As antibiotic-resistant bacteria in water environments are increasingly recognized as contributors to the global antibiotic resistance crisis, the need for a monitoring subject that captures antibiotic resistance trends on a global scale increases. The World Health Organization TriCycle Protocol proposes the use of cefotaxime-resistant
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Semrau, Jeremy D (Ed.)more » « less
ABSTRACT Escherichia coli is a promising subject for globally coordinated surveillance of antimicrobial resistance (AMR) in water environments due to its clinical relevance and widespread use as an indicator of fecal contamination. Cefotaxime-resistantE. coli was recently evaluated favorably for this purpose by the World Health Organization TriCycle Protocol, which specifies tryptone bile x-glucuronide (TBX) medium and incubation at 35°C. We assessed comparability with the U.S. Environmental Protection Agency-approved method forE. coli quantification, which uses membrane-thermotolerantE. coli (mTEC) agar and incubation at 44.5°C, in terms of recovery ofE. coli and cefotaxime-resistantE. coli from wastewater influent and surface waters. TotalE. coli concentrations in wastewater influent were 106–108CFU/100 mL, while cefotaxime-resistantE. coli were ~100-fold lower. TotalE. coli in surface waters were ~102CFU/100 mL, and cefotaxime-resistant isolates were near the limit of detection (0.4 CFU/100 mL). Total and putative cefotaxime-resistantE. coli concentrations did not differ significantly between media or by incubation method; however, colonies isolated on mTEC were more frequently confirmed to species (97.1%) compared to those from TBX (92.5%). Incubation in a water bath at 44.5°C significantly decreased non-specific background growth and improved confirmation frequency on both media (97.4%) compared to incubation at 35°C (92.3%). This study helps to advance globally coordinated AMR in water environments and suggests that the TriCycle Protocol is adaptable to other standard methods that may be required in different locales, while also offering a means to improve specificity by decreasing the frequency of false-positive identification of cefotaxime-resistantE. coli by modifying incubation conditions.IMPORTANCE Escherichia coli isolated on tryptone bile x-glucuronide agar. The U.S. Environmental Protection Agency (USEPA) criteria for safe recreational waters also useE. coli as an indicator but specify the use of mTEC agar at a higher incubation temperature (44.5°C vs 35°C). We assessed the comparability of these methods for isolating total and cefotaxime-resistantE. coli , finding overall good agreement and performance, but significantly higher specificity towardE. coli selection with the use of the USEPA incubation protocol and mTEC agar. This study is the first to directly compare these methods and provides evidence that the methods may be used interchangeably for global surveillance of antibiotic resistance in the environment.Free, publicly-accessible full text available May 21, 2025 -
Free, publicly-accessible full text available May 1, 2025
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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.
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Abstract Purpose of Review Mounting evidence indicates that habitats such as wastewater and environmental waters are pathways for the spread of antibiotic-resistant bacteria (ARB) and mobile antibiotic resistance genes (ARGs). We identified antibiotic-resistant members of the genera
Acinetobacter ,Aeromonas , andPseudomonas as key opportunistic pathogens that grow or persist in built (e.g., wastewater) or natural aquatic environments. Effective methods for monitoring these ARB in the environment are needed to understand their influence on dissemination of ARB and ARGs, but standard methods have not been developed. This systematic review considers peer-reviewed papers where the ARB above were cultured from wastewater or surface water, focusing on the accuracy of current methodologies.Recent Findings Recent studies suggest that many clinically important ARGs were originally acquired from environmental microorganisms.
Acinetobacter ,Aeromonas, andPseudomonas species are of interest because their ability to persist and grow in the environment provides opportunities to engage in horizontal gene transfer with other environmental bacteria. Pathogenic strains of these organisms resistant to multiple, clinically relevant drug classes have been identified as an urgent threat. However, culture methods for these bacteria were generally developed for clinical samples and are not well-vetted for environmental samples.Summary The search criteria yielded 60 peer-reviewed articles over the past 20 years, which reported a wide variety of methods for isolation, confirmation, and antibiotic resistance assays. Based on a systematic comparison of the reported methods, we suggest a path forward for standardizing methodologies for monitoring antibiotic resistant strains of these bacteria in water environments.
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Abstract 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.
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Temperature constrains the transmission of many pathogens. Interventions that target temperature-sensitive life stages, such as vector control measures that kill intermediate hosts, could shift the thermal optimum of transmission, thereby altering seasonal disease dynamics and rendering interventions less effective at certain times of the year and with global climate change. To test these hypotheses, we integrated an epidemiological model of schistosomiasis with empirically determined temperature-dependent traits of the human parasite
Schistosoma mansoni and its intermediate snail host (Biomphalaria spp.). We show that transmission risk peaks at 21.7 °C (T opt ), and simulated interventions targeting snails and free-living parasite larvae increasedT opt by up to 1.3 °C because intervention-related mortality overrode thermal constraints on transmission. ThisT opt shift suggests that snail control is more effective at lower temperatures, and global climate change will increase schistosomiasis risk in regions that move closer toT opt . Considering regional transmission phenologies and timing of interventions when local conditions approachT opt will maximize human health outcomes.
