- Clostridium botulinum is a common anaerobic gram-positive bacillus that produces spores in the environment.
- Rarely, C. botulinum, C. butyricum, and C. baratii are capable of producing neurotoxins that are among the most potent known to man.
- C. botulinum produces classes A–G of botulinum neurotoxins (BoNTs). Human botulism is typically associated with types A, B, and E, while animal botulism is typically related with types C and D.
- Type G is now C. argentinense, which has been linked to rapid mortality but not neuroparalytic disease.
- Type G disease is unknown in mammals. C. botulinum can be separated into four subgroups I–IV based on their biochemical properties.
- Group I is proteolytic, grows between 10 and 45 degrees Celsius, and produces toxin types A, B, and F.
- Group II is nonproteolytic, grows between 3 and 45 degrees Celsius, and is capable of producing toxin types B, E, and F.
- Group II organisms are psychrotrophic because they can thrive at temperatures below 5 degrees Celsius.
- Groups I and II are the primary groups connected with human sickness, with group I being responsible for nearly all occurrences of baby botulism.
- Group III is nonproteolytic and produces toxin types C and D, whereas group IV is slightly proteolytic and only produces toxin type G.
Types of Botulism
There are three primary forms of botulism: baby, wound, and foodborne. Additional clinical classifications include adult intestinal toxaemia and iatrogenic botulism.
1. Foodborne botulism
- Ingestion of foods containing C. botulinum toxin causes the most prevalent form of botulism, which is foodborne botulism.
- In the case of foodborne botulism, contaminated items may be widely consumed, so exposing a large number of individuals to the risk, which may result in a medical and public health crisis.
2. Infant botulism
- Infant botulism affects newborns younger than one year and is caused by the ingestion of C. botulinum spores, which germinate and create toxin in the intestine.
- Spores may originate from the environment or food products.
- Honey has already been linked to the condition and is no longer advised for infants younger than 12 months.
- Almost all newborn botulism cases have been caused by types A and B.
3. Wound botulism
- Botulism of the wound develops when C. botulinum infects a wound and produces toxin.
- It has been linked to significant soil pollution via compound fractures or severe traumas/lacerations.
- Botulism has become significantly more prevalent in recent years, particularly among heroin injectors who use black tar.
4. Adult intestinal toxemi
- Comparable to newborn botulism, adult intestinal toxaemia is the colonisation of the intestine with C. botulinum following consumption of spores.
- Germination, vegetation, and toxin generation by C. botulinum are typically not sustained in the normal adult intestine; nevertheless, colonisation can occur in a small number of individuals with a bowel anomaly or bowel disease or who are taking antimicrobial medication.
5. Iatrogenic botulism
- Iatrogenic botulism is a toxin overdose caused by human error (e.g., via inhalation by laboratory workers or via injection for therapeutic or cosmetic purposes).
Morphology of Clostridium botulinum
C. botulinum has the following characteristics:
- C. botulinum is Gram-positive in cultures that are younger than 18 hours. In cultures, the organism may be Gram-negative after 18 hours of incubation.
- The bacillus is 5.01 metres long.
- The bacillus has motile peritrichous flagella and subterminal oval bulging spores.
- The bacteria lack cell walls.
Habitat of Clostridium botulinum
- C. botulinum is ubiquitous. It is found worldwide in soil and water.
- C. botulinum type A or type B spores are widespread in the soil and have been discovered all over the world.
Geographical distribution of Clostridium botulinum
- Toxins from C. botulinum types A, B, E, and occasionally F and G cause human disease. Types C and D cause disease in birds and nonhuman mammals. Infant botulism is more prevalent than botulism transmitted via food. Food-borne botulism is caused by C. botulinum types A, B, and F; E forms are extremely uncommon causes.
- Botulism of the wounds is extremely uncommon and is caused by C. botulinum type A strains.
Reservoir, source, and transmission of infection
Botulism is caused by the spores of C. botulinum:
- Preserved food, particularly home-canned meat and meat products in Europe, canned vegetables in the United States, and preserved seafood in Japan, which are contaminated with preformed toxin, are the leading causes of food-borne botulism illnesses.
