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

Sadhana Shrestha; Bikash Malla; Eiji Haramoto

doi:10.1016/j.envres.2024.119156

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

Environ Res. 2024 May 15:255:119156. doi: 10.1016/j.envres.2024.119156. Online ahead of print.

Authors

Sadhana Shrestha¹, Bikash Malla², Eiji Haramoto³

Affiliations

¹ Interdisciplinary Center for River Basin Environment, University of Yamanashi, Yamanashi, 400-8511, Japan. Electronic address: gosadhana@gmail.com.
² Interdisciplinary Center for River Basin Environment, University of Yamanashi, Yamanashi, 400-8511, Japan. Electronic address: mallabikash@hotmail.com.
³ Interdisciplinary Center for River Basin Environment, University of Yamanashi, Yamanashi, 400-8511, Japan. Electronic address: eharamoto@yamanashi.ac.jp.

PMID: 38759773
DOI: 10.1016/j.envres.2024.119156

Abstract

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 bla_CTX-M, irrespective of season. The less explored bla_NDM-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 bla_CTX-M varied seasonally. int1 was significantly correlated with bla_CTX-M in autumn and spring, whereas it showed no correlation with bla_NDM-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.

Keywords: Antibiotic resistance gene; Class 1 integron-integrase; High-throughput qPCR; Pathogenic bacterial gene; Pathogenic virus; Wastewater-based epidemiology.