REVIEWS OF INFECTIOUS DISEASES. VOL. 1, NO.4. JULY-AUGUST 1979 © 1979 by The University of Chicago. 0162-0886179/0104-0006-$00.75

Clostridium botulinum: Characteristics and Occurrence From the Department of Microbiology, University of Hawaii at Manoa, Honolulu, Hawaii

I..ouis DS. Smith

Clostridium botulinum is not really a single species of bacterium. Instead, it is a conglomerate of four quite distinct groups of bacteria with different cultural and antigenic characteristics. These bacteria are alike in a few respects, however; they are clostridia, forming spores and growing only under anaerobic conditions, and they produce toxins with similar pharmacological action [1]. Each cultural group consists of several toxin types. The toxin that each type produces is serologically distinct, however, and six types are separated on that basis. Only two types, A and B, have been implicated in infant botulism [2]. Type A and B organisms have the soil as their habitat and produce spores that are highly resistant to heat, light, drying, and radiation. Type B strains may be proteolytic or nonproteolytic. The proteolytic strains of type B and all type A strains have identical cultural characteristics, similar bacteriocin specificity, and even high nucleic-acid homology [3]; they differ only in the serological specificity of the toxins that they produce. Proteolytic strains of type B occur in the soil of the United States and appear to produce toxin that is more lethal to humans than that produced by the nonproteolytic type B strains, which occur in northern Europe and cause the great majority of cases of botulism there. However, even the toxin produced by the proteolytic type B

strains is not as lethal to humans as is that produced by type A strains. In the period 1959-1973 in the United States, the fatality rate from type A food-borne botulism was 32%; that from type B food-borne botulism was 16% [1]. In a recent investigation of the occurrence of C. botulinum in the soil of the United States, this organism was found in 18.5% of the specimens investigated [4]. Almost all of the type A strains were found in specimens of soil taken west of the north-south line that marks the rise of the Rocky Mountain cordillera out of the Great Plains (figure 1). Type B strains were more generally distributed, with a higher proportion in soil from the mid-Atlantic seaboard; Kentucky had a particularly high frequency of this type (figure 2). The incidence of food-borne botulism is highest in those portions of the country in which the frequency of occurrence of the organisms in the soil is highest. The number of type A spores in some specimens varied from 10 to 200/g of soil. The germination of C. botulinum spores in vitro has been studied extensively because of the relation of this phenomenon to food-borne botulism, but nothing is known about the germination of ingested spores. C. botulinum type A spores require a combination of several triggering factors for germination-anyone of several organic acids, either alanine or cysteine, and either lactate or bicarbonate. The oxidation-reduction potential (Eh) is not critical, the pH may range from 5 to 9, .and the temperature may range from 4 C to 70 C [5,6]. The conditions

Please address requests for reprints to Dr. Louis DS. Smith, 3605 Burchvale Road, Wenatchee, Washington 98801.

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Clostridium botulinum is not a well-defined species of bacterium. Instead, it is a conglomerate of four culturally distinct groups of organisms that, among them, produce seven serologically distinct toxins, all with similar pharmacological action. The principal habitat of C. botulinum is the soil, although its distribution in the soil is sometimes highly regional. Infant botulism is caused by two types of C. botulinum: type A and the proteolytic strains of type B. Type A strains, to whose toxin humans seem most susceptible, are found most frequently in the soil of the western United States; type B strains are somewhat more universally distributed, with a higher frequency of isolation from the soil of some Appalachian areas. The frequency of occurrence of type A and type B food-borne botulism parallels the distribution of these types in the soil.

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First, the soil of this country contains the cells of type A and proteolytic type B strains, i.e., the organisms producing toxin that is the most lethal to humans. Second, the incidence of food-borne botulism is highest where the organism is found most frequently in the soil; we may expect the incidence of infant botulism to follow a similar pattern. Third, we are singularly ignorant of the microbial ecology of the human gut; we need to know much more than we do if we are to understand the factors that affect growth and production of toxin by C. botulinum in the intestine of the human infant. It would certainly be worthwhile (1) to follow the development of the intestinal flora in 20 human infants and the changes in that flora during the first year of life, (2) to identify the organisms in the intestinal microflora of human adults that prevent the establishment of C. botulinum in the microbial community of the intestine, (3) to determine the mechanism by which this inhibition is accomplished, and (4) to work out methods of establishing such inhibiting or-

Figure 1. Sites at which specimens of soil containing Clostridium botulinum type A were taken. The dotted line indicates the 500-m altitude level.

