Bacillus cytotoxicus, a member of the Bacillus cereus group, is a thermotolerant species originally reported from a lethal foodborne infection in France in 1998. The strain NVH391-98, isolated from this outbreak, produces cytotoxin K1, a potential cytotoxic enterotoxin. However, the habitat and diversity of B. cytotoxicus isolates so far have been poorly explored. The objective of this study was to assess the prevalence of this bacterium in different food products (mainly dried) and to estimate its diversity. Among the 210 samples analyzed, all potato flakes contained the bacterium at low concentrations (≤102 CFU/g). However, prepared and kept at room temperature for 2 days, the puree contained ca. 105 CFU/g B. cytotoxicus. Besides potato flakes, some samples of millet flour, salted potato chips, and soups also contained B. cytotoxicus. From these samples, 55 thermotolerant B. cytotoxicus isolates were obtained. When classified into six distinct random amplified polymorphism DNA patterns, they showed the existence of 11 distinct plasmid profiles. Although most isolates (including the reference strains NVH391-98 and NVH883-00) contained no detectable plasmid, some displayed one to three plasmids with sizes from ca. 8 to 90 kb. It also emerged from this study that a single food sample could contain B. cytotoxicus isolates with different genetic profiles. HIGHLIGHTS

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The thermotolerant representative of the Bacillus cereus group, Bacillus cytotoxicus, reliably harbors the coding gene of cytotoxin K-1 (CytK-1). This protein is a highly cytotoxic variant of CytK toxin, initially recovered from a diarrheal foodborne outbreak that caused the death of three people. In recent years, the cytotoxicity of B. cytotoxicus has become controversial, with some strains displaying a high cytotoxicity while others show no cytotoxicity towards cell lines. In order to better circumscribe the potential pathogenic role of CytK-1, knockout (KO) mutants were constructed in two B. cytotoxicus strains, E8.1 and E28.3. The complementation of the cytK-1 KO mutation was implemented in a mutant strain lacking in the cytK-1 gene. Using the tetrazolium salt (MTT) method, cytotoxicity tests of the cytK-1 KO and complemented mutants, as well as those of their wild-type strains, were carried out on Caco-2 cells. The results showed that cytK-1 KO mutants were significantly less cytotoxic than the parental wild-type strains. However, the complemented mutant was as cytotoxic as the wild-type, suggesting that CytK-1 is the major cytotoxicity factor in B. cytotoxicus.

Bacillus cytotoxicus is the thermotolerant representative of the Bacillus cereus group. This group, also known as B. cereus sensu lato, comprises both beneficial and pathogenic members and includes psychrotolerant and thermotolerant species. Bacillus cytotoxicus was originally recovered from a fatal outbreak in France in 1998. This species forms a remote cluster from the B. cereus group members and reliably contains the cytk-1 gene, coding for a cytotoxic variant of cytotoxin K. Although this species was originally thought to be homogenous, intra-species diversity has been recently described with four clades, six random amplified polymorphic DNA (RAPD) patterns, and 11 plasmids profiles. This study aimed to get new insights into the genomic diversity of B. cytotoxicus and to decipher the underlying chromosomal and plasmidial variations among six representative isolates through whole genome sequencing (WGS). Among the six sequenced strains, four fitted the previously described genomic clades A and D, while the remaining two constituted new distinct branches. As for the plasmid content of these strains, three large plasmids were putatively conjugative and three small ones potentially mobilizable, harboring coding genes for putative leaderless bacteriocins. Mobile genetic elements, such as prophages, Insertion Sequences (IS), and Bacillus cereus repeats (bcr) greatly contributed to the B. cytotoxicus diversity. As for IS elements and bcr, IS3 and bcr1 were the most abundant elements and, along with the group II intron B.c.I8, were found in all analyzed B. cytotoxicus strains. When compared to other B. cytotoxicus strains, the type-strain NVH 391-98 displayed a relatively low number of IS. Our results shed new light on the contribution of mobile genetic elements to the genome plasticity of B. cytotoxicus and their potential role in horizontal gene transfer.