Clostridium botulinum is a Gram-positive, rod-shaped bacterium that produces several toxins. The best known are its neurotoxins, subdivided in types A-H, that cause the flaccid muscular paralysis seen in botulism. They are also the main paralytic agent in Botox. C. botulinum is an anaerobic spore-former, which produces oval, subterminal endospores and is commonly found in soil.
Clostridium botulinum is a rod-shaped microorganism. It is an obligate anaerobe, meaning that oxygen is poisonous to the cells. However, C. botulinum tolerates traces of oxygen due to the enzyme superoxide dismutase (SOD) which is an important antioxidant defense in nearly all cells exposed to oxygen. C. botulinum is only able to produce the neurotoxin during sporulation, which can only happen in an anaerobic environment. Other bacterial species produce spores in an unfavorable growth environment to preserve the organism's viability and permit survival in a dormant state until the spores are exposed to favorable conditions.
In the laboratory Clostridium botulinum is usually isolated in tryptose sulfite cycloserine (TSC) growth media in an anaerobic environment with less than 2% of oxygen. This can be achieved by several commercial kits that use a chemical reaction to replace O2 with CO2. C. botulinum is a lipase positive microorganism that grows between pH of 4.8 and 7 and can not use lactose as a primary carbon source, characteristics important during a biochemical identification.
Clostridium botulinum was first recognized and isolated in 1895 by Emile van Ermengem from home cured ham implicated in a botulism outbreak. The isolate was originally named Bacillus botulinus, after the Latin word for sausage, botulus. ("Sausage poisoning" was a common problem in 18th and 19th century Germany, and was most likely caused by botulism) However, isolates from subsequent outbreaks were always found to be anaerobic spore formers, so Ida Bengston proposed that the organism be placed into the genus Clostridium as the Bacillus genus was restricted to aerobic spore-forming rods.
Since 1959 all species producing the botulinum neurotoxins (types A-G) have been designated C. botulinum. Substantial phenotypic and genotypic evidence exists to demonstrate heterogeneity within the species. This has led to the reclassification of C. botulinum type G strains as a new species Clostridium argentinense.
The complete genome of C. botulinum has been sequenced at Sanger.
The current nomenclature for C. botulinum recognises four physiological groups (I-IV). The classification is based on the ability of the organism to digest complex proteins. Studies at the DNA and rRNA level support the subdivision of the species into groups I-IV. Most outbreaks of human botulism are caused by group I (proteolytic) or II (non-proteolytic) C. botulinum. Group III organisms mainly cause diseases in animals. There has been no record of Group IV C. botulinum causing human or animal disease.
Botulism poisoning can occur due to improperly preserved or home-canned, low-acid food that was not processed using correct preservation times and/or pressure.
Neurotoxin production is the unifying feature of the species C. botulinum. Eight types of toxins have been identified and allocated a letter (A-H). Most strains produce one type of neurotoxin but strains producing multiple toxins have been described. Clostridium botulinum producing B and F toxin types have been isolated from human botulism cases in New Mexico and California. The toxin type has been designated Bf as the type B toxin was found in excess to the type F. Similarly, strains producing Ab and Af toxins have been reported. There is evidence that the neurotoxin genes have been the subject of horizontal gene transfer, possibly from a viral source. This theory is supported by the presence of integration sites flanking the toxin in some strains of C. botulinum. However, these integrations sites are degraded indicating that the C. botulinum acquired the toxin genes quite far into the evolutionary past.
Only types A, B, E, and F cause disease in humans while types C and D cause disease in cows, birds, and other animals but not in humans. The "gold standard" for determining toxin type is a mouse bioassay, but the genes for types A, B, E, and F can now be readily differentiated using quantitative PCR (PCR).
A few strains from organisms genetically identified as other Clostridium species have caused human botulism: Clostridium butyricum has produced type E toxin and Clostridium baratii had produced type F toxin. The ability of C. botulinum to naturally transfer neurotoxin genes to other clostridia is concerning, especially in the food industry where preservation systems are designed to destroy or inhibit only C. botulinum but not other Clostridium species.
