Selective shifts in mobile antibiotic resistance genes under carbamazepine exposure in wastewater microbiomes

Carbamazepine (CBZ) is a widely used non-antibiotic pharmaceutical that frequently persists through wastewater treatment and enters aquatic environments. CBZ has been, in simplified experimental systems, reported to stimulate horizontal gene transfer (HGT), a major process of antimicrobial resistance gene (ARG) dissemination in microbial communities. Moreover, it may facilitate selection for ARGs, directly or via co-selection. However, whether CBZ significantly modulates these processes in complex microbiomes remains insufficiently characterised. To address this gap, we exposed wastewater microbial communities to a gradient of CBZ concentrations for three days to evaluate early responses in community composition, ARG, and mobile genetic element (MGE) dynamics. Community structure remained largely unchanged across environmentally relevant CBZ concentrations. Most ARGs showed no consistent concentration-dependent response. However, a subset of clinically relevant ARGs increased in relative abundance in a dose-dependent manner. For the beta-lactam ARGs (blaCMY, blaOXA-48, blaCTX-M), and the trimethoprim ARG dfrA1, this increase was significantly correlated with IncP and IncW plasmid markers and the transposable element IS26, consistent with enhanced HGT-mediated dissemination. By contrast, the macrolide resistance gene ermF increased independently of the tested MGE markers, suggesting direct or host-specific selection or association with a non-tested MGE. The strongest shifts occurred at sub-inhibitory CBZ concentrations within the upper range of concentrations reported in wastewater-impacted environments. These findings show that CBZ exposure can indeed influence the dissemination of selected ARGs in complex microbial communities without major effects on overall community composition, highlighting the potential for non-antibiotic pharmaceuticals to shape early resistome responses to pollutants in the environment.