Clostridium Botulinum

EA Johnson, University of Wisconsin, Madison, WI, USA

Introduction South America. The principal habitat of type E spores appears to be freshwater and brackish marine habitats. It commonly Botulism is a neuroparalytic disease in humans and animals, has been found in the Great Lakes of the United States and in resulting from the actions of neurotoxins produced by Clos- the western seacoasts of Washington state and Alaska. Type C tridium botulinum and rare strains of Clostridium butyricum strains occur worldwide, whereas the distribution of type D is and Clostridium baratii. Botulinum neurotoxins (BoNTs) are more limited and is especially common in certain regions of the most poisonous toxins known, and are toxic by the oral, Africa. intravenous, and inhalational routes. It is estimated that Clostridium botulinum is a diverse species including organ- 0.1–1 mg of BoNT is sufficient to kill a human and the lethal isms differing widely in physiological properties and genetic dose for most animals is w1ngkg 1 body weight. Foodborne relatedness. They all share the ability to produce BoNT and botulism occurs following ingestion of BoNT preformed in cause botulism in humans and animals. The neurotoxins are foods. Botulism also can result from ingestion of spores and distinguished serologically by homologous antisera and growth and BoNT production by C. botulinum in the intestine, designated as serotypes A to G. C. botulinum types A, B, and E which is absorbed into circulation (infant botulism and adult most commonly cause botulism in humans, whereas types B, intestinal botulism). C, and D cause the disease in various animal species. Clos- Since the early 1900s, botulism has been a serious concern tridium botulinum consists of four physiological groups (I–IV) of the food industry and regulatory agencies because of the with diverse physiological and genetic characteristics. Group IV resistance properties of the pathogen, its ability to survive and C. botulinum is the only group that has not been demonstrated grow in many foods, and the severity of the disease. Resistant to cause botulism in humans or animals and has been assigned endospores produced by C. botulinum are distributed widely in to the species Clostridium argentinense. The organisms are soils and contaminate many foods. In improperly processed morphologically large rods, typically 1 4–6 mm with oval, and preserved foods, the endospores can germinate and vege- subterminal spores that swell the rod giving the characteristic tative cells proliferate to form BoNTs, which cause botulism on ‘tennis-racket’ or spindle-shaped cells (Figure 1). Spores of ingestion. Consequently, a major goal of the food industry and most pathogenic species of clostridia can be produced in of regulatory agencies is to prevent survival of spores and complex laboratory media, such as chopped meat broth or proliferation of vegetative cells in foods, and certain food tryptose–peptone–glucose broth. regulations and industry practices have been designed specifi- Groups I and II are the cause of human botulism, whereas cally to prevent growth and toxin formation by C. botulinum. group III causes botulism in various taxa of animals. The The importance of C. botulinum and its neurotoxins in food primary properties and limiting growth parameters of safety has contributed to unique research approaches and C. botulinum groups I and II pertaining to foods are presented preventative measures in food microbiology. in Table 1. Organisms in group I are proteolytic, and may produce type A, B, or F BoNT. They may form highly heat- resistant spores, have an optimum growth temperature of Characteristics of C. botulinum 30–40 C, and are inhibited by 10% NaCl. Organisms in group II commonly are referred to as nonproteolytic, require The genus Clostridium is a large and diverse group with more sugars for growth, and may produce either type B, E, or F than 120 species. It includes anaerobic or aerotolerant rod- BoNT. They have a lower optimum temperature for growth shaped bacteria that produce endospores and obtain their (20–30 C), and some strains of types B and E can grow slowly energy for growth by fermentation. Clostridia are classified on in foods at temperatures as low as 3.3 C. Consequently, there the basis of morphology, disease association, physiology, serologic properties, DNA relatedness, and ribosomal RNA gene sequence homologies. Many species of clostridia produce protein toxins that are lethal to animals and are responsible for their pathogenicity. Botulinogenic clostridia are distributed widely in nature by virtue of their ability to form resistant endospores. The two principal habitats are soils, including marine and freshwater sediments, and the gastrointestinal tracts of certain animals (but not healthy humans). The incidence of spores of C. botulinum varies according to geographic region. In the United States, type A is found most commonly west of the Rocky Mountains, and type B is found in certain regions of the eastern United States. Type B from non- Figure 1 Characteristic spindle morphology of C. botulinum. The proteolytic strains of C. botulinum also frequently is found in photograph shows a transmission electron micrograph (50 000) of Europe. Type A is found infrequently in the soils of England. a longitudinal section through a spore and sporangium of C. botulinum Type A spores have also been detected in soils of China and type A.

