Probiotic Bacillus paramycoides and Bacillus subtilis from Chinese Shanlan rice wine inhibit biofilm formation by Burkholderia thailandensis and Staphylococcus aureus

The intrinsic bacterial probiotic properties of Chinese Shanlan rice wine have not been explored previously. In this study, we isolated eighteen bacteria from Shanlan rice wine and selected promising probiotics to evaluate their antibiofilm efficacy. Most isolates survived in extreme simulated conditions mimicking the gastric digestive tract (acidic pH 1.0–3.0, bile salt, and gastric juice). Higher auto-aggregation, hydrophobicity, and bile-salt hydrolytic activity were associated with effective colonization and competition against pathogens. Neutralized cell-free supernatants (CFSs) from isolates exhibited antipathogenic effects against Burkholderia thailandensis and methicillin-resistant Staphylococcus aureus (MRSA). SDS-PAGE exhibited low-molecular-weight bands (approximately 10 kDa) resembling bacteriocins. Most isolates were positive for protease, amylase, lipase, and cellulase, whereas a few of them produced β-galactosidase, phytase, and exopolysaccharides. The maximum number of isolates exhibited cholesterol-lowering and antioxidant abilities. The antibiotic sensitivity and non-hemolytic properties of these isolates rendered them safe. Bacillus paramycoides SW9 and Bacillus subtilis SW12 were selected as promising probiotic isolates. CFSs from these isolates dose-dependently inhibited biofilm formation by B. thailandensis and MRSA, further confirmed by CLSM and SEM analyses. RT-qPCR gene expression profiling revealed the differential regulation of virulence genes. These probiotic CFSs could be used in the future to identify new antibiofilm peptides against antibiotic-resistant pathogens. • Eighteen isolates survived in vitro harsh conditions and enhanced gut colonization. • Isolates have better antibacterial, antioxidant, and cholesterol-lowering properties. • They are safe, non-hemolytic, sensitive to antibiotics and produce EPS and enzymes. • B. paramycoides SW9 & B. subtilis SW12 are the most promising probiotic candidates. • SW9 and SW12 CFSs inhibited the biofilm formation of tested bacterial pathogens.