Can extracellular particles from different Bacillus spp. strains contribute to the inhibition and synergistic effect against Salmonella Heidelberg strains isolated from poultry?

Abstract

The role of extracellular particles (EPs) in bacterial communication and pathogenesis has gained increasing attention, and studies related to probiotic strains remain scarce. This study evaluated the strain-dependent inhibitory and synergistic effects of EPs produced by Bacillus cereus (BC), Bacillus subtilis (BS), and Bacillus licheniformis (BL) against Salmonella enterica subsp. enterica serovar Heidelberg (SH). EPs were separated from the water-soluble, non-particulate (WS-NP) fraction of cell-free supernatants (CFS) obtained from Bacillus strains, cultured individually and in combination, using ultracentrifugation and ultrafiltration. EPs were characterized for particle concentration, size distribution, protein content, and metabolomic profiling to identify strain- and combination-specific metabolic signatures. Finally, the antimicrobial activity of EPs against SH was assessed. The EPs exhibited pronounced heterogeneity, with particle diameters ranging from 142.3 to 367.4 nm and high yields exceeding 10¹⁰ particles/mL after concentration. Transmission electron microscopy is consistent with the coexistence of vesicular and non-vesicular particle subpopulations. Functionally, dual EP combinations (BL+BS and BL+BC) showed synergistic effects against SH strain 13, achieving up to 99% growth inhibition. All EPs treatments reduced SH strain 18 by approximately 2 log CFU/mL, while inhibition of SH strain 06 was more limited (~1 log CFU/mL). Metabolomic profiling identified 61 metabolites, 23 of which showed differential abundance across groups, particularly those with antimicrobial and quorum-sensing activity, providing insight into the metabolic basis underlying EP-mediated interactions. Overall, these findings demonstrate strain-dependent EP-mediated inhibition of SH and their synergistic effects, highlighting the critical role of probiotic bacteria's EPs in inhibition and the contribution of their active components to this process.