Purpose: This study describes a strategy that was developed to characterize diarrheagenic Bc isolates representing different enterotoxin and allelic profiles based on PCR, multi-loci analysis (MLA), and whole genome sequencing (WGS).
Methods: Seventy-nine Bc isolates were recovered from dried spices, infant formula, dietary supplements, and medicated animal feed. An isolate’s toxin profile was determined by a multiplex end point PCR assay using primers derived from the hemolysin BL (hbl), nonhemolytic enterotoxin (hhe), cytotoxin K (cytK), and enterotoxin FM (entFM ) genes. The genomic diversity of the Bc isolates was characterized using our comprehensive MLA consisting of sequences of all known MLST and enterotoxin genes. Genomes were obtained from NCBI and by using WGS on MiSeq using Nexteria XT chemistry. NCBI Blast++, MEGA6 and in-house perl scripts were applied to identify alleles of target genes.
Results: Alleles for 13 enterotoxin loci from these food and feed isolates were identified using MLA, and by PCR. Phylogenetic analysis of these sequences revealed distinct patterns of sequence polymorphisms associated with isolates of specific genome groups. Comparison of the divergence of these enterotoxin gene sequences identified certain genomic landmarks. Three recently described alleles of the entABC toxin were also found in these isolates.
Significance: A comprehensive strategy utilizing bioinformatics with new sequence assemblies and comparing the genome sequence analysis with endpoint PCR proved to be an effective scheme to rapidly determine genome-type and enterotoxin content of diarrheagenic isolates found in dried foods and feeds. This study also demonstrates a powerful genomic strategy for further application to the phylogenetically diverse B. cereus group, a prerequisite towards development of future countermeasures against this important foodborne pathogen.