Multiplexed Amplicon Sequencing Reveals High Sequence Diversity of Antibiotic Resistance Genes in Quebec Sewers

The United Nations Environment Assembly (UNEA-3) have recognised the importance of the environment in the development, spread and transmission of antimicrobial resistance (AMR) to humans and animals. Such recognition calls for wider surveillance of antimicrobial resistance genes (ARG) in wastewater and other environmental reservoirs. For ARG surveillance to be valuable to regulators, it must enable source tracking and risk assessment. Adequate surveillance also requires the processing of a large number of samples at a relatively low cost, and a low detection limit to allow quantification of the riskiest ARGs. However, current methods for tracking ARGs have various limitations. The current study presents a multiplexed targeted amplicon sequencing approach for the detection of sequence variants of ARGs in environmental samples. To demonstrate the application of this technique, wastewater samples collected from the inlet to 16 treatment plants located along a 440-km transect of the St-Lawrence river in the province of Quebec (Canada) were analysed. Among the ARGs examined, between 3 and 45 nucleic acid sequence variants were detected demonstrating the high sequence diversity that occurs within genes originating from a single sample type and the information that is missed using traditional techniques. Using the PLSDB and Comprehensive Antibiotic Resistance Database (CARD), the risk of ARG sequence variants was inferred based upon their reported mobility and detection in pathogens. Results suggest that sequence variants within a single ARG class present different risks to public health. In the future, targeted amplicon sequencing could be a valuable tool in environmental studies for both risk assessment purposes and in AMR source tracking.