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for outgrowth after germination are more rigorous [7], including the presence of nine amino acids and three vitamins, a low Eh, a pH in the neutral range, and a temperature between 15 C and 45 C. These conditions exist in the human intestine. C. botulinum, however, does not compete well with other bacteria. Food-borne botulism, for example, always occurs because of improperly preserved food in which the ordinary spoilage bacteria and molds have been killed or their growth has been inhibited so that C. botulinum can grow with little or no competition. On this basis, we would expect the microflora of the intestine to be a critical factor in allowing or repressing growth and production of toxin by C. botulinum. We do not know what intestinal organisms can inhibit the growth of C. botulinum, although strains of three organisms generally found in soil -Clostridium perfringens, Clostridium sporogenes, and Bacillus cereus-have been found to do so [8]. In considering infant botulism in the United States, several points should be kept in mind.

Smith

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Characteristics of C. botulinum

Sites at which specimens of soil containing Clostridium botulinum type B were taken.

ganisms in the infant gut. To put it briefly, we need to determine the factors that govern the microbial flora of the intestine.

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References 1. DasGupta, B. R., Sugiyama, H. Biochemistry and phar-

macology of botulinum and tetanus neurotoxins. In A. W. Bernheimer [ed.]. Perspectives in toxinology. Wiley and Sons, New York, 1977, p. 88-103. 2. Arnon, S. S., Midura, T. F., Clay, S. A., Wood, R. M., Chin, J. Infant botulism: epidemiological, clinical, and laboratory aspects. lA.M.A. 237:1946-1951,1977. 3. Johnson,.J. L., Francis, B. S. Taxonomy of the clostridia: ribosomal ribonucleic acid homologies among the species. J. Gen. Microbio1. 88:229-244,1975. 4. Smith, L. DS. The occurrence of Clostridium botuli·

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num and Clostridium tetani in the soil of the United States. Health Lab. Sci. 15:74-80,1978. Ando, Y. Studies on germination of spores of clostridial species capable of causing food poisoning. I. Factors affecting the germination of spores of Clostridium botulinum type A in a chemically defined medium. Journal of the Food and Hygiene Society of Japan 14:457-461,1973. Rowley, D. B., Feeherry, F. Conditions affecting germination of Clostridium botulinum 62A spores in a chemically defined medium. J. Bacteriol. 104:11511157,1970. Smith, L. DS. Botulism: the organism, its toxins, the disease. Charles C Thomas, Springfield, 111., 1977, p. 20-25. Smith, L. DS. Inhibition of Clostridium botulinum by strains of Clostridium perfringens isolated from soil. Appl. Microbiol. 30:319-323, 1975.

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Figure 2.

Discussion

mid- or a phage-mediated conversion, become a toxigenic organism. DR. SMITH. That possibility exists; however, no one has been able to demonstrate phage- or plasmid-induced toxigenicity. DR. NELSON. You mentioned bacteriocins-do they fulfill the usual criteria of bacteriocins? DR. SMITH. Yes, they are heat-stable, digestible by trypsin, and absorbed on cellulose membranes. DR. THADDEUS MIDURA. How do you differentiate between the nontoxigenic C. botulinum and C. sporogenes? lVlany times all we get from the stool cultures is C. sporogenes. DR. SMITH. Nakamura and Yashida found a correlation of

Clostridium botulinum: characteristics and occurrence.

REVIEWS OF INFECTIOUS DISEASES. VOL. 1, NO.4. JULY-AUGUST 1979 © 1979 by The University of Chicago. 0162-0886179/0104-0006-$00.75 Clostridium botulin...
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