Close interactions between prokaryotes and plasmids or viruses highlight a pivotal role of horizontal gene transfer in shaping antibiotic/metal(loid) resistome and their prokaryotic supercarriers in untreated hospital sewage.

Unveiling horizontal gene transfer (HGT) of antibiotic (ARGs) and metal(loid) resistance genes (MRGs) in hospital sewage is critical for surveilling antimicrobial resistance (AMR) mobility that poses huge threats to public health. Using metagenomic shotgun sequencing, we provided an integrate insight into AMR characters and the relevant HGT in untreated sewage from one of the world's largest comprehensive hospitals from Oct 2022 to Aug 2023. We uncovered higher richness and diversity of ARGs or MRGs than mobile genetic elements (MGEs), while MGEs exhibited the highest abundance, suggesting great HGT potentials. Higher number of ARG, MRG, and MGE subtypes and abundances of putative human pathogens were found in autumn-winter than in spring-summer. ARG- and MGE-carrying prokaryotes outcompeted non-carriers in abundances, and multi-ARG and MGE carriers outcompeted single ones. Resistome supercarriers occupying 25 % of prokaryotic abundance harbored higher functional diversity and more metabolic capacity than other prokaryotes, which could be related to more predicted HGT events. Notably, 30 %, 22 %, and 40 % of prokaryote-carrying ARGs, MRGs, and MGEs were associated with HGTs. Diversity variation of plasmids as a critical contributor to HGT was positively correlated with those of prokaryotes and ARGs or MRGs. Plasmids carrying high-risk ARGs (e.g., multidrug and tetracycline types) showed higher abundances than prokaryotes and viruses. Most bacterial taxa may undergo high levels of active viral replication (phylum-specific virus/host abundance ratios >12). Hundreds of virulent viruses could lyse abundant ARG or MRG supercarriers and hosts of multidrug, multi-metals, and As resistome, whilst one temperate virus infecting multiple Azonexus supercarriers may contribute the HGT of Hg resistome. We found the dominance of stochasticity in assembling of ARGs/MGEs rather than prokaryotes or viruses, which was likely owed to functional redundancy led by HGT. Overall, this study sheds lights on a pivotal role of HGT in driving microbial community and functionality, and provides a guidance for the optimization of the treatment strategies particularly on MGEs.