Clostridium botulinum is a group of bacteria best known as the main source of botulinum neurotoxin, the cause of botulism in humans. There are several types of botulism. Foodborne botulism is caused by consumption of pre-formed toxin, whereas infant/intestinal (adult) botulism and wound botulism are infections involving toxin formation in situ.
The symptoms of botulism are primarily neurological and frequently begin with blurred vision, continuing to a descending bilateral flaccid paralysis, and in severe cases a flaccid paralysis of the respiratory or cardiac muscles. In many countries, equine antitoxin is administered to adults suffering from botulism, although in severe cases full recovery may take months or even years. The fatality rate is approximately 5 to 10% of cases. The economic and medical costs associated with foodborne botulism are extremely high.
Clostridium botulinum is defined solely on the basis of an ability to form botulinum neurotoxin, rather than on phylogenetic relationships. This approach is taken in order to emphasize the importance of neurotoxin formation. As a consequence, however, C. botulinum is a heterogeneous "species" that actually comprises four phylogenetically and physiologically distinct clades, known as C. botulinum Groups I to IV, with the distinction among the groups strong enough to merit treatment as four different species. Proteolytic C. botulinum (C. botulinum Group I) and non-proteolytic C. botulinum (C. botulinum Group II) are responsible for most cases of foodborne botulism.
(Peck et al. 2011 and references therein)
Based on research begun in the 1980s, botulinum toxin has been found useful in an expanding list of medical applications that require the blocking of involuntary muscle contractions, including eyelid twitching and other neurological problems. In the late 1980s, it was found that injection of botulinum toxin had cosmetic dermatological applications (wrinkle reduction) as well. Botulinum toxin entered the cultural mainstream in the United States in the formulation known as Botox®, establishing a new and highly profitable market for a product once known only as the cause of death for unfortunate individuals consuming improperly prepared sausages. (Kopera 2011)
More information on botulism is available from the U.S.Centers for Disease Control and Prevention.
Uses for botulinum toxin
Clinical Applications of Clostridium botulinum
Botulinum toxin is produced by anaerobic Clostridium botulinum bacteria and causes a paralysis called botulism. It was known as a deadly form of food poisoning but while this toxin is one of the deadliest on the planet, it also has a variety of clinical indications. In the 1950’s doctors discovered that injecting overactive muscles with small doses of toxin resulted in reduced muscle activity by blocking the release of acetylcholine at the neuromuscular junction3 and thus weakening the affected muscle for 4-6 months. It has since developed into a therapeutic tool. Since its discovery as a muscle inhibitor a variety of uses have been found for botulinum toxin ranging from cosmetic and medicinal aids to biological weapons. The most popular and recognizable use is a cosmetic injectable form (Botox or Dysport) that reduces wrinkles and signs of aging in the face. Local injection of botulinum toxin is also used to treat excessive sweating, cervical and laryngeal dystonia, excessive blinking and crossed eyes, Tardive dystonia, as well as other disorders of the central nervous system which result in lack of muscle control such as Parkinson’s, stroke and multiple sclerosis2. Botulinum toxin is the most common treatment for symptoms of movement disorders2 such as spasmodic torticollis, tics, tremors and dysphonia. Botulinum Toxin A has been effective in treating migraines and headaches and has been shown to improve gait patterns in patients with cerebral palsy who suffer from foot deformities4. Botulinum toxin has a multitude of medical uses but it must used carefully and with control to avoid permanent paralysis or poisoning.
Botulinum toxin has some unfavorable indications as well. Due to its high toxicity and potency, botulinum toxin has great potential for use as a biological weapon1. Great care need be taken when handling or using Botulinum toxin. A single gram of crystalline toxin, evenly dispersed, can kill over 1 million people1. Also due to its deadly toxicity, botulinum toxin can be used as a powerful poison if misused or ingested. As mentioned above, the toxin can cause a deadly form of food poisoning because it is found in soil and thennwhen transmitted in food sources. . Marine animals have even been shown to ingest C. botulinum from shellfish, resulting in contamination of the intestinal tract5 that is then spread to humans through onshore butchering and poor food preparation precautions.