458 Encyclopedia of Food Microbiology, Volume 1 http://dx.doi.org/10.1016/B978-0-12-384730-0.00072-0 CLOSTRIDIUM j Clostridium botulinum 459

Table 1 Factors controlling growth and inactivation of C. botulinum an F0 of 3 min since other factors control their safety from in foods C. botulinum. In preserved food products, C. botulinum growth can be C. botulinum group prevented by a single factor, such as extensive thermal pro- Factor IIIcessing (a ‘bot cook’). Often, a combination of factors is used to prevent C. botulinum growth in low-acid foods (pH4.6). For Minimal pH 4.6 5.0 example, in cured meats, the combination of a mild heat Minimal a 0.94 0.97 w treatment, and the presence of nitrite and salt prevents growth. Required brine concentration for 10 5 growth inhibition (%) Challenging foods with spores of C. botulinum and determining Minimum temperature (C) 10 3.3 whether BoNT is produced in optimal conditions or on Maximum temperature (C) 50 45 temperature abuse is often a desired procedure to evaluate the D100 of spores (min) 30 <0.2 botulinogenic safety of a food, particularly in new products or D121 of spores (min) 0.2 – new formulations. Because of the severity of botulinum poisoning, the food industry has devoted considerable research and resources to has been considerable concern that group II organisms can prevent botulism outbreaks in foods. The control of this grow and produce toxin in refrigerated foods that receive organism is of such paramount importance to the safety of minimal processing and have extended shelf life. Strains that foods that certain food laws and definitions such as thermal produce type E toxin commonly are associated with food- processing of low-acid foods in hermetically sealed containers borne botulism transmitted in contaminated fish or marine were designed specifically to control C. botulinum. The products. Group II strains that produce type B toxin com- organism has served as a ‘barometer’ by which to gauge certain monly are found in Europe and are associated with botulism advances in food formulation and processing. Thus, newly from salt-cured meats. developed foods and food processes may need to be evaluated The D value is the time at a specified temperature to inac- for their impact on C. botulinum growth and toxin formation. tivate 90% of spores. An industry ‘bot cook’ is typically These efforts and vigilance by the food industry have contrib- designed to inactivate 1012 of spores (see below). uted to a safe food supply.

Control of C. botulinum in Foods Clinical Features of Botulism

The primary factors controlling growth of C. botulinum in foods Botulism is categorized according to the route by which BoNT are temperature, pH, water activity, redox potential, oxygen enters the human circulation. Classical foodborne botulism level, presence of preservatives, and competing microflora. In results from the ingestion of neurotoxin preformed in foods. the commercial setting, botulism can occur when a food is Botulism caused by food poisoning generally has an incuba- exposed inadvertently to temperatures that allow growth and tion period of 12–36 h after consumption of a toxic food. toxin formation. Because BoNT is extremely potent, quantities Wound botulism is analogous to tetanus and occurs when sufficient to cause botulism can be formed without obvious C. botulinum grows and produces toxin in the infected tissue. spoilage of foods. In most foods, C. botulinum is a poor Intestinal botulism results from the growth and toxin produc- competitor and other microorganisms, such as lactic acid tion by C. botulinum in the intestine (infant botulism and adult bacteria, often grow more rapidly, commonly lowering the pH, intestinal botulism). Because BoNT is entirely responsible for producing inhibitory metabolites, and preventing growth. the clinical symptoms, the three types of botulism exhibit Spores of C. botulinum, however, are more resistant to heat, similar clinical symptoms. The characteristic symptomatology irradiation, and other processing methods than are vegetative of botulism poisoning is a progressive descending symmetrical cells of competing organisms. Therefore, minimal processing of flaccid paralysis initially affecting musculature innervated by foods can eliminate or reduce the numbers of competing cranial nerves. The first signs are typically disturbances in ocular microflora and increase the probability of C. botulinum growing function, including blurred and double vision, and the pupils and producing toxin. The critical level of oxygen that will become enlarged and unresponsive to light. As intoxication permit growth of group I C. botulinum is 1–2%, but this proceeds, a flaccid paralysis occurs in the facial and head depends on other conditions, such as aw and pH. region, characterized by weakness and drooping of the eyelids Spores of group I C. botulinum have heat resistances ranging and facial muscles (Figure 2). Speech becomes slurred, and from D121 ¼ 0.03–0.23 min and D100 w30 min. Certain swallowing and breathing become difficult. In severe cases, strains of Clostridium sporogenes, which are related genetically extreme muscular weakness causes the patient to become weak, to group I C. botulinum, can produce spores with much higher fatigued, and unable to lift their head and limbs. Death can heat resistance (maximum D121 w1.0 min) than C. botulinum, occur, usually by respiratory failure or possibly by cardiac and these strains may be used to determine the heat treatment arrest. Because BoNT affects alpha motor nerves and does not required for obtaining a 12D inactivation or total lethality (F0) enter the central nervous system in toxic concentrations, as is recommended for shelf-stable low acid foods in cans, sensory responses, mental function, and consciousness gener- glass jars, or pouches. The required treatment for achieving F0 ally are maintained. The inability of the patient to communi- of a food from C. botulinum spores is w3 min at 121 Cor cate the symptoms and the awareness of the progression of the higher. The commercial processing of many foods is less than disease can cause mental depression and anxiety. In severe 460 CLOSTRIDIUM j Clostridium botulinum

