The Impact of Antioxidant-Ciprofloxacin Combinations on the Evolution of Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

Abstract Antibiotic resistance in biofilms is a critical challenge driven by mechanisms such as increased oxidative stress, which could lead to enhanced mutagenesis. Experimental evolution studies have shown that biofilm growth fosters antimicrobial resistance and tolerance. The bactericidal antibiotic ciprofloxacin (CIP) exacerbates oxidative stress, enhancing reactive oxygen species (ROS) production and mutagenesis at sub-inhibitory concentrations. This study tests the hypothesis that antioxidants (AOs) such as N-acetyl-cysteine (NAC), and Edaravone (ED) could serve as anti-evolutionary drugs to prevent or reduce antimicrobial resistance in P. aeruginosa biofilms exposed to sub-inhibitory concentrations of ciprofloxacin (CIP), with Thiourea (THU) used as a control. In vitro experimental evolution studies using the monitor strain PAO1-mCherry-PCD-gfp + were conducted in flow cells and glass beads biofilm models. The harvested biofilm populations underwent population analysis and the flow-cell populations were also whole genome sequenced (WGS). The results showed that the CIP-AO combination of CIP-THU could reduce the fast increase of mutagenic resistance in both biofilm models. The confocal laser microscopy of flow cells biofilms showed a reduction in the percentage of nfxB mutants in biofilms treated with all the three CIP-AO combinations compared to the CIP treatment alone following 24h of treatment. The population analysis revealed a significant reduction of the subpopulations growing on 0.5 mg/L of CIP-supplemented plates in all the combined treatments of CIP-AO as compared to CIP populations. The WGS results showed CIP-specific mutants in the CIP-treated populations including nfxB and nalC mutations which were not observed in CIP-AO-treated ones. Flagellar gene mutations appeared in CIP-ED (fliG) and CIP-NAC (flhA) treatments. Additionally, wspA, which regulates biofilm formation, had a transversion mutation in CIP-ED and a deletion mutation in CIP-NAC. The minimum inhibitory concentrations (MICs) level for CIP-treated isolates was significantly higher than all isolates from CIP-AO treatments in both tested biofilm models of flow cells and glass beads. Overall, these findings suggest that the two clinical antioxidants may have a potential role in mitigating the development of antibiotic resistance in biofilms.