Clostridium botulinum

Clostridium botulinum

Overview
Clostridium botulinum is a Gram-positive, rod-shaped bacterium that produces several toxins. The best known are its neurotoxin
Neurotoxin
A neurotoxin is a toxin that acts specifically on nerve cells , usually by interacting with membrane proteins such as ion channels. Some sources are more general, and define the effect of neurotoxins as occurring at nerve tissue...

s, subdivided in types A-G, that cause the flaccid muscular paralysis
Paralysis
Paralysis is loss of muscle function for one or more muscles. Paralysis can be accompanied by a loss of feeling in the affected area if there is sensory damage as well as motor. A study conducted by the Christopher & Dana Reeve Foundation, suggests that about 1 in 50 people have been diagnosed...

 seen in botulism
Botulism
Botulism also known as botulinus intoxication is a rare but serious paralytic illness caused by botulinum toxin which is metabolic waste produced under anaerobic conditions by the bacterium Clostridium botulinum, and affecting a wide range of mammals, birds and fish...

. It is also the main paralytic agent in botox. C. botulinum is an anaerobic
Anaerobic organism
An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. It could possibly react negatively and may even die if oxygen is present...

 spore-former, which produces oval, subterminal endospores and is commonly found in soil
Soil
Soil is a natural body consisting of layers of mineral constituents of variable thicknesses, which differ from the parent materials in their morphological, physical, chemical, and mineralogical characteristics...

. In addition it produces two ADP-ribosyltransferases: C2 and Clostridium botulinum C3 toxin
Clostridium botulinum C3 toxin
Clostridium botulinum C3 exoenzyme is a toxin that causes the ADP-ribosylate of Rho-like proteins. Many bacterial toxins nucleotide-binding modify by ADP-ribosylation proteins involved in essential cell functions....

.
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Encyclopedia
Clostridium botulinum is a Gram-positive, rod-shaped bacterium that produces several toxins. The best known are its neurotoxin
Neurotoxin
A neurotoxin is a toxin that acts specifically on nerve cells , usually by interacting with membrane proteins such as ion channels. Some sources are more general, and define the effect of neurotoxins as occurring at nerve tissue...

s, subdivided in types A-G, that cause the flaccid muscular paralysis
Paralysis
Paralysis is loss of muscle function for one or more muscles. Paralysis can be accompanied by a loss of feeling in the affected area if there is sensory damage as well as motor. A study conducted by the Christopher & Dana Reeve Foundation, suggests that about 1 in 50 people have been diagnosed...

 seen in botulism
Botulism
Botulism also known as botulinus intoxication is a rare but serious paralytic illness caused by botulinum toxin which is metabolic waste produced under anaerobic conditions by the bacterium Clostridium botulinum, and affecting a wide range of mammals, birds and fish...

. It is also the main paralytic agent in botox. C. botulinum is an anaerobic
Anaerobic organism
An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. It could possibly react negatively and may even die if oxygen is present...

 spore-former, which produces oval, subterminal endospores and is commonly found in soil
Soil
Soil is a natural body consisting of layers of mineral constituents of variable thicknesses, which differ from the parent materials in their morphological, physical, chemical, and mineralogical characteristics...

. In addition it produces two ADP-ribosyltransferases: C2 and Clostridium botulinum C3 toxin
Clostridium botulinum C3 toxin
Clostridium botulinum C3 exoenzyme is a toxin that causes the ADP-ribosylate of Rho-like proteins. Many bacterial toxins nucleotide-binding modify by ADP-ribosylation proteins involved in essential cell functions....

. C2 acts on monomeric G-actin, while C3 causes the ADP-ribosylation
ADP-ribosylation
ADP-ribosylation is the addition of one or more ADP-ribose moieties to a protein. These reactions are involved in cell signaling and the control of many cell processes, including DNA repair and apoptosis.-ADP-ribosylation enzymes:...

 of rho G-proteins with dramatic consequences for cellular integrity.

Biology


Clostridium botulinum is a rod-shaped microorganism. It is an obligate anaerobe
Obligate anaerobe
Obligate anaerobes are microorganisms that live and grow in the absence of molecular oxygen; some of these are killed by oxygen. -Metabolism:...

, meaning that oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...

 is poisonous to the cells. However, C. botulinum tolerates traces of oxygen due to the enzyme superoxide dismutase
Superoxide dismutase
Superoxide dismutases are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. As such, they are an important antioxidant defense in nearly all cells exposed to oxygen...

 (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
Cycloserine
Cycloserine is an antibiotic effective against Mycobacterium tuberculosis. For the treatment of tuberculosis, it is classified as a second line drug, i.e...

