Modulation of Bacillus subtilis anti-Listeria activity via the ComQXPA quorum sensing system

Listeria monocytogenes causes listeriosis, a severe foodborne disease with a high hospitalization rate. This pathogen forms robust biofilms in food environments and poses significant health risks. Bacillus subtilis PS-216, a proven surfactin producer with antimicrobial activity against Gram-negative pathogens, was investigated for its anti- Listeria effects. We analyzed the antimicrobial and antibiofilm activities of wild-type B. subtilis PS-216 and knockout mutants lacking quorum sensing genes (Δ comQ , Δ comA ) and QS-regulated antimicrobials (Δ srfAA , Δ bacA , Δ srfAA Δ bacA ) against L. monocytogenes and L. innocua . Gene expression of comQ , srfAA , and bacA was analyzed using YFP promoter fusions in mono- and cocultures. B. subtilis PS-216 demonstrated potent antimicrobial activity against Listeria sp., achieving a strong log reduction (5.9 and 6.7 log for L. monocytogenes and L. innocua , respectively) in coculture. The ComQXPA quorum sensing system centrally regulates this anti- Listeria activity by coordinated surfactin and bacilysin production. Both compounds are essential for a strong antagonistic effect, with coculture triggering enhanced expression. Additionally, B. subtilis PS-216 effectively inhibits Listeria biofilm formation, with surfactin preventing lawn-type biofilms. The similar response patterns between L. monocytogenes and L. innocua support the use of L. innocua as a suitable surrogate, though species-specific differences exist. These findings highlight B. subtilis PS-216's broad-spectrum probiotic potential and provide mechanistic insights for optimizing strategies to control L. monocytogenes in food processing environments. • Bacillus subtilis PS-216 shows strong antagonism towards pathogenic Listeria spp. • Modes of action include bacilysin and surfactin • bacA and srfAA activity is increased in coculture with Listeria • Listeria biofilm structure is modified by B. subtilis PS-216 cell free supernatant • Listeria adhesion to polystyrene is reduced by B. subtilis PS-216 cell free supernatant