Several Bacillus cereus strains possess the genetic fittings to produce two different types of toxins, the heat-stable cereulide or different heat-labile proteins with enterotoxigenic potential. Unlike the diarrheal toxins, cereulide is (pre-)formed in food and can cause foodborne intoxications shortly after ingestion of contaminated food. Based on the widely self-limiting character of cereulide intoxications and rarely performed differential diagnostic in routine laboratories, the real incidence is largely unknown. Therefore, during a 7-year period about 4.300 food samples linked to foodborne illness with a preliminary report of vomiting as well as food analysed in the context of monitoring programs were investigated to determine the prevalence of emetic B. cereus in food environments. In addition, a lux-based real-time monitoring system was employed to assess the significance of the detection of emetic strains in different food matrices and to determine the actual risk of cereulide toxin production in different types of food. This comprehensive study showed that emetic strains are much more volatile than previously thought. Our survey highlights the importance and need of novel strategies to move from the currently taxonomic-driven diagnostic to more risk orientated diagnostics to improve food and consumer safety.

Cereulide, the emetic Bacillus cereus toxin, is synthesized by cereulide synthetase via a nonribosomal peptide synthetase (NRPS) mechanism. Previous studies focused on the identification, structural organization, and biochemical characterization of the ces gene locus encoding cereulide synthetase; however, detailed information about the transcriptional organization of the ces genes was lacking. The present study shows that the ces-PTABCD genes are transcribed as a 23-kb polycistronic transcript, while cesH, encoding a putative hydrolase, is transcribed from its own promoter. Transcription initiation was mapped by primer extension and rapid amplification of cDNA ends (RACE). Deletion analysis of promoter elements revealed a main promoter located upstream of the cesP coding sequence, encoding a 4-phosphopantetheinyl transferase. This promoter drives transcription of cesPTABCD. In addition, intracistronic promoter regions in proximity to the translational start sites of cesB and cesT were identified but were only weakly active under the chosen assay conditions. The identified main promoter was amplified from the emetic reference strain B. cereus F4810/72 and fused to luciferase genes in order to study promoter activity in complex environments and to establish a biomonitoring system to assess cereulide production in different types of foods. ces promoter activity was strongly influenced by the food matrix and varied by 5 orders of magnitude. The amount of cereulide toxin extracted from spiked foods correlated well with the bioluminescence data, thus illustrating the potential of the established reporter system for monitoring of ces gene expression in complex matrices.Bacillus cereus is the causative agent of two types of food poisoning: diarrhea and emesis. The toxicoinfection referred to as diarrheal disease occurs after consumption of B. cereus spores or vegetative cells, most likely due to the action of heat-labile enterotoxins, which are produced by cells multiplying in the small intestine (4,22,30,39). Contrarily, the emetic type of food-borne illness is caused by intoxication with the heat-stable peptide cereulide, which is preformed in foods and elicits vomiting a few hours after ingestion (18,51,58).Regulation of enterotoxin expression of B. cereus has been studied in some detail (e.g., references 16, 29, and 46; for a review, see reference 58), revealing a major role of the pleiotropic transcription regulator PlcR in activation of enterotoxin genes and other virulence factors (1,29,45). Cereulide synthesis in emetic B. cereus, in contrast to synthesis of B. cereus enterotoxins, is not controlled by PlcR but by the Spo0A phosphorelay. The global transition state factor AbrB was identified as one factor repressing cereulide production in early exponential phase (38). Although B. cereus enterotoxins and the emetic toxin cereulide seem to belong to completely different regulatory networks, production of both types of toxins depends substantially on nutritional and environmental factors (3,46,55), and it is expe...