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In this article we will discuss about:- 1. Introduction to Clostridium Botulinum 2. The Organism of Clostridium Botulinum and its Characteristics 3. Pathogenesis and Clinical Features 4. Isolation and Identification 5. Association with Foods.
Contents:
- Introduction to Clostridium Botulinum
- The Organism of Clostridium Botulinum and its Characteristics
- Pathogenesis and Clinical Features of Clostridium Botulinum
- Isolation and Identification of Clostridium Botulinum
- Clostridium Botulinum’s Association with Foods
1. Introduction to Clostridium Botulinum:
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Because of its severity and distinctive symptoms, botulism is the form of bacterial food poisoning for which we have the earliest reliable reports.
In 1793 in Wildbad, Wurttemburg, 13 people fell ill and 6 later died after eating Blunzen, a type of sausage made by packing blood and other ingredients into a pig’s stomach. The sausage had been boiled and then smoked, after which it was considered stable at room temperature for several weeks and suitable for consumption without reheating.
Several further incidents of Wurstvergiftung, or sausage poisoning, were recorded in the years that followed, usually associated with sausages that contained animal components other than muscle tissue. This prompted a local district medical officer, Justinius Kerner, to undertake a study of the disease which became known as botulism (Latin: botulus = sausage).
Kerner noted several important features including the facts that heating was an essential precondition for the development of toxicity in sausages and that small sausages or those containing air pockets were less likely to become toxic.
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It was not until 1896 that the micro-organism responsible was isolated and described by van Ermengem, Professor of Bacteriology at the University of Ghent and former pupil of Robert Koch.
This was a result of his investigation into an outbreak of botulism where 34 members of a music club in Belgium ate raw, un-smoked ham. Several noted that the ham had a slightly ‘off’ flavour akin to rancid butter but was otherwise unremarkable. About a day later, 23 of the group fell ill and 3 died within a week.
Van Ermengem established that botulism resulted from the consumption of food containing a heat-labile toxin produced by an obligately anaerobic, spore-forming bacillus which he called Bacillus botulinus. He further demonstrated that toxin would not be produced in the presence of sufficient salt, that it was resistant to mild chemical agents and was not uniformly active against all animal species.
Although much of the early evidence suggested that botulism was confined to meat products, it was later found to occur wherever foods and their processing offer conditions suitable for survival and growth of the causative organism.
It was identified with icthyism, a paralytic illness associated with the consumption of raw, salted fish, known in Russia since 1880, and in 1904 an outbreak of botulism in Darmstadt, Germany was caused by canned white beans.
2. The Organism of Clostridium Botulinum and its Characteristics:
Van Ermengem’s original designation was superseded in 1923 when the organism responsible for botulism was reclassified as Clostridium botulinum. The cells are Gram- positive, motile with peritrichous flagella, obligately anaerobic, straight or slightly curved rods 2-10 µm long, and form central or sub-terminal oval spores.
Strains of Clostridium Botulinum display sufficient variety of physiological and biochemical characteristics to be inconsistent with their inclusion in a single species. In this instance however, taxonomic rectitude has been sacrificed to avoid any possibility of confusion over nomenclature with potentially fatal consequences.
The most important common feature of the species is the production of pharmacologically-similar neurotoxins responsible for botulism. Eight serologically distinct toxins are recognized (A, B, C1, C2, D, E, F, and G), a single strain of Clostridium Botulinum will usually only produce one type, although there are exceptions.
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In 1985, certain strains of C. barati and C. butyricum responsible for cases of infant botulism were found to produce similar neurotoxins, although they have not been implicated in any foodborne cases of botulism.
Physiological diversity within the species Clostridium Botulinum is recognized by its division into four groups (Table 7.4). Group I strains are culturally indistinguishable from the non-toxigenic species Clostridium sporogenes which can sometimes serve as a useful and safe model in laboratory studies.
They are strongly proteolytic and will often betray their presence in food by partial disintegration of the product and a slight rancid or cheesy odour.
Unfortunately despite these warning signs the potency of the toxin is such that the amount ingested on sampling the food has often proved sufficient to cause illness. Group I strains are not psychrotrophic and are therefore of little concern in adequately refrigerated products.
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They do, however, produce the most heat-resistant spores and can pose a problem when foods that depend upon a heating step for their stability and safety are under processed.
In contrast, Group II strains represent a greater potential hazard in chilled foods. They are non-proteolytic with native protein, can grow and produce toxin down to about 3°C and produce spores with a low resistance to heat. They also tend to be more susceptible to inhibition by salt (Table 7.4).
