A framework for standardized qPCR-targets and protocols for quantifying antibiotic resistance in surface water, recycled water and wastewater

dc.contributor.authorKeenum, Ishi M.en
dc.contributor.authorLiguori, Kristaen
dc.contributor.authorCalarco, Jeanetteen
dc.contributor.authorDavis, Benjamin C.en
dc.contributor.authorMilligan, Erinen
dc.contributor.authorHarwood, Valerie J.en
dc.contributor.authorPruden, Amyen
dc.description.abstractWater 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.en
dc.description.sponsorshipThis work was supported by the Water Research Foundation (WRF) Project 5052: Standardizing Methods with QA/QC Standards for Investigating the Occurrence and Removal of Antibiotic Resistant Bacteria/Antibiotic Resistance Genes (ARB/ARGs) in Surface Water, Wastewater, and Recycled Water. Support was also provided by National Science Foundation Awards OAC 2004751, OISE 1545756, NRT 2125798, and ECCS 2025151.en
dc.description.versionPublished versionen
dc.identifier.citationIshi Keenum, Krista Liguori, Jeanette Calarco, Benjamin C. Davis, Erin Milligan, Valerie J. Harwood & Amy Pruden (2022) A framework for standardized qPCR-targets and protocols for quantifying antibiotic resistance in surface water, recycled water and wastewater, Critical Reviews in Environmental Science and Technology, DOI: 10.1080/10643389.2021.2024739en
dc.publisherTaylor & Francisen
dc.rightsCreative Commons Attribution-NonCommercial 4.0 Internationalen
dc.titleA framework for standardized qPCR-targets and protocols for quantifying antibiotic resistance in surface water, recycled water and wastewateren
dc.title.serialCritical Reviews in Environmental Science and Technologyen
dc.typeArticle - Refereeden
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