Description of Clostridium botulinum
Clostridium botulinum is a Gram-positive, rod-shaped, anaerobic, spore-forming, motile bacterium with the ability to produce the neurotoxin botulinum. The botulinum toxin can cause a severe flaccid paralytic disease in humans and animals and is the most potent toxin known to humankind, natural or synthetic, with a lethal dose of less than 1 μg in humans.
C. botulinum is a diverse group of bacteria initially grouped together by their ability to produce botulinum toxin and now known as four distinct groups, C. botulinum groups I-IV. C. botulinum groups I-IV, as well as some strains of Clostridium butyricum and Clostridium baratii, are the bacteria responsible for producing botulinum toxin.
C. botulinum is responsible for foodborne 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 allow for survival in adverse conditions.
C. botulinum is a Gram-positive, rod-shaped, spore-forming bacterium. 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, 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.
C. botulinum is divided in to four distinct phenotypic groups (I-IV) and is also classified into seven serotypes (A-G) based on the antigenicity of the botulinum toxin produced. Recently, an eighth serotype (H) has been described.
The classification into groups 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. Group IV C. botulinum has not been shown to cause human or animal disease.
Neurotoxin production is the unifying feature of the species. Eight types of toxins have been identified (including the recently described type H) that are allocated a letter (A-H). All toxins are rapidly destroyed at 100°C, but they are resistant to degradation by enzymes found in the gastrointestinal tract. This allows for ingested toxin to be absorbed from the intestines into the bloodstream.
Most strains produce one type of neurotoxin, 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 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 in the evolutionary past.
Botulinum toxin types
Only botulinum toxin types A, B, E, F, and H cause disease in humans. Types A, B, and E are associated with foodborne illness, with type E specifically associated with fish products. Type C produces limberneck 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.
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 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.
|A, B, F||B, E, F||C, D||G|
|C. sporogenes, C. putrificum||C. butyricum, C. beijerinickii||C. haemolyticum, C. novyi type A||C. subterminale, C. haemolyticum|
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 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.
C. 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 A. Bengtson 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, C. argentinense.
The complete genome of C. botulinum has been sequenced at Sanger.
Botulism poisoning can occur due to preserved or home-canned, low-acid food that was not processed using correct preservation times and/or pressure.
Foodborne botulism "Signs and symptoms of foodborne botulism typically begin between 18 and 36 hours after the toxin gets into your body, but can range from a few hours to several days, depending on the amount of toxin ingested."
- Double vision
- Blurred vision
- Drooping eyelid
- Nausea, vomiting, and abdominal cramps
- Slurred speech
- Trouble breathing
- Difficulty in swallowing
- Dry mouth
- Muscle weakness
- Reduced or absent deep tendon reactions, such as in the knee.
Wound botulism Most people who develop wound botulism inject drugs several times a day, so it's difficult to determine how long it takes for signs and symptoms to develop after the toxin enters the body. Most common in people who inject black tar heroin, wound botulism signs and symptoms include:
- Difficulty swallowing or speaking
- Facial weakness on both sides of the face
- Blurred or double vision
- Drooping eyelids
- Trouble breathing
Infant botulism If infant botulism is related to food, such as honey, problems generally begin within 18 to 36 hours after the toxin enters the baby's body. Signs and symptoms include:
- Constipation (often the first sign)
- Floppy movements due to muscle weakness and trouble controlling the head
- Weak cry
- Drooping eyelids
- Difficulty sucking or feeding
Beneficial effects of botulinum toxin: Purified botulinum toxin is diluted by a physician for treatment:
- Congenital pelvic tilt
- Spasmodic dysphasia (the inability of the muscles of the larynx)
- Achalasia (esophageal stricture)
- Strabismus (crossed eyes)
- Paralysis of the facial muscles
- Failure of the cervix
- Blinking frequently
- 8 - According to the BBC, an American group is trying Clostridium type E to deliver anticancer drugs directly to the tumor.
C. 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 Lakes region were surveyed after outbreaks of botulism among commercially reared fish, and only type E spores were detected. In a survey, 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).
C. botulinum type E is prevalent in aquatic sediments in Norway and Sweden, Denmark, the Netherlands, the Baltic coast of Poland, and Russia. The type-E C. botulinum was suggested to be 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 conducted in the vicinity of Rome found a low level of contamination; all strains were proteolytic C. botulinum types A or B.
C. 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 has 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.
C. 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.
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.
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 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. 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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