Identification of small-molecule adjuvants that enhance the sensitivity of Escherichia coli to nitrofurantoin: Roles of Lon and MarA

The increasing cases of resistance among UTI pathogens pose a significant threat to the continued clinical use of nitrofurantoin. In this study, we explored the molecular mechanisms underlying nitrofurantoin resistance and investigated the potential of synergistic activity of salicylates in enhancing the antibacterial activity of nitrofurantoin. In our initial observation, deletion of lon ({Delta}lon) conferred enhanced susceptibility to nitrofurantoin. We identified the critical role of Lon protease in regulating the sensitivity to nitrofurantoin. Investigation into the mechanisms revealed that the lon deletion strains show a higher level of marA and nfsA, which is likely to facilitate the conversion of nitrofurantoin from its pro form to its active form. The {Delta}lon strains displayed an elevated level of ROS, membrane alteration and filamentation upon treatment with nitrofurantoin. Higher ROS levels and membrane alteration were reversed upon treatment with glutathione, further confirming the role of oxidative stress in mediating the sensitivity to nitrofurantoin. Building on these mechanistic insights, we tested salicylates to synergistically enhance the efficacy of nitrofurantoin by indirectly inducing marA through the repression of the mar operon, thereby enhancing nfsA transcription. Both sodium salicylate and acetyl salicylate enhanced the efficacy of nitrofurantoin and lowered the dose of nitrofurantoin required to inhibit the growth of the WT strain. Importantly, this synergistic effect with acetyl salicylate was also observed in nitrofurantoin-resistant clinical isolates, where the combination reduced the effective nitrofurantoin concentration required for growth inhibition. This work provides novel insights into the roles of transcriptional regulators and proteolysis in antibiotic susceptibility, advancing the notion that antibiotic adjuvants are a reliable means of reviving the efficacy of antibiotics. ImportanceThis study unravels the uncharacterised role of Lon protease in nitrofurantoin susceptibility and illustrates the enhanced efficacy of nitrofurantoin-salicylate combinations as a promising therapeutic strategy to overcome emerging resistance in UTI pathogens. This study highlights the importance of investigating the repurposing of other FDA-approved molecules to combat resistance.