Abstract

Water environments are increasingly recognized as a conduit for the spread of antibiotic resistance, but there is need to standardize antibiotic resistance monitoring protocols to ensure comparability across studies. Quantitative polymerase chain reaction (qPCR) is attractive as a sensitive means of quantifying antibiotic resistance genes (ARGs) and has been applied broadly over the past two decades to various water matrices. QPCR avoids challenges and biases associated with culture-based methods, providing a reproducible and highly sensitive measure of ARGs carried across a bacterial community. However, there are numerous quality assurance and other aspects of protocols that need to be addressed to ensure that measurements are representative and comparable across studies. Here we conducted a critical review to identify gene targets that are most commonly measured by qPCR to quantify antibiotic resistance in surface water, recycled water, and wastewater and to assess corresponding protocols. Identified targets monitored in water samples included sul1, tetA, and intI1, given their abundance and tendency to correlate with anthropogenic inputs, and vanA and blaCTX-M, as more rarely detected, but highly clinically-relevant targets. We identified 117 peer-reviewed studies meeting search criteria for application of these assays to water matrices of interest and systematically assessed the corresponding protocols, including sample collection and concentration, DNA extraction, primer/probe specificity, amplification conditions, amplicon length, PCR inhibition evaluation, and limit of detection/quantification. Gene copy numbers reported across studies were further compared by assay and water matrix. Based on this comprehensive evaluation, we recommend assays, standardized workflows, and reporting for the five target genes.

摘要

水环境越来越被认为是抗生素耐药性传播的渠道，但需要标准化抗生素耐药性监测协议，以确保研究之间的可比性。定量聚合酶链反应 (qPCR) 作为一种量化抗生素抗性基因 (ARG) 的敏感方法很有吸引力，并且在过去的二十年中已广泛应用于各种水基质。QPCR 避免了与基于培养的方法相关的挑战和偏见，提供了一种可重复且高度敏感的 ARG 测量方法，该测量方法在细菌群落中携带。然而，有许多质量保证和协议的其他方面需要解决，以确保测量在研究中具有代表性和可比性。在这里，我们进行了一项重要的审查，以确定最常通过 qPCR 测量的基因靶标，以量化地表水、循环水和废水中的抗生素耐药性，并评估相应的协议。包括在水样中监测的已识别目标sul 1 、tet A 和int I1，考虑到它们的丰富性和与人为输入相关的趋势，以及van A 和blaCTX-M，很少检测到，但与临床高度相关。我们确定了 117 项符合搜索标准的同行评审研究，将这些测定应用于感兴趣的水基质，并系统地评估了相应的方案，包括样品收集和浓缩、DNA 提取、引物/探针特异性、扩增条件、扩增子长度、PCR 抑制评估，和检测/量化的限制。通过测定和水基质进一步比较了跨研究报告的基因拷贝数。基于这一综合评估，我们推荐五个靶基因的检测、标准化工作流程和报告。