National-scale antimicrobial resistance surveillance in wastewater: A comparative analysis of HT qPCR and metagenomic approaches

• Published in: Water research (Oxford) 2024-09, Vol.262, p.121989, Article 121989
• Main Authors: Knight, Margaret E., Webster, Gordon, Perry, William B., Baldwin, Amy, Rushton, Laura, Pass, Daniel A., Cross, Gareth, Durance, Isabelle, Muziasari, Windi, Kille, Peter, Farkas, Kata, Weightman, Andrew J., Jones, Davey L.
• Format: Article
• Language: English
• Subjects: ARG; Bacteria - drug effects; Bacteria - genetics; Drug Resistance, Bacterial - genetics; Environmental Monitoring - methods; Environmental reservoir; Metagenomics - methods; One Health approach; Real-Time Polymerase Chain Reaction; Wastewater - microbiology; Wastewater-based epidemiology; WBE strategy
• ISSN: 0043-1354; 1879-2448; 1879-2448
• DOI: 10.1016/j.watres.2024.121989

•HT qPCR detected all 73 targeted ARGs, while metagenomics detected 491 ARGs.•HT qPCR was more sensitive to low abundance genes, detecting all target ARGs.•Both methods enabled the spatiotemporal separation of hospital and WWTP resistomes.•Metagenomics provided contextual data making it more suitable for risk assessment.•HT qPCR permitted more sensitive quantification of clinically relevant AMR genes. Wastewater serves as an important reservoir of antimicrobial resistance (AMR), and its surveillance can provide insights into population-level trends in AMR to inform public health policy. This study compared two common high-throughput screening approaches, namely (i) high-throughput quantitative PCR (HT qPCR), targeting 73 antimicrobial resistance genes, and (ii) metagenomic sequencing. Weekly composite samples of wastewater influent were taken from 47 wastewater treatment plants (WWTPs) across Wales, as part of a national AMR surveillance programme, alongside 4 weeks of daily wastewater effluent samples from a large municipal hospital. Metagenomic analysis provided more comprehensive resistome coverage, detecting 545 genes compared to the targeted 73 genes by HT qPCR. It further provided contextual information critical to risk assessment (i.e. potential bacterial hosts). In contrast, HT qPCR exhibited higher sensitivity, quantifying all targeted genes including those of clinical relevance present at low abundance. When limited to the HT qPCR target genes, both methods were able to reflect the spatiotemporal dynamics of the complete metagenomic resistome, distinguishing that of the hospital and the WWTPs. Both approaches revealed correlations between resistome compositional shifts and environmental variables like ammonium wastewater concentration, though differed in their interpretation of some potential influencing factors. Overall, metagenomics provides more comprehensive resistome profiling, while qPCR permits sensitive quantification of genes significant to clinical resistance. We highlight the importance of selecting appropriate methodologies aligned to surveillance aims to guide the development of effective wastewater-based AMR monitoring programmes. [Display omitted]