3 Clostridium botulinum and Clostridium perfringens Jim McLauchlin and Kathie A. Grant 1. INTRODUCTION Clostridium is a diverse genus of Gram-positive, endospore-bearing obligate anaerobes that are widespread in the environment. This genus includes more than 100 species, and the overall range in the G+C content (22–55 mol%) reflects the enormous phylogenetic variation encompassed within this group. The principal foodborne pathogens are Clostridium botulinum and Clostridium perfringens that cause toxin-mediated disease either by preformed toxin (foodborne botulism) or by the formation of toxin in the enteric tract (infant botulism and C. perfringens diarrhea). These two bacteria and their foodborne diseases will be discussed here. 2. CLOSTRIDIUM BOTULINUM 2.1. Introduction Botulism is a rare but potentially fatal disease caused by neurotoxins (BoNTs) usually produced by C. botulinum. Disease symptoms result from paralysis owing to the inhibition of neurotransmitter release by BoNTs. Human foodborne disease results from either ingestion of preformed BoNT in foods contaminated by C. botulinum, or production of BoNT following intestinal colonization by C. botu- linum (usually in infants). The disease can also be transmitted by nonfoodborne routes including the production of BoNT from C. botulinum-infected wounds as well as accidental and deliberate release of BoNT (1). Although botulism has been reported as causing a tooth abscess, and hence was probably foodborne (2), wound botulism is most often associated with the trauma at other sites (especially at injec- tion sites to illegal drug users) and are outside the scope of this work and will not be discussed here. Foodborne botulism represents a potential national (or international) public health emergency. Prompt diagnosis and early treatment are essential to reduce the considerable morbidity and mortality of this disease. The laboratory investigation of botulism is highly specialized, but is essential for the rapid identification of reservoirs of infection and provides vital information allowing the most appropriate interventions to be implemented. Key reviews on C. botulinum can be found in the literature(1,3–17). From: Infectious Disease: Foodborne Diseases Edited by: S. Simjee © Humana Press Inc., Totowa, NJ 41 42 McLauchlin and Grant 2.2. Classification, Identification, Isolation and Diagnosis 2.2.1. Taxonomy of C. botulinum The species C. botulinum is defined on the basis of a single phenotypic characteristic, i.e., neurotoxin production, and is classified into four distinct taxonomic lineages (des- ignated groups I–IV; [18]). Characteristics of these four groups are shown in Table 1. This grouping combines highly diverse phylogenetic organisms that consequently show considerable differences in genotype and phenotype. Indeed the genetic (phylogenetic) differences within C. botulinum are greater than that between Bacillus subtilis and Staphylococcus aureus (18). This situation is further, and somewhat inconsistently, complicated because there are genetically very closely related Clostridium species that do not possess neurotoxins and are, therefore, not named C. botulinum (i.e., Clostridium sporogenes, Clostridium novyi, and un-named organisms) and neurotoxin containing Clostridium baratii and Clostridium butyricum (18). Further complications exist becaue there is evidence for instability and horizontal gene transfer of BoNT genes as well as nontoxigenic C. botulinum variants containing cryptic or fragmentary copies of BoNTs. It has been proposed (18) that the four taxonomic lineages should be reclassified into separate species, and a precedence has been set by a proposal to rename C. botulinum Group IV (as well as some Clostridium subterminale and Clostridium hastiforme) as Clostridium argentinense (148). However, this proposed revision for the classification of C. botulinum has not found common use and the taxonomy remains unresolved. The genome size of both C. botulinum groups I and II has been estimated at between 3.6 and 4.1 Mbp by pulsed-field gel electrophoresis analysis (19,149). A complete genome sequence for a single C. botulinum Type A (Hall strain, ATCC 3502) is available, and at the time of writing the annotation is not yet complete: the genome size is 3,886,916 bp in size, with a G+C content of 28.2 mol %, and one 16,344 bp plasmid was detected (20). 