Use of gyrB and rpoB gene sequencing for identification of Bacillus subtilis group strains isolated from an immunobiological industry facility

Introduction: Microbiological quality control is essential in the pharmaceutical industry, as contamination can compromise medicine safety and efficacy.Precise microorganism identification is required in various samples during production.Bacillus subtilis, a Gram-positive spore-forming bacterium, is frequently found in pharmaceutical environments.Genetic similarity among related species in the B. subtilis group complicates identification.Genotypic analyses, such as gyrB and rpoB sequencing, which offer greater variability than 16S rRNA sequencing, enhance species discrimination and improve microbiological control. Objectives:The aim of this study was to evaluate gyrB and rpoB gene sequencing for identification of B. subtilis group strains isolated from an immunobiological industry facility.Methodology: Twenty strains isolated from the immunobiological production facility during 2020-2022, previously identified as belonging to the B. subtilis group by VITEK 2, were analyzed.The 16S rRNA, gyrB and rpoB genes were amplified by PCR and sequenced by Sanger methodology.Sequences from each gene were used to build phylogenetic trees using Maximum likelihood method. Results:The rpoB and gyrB genes showed greater efficacy than 16S rRNA in discriminating genetically close species.The phylogenetic analysis allowed the differentiation of all strains species: B. subtilis (rpoB n=7; gyrB n=7), B. stercoris (rpoB n=3; gyrB n=3), B. spizizenii (rpoB n=3; gyrB n=3), B. siamensis (rpoB n=3; gyrB n=2), B. velezensis (rpoB n=4; gyrB n=4), B. amyloliquefaciens (rpoB n=0; gyrB n= 1).Only one (5%) strain showed divergent result (B.siamensis/B.amyloliquefaciens). Conclusion:The differentiation of B. subtilis group species requires a polyphasic approach, including the sequencing of constitutive genes for a multilocus sequence analysis.However, the absence of sequences of some species in public database still limits the identification of some strains.Moreover, genotypic methods are laborious and time-consuming to be used routinely in the microbiological control of a pharmaceutical industry.So, faster and cheaper methods are needed for speciation of this group.