly by gastric proteases) to produce two chains: a light chain (the A fragment) with a molecular weight of 50 kDa; and a heavy chain (the B fragment), with a mw of 100kDa. As with tetanospasmin, the chains remain connected by a disulfide bond. The A fragment of the nicked toxin, on a molecular weight basis, becomes the most potent toxin found in nature.

Toxin Action

The botulinum toxin is specific for peripheral nerve endings at the point where a motor neuron stimulates a muscle. The toxin binds to the neuron and prevents the release of acetylcholine across the synaptic cleft.

The heavy chain of the toxin mediates binding to presynaptic receptors. The nature of these receptors is uncertain; different toxin types seem to utilize slightly different receptors. The binding region of the toxin molecule is located near the carboxy terminus of the heavy chain. The amino terminus of the heavy chain is thought to form a channel through the membrane of the neuron allowing the light chain to enter. The toxin (A fragment) enters the cell by receptor mediated endocytosis. Once inside a neuron, the toxin types probably differ in mechanisms by which they inhibit acetylcholine release, but a mechanism similar to or identical to tetanospasmin has been reported (i.e., proteolytic cleavage of synaptobrevin II). The affected cells fail to release a neurotransmitter, thus producing paralysis of the motor system. Once damaged, the synapse is rendered permanently useless. The recovery of function requires sprouting of a new presynaptic axon and the subsequent formation of a new synapse.

As stated above, the mechanism by which acetylcholine release is prevented is not known. However, recent evidence suggests that both botulinum toxin as well as tetanus toxin are zinc-dependent endopeptidases that cleave specific proteins that are involved in excretion of neurotransmitters. Both toxins cleave a set of proteins called synaptobrevins. Synaptobrevins are a set of proteins found in synaptic vesicle of neurons, the vesicles responsible for release of neurotransmitters. Presumably, proteolytic cleavage of synaptobrevin II would interfere with vesicle function and release of neurotransmitters.

Immunity

On the average there are about 25 cases of botulism annually in the U.S. Prior to the advent of critical care, the case fatality rate exceeded 60%, but currently it is about 20%. The first (or only) patient in an outbreak has a 25% chance of death, whereas subsequent cases which are diagnosed and treated more quickly, carry only a 4% risk.

The toxins that cause botulism are each specifically neutralized by its antitoxin. Botulinum toxins can be toxoided and make good antigens for inducing protective antibody. As with tetanus, immunity to botulism does not develop, even with severe disease, because the amount of toxin necessary to induce an immune response is toxic. Repeated occurrence of botulism has been reported.

Once the botulinum toxin has bound to nerve endings, its activity is unaffected by antitoxin. Any circulating ("unfixed") toxin can be neutralized by intravenous injection of antitoxin. Individuals known to have ingested food with botulism should be treated immediately with antiserum.

A multivalent toxoid evokes good protective antibiody response but its use is unjustified due to the infrequency of the disease. An experimental vaccine exists for laboratory workers.

Prevention

The most important aspect of botulism prevention is proper food handling and preparation. The spores of C. botulinum can survive boiling (100 degrees at 1 atm) for more than one hour although they are killed by autoclaving. Because the toxin is heat-labile boiling or intense heating (cooking) of contaminated food will inactivate the toxin. Food containers that bulge may contain gas produced by C. botulinum and should not be opened or tasted. Other foods that appear to be spoiled should not be tasted.