2.2.2. Neurotoxin Types BoNTs are proteins that can be divided into seven antigenically distinct types, designated A to G, and are among the most potent toxins known (1). The distribution of BoNTs among the four taxonomic groups is shown in Table 1. Group I includes the proteolytic strains producing BoNTs A, B, and F, either singly or as dual toxin types AB, AF, and BF. Group II includes the nonproteolytic strains producing toxins B, F, and E. Group III organisms produce either type C or D toxins and are generally nonproteolytic. Group IV strains produce type G toxin and differ from other groups in not producing lipase. Important C. botulinum for human foodborne diseases are in groups I (BoNTs A, B, and F) and II (BoNTs B, E, and F), with type F being the least common (1). However human infection resulting from type C has been reported (21). Organisms from groups I–III cause diseases among animals especially aquatic birds, horses, and cattle. Group IV organisms producing BoNTG have been isolated from autopsy specimens, although evidence that botulism was the cause of death in these cases was not demonstrated (22). BoNTG has not been associated with any other naturally occurring cases of botulism in humans or any other animals (9). BoNT production, pre- sumably as a result of horizontal gene transfer, has also been detected in C. baratii (type F) and C. butyricum (type E), both of which have been associated with human diseases (23–26). Clostridium 43 Table 1 Characteristics of C. botulinum Groups Group I II III IV Neurotoxin types A, B, F B, E, F C, D G Human disease Yes Yes Noa No Growth temperatures Minimum 10 3.3 15 ND Optimum 35–40 18–25 40 37 Minimum pH for growth 4.6 5.0 5.0 ND Minimum aw for growth 0.94 0.97 ND ND Inhibition by NaCl 10% 5% 2% ND D100°C of spores (min) 25 <0.1 0.1–0.9 0.8–1.12 D121°C of spores (min) 0.1–0.2 <0.001 ND ND Proteolytic activity Yes No No Yes (weak) Lipolytic activity Yes Yes Yes No Saccharolytic activity No Yes Yes (weak) No Related non-neurotoxigenic C. sporogenes C. beijerinkii C. novyi C. subterminale Clostridium species C. putrificum C. haemolyticum C. histolyticum C. linosium Location of neurotoxin Chromosomal Chromosomal Phage Plasmid genes Overall G+C content 26–29 27–29 26–28 28–30 (mol%) aOne case of infant botulism due to type C has been reported (21). ND, not determined. 2.2.3. Detection of BoNTs By Mouse Bioassay Detection of BoNT production is the cardinal feature for both the classification and identification of C. botulinum as well as providing confirmation of a clinical diagnosis of botulism. Traditionally this has relied on the use of an in vivo mouse bioassay to detect BoNT activity (8,11). Toxin extracts for bioassays are prepared as described here and injected intra- peritoneally into mice. The biological action of the toxin is generally observed within 24 h and initially often presents with hyperactivity followed by reduced mobility, ruffled fur, labored breathing, contraction of the abdominal muscles (wasp waist), and total paralysis. Respiratory failure and death follow unless animals are humanely euthanized. Animals are observed initially after injection and then at 2, 4, 8, 12, and 24 h, and then daily for up to 4 d. Those showing signs within 2–6 h usually die within 24 h. The rapidity of symptoms (and time of death) is proportional to the amount of toxin present in the sample. Confirmation of the toxin (together with the determination of the toxin type) is achieved by an absence of the above symptoms in the mice following injection of the inoculum after preincubation with specific antitoxin (8,11). Extracts from food or feces are generally prepared by homogenization in gelatin- phosphate buffer (pH 6.2) which is centrifuged and filtered (to prevent infection) prior to injection. When low levels of toxin or nonproteolytic strains are suspected, the preparation can be pretreated with trypsin to activate the neurotoxin. 44 McLauchlin and Grant Assays for toxin are applied to direct analysis of clinical material (serum), extracts from clinical material (feces, gastric contents, and vomitus) and food, as well as to the supernatants from enrichment and pure cultures growing in broths. In vitro growth of C. botulinum in enrichment cultures is achieved by inoculation of broths with feces, gastric contents, and vomitus as well as food samples. For nonfoodborne infections (i.e., material from infected wounds), additional toxin tests are performed to the extracts from pus or tissue, together with application of cultural methods in these samples. It should be noted that the detection of toxin by bioassays is highly specialized and is likely to require specific authority (including ethical considerations) for their performance. 2.2.4. Identification and Isolation of C. botulinum As stated earlier, C. botulinum is defined on the basis of a single phenotypic characteristic, i.e., neurotoxin production, and hence more conventional phenotypic reactions such as the fermentation of carbohydrates, cellular fatty-acid analysis, and 16S rDNA gene sequence in not sufficient to provide an unequivocal identification (18,27,28). The identification process, therefore, relies on the detection of neurotoxins (or their genes). The initial process for culture of C. botulinum is the enrichment in prereduced cooked meat medium with or without glucose, chopped-meat glucose-starch medium, or tryptone–peptone glucose yeast extract broth. Trypsin may be added to inactivate bacteriocins and to activate neurotoxin. Replicate inoculated broths are treated either with or without a heat shock (60–80°C for 10–20 min).