- Honey and other foods infected with bacterial spores are the origins of newborn botulism illness.
- Soil and water contaminated with spores are sources of botulism infection in wounds.
Transmission of infection
Botulism is transmitted via the subsequent means:
- Botulism of the wound is caused by contamination with spore-forming C. botulinum. The condition occurs in (a) individuals with trauma including soil contamination, (b) individuals with chronic intravenous drug addiction (e.g., black tar heroin), and (c) women after caesarean delivery (very rare). Even if medicines are taken to prevent wound infection, the disease can still arise.
- Food-borne botulism is caused by the intake of neurotoxins already produced in food. The use of food contaminated with even minute levels of these chemicals has resulted in the development of full-blown illness. Toxin A has been the leading cause of food-borne epidemics during the past two decades, followed by toxins B and E. High-risk foods consist of home-canned or -prepared low-acid fruits and vegetables, fish and fish products, and condiments such as relish and chilli peppers. Foods that have been prepared commercially and fresh foods that have been carelessly handled can occasionally produce outbreaks of botulism.
- Infant botulism is caused by the intake of C. botulinum spores found in infant foods like honey. The spores then develop into vegetative forms that produce toxins and invade the newborn stomach. The clinical condition is caused by a toxin generated in and absorbed by the gut.
- All generated toxins are 130–150 kDa mono polypeptides with a similar structure. A single disulfide bond connects a 100 kDa heavy chain and a 50 kDa light chain produced by enzymatic cleavage.
- In proteolytic strains, proteases produced by the cell are responsible for enzymatic cleavage, whereas in nonproteolytic strains, an external protease such as trypsin is involved.
- Either the toxin enters the bloodstream immediately or it is completely absorbed from the gastrointestinal system.
- The toxin attaches to high-affinity presynaptic receptors via gangliosides. The toxin is then delivered into the nerve cell by a receptor-mediated endocytosis pathway, which is typical of dichain poisons.
- The toxin inhibits the release of the neurotransmitter acetylcholine, which, under normal conditions, causes skeletal muscle contractions.
- The poison binds irreversibly, and the recovery of function is contingent upon ultraterminal neuron sprouting to produce new motor endplates.
- Each of the seven poisons (A–F) exhibits distinct toxicities and persistence lengths within nerve cells.
Culture of Clostridium botulinum
- C. botulinum is anaerobe in nature.
- The optimal growth temperature for bacteria is 35°C.
- On a variety of medium, including blood agar, Mueller–Hinton agar, and RCM media, the bacteria can thrive.
- C. botulinum develops big, irregular, semitransparent colonies with an irregular fimbriated border on blood agar.
- They develop spores when cultured at 20–25 degrees Celsius in alkaline glucose gelatin media.
Biochemical reactions of Clostridium botulinum
C. botulinum causes the following responses:
- C. botulinum can ferment glucose, hydrolyze gelatin, digest protein, and create the enzyme lipase.
- The production of the enzyme lipase is evidenced by the creation of an iridescent film on egg yolk agar-grown C. botulinum colonies.
Susceptibility to physical and chemical agents
- Both C. botulinum organisms and spores are extremely resistant.
- They are resistant to several hours of boiling at 100°C, but succumb to 10 minutes of pressure cooking at 120°C. The spores of C. botulinum types B, E, and F are substantially less resistant to heat.
- Boiling for 10 minutes or cooking at 80°C for 30 minutes destroys the poisons.
- C. botulinum is a heterogeneous collection of anaerobic, Gram-positive, spore-forming microbes.
- Based on the antigenic specificities of their poisons, they are categorised into seven kinds (A through G).
Virulence factors of Clostridium botulinum
The molecular weight of botulinum toxin is 70,000 daltons. The poison is relatively stable against heat. At 80°C for 30–40 minutes and 100°C for 10 minutes, it is inactivated. The toxin is structurally and functionally similar to tetanus toxin, with the exception of its location of action.
- Botulinum toxin is created only upon the death and autolysis of the bacteria, but not when the bacteria are alive.
