Genotyping of dairy Bacillus licheniformis isolates by high resolution melt analysis of multiple variable number tandem repeat loci.

In dairy foods, the sporeformer Bacillus licheniformis can be the cause of spoilage or specification compliance issues. Currently used methods for genotyping B. licheniformis have limited discrimination with only 2 or 3 different subgroups being identified. Here, we have developed a multi-locus variable number tandem repeat analysis (MLVA) method and combined it with high resolution melt analysis (MLV-HRMA) for genotyping B. licheniformis. Five repetitive loci were identified and used as markers for genotyping 52 isolates from two milk powder processing plants and retail samples. Nineteen genotypes could be identified using both MLVA and MLV-HRMA leading to Hunter-Gaston discrimination indices (D-value) of 0.93 each. It was found that all 5 MLVA loci were stable following 10 days of sub-culturing of 8 representative isolates. All isolates were also genotyped using previously used methods including randomly amplified polymorphic DNA-PCR (RAPD) and partial rpoB sequencing. Five different RAPD profiles and 5 different partial rpoB sequence types were identified resulting in corresponding D-values of 0.6 and 0.46, respectively. Analysis of the genotypes from dairy samples revealed that dairy B. licheniformis isolates are more heterogeneous than previously thought and that this new method can potentially allow for more discriminatory tracking and monitoring of specific genotypes.