Purpose: This study describes a strategy that was developed to characterize diarrheagenic Bc strains obtained from food and feed, as well as to evaluate the reliability of using whole genome sequencing (WGS) and a 25 allele MLSA analysis as a viable bioinformatics tool to compare phylogenetic relatedness among strains.
Methods: Bc strains were recovered from dried foods and animal feed. Toxin profiles were determined by a multiplex end point PCR assay using primers derived from the hemolysin BL (hbl), nonhemolytic enterotoxin (nhe), cytotoxin K (cytK), and enterotoxin FM (entFM) genes. The genomic diversity of the Bc strains was characterized using MLST analysis of genomes from NCBI and by WGS using MiSeq Nextera XT chemistry.
Results: A single local database consisting of genomes from 36 FDA isolates, 13 Dairy, and 21 Genome groups from NCBI were used in the analysis. A comprehensive 25-gene MLSA strategy was designed to capture the genomic diversity of these isolates. Our analysis, using 25 housekeeping genes, resulted in the identification of alleles in 2,179 positions within the 70 genomes. Phylogenetic analysis showed that only 11 of the FDA genomes clustered with 16 genomes of the NCBI Genome group; the other 25 FDA genomes clustered together in a second group, suggesting a greater intra-species genomic diversity, not previously recognized.
Significance: A comprehensive strategy utilizing bioinformatics showed that the current approach of using MLSA with 25 housekeeping genes and WGS was sufficient to capture the intra-species genomic diversity of Bc isolates. This study establishes an effective protocol for further genomics research of the phylogenetically diverse Bc group.