- Botulinum toxin is an intracellular progenitor protein produced by bacteria. It comprises of A and B subunits. The light chain, subunit A, is neurotoxic. Subunit B is a hefty chain that inhibits the neurotoxic portion (chain A) from being deactivated by stomach acidity. There may be more than three B subunits in the toxin.
- Botulinum toxin is the world’s most potent naturally occurring toxin. The deadly dose for mice is 0.00000000033 mg, while the lethal dose for humans is 1–2 g. At femtomolar concentrations of 1012 g/kg, botulinum toxin is fatal, making it 15,000–100,000 times more toxic than serin gas.
- Toxoids can also be created from the toxin.
Eight antigenically distinct botulinum toxins (types A, B, C1, C2, D, E, and F, G) have been described based on the immunological distinctions between the toxins produced by C. botulinum. The biological actions of these many poisons appear identical, however they differ immunologically. The only antiserum capable of neutralising the poisons is a homologous one.
- A single bacterial strain produces a single toxin.
- Occasionally, a single strain may release many toxins. All toxins except C2 are neurotoxins.
The neurotoxin only affects cholinergic nerves. It inhibits the release of acetylcholine, a neurotransmitter, at synapses and neuromuscular junctions. It causes muscular atrophy at the site of the infection, but the neurons repair within 2–4 months. Toxins A and B have the highest toxicity. The generation of toxins by types C and D of C. perfringens appears to be mediated by bacteriophages.
Pathogenesis of botulism
Botulism is induced by numerous factors:
- Food-borne botulism is induced by ingesting botulinum toxin directly from a contaminated food.
- Infant botulism is caused by C. botulinum toxin production in the gut.
- Botulism of the wound is caused by toxins produced by C. botulinum infected wounds.
Toxins are absorbed from the stomach and small intestine because they are not denatured by digestive enzymes. The toxin is delivered to peripheral cholinergic nerve terminals, including neuromuscular junctions, cholinergic parasympathetic nerve ends, and certain peripheral ganglia. The toxin binds to receptor sites on presynaptic motor nerve terminals, causing neuromuscular transmission to be blocked. After entering the nerve terminal, the toxin blocks the release and transmission of acetylcholine in cholinergic nerve fibres. The poison attaches irreversibly to neurons. Principally affected are the neurological, gastrointestinal, and endocrine-metabolic systems. Due to the fact that the motor end plate responds to acetylcholine, the consumption of botulinum toxin causes hypotonia, which manifests as descending symmetric flaccid paralysis of the respiratory muscles.
Clinical Syndromes of Clostridium botulinum
The following types of botulism are caused by C. botulinum: (a) food-borne botulism, (b) baby botulism, and (c) wound botulism.
Ingestion of preformed toxins in C. botulinum-contaminated food causes food poisoning. The severity of the disease ranges from minor to quite severe, resulting in death within 24 hours. The incubation period is brief, lasting between 12 and 36 hours after the consumption of contaminated food.
- Initial symptoms include vomiting, nausea, dry mouth, constipation, stomach pain, and impaired vision with fixedly dilated pupils. Typically, fever is absent.
- The condition advances to bilateral, descending muscular weakening in the periphery, resulting in flaccid paralysis.
- Typically, fever is absent.
In 1–7 days following the commencement of the disease, death results from respiratory paralysis. Case fatality ranges between 10% and 25%. Prior to the implementation of specialised medical support services in hospitals, the mortality rate was as high as 20 percent.
- In contrast to food poisoning, newborn botulism is caused by neurotoxins produced in vivo by C. botulinum that have colonised the infants’ gastrointestinal system.
- Initial symptoms include constipation, tiredness, weakness, a weak and distorted scream, loss of head control, and so on.
- The illness has the potential to escalate to flaccid paralysis and respiratory arrest.
- The infants’ excrement contain toxic substances.
- Infant botulism causes a relatively low infant mortality rate (11–20%).
- Botulism of the wound occurs when wounds are heavily contaminated with soil or water carrying C. botulinum spores.
- The average incubation period is 10 days, ranging from 4 to 14 days.