The rate of growth and toxin production at the lower temperature limit is slow and will be reduced still further by any other factors adverse to growth.
Experimental studies have indicated that storage periods of 1-3 months are necessary for toxin production at 3.3 °C, although this period can be markedly reduced at higher temperatures still within the chill range. Vacuum-packed herrings inoculated with 100 spores per pack became toxic after 15 days storage at 5 °C.
Most cases of botulism in humans are due to types A, B or E. The incidence of other toxin types in human illness is extremely rare and the incrimination of type G has come largely from its isolation at autopsy from people who had died suddenly and unexpectedly. Types C and D are usually associated with illness in animals and birds.
Although it is found, occasionally, growing in the alimentary tract of birds and mammals, Clostridium Botulinum is essentially a soil saprophyte. It occurs widely, although the geographical distribution is not uniform. Surveys conducted in the United States found type A to be the most common in the Western States, rare in the Mississippi Valley but less so along the Eastern Seaboard where type B was predominant.
This distribution was reflected in outbreaks of botulism in the United States in the period 1950-1979; when 85% of those west of the Mississippi were due to type A toxin and 63% of those to the east were due to type B. In European soils type B tends to be more common than type A.
Aquatic muds provide a moist, anaerobic, nutrient-rich environment in which Clostridia can flourish, so isolation of Clostridium Botulinum from these sources is more frequent than from soils.
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The psychrotrophic type E has been particularly associated with this environment in regions such as western North America, Japan and the Baltic sea coasts. As a consequence, type E is often responsible for outbreaks of botulism where fish is the vehicle.
The minimum pH at which Clostridium Botulinum will grow depends very much on factors such as temperature, water activity and the acid used to adjust the pH. The consensus has long been that a pH around 4.7 represents an absolute minimum and this fact has had important practical implications for the canning industry.
Non-proteolytic strains have a lower acid tolerance and are generally inhibited at pH 5.0-5.2. Reports have appeared of growth and toxin production at pH values as low as 4.0 in protective, high-protein containing media but this does not reflect the situation in acid canned foods which are generally low in protein.
In cases where botulism has occurred in foods where acidity is an important protective hurdle, such as canned fruits, it has been as a result of other organisms, yeasts or moulds, growing in the product and increasing the pH.
The maximum pH for growth is 8.5-8.9 and the toxin is unstable at alkaline pH values. This is generally an unimportant feature of the organism’s physiology since nearly all foods are slightly acidic. It may be significant however in some North American fermented fish products occasionally associated with botulism where the usual increase in pH on fermentation would be a protective factor.
3. Pathogenesis and Clinical Features of Clostridium Botulinum:
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Botulism is an example of bacterial food poisoning in its strictest sense: it results from the ingestion of an exotoxin produced by Clostridium botulinum growing in the food. The botulinum toxins are neurotoxins; unlike enterotoxins, which act locally in the gut, they affect primarily the cholinergic nerves of the peripheral nervous system.
Experiments in animals have shown that toxin ingested with food and surviving inactivation is absorbed in the upper part of the small intestine and reaches the bloodstream via the lymphatics. It binds to the nerve ending at the nerve-muscle junction, blocking release of the acetylcholine responsible for transmission of stimuli, thus producing a flaccid paralysis.
Initial symptoms of botulism occur anything from 8 h to 8 days, most commonly 12-48 h, after consumption of the toxin-containing food. Symptoms include vomiting, constipation, urine retention, double vision, difficulty in swallowing (dysphagia), dry mouth and difficulty in speaking (dysphonia).
The patient remains conscious until, in fatal cases, shortly before the end when the progressive weakness results in respiratory or heart failure. This usually occurs 1-7 days after the onset of symptoms. Surviving patients may take as long as 8 months to recover fully.
The clinician can do little to mitigate the effect of toxin already adsorbed at the neuromuscular junction, although neuromuscular blockade antagonists such as 4- amino-pyridine have produced transient improvements.
Survival is therefore critically dependent on early diagnosis and treatment, principally by alkaline stomach washing to remove any remaining toxic food, intravenous administration of specific or polyvalent anti-toxins to neutralize circulating toxin, and mechanical respiratory support where necessary.
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The mortality rate is usually high (20-50%), but will depend on a variety of factors such as the type of toxin (type A usually produces a higher mortality than B or E), the amount ingested, the type of food and the speed of treatment.
The botulinum toxins are the most toxic substances known, with a lethal dose for an adult human in the order of 10-8g. They are high molecular mass (150 kDa) proteins and can be inactivated by heating at 80 °C for 10 min. In culture, they are produced during logarithmic growth as complexes and released into the surrounding medium on cell lysis.
