Characterization of Bacillus sporothermodurans Isolated from Ultra High Temperature (UHT) Milk

Introduction: Some strains of Bacillus sporothermodurans produce highly resistant spores during unfavorable conditions thereby having implications on the quality of ultra-high temperature (UHT) processed milk and other milk products. Identification at the strain level of vegetative cells is mainly through sequencing of the 16S rRNA gene; however, multiple copies of the 16S rRNA gene which differ in sequence are usually present in a bacterium leading to the identification of multiple ribotypes. Protein coding genes (including rpoB), single copy in nature may afford better discrimination especially at the species and sub-species level.

Purpose: To determine the genetic diversity of B. sporothermodurans strains through sequencing of the 16S rRNA gene and rpoB gene with the aim of improving molecular characterization to the strain level. 

Methods: Genomic DNA was extracted from bacterial cells grown on brain heart infusion agar and amplified through PCR. Sequencing reactions were performed using the 16S rRNA and rpoB genes at sequencing read lengths of 750 bp and 1000 bp. Phylogenetic analysis were performed to determine genetic discrimination levels. 

Results: Partial 16S rDNA sequencing showed a wide variation of B. sporothermodurans strains isolated from different locations. Eight clusters for the target organisms were identified at similarity level > 90%. Partial rpoB sequencing showed 10 clusters for the target organism at similarity level of > 90%. For a total of 10 clusters, the rpoB gene provided more phylogenetic resolution than the 16S rRNA gene in 6 clusters with observed differences, with equal resolution in 1 cluster and lower resolution in 3 clusters. 

Significance: In the context of the growing number of studies focusing on sub-species diversity in the food and biomedical industries, this research would help develop rapid, efficient molecular detection and characterization protocols with higher resolution levels for food spoilage organisms and microbes of public health importance.