- Botulism transmitted through wounds is identical to botulism transmitted through food, except that the incubation period is longer and there are no gastrointestinal symptoms.
- Typically, a wound seems to be relatively benign.
Laboratory Diagnosis of Clostridium botulinum
A significant degree of clinical suspicion is required for the clinical diagnosis of botulism. The diagnosis is suspected in a patient with afebrile progressive descending paralysis, especially if gastrointestinal signs are present. Laboratory diagnosis of the disease depends on the presence of C. botulinum bacilli or toxin:
- Food-borne botulism is diagnosed by demonstrating the presence of C. botulinum in food by culture and by demonstrating the presence of toxin in food or faeces.
- Infant botulism is established by the isolation of bacilli and the detection of botulinum toxin in the patient’s stool.
- Botulism of the wound is diagnosed by isolating or detecting botulinum toxin in wound pus and exudates.
- For the diagnosis of all types of botulism, faeces, vomitus, and stomach aspirate are collected.
- Wound tissue is collected for the diagnosis of wound botulism.
- Gram staining of food and other material smears may reveal C. botulinum with Gram-positive spores.
Demonstration of toxin
- Using serum toxin bioassay, enzyme-linked immunosorbent assay, and polymerase chain reaction, C. botulinum toxin can be detected in meals, faeces, and other specimens.
Serum toxin bioassay
- The presence of the toxin in food or excrement is demonstrated using a neutralisation test with mice.
- In this approach, two mice are infected intraperitoneally with food filtrate in sterile saline.
- One mouse is protected by a polyvalent antitoxin against botulinum toxin (the control animal), while another is not protected by any antitoxin (test animal).
- If the test animal dies but the control animal survives, the test is considered positive and suggests the presence of a poison in the specimen.
- This test is excellent for detecting toxin at the first stages of food botulism.
- Enzyme-linked immunoassays and polymerase chain reaction are, however, still in the experimental phase.
- C. botulinum can be cultured from suspected contaminated food and excrement in cases of food-borne botulism.
- The specimens are heated at 80 °C for 10 minutes to initiate the food culture procedure. This is done to eliminate all bacterial vegetative forms.
- The specimens are cultivated on RCM media, incubated in anaerobic conditions, and then subcultured on blood agar.
- Anaerobic incubation accelerates the transformation of bacterial spores into vegetative forms.
Identifying features of Clostridium botulinum
- On blood agar, this organism produces big, irregular, semitransparent colonies with an irregular fimbriate border.
- In the Gram-stained smear of the colony, subterminal and oval bulging spores are present on Gram-positive bacilli.
- Capsulated and mobile bacteria
- Ferments glucose, hydrolyze gelatin, and digest protein.
- Produces enzyme lipase.
- Serum toxin bioassay in mice is a reliable approach for detecting C. botulinum toxin.
Treatment of Clostridium botulinum
Botulism is treated with (a) initial supportive therapy, (b) specific antitoxins to neutralise unbound toxin, and (c) antibiotics to stop toxin production.
- A trivalent A-B-E botulinum antitoxin serum is used to treat botulism specifically.
- The sera contain antibodies against types A, B, and E of Clostridium botulinum that bind and neutralise the toxins contained in the serum.
- Immediate administration of trivalent botulinum antitoxin to symptomatic patients with a high clinical suspicion of food-borne botulism and wound botulism.
- Antitoxin is effective even when administered many weeks after toxin ingestion, as circulating toxin has been identified in serum up to four weeks after ingestion.
- Antitoxins do not, however, neutralise toxin already linked to neuromuscular junctions.
- Antitoxin can reduce the progression of a disease, but it has little effect on established neurologic pathology.
- Antibiotics are used to inhibit C. botulinum from multiplying in the gastrointestinal system and the wound, so stopping the generation and release of toxins.
- The current antimicrobial medicine of choice is metronidazole, with penicillin serving as an option.
Prevention and Control of Clostridium botulinum
- The most effective method for preventing food-borne botulism is high-temperature pressure cooking, which destroys spores on fruits and vegetables.