In the smallest of these complexes, the M complex, neurotoxin is accompanied by a similar-sized protein with no apparent biological activity, while in the larger L complex, an additional haemagglutinin component is also present.
It appears that the neurotoxin is synthesized as a single chain pro-toxin which is activated by proteolytic cleavage to produce a molecule consisting of light (Mr 50 kDa) and heavy (Mr 100 kDa) chains linked, with the exception of type C2, by a disulfide bridge.
The heavy chain is responsible for specific binding to neuronal cells and cell penetration by the light chain. It is thought that the light chain is a zinc endopeptidase which is activated by reduction of the inter-chain disulfide bond.
The endopeptidase then cleaves synaptobrevin, an integral membrane protein of the small synaptic vesicles thus blocking neurotransmitter release (Figure 7.2). Where the organism does not itself produce appropriate proteolytic enzymes, pro-toxin can be activated by the gut enzyme trypsin.
More extensive proteolysis will lead to toxin inactivation so that, although the structure of the natural complex affords some protection, the lethal oral dose of toxin A in mice is 104-105 times that observed when administered intraperitoneally.
It has been shown, at least for types C and D, that the genetic information coding for toxin production is associated with a temperate bacteriophage. This persists in the bacterial cell as a prophage; its DNA incorporated and replicating with the bacterial chromosome without causing lysis.
This lysogenic state occurs widely among bacteria in nature, usually without changing the micro-organism’s characteristics, but sometimes, as here, it is associated with the production of toxins. Another example is the production of diphtheria toxin by Corynebacterium diphtheriae.
Infant botulism differs from the classical syndrome in that it results from colonization of the infant’s gut with Clostridium Botulinum and production of toxin in situ.
It was first described (in 1976) and is most frequently reported in the United States, although cases have occurred in Australia, Canada, Europe and South America. Up until 1987 only two cases had been reported in the UK, one type A and the other involving the rare type F toxin.
It occurs mostly in infants aged 2 weeks to 6 months, particularly around the time that non-milk feeds are introduced. At this stage the infant’s gut microflora is not fully developed and is less able to out-compete and exclude Clostridium Botulinum.
Since it only requires the ingestion of viable spores, environmental sources other than food may be involved and those foods that do act as vehicles need not be capable of supporting growth of the organism.
Honey has been associated with several cases of infant botulism in the USA and some surveys have found viable spores of Clostridium Botulinum in 10% of the samples examined. Consequently it is thought inadvisable to feed honey to children less than a year old.
The illness is characterized by neuromuscular symptoms related to those of classical botulism and diagnosis can be confirmed by the isolation of the organism and its toxin from the faeces. Although implicated in a small proportion (4%) of cases of sudden infant death syndrome in the United States, the mortality rate is low in treated cases.
4. Isolation and Identification of Clostridium Botulinum:
In view of the metabolic diversity within the species selective media are of limited use in the isolation of Clostridium Botulinum and identification is based on the ability of typical colonies to produce toxin in culture. Clostridium Botulinum will often constitute only a small proportion of the total microflora so enrichment or pre-incubation is necessary to improve the chances of isolation.
Sometimes enrichment cultures are heated prior to incubation to eliminate non-spore-forming anaerobes. However, depending on the heating regime used, 80 °C for 10 min is commonly cited, this may also eliminate the less heat resistant strains of Clostridium Botulinum and is therefore often omitted.
After enrichment in a medium such as cooked meat broth at 30°C for 7 days, the culture is streaked on to fresh horse-blood or egg yolk agar and incubated anaerobically for 3 days. Characteristic smooth colonies, 2-3 mm in diameter with an irregular edge and showing lipolytic activity on egg-yolk agar (type G excepted) are transferred into a broth medium to check for toxin production.
A technique has been described that simplifies this procedure by incorporating antitoxin into the agar medium so that toxin-producing colonies are surrounded by a zone of toxin-antitoxin precipitate. Despite the development of a range of in vitro immunoassay procedures for toxin, the mouse neutralization test (Figure 7.3), remains the most sensitive (a typical lethal dose of toxin for a mouse is a few picograms).
However, the distressing nature of the test guarantees its eventual replacement as soon as immunoassay amplification systems have been sufficiently improved.
A suspect toxin extract is divided into three portions: one, to serve as control, is heated at 100 °C for 10 min to destroy any toxin present; a second is treated with trypsin to activate any pro-toxin that may be there; and a third is untreated.
