Clostridium Botulinum and Botulism JW Austin, Tunney’S Pasture, Ottawa, ON, Canada

Clostridium botulinum and Botulism JW Austin, Tunney’s Pasture, Ottawa, ON, Canada

Botulism the microbiological safety of meat, further discussion will be limited to that type of illness. Foodborne botulism is an intoxication resulting from con- Foodborne botulism is likely as old as food preservation. sumption of foods in which Clostridium botulinum has grown ‘Sausage poisoning’ was first seriously studied following an and produced toxin. Depending on the amount of toxin in- outbreak in Wildbad, Germany, in 1793, which resulted in 13 gested, the onset of botulism usually begins 18–36 h after cases with 6 deaths. Although originally attributed to bella- ingesting toxin-containing food. As with other bacterial food- donna poisoning (caused by consumption of leaves or berries borne illnesses, the initial symptoms may include diarrhea and of the Nightshade plant), it was more widely accepted that the vomiting. Botulinum intoxication results in a descending cause was consumption of a locally produced blood sausage symmetrical flaccid paralysis. Cranial nerves are affected ini- ‘Blunzen,’ which was a pig stomach filled with blood and tially, resulting in one or more of blurred and double vision, other ingredients, briefly boiled, and preserved by smoking. fixed and dilated pupils, ptosis (drooping eyelids), dysphonia Following the Wildbad outbreak, the number of reported cases (difficulty swallowing), dysphagia (difficulty speaking), and of sausage poisoning rapidly increased, prompting an 1829 dry mouth. Paralysis may descend causing muscle weakness study of the disease by the local health officer, Justinius Kerner, and paralysis affecting the diaphragm. Fatal cases are normally who described 230 cases of sausage poisoning over a period of the result of respiratory failure. Botulism is frequently con- 25 years. The illness became known as ‘botulism’ after ‘botulus,’ fused with other illnesses, especially in geographical areas the Latin word for sausage. Kerner's report was the first pub- where the disease has a low incidence. The symptoms of lished complete description of the clinical symptoms of botulism are commonly confused with Guillain–Barré syn- foodborne botulism. drome, stroke, and myasthenia gravis. An outbreak of botulism involving 34 cases caused by A diagnosis of botulism can be made by demonstration of salted ham served at a gathering of amateur musicians in botulinum neurotoxin in the patient's serum or the toxin and/ Ellezelles, Belgium, occurred in December 1895. The outbreak or the organism in feces. As C. botulinum is not normally part was investigated by Prof. E. Van Ermengem of the University of of the human intestinal microflora, its isolation from feces or Ghent. Van Ermengem reported that portions of ham fed to gastric contents, in association with classic symptoms of the mice, guinea pigs, and monkeys caused flaccid paralysis and disease, is diagnostic. Owing to improved medical care, such death. Filtered extracts of ham had the same effects as ma- as the use of respiratory support systems and timely ad- cerated ham. Van Ermengem found an anaerobic sporulating ministration of antitoxin, the fatality rate due to botulism has bacillus, which was named Bacillus botulinus, in cultures of the fallen significantly in recent years. ham as well as a culture of spleen from a deceased victim. In Europe, more than 2500 cases of foodborne botulism Cultures and culture filtrates had the same effects as the ma- were reported in 1999–2000. Countries with a high reported cerated ham and ham filtrates, indicating involvement of a incidence include Armenia, Azerbaijan, Belarus, Georgia, Pol- filterable toxin that was produced by the bacterium. In the and, Russia, Turkey, and Uzbekistan. A smaller but significant investigation of this outbreak, Van Ermengem established number of cases are reported annually in France, Germany, that botulism was an intoxication, not an infection, and the Italy, China, and the USA. It should be noted that the true toxin was produced by a spore-forming obligately anaerobic incidence of foodborne botulism is likely to be much higher, bacterium. with underreporting being an issue. Foodborne botulism is fi not reportable in all countries and the ef ciency with which C. botulinum potential outbreaks are investigated varies from country to country. Clostridium botulinum is the ‘species’ name assigned to an as- Being described first in 1976, infant botulism has become semblage of anaerobic, spore-forming, rod-shaped bacteria, all the most commonly reported form of botulism in the US, with of which produce botulinum neurotoxin that causes the severe approximately 80–100 cases occurring annually. Unlike neuroparalytic condition known as botulism. Morpho- foodborne botulism, infant botulism is caused by ingestion of