Impact of Bacillus subtilis Antibiotic Bacilysin and Campylobacter jejuni Efflux Pumps on Pathogen Survival in Mixed Biofilms.

The foodborne pathogen Campylobacter jejuni is typically found in an agricultural environment; in animals, such as birds, as an intestinal commensal; and also in food products, especially fresh poultry meat. Campylobacter interactions within mixed species biofilms are poorly understood, especially at the microscale. We have recently shown that the beneficial bacterium Bacillus subtilis reduces C. jejuni survival and biofilm formation in coculture by secreting the antibiotic bacillaene. We extend these studies here by providing evidence that besides bacillaene, the antagonistic effect of B. subtilis involves a nonribosomal peptide bacilysin and that the fully functional antagonism depends on the quorum-sensing transcriptional regulator ComA. Using confocal laser scanning microscopy, we also show that secreted antibiotics influence the distribution of C. jejuni and B. subtilis cells in the submerged biofilm and decrease the thickness of the pathogen's biofilm. Furthermore, we demonstrate that genes encoding structural or regulatory proteins of the efflux apparatus system (cmeF and cmeR), respectively, contribute to the survival of C. jejuni during interaction with B. subtilis PS-216. In conclusion, this study demonstrates a strong potential of B. subtilis PS-216 to reduce C. jejuni biofilm growth, which supports the application of the PS-216 strain to pathogen biofilm control. IMPORTANCE Campylobacter jejuni is a prevalent cause of foodborne infections worldwide, while Bacillus subtilis as a potential probiotic represents an alternative strategy to control this alimentary infection. However, only limited literature exists on the specific mechanisms that shape interactions between B. subtilis and C. jejuni in biofilms. This study shows that in the two species biofilms, B. subtilis produces two antibiotics, bacillaene and bacilysin, that inhibit C. jejuni growth. In addition, we provide the first evidence that specific pathogen efflux pumps contribute to the defense against B. subtilis attack. Specifically, the CmeDEF pump acts during the defense against bacilysin, while CmeR-dependent overexpression of CmeABC nullifies the bacillaene attack. The role of specific B. subtilis antibiotics and these polyspecific pumps, known for providing resistance against medically relevant antibiotics, has not been studied during bacterial competition in biofilms before. Hence, this work broadens our understanding of mechanisms that shape antagonisms and defense during probiotic-pathogen interactions.

Bacillaene Mediates the Inhibitory Effect of Bacillus subtilis on Campylobacter jejuni Biofilms.

Biofilms are the predominant bacterial lifestyle and can protect microorganisms from environmental stresses. Multispecies biofilms can affect the survival of enteric pathogens that contaminate food products, and thus, investigating the underlying mechanisms of multispecies biofilms is essential for food safety and human health. In this study, we investigated the ability of the natural isolate Bacillus subtilis PS-216 to restrain Campylobacter jejuni biofilm formation and adhesion to abiotic surfaces as well as to disrupt preestablished C. jejuni biofilms. Using confocal laser scanning microscopy and colony counts, we demonstrate that the presence of B. subtilis PS-216 prevents C. jejuni biofilm formation, decreases growth of the pathogen by 4.2 log10, and disperses 26-h-old preestablished C. jejuni biofilms. Furthermore, the coinoculation of B. subtilis and C. jejuni interferes with the adhesion of C. jejuni to abiotic surfaces, reducing it by 2.4 log10. We also show that contact-independent mechanisms contribute to the inhibitory effect of B. subtilis PS-216 on C. jejuni biofilm. Using B. subtilis mutants in genes coding for nonribosomal peptides and polyketides revealed that bacillaene significantly contributes to the inhibitory effect of B. subtilis PS-216. In summary, we show a strong potential for the use of B. subtilis PS-216 against C. jejuni biofilm formation and adhesion to abiotic surfaces. Our research could bring forward novel applications of B. subtilis in animal production and thus contribute to food safety. IMPORTANCE Campylobacter jejuni is an intestinal commensal in animals (including broiler chickens) but also the most frequent cause of bacterial foodborne infection in humans. This pathogen forms biofilms which enhance survival of C. jejuni in food processing and thus threaten human health. Probiotic bacteria represent a potential alternative in the prevention and control of foodborne infections. The beneficial bacterium Bacillus subtilis has an excellent probiotic potential to reduce C. jejuni in the animal gastrointestinal tract. However, data on the effect of B. subtilis on C. jejuni biofilms are scarce. Our study shows that the B. subtilis natural isolate PS-216 prevents adhesion to the abiotic surfaces and the development of submerged C. jejuni biofilm during coculture and destroys the preestablished C. jejuni biofilm. These insights are important for development of novel applications of B. subtilis that will reduce the use of antibiotics in human and animal health and increase productivity in animal breeding.