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Awareness of the need for surveillance of antimicrobial resistance (AMR) in water environments is growing, but there is uncertainty regarding appropriate monitoring targets. Adapting culture-based fecal indicator monitoring to include antibiotics in the media provides a potentially low-tech and accessible option, while quantitative polymerase chain reaction (qPCR) targeting key genes of interest provides a broad, quantitative measure across the microbial community. The purpose of this study was to compare findings obtained from the culture of cefotaxime-resistant (cefR) Escherichia coli with two qPCR methods for quantification of antibiotic resistance genes across wastewater, recycled water, and surface waters. The culture method was a modification of US EPA Method 1603 for E. coli, in which cefotaxime is included in the medium to capture cefR strains, while qPCR methods quantified sul1 and intI1. A common standard operating procedure for each target was applied to samples collected by six water utilities across the United States and processed by two laboratories. The methods performed consistently, and all three measures reflected the same overarching trends across water types. The qPCR detection of sul1 yielded the widest dynamic range of measurement as an AMR indicator (7-log versus 3.5-log for cefR E. coli), while intI1 was the most frequently detected target (99% versus 96.5% and 50.8% for sul1 and cefR E. coli, respectively). All methods produced comparable measurements between labs (p < 0.05, Kruskal–Wallis). Further study is needed to consider how relevant each measure is to capturing hot spots for the evolution and dissemination of AMR in the environment and as indicators of AMR-associated human health risk. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aims: Beach water quality is regulated by faecal indicator bacteria levels, sand is not, despite known human health risk from exposure to beach sand. We compared the performance of three methods to extract bacterial DNA from beach sand as a step toward a standard method. Methods and results: The analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus direct extraction using PowerSoil™ or PowerMax Soil™ kits. The slurry method had the lowest limit of detection at 20–80 gene copies g−1, recovered significantly more DNA, and the only method that detected Enterococcus by qPCR in all samples; therefore, the only method used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the microbial source tracking marker HF183 from wastewater spiked marine and freshwater beach sand was 100.8% and 64.1%, respectively, but varied, indicating that the mixing protocol needs improvement. Conclusions: Among the three methods, the slurry method had the best analytical sensitivity and produced extracts that were useful for culture or molecular analysis. Significance and impact of study: Standardization of methods for extraction of bacterial DNA from sand facilitates comparisons among studies, and ultimately contributes to the safety of recreational beaches. [ABSTRACT FROM AUTHOR]