Clostridium botulinum

Clostridium botulinum is a gram-positive, rod-shaped, anaerobic, spore-forming, motile bacterium that has the ability to produce botulinum toxin, which is a neurotoxin known to be the deadliest substance ever recorded in the chemical literature.

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.

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. 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,

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. 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.

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. Hall A strain of C. botulinum has an active chitinolytic system to aid in the breakdown of chitin. 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+, 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.

| chromosome

! Close relatives

C. beijerinckii
C. butyricum

C. novyi type A
C. haemolyticum

| colspan=3 | 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. Group III organisms mainly cause diseases in non-human animals.

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. Within each group, different strains will use different strategies to adapt to their environment to survive. C. botulinum relies on quorum-sensing to initiate the sporulation process. 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.

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.

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.

The control of food-borne botulism caused by C. botulinum is based almost entirely on thermal destruction (heating) of the spores or inhibiting spore germination into bacteria and allowing cells to grow and produce toxins in foods. Conditions conducive of growth are dependent on various environmental factors.

Growth of C. botulinum is a risk in low acid foods as defined by having a pH greater than 4.6 although growth is significantly retarded for pH below 4.9.

Mechanisms of tolerance

C. botulinum is very resilient against many mechanisms of food processing because of the endospores that it produces. These endospores allow the bacteria to survive extreme conditions such as heat, cold, and high or low acidity. These endospores have multiple thick protective layers which allow the bacteria to survive in a dormant state for very long periods of time. The endospores can remain dormant in the bacteria, and in this state they are harmless. As endospores germinate when they are subject to environments with low oxygen, they multiply producing the neurotoxin that causes botulism.

Taxonomic history

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 both be placed into the genus Clostridium, as the genus Bacillus 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, with at least four clearly defined "groups" (see ) straddling other species, implying that they each deserve to be a genospecies.

The situation as of 2018 is as follows:

Group I C. botulinum strains that do not produce a botulin toxin are referred to as C. sporogenes. Both names are conserved names since 1999. Group I also contains C. combesii.
All other botulinum toxin-producing bacteria, not otherwise classified as C. baratii or C. butyricum, is called C. botulinum. This group still contains three genogroups.

The complete genome of C. botulinum ATCC 3502 has been sequenced at Wellcome Trust Sanger Institute in 2007. This strain encodes a type "A" toxin.

Diagnosis

Physicians may consider the diagnosis of botulism based on a patient's clinical presentation, which classically includes an acute onset of bilateral cranial neuropathies and symmetric descending weakness. Other key features of botulism include an absence of fever, symmetric neurologic deficits, normal or slow heart rate and normal blood pressure, and no sensory deficits except for blurred vision. A careful history and physical examination is paramount to diagnose the type of botulism, as well as to rule out other conditions with similar findings, such as Guillain–Barré syndrome, stroke, and myasthenia gravis. Depending on the type of botulism considered, different tests for diagnosis may be indicated.

Foodborne botulism: serum analysis for toxins by bioassay in mice should be done, as the demonstration of the toxins is diagnostic.
Wound botulism: isolation of C. botulinum from the wound site should be attempted, as growth of the bacteria is diagnostic.
Adult enteric and infant botulism: isolation and growth of C. botulinum from stool samples is diagnostic. Infant botulism is a diagnosis which is often missed in the emergency room.

Other tests that may be helpful in ruling out other conditions are:

Electromyography (EMG) or antibody studies may help with the exclusion of myasthenia gravis and Lambert–Eaton myasthenic syndrome (LEMS).
Collection of cerebrospinal fluid (CSF) protein and blood assist with the exclusion of Guillan-Barre syndrome and stroke.
Detailed physical examination of the patient for any rash or tick presence helps with the exclusion of any tick transmitted tick paralysis.

Pathology

Foodborne botulism

Signs and symptoms of foodborne botulism typically begin between 18 and 36 hours after the toxin gets into the body, but can range from a few hours to several days, depending on the amount of toxin ingested. Symptoms include:

Double vision
Blurred vision
Ptosis
Nausea, vomiting, and abdominal cramps
Slurred speech
Trouble breathing
Difficulty in swallowing
Dry mouth
Muscle weakness
Constipation
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 determining a timeline of when onset symptoms first occurred and when the toxin entered the body can be difficult. It is more 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
Ptosis
Trouble breathing
Paralysis

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:

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
Anti-cancer drug delivery

Adult intestinal toxemia

A very rare form of botulism that occurs by the same route as infant botulism but is among adults. Occurs rarely and sporadically. Signs and symptoms include:

Abdominal pain
Blurred vision
Diarrhea
Dysarthria
Imbalance
Weakness in arms and hand area

Veterinary pathology

Most cases of botulism in animals come from foodborne botulism, though wound botulism is also common. Foodborne botulism comes from ingestion of infected material, like raw or decaying vegetation, carcasses, or larvae that have consumed infected material.

In North America, an equine-derived heptavalent botulinum antitoxin is used to treat all serotypes of non-infant naturally occurring botulism. For infants less than one year of age, botulism immune globulin is used to treat type A or type B.

Outcomes vary between one and three months, but with prompt interventions, mortality from botulism ranges from less than 5 percent to 8 percent.

Despite these interventions, there is currently no approved pharmacological treatment that reverses botulinum neurotoxin once it has entered neurons. Due to this limitation, early antitoxin administration and supportive care is critical. Ongoing research investigates several therapeutic strategies; for example, recombinant vaccines based on the heavy-chain (HC) domains of BoNTs have provided protective immunity in animals and shown promise in early clinical trials. However, no human vaccine is currently licensed. Veterinary vaccines are available to prevent outbreaks in livestock.

Antibody-based therapies are also under development. Traditional equine antitoxins, though effective, carry risks of serum sickness. Human polyclonal antibodies isolated from immunized volunteers and novel camelid single-domain antibodies are two alternatives, which may provide safer and longer-lasting protection. Additionally, small-molecule inhibitors targeting the metalloprotease activity of the toxin’s light chain are being explored as potential post-exposure therapies.

Use and detection

C. botulinum is 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.

Botulinum 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, and quantitative PCR can detect the toxin genes in the organism. The type-B organisms were of the proteolytic Group 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 6.23).

|Europe

|C. botulinum type E is prevalent in aquatic sediments in Norway and Sweden, Denmark, the Netherlands, the Baltic coast of Poland, and Russia.

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 (Group I) C. botulinum types A or B.

|Australia

|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.