logically, members of the C. botulinum assemblage are Gram- spores, followed by colonization of the intestinal tract by C. positive, anaerobic, motile rods, 4–6 mm by 0.9–1.2 mm in size botulinum, with in situ production of neurotoxin. A third form with oval, subterminal spores (Figure 1). is wound botulism, wherein the organism infects tissue and Originally, all organisms known to produce botulinum produces toxin at a wound site. With no cases being reported neurotoxin were included in this species. There are seven types in the UK before 2000, wound botulism has now become the of C. botulinum, A through G, based on the serological speci- most common form of botulism in the UK as a result of in- ficity of the neurotoxin produced. Human botulism, including jection of illicit drugs. As only foodborne botulism relates to foodborne, wound, and infant botulism, is associated with

330 Encyclopedia of Meat Sciences, Volume 2 doi:10.1016/B978-0-12-384731-7.00036-2 Microbiological Safety of Meat | Clostridium botulinum and Botulism 331

cause animal botulism. These strains are nonproteolytic, grow optimally at 40 °C, and do not grow at temperatures less than 15 °C. Group IV strains, which produce type G neurotoxin, grow optimally at 37 °C and have a minimal growth temperature of 10 °C. These four groups, plus neurotoxin-producing strains of Clostridium butyricum type E and Clostridium baratii type F, make a total of six distinct genomic groups that produce botulinum neurotoxin. With a few exceptions, infant botulism is caused by proteolytic strains of C. botulinum. In the few exceptional cases, other clostridia (C. baratii producing type F toxin and C. butyricum producing type E toxin) have been implicated.

Figure 1 A transmission electron microsgraph of a thin section Isolation and Characterization of C. botulinum through several vegetative cells and spores of Clostridium botulinum. Bar represents 2 mm. The diagnosis of foodborne botulism is generally confirmed if, in addition to the clinical syndrome, botulinum toxin and/or types A, B, E, and F. Types C and D cause botulism in animals. viable C. botulinum are detected in a suspect food or a clinical To date, there is no direct evidence linking type G to the specimen. Suitable specimens for toxin analysis are serum, disease. feces, enema fluid, and stomach contents. The same speci- The species is also divided into four groups on the basis of mens, except serum, are also suitable for the detection of C. physiological and genomic differences. Strains belonging to botulinum. Botulinum toxin is detected by injecting serum or group I include all type A strains and proteolytic strains of extracts from foods and clinical specimens into mice, and then types B and F. Clostridium botulinum Group II includes all type observing them for characteristic symptoms including ruffled E strains and nonproteolytic strains of types B and F. Clos- fur, labored breathing, and a pinched waist. These symptoms, tridium botulinum Group III includes all type C and D strains. combined with the absence of these symptoms in specimens Clostridium botulinum group IV, or C. botulinum type G, has neutralized with specific antisera, can be considered an earlier been renamed Clostridium argentinense. A high degree of re- endpoint for the mouse assay negating the requirement for latedness exists among strains within each group, but there is death as an endpoint. little relatedness between groups. The detection of C. botulinum involves anaerobic incu- Group I strains are proteolytic and are typified by strains bation of foods and clinical specimens in liquid media, and that produce neurotoxin type A. The optimal temperature for then subsequent toxin analysis. In major outbreaks or in the growth is 37 °C, with growth occurring between 10 °C and isolation of uncommon serotypes, the analyses are followed 48 °C. High levels of neurotoxin are typically produced in by the isolation of the incriminated microorganism. The cus- cultures. Spores of proteolytic strains are resistant to heat, with tomary procedure for isolation of C. botulinum from food is D °C values of approximately 25 min (the D value is the 100 based on an enrichment procedure followed by detection and time required to inactivate 90% of the population at a given serotyping of toxin in culture supernatant fluid. Commonly temperature). To inhibit growth, the pH must be less than 4.6, used enrichment procedures include preliminary heat or salt concentration more than 10%, or the water activity (a ) w ethanol treatments to destroy competing nonsporing bacteria, less than 0.94. followed by anaerobic culture in media such as cooked meat Group II strains are nonproteolytic, have a lower optimum medium or trypticase-peptone-glucose-yeast extract broth. growth temperature (30 °C), and are psychrotolerant, that is to Enrichment incubation is normally at temperatures between