 (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
Lipase
A lipase is an enzyme that catalyzes the formation or cleavage of fats . Lipases are a subclass of the esterases.Lipases perform essential roles in the digestion, transport and processing of dietary lipids in most, if not all, living organisms...

 negative microorganism that grows between pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...

 of 4.8 and 7 and it can't use lactose
Lactose
Lactose is a disaccharide sugar that is found most notably in milk and is formed from galactose and glucose. Lactose makes up around 2~8% of milk , although the amount varies among species and individuals. It is extracted from sweet or sour whey. The name comes from or , the Latin word for milk,...

 as a primary carbon source, characteristics important during a biochemical identification.

Taxonomy history


Clostridium botulinum was first recognized and isolated in 1895 by Emile van Ermengem
Émile van Ermengem
Émile Pierre-Marie van Ermengem was a Belgian bacteriologist who, in 1895, isolated Clostridium botulinum, the bacterium that causes botulism, from a piece of ham that had poisoned thirty four people....

 from home cured ham
Ham
Ham is a cut of meat from the thigh of the hind leg of certain animals, especiallypigs. Nearly all hams sold today are fully cooked or cured.-Etymology:...

 implicated in a botulism
Botulism
Botulism also known as botulinus intoxication is a rare but serious paralytic illness caused by botulinum toxin which is metabolic waste produced under anaerobic conditions by the bacterium Clostridium botulinum, and affecting a wide range of mammals, birds and fish...

 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
Anaerobic organism
An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. It could possibly react negatively and may even die if oxygen is present...

 spore formers, so Ida Bengston proposed that the organism be placed into the genus Clostridium as the Bacillus
Bacillus
Bacillus is a genus of Gram-positive, rod-shaped bacteria and a member of the division Firmicutes. Bacillus species can be obligate aerobes or facultative anaerobes, and test positive for the enzyme catalase. Ubiquitous in nature, Bacillus includes both free-living and pathogenic species...

genus was restricted to aerobic
Aerobic organism
An aerobic organism or aerobe is an organism that can survive and grow in an oxygenated environment.Faculitative anaerobes grow and survive in an oxygenated environment and so do aerotolerant anaerobes.-Glucose:...

 spore-forming rods.

Since 1959 all species producing the botulinum neurotoxins
Botulinum toxin
Botulinum toxin is a protein produced by the bacterium Clostridium botulinum, and is considered the most powerful neurotoxin ever discovered. Botulinum toxin causes Botulism poisoning, a serious and life-threatening illness in humans and animals...

 (types A-G) have been designated C. botulinum. Substantial phenotypic and genotypic evidence exists to demonstrate heterogeneity within the species
Species
In biology, a species is one of the basic units of biological classification and a taxonomic rank. A species is often defined as a group of organisms capable of interbreeding and producing fertile offspring. While in many cases this definition is adequate, more precise or differing measures are...

. This has led to the reclassification of C. botulinum type G strains as a new species Clostridium argentinense.

Group I Clostridium botulinum strains that do not produce a botulin toxin are referred to as Clostridium sporogenes
Clostridium sporogenes
Clostridium sporogenes is the name given to strains of Clostridium botulinum that do not produce botulinum neurotoxins. Like other strains of C. botulinum, it is an anaerobic Gram-positive, rod-shaped bacterium that produces oval, subterminal endospores and is commonly found in soil...

.

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

Phenotypes


The current nomenclature
Nomenclature
Nomenclature is a term that applies to either a list of names or terms, or to the system of principles, procedures and terms related to naming - which is the assigning of a word or phrase to a particular object or property...

 for C. botulinum recognises four physiological groups (I-IV). The classification is based on the ability of the organism to digest
Digest
Digest can refer to any of the following:*A condensed collection or compendium of writings:**Pandects, or "The Digest", a digest of Roman law**A tax digest...

 complex proteins. Studies at the DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 and rRNA level support the subdivision of the species into groups I-IV. Most outbreaks of human botulism
Botulism
Botulism also known as botulinus intoxication is a rare but serious paralytic illness caused by botulinum toxin which is metabolic waste produced under anaerobic conditions by the bacterium Clostridium botulinum, and affecting a wide range of mammals, birds and fish...

 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
Neurotoxin
A neurotoxin is a toxin that acts specifically on nerve cells , usually by interacting with membrane proteins such as ion channels. Some sources are more general, and define the effect of neurotoxins as occurring at nerve tissue...

 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
Neurotoxin
A neurotoxin is a toxin that acts specifically on nerve cells , usually by interacting with membrane proteins such as ion channels. Some sources are more general, and define the effect of neurotoxins as occurring at nerve tissue...

 but strains producing multiple toxins have been described. Clostridium botulinum producing B and F toxin types have been isolated from human botulism
Botulism
Botulism also known as botulinus intoxication is a rare but serious paralytic illness caused by botulinum toxin which is metabolic waste produced under anaerobic conditions by the bacterium Clostridium botulinum, and affecting a wide range of mammals, birds and fish...

 cases in New Mexico
New Mexico
New Mexico is a state located in the southwest and western regions of the United States. New Mexico is also usually considered one of the Mountain States. With a population density of 16 per square mile, New Mexico is the sixth-most sparsely inhabited U.S...

 and California
California
California is a state located on the West Coast of the United States. It is by far the most populous U.S. state, and the third-largest by land area...

