Nontarget screening uncovers the overlooked impact of antibiotic transformation products on riverine resistomes.

Antibiotics, their transformation products (TPs), and antibiotic resistance genes (ARGs) pose a major environmental threat, yet the influence of TPs on ARGs remains poorly understood. This study combined target, suspect and nontarget analysis via high-resolution mass spectrometry with metagenomic analysis to systematically profile the occurrence of antibiotics, TPs, and ARGs in an urban river. A total of 32 parent antibiotics and 49 TPs were identified, with concentrations ranging from 0.002193 ng/L and not detected145 ng/L, respectively. TPs accounted for a substantial portion of total antibiotic loads (41.3 % in spring and 31.9 % in summer). Metagenomic sequencing revealed 1599 ARG subtypes conferring resistance to 28 classes of antibiotics. TPs can drive antimicrobial resistance directly by exerting selective pressure through their residual antibacterial activity, and indirectly through reverting to parent compounds, thereby reintroducing bioactive antibiotics into the environment. Therefore, the inclusion of TPs improved ARG-antibiotic correlations. Multivariate analyses revealed that TPs exerted comparable or greater influence on ARG profiles than parent antibiotics. Specific TPs, particularly β-lactams, macrolides, and quinolones, significantly affected the abundance of multiple ARGs, underscoring their substantial and non-negligible impact. This study highlights the previously underestimated role of antibiotic TPs in shaping ARG profiles and calls for an urgent update of risk assessment frameworks to incorporate TPs for comprehensive environmental and public health evaluation.