Antibiotic-degrading bacteria shape resistome dynamics and horizontal gene transfer potential in soils with contrasting properties

Soils act as both reservoirs and filters of antimicrobial resistance genes (ARGs); however, the ecological and genetic traits of antibiotic-degrading bacteria (ADB) and their interactions with nondegrading bacteria (NADB) across soil types remain poorly understood. In particular, the role of ADB in ARG dynamics and their potential contribution to horizontal gene transfer (HGT) are still underexplored. Here, we applied ¹³C-DNA stable isotope probing (DNA-SIP) combined with metagenomic sequencing to resolve active ADB from NADB in two contrasting soils: Ultisol and Mollisol. ADB harbored significantly more abundant and diverse chromosomal ARGs - especially multidrug and tetracycline resistance genes - often co-localized with mobile genetic elements (MGEs) and degradation genes, suggesting robust and regulated resistance strategies. In contrast, NADB relied more on plasmid-borne ARGs, reflecting flexible but potentially transient adaptation. Soil properties shaped both resistome composition and host taxa. Mollisol enriched enzymatic degraders such as <i>Lysobacter</i> and <i>Nocardioides</i>, while Ultisol favored stress-tolerant <i>Burkholderia</i>, which carried up to 34 ARGs and exhibited membrane-associated resistance. Notably, 89 ARGs or MGEs were found co-localized with degradation genes on assembled contigs, highlighting a strong potential for HGT. In addition, 24 high-potential ARG hosts were identified, including <i>Ralstonia pickettii</i> and <i>Saccharomonospora viridis</i>. These findings reveal that antibiotic degradation is embedded within complex, soil-specific resistome networks. This work enhances our understanding of ARG ecology and supports targeted mitigation strategies based on soil microbiome characteristics.