Phages and plasmids mediate antibiotic resistance gene transfer in urban airborne bacteria

Airborne antibiotic resistance genes (ARGs) pose emerging public health risks, particularly in urban settings, yet their dissemination mechanisms remain unclear. Here we cultured airborne bacteria from diverse urban environments and performed metagenomic sequencing to reconstruct 931 non-redundant metagenome-assembled genomes (MAGs), integrating horizontal gene transfer (HGT) detection, machine learning, and causal inference analyses. We identified hospitals, pharmaceutical factories, and railway stations as major sources of mobile ARGs. Both plasmids and phages actively mediate ARG transfer, promoting gene dissemination across broad phylogenetic distances. Machine learning revealed key phage functional modules related to structure, host attachment, lysis, DNA entry, and regulation that facilitate virus-mediated HGT, with synergistic interactions observed between plasmids and phages. These findings elucidate the dynamic resistome and mobility potential of metabolically active airborne bacteria, informing environmental surveillance and mitigation strategies to address airborne antimicrobial resistance within the One Health framework.