Heavy metals and microbiome are negligible drivers than mobile genetic elements in determining particle-attached and free-living resistomes in the Yellow River

Suspended particles in water can shelter both microorganisms and contaminants. However, the emerging pol-lutants antibiotic resistance genes (ARGs) in free-living (FL) or particle-attached (PA) bacteria in aquatic envi-ronments are less explored. In this study, we compared the free-living and particle-attached ARGs during four seasons in the Yellow River using high-throughput quantitative PCR techniques and 16S rRNA gene sequencing. Our results demonstrated that both the free-living water and particles were dominated by tetracycline and beta-lactamase resistance genes. The PA-ARGs had a higher absolute abundance than FL-ARGs in the Yellow River, regardless of the season. Both PA-ARGs and FL-ARGs had the highest absolute abundance and diversity during winter. Mobile genetic elements (MGEs) were the dominant driver for both size-fractionated ARGs. However, the microbiome had less influence on PA-ARG profiles than the FL-ARG profiles, while the effects of the heavy metals on ARGs were negligible. The community assembly of both FL-ARG and PA-ARG can be explained by neutral processes. Several opportunistic pathogens (e.g., Escherichia coli) associated with human health exhibited a higher relative abundance in the particles than during a free-living lifestyle. Parts of these pathogens were po-tential ARG hosts. As such, it is important to monitor the ARGs and opportunistic pathogens from size-fractionated bacteria and develop targeted strategies to manage ARG dissemination and opportunistic patho-gens to ensure public health.