Clostridium Botulinum

Ecology and Control in Foods

Hauschild

• Publisher: CRC Press
• Year: 2018
• Print ISBN:
  9780367402624
• eBook ISBN:
  9781351460033
• Topic: Tecnologia e ingegneria
• Subtopic: Scienze dell'alimentazione

An in-depth resource addressing the ecology of Clostridium botulinum which affects the degree of food contamination, and its control in various foods. The text summarizes worldwide data on this organism in food and the environment and the principles of its control in specific foods and products.

I

ECOLOGY

1

Clostridium botulinum and Other Clostridia that Produce Botulinum Neurotoxin

Charles L. Hatheway

Centers for Disease Control, Atlanta, Georgia

I. INTRODUCTION

Van Ermengem (1897) first established that botulism is caused by a toxin consumed in a food and then isolated the organism responsible for the toxin from the food. He named the organism Bacillus botulinus. As subsequent incidents of botulism were investigated, it was found that sometimes the toxins had different serological properties and that the causative organisms had varying physiological characteristics. The organisms were always anaerobic and formed spores, and they were later placed into the genus Clostridium (Bengtson, 1924); the genus Bacillus was restricted to aerobic spore-forming rods.

The genus Clostridium includes all anaerobic spore-forming rods except for those classified as Desulfotomaculum (Cato et al., 1986). Clostridia are considered gram-positive, although many strains stain positive only in very young cultures and may appear as gram-negative cells in more mature cultures. Clostridia can be distinguished from non–spore-forming organisms by visualizing the spores on a stained smear or wet mount or by demonstrating heat resistance. The morphology of a typical strain of Clostridium botulinum is shown in the photomicrographs in Figure 1. Most species of Clostridium will not grow on agar surfaces in the presence of oxygen; some, however, are aerotolerant and could be judged to be aerobic organisms, i.e., Bacillus species, but they can be distinguished from these by their failure to produce catalase.

Figure 1 Photomicrographs of egg yolk agar culture of a typical strain of Clostridium botulinum type A (CDC strain 5356). (A) Gram-stained smear from 3-day culture. (B) Phase-contrast view of wet-mount suspension from 4-day culture. Scale for both photomicrographs is indicated by bar in B.

C. botulinum was defined in 1953 as the species designation for all organisms known to produce botulinum neurotoxin and thereby having the capability of causing botulism in humans or animals (Prévot, 1953). This nomenclature eliminated the use of the name C. parabotulinum to distinguish the proteolytic group of toxigenic organisms from the nonproteolytic group. There had been a problem of other ambiguous uses of that species name. The unifying feature of the organisms known as C. botulinum, then, has been their toxigenicity. It will be seen that C. botulinum, as presently constituted, can be separated into four different species. In the investigation of some recent botulism incidents, the causative organisms have been found to be other established species of Clostridium that possess the ability to produce botulinum neurotoxin and to cause botulism. This illustrates the difficulty inherent in a nomenclature rigidly based on the ability to produce a toxin (Hatheway and McCroskey, 1989).

Thus, botulinum toxin can be produced by a variety of clostridia. The neurotoxins produced by various organisms can be differentiated serologically by specific neutralization into seven toxin types: A, B, C, D, E, F, and G. A given toxin type is not necessarily restricted to one phenotypic group of organisms. Regardless of serological type, the toxins are structurally and pharmacologically similar, causing the same flaccid paralysis of botulism in susceptible animal species.

II. TOXIN TYPES

Nine years after van Ermengem (1897) established that botulism was caused by a bacterial toxin, a second outbreak due to bean salad was investigated and confirmed in a similar manner by Landmann (1904) in Darmstadt, Germany. Leuchs (1910) compared the organisms isolated from the Belgian and the German outbreaks and, using the method of Kempner (1897) for preparing antitoxins against botulinum toxin, found that the toxins were serologically distinct, each being neutralized only by its homologous antiserum. Letter designations were not assigned to toxin types until Burke (1919)reported the results of studies on the characteristics and toxins of strains recovered from botulism outbreaks in the United States. Toxins produced by these organisms were identified as either type A or type B on the basis of neutralization with rabbit antitoxins. Although the van Ermengem and the Landmann strains were no longer available for testing with the typing reagents, it was apparent from the descriptions of the cultural characteristics of the organisms in the earlier literature that the former would correspond to type B and the latter to type A. Additional types were established as other organisms were recovered from incidents of botulism in animals and humans that produced toxins that did not correspond to existing types.

