Phylogenetic Analysis of Bacillus cereus sensu lato Isolates from Commercial Bee Pollen Using tRNA^Cys-PCR

Endospore-forming bacteria related to the Bacillus cereus group produce toxins that cause illnesses in organisms from invertebrates to mammals, including foodborne illnesses in humans. As commercial bee pollen can be contaminated with these bacteria, a comprehensive microbiological risk assessment of commercial bee pollen must be incorporated into the relevant regulatory requirements, including those that apply in Mexico. To facilitate detection of members of this group of bacteria, we have developed a PCR strategy that is based on the amplification of the single-copy tRNA^Cys gene and specific genes associated with tRNA^Cys to detect Bacillus cereus sensu lato (B. cereus s.l.). This tRNA^Cys-PCR-based approach was used to examine commercial bee pollen for endospore-forming bacteria. Our analysis revealed that 3% of the endospore-forming colonies isolated from a commercial source of bee pollen were related to B. cereus s.l., and this result was corroborated by phylogenetic analysis, bacterial identification via MALDI-TOF MS, and detection of enterotoxin genes encoding the HBL and NHE complexes. The results show that the isolated colonies are closely related phylogenetically to B. cereus, B. thuringiensis, and B. bombysepticus. Our results indicate that the tRNA^Cys-PCR, combined with other molecular tools, will be a useful approach for identifying B. cereus s.l. and will assist in controlling the spread of potential pathogens.