- If spores are present, storing the meal in a refrigerator at 4°C or at an acidic pH prevents their germination into vegetative bacteria.
- It is vital to strictly adhere to suggested home-canning processes.
- Botulism of the wound is prevented by the prompt and complete debridement of infected wounds.
- Botulism caused by intravenous drug consumption can be avoided if drug addicts stop using drugs.
- Avoiding the administration of honey to infants prevents infant botulism.
Factors Affecting Growth and Toxin Production in Foods
Temperature, pH, water activity (aw), redox potential, additional preservatives, and other microbes are the primary elements that influence the growth of C. botulinum in food. The growth of groups I and II types A, B, E, and F has been thoroughly investigated. Historically, food microbiologists have defined maximum and/or minimum limits for certain characteristics that would permit the growth of C. botulinum, and these limits have frequently been employed to regulate C. botulinum. However, these elements rarely operate separately; often, they work in tandem, frequently exhibiting synergistic or cumulative effects. There exist models that incorporate pH, aw, and temperature to forecast the growth of C. botulinum under various combinations of environmental variables.
- Due to the fact that foods are typically stored at low temperatures, research has centred on establishing the minimal temperatures that permit growth.
- The specified bottom limits for group I are 10 C, whereas those for group II are 3.0 C.
- However, these limits only apply to a small number of strains and are contingent on optimal growth conditions.
- Generally, toxin production requires many weeks at the temperature limitations.
- The optimal growth temperature range for group I organisms is 35–40 degrees Celsius, and for group II organisms it is 25–30 degrees Celsius.
- Approximately 45–50 C and 40–45 C are the highest temperature limits for group I and group II species, respectively.
- It is widely believed that the minimal pH allowing growth of C. botulinum group I is 4.6, and many policies around the world utilise this threshold.
- The limit for group II is around pH 5.0. The upper pH limits for plant growth are in the 8–9 pH range, however they have little practical significance.
- Many fruits and vegetables are sufficiently acidic to inhibit C. botulinum based only on their pH, however other goods, such as vegetables marinated in vinegar, require the addition of acidulants for preservation.
- C. botulinum’s acid tolerance is affected by a number of variables, including strain, substrate, temperature, type of the acidulant, presence of preservatives, aw, and redox potential.
- Acid-tolerant microorganisms, such as yeasts and moulds, may elevate the pH in their immediate surroundings to a level that allows C. botulinum to flourish. C. botulinum can also grow in certain acidified meals if pH equilibration occurs too slowly.
- While high protein concentrations in laboratory conditions appear to protect C. botulinum and permit growth at pH levels below 4.6, this is not the case with acid-preserved foods.
- Therefore, the current standards requiring a minimum acidity of 4.6 pH for the control of C. botulinum are valid.
3. Salt and aw
- Salt (NaCl) is one of the most important food preservatives against C. botulinum. Its inhibitory impact is caused primarily by the reduction of aw and, subsequently, its concentration in the aqueous phase, also known as the brine concentration (%brine = NaCl x 100(%H2O + %NaCl)).
- Under otherwise optimum conditions, the growth-limiting brine concentrations for group I strains are approximately 10% and for group II strains, 5%.
- These concentrations correspond well to the aw limit of 0.94 for group I and 0.97 for group II in foods where NaCl is the predominant awdepressant.
- These limits may be affected by the type of solute employed to control aw. In general, NaCl, potassium chloride, glucose, and sucrose exhibit similar patterns, although glycerol can reduce the growth-limiting aw concentration by up to 0.03 units.
- Other factors, such as an increase in acidity or preservatives, may greatly increase the aw limit.
4. Redox Potential
- C. botulinum grows optimally at an oxidation–reduction potential (Eh) of 350 mV, however growth start can occur between 30 and 250 mV Eh.
- The inclusion of additional inhibiting variables reduces this upper limit. Once growth has begun, Eh decreases fast.
- Increasingly, modified environment packaging is utilised to lengthen the shelf life and improve the quality of food.
- Depending on the environment and the food, C. botulinum development might be prevented or increased.