An eighth toxin, type H, was discovered by researchers at the California Department of Public Health in 2013. With a lethal dose of 2 ng by injection or 13 ng by inhalation, it was deemed the most toxic substance on Earth.
|Properties||Group I||Group II||Group III||Group IV|
|Toxin Types||A, B, F||B, E, F||C, D||G|
|Close relatives||C. sporogenes, C. putrificum||C. butyricum, C. beijerinickii||C. haemolyticum, C. novyi type A||C. subterminale, C. haemolyticum|
Clostridium botulinum in different geographical locations
Type A C. botulinum predominates the soil samples from the western regions while type B is the major type found in eastern areas. The type B organisms were of the proteolytic type I. Sediments from the Great Lake regions were surveyed after outbreaks of botulism among commercially reared fish and only type E spores were detected. It has been noted in a survey that type A strains were isolated from soils that were neutral to alkaline (average pH 7.5) while type B strains were isolated from slightly acidic soils (average pH 6.25).
Clostridium botulinum type E is prevalent in aquatic sediments in Norway and Sweden, Denmark, the Netherlands, the Baltic coast of Poland and Russia. It was then suggested that the type E C. botulinum is a true aquatic organism, which was indicated by the correlation between the level of type E contamination and flooding of the land with seawater. As the land dried, the level of type E decreased and type B became dominant.
In soil and sediment from the United Kingdom, C. botulinum type B predominates. In general, the incidence is usually lower in soil than in sediment. In Italy, a survey was conducted in the vicinity of Rome, and a low level of contamination was found; all strains were proteolytic C. botulinum type A or B.
Clostridium botulinum type A was found to be present in soil samples from mountain areas of Victoria. Type B organisms were detected in marine mud from Tasmania. Type A C. botulinum have been found in Sydney suburbs and types A and B were isolated from urban areas. In a well defined area of the Darling-Downs region of Queensland, a study showed the prevalence and persistence of C. botulinum type B after many cases of botulism in horses.
A "mouse protection" or "mouse bioassay" test determines the type of C. botulinum toxin present using monoclonal antibodies. An enzyme-linked immunosorbent assay (ELISA)with digoxigenin-labeled antibodies can also be used to detect the toxin, and quantitative PCR can detect the toxin genes in the organism.
Clostridium botulinum is also used to prepare the medicaments Botox, Dysport, Xeomin, and Neurobloc used to selectively paralyze muscles to temporarily relieve muscle function. It has other "off-label" medical purposes, such as treating severe facial pain, such as that caused by trigeminal neuralgia.
Botulin toxin produced by C. botulinum is often believed to be a potential bioweapon as it is so potent that it takes about 75 nanograms to kill a person (LD50 of 1 ng/kg, assuming an average person weighs ~75 kg); 1 kilogram of it would be enough to kill the entire human population. For comparative purposes, a quarter of a typical grain of sand's weight (350 ng) of botulinum toxin would constitute a lethal dose for humans.
Clostridium botulinum is a soil bacterium. The spores can survive in most environments and are very hard to kill. They can survive the temperature of boiling water at sea level, thus many foods are canned with a pressurized boil that achieves an even higher temperature, sufficient to kill the spores.
Growth of the bacterium can be prevented by high acidity, high ratio of dissolved sugar, high levels of oxygen, very low levels of moisture or storage at temperatures below 3°C (38°F) for type A. For example in a low acid, canned vegetable such as green beans that are not heated hot enough to kill the spores (i.e., a pressurized environment) may provide an oxygen free medium for the spores to grow and produce the toxin. On the other hand, pickles are sufficiently acidic to prevent growth; even if the spores are present, they pose no danger to the consumer. Honey, corn syrup, and other sweeteners may contain spores but the spores cannot grow in a highly concentrated sugar solution; however, when a sweetener is diluted in the low oxygen, low acid digestive system of an infant, the spores can grow and produce toxin. As soon as infants begin eating solid food, the digestive juices become too acidic for the bacterium to grow.
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