. 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
Horizontal gene transfer
Horizontal gene transfer , also lateral gene transfer , is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism...

, 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
Real-time polymerase chain reaction
In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction or kinetic polymerase chain reaction , is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule...

 (PCR).

Organisms genetically as they identified as other Clostridium species have caused human botulism; Clostridium butyricum producing type E toxin and Clostridium baratii producing type F toxin. The ability of C. botulinum to naturally transfer
Horizontal gene transfer
Horizontal gene transfer , also lateral gene transfer , is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism...

 neurotoxin genes to other clostridia is concerning, especially in the food industry
Food industry
The food production is a complex, global collective of diverse businesses that together supply much of the food energy consumed by the world population...

 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
Clostridium beijerinckii
Clostridium beijerinckii is a gram positive, rod shaped, motile bacterium of the genus Clostridium. It has been isolated from feces and soil...

C. haemolyticum, C. novyi type A C. subterminale, C. haemolyticum

Clostridium botulinum in different geographical locations


A number of quantitative
Numerical data
Numerical data is data measured or identified on a numerical scale. Numerical data can be analyzed using statistical methods, and results can be displayed using tables, charts, histograms and graphs. For example, a researcher will ask a questions to a participant that include words how often, how...

 surveys
Statistical survey
Survey methodology is the field that studies surveys, that is, the sample of individuals from a population with a view towards making statistical inferences about the population using the sample. Polls about public opinion, such as political beliefs, are reported in the news media in democracies....

 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
Soil
Soil is a natural body consisting of layers of mineral constituents of variable thicknesses, which differ from the parent materials in their morphological, physical, chemical, and mineralogical characteristics...

 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
Fish
Fish are a paraphyletic group of organisms that consist of all gill-bearing aquatic vertebrate animals that lack limbs with digits. Included in this definition are the living hagfish, lampreys, and cartilaginous and bony fish, as well as various extinct related groups...

 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).

Europe


Clostridium botulinum type E is prevalent in aquatic sediments in Norway
Norway
Norway , officially the Kingdom of Norway, is a Nordic unitary constitutional monarchy whose territory comprises the western portion of the Scandinavian Peninsula, Jan Mayen, and the Arctic archipelago of Svalbard and Bouvet Island. Norway has a total area of and a population of about 4.9 million...

 and Sweden
Sweden
Sweden , officially the Kingdom of Sweden , is a Nordic country on the Scandinavian Peninsula in Northern Europe. Sweden borders with Norway and Finland and is connected to Denmark by a bridge-tunnel across the Öresund....

, Denmark
Denmark
Denmark is a Scandinavian country in Northern Europe. The countries of Denmark and Greenland, as well as the Faroe Islands, constitute the Kingdom of Denmark . It is the southernmost of the Nordic countries, southwest of Sweden and south of Norway, and bordered to the south by Germany. Denmark...

, the Netherlands
Netherlands
The Netherlands is a constituent country of the Kingdom of the Netherlands, located mainly in North-West Europe and with several islands in the Caribbean. Mainland Netherlands borders the North Sea to the north and west, Belgium to the south, and Germany to the east, and shares maritime borders...

, the Baltic
Baltic Sea
The Baltic Sea is a brackish mediterranean sea located in Northern Europe, from 53°N to 66°N latitude and from 20°E to 26°E longitude. It is bounded by the Scandinavian Peninsula, the mainland of Europe, and the Danish islands. It drains into the Kattegat by way of the Øresund, the Great Belt and...

 coast of Poland
Poland
Poland , officially the Republic of Poland , is a country in Central Europe bordered by Germany to the west; the Czech Republic and Slovakia to the south; Ukraine, Belarus and Lithuania to the east; and the Baltic Sea and Kaliningrad Oblast, a Russian exclave, to the north...

 and Russia
Russia
Russia or , officially known as both Russia and the Russian Federation , is a country in northern Eurasia. It is a federal semi-presidential republic, comprising 83 federal subjects...

. It was then suggested that the type E C. botulinum is a true aquatic
Aquatic ecosystem
An aquatic ecosystem is an ecosystem in a body of water. Communities of organisms that are dependent on each other and on their environment live in aquatic ecosystems. The two main types of aquatic ecosystems are marine ecosystems and freshwater ecosystems....

 organism, which was indicated by the correlation between the level of type E contamination and flooding of the land with seawater
Seawater
Seawater is water from a sea or ocean. On average, seawater in the world's oceans has a salinity of about 3.5% . This means that every kilogram of seawater has approximately of dissolved salts . The average density of seawater at the ocean surface is 1.025 g/ml...

