缺乏有关腹泻的细菌和病毒病原体的综合数据,以及应用非培养方法检查废水中抗生素耐药性的研究。这项研究旨在同时量化抗生素抗性基因(ARGs),1类整合子-整合酶(int1),腹泻的细菌和病毒病原体,16SrRNA,等指标采用高通量定量PCR(HT-qPCR)系统。在2022年8月至2023年7月之间,每月三次对来自日本废水处理厂的36个抓取废水样品进行离心,然后是核酸提取,逆转录,和HT-qPCR。包括14个目标,和HT-qPCR在BiomarkX9™系统(标准BioTools)上进行。对于所有qPCR测定,R2≥0.978,效率从90.5%到117.7%,表现出高性能。在36个样本中,20例(56%)诺如病毒基因组II(NoV-GII)阳性,而沙门氏菌属。空肠弯曲菌24例(67%),大肠弯曲菌13例(36%),平均浓度范围为3.2±0.8至4.7±0.3log10拷贝/L。冬季和春季NoV-GII检出率和浓度较高。腹泻的病原体与急性胃肠炎病例无关,除了NoV-GII,这表明需要有关特定细菌感染的数据来验证基于细菌废水的流行病学(WBE)。所有样本的sul1、int1和blaCTX-M检测呈阳性,不管季节。较少探索的blaNDM-1在所有季节显示广泛的患病率(>83%)和一致的丰度,范围为4.3±1.0至4.9±0.2log10拷贝/L。sul1是主要的ARG,而16SrRNA的绝对丰度,int1和blaCTX-M随季节变化。在秋季和春季,int1与blaCTX-M显著相关,尽管它与blaNDM-1没有相关性,但质疑int1作为整体抗性决定因素的唯一指标的适用性。这项研究表明,HT-qPCR系统对WBE至关重要。
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.