say, being capable of growth at temperatures as low as 3.0 °C. 26 °C and 35 °C for 5–10 days. A selective and differential agar The spores have a much lower heat resistance than those of medium containing antibiotics and egg yolk may be used for group I strains, with D °C values less than 0.1 min. Group II 100 isolation of the organism from enrichment broth. strains are inhibited at pH less than 5.0, salt concentrations more than 5%, or water activities (aw) less than 0.97. The original strain isolated by van Ermengem in 1895 was prob- ably nonproteolytic C. botulinum type B, and many type B cases Botulinum Neurotoxin in Europe are still due to strains of nonproteolytic C. botulinum. Recorded outbreaks of botulism involving non- The specificity of botulinum toxin for neurons makes it ex- proteolytic C. botulinum have most frequently been associated quisitely toxic. The lethal dose of botulinum toxin for a 70 kg with meat, fish (e.g., salted, dried, vacuum, or smoked), and human is estimated to be approximately 0.09–0.15 mgintra- homemade foods prepared by the peoples of Alaska and venously or intramuscularly and 70 mg orally. Following in- northern Canada, such as aged beaver tail and paw, aged sal- gestion, the toxin is absorbed through the gastric and upper mon roe and ‘muktuk’ (i.e., whale skin and fat). intestinal mucosa from where it enters the bloodstream. Botu- Group III includes strains producing types C and D linum neurotoxin then gains access to the nervous system, neurotoxins, which are not involved in human botulism but where it blocks the release of the neurotransmitter acetylcholine, 332 Microbiological Safety of Meat | Clostridium botulinum and Botulism preventing muscle contraction and thereby causing flaccid the consumption of home-prepared pork products (ham, paralysis. bacon, blood pudding, mosaic salami, raw sausages, and The active toxins are first synthesized as single polypeptides smoked-dried meat), whereas commercially canned products with low activity that require posttranslational proteolytic were involved in 20 cases (18.9%). cleavage to form the active dichain molecule consisting of Most cases of botulism due to meat products in North heavy and light chains joined together by a disulfide bridge. America result from consumption of traditional northern Strains of Group I C. botulinum are proteolytic and self-activate foods by Inuit or Eskimos. Peccant foods include meat and fat the toxin with endogenous proteases. However, Group II from seal, whale, walrus, and beaver. Other than these strains, all of which are considered nonproteolytic, need ex- northern foods, botulism from meat products is rare in Can- ogenous proteolytic enzymes or trypsin treatment to activate ada and the US. Commercial pate was the cause of two cases of the toxin. The toxin is stable in acidic conditions (pH 3.5) but botulism in Quebec in 1995, whereas commercial jarred pork is easily inactivated in slightly alkaline conditions. The toxin is caused a single case in Quebec in 2001. In July of 2007, an heat labile; consequently, normal cooking procedures should outbreak from commercially canned hotdog chili sauce caused ensure a food's safety with respect to botulism. However, 10 cases of botulism in Texas and Indiana. A 15-case botulism freezing does not destroy the toxin. outbreak, also caused by chili, occurred in Texas in 2001. As both these cases involved chili, a food with several ingredients, it was not determined whether meat, or another ingredient, Incidence of C. botulinum in Meats contributed the spores. Occasional single cases were reported in the US from home-prepared foods including stew and Spores of proteolytic and nonproteolytic C. botulinum are home-canned beef and peas; however, in these cases, it is also widespread in nature and are found in soils, aquatic sedi- impossible to determine whether meat was the responsible ments, and the gastrointestinal tracts of animals. Given the ingredient. ubiquitous nature of C. botulinum spores, their presence in Although home and artisanal production remain the meats must be assumed and appropriate controls to prevent principal causes of botulism outbreaks, the proportion of cases growth and toxin production must be applied. Information attributable to commercial products is increasing, especially in regarding the prevalence rates for C. botulinum on meat is not Europe, where recent outbreaks have been linked to widely readily available, mainly because of the cost and difficulties of distributed, commercially produced foods. Methods for pre- detecting C. botulinum in foods. However, the studies that have serving food products are changing, and fresh products that are been done indicate that the incidence and levels of C. botuli- vacuum-packed and refrigerated with extended shelf-lives, or num in meats are low, compared with fish and fishery prod- products that are heat