Characterization and Removal of Multispecies Biofilms Formed by <i>L. monocytogenes</i> , <i>S. aureus</i> , and <i>E. coli</i>

ABSTRACT This study aimed to characterize mono‐, dual‐, and triple‐species biofilms formed by the bacteria Listeria monocytogenes , Staphylococcus aureus , and Escherichia coli . This was achieved by assessing biofilm formation, planktonic cell growth, biomass, and metabolic activity. In total, 111 biofilm combinations were analyzed. Significant synergistic interactions were observed between S. aureus and L. monocytogenes in dual biofilms. In contrast, E. coli showed no synergy and frequently inhibited biofilm formation when co‐cultured with the other species. In triple‐species biofilms containing E. coli , biofilm formation decreased by up to 47%, and metabolic activity decreased by over 75%. Furthermore, the effectiveness of selected common disinfectants was evaluated. The effectiveness of solutions containing 0.47% NaClO (chlorine bleach), 8% acetic acid, and 0.5% lactic acid against 24‐h biofilms was tested. All disinfectants were effective, with bleach demonstrating the greatest reduction in biomass and metabolic activity. Notably, higher resistance to disinfectants was exhibited by mono‐species biofilms than multi‐species biofilms in this study. Inter‐species interactions strongly modulate biofilm behavior. E. coli tends to suppress biofilm formation, whereas S. aureus and L. monocytogenes consortia promote it. While mono‐species biofilms exhibit greater resistance in this study, bleach remains the most effective disinfectant. These results improve our understanding of the dynamics of mixed biofilms and emphasize the importance of selecting appropriate disinfectants for controlling biofilms in the food and healthcare industries.