Proline mitigates antibiotic resistance evolution induced by ciprofloxacin at environmental concentrations

Antibiotics-induced resistance development in the environment has emerged as a critical issue under the 'one health' framework. Although there have been approaches to control antibiotic resistance evolution in clinical settings, they are rarely applicable in environmental contexts. Amino acids can affect the metabolic states of bacteria and potentially influence their resistance evolution. In this study, we screened 18 amino acids and identified proline as an efficient agent capable of mitigating ciprofloxacin-induced resistance of a soil-isolated Escherichia coli by over 50 % during a 24-day evolutionary experiment. Using transcriptomics and ¹³C metabolic flux analysis, we revealed the evolution mitigation mechanism of proline, which mainly involves down-regulated gene expressions and reduced metabolic flux of the TCA cycle, thereby decreasing NADH production, proton motive force, and uptake of ciprofloxacin. Based on single-cell RNA-seq, proline also reduced the size of resistant subgroups in the evolved E. coli population. Based on soil microcosm experiments, proline not only reduced the overall antibiotic resistance but also increased community diversity and robustness (optimal dosage: 5 mg/kg). Moreover, proline's evolution mitigation potentials likely extend to other antibiotics (e.g., streptomycin) and populations (e.g., Pseudomonas and Serratia spp.). Overall, proline addition holds promising potentials for mitigating antibiotic resistance in diverse antibiotics-polluted environments.