# Evolutionary adaptations of Pseudomonas aeruginosa biofilms to ciprofloxacin and antioxidant co-treatment in synthetic sputum medium

## TL;DR

This study shows that antioxidants can slow the development of antibiotic resistance in Pseudomonas aeruginosa biofilms under conditions mimicking cystic fibrosis lung infections.

## Contribution

The study reveals that antioxidants suppress resistance evolution in Pseudomonas biofilms by altering mutation patterns under CF-like conditions.

## Key findings

- Ciprofloxacin alone led to high resistance (MIC 8–32 mg/L), while ciprofloxacin plus antioxidants suppressed resistance (MIC 0.75–2 mg/L).
- Genome sequencing showed distinct mutations in ciprofloxacin-only isolates versus those treated with ciprofloxacin and antioxidants.
- Antioxidants like edaravone altered resistance mechanisms, suggesting a role for reactive oxygen species in resistance evolution.

## Abstract

Antimicrobial resistance (AMR) is a growing public health concern, particularly in biofilm-related infections, where microbial aggregates display high levels of tolerance. Oxidative stress has been hypothesized to accelerate the development of resistance, whereas antioxidants (AOs) may mitigate this process. In this study, we investigated the impact of AOs on the evolution of ciprofloxacin (CIP) resistance in Pseudomonas aeruginosa PAO1 using synthetic cystic fibrosis sputum medium (SCFM2), which mimics the physiochemical conditions of cystic fibrosis (CF) respiratory infections. Experimental evolution was performed over six passages with CIP alone or in combination with edaravone (ED), N-acetyl-cysteine (NAC), or thiourea (THU). Population analysis profiles and minimum inhibitory concentration (MIC) assays demonstrated that CIP treatment produced high-level resistance (MIC 8–32 mg/L), whereas CIP + AO treatments markedly suppressed resistance development (MIC 0.75–2 mg/L). Whole-genome sequencing revealed distinct mutational patterns. CIP-treated isolates carried mutations in mexR and nalC (efflux pump regulators), and gyrA (fluoroquinolone target), consistent with elevated resistance, along with additional mutations in rocR and dnaX. In contrast, evolved isolates in the presence of CIP + AO harbored nfxB mutations associated with lower resistance, while CIP + ED uniquely produced a mutation in parS. These findings support the role of reactive oxygen species (ROS) in driving resistance evolution under CF-like conditions and suggest that antioxidants can suppress this process, providing a potential strategy for limiting antimicrobial resistance in biofilm-associated infections.

Fighting antimicrobial resistance (AMR) is one of the greatest health challenges of our time. To find new ways to stop it, we need to better understand how resistance develops. Our study suggests that antioxidants may help slow down the process that allows bacteria to become resistant. We also show that resistance develops more quickly, and in a different way, when bacteria grow in conditions that resemble the human body rather than in standard laboratory media. In particular, the synthetic sputum medium promoted the formation of aggregated biofilms—sticky communities of cells that often occur in chronic and hard-to-treat infections.