Clostridium botulinum is a gram-positive, rod-shaped, anaerobic, spore-forming, motile bacterium that has the ability to produce botulinum toxin, which is a highly potent neurotoxin.
C. botulinum is a diverse group of pathogenic bacteria. Initially, they were grouped together by their ability to produce botulinum toxin and are now known as four distinct groups, C. botulinum groups I–IV. Along with some strains of Clostridium butyricum and Clostridium baratii, these bacteria all produce the toxin.
Botulinum toxin can cause botulism, a severe flaccid paralytic disease in humans and other animals. The botulinum toxin is the most potent toxin known in scientific literature, natural or synthetic, with a lethal dose of 1.3–2.1 ng/kg in humans.
C. botulinum is commonly associated with bulging canned food; misshapen cans can be due to an internal increase in pressure caused by gas produced by bacteria. C. botulinum can also grow in traditionally aged meats (such as igunaq) and carrion (such as a dead beached whale).
C. botulinum is responsible for food-borne botulism (ingestion of preformed toxin), infant botulism (intestinal infection with toxin-forming C. botulinum), and wound botulism (infection of a wound with C. botulinum). C. botulinum produces heat-resistant endospores that are commonly found in soil and are able to survive under adverse conditions.
Microbiology
C. botulinum is an obligate anaerobe, requiring an environment that lacks oxygen. However, C. botulinum tolerates traces of oxygen due to the enzyme superoxide dismutase, which is an important antioxidant defense in nearly all cells exposed to oxygen. C. botulinum is able to produce the neurotoxin only during sporulation, which can happen only in an anaerobic environment.
C. botulinum is divided into four distinct phenotypic groups (I-IV) and is also classified into seven serotypes (A–G) based on the antigenicity of the botulinum toxin produced. On the level visible to DNA sequences, the phenotypic grouping matches the results of whole-genome and rRNA analyses, and setotype grouping approximates the result of analyses focused specifically on the toxin sequence. The two phylogenetic trees do not match because of the ability of the toxin gene cluster to be horizontally transferred.
Serotypes
Botulinum neurotoxin (BoNT) production is the unifying feature of the species. Seven serotypes of toxins have been identified that are allocated a letter (A–G), several of which can cause disease in humans. They are resistant to degradation by enzymes found in the gastrointestinal tract. This allows for ingested toxins to be absorbed from the intestines into the bloodstream. Toxins can be further differentiated into subtypes on the bases of smaller variations. However, all types of botulinum toxin are rapidly destroyed by heating to for 15 minutes (900 seconds). for 30 minutes also destroys BoNT.
Most strains produce one type of BoNT, but strains producing multiple toxins have been described. C. 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.
Evidence indicates the neurotoxin genes have been the subject of horizontal gene transfer, possibly from a viral (bacteriophage) 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 (except for the C and D types), indicating that the C. botulinum acquired the toxin genes quite far in the evolutionary past. Nevertheless, further transfers still happen via the plasmids and other mobile elements the genes are located on.
Toxin types in disease Botulinum toxin types A, B, E, F and H (FA) cause disease in humans. Types A, B, and E are associated with food-borne illness, while type E is specifically associated with fish products. Type C produces limber-neck in birds and type D causes botulism in other mammals. No disease is associated with type G. 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. Type "H" is in fact a recombinant toxin from types A and F. It can be neutralized by type A antitoxin and no longer is considered a distinct type.
A few strains from organisms genetically identified as other Clostridium species have caused human botulism: C. butyricum has produced type E toxin and C. baratii has 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.
Metabolism Many C. botulinum genes play a role in the breakdown of essential carbohydrates and the metabolism of sugars. Chitin is the preferred source of carbon and nitrogen for C. botulinum. Hall A strain of C. botulinum has an active chitinolytic system to aid in the breakdown of chitin. Type A and B of C. botulinum production of BoNT is affected by nitrogen and carbon nutrition. There is evidence that these processes are also under catabolite repression.
Proteolytic Clostridium often rely on amino acids as carbon and energy sources. They carry out a unique metabolic process called Stickland Fermentation. In this process, two amino acids are used in complementary roles. One will serve as an electron donor, and the other amino acid serves as an electron acceptor. Not only does this reaction create precursors for other metabolic pathways, but it also regenerates NAD+, an important oxidizing agent in the Embden-Meyerhof-Parnas pathway. Utilizing amino acids as a carbon source and regenerating NAD+ allows C. botulinum to thrive in anaerobic environments.
