Zinc differentially modulates resistome responses to ciprofloxacin and sulfamethazine in activated sludge.

Antibiotics and metals frequently coexist in wastewater, but whether metals uniformly intensify antibiotic-driven resistome selection remains unclear. Here, activated sludge reactors were exposed to zinc (Zn), ciprofloxacin (CIP), sulfamethazine (SMZ), and their combinations across four stepwise exposure stages. Read-based, assembly-based, and genome-resolved metagenomics were integrated to characterize antibiotic resistance gene (ARG) abundance, composition, mobility-associated genetic contexts, and putative hosts. Antibiotics primarily drove resistome restructuring, whereas Zn alone had limited effects. CIP strongly enriched ARGs, while Zn co-exposure attenuated this response and caused late-stage declines in total ARG abundance and the relative abundance of ARG-hosting metagenome-assembled genomes (MAGs). In contrast, ARG abundance remained comparatively stable under SMZ but increased progressively under Zn+SMZ. Because antibiotic concentrations increased sequentially over time, these patterns represent stage-dependent responses rather than independent concentration effects. Antibiotic-related treatments showed higher contributions of ARGs on predicted plasmid-like contigs and more frequent ARG-mobile genetic element co-localization. Pseudomonadota dominated putative ARG hosts, and CIP showed broader ARG-host profiles. Approximately 67% of ARG-hosting MAGs carried virulence factor genes, indicating co-occurring resistance and virulence-associated genomic potential. These findings demonstrate that Zn altered activated sludge resistome in an antibiotic-dependent manner, highlighting that mixture effects cannot be inferred from single-contaminant responses alone.