Comparison of three sequencing methods for identifying and quantifying antibiotic. resistance genes (ARGs) in sewage.

Abstract Background Globally, antimicrobial resistance (AMR) poses a critical threat, requiring robust surveillance methodologies to tackle the growing challenge of drug-resistant microbes. AMR is a huge challenge in India due to high disease burden, lack of etiology-based diagnostic tests and over the counter availability of antibiotics and inadequate treatment of wastewaters are important drivers of AMR in India. There is lack of effective surveillance platforms that monitor health-associated infections. This include developing an understanding on background levels of AMR in the environment and comparison of AMR monitoring methods. Objectives This study evaluated the performance of three AMR sequencing methods, Illumina AmpliSeq AMR panel, QIAseq xHYB AMR panel and shotgun sequencing method for the detection of antimicrobial resistance genes (ARGs) in urban sewage. Our goal is to provide insights into the application and robustness of each sequencing method. Methods We compare the prevalence, diversity, and composition of ARGs across sequencing method and by sample type (inlet vs outlet) in four sewage treatment plants (STP). Results Regardless of the sequencing method used the dominant ARGs remained consistent, and their differential analysis showed consistent trends in detection of epidemiologically relevant ARGs. The cost-effectiveness analysis revealed comparable per-sample costs, with amplicon-based sequencing offering specificity for targeted genes, and shotgun sequencing uses a whole-genome sequencing approach that provides high-resolution taxonomic information for the characterisation of pathogens. This methodology can only detect ARGs that have been annotated in the reference database (e.g., CARD). Therefore, some novel types of ARGs present in the samples may be missed since the analysis is based on a similarity search. Differential abundance analysis to understand change in abundance in dominant ARGs from inlet to outlet of STP showed consistent trends across methods. However, with caution raised regarding potential artifacts introduced by enrichment steps in QIAseq xHYB AMR panel. Conclusion The choice of panel used would be governed by the context of the study. Nonetheless, our exploratory study shows that the data gathered using different sequencing pipelines helps in quantifying the ARG burden in the environment. This information is crucial in understanding the spatio-temporal distribution of ARGs in different environment and could help in developing PCR-based approaches for targeted surveillance.