Feed additives increase soil risk from antibiotic resistance genes via distinct horizontal gene transfer pathways

Non-antibiotic components of feed additives can enter farmland soils via livestock manure and accumulate persistently in agroecosystems, presenting potential environmental risks. We established soil microcosms, integrated metagenomes with viromes, and applied a contig-based horizontal gene transfer (HGT)-resolution pipeline to partition vector-level contributions, to assess how saccharin, copper, and their co-contamination affect soil gene flow and health risk. Results indicate divergent vector responses under additive stress: phage-host associations increased under saccharin (82 pairs vs. control 29 pairs), whereas copper strengthened plasmid-host associations. With saccharin, phage nucleotide diversity rose while synonymous nucleotide diversity declined, consistent with stronger purifying selection atop enhanced mutation supply, whereas copper increased lysogeny. Saccharin significantly elevated HGT frequency (∼50% increase), expanded donor-recipient phylogenetic span (class-level P < 0.05), and raised the phage-mediated share (∼100% increase). Copper primarily modestly increased the plasmid-mediated contribution (Cu 2.7%, HS 1.9%). Two-factor analyses revealed a significant antagonistic interaction between saccharin and copper, reducing overall HGT across taxonomic ranks under co-exposure. Although total ARG abundance did not change significantly, the health-risk index increased under saccharin, driven by enhanced ARG-MGE co-occurrence. Under co-contamination, auxiliary metabolic genes were enriched, suggesting phage-conferred metabolic empowerment that mitigates stress, partly explaining the antagonism. Altogether, our findings reveal that feed additives reshape vector-specific gene mobility and ARG risk, and they underpin a three-tiered risk-assessment framework that progresses from mere abundance to network-structured mobility and finally to mobility drivers incorporating phylogenetic transfer distance, offering a more mechanistic basis for soil-health management.