Antibiotic resistance is one of the most challenging issues in public health. Antibiotics have been increasingly used not only for humans and animals but also for crop protection as pesticides. Thus, antibiotics often coexist with pesticides in some environments. To investigate the effects of the co-occurring, nonantibiotic pesticides on the development of antibiotic resistance, we conducted long-term exposure experiments using an Escherichia coli K-12 model strain. The results reveal that (1) the exposure to pesticides (in mg/L) alone led to the emergence of mutants with significantly higher resistance to streptomycin; (2) the exposure to pesticides (in μg/L) together with a subinhibitory level (in high μg/L) of ampicillin synergistically stimulated the selection of ampicillin resistance and the cross-resistance to other antibiotics (i.e., ciprofloxacin, chloramphenicol, and tetracycline). Distinct and diversified genetic mutations emerged in the resistant mutants selected from the coexposure to both pesticides and ampicillin. The genetic mutations likely caused a holistic transcriptional regulation (e.g., biofilm formation, oxidative stress defense) when grown under antibiotic stress and led to increased antibiotic resistance. Together, these findings provide important fundamental insights into the development of antibiotic resistance and the resistance mechanisms under environmentally relevant conditions where antibiotics and nonantibiotic micropollutants coexist.

To explore how co-occurring non-antibiotic environmental stressors affect evolutionary trajectories toward antibiotic resistance, we exposed susceptible Escherichia coli K-12 populations to environmentally relevant levels of pesticides and streptomycin for 500 generations. The coexposure substantially changed the phenotypic, genotypic, and fitness evolutionary trajectories, resulting in much stronger streptomycin resistance (>15-fold increase) of the populations. Antibiotic target modification mutations in rpsL and rsmG, which emerged and dominated at late stages of evolution, conferred the strong resistance even with less than 1% abundance, while the off-target mutations in nuoG, nuoL, glnE, and yaiW dominated at early stages only led to mild resistance (2.5-6-fold increase). Moreover, the strongly resistant mutants exhibited lower fitness costs even without the selective pressure and had lower minimal selection concentrations than the mildly resistant ones. Removal of the selective pressure did not reverse the strong resistance of coexposed populations at a later evolutionary stage. The findings suggest higher risks of the selection and propagation of strong antibiotic resistance in environments potentially impacted by antibiotics and pesticides.

Enzymatic cleavage of C─F bonds in per- and polyfluoroalkyl substances (PFAS) is largely unknown but avidly sought to promote systems biology for PFAS bioremediation. Here, we report the reductive defluorination of α, β-unsaturated per- and polyfluorocarboxylic acids by Acetobacterium spp. The microbial defluorination products were structurally confirmed and showed regiospecificity and stereospecificity, consistent with their formation by enzymatic reactions. A comparison of defluorination activities among several Acetobacterium species indicated that a functional fluoride exporter was required for the detoxification of the released fluoride. Results from both in vivo inhibition tests and in silico enzyme modeling suggested the involvement of enzymes of the flavin-based electron-bifurcating caffeate reduction pathway [caffeoyl-CoA reductase (CarABCDE)] in the reductive defluorination. This is a report on specific microorganisms carrying out enzymatic reductive defluorination of PFAS, which could be linked to electron-bifurcating reductases that are environmentally widespread.