treated at temperatures and times too ucts. Several studies indicate that the incidence of C. botulinum mild to inactivate C. botulinum spores, are increasing the risk of in meat samples is often less than 10%, with levels at ap- botulism from commercial foods. proximately 1 spore per kg. The more frequent occurrence of botulism acquired from pork than from other meat products suggests more frequent contamination of pork than that of Control of C. botulinum in Meats beef, lamb, and other meats. Toxin type B is typically implicated in cases of meat-borne botulism. Survey results reveal Safe food production is based on either destroying spores of that nearly all toxin types identified from raw and semi- C. botulinum using a thermal process or inhibiting growth of preserved meats were either A or B. The finding of low num- the organism in foods, in combination with low storage bers of spores of C. botulinum in pig and cattle's feces indicates temperatures and limited storage times. Incorporation of a that clinically healthy animals occasionally carry C. botulinum combination of inhibitory factors such as reduced pH or water spores. Thus, the contamination of carcasses with C. botulinum activity, added preservatives, and competing microflora pro- spores during carcass dressing seems likely. vide multiple barriers and help processors to achieve a safe product. In addition to inhibition of growth of C. botulinum, the microbiological safety of foods also relies on good control Botulism Incidents Involving Meat Products and monitoring of processes throughout manufacture and distribution. Home processing of meat is more common in continental Europe than in the UK or North America and an increased incidence of foodborne botulism, most frequently associated Refrigeration with meat products, has been observed in eastern European countries. Nonproteolytic type B strains are the etiological The psychrotolerant nature of nonproteolytic strains of C. agents in most outbreaks of botulism in Germany, France, and botulinum makes these strains of particular concern in re- Portugal that have been caused by home-prepared salted or frigerated products. Psychrotrophic C. botulinum has the ability cured hams. Homemade bottled pork meat is the main vehicle to survive mild heat treatment of minimally processed foods of botulism in Poland. Three incidents occurring in the UK and may grow during cold storage. Growth of psychrotrophic and Ireland involved Polish nationals and were associated C. botulinum occurs in response to extrinsic parameters such as with the consumption of home-prepared meat products ori- storage at temperatures and redox potentials permissive for ginating from Poland. Of 106 cases of botulism in Romania growth and parameters intrinsic to the food, such as pH and diagnosed during 2007–09, 86 (81.1%) were associated with water activity. Microbiological Safety of Meat | Clostridium botulinum and Botulism 333

Toxin formation by C. botulinum is both time and tem- the case of underprocessing, it is the heat-resistant proteolytic perature dependent. At its lowest growth temperatures, it takes strains that are of concern with regard to product safety. a C. botulinum strain many weeks to produce toxin. However, a C. botulinum is controlled in shelf-stable canned meats using slight rise in incubation temperature is accompanied by a thermal processing. rapid increase in growth rate of C. botulinum and a reduction in time to toxin detection. Storage of foods for several weeks at refrigeration temperatures is needed for toxigenesis by non- Redox Potential and Atmosphere proteolytic strains. Thus, refrigerated products with a short shelf-life normally would not present significant risk. How- C. botulinum is a strict anaerobe. However, even within foods ever, products with extended shelf-lives stored at refrigeration exposed to oxygen, the redox potential is often low enough to temperatures for extended periods may produce toxin by support the growth of this organism. Vacuum packaging and nonproteolytic strains of C. botulinum. modified atmosphere packaging were developed for the ex- As strict temperature control of refrigerated products is tension of product shelf-life, but concerns have been raised often not maintained during their storage, distribution, dis- about risks from these products with respect to botulism. In play, and subsequent handling in domestic, food service, or modified atmosphere packaging of certain meat products, the institutional facilities, the likelihood of toxin production by air in the headspace of the package is replaced by CO2 and N2, nonproteolytic strains of C. botulinum in extended shelf-life resulting in a low O2 environment. There is concern about the products is increased. However, low-temperature storage can- safety of these products with respect to the potential for not be reliably used as the sole means of controlling growth growth and toxin production by nonproteolytic C. botulinum. and toxin production by C. botulinum. Vacuum or modified atmosphere packaging in atmospheres

