Source identification of antibiotic resistance genes in a peri-urban river using novel crAssphage marker genes and metagenomic signatures

Antimicrobial resistance is a growing public health concern, and environment is regarded as an important reservoir and dissemination route for antibiotic resistance genes (ARGs). To prevent and control ARG pollution, it is essential to correctly disentangle source-sink relationship of ARGs in the environment. However, accurately apportioning sources of ARGs is still a big challenge due to the complex interaction of multiple sources and contaminants in the environment with changing dynamics. In this study, we addressed this problem and focused on identifying the potential sources of ARGs in a peri-urban river by jointly utilizing two novel microbial source tracking methods. To attain the objective, sediment/water samples were collected from the peri-urban river and four ARG-associated ecotypes including effluents of sewage treatment plants (STPs), STP influent, chicken manures and pig manures. The high-throughput profilings of ARGs and microbial taxa in the river sediments and the four ecotypes were comprehensively characterized in combination of shotgun sequencing and metagenomic assembly analysis. CrAssphage, a recently-discovered DNA bacteriophage, was employed to track the impact of human fecal pollution on ARGs in the river sediments. Further, SourceTracker, a machine-learning classification tool, was used for quantifying the contributions of potential sources to ARGs in the river sediments based on the metagenomic signatures of ARGs and microbial taxa. In total, 888 ARG subtypes belonging to 29 ARG types were detected across all samples, including mcr-1 and a range of carbapenemases types. Statistical analyses suggested different ecotypes generally had distinct profiles of both ARGs and microbial taxa, while the ARG compositions were significantly correlated with the microbial community. Source tracking with crAssphage showed the presence of ARGs in the river sediments might be largely impacted by the extent of human fecal pollution, which was also confirmed by the analyses of SourceTracker that the discharge from STPs was the largest contributor of ARGs (81.6-92.1%) and microbes (49.3-68.1%) in the river sediments. Results of the study can help us to better understand the characterization of ARGs in the peri-urban ecosystem and to design effective prevention and control strategies for reducing ARG dissemination.