The toxin produced by organisms causing botulism in chickens in the United States (Bengtson, 1922) and in cattle in Australia (Seddon, 1922) was designated as type C. Upon observing that antitoxin produced against the Bengtson strain neutralized the toxin of either strain, while antitoxin against the Seddon strain neutralized only toxin from the homologous strain, the former was designated C_α, and the latter strain, C_β (Gunnison and Meyer, 1929). Another outbreak in cattle in South Africa was caused by an organism subsequently designated type D because its toxin was serologically distinct from types A, B, and C (Meyer and Gunnison, 1928–29). Type E was established to designate the toxin type of organisms causing botulism in the United States and in the Soviet Union caused by consumption of fish (Gunnison et al., 1936–37). An outbreak of botulism caused by a liver paste in Denmark involved a sixth type of toxin, known as F (Moller and Scheibel, 1960). Finally, the toxin produced by an organism isolated from soil in Argentina was designated type G (Giménez and Ciccarelli, 1970b).

The distinction between the C_α and the C_β toxin types is a source of confusion. C_α strains produce C₁, a neurotoxin, and C₂, which is not a neurotoxin but is lethal in mice, ducks, and geese; congestion and hemorrhage in the lung and dripping of the nares are observed in birds injected with C₂ toxin, but paralytic signs of botulism are absent (Jensen and Duncan, 1980; Ohishi and DasGupta,1987). C_β strains produce only C₂ toxin.

Thus, there are seven serologically differentiable types of botulinum neurotoxin. Although, in general, each type is serologically distinct, slight partial cross-neutralizations of the neurotoxins produced by types C and D strains occur because type C strains produce a minor amount of type D neurotoxin, and type D strains produce a trace of type C neurotoxin (Jansen, 1971a, b, 1987). Oguma et al. (1984) have also found evidence for common antigenic sites on types C and D toxin molecules. The initial studies of C. botulinum type F showed that its toxin was neutralized by a large excess of type E antitoxin (Moller and Scheibel, 1960; Dolman and Murakami, 1961). A later study indicates that the crossneutralization between type E and type F is reciprocal (Yang and Sugiyama, 1975). There are also subtle differences between toxins produced by strains of the same toxin type (Ciccarelli and Giménez, 1971; Giménez and Ciccarelli, 1972; (Hatheway et al., 1981). Strains have been found that produce mixtures of two types of toxin, A + F (Giménez and Ciccarelli, 1970a), A + B (Poumeyrol et al., 1983), and B + F (Hatheway and McCroskey, 1989).

III. PHYSIOLOGICAL AND GENETIC DIFFERENCES AMONG ORGANISMS PRODUCING BOTULINUM NEUROTOXIN

A. Physiological Groups

Since botulism had always been associated with meat products, as denoted by its name (derived from botulus, the Latin word for sausage), it was surprising that the illness in Darmstadt (Landmann, 1904) due to consumption of bean salad was also confirmed as botulism. Leuchs (1910) noted that the Ellezelles strain (van Ermengem, 1897)and the Darmstadt strain were similar in their morphological and cultural characteristics but differed in their optimal temperatures for growth and toxin production; for the former it was 18–25°C, and for the latter, 35–37°C. Burke (1919) found that the strains of both toxin variants (type A and type B) of organisms isolated from botulism incidents in the United States resembled the Darmstadt strain, with 37°C as their optimum temperature for growth and toxin production. Bengtson (1924) found that all the American isolates of either toxin type were strongly proteolytic, i.e., they digested meat particles, casein, and coagulated egg albumin, while a European isolate that produced type B toxin clearly was not proteolytic. The latter corresponded very closely to the organism isolated and described by van Ermengem (1897) in all of its cultural characteristics.

In examining an international collection of strains, Gunnison and Meyer (1929) confirmed the findings of Bengtson (1924). They found that two of three European toxin type B strains were nonproteolytic, as well as all strains of toxin type C and one strain (the only one tested) of toxin type D. Although the type C and D strains are nonproteolytic (or weakly so according to some researchers), they are distinguishable from the nonproteolytic type B strains by their high optimum growth temperature of 40°C (Smith and Sugiyama, 1988).

Type E strains resemble European strains of toxin type B in that they are clearly nonproteolytic (Gunnison et al., 1936–1937). Although they may grow well at 37°C, toxigenicity is more constant at room temperature, and they are capable of growing and producing toxin at lower temperatures than the proteolytic strains. Nonproteolytic type B strains have been shown to grow and produce toxin at temperatures as low as 3.3°C (Eklund et al., 1967).

The initial isolate of C. botulinum type F (Moller and Scheibel, 1960) was proteolytic and resembled the American strains of toxin types A and B (Burke, 1919; Bengtson, 1924). A strain isolated from soil in Argentina also belongs to the proteolytic group (Giménez and ...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Preface
• Contributors
• PART I: ECOLOGY
• PART II: CONTROL OF CLOSTRIDIUM BOTULINUM IN FOODS
• Index