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Clostridium perfringens type A isolates producing enterotoxin (CPE) are an
important cause of both food poisoning (FP) and non food borne gastrointestinal
diseases (NFBGID) in both humans and animals. C. perfringens type A food
poisoning is caused by isolates carrying the CPE encoding gene (cpe) on the
chromosome while the NFBGID isolates carry cpe on a large plasmid. Recent
studies indicate that the association between chromosomal cpe isolates and food
poisoning is, in part, because spores of these isolates exhibit heat resistance which
enhances their survival in incompletely cooked or inadequately warmed foods. The
molecular basis for resistance of spores to heat and other environmental stress
factors remains unknown. Since in Bacillus subtilis, small acid soluble spore
proteins (SASPs) have been shown to play a major role in spore resistance to heat
and other environmental factors, in the current study, we hypothesized that α/β-type
SASPs in C. perfringens play a similar role. Results of this study showed that the
SASP encoding genes, ssp1, 2 and 3 are present and expressed in food poisoning
isolates of C. perfringens. Consistent with the results in B. subtilis, α/β-type SASPs
in C. perfringens protect the spores by binding to spore DNA. A single C.
perfringens ssp3 knock out mutant showed a slightly decreased spore heat resistance
compared to wild type. Since there is no genetic tool available to make a triple ssp
(ssp1,2 and 3) knock out mutant in C. perfringens, the antisense RNA strategy was
used to down regulate the production of α/β-type SASPs in a food poisoning isolate.
The strains with lower α/β-type SASP production had lower resistance to moist heat
and UV radiation. These results support our hypothesis that C. perfringens α/β-type
SASPs play a major role in spore resistance to heat and other environmental factors.
The second hypothesis in this study was that the NFBGID isolates carrying
cpe on a plasmid exhibit lower heat resistance than the FP isolates because these
isolates produce less α/β-type SASPs. PCR and nucleotide sequencing analysis
demonstrated that all three ssp genes are present in NFBGID isolates. Quantitative
analysis of SASPs production demonstrated that NFBGID isolates produce similar
levels of SASPs as the FP isolates. Further studies showed that the cpe plasmid does
not encode any factors responsible for heat sensitivity in NFBGID isolates. The cpe
plasmid cured strain of these isolates showed a similar heat resistance to that of the
wild type strain carrying the cpe plasmid. However, it was observed that the dry heat
resistance of the plasmid cpe isolates was similar to that of the chromosomal cpe
isolates indicating that there might be additional factors (eg: spore water content)
involved in spore resistance to dry heat. Future studies on identifying the genes
encoding factors that affect spore water content should help in understanding the
mechanism of resistance to dry heat.
Collectively, these studies give an insight into the molecular basis for C.
perfringens spore resistance to heat and other environmental factors. Further
understanding of the mechanism of action of SASPs should help in designing and
developing a preventive measure for C. perfringens type A food poisoning. |
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