Each of the portions is injected intraperitoneally into 2 mice and the mice observed over 4 days for the development of typical symptoms of laboured breathing and the characteristic ‘wasp-waist’ appearance. The presence of toxin is confirmed by protection of mice with polyvalent antitoxin and the toxin type can be identified using monovalent antisera.
5. Clostridium Botulinum’s Association with Foods:
Four common features are discernible in outbreaks of botulism:
1. The food has been contaminated at source or during processing, with spores or vegetative cells of Clostridium Botulinum.
2. The food receives some treatment that restricts the competitive microflora and, in normal circumstances, should also control Clostridium Botulinum.
3. Conditions in the food (temperature, pH, Eh, aw) are suitable for the growth of Clostridium Botulinum.
4. The food is consumed cold or after a mild heat treatment insufficient to inactivate toxin.
Since low-acid canned foods can fulfill all the above criteria, it has been necessary for the canning industry to introduce stringent process control measures to ensure safety. When canned foods are produced as a small-scale, domestic activity however, greater variability and less rigorous control are clearly potential sources of problems.
In the United States, where home-canning is more widely practiced than elsewhere, inadequately processed products, particularly vegetables, are the most common cause of botulism.
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Between 1899 and 1981 there were 522 outbreaks associated with home-canned products, including 432 involving vegetables. This compares with 55 outbreaks over the same period caused by commercially canned products; the majority occurring before 1925.
Fish can be contaminated with Clostridium Botulinum, particularly type E, from the aquatic environment and uncooked fish products have been responsible for several outbreaks of type E botulism. Smoked fish consumed without reheating has generally been hot-smoked so control of Clostridium Botulinum depends on microbial inactivation by heat plus the inhibitory effects of salt, smoke constituents and surface drying.
With the advent of refrigeration, the severity of the salting and smoking stages has been reduced in line with the perceived consumer preference for a less strongly flavoured product.
In the early 1960s, two outbreaks of type E botulism in North America associated with vacuum-packed, hot-smoked fish caused considerable alarm and led to Canada banning the importation of all types of packaged fish.
A similar outbreak in Germany in 1970 was caused by smoked trout from a fish farm. At first it was feared that vacuum packing, an emerging technology at that time, was responsible by providing an anaerobic environment in which Clostridium Botulinum could flourish.
It transpired that the problem was compounded of several factors. The salting and smoking treatments had been insufficient to eliminate Clostridium Botulinum or inhibit its growth during storage. A minimum salt concentration (in the water phase) of 3% and an internal temperature not less than 63 °C during smoking are recommended.
The product had also been subjected to severe temperature/time abuse allowing Clostridium Botulinum to grow and produce toxin. The product should have been stored at temperatures below 4 °C. Finally, vacuum packing had improved the product shelf- life by inhibiting the normal spoilage microflora of bacteria and moulds which would have indicated that the product was inedible.
Fish products that are consumed raw after a fermentation process have also caused occasional problems, for example I-sushi. In 1986 in the Canadian Northwest Territories an outbreak of type E botulism was recorded after consumption of a meal comprising raw fish, seal meat and fermented seal flipper.
The latter had been prepared by packing the product in a plastic bucket, covering with seal fat and leaving it outside the house to ferment.
The process differed from normal in that the product was stored for 7 days instead of the usual three and the weather had been unseasonably warm. It was claimed that the seal flipper had an unusual taste and subsequent investigation established the presence of Clostridium Botulinum type E in the product. In Europe, the Norwegian fermented trout rakorret has also been responsible for outbreaks of botulism.
The long association of botulism with meat products in Europe has already been noted and inadequate curing of meats still gives rise to occasional problems in some European countries. Outbreaks of botulism in the UK are relatively infrequent. The largest outbreak this Century occurred in 1989 when 27 people fell ill and one died.
In this outbreak the vehicle was hazelnut yoghurt. The pH of yoghurt is too low for toxin production in situ, but the toxin (type B) had been produced in the hazelnut puree which was inadequately heat processed.
Soil contamination is a major source of Clostridium Botulinum in foods and one to which vegetables, particularly root crops, are inevitably prone. Three outbreaks of type A botulism in the United States have been attributed to potato salad where cooked or partly cooked potatoes had been stored for several days at ambient temperatures and under anaerobic conditions before further processing.
In 1988 an airline passenger in Europe contracted type A botulism from a pre-packed vegetable salad. Important features in these outbreaks were temperature abuse and anaerobiosis created by vacuum packing or wrapping in aluminium foil.
They further indicate the importance of ensuring that vegetables, particularly those such as mushrooms that are frequently eaten raw, are not stored at ambient temperatures in hermetically sealed packs.
