Unraveling the ecological interactions between dairy strains Bacillus licheniformis and Bacillus cereus during the dual-species biofilm formation.

Bacillus cereus and Bacillus licheniformis are widely presented in dairy products. They can form thick biofilms on surfaces of dairy processing equipment, which may pose serious safety issues and spoilage of final dairy products. However, how ecological interactions between B. cereus and B. licheniformis affect the functions and stability of mixed-species biofilm remains uncovered. In this work, the altered profiles of a dual-species biofilm by dairy-derived B. cereus 121 and B. licheniformis 919 were investigated by RNA-sequencing analysis in combined with phenotype validation (bacterial growth, biofilm-forming capacity, biofilm EPS production, and biofilm structures). The results confirmed that the presence of B. cereus 121 reduced the growth of B. licheniformis 919 planktonic cells, and decreased the biofilm cell numbers of B. licheniformis 919 in the dual-species biofilm when compared to that in its single-species biofilm. The bacterial interaction also reduced the amount of proteins and carbohydrates in the biofilm matrix, and decreased the coverage, average thickness, and total biomass of biofilms. In addition, results from RNA-sequencing analysis showed that the bacterial interaction caused a total of 128 (B. licheniformis 919) and 216 (B. cereus 121) differentially expressed genes (DEGs) during the co-culture of planktonic cells. Functional annotation revealed that the DEGs of B. licheniformis 919 were mainly involved in 10 downregulated pathways including citrate cycle, pyruvate metabolism, nonribosomal peptide structures, glycolysis/gluconeogenesis, quorum sensing, alanine, aspartate and glutamate metabolism, oxidative phosphorylation, beta-Lactam resistance, arginine and proline metabolism, and beta-Alanine metabolism when co-cultured with B. cereus 121. On the other hand, the DEGs from B. cereus 121 were significantly enriched for two downregulated pathways (cysteine and methionine metabolism, and inositol phosphate metabolism) and four upregulated pathways (nitrogen metabolism, glyoxylate and dicarboxylate metabolism, glycine, serine and threonine metabolism, and propanoate metabolism). Results of this study facilitate updated knowledge of how bacterial interaction during the biofilm formation shapes the features of the mixed-species biofilm.