Probiotic Bacillus strains inhibit growth, biofilm formation, and virulence gene expression of Listeria monocytogenes

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This research aimed to evaluate the probiotic potential and anti-biofilm properties of Bacillus species isolated from dairy sludge, namely Bacillus subtilis GS3 (DS-1), Bacillus cereus BF2 (DS-2), Bacillus velezensis DM (DS-3), and Bacillus thuringiensis JB (DS-4). These strains exhibited tolerance to gastrointestinal fluids, bile salt, phenol, NaCl, and temperature variations. Moreover, they demonstrated notable cell surface hydrophobicity (70.80–89.30%), auto-aggregation (79.30–92.70%), and cholesterol removal (20.12–59.46%). Additionally, the strains displayed antioxidant activity and susceptibility to a wide range of antibiotics. The cell-free supernatants (CFS) of the strains exhibited inhibitory effects against Listeria monocytogenes ATCC 19115, with minimum inhibitory concentrations (MIC) observed at 12.5%, 25%, 25%, and 50% v/v for DS-1, DS-2, DS-3, and DS-4, respectively. The CFS also showed degradation and inhibition of L. monocytogenes biofilms, affecting adhesion, hydrophobicity, auto-aggregation, and exopolysaccharide production. Notably, DS-1 CFS suppressed the expression of genes associated with biofilm formation in L. monocytogenes, including those related to quorum sensing, virulence factors, and flagella. The antibacterial activity of CFS against L. monocytogenes was further confirmed by scanning electron microscopy. Particularly, the CFS of Bacillus spp., especially B. subtilis GS3, exhibited a strong inhibitory effect on the biofilm formation of L. monocytogenes.