Resolving the Genomic Context of Clinically Relevant Antibiotic Resistance Genes in Wastewater with Ligation-Mediated PCR
- 1University of Washington
Abstract
Antimicrobial resistance (AMR) is a pressing global public health challenge. AMR is driven in part by the spread of antibiotic resistance genes (ARGs) through bacterial communities via mobile genetic elements. Influent wastewater is a promising sample type for monitoring AMR because it pools biological inputs shed by individuals across a population. However, untargeted sequencing approaches, such as metagenomic sequencing, often miss low-abundance targets such as clinically important ARGs. In this work, we develop a ligation-mediated PCR (LM-PCR) enrichment strategy that can directionally capture the genomic context surrounding an ARG using long-read sequencing. We applied this method to study the natural genomic context diversity of four clinically relevant ARGs (blaCTX-M, blaKPC, and blaOXA-48-like, and qnrS) across 13 wastewater treatment plants in Washington state, each sampled at two timepoints. Across all timepoints, LM-PCR identified distinct genomic context cluster families associated with each ARG, including seven for blaKPC, 11 for blaCTX-M, 24 for qnrS, and one for blaOXA-48-like. Notably, 11 of the 24 qnrS containing clusters were putatively novel, with no matches to existing sequences in public databases. Genomic contexts associated with blaCTX-M and blaKPC were comparatively conserved across clusters, whereas qnrS was associated with a more diverse set of genetic sequences. Together, these results demonstrate that LM-PCR can resolve low-abundance, ARG-associated genomic variation in complex wastewater samples and provide a scalable framework for tracking the dissemination of clinically relevant AMR determinants.
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BibTeX
@misc{obrien2026resolving,
title = {{Resolving the Genomic Context of Clinically Relevant Antibiotic Resistance Genes in Wastewater with Ligation-Mediated PCR}},
author = {M. E. O'Brien and B. Ahern and P. Brase and S. Kim and C. E. M. McCormack and D. Garcia and E. Keim and E. Dahl and M. Feck and K. Kauber and J. A. Marchand and R. S. Kantor and A. J. Pickering and B. McArdle and E. R. Fuhrmeister},
howpublished = {medRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.09.26361541},
url = {https://www.medrxiv.org/content/10.64898/2026.09.09.26361541v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Resolving the Genomic Context of Clinically Relevant Antibiotic Resistance Genes in Wastewater with Ligation-Mediated PCR
AU - M. E. O'Brien
AU - B. Ahern
AU - P. Brase
AU - S. Kim
AU - C. E. M. McCormack
AU - D. Garcia
AU - E. Keim
AU - E. Dahl
AU - M. Feck
AU - K. Kauber
AU - J. A. Marchand
AU - R. S. Kantor
AU - A. J. Pickering
AU - B. McArdle
AU - E. R. Fuhrmeister
PY - 2026
DA - 2026/09/09
PB - medRxiv
DO - 10.64898/2026.09.09.26361541
UR - https://www.medrxiv.org/content/10.64898/2026.09.09.26361541v1
AB - Antimicrobial resistance (AMR) is a pressing global public health challenge. AMR is driven in part by the spread of antibiotic resistance genes (ARGs) through bacterial communities via mobile genetic elements. Influent wastewater is a promising sample type for monitoring AMR because it pools biological inputs shed by individuals across a population. However, untargeted sequencing approaches, such as metagenomic sequencing, often miss low-abundance targets such as clinically important ARGs. In this work, we develop a ligation-mediated PCR (LM-PCR) enrichment strategy that can directionally capture the genomic context surrounding an ARG using long-read sequencing. We applied this method to study the natural genomic context diversity of four clinically relevant ARGs (blaCTX-M, blaKPC, and blaOXA-48-like, and qnrS) across 13 wastewater treatment plants in Washington state, each sampled at two timepoints. Across all timepoints, LM-PCR identified distinct genomic context cluster families associated with each ARG, including seven for blaKPC, 11 for blaCTX-M, 24 for qnrS, and one for blaOXA-48-like. Notably, 11 of the 24 qnrS containing clusters were putatively novel, with no matches to existing sequences in public databases. Genomic contexts associated with blaCTX-M and blaKPC were comparatively conserved across clusters, whereas qnrS was associated with a more diverse set of genetic sequences. Together, these results demonstrate that LM-PCR can resolve low-abundance, ARG-associated genomic variation in complex wastewater samples and provide a scalable framework for tracking the dissemination of clinically relevant AMR determinants.
ER -