The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France. A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C. botulinum spores per 25 g and 100% of samples inoculated with more than 30 C. botulinum spores per 25 g. The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did. The prevalence of C. botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C. botulinum most-probable-number counts, respectively, and is in the low range of C. botulinum contamination reported elsewhere. The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E. Type F was not detected. The high prevalence of C. botulinum type B in fish samples is relatively unusual compared with the high prevalence of C. botulinum type E reported in many worldwide and northern European surveys. However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.

The mechanisms involved in the ability of Bacillus cereus to multiply at low temperatures were investigated. It was assumed that many genes involved in cold acclimation would be upregulated at low temperatures. Recombinase-based in vivo expression technology (IVET) was adapted to the detection of the transient activation of B. cereus promoters during growth at 10°C. Four independent screenings of a promoter library from type strain ATCC 14579 were performed, and 17 clones were isolated. They corresponded to 17 promoter regions that displayed reproducibly elevated expression at 10°C relative to expression at 30°C. This analysis revealed several genes that may be important for B. cereus to grow successfully under the restrictive conditions of cold habitats. Among them, a locus corresponding to open reading frames BC5402 to BC5398, harboring a lipase-encoding gene and a putative transcriptional regulator, was identified three times. While a mutation in the putative regulator-encoding gene did not cause any particular phenotype, a mutant deficient in the lipase-encoding gene showed reduced growth abilities at low temperatures compared with the parental strain. The mutant did not change its fatty acid profiles in the same way as the wild type when grown at 12°C instead of 37°C. This study demonstrates the feasibility of a promoter trap strategy for identifying cold-induced genes. It outlines a first picture of the different processes involved in B. cereus cold acclimation.The food-borne disease agent Bacillus cereus is an endospore-forming bacterium belonging to the B. cereus group (B. cereus sensu lato). B. cereus sensu lato has recently been divided into seven major phylogenetic groups (I to VII) with clear-cut differences in growth temperature ranges, suggesting that the genetic structure corresponds to "thermotypes" and showing the emergence of multiple psychrotrophic groups within B. cereus sensu lato (26). Temperature adaptation has thus presumably played a major role in B. cereus evolution. B. cereus is also a human pathogen, causing local and systemic infections. Most outbreaks of food-borne poisoning have been caused by mesophilic strains (26) that can grow at temperatures as low as 10°C. This characteristic enables initially relatively low levels of B. cereus in foods to increase greatly under commonly reported suboptimal refrigeration conditions (20). Understanding the ability of B. cereus to grow at low temperatures will help to control multiplication in refrigerated food and prevent outbreaks of food-borne poisoning.At low temperatures, bacteria undergo various modifications in cellular physiology, with effects such as decreased membrane fluidity and inefficient folding of proteins and secondary structures of RNA and DNA (43). Bacterial responses can be divided into low-temperature responses (or acclimation, also called low-temperature adaptation) and cold shock responses (43). Both types of responses include a vast array of structural and physiological adjustments to cope with the reduction in biochemic...

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