Consumer demands for ready-to-eat convenience foods, without O2 restricts the growth of aerobic spoilage bacteria but combined with an emphasis on little or no preservatives and not of clostridia or other anaerobic bacteria. There have been a less processing, have placed a new focus on C. botulinum. The number of reports describing low temperature spoilage of lack of a heat treatment sufficient to destroy spores of C. vacuum-packaged meats caused by psychrotolerant Clostridium botulinum, use of packaging that mostly or totally exclude spp. These incidents demonstrate that conditions within a oxygen, prolonged storage at chill temperatures, and the lack chilled vacuum pack may select for the growth of psychroto- of heating before consumption combine to increase the risk of lerant clostridia. Outbreaks of type E botulism in the 1960s botulism from these foods. These products often do not use were linked to vacuum-packed smoked fish. Studies have additional preservative systems such as reduced pH or water shown no difference in the rate of toxin production in high- activity. In those foods where such treatment would adversely and low-oxygen barrier films. However, it is of concern that affect product quality, other controls, such as reduced water the use of high-barrier oxygen film may increase the organo- activity or acidification, should be considered. Subinhibitory leptic acceptability of a toxic product. Inhibition of competing levels of several factors can be combined to control growth of organisms may permit toxin production by C. botulinum in C. botulinum by application of what is known as the hurdle refrigerated modified atmosphere packaged foods without concept. obvious sensory evidence of spoilage.

Thermal Processing pH

A great number of botulism outbreaks during the early twen- C. botulinum will not grow in highly acidic foods that have a tieth century in Europe and North America were associated pH value less than 4.6. Consequently, acidiﬁcation is widely with the widening use of canning and bottling processes to used to control the growth of C. botulinum in food products. extend shelf-life. This led to the development and enforcement Cases of botulism have been linked to the consumption of of the ‘botulinum cook’ for commercial processing. The high-acid foods in which the growth of yeasts or molds has ‘botulinum cook’ was developed as a heat process for low acid raised the pH sufﬁciently to allow the growth of C. botulinum. foods (4pH 4.6) in order to destroy the spores of C. botuli- In general, proteolytic strains of C. botulinum are more tolerant num, and its widespread use led to substantial reductions in of acidic conditions than the nonproteolytic strains. the number of botulism outbreaks. Spores are a dormant form of the organism that are resistant to high temperatures, high pressure, UV light, and desiccation. The ‘botulinum cook’ Water Activity process is designed to reduce the population of the most re- –12 sistant C. botulinum spores to 10 of their original numbers. The generally accepted minimum aw for growth of C. botulinum The decimal reduction time (D value) is the time in minutes at in foods, under otherwise optimal conditions, is 0.94 and 0.97 a given temperature in order to produce a one log reduction in for proteolytic and nonproteolytic strains, respectively. The C. botulinum spore number. Much higher temperatures are values 0.94 and 0.97 correspond to salt concentrations of required to inactivate spores of Group I strains, compared with approximately 10% and 5%, respectively. Today, high salt those of Group II. Group I strains typically have D values in concentrations in foods are unacceptable to most consumers. the range of 0.21 min at 121 °C, whereas Group II strains have Lower salt concentrations can, however, be effective when used D values of 2.4 min at 82.2 °C. Botulism in canned foods re- in combination with other inhibitory factors such as pH and sults from underprocessing or postprocess contamination. In nitrite. 334 Microbiological Safety of Meat | Clostridium botulinum and Botulism

