Molecular characterization of Bacillus sporothermodurans in Ultra High Temperature (UHT) milk

Abstract

Bacillus sporothermodurans spores are known to withstand Ultra high temperature (UHT) processing. The surviving spores germinate and cause the technological problem of non-sterility in milk and milk products. This high heat resistance has been attributed to clonal effects, with the highly heat resistant spore (HRS) responsible. This study sought to use whole genome sequencing (WGS) to characterize strains of B. sporothermodurans isolated from UHT milk and to determine and compare heat inactivation dynamics of an HRS and non-HRS strain. Subsequent comparative genetic analysis was undertaken to compare B. sporothermodurans strains with other closely related Bacillus sp. and determine the pangenome of B. sporothermodurans. The closest phylogenetic neighbour of B. sporothermodurans was identified as B. oleronius using WGS. Further, the closest relatives besides B. oleronius were identified as B. acidicola and B. coagulans rather than B. lentus and B. firmus as previously thought. The B. sporothermodurans strains had genome sizes ranging from 3.4 Mbp – 4.0 Mbp and an average G + C content of 36% as compared to 5.1 Mbp and 35% respectively for B. oleronius. The number of mobile element proteins in B. sporothermodurans strains ranged from 61 – 141, with the HRS strains SA01 and BR12 generally having twice as much as compared to the non-HRS strains. This will probably make the HRS strains more prone to horizontal gene transfer (HGT). Additionally, the HRS strains exhibited the Tn552 and Tn554 transposons, responsible for coding for beta-lactamases and erythromycin and kanamycin resistance and possibly also affecting the control of heat resistance in B. sporothermodurans. Heat inactivation dynamics determined for the two South African strains SA01 (HRS) and SAD (non-HRS) showed similar inactivation data. Data from the study suggests spores of B. sporothermodurans strains can survive UHT temperatures for over 6 s at 140 °C. The Weibull inactivation model gave a superior fit as compared to especially the linear inactivation model. Comparative genetic analysis of B. sporothermodurans strains show no observable difference in the nucleotide sequence of the GrpE heat shock protein and other proteins in the DnaK cluster of heat resistance related proteins, suggesting the influence of mobile genetic elements (MGEs) in their varying heat resistance. Pangenome analysis of B. sporothermodurans strains exhibited a core genome covering 69.9 – 92.2% and the accessory genome covering 7.8 – 30.1%. B. sporothermodurans core/pangenome ratio of 55% suggests an open pangenome thereby making it more prone to HGT events. Results from this study suggests that while HRS and non-HRS strains have a similar complement of heat resistant genes, their differing heat resistance may be as a result of MGEs, since B. sporothermodurans has an especially open pangenome.