Investigation of Antimicrobial and Antivirulence Effects of Lactobionic Acid and Carvacrol, Individually and in Combination, Against Escherichia coli Isolates

Background: The proliferation of multidrug-resistant (MDR) Escherichia coli represents a critical global health challenge, driven primarily by the bacterium's robust biofilm-forming capacity, which facilitates antimicrobial tolerance and infection persistence. This study aimed to investigate the individual and synergistic antibiofilm and antivirulence activities of lactobionic acid (LBA) and carvacrol (CAR) against MDR E. coli clinical isolates. Methods and Results: Biofilm formation was characterized in 51 clinical isolates, including the reference strain E. coli ATCC 25922. The antimicrobial efficacy of LBA and CAR was determined via Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays. For 43 isolates identified as strong or moderate biofilm producers, the Minimum Biofilm Eradication Concentration (MBEC) was established. Synergistic effects were evaluated using the checkerboard method. Furthermore, RT-qPCR was employed to quantify the expression of luxS, csgA, pfs, and rpoS genes critical to quorum sensing, adhesion, and stress response. MIC values for CAR and LBA ranged from 256-4096 µg/mL and 2048-16,384 µg/mL, respectively, with MBC/MIC ratios ≤ 4, indicating potent bactericidal activity. Individually, CAR and LBA inhibited biofilm formation by 24.12% and 57.7%, respectively; however, their combination achieved up to 87.7% inhibition in strong biofilm-formers. Checkerboard assays demonstrated synergy (FICI ≤ 0.5) in 85% of the tested isolates, resulting in a 32-fold reduction in the MIC. LBA was found to be more effective than CAR for the eradication of biofilm structures. Molecular analysis revealed a significant downregulation of all target genes, with the combined treatment resulting in a 10-fold reduction in csgA expression. Conclusions: These results demonstrate that the combination of LBA and CAR effectively suppresses the growth, biofilm maturity, and virulence of MDR E. coli . This synergistic approach represents a promising alternative therapeutic strategy for managing persistent infections caused by multidrug-resistant pathogens.