In recent years Bacillus cereus has gained increasing importance as a food poisoning pathogen. It is the eponymous member of the B. cereus sensu lato group that consists of eight closely related species showing impressive diversity of their pathogenicity. The high variability of cytotoxicity and the complex regulatory network of enterotoxin expression have complicated efforts to predict the toxic potential of new B. cereus isolates. In this study, comprehensive analyses of enterotoxin gene sequences, transcription, toxin secretion and cytotoxicity were performed. For the first time, these parameters were compared in a whole set of B. cereus strains representing isolates of different origin (food or food poisoning outbreaks) and of different toxic potential (enteropathogenic and apathogenic) to elucidate potential starting points of strain-specific differential toxicity. While toxin gene sequences were highly conserved and did not allow for differentiation between high and low toxicity strains, comparison of nheB and hblD enterotoxin gene transcription and Nhe and Hbl protein titers revealed not only strain-specific differences but also incongruence between toxin gene transcripts and toxin protein levels. With one exception all strains showed comparable capability of protein secretion and so far, no secretion patterns specific for high and low toxicity strains were identified. These results indicate that enterotoxin expression is more complex than expected, possibly involving the orchestrated interplay of different transcriptional regulator proteins, as well as posttranscriptional and posttranslational regulatory mechanisms plus additional influences of environmental conditions.

Bacillus cereus is a ubiquitous bacterial pathogen increasingly reported to be the causative agent of foodborne infections and intoxications. Since the enterotoxins linked to the diarrheal form of food poising are foremost produced in the human intestine, the toxic potential of enteropathogenic B. cereus strains is difficult to predict from studies carried out under routine cultivation procedures. In this study, toxigenic properties of a panel of strains (n = 19) of diverse origin were compared using cell culture medium pre-incubated with CaCo-2 cells to mimic intestinal growth conditions. Shortly after contact of the bacteria with the simulated host environment, enterotoxin gene expression was activated and total protein secretion of all strains was accelerated. Although the signal stimulating enterotoxin production still needs to be elucidated, it could be shown that it originated from the CaCo-2 cells. Overall, our study demonstrates that the currently used methods in B. cereus diagnostics, based on standard culture medium, are not allowing a conclusive prediction of the potential health risk related to a certain strain. Thus, these methods should be complemented by cultivation procedures that are simulating intestinal host conditions.

Enteropathogenic Bacillus cereus causes foodborne infections due to the production of pore-forming enterotoxins in the intestine. Before that, spores have to be ingested, survive the stomach passage, and germinate. Thus, before reaching epithelial cells, B. cereus comes in contact with the intestinal mucus layer. In the present study, different aspects of this interaction were analyzed. Total RNA sequencing revealed major transcriptional changes of B. cereus strain F837/76 upon incubation with porcine gastric mucin (PGM), comprising genes encoding enterotoxins and further putative virulence factors, as well as proteins involved in adhesion to and degradation of mucin. Indeed, PGM was partially degraded by B. cereus via secreted, EDTA-sensitive proteases. The amount of enterotoxins detectable in culture media supplemented with PGM was also clearly increased. Tests of further strains revealed that enhancement of enterotoxin production upon contact with PGM is broadly distributed among B. cereus strains. Interestingly, evidence was found that PGM can also strain-specifically trigger germination of B. cereus spores and that vegetative cells actively move toward mucin. Overall, our data suggest that B. cereus is well adapted to the host environment due to massive transcriptome changes upon contact with PGM, attributing mucin an important and, thus far, neglected role in pathogenesis.M ucus is a highly complex viscous substance that covers gastrointestinal cells.Besides salts, lipids, and several proteins involved in defense mechanisms, it consists mainly of water (approximately 95%) and the glycoprotein mucin (1, 2). A large number of different mucins has been discovered. These can be divided into membranebound and secretory mucins, which form the mucus layers (3, 4). Mucins have a molecular weight of 0.5 to 20 MDa and consist of ϳ20% proteins and ϳ80% carbohydrates (1). The protein core is organized in tandem repeats of serine-, threonine-, and proline-rich regions, which are mostly O-glycosylated. At the C and N termini, large amounts of cysteine are found. The carbohydrates include N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid, fucose, galactose, and mannose. Mucin monomers dimerize and multimerize via disulfide bonds (1).Mucus functions as a lubricant in the gastrointestinal tract, facilitating the passage of food. It is also important for growth, adhesion, and protection of the intestinal microbiota (2). Furthermore, it represents a biophysical barrier between epithelial cells and the environment, including chemical and mechanical insults as well as the commensal microbiota and pathogens (2,5,6). The mucus layer is a reservoir for many antimicrobial molecules, and various mucin oligosaccharides themselves show antimicrobial activity (5). By adhesion to mucin oligosaccharides, pathogens are trapped and removed due to the constant renewal of the mucus layer (5, 6). Cell surface-bound mucins can even initiate intracellular signaling in response to bacteria (5).On the other hand, pathogens, espe...

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