- Numerous studies have demonstrated that C. botulinum grows equally well in air-packed and vacuum-packed foods; the oxygen in the package head space can be rapidly depleted by respiration of background microorganisms or, in the case of vegetables packaged in modified atmosphere, by the plant tissue itself.
- Before using different atmospheres, the safety of C. botulinum should be thoroughly examined.
- Nitrite is essential for providing the unique colour and flavour of preserved foods, but its most important function is inhibiting the growth of Clostridium botulinum.
- It is more effective when the pH is decreased, the NaCl content is increased, and ascorbate or isoascorbate is added to the food product.
- Nitrite appears to inhibit C. botulinum via multiple mechanisms, one of which is presumably its reactivity with iron-sulfur proteins necessary to the cell’s energy-producing phosphoroclastic system.
- The interactions of nitrite or nitric oxide with secondary amines in meats to form nitrosamines, some of which are carcinogenic, have led to restrictions banning the use of nitrite.
- In addition to sorbates, parabens, nisin and other bacteriocins, phenolic antioxidants, polyphosphates, ascorbates, ethylenediaminetetraacetic acid (EDTA), metabisulfite, n-monoalkyl maleates and fumarates, and lactate salts are also active against C. botulinum.
- Certain food extracts, such as mace, bay leaf, and nutmeg extracts, as well as oils from garlic, onion, black pepper, clove, cinnamon, and origanum, impede the growth of C. botulinum.
- Natural or liquid smoke had a substantial inhibitory impact on C. botulinum in hot-smoked fish, decreasing the inhibitory water-phase NaCl content from 3.7% to 2.9%–3.0%.
6. Other Microorganisms
- Other bacteria have an important role in controlling C. botulinum in food. Acid-tolerant yeasts and fungi may make the environment more conducive to C. botulinum growth.
- Other microbes may suppress C. botulinum by altering the environment, secreting inhibitory chemicals, or both.
- Lactic acid bacteria, such as Lactobacillus, Pediococcus, and Streptococcus spp., can limit the growth of Clostridium botulinum in meat products by lowering the pH and possibly by producing bacteriocins.
- It has been demonstrated that Bacillus spp. that produce antimicrobials hinder the growth of group II C. botulinum in meals.
- In the United States, the combination of lactic acid bacteria and a fermentable carbohydrate, known as the ‘Wisconsin process,’ has been authorised for the production of bacon with a reduced amount of nitrite.
- The proliferation of additional bacteria may also safeguard the user by producing spoiling, which reduces the likelihood that a dangerous product would be consumed.
Therapeutic/Cosmetic Use of Botulinum Toxin
- Despite the potency of C. botulinum neurotoxins and their association with serious diseases, their usage for medicinal and aesthetic purposes (especially BoNT type A) is on the rise.
- Use of neurotoxins for cosmetic objectives, such as lowering the appearance of wrinkles and frown lines, is well-established, but the results are only temporary. C. botulinum neurotoxin is utilised in a wide range of medical diseases involving muscle hyperactivity, glandular hypersecretions, and discomfort.
- These include focal dystonias, spasticity, nondystonic diseases, strabismus, chronic pain and localised muscle spasm disorders, smooth muscle hyperactivity disorders, and sweating, salivary, and allergy disorders.
- Neurotoxin does not cure the underlying illness, although it temporarily alleviates clinical symptoms.
- The duration and quality of the improvement vary according on the dose, dilution, and injection technique.
- The effects typically last around three months, after which further injections are required.
- There is a possibility that the patient will develop resistance, however this danger has been mitigated by administering the lowest effective dose and maintaining sufficient intervals between injections.
- Due to the rarity of intestinal carriage, the detection of C. botulinum in faeces, vomitus, or gastric aspirate by culture strongly suggests food-borne botulism. In over 60% of cases, culture is beneficial. For the diagnosis of foodborne botulism, toxin can potentially be detected in food or faeces.
- Botulism in a wound is strongly suggested by the presence of C. botulinum in pus culture and in wound pus and exudates.
- The diagnosis of newborn botulism is confirmed by the isolation of bacilli and detection of botulinum toxin in the infant’s stool.
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