Bacillus cereus is a Gram-positive endospore forming bacterium known for its wide spectrum of phenotypic traits, enabling it to occupy diverse ecological niches. Although the population structure of B. cereus is highly dynamic and rather panmictic, production of the emetic B. cereus toxin cereulide is restricted to strains with specific genotypic traits, associated with distinct environmental habitats. Cereulide is an ionophoric dodecadepsipeptide that is produced non-ribosomally by an enzyme complex with an unusual modular structure, named cereulide synthetase (Ces non-ribosomal peptide synthetase). The ces gene locus is encoded on a mega virulence plasmid related to the B. anthracis toxin plasmid pXO1. Cereulide, a highly thermo- and pH- resistant molecule, is preformed in food, evokes vomiting a few hours after ingestion, and was shown to be the direct cause of gastroenteritis symptoms; occasionally it is implicated in severe clinical manifestations including acute liver failures. Control of toxin gene expression in emetic B. cereus involves central transcriptional regulators, such as CodY and AbrB, thereby inextricably linking toxin gene expression to life cycle phases and specific conditions, such as the nutrient supply encountered in food matrices. While in recent years considerable progress has been made in the molecular and biochemical characterization of cereulide toxin synthesis, far less is known about the embedment of toxin synthesis in the life cycle of B. cereus. Information about signals acting on toxin production in the food environment is lacking. We summarize the data available on the complex regulatory network controlling cereulide toxin synthesis, discuss the role of intrinsic and extrinsic factors acting on toxin biosynthesis in emetic B. cereus and stress how unraveling these processes can lead to the development of novel effective strategies to prevent toxin synthesis in the food production and processing chain.

Food-borne intoxications are increasingly caused by the dodecadepsipeptide cereulide, the emetic toxin produced by Bacillus cereus. As such intoxications pose a health risk to humans, a more detailed understanding on the chemodiversity of this toxin is mandatory for the reliable risk assessment of B. cereus toxins in foods. Mass spectrometric screening now shows a series of at least 18 cereulide variants, among which the previously unknown isocereulides A-G were determined for the first time by means of UPLC-TOF MS and ion-trap MS(n) sequencing, (13)C-labeling experiments, and post-hydrolytic dipeptide and enantioselective amino acid analysis. The data demonstrate a high microheterogeneity in cereulide and show evidence for a relaxed proof reading function of the non-ribosomal cereulide peptide synthetase complex giving rise to an enhanced cereulide chemodiversity. Most intriguingly, the isocereulides were found to differ widely in their cell toxicity correlating with their ionophoric properties (e.g., purified isocereulide A showed about 8-fold higher cytotoxicity than purified cereulide in the HEp-2 assay and induced an immediate breakdown of bilayer membranes). These findings provide a substantial contribution to the knowledge-based risk assessment of B. cereus toxins in foods, representing a still unsolved challenge in the field of food intoxications.

SummaryBacillus cereus causes gastrointestinal diseases and local and systemic infections elicited by the depsipeptide cereulide, enterotoxins, phospholipases, cytolysins and proteases. The PlcR-PapR quorum sensing system activates the expression of several virulence factors, whereas the Spo0A-AbrB regulatory circuit partially controls the plasmid-borne cereulide synthetase (ces) operon. Here, we show that CodY, a nutrient-responsive regulator of Gram-positive bacteria, has a profound effect on both regulatory systems, which have been assumed to operate independently of each other. Deletion of codY resulted in downregulation of virulence genes belonging to the PlcR regulon and a concomitant upregulation of the ces genes. CodY was found to be a repressor of the ces operon, but did not interact with the promoter regions of PlcRdependent virulence genes in vitro, suggesting an indirect regulation of the latter. Furthermore, CodY binds to the promoter of the immune inhibitor metalloprotease InhA1, demonstrating that CodY directly links B. cereus metabolism to virulence. In vivo studies using a Galleria mellonella infection model, showed that the codY mutant was substantially attenuated, highlighting the importance of CodY as a key regulator of pathogenicity. Our results demonstrate that CodY profoundly modulates the virulence of B. cereus, possibly controlling the development of pathogenic traits in suitable host environments.

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.