Preservatives Hurdle Technology Although hurdles to growth of C. botulinum have been re- Nitrite viewed separately, they are typically used in combination. Sodium nitrite is a multifunctional food additive, responsible Thermal processing resulting in a 12 log reduction of C. botu- for the characteristic color and flavor associated with cured linum spores, although acceptable for canned foods, would meats and at the same time providing protection against render many meat products inedible. Reliance on refrigerated growth and toxin formation by C. botulinum in cured meats storage alone results in the risk that a food may be temperature subjected to temperature abuse. The exact mechanism of abused. botulinum inhibition by nitrite is not known. Its efficacy de- Perishable meat products rely on chilling, in combination pends on interactions involving pH, salt, heat treatment, with intrinsic factors, in order to control growth of C. botuli- storage temperature and time, and the composition of the num. Products such as luncheon meats in hermetically sealed food matrix. It has been shown that nitrite interacts with ni- containers receive a mild heat treatment but remain shelf trogenous compounds to form carcinogenic nitrosamines and stable due to refrigerated storage, and nitrites and salt are intestinal bacteria mediate the formation of nitrosamines in added to inhibit growth of C. botulinum. Bacon and some the body. As a result, in recent years, consumer and regulatory fermented, undried sausages are preserved by chilling, in pressure has mounted to reduce the levels of nitrite in pro- combination with nitrite, and reduced water activity and pH. cessed meats. The maximum concentration of sodium nitrite Shelf-stable meat products rely on intrinsic factors to pre- –1 (NaNO2) in meat products typically is 150 mg kg . C. botu- vent growth of C. botulinum. For example, Italian mortadella linum in cured meat products is primarily controlled by re- relies on a combination of a thermal process, reduced water frigeration and the use of nitrite. The control measure to apply activity, low pH, and the addition of nitrite and smoke. Other is the addition of a sufficient quantity of nitrite salt (defined by meat products rely more on a limited number of hurdles. For the sodium nitrite (NaNO2) concentration). example, canned unsalted meat relies only on the thermal process, whereas Parma-type ham relies primarily on reduction of water activity with salt, with a lesser reliance on reduced pH. Nisin Bacteriocins, antimicrobial peptides that inhibit the growth of a broad spectrum of Gram-positive microorganisms, offer a See also: Biopreservation. Canning. Chemical Analysis for natural alternative as biopreservatives for safeguarding min- Specific Components: Curing Agents. Microbiological Safety of imally processed foods. Nisin is a peptide antibiotic produced Meat: Hurdle Technology by some strains of Lactococcus lactis. It has been used as a food preservative in many countries for more than 50 years and has been granted generally regarded as safe status. Nisin is not only Further Reading active against closely related lactic strains but is also effective for inhibiting the growth of the foodborne pathogens Listeria Austin, J.W., Leclair, D., 2011. Botulism in the North: a disease without borders. monocytogenes, Staphylococcus aureus, and C. botulinum under Clinical Infectious Diseases 52, 593–594. certain conditions. Nisin sensitivity varies among C. botulinum Emanuelle, E., Vaillant, V., de Valk, H., Popoff, M.R., 2003. France recalls strains. Growth conditions and food components also affect internationally distributed halal meat products from the plant implicated as the nisin's effectiveness. Factors decreasing nisin's ability to inhibit source of a type B botulism outbreak. Eurosurveillance 7 (38), 2295. Fagan, R.P., McLaughlin, J.B., Castrodale, L.J., et al., 2011. Endemic foodborne C. botulinum growth include a low-acid environment and high botulism among Alaska Native persons − Alaska, 1947–2007. Clinical Infectious protein and phospholipid concentrations in foods. Diseases 52, 585–592. Lucke, F.-K., Roberts, T.A., 1993. Control in meat and meat products. In: Hauschild, A.H.W., Dodds, K.L. (Eds.), Clostridium botulinum. Ecology and Control in Competing Microorganisms Foods. New York: Marcel Dekker, pp. 177–207. Neghina, A.M., Marincu, I., Moldovan, R., Iacobiciu, I., Neghina, R., 2010. In most foods, competing organisms, when present, often Foodborne botulism in southwest Romania during the post-communism period grow more rapidly than C. botulinum, as this microorganism is 1990−2007. International Journal ofInfectious Diseases 14, 96–101. a poor competitor. The growth of organisms such as the lactic Neghina, A.M., Neghina, R., 2011. Epidemiology of foodborne botulism in Romania − – acid bacteria lowers the pH and, consequently, inhibits the 1980 2009. Foodborne Pathogens and Diseases 8, 907 911. Peck, M.W., Stringer, S.C., 2005. The safety of pasteurised in-pack chilled meat growth of C. botulinum. Strains of lactic acid bacteria have been products with respect to the foodborne botulism hazard. Meat Science 70 (3), found to produce bacteriocins (low molecular weight pro- 461–475. teins) that are inhibitory to C. botulinum. These strains hold Peck, M.W., Stringer, S.C., Carter, A.T., 2011. Clostridium botulinum in the post- promise for use in the food industry, where they may provide genomic era. Food Microbiology 28, 183–191. ‘natural’ protection against C. botulinum hazards.