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
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.
more » « less- PAR ID:
- 10538528
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- Antibiotics
- Volume:
- 12
- Issue:
- 8
- ISSN:
- 2079-6382
- Page Range / eLocation ID:
- 1252
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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. -
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/antibiotics12081252
