Abstract: Objective: To investigate the effect and underlying mechanism of ethyl acetate extract from Lactiplantibacillus plantarum Z139 on the single- and mixed-species biofilms of Escherichia coli and Staphylococcus aureus. Methods: The minimum inhibitory concentrations (MICs) of the extract against single and mixed cultures were determined using the two-fold broth dilution method. We investigated the effect of the extract on the formation of single and mixed-species biofilms by E. coli and S. aureus, the eradication of mature biofilms, cell membrane permeability, bacterial nucleic acid leakage, extracellular polysaccharide production, and bacterial survival in biofilms. Results: The MIC of the extract against S. aureus, E. coli and mixed cultures was 3.2 mg/mL. When incubated at the sub-inhibitory concentration (1/2 MIC, 1.6 mg/mL) for 24 h, the inhibition rates against the single- and mixed-species biofilms of S. aureus and E. coli were 49.70%, 51.36%, and 50.94%, respectively, while eradication rates after 48 h incubation at 12.8 mg/mL were 65.55%, 75.26%, and 65.56%, respectively. In addition, the extract effectively inhibited extracellular polysaccharide production. Conclusion: The extract exhibits strong inhibitory effects on E. coli, S. aureus, and their mixed cultures, suggesting its potential as a novel lactic acid bacteria-derived biocontrol agent against mixed biofilms. This study provides data support for the prevention and control of pathogenic biofilm contamination and the development of novel antibacterial agents.

Key words: Lactiplantibacillus plantarum; Escherichia coli; Staphylococcus aureus; mixed-species biofilms; anti-biofilm effect