Coexisting pollutants modulate cephalosporin bioavailability and shape antibiotic resistance evolution under co-exposure conditions.

Antibiotic resistance is one of the most challenging issues in public health. Our understanding of microbial responses to multiple pollutants (e.g., pharmaceutical and personal care products) is largely based on single or dual-stressor exposures. The impacts of three or more stressors on antibiotic resistance development remain largely unknown. Cephalosporins are widely detected in wastewater and play an important role in driving bacterial resistance evolution. Based on a literature meta-analysis, we identified 75 pollutants frequently coexisted with a representative cephalosporin (cefotaxime) in wastewater. A wastewater isolated E. coli strain was exposed to cefotaxime and 10 high-frequency coexisting pollutants (in combinations of one to six) and evolved for 30 days. Three- and four-stressor combined exposures resulted in the most pronounced increase in resistance (> 7-fold). Five pollutants exhibited synergistic effects with cefotaxime, among which ofloxacin exhibited the strongest contribution. The increased bioavailability of cefotaxime was consistent with increased outer membrane permeability and reduced efflux pump activity. The magnitude of synergistic effect was positively correlated with their interaction energy with cefotaxime and bioavailability of cefotaxime. Pollutants exerting antagonistic effects, e.g., tetracycline, regulated outer membrane permeability and efflux pump activity in an opposite manner to synergistic pollutants. High-level resistant clones evolved under three- and four-stressor exposures accumulated the most mutations in efflux pump genes. These findings highlight the primary mechanism behind coexisting pollutants in synergistically driving antibiotic resistance development by increasing antibiotic bioavailability. These pollutants should also be of priority for resistance risk assessment and control in polluted environment such as wastewater treatment systems.