Sensitivity and consistency of long- and short-read metagenomics and epicPCR for the detection of antibiotic resistance genes and their bacterial hosts in wastewater

Wastewater surveillance is a powerful tool to assess the risks associated with antibiotic resistance in communities. One challenge is selecting which analytical tool to deploy to measure risk indicators, such as antibiotic resistance genes (ARGs) and their respective bacterial hosts. Although metagenomics is frequently used for analyzing ARGs, few studies have compared the performance of long-read and short-read metagenomics in identifying which bacteria harbor ARGs in wastewater. Furthermore, for ARG host detection, untargeted metagenomics has not been compared to targeted methods such as epicPCR. Here, we 1) evaluated long-read and short-read metagenomics as well as epicPCR for detecting ARG hosts in wastewater, and 2) investigated the host range of ARGs across the wastewater treatment plant (WWTP) to evaluate host proliferation. Results highlighted long-read revealed a wider range of ARG hosts compared to short-read metagenomics. Nonetheless, the ARG host range detected by long-read metagenomics only represented a subset of the hosts detected by epicPCR. The ARG-host linkages across the influent and effluent of the WWTP were characterized. Results showed the ARG-host phylum linkages were relatively consistent across the WWTP, whereas new ARG-host species linkages appeared in the WWTP effluent. The ARG-host linkages of several clinically relevant species found in the effluent were identified. Environmental Implication Wastewater treatment plants are a major barrier to the environmental dissemination of antibiotic resistance genes (ARGs). In the environment, ARGs can persist, be transferred between microorganisms, and evolve through interactions with the host genomes. The bacterial host of an ARG is directly related to the risk it poses to human health. Therefore, identifying ARG hosts and how they shift across wastewater treatment processes is critical to assessing the risk of ARGs in wastewater effluents. This study compares three state-of-the-art approaches for identifying ARG hosts. This study shows the benefits and challenges of different methods and addresses the impact of method selection on the scope of environmental evaluation of ARG hosts.