. As the land dried, the level of type E decreased and type B became dominant.

In soil and sediment from the United Kingdom
United Kingdom
The United Kingdom of Great Britain and Northern IrelandIn the United Kingdom and Dependencies, other languages have been officially recognised as legitimate autochthonous languages under the European Charter for Regional or Minority Languages...

, C. botulinum type B predominates. In general, the incidence is usually lower in soil than in sediment
Sediment
Sediment is naturally occurring material that is broken down by processes of weathering and erosion, and is subsequently transported by the action of fluids such as wind, water, or ice, and/or by the force of gravity acting on the particle itself....

. In Italy
Italy
Italy , officially the Italian Republic languages]] under the European Charter for Regional or Minority Languages. In each of these, Italy's official name is as follows:;;;;;;;;), is a unitary parliamentary republic in South-Central Europe. To the north it borders France, Switzerland, Austria and...

, a survey was conducted in the vicinity of Rome
Rome
Rome is the capital of Italy and the country's largest and most populated city and comune, with over 2.7 million residents in . The city is located in the central-western portion of the Italian Peninsula, on the Tiber River within the Lazio region of Italy.Rome's history spans two and a half...

, and a low level of contamination was found; all strains were proteolytic C. botulinum type A or B.

Australia


Clostridium botulinum type A was found to be present in soil samples from mountain
Mountain
Image:Himalaya_annotated.jpg|thumb|right|The Himalayan mountain range with Mount Everestrect 58 14 160 49 Chomo Lonzorect 200 28 335 52 Makalurect 378 24 566 45 Mount Everestrect 188 581 920 656 Tibetan Plateaurect 250 406 340 427 Rong River...

 areas of Victoria
Victoria (Australia)
Victoria is the second most populous state in Australia. Geographically the smallest mainland state, Victoria is bordered by New South Wales, South Australia, and Tasmania on Boundary Islet to the north, west and south respectively....

. Type B organisms were detected in marine mud from Tasmania
Tasmania
Tasmania is an Australian island and state. It is south of the continent, separated by Bass Strait. The state includes the island of Tasmania—the 26th largest island in the world—and the surrounding islands. The state has a population of 507,626 , of whom almost half reside in the greater Hobart...

. Type A C. botulinum have been found in Sydney
Sydney
Sydney is the most populous city in Australia and the state capital of New South Wales. Sydney is located on Australia's south-east coast of the Tasman Sea. As of June 2010, the greater metropolitan area had an approximate population of 4.6 million people...

 suburbs and types A and B were isolated from urban
Urban area
An urban area is characterized by higher population density and vast human features in comparison to areas surrounding it. Urban areas may be cities, towns or conurbations, but the term is not commonly extended to rural settlements such as villages and hamlets.Urban areas are created and further...

 areas. In a well defined area of the Darling-Downs region of Queensland
Queensland
Queensland is a state of Australia, occupying the north-eastern section of the mainland continent. It is bordered by the Northern Territory, South Australia and New South Wales to the west, south-west and south respectively. To the east, Queensland is bordered by the Coral Sea and Pacific Ocean...

, 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.

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
Trigeminal neuralgia
Trigeminal neuralgia , tic douloureux is a neuropathic disorder characterized by episodes of intense pain in the face, originating from the trigeminal nerve. It has been described as among the most painful conditions known...

.

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 ( of 1 ng/kg, assuming an average person weighs ~75 kg); 500 grams of it would be enough to kill half of the entire human population
World population
The world population is the total number of living humans on the planet Earth. As of today, it is estimated to be  billion by the United States Census Bureau...

.

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
Sugar
Sugar is a class of edible crystalline carbohydrates, mainly sucrose, lactose, and fructose, characterized by a sweet flavor.Sucrose in its refined form primarily comes from sugar cane and sugar beet...

, 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 bean
Green bean
Green beans , also known as French beans , are the unripe fruit of any kind of bean, including the yardlong bean, the hyacinth bean, the winged bean, and especially the common bean , whose pods are also usually called string beans in the northeastern and western United States, but can also be...

s 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
Honey
Honey is a sweet food made by bees using nectar from flowers. The variety produced by honey bees is the one most commonly referred to and is the type of honey collected by beekeepers and consumed by humans...

, corn syrup
Corn syrup
Corn syrup is a food syrup, which is made from the starch of maize and contains varying amounts of maltose and higher oligosaccharides, depending on the grade. Corn syrup is used in foods to soften texture, add volume, prevent crystallization of sugar, and enhance flavor...

, 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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