Intraclade Variability in Toxin Production and Cytotoxicity of Bacillus cereus Group Type Strains and Dairy-Associated Isolates

While some species in the group are well-characterized human pathogens (e.g., and ), the pathogenicity of other species (e.g., ) either has not been characterized or is presently not well understood. To provide an updated characterization of the pathogenic potential of species in the group, we classified a set of 52 isolates, including 8 type strains and 44 isolates from dairy-associated sources, into 7 phylogenetic clades and characterized them for (i) the presence of toxin genes, (ii) phenotypic characteristics used for identification, and (iii) cytotoxicity to human epithelial cells. Overall, we found that toxin genes are broadly distributed but are not consistently present within individual species and/or clades. After growth at 37°C, isolates within a clade did not typically show a consistent cytotoxicity phenotype, except for isolates in clade VI ( / ), where none of the isolates were cytotoxic, and isolates in clade I ( ), which consistently displayed cytotoxic activity. Importantly, our study highlights that is cytotoxic toward human cells. Our results indicate that the detection of toxin genes does not provide a reliable approach to predict the pathogenic potential of group isolates, as the presence of toxin genes is not always consistent with cytotoxicity phenotype. Overall, our results suggest that isolates from multiple group clades have the potential to cause foodborne illness, although cytotoxicity is not always consistently found among isolates within each clade. Despite the importance of the group as a foodborne pathogen, characterizations of the pathogenic potential of all group species were lacking. We show here that (clade I), which has been considered a harmless environmental microorganism, produces toxins and exhibits a phenotype consistent with the production of pore-forming toxins. Furthermore, isolates (clade VI) did not show cytotoxicity when grown at 37°C, despite carrying multiple toxin genes. Overall, we show that the current standard methods to characterize group isolates and to detect the presence of toxin genes are not reliable indicators of species, phylogenetic clades, or an isolate's cytotoxic capacity, suggesting that novel methods are still needed for differentiating pathogenic from nonpathogenic species within the group. Our results also contribute data that are necessary to facilitate risk assessments and a better understanding as to which group species are likely to cause foodborne illness.