Clostridium botulinum

Clostridium botulinum
Clostridium botulinum stained with gentian violet.
Scientific classification
Domain:	Bacteria
Class:	Clostridia
Order:	Clostridiales
Family:	Clostridiaceae
Genus:	Clostridium
Species:	C. botulinum
Binomial name
Clostridium botulinum van Ermengem, 1896

Clostridium botulinum is a Gram-positive, rod-shaped bacterium that produces several toxins. The best known are its neurotoxins, subdivided in types A-G, that cause the flaccid muscular paralysis seen in botulism. It is 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. In addition it produces two ADP-ribosyltransferases: C2 and Clostridium botulinum C3 toxin. C2 acts on monomeric G-actin, while C3 causes the ADP-ribosylation of rho G-proteins with dramatic consequences for cellular integrity.

Wittebiology

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.^[1] 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 (E.J. GasPak System). C. botulinum is a lipase negative microorganism that grows between pH of 4.8 and 7 and it can't use lactose as a primary carbon source, characteristics important during a biochemical identification.^[2]

Taxonomy history

Clostridium botulinum was first recognized and isolated in 1895 by Emile van Ermengem from home cured ham implicated in a botulism outbreak.^[3] 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)^[4] 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.^[5]

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.^[6]

Group I Clostridium botulinum strains that do not produce a botulin toxin are referred to as Clostridium sporogenes.^[7]

The complete genome of C. botulinum has been sequenced Sanger.

Phenotypes

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.^[8][9] 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.

Pathology

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 types

Neurotoxin production is the unifying feature of the species C. botulinum. Seven types of toxins have been identified and allocated a letter (A-G). 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.^[10] 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 Real-time polymerase chain reaction (PCR).^[11]

Organisms genetically as they identified as other Clostridium species have caused human botulism; Clostridium butyricum producing type E toxin^[12] and Clostridium baratii producing type F toxin.^[13][14] 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.

Phenotypic groups of Clostridium botulinum

Properties	Group I	Group II	Group III	Group IV
Toxin Types	A, B, F	B, E, F	C, D	G
Proteolysis	+	-	weak	-
Saccharolysis	-	+	-	-
Disease host	human	human	animal	-
Toxin gene	chromosome	chromosome	bacteriophage	plasmid
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

A number of quantitative surveys for C. botulinum spores in the environment have suggested a prevalence of specific toxin types in given geographic areas, which remain unexplained.

North America

Type A C. botulinum predominates the soil samples from the western regions while type B is the major type found in eastern areas.^[15] 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.^[16][17][18] 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).

Europe

Clostridium botulinum type E is prevalent in aquatic sediments in Norway and Sweden,^[19] Denmark,^[20] the Netherlands, the Baltic coast of Poland and Russia.^[15] 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.^[21]

Australia

Clostridium botulinum type A was found to be present in soil samples from mountain areas of Victoria.^[22] Type B organisms were detected in marine mud from Tasmania.^[23] 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.

Other

A "mouse protection" or "mouse bioassay" test determines the type of C. botulinum present using monoclonal antibodies. This can now also be accomplished using real-time PCR.^[11]

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,^[24] assuming an average person weighs ~75 kg); 500 grams of it would be enough to kill half of the entire human population.

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.

