Insights into the genomic and phenotypic characteristics of <i>Bacillus</i> spp. strains isolated from biofilms in broiler farms

The characterization of surface microbiota living in biofilms within livestock buildings has been relatively unexplored, despite its potential impact on animal health. To enhance our understanding of these microbial communities, we characterized 11 spore-forming strains isolated from two commercial broiler chicken farms. Sequencing of the strains revealed them to belong to three species <i>Bacillus velezensis</i>, <i>Bacillus subtilis</i>, and <i>Bacillus licheniformis</i>. Genomic analysis revealed the presence of antimicrobial resistance genes and genes associated with antimicrobial secretion specific to each species. We conducted a comprehensive characterization of the biofilm formed by these strains under various conditions, and we revealed significant structural heterogeneity across the different strains. A macro-colony interaction model was employed to assess the compatibility of these strains to coexist in mixed biofilms. We identified highly competitive <i>B. velezensis</i> strains, which cannot coexist with other <i>Bacillus</i> spp. Using confocal laser scanning microscopy along with a specific dye for extracellular DNA, we uncovered the importance of extracellular DNA for the formation of <i>B. licheniformis</i> biofilms. Altogether, the results highlight the heterogeneity in both genome and biofilm structure among <i>Bacillus</i> spp. isolated from biofilms present within livestock buildings.IMPORTANCELittle is known about the microbial communities that develop on farms in direct contact with animals. Nonpathogenic strains of <i>Bacillus velezensis</i>, <i>Bacillus subtilis</i>, and <i>Bacillus licheniformis</i> were found in biofilm samples collected from surfaces in contact with animals. Significant genetic and phenotypic diversity was described among these <i>Bacillus</i> strains. The strains do not possess mobile antibiotic resistance genes in their genomes and have a strong capacity to form structured biofilms. Among these species, <i>B. velezensis</i> was noted for its high competitiveness compared with the other <i>Bacillus</i> spp. Additionally, the importance of extracellular DNA in the formation of <i>B. licheniformis</i> biofilms was observed. These findings provide insights for the management of these surface microbiota that can influence animal health, such as the use of competitive strains to minimize the establishment of undesirable bacteria or enzymes capable of specifically deconstructing biofilms.