Groups Physiological differences and genome sequencing at 16S rRNA level support the subdivision of the C. botulinum species into groups I-IV. Some authors have briefly used groups V and VI, corresponding to toxin-producing C. baratii and C. butyricum. What used to be group IV is now C. argentinense. +Phenotypic groups of toxin-producing Clostridium Property Group I Group II Group III C. argentinense C. baratii C. butyricum Proteolysis (casein) Saccharolysis Lipase Toxin Types A, B, F B, E, F C, D G F E Toxin gene chromosome/plasmid chromosome/plasmid bacteriophage plasmid chromosome chromosome Close relatives C. beijerinckii C. butyricum C. novyi type A C. haemolyticum N/A (already a species)
Although group II cannot degrade native protein such as casein, coagulated egg white, and cooked meat particles, it is able to degrade gelatin.
Human botulism is predominantly caused by group I or II C. botulinum. Group III organisms mainly cause diseases in non-human animals.
Laboratory isolation In the laboratory, C. botulinum is usually isolated in tryptose sulfite cycloserine (TSC) growth medium in an anaerobic environment with less than 2% oxygen. This can be achieved by several commercial kits that use a chemical reaction to replace O2 with CO2. C. botulinum (groups I through III) is a lipase-positive microorganism that grows between pH of 4.8 and 7.0 and cannot use lactose as a primary carbon source, characteristics important for biochemical identification.
Transmission, sporulation, and germination Sporulation and germination in Clostridium botulinum is a major virulence factor, allowing the bacteria to be prevalent in a wide variety of environments, and botulism to exist in many forms. The exact mechanism behind sporulation and germination of C. botulinum is not known, but the study of Bacillus subtilus can be used to give a general prediction of the mechanism.
Different strains of C. botulinum can be divided into three different groups, group I, II, and III, based on environmental conditions like heat resistance, temperature, and biome. Within each group, different strains will use different strategies to adapt to their environment to survive. Unlike other clostridial species, C. botulinum spores will sporulate as it enters the stationary phase. C. botulinum relies on quorum-sensing to initiate the sporulation process.
C. botulinum begins sporulation once its sporulation protein becomes phosphorylated. The phosphorylation activates the stage I and II sporulation genes that halt cell division, and begin creation of an asymmetric septum and the spore. The cell forms spore layers until the spore is fully mature, after which, the cell is covered in exosporium. Group I cells have a thick yet loose exosporium layer that allows the spore to be heat-resistant, while group III have a thin yet tight exosporium layer that leaves the cell with a moderate amount of heat-resistance.
Germination will begin if specific nutrients called germinants interact with receptors found within the spore. Once the germinants are bound to the cell, the cell begins to hydrolyze itself to rehydrate its core and create a vegetative cell. C. botulinum spores are not found in human feces unless the individual has contracted botulism, but C. botulinum cannot spread from person to person.
Motility structures The most common motility structure for C. botulinum is a flagellum. Though this structure is not found in all strains of C. botulinum, most produce peritrichous flagella. When comparing the different strains, there are also differences in the length of the flagella and how many are present on the cell.
Genomic analyses of C. botulinum have shown many strains to harbor mutations in flagellar genes, including flaA, which encodes the flagellin protein, and flhA and flgN, which are involved in flagellar biosynthesis. These mutations can change motility to varying extents, and in some cases, may modify the structure of the flagellum without fully impairing its function. Flagella are important in colonization and movement in gut environments, but also have immunogenic properties that make the bacterium more visible to host defenses. As a result, strains may downregulating flagellar production or acquire mutations that change flagellar assembly, so they can be used for motility but also evade the immune system during colonization.
Growth conditions and prevention
C. 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 even higher temperatures, sufficient to kill the spores. This bacteria is widely distributed in nature and can be assumed to be present on all food surfaces. Its optimum growth temperature is within the mesophilic range. In spore form, it is a heat resistant pathogen that can survive in low acid foods and grow to produce toxins. The toxin attacks the nervous system and will kill an adult at a dose of around 75 ng. Botulinum toxin can be destroyed by holding food at for 10 minutes; however, because of its potency, this is not recommended by the USA's FDA as a means of control.
Botulism poisoning can occur due to preserved or home-canned, low-acid food that was not processed using correct preservation times and/or pressure. 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 for type A. For example, low-acid, canned vegetables (such as green beans) that are not heated 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. However, pickles are sufficiently acidic to prevent growth; even if the spores are present, they pose no danger to the consumer.