References

1. ^ Doyle, Michael P. (2007). Food Microbiology: Fundamentals and Frontiers. ASM Press. ISBN 1555812082. 
2. ^ . (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall. ISBN 0131443291. 
3. ^ E. van Ergmengem. 1897. Über einen neuen anaeroben Bacillus und seine Beziehungen Zum Botulismus. Zentralbl. Hyg. Infektionskr. 26:1–8.
4. ^ Frank J. Erbguth. Historical notes on botulism, Clostridium botulinum, botulinum toxin, and the idea of the therapeutic use of the toxin. Movement Disorders. Volume 19, Issue S8, pages S2-S6, March 2004.
5. ^ I. A. Bengston. 1924. Studies on organisms concerned as causative factors in botulism. Hyg. Lab. Bull. 136:101
6. ^ J. C. Suen, C. L. Hatheway, A. G. Steigerwalt, D. J. Brenner. 1988, Clostridium argentinense sp.nov.: a genetically homogeneous group composed of all strains of Clostridium botulinum type G and some nontoxigenic strains previously identified as Clostridium subterminale or Clostridium hastiforme. Int. J. Sys. Bacteriol. 38:375–381.
7. ^ Judicial Commission of the International Committee on Systematic Bacteriology (1999) Rejection of Clostridium putrificum and conservation of Clostridium botulinum and Clostridium sporogenes Opinion 69. International Journal of Systematic Bacteriology. 49:339.
8. ^ L. V. Holdeman, J. B. Brooks. 1970. Variation among strains of Clostridium botulinum and related clostridia. Protocols of the first U.S-Japan conference on Toxic Microorganisms. pp. 278–286
9. ^ L. D. S. Smith, G. Hobbs. 1974. Genus III Clostridium Prazmowski 1880, 23. In R. E. Buchanan, N. E. gibbons (eds.), Bergey’s Manual of Determinative Bacteriology, 8th edition. William & Wilkins, Baltimore. pp. 551–572.
10. ^ Hatheway C. L., McCroskey L. M. (1987). "Examination of faeces for diagnosis of infant botulism in 336 patients". J. Clin. Microbiol 25 (12): 2334–2338. PMC 269483. PMID 3323228. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=269483. 
11. ^ ^{a b} Satterfield, B. A.; Stewart, A. F.; Lew, C. S.; Pickett, D. O.; Cohen, M. N.; Moore, E. A.; Luedtke, P. F.; O'Neill, K. L. et al. (2010). "A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F". J Med Microbiol 59 (Pt 1): 55–64. doi:10.1099/jmm.0.012567-0. PMID 19779029. 
12. ^ Aureli, P.; Fenicia, L.; Pasolini, B.; Gianfrancesche, M.; Mccroskey, J. M.; Hatheway, C. L. (1986). "Two cases of type E infant botulism caused by neurotoxigenic Clostridium botulinum in Italy". J. Infect. Dis. 154 (2): 207–211. PMID 3722863. 
13. ^ Hall, J. D.; McCroskey, L. M.; Pincomb, B. J.; Hatheway, C. L. (1985). "Isolation of an organism resembling Clostridium baratii which produces a type F botulinal toxin from an infant with botulism". J. Clin. Microbiol 21 (4): 654–655. PMC 271744. PMID 3988908. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=271744. 
14. ^ Notermans S., Havellar A. H. (1980). "Removal and inactivation of botulinum toxin during production of drinking water from surface water". Antonie van Leeuwenhoek 46: 511–514. doi:10.1007/BF00395840. 
15. ^ ^{a b} A. H. W. Hauschild. 1989. Clostridium botulinum. In M. P. Doyle (ed.), Food-borne Bacterial Pathogens. Marcel Dekker, New York. Pp. 111–189
16. ^ Bott, T. L.; Johnson, J.; Foster, E. M.; Sugiyama, H. (1968). "Possible origin of the fish incidences of Clostridium botulinum type E in an inland bay (Green Bay of Lake Michigan)". J. Bacteriol 95: 1542. 
17. ^ M. W. Eklund, M. E. Peterson, F. T. Poysky, L. W. Peck, J. F. Conrad. 1982. Botulism in juvenile Coho salmon (Onocorhynchus kisutch) in the United States. Aquaculture 27:1–11
18. ^ M. W. Eklund, F. T. Poysky M. E. Peterson, L. W. Peck, Brunson. 1984. Type E botulism in salmonids and conditions contributing to outbreaks. Aquaculture 41:293–309.
19. ^ A. Johannsen. 1963. Clostridium botulinum in Sweden and the adjacent waters. J. Appl. Bacteriol. 26:43–47.
20. ^ Huss, H. H. (1980). "Distribution of Clostridium botulinum". Appl. Environ. Microbiol 39 (4): 764–769. PMC 291416. PMID 6990867. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=291416. 
21. ^ Creti, R.; Fenicia, J.; Aureli, P. (1990). "Occurrence of Clostridium botulinum in the soil of the vicinity of Rome". Curr. Microbiol 20: 317. 
22. ^ C. E. Eales, J. M. Gillespie. 1947. the isolation of Clostridium botulinum type A from Victorian soils. Aust. J. Sci. 10:20–21.
23. ^ D. f. Ohye, W. J. Scott. 1957. Studies in the physiology of Clostridium botulinum type E. Aust. L. Biol. Sci. 10:85–94.
24. ^ Fleming, Diane O.. "Biological Safety: principles and practices". ASM Press, 2000: 267. 

External links

• Pathema-Clostridium Resource
• Jeremy Sobel (2005). "Botulism". Clinical Infectious Diseases 41 (8): 1167–1173. doi:10.1086/444507. PMID 16163636. http://www.journals.uchicago.edu/doi/full/10.1086/444507. 
• Current research on Clostridium botulinum at the Norwich Research Park