botulism in commercial or restaurant-prepared foods have been summarized (see further reading). Infant botulism differs from foodborne and wound botulism in the ages of the affected persons, and usually the first symptom is constipation, indicated by not passing stool for 3 days or more. As the neurotoxin binds to motor nerves, the characteristic flaccid paralysis affects the baby’s musculature in the head and neck regions. The baby has a weak cry and suck and the paralysis may render the baby unable to hold its head and limbs erect. Infants should receive intubation and respiratory assistance to prevent respiratory arrest. Recent studies have shown that administration of human antibotulinal antibodies shortened the hospital stay of infants with botulism. Botulism may be difficult to recognize in infants because of the baby’s inability to communicate its symptoms, the rarity of the disease and inexperience of many doctors, and misdiagnosis of other neurological diseases such as Guillain–Barré syndrome, tick paralysis, drug reactions, or viral and bacterial infections of the nervous system. Infant botulism has been reported from various countries around the world, including all continents except for Africa. Infant botulism is rare in most countries and the majority of cases have been detected in the United States. Within the United States, infant botulism occurs in clustered geographic regions with about half of the diagnosed cases occurring in California. The clustered geographic distribution of infant botulism may be related to the prevalence and type of spores in the environment. Nearly all cases of infant botulism have been caused by proteolytic strains of C. botulinum types A and B. BoNT-producing strains of C. butyricum and C. baratii also Figure 2 Patient with botulism. Photograph courtesy of Charles L. successfully colonized the intestine of babies and caused fi Hatheway (deceased), Centers for Disease Control and Prevention, botulism. The only food de nitively shown to be associated Atlanta, GA, USA. with infant botulism is honey, and babies under 1 year of age should not be given this food. Most cases probably occur from environmental exposure to dust and other sources. cases, intubation and respiratory assistance are required. If Botulism is rare compared with many other foodborne diagnosed early, administration of antibodies can scavenge the microbial diseases, but it has a relatively high fatality rate in free toxin in the blood and prevent the disease from progress- humans and animals. Human botulism outbreaks can have ing to more severe symptoms. Equine antibodies are available a dramatic impact on communities in which they occur and can from the Centers for Disease Control and Prevention in the lead to the demise of food companies, and outbreaks of animal United States and in various other public health laboratories botulism periodically devastate populations of domestic and throughout the world. Recovery from botulism generally is wild animals. To prevent outbreaks, it is necessary for the food prolonged, requiring weeks to months for muscle activity to industry to properly formulate and process foods to prevent return to a normal level, but complete recovery usually is growth and toxin formation. attained. Foodborne botulism is rare in most areas of the world, although the actual incidence probably is higher than reported Properties and Detection of BoNT because mild cases often are not diagnosed, and botulism may be misdiagnosed as another neurological disorder. The preva- The outstanding feature of C. botulinum is its ability to lence of foodborne botulism throughout the world probably is synthesize a neurotoxin of extraordinary potency. BoNTs associated with the prevalence of spores in the environment. include a family of pharmacologically similar toxins that bind The primary geographic regions of the world with reported to peripheral cholinergic synapses and block acetylcholine foodborne botulism are East Asia (China, Japan), North exocytosis at the neuromuscular junctions, resulting in America, certain countries in Europe (Poland, Germany, acharacteristicflaccid paralysis. BoNTs are produced in foods, France, Italy, Spain, Denmark, Finland, Norway), the Middle in the intestine, and in culture as progenitor toxin complexes East (Iran), Latin America, Russia, and South Africa. Foodborne that consist of BoNT associated with nontoxic proteins. The botulism is rare in the United Kingdom, although certain nontoxic components of the complexes have been demon- outbreaks such as the Loch Maree incident, the Birmingham strated to impart stability to the neurotoxin in culture and outbreak, and the hazelnut yogurt incident have attracted in foods and to prevent inactivation by digestive enzymes in much attention and publicity. Recent examples of outbreaks of the gut. CLOSTRIDIUM j Clostridium botulinum 461

