Molecular characterization and biofilm formation potential of Bacillus subtilis and Bacillus velezensis in extended shelf-life milk processing line

Abstract

This study aims to characterize Bacillus subtilis complex group from raw, pasteurized, and packaged extended shelf-life (ESL) milk samples, to determine their biofilm potential and source-track the microbial contaminants to control their presence during processing. Isolates were characterized using multi-locus sequence typing (MLST) with 7 housekeeping genes. The primers used were designed from the coding regions with the highest number of polymorphic sites. The heat resistance profile indicated that all 12 isolates are psychrotolerant as well as thermophilic, with temperature ranges of 6°C to 55°C (B43, B44, B52, B54, B55, B56, B57), 6°C to 60°C (B46, B47, B48), and 15°C to 60°C (B49, B50). A general linear model 2-way repeated-measure ANOVA of the biofilm-forming potential of the isolates shows a statistically significant difference across the time of incubation (6, 12, 18, and 24 h). All isolates except 2 formed moderate to strong biofilms, with B44 having the most robust biofilm formation (3.14 ± 0.60). Scanning electron and confocal microscopy images reveal the strain specificity of the biofilm structure. The MLST analysis identified all isolates as belonging to either B. subtilis or Bacillus velezensis. All the isolates were novel sequence types (ST) when compared with the PubMLST database (https://pubmlst.org/) but showed relatedness to isolates in the raw milk that was processed. The closest ST are 96 for B. velezensis and 128 for B. subtilis, mostly isolated from soil. This study presents the significance of biofilms of thermophilic B. subtilis and B. velezensis and their possible perpetuation in the dairy processing plant. The information provided is a call for an innovative food contact surface or any other intervention that can minimize or prevent microbial adhesion in the processing plant, to prevent negative effects in ESL milk.

Keywords: Bacillus subtilis complex groups; biofilm; extended shelf-life (ESL) milk; multi-locus sequence typing (MLST).