Further comparison of temperature effects on growth and survival of Clostridium perfringens type A isolates carrying a chromosomal or plasmid-borne enterotoxin gene.

Clostridium perfringens isolates are commonly classified into five types (types A to E) based on the production of four typing toxins (alpha, beta, epsilon, and iota toxins) (24). Type A isolates, the most abundant toxinotype, produce alpha toxin, but not beta, epsilon, or iota toxin (11, 14, 19). Some type A isolates also produce another toxin, C. perfringens enterotoxin (CPE). These enterotoxigenic type A strains cause several human enteric diseases, including C. perfringens type A food poisoning, which is among the three most common food-borne illnesses in the United States, and some cases of non-food-borne human gastrointestinal disease, including antibiotic-associated diarrhea and sporadic diarrhea (5, 6). In C. perfringens type A isolates, the enterotoxin gene (cpe) can be located on either the chromosome or a large plasmid (6, 7). Interestingly, in food poisoning isolates the cpe gene is typically on the chromosome, perhaps due to integration of a 6.3-kb cpe-carrying transposon that is flanked by IS1470 sequences (3). In contrast, the cpe gene is usually plasmid borne in non-food-borne human gastrointestinal disease isolates, and it typically is present on either a 75.3-kb plasmid that also encodes beta2 toxin or a 70.5-kb plasmid that lacks the beta2 toxin gene (10, 16, 17). Both cpe plasmids found in type A isolates may be capable of conjugative transfer due to the presence of Tn916-related open reading frames (4, 17). To date, there are at least two explanations for the strong association between type A isolates carrying a chromosomal cpe gene and food poisoning. First, a recent study (26) that evaluated the presence of cpe-positive isolates in American retail foods showed that all 13 cpe-positive type A isolates recovered from the foods surveyed had a chromosomal cpe gene. The findings indicated that, at least in part, chromosomal cpe isolates are the predominant cause of food poisoning because they are the cpe-positive type A isolates that are most often present in food. However, other evidence suggests that the strong association between chromosomal cpe isolates and food poisoning may involve additional factors. Several labs (1, 2, 22) have determined that the vegetative cells or spores of type A chromosomal cpe isolates obtained from food poisoning outbreaks survive heating much better than the cells or spores of type A plasmid cpe isolates survive heating. For example, one study (22) showed that the average decimal reduction value (D value) at 55°C for vegetative cells of chromosomal cpe food poisoning isolates was twofold higher than the average D value at 55°C for vegetative cells of plasmid cpe isolates. Furthermore, the average D value at 100°C for spores of chromosomal cpe food poisoning isolates was nearly 60-fold higher than the average D value at 100°C for spores of plasmid cpe isolates. These cpe genotype-related heat resistance differences appear to be important since survival at elevated temperatures should be helpful for a food-borne pathogen like C. perfringens that is primarily transmitted via temperature-abused foods. When spores of the chromosomal cpe isolates recovered from retail foods during a recent survey (26) were tested for heat sensitivity, they also exhibited considerably greater heat resistance than the spores of type A plasmid cpe isolates exhibited. Since the chromosomal cpe isolates were recovered from raw meat that was never cooked, heat resistance appears to be an intrinsic trait of chromosomal cpe isolates rather than merely a trait selected for in food poisoning isolates that survive cooking. Little is known about the mechanisms responsible for the specific heat resistance of chromosomal cpe isolates, although a recent study (20) showed that (i) the heat resistance of chromosomal cpe isolates is not dependent on the presence of a functional cpe gene and (ii) the heat sensitivity of plasmid cpe isolates is not dependent on the presence of a cpe plasmid. In the food environment, pathogens often encounter many lethal or inhibitory environmental stresses in addition to heat. One of the most important such food stresses facing a pathogen is low temperature, which is universally used for food preservation in the form of either refrigeration or freezing (18). Since understanding the strong association between type A isolates carrying a chromosomal cpe gene and food poisoning has implications for controlling C. perfringens type A food poisoning, in the current study we compared the effects of temperature, especially refrigeration and freezer temperatures, on both the survival and the growth of type A isolates carrying a chromosomal cpe gene and type A isolates carrying a plasmid-borne cpe gene.