The diagnosis of botulism generally is accomplished by mouse bioassay, and they also have the potential drawback of assessment of clinical symptoms in patients, and for foodborne detecting biologically inactive BoNT. Several advances in outbreaks, on the clustering of cases involving a group of enzyme-linked immunosorbent assays (ELISA) have been people who have eaten a common food. In most investigations made to alleviate these drawbacks, and it is likely that ELISA of botulism, the primary goal is to detect the presence of BoNT will be used to complement but not replace the mouse because spores of C. botulinum are widespread in the environ- bioassay. Recently, cell-based assays using neuronal cells have ment and contaminate many foods. The detection of BoNT in been developed and hold much potential for replacement of the blood, gastric contents, and food provides confirmation of the mouse bioassay. botulism. Isolation of C. botulinum from a suspect food, from feces of infants with botulism symptoms, or from wounds provides supporting evidence for the diagnosis of botulism. Botulinum Toxin as a Pharmaceutical BoNT preferably is detected using a bioassay of the toxin extracted from a food or clinical sample. The extract is injected One of the most remarkable recent developments in medicine is intraperitoneally into mice and the animals are observed the use of BoNT to treat humans who suffer from dystonias, periodically for typical signs of botulism for up to 4 days. hyperactive muscle disorders, inflammatory conditions, and Depending on the quantity of BoNT present, symptoms of pain. Botulinum toxin increasingly is being used to treat humans botulism generally are observed within 4–24 h. Characteristic suffering from a number of neurological diseases. Botulinum symptoms include decreased mobility of the animals, ruffling toxin frequently is used for the treatment of a number of dys- of the fur, difficulty in breathing, contraction of abdominal tonias, movement disorders, cosmetic problems, and pain muscles giving the ‘wasp’ morphology, followed by convul- disorders, all of which have been difficult to treat by traditional sions and death. Animals demonstrating these signs usually die therapies. The important properties of BoNT as a therapeutic are within 24–48 h. Animals that die sooner than 2 h or after 48 h its high specificity for motor neurons, its very high toxicity that should be considered as succumbing to substances other than enables the injection of extremely low quantities, thereby BoNT. Death resulting BoNT is confirmed by neutralization avoiding side effects and an immunological response, and its with serotype-specific antitoxins. long (several months) duration of action. The treatment of Complications often are encountered in the mouse bioassay neurological disorders with BoNT stemmed from its properties of BoNT from clinical specimens and from certain foods. In as a food poison and its study as a potential biological terrorism particular, deaths caused by non-BoNT interfering substances are agent. The use of the toxin as a drug has enabled thousands of common. These nonspecific fatalities generally can be avoided humans to lead an enjoyable and productive life and also has by diluting out the interfering lethal substance to an end point at opened a new field of investigation in the application of BoNT which death is caused by the more potent BoNT. Occasionally, to nerve and muscle tissue in the human body. more than one serotype of BoNT may be present in a sample being analyzed, and confirmation would require neutralization by a mixture of antitoxins. With foods or clinical specimens, See also: Clostridium; Ecology of Bacteria and Fungi: Influence nonbotulinum deaths can occur by infection or by the presence of Available Water; Ecology of Bacteria and Fungi in Foods: of endotoxins. Infectious agents can be removed by membrane Influence of Temperature; Ecology of Bacteria and Fungi in filtration or by the addition of antibiotics to the extract being Foods: Influence of Redox Potential; Food Poisoning tested. Extracts containing endotoxins generally can be diluted to Outbreaks; Hazard Analysis and Critical Control Point (HACCP): a proper end point, or the endotoxins can be removed by Critical Control Points; Hazard Appraisal (HACCP): Involvement adsorption. With extracts from nonproteolytic strains of of Regulatory Bodies; Hazard Appraisal (HACCP): C. botulinum (group II), toxicity is increased by activation by Establishment of Performance Criteria; Heat Treatment of a protease such as trypsin. In some foods, trypsin can generate Foods: Spoilage Problems Associated with Canning; National toxic peptides, and therefore the reaction should be terminated Legislation, Guidelines, and Standards Governing by addition of soybean trypsin inhibitor after 30–60 min. Microbiology: Canada; National Legislation, Guidelines, and Viable C. botulinum can be isolated from foods by enrich- Standards Governing Microbiology: European Union; National ment in a suitable growth medium, such as cooked meat– Legislation, Guidelines, and Standards Governing glucose broth or media containing peptones, yeast extract, and Microbiology: US; Processing Resistance; Modified glucose. Clostridium botulinum has complex nutrient require- Atmosphere Packaging of Foods; Ecology of Bacteria and ments and requires a rich medium for growth. For isolation, it Fungi in Foods: Effects of pH. often is useful to heat a portion of the food or clinical specimen at 80 or 60 C to select for spores of groups I and II C. botulinum, respectively. Occasionally, 50% ethanol or chloroform is used Further Reading to inactivate vegetative cells in food samples analyzed for group II C. botulinum. Following enrichment, the presence of BoNT is Hatheway, C.L., Johnson, E.A., 1998. Clostridium: the spore-bearing anaerobes. In: assayed by mouse bioassay as described previously. Selective Collier, L., Balows, A., Sussman, M. (Eds.), Topley and Wilson’s Microbiology isolation agars containing antibiotics, including cycloserine, and Microbial Infections, Systematic Bacteriology, ninth ed., vol. 2. Arnold, sulphamethoxazole, and trimethoprim, have been used for the London, p. 731. Hauschild, A.H.W., 1989. Clostridium botulinum. In: Doyle, M.P. (Ed.), Foodborne isolation of group I C. botulinum from clinical samples. Bacterial Pathogens. Marcel Dekker, New York, p. 111. A variety of immunological methods have been developed Hauschild, A.H.W., Dodds, K.L., 1993. Clostridium botulinum. Ecology and Control in for the detection of BoNT but most are not as sensitive as the Foods. Marcel Dekker, New York. 462 CLOSTRIDIUM j Clostridium botulinum

