High-throughput microfluidic quantitative PCR system for the simultaneous detection of antibiotic resistance genes and bacterial and viral pathogens in wastewater

• Published in: Environmental research 2024-08, Vol.255, p.119156-119156, Article 119156
• Main Authors: Shrestha, Sadhana, Malla, Bikash, Haramoto, Eiji
• Format: Article
• Language: English
• Subjects: Antibiotic resistance gene; Bacteria - drug effects; Bacteria - genetics; Bacteria - isolation & purification; Class 1 integron-integrase; Drug Resistance, Bacterial - genetics; Drug Resistance, Microbial - genetics; Genes, Bacterial; High-throughput qPCR; Japan; Microfluidics - methods; Pathogenic bacterial gene; Pathogenic virus; Real-Time Polymerase Chain Reaction - methods; RNA, Ribosomal, 16S - analysis; RNA, Ribosomal, 16S - genetics; Viruses - drug effects; Viruses - genetics; Viruses - isolation & purification; Wastewater - microbiology; Wastewater - virology; Wastewater-based epidemiology
• ISSN: 0013-9351; 1096-0953
• DOI: 10.1016/j.envres.2024.119156

Comprehensive data on bacterial and viral pathogens of diarrhea and studies applying culture-independent methods for examining antibiotic resistance in wastewater are lacking. This study aimed to simultaneously quantify antibiotic resistance genes (ARGs), class 1 integron-integrase (int1), bacterial and viral pathogens of diarrhea, 16S rRNA, and other indicators using a high-throughput quantitative PCR (HT-qPCR) system. Thirty-six grab wastewater samples from a wastewater treatment plant in Japan, collected three times a month between August 2022 and July 2023, were centrifuged, followed by nucleic acid extraction, reverse transcription, and HT-qPCR. Fourteen targets were included, and HT-qPCR was performed on the Biomark X9™ System (Standard BioTools). For all qPCR assays, R2 was ≥0.978 and the efficiencies ranged from 90.5% to 117.7%, exhibiting high performance. Of the 36 samples, 20 (56%) were positive for Norovirus genogroup II (NoV-GII), whereas Salmonella spp. and Campylobacter jejuni were detected in 24 (67%) and Campylobacter coli in 13 (36%) samples, with mean concentrations ranging from 3.2 ± 0.8 to 4.7 ± 0.3 log10 copies/L. NoV-GII detection ratios and concentrations were higher in winter and spring. None of the pathogens of diarrhea correlated with acute gastroenteritis cases, except for NoV-GII, suggesting the need for data on specific bacterial infections to validate bacterial wastewater-based epidemiology (WBE). All samples tested positive for sul1, int1, and blaCTX-M, irrespective of season. The less explored blaNDM-1 showed a wide prevalence (>83%) and consistent abundance ranging from 4.3 ± 1.0 to 4.9 ± 0.2 log10 copies/L in all seasons. sul1 was the predominant ARG, whereas absolute abundances of 16S rRNA, int1, and blaCTX-M varied seasonally. int1 was significantly correlated with blaCTX-M in autumn and spring, whereas it showed no correlation with blaNDM-1, questioning the applicability of int1 as a sole indicator of overall resistance determinants. This study exhibited that the HT-qPCR system is pivotal for WBE. [Display omitted] •HT-qPCR system successfully analyzed pathogens and ARGs in a single run.•NoV-GII concentration was linked with AGE cases but not bacterial pathogens.•Specific bacterial infection data could be useful for validation of bacterial WBE.•sul1, blaCTX-M, an ESBL, and the less explored blaNDM-1 were widely prevalent.•int1 correlated with blaCTX-M in some seasons and did not correlate with blaNDM-1.