Antibiotic resistance genes in river biofilms: a metagenomic approach toward the identification of sources and candidate hosts

Treated wastewater is a major pathway by which antibiotic resistance genes (ARG) enter aquatic ecosystems. However, knowledge gaps remain concerning the dissemination of specific ARG and their association with bacterial hosts. Here, we employed shotgun metagenomics to track ARG and taxonomic markers in river biofilms along a gradient of fecal pollution depicted by crAssphage signatures. We found strong evidence for an impact of wastewater effluents on both community composition and resistomes. In the light of such simultaneity, we employed a model comparison technique to identify ARG-host relationships from nonassembled metagenomic DNA. Hereby, a major cause of spurious associations otherwise encountered in correlation-based ARG-host analyses was suppressed. For several families of ARG, namely those conferring resistance to beta-lactams, particular bacterial orders were identified as candidate hosts. The found associations of (bla)FOX and (cpf)A with Aeromonadales or (bla)PER with Chromatiales support the outcome of independent evolutionary analyses and thus confirm the potential of the methodology. For other ARG families including (bla)IMP or (tet), clusters of bacterial orders were identified which potentially harbor a major proportion of host species. For yet other ARG, like, for example, ant or erm, no particular host candidates were identifiable, indicating their spread across various taxonomic groups.