Johnson, 2013. Clostridium botulinum. In: Doyle, M.P., Buchanan, R.L. (Eds.), Food Setlow, Johnson, 2013. Spores and their signiﬁcance. In: Doyle, M.P., Buchanan, R.L. Microbiology: Fundamentals and Frontiers. 4th ASM Press, Washington (Eds.), Food Microbiology. Fundamentals and Frontiers, fourth ed. ASM Press, DC, p. 441. Washington DC, p. 45. Johnson, E.A., Montecucco, C., 2008. Botulism. In: Engel, A.G. (Ed.), Handbook of Smith, L.D.S., Sugiyama, H., 1988. Botulism, second ed. Charles C. Thomas, Clinical Neurology. vol. 91. Elsevier Inc., pp. 333–368. Springﬁeld, Illinois. Peck, M.W., 2009. Biology and genomic analysis of Clostridium botulinum. Advances van Ermengem, E., 1897. Ueber einen neuenn anaeroben Bacillus and seine Bezie- in Microbial Physiology 55, 183–265. hungen zum Botulisms. Zeitschrift fuer Hygiene und Infektionskrankheiten 26, Schantz, E.J., Johnson, E.A., 1992. Properties and use of botulinum toxin and other 1–56. English reprinting: van Ermengem, E., 1979. A new anaerobic bacillus and microbial neurotoxins in medicine. Microbiological Reviews 56, 80–99. its relation to botulism. Journal of Infectious Diseases 1, 701–719.