Source-oriented risks of heavy metals and their effects on resistance genes in natural biofilms

Heavy metal (HM) introduction from various land-use patterns can be a major source of metal resistance genes (MRGs) entering river environments. This influx can trigger the occurrence of other resistomes, such as antibiotic resistance genes (ARGs), by improving co-resistant conjugative transfer. Biofilms, which form at water-solid interfaces, could serve as potential hotspots for HMs and resistance genes. However, the enrichment of HMs from various sources within biofilms and their effect on resistomes remain undocumented. This study aims to investigate the physicochemical properties of biofilm samples collected from the Heihui River, a tributary of the Lancang River, and to analyze the concentrations of nine HMs (As, Cd, Co, Cr, Cu, Ni, Pb, V, and Zn) within these biofilms. The 16S rRNA gene and metagenomic high-throughput sequencing techniques were integrated to uncover the association between HM accumulation levels in biofilms and ecological and health risks, considering the presence of two resistance genes. Natural sources (Co, Cr), industrial (As, Cu, V), agricultural (Cd, Ni), and transportation activities (Pb, Zn) markedly contributed to HM presence within biofilms, with industrial activities posing higher noncarcinogenic and carcinogenic risks than other sources. The network-correlation analyses revealed higher levels of ARG-MRG coexistence in biofilms, with the ecological and health risk index of HMs in biofilms closely associated with the abundance of both resistance genes. Furthermore, the biofilm As concentration markedly affected the abundance and expression of ARGs and MRGs, with elevated As levels within biofilms significantly and positively influencing all four functional categories of MRGs. Water pH also indirectly impacted these functional types by modulating the ionic form of HMs within the biofilm matrix. Our findings underscore the significance of integrating biofilms into environmental management practices and standards for assessing environmental quality.