Interspecies interactions in biofilms: co-regulation of Pseudomonas fluorescens and Listeria monocytogenes isolated from chilled beef processing

Mixed biofilms formed with the coexistence of spoilage bacteria and foodborne pathogens represent a significant cause for persistent microbial contamination in the food industry. Deciphering the interspecies interaction mechanisms is a critical prerequisite for developing effective control strategies. This study systematically investigated the interaction mechanisms within dual-species biofilms of Pseudomonas fluorescens and Listeria monocytogenes. Results found that the dual-species biofilms exhibited significantly enhanced tolerance to sodium hypochlorite, with a higher survival rate than mono-species biofilms after 20 min treatment (P < 0.05). Dual RNA-Seq analysis revealed that P. fluorescens activated pathways related to the two-component system, flagellar assembly, fatty acid metabolism, and quorum sensing (QS) in the co-culture system. In response, L. monocytogenes regulated numerous genes and pathways, such as chemotaxis, two-component system, flagellar assembly, phosphotransferase system, and amino acid biosynthesis. The cell-free supernatant (CFS) from the co-culture significantly promoted biomass accumulation, increased matrix thickness, and reduced roughness in mono-species biofilms (P < 0.05), suggesting that metabolites could act as positive signaling molecules in this process. The autoinducer-2 (AI-2) concentration in the co-cultured CFS was significantly higher than that in mono-cultured CFS (P < 0.05). In addition, exogenous addition of AI-2 enhanced the biofilm formation of P. fluorescens and dual-species in a concentration-dependent manner (P < 0.05), confirming the key regulatory role of QS in this process. This study provided new insights into the synergistic mechanisms of dual-species biofilms and the development of control strategies.