Environmental co-exposure to roxarsone and heavy metals drives bacterial antibiotic multidrug resistance via genetic and phenotypic adaptations
Agricultural wastewater, a significant component of the anthropogenic water cycle, often contains complex mixtures of contaminants, including non-antibiotic feed additives, posing largely uncharacterized risks for antimicrobial resistance (AMR) evolution. This study addresses the critical knowledge gap regarding the combined impact of commonly co-occurring roxarsone (ROX) and heavy metals (Zn, Cu) at environmentally relevant concentrations on the de novo evolution and persistence of multidrug resistance in aquatic environments. Using a 90-day laboratory evolution experiment with Escherichia coli , we demonstrate that while ROX alone induced moderate resistance (e.g., 6.2-fold increase in minimum inhibitory concentration (MIC) for chloramphenicol), co-exposure with Zn and Cu synergistically drove multidrug tolerance to nine antibiotics and selected for stable, heritable resistance to chloramphenicol, tetracycline, and kanamycin (up to 8.2-fold MIC increase). This robust resistance persisted even after pollutant removal, highlighting a significant long-term threat to water quality and public health via the dissemination of resilient AMR bacteria from agricultural sources. Co-exposure intensified oxidative stress-induced mutagenesis and selected for key adaptive strategies enhancing bacterial survival and AMR persistence in contaminated water. These include upregulated efflux pumps, increased secretion of extracellular polymeric substances (1.1-3.2-fold), enhanced motility (1.1-1.5-fold), and cell filamentation (lengths 2.4-8.2-fold greater). These findings illuminate a potent, previously underestimated environmental pathway where mixtures of common agricultural pollutants in wastewater synergistically select for persistent multidrug resistance. This research underscores the urgent need to revise water quality criteria and wastewater treatment paradigms to address the co-selection pressures exerted by non-antibiotic chemical mixtures in aquatic ecosystems.
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