Abstract
Antimicrobial resistance can be an indicator of anthropogenic contamination in surface waters and is a potential public health threat. Methodological standardization for characterization of antimicrobial resistance in the environment is lacking. Quantitative PCR (qPCR) is used for rapid assessment of antibiotic resistance genes (ARGs) from environmental sources, including surface water. Here we describe the validation and application of a qPCR assay for 47 bacterial gene targets intended for surface water samples. The qPCR assay displayed excellent sensitivity (97.66%) and specificity (98.71%) for detecting ARGs when compared to whole genome sequencing of bacterial isolates. The qPCR assay was able to detect up to 6/8 (75.0%) of ARGs spiked into sterile water at varying concentrations and four sample ultrafiltration volumes. Nineteen different ARGs were detected across six samples sites at three national parks in Alaska using ultrafiltered surface water samples. The number of unique ARGs detected was higher at sites within parks with greater visitation. The relative abundance of ARGs/16S from Exit Creek in Kenai Fjords National Park, downstream from a visitor center was greater than all other sampled sites. We have demonstrated a robust qPCR assay for monitoring ARGs in surface waters, including those that are minimally human impacted.
Data availability
Quantitative PCR data presented in this paper are publicly available at [https://doi.org/10.5066/P14G9MJW]60 . The sequencing data generated during the current study are available in NCBI (BioProject ID: PRJNA1214890).
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Acknowledgements
We thank Ora Russ and the U.S. Fish and Wildlife Service Conservation Genetics for the use of their equipment and space. We appreciate constructive reviews provided by Ora Russ and John Pearce. We thank Eleni Petrou for helpful conversations about the illustration of data. This research used resources of the Core Science Analytics and Synthesis Advanced Research Computing program at the U.S. Geological Survey.
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All authors contributed to study design. LCS, CA, CMM, MEM, and AMR conducted field work. LCS, HW, JB, and CMM conducted lab work. LCS, CA, and HW conducted data analysis. LCS, CA, and CMM generated figures. LCS, CA, and HW contributed to manuscript writing. All authors reviewed the manuscript.
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Scott, L.C., Ahlstrom, C.A., Woksepp, H. et al. Validation and application of a standardized quantitative PCR assay for the assessment of antimicrobial resistance genes in surface water. Sci Rep (2026). https://doi.org/10.1038/s41598-026-35635-x
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DOI: https://doi.org/10.1038/s41598-026-35635-x
