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

Abstract

Clostridium perfringens type A isolates can carry the enterotoxin gene (cpe) on either their chromosome or a plasmid, but food poisoning isolates usually have a chromosomal cpe gene. This linkage between chromosomal cpe isolates and food poisoning has previously been attributed, at least in part, to better high-temperature survival of chromosomal cpe isolates than of plasmid cpe isolates. In the current study we assessed whether vegetative cells and spores of chromosomal cpe isolates also survive better than vegetative cells and spores of plasmid cpe isolates survive when the vegetative cells and spores are subjected to low temperatures. Vegetative cells of chromosomal cpe isolates exhibited about eightfold-higher decimal reduction values (D values) at 4 degrees C and threefold-higher D values at -20 degrees C than vegetative cells of plasmid cpe isolates exhibited. After 6 months of incubation at 4 degrees C and -20 degrees C, the average log reductions in viability for spores of plasmid cpe isolates were about fourfold and about threefold greater, respectively, than the average log reductions in viability for spores from chromosomal cpe isolates. C. perfringens type A isolates carrying a chromosomal cpe gene also grew significantly faster than plasmid cpe isolates grew at 25 degrees C, 37 degrees C, or 43 degrees C. In addition, chromosomal cpe isolates grew at higher maximum and lower minimum temperatures than plasmid cpe isolates grew. Collectively, these results suggest that chromosomal cpe isolates are commonly involved in food poisoning because of their greater resistance to low (as well as high) temperatures for both survival and growth. They also indicate the importance of proper low-temperature storage conditions, as well as heating, for prevention of C. perfringens type A food poisoning.

FIG. 1.

Thermal death curves at low temperatures for vegetative cells of NCTC10239, a type A isolate carrying a chromosomal cpe gene, and isolate 458, a type A isolate carrying a plasmid cpe gene. Vegetative cultures of NCTC10239 or isolate 458 were incubated at 4°C (A) or −20°C (B) for different times, and then aliquots were removed and plated onto BHI agar to determine the number of viable bacteria per milliliter of culture. The data are the results of a representative experiment; similar results were obtained when the experiment was repeated.

FIG. 2.

Survival of cpe-positive C. perfringens type A vegetative cells at low temperatures: average D values for vegetative cells incubated at 4°C or −20°C for eight type A chromosomal cpe isolates and seven type A plasmid cpe isolates. At each temperature, D values were determined in two independent experiments for each isolate, as described in Materials and Methods. The error bars indicate standard deviations, and two asterisks indicate that the difference is statistically significant at a P value of <0.01.

FIG. 3.

Thermal death curves at low temperatures for spores from NCTC10239, a type A isolate carrying a chromosomal cpe gene, and isolate 458, a type A isolate carrying a plasmid cpe gene. Sporulating cultures of NCTC10239 or isolate 458 were incubated at 4°C (A) and −20°C (B) for different times, and then aliquots were removed, heat shocked, and plated onto BHI agar to determine the number of viable bacteria per milliliter of culture. The data are the results of a representative experiment; similar results were obtained when the experiment was repeated.

FIG. 4.

Survival of cpe-positive C. perfringens type A spores at low temperatures: average log reductions in the number of viable spores after incubation at 4°C or −20°C for eight type A chromosomal cpe isolates and seven type A plasmid cpe isolates. At each temperature, log reduction values were determined in two independent experiments for each isolate, as described in Materials and Methods. The error bars indicate standard deviations, and two asterisks indicate that the difference is statistically significant at a P value of <0.01.

FIG. 5.

Average growth rate constants (A) and mean generation times (B) for cpe-positive C. perfringens type A isolates at various temperatures. Eight type A chromosomal cpe isolates and seven type A plasmid cpe isolates were grown at different temperatures, aliquots of each culture were removed periodically, and growth was measured spectrophotometrically, as described in Materials and Methods. At each temperature, the growth of each isolate was determined for two independent cultures. The error bars indicate standard deviations; two asterisks indicate that the difference is statistically significant at a P value of <0.01, and one asterisk indicates that the difference is statistically significant at a P value of <0.05.

FIG. 6.

Maximum and minimum growth temperatures for cpe-positive C. perfringens type A isolates. Eight type A chromosomal cpe isolates and seven type A plasmid cpe isolates were inoculated into cultures that were then incubated at 10 to 20°C and at 49 to 54°C. Aliquots of each culture were removed periodically, and growth was measured spectrophotometrically, as described in Materials and Methods. At each temperature, the growth of each isolate was measured for two independent cultures. The error bars indicate standard deviations, and two asterisks indicate that the difference is statistically significant at a P value of <0.01.