Characterization of biofilm and exopolysaccharide production in Bacillus strains isolated from the bovine uterus.

The bovine uterus hosts a diverse microbiome whose role in reproductive physiology and pathology is increasingly recognized. While Bacillus species have been occasionally isolated from the uterus, their biofilm and exopolysaccharide (EPS) forming capabilities have not been systematically characterized. In this study, four Bacillus strains (BU13, BU14, BU15, and BU16) were isolated from the bovine uteri and examined for their taxonomic affiliation, phenotypic characteristics, EPS production, biofilm formation, and antibiotic susceptibility. Phylogenetic analyses based on nearly full-length 16S rRNA gene sequences revealed that all isolates belonged to the B. subtilis group, with BU13, BU14, and BU16 closely related to B. licheniformis, and BU15 related to B. amyloliquefaciens and B. siamensis. BU13 and BU16 demonstrated high levels of EPS and biofilm production, especially in sucrose-supplemented media and under nutrient-rich conditions. Notably, these strains also exhibited relatively smaller inhibition zones against β-lactam antibiotics, which may be associated with their robust EPS-biofilm phenotypes. In contrast, larger inhibition zones were observed with gentamicin, enrofloxacin, and trimethoprim. These findings underscore the importance of characterizing commensal Bacillus spp. in the uterus and highlight that certain strains may possess traits that facilitate persistence and reduce antimicrobial responsiveness. This is the first study to comprehensively evaluate the biofilm-forming potential of uterine Bacillus isolates and provides a foundation for future investigations into their role in reproductive health and disease.