Cytotoxicity and Genome Characteristics of an Emetic Toxin-Producing Bacillus cereus Group sp. Isolated from Raw Milk

The <i>Bacillus cereus</i> group frequently contaminates milk and dairy products. Some members of this group can produce the heat-stable pre-formed toxin cereulide, which causes emetic foodborne intoxication. This study characterised emetic <i>B. cereus</i> group isolates from raw cow's milk in the biochemical, genetic, and toxigenic aspects. Of the 158 <i>B. cereus</i> group isolates derived from 99 raw milk samples, 7 (4.43%) harboured <i>cereulide synthetase A</i> (<i>cesA</i>), which encodes a cereulide synthetase associated with the emetic phenotype. Heat-treated culture filtrates from the <i>cesA</i>-positive isolates demonstrated cytotoxicity to HepG2 and Caco-2 cells, resulting in cell viabilities of 32.22-36.57% and 44.41-47.08%, respectively. The cytotoxicity levels were comparable to those of the reference emetic strain, F4810/72 (alternately termed AH187). Genome analysis of a representative isolate, CSB98, revealed the complete <i>ces</i> gene cluster with additional virulence factors such as non-haemolytic enterotoxin, haemolysins and phospholipases, suggesting that the isolate could be both emetic and diarrhoeagenic. CSB98 exhibited a closer relationship to the type strain of <i>B. paranthracis</i> than to that of <i>B. cereus sensu stricto</i> (ATCC 14579). The genomes of CSB98 and AH187 were indistinguishable through OrthoANI analysis, but 13 variants were identified via SNP calling. These results affirm genetic conservation among the emetic traits.