Clostridium botulinum EA Johnson, University of Wisconsin, Madison, WI, USA Ó 2014 Elsevier Ltd. All rights reserved. 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 À 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 Â 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 inci- dence 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 (Â50 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.