Overcoming challenges in metagenomic AMR surveillance with nanopore sequencing: a case study on fluoroquinolone resistance
IntroductionAntimicrobial resistance is an alarming public health problem, and comprehensive surveillance across environments is required to reduce its impact. Phenotypic testing and whole-genome sequencing of isolates are efficient, but culture-free approaches like metagenomic sequencing potentiall...
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Frontiers Media S.A.
2025-07-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1614301/full |
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| author | Bram Bloemen Bram Bloemen Mathieu Gand Moniek Ringenier Bert Bogaerts Kevin Vanneste Kathleen Marchal Kathleen Marchal Nancy H. C. Roosens Jeroen Dewulf Filip Boyen Sigrid C. J. De Keersmaecker |
| author_facet | Bram Bloemen Bram Bloemen Mathieu Gand Moniek Ringenier Bert Bogaerts Kevin Vanneste Kathleen Marchal Kathleen Marchal Nancy H. C. Roosens Jeroen Dewulf Filip Boyen Sigrid C. J. De Keersmaecker |
| author_sort | Bram Bloemen |
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| description | IntroductionAntimicrobial resistance is an alarming public health problem, and comprehensive surveillance across environments is required to reduce its impact. Phenotypic testing and whole-genome sequencing of isolates are efficient, but culture-free approaches like metagenomic sequencing potentially allow for broader investigation of resistance gene occurrence, evolution and spread. However, technical challenges such as difficulties in associating antimicrobial resistance genes with their bacterial hosts and the collapse of strain-level variation during metagenome assembly, hinder its implementation.MethodsTo illustrate how these challenges can be overcome, we applied Oxford Nanopore Technologies long-read metagenomic sequencing and novel bioinformatic methods to a case study focused on fluoroquinolone resistance in chicken fecal samples.ResultsWe demonstrate plasmid-host linking based on detecting common DNA methylation signatures. Additionally, we use new bioinformatic approaches for strain haplotyping, enabling phylogenomic comparison and uncovering fluoroquinolone resistance determining point mutations in metagenomic datasets.DiscussionWe leverage long-read sequencing, including DNA methylation profiling and strain-level haplotyping, to identify antimicrobial resistance gene hosts, link plasmids to their bacterial carriers, and detect resistance-associated point mutations. Although some limitations remain, our work demonstrates how these improvements in metagenomic sequencing can enhance antimicrobial resistance surveillance. |
| format | Article |
| id | doaj-art-2b65ccfb95a24a5daa28f88dad48d2c2 |
| institution | DOAJ |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj-art-2b65ccfb95a24a5daa28f88dad48d2c22025-08-20T02:46:00ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-07-011610.3389/fmicb.2025.16143011614301Overcoming challenges in metagenomic AMR surveillance with nanopore sequencing: a case study on fluoroquinolone resistanceBram Bloemen0Bram Bloemen1Mathieu Gand2Moniek Ringenier3Bert Bogaerts4Kevin Vanneste5Kathleen Marchal6Kathleen Marchal7Nancy H. C. Roosens8Jeroen Dewulf9Filip Boyen10Sigrid C. J. De Keersmaecker11Transversal Activities in Applied Genomics, Sciensano, Elsene, BelgiumDepartment of Plant Biotechnology and Bioinformatics, Ghent University, Zwijnaarde, BelgiumTransversal Activities in Applied Genomics, Sciensano, Elsene, BelgiumDepartment of Internal Medicine, Reproduction and population medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, BelgiumTransversal Activities in Applied Genomics, Sciensano, Elsene, BelgiumTransversal Activities in Applied Genomics, Sciensano, Elsene, BelgiumDepartment of Plant Biotechnology and Bioinformatics, Ghent University, Zwijnaarde, BelgiumDepartment of Information Technology, Ghent University, Zwijnaarde, BelgiumTransversal Activities in Applied Genomics, Sciensano, Elsene, BelgiumDepartment of Internal Medicine, Reproduction and population medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, BelgiumDepartment of Pathobiology, Pharmacology and Zoological Medicine, Faculty of Veterinary Medicine, Ghent University, Merelbeke, BelgiumTransversal Activities in Applied Genomics, Sciensano, Elsene, BelgiumIntroductionAntimicrobial resistance is an alarming public health problem, and comprehensive surveillance across environments is required to reduce its impact. Phenotypic testing and whole-genome sequencing of isolates are efficient, but culture-free approaches like metagenomic sequencing potentially allow for broader investigation of resistance gene occurrence, evolution and spread. However, technical challenges such as difficulties in associating antimicrobial resistance genes with their bacterial hosts and the collapse of strain-level variation during metagenome assembly, hinder its implementation.MethodsTo illustrate how these challenges can be overcome, we applied Oxford Nanopore Technologies long-read metagenomic sequencing and novel bioinformatic methods to a case study focused on fluoroquinolone resistance in chicken fecal samples.ResultsWe demonstrate plasmid-host linking based on detecting common DNA methylation signatures. Additionally, we use new bioinformatic approaches for strain haplotyping, enabling phylogenomic comparison and uncovering fluoroquinolone resistance determining point mutations in metagenomic datasets.DiscussionWe leverage long-read sequencing, including DNA methylation profiling and strain-level haplotyping, to identify antimicrobial resistance gene hosts, link plasmids to their bacterial carriers, and detect resistance-associated point mutations. Although some limitations remain, our work demonstrates how these improvements in metagenomic sequencing can enhance antimicrobial resistance surveillance.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1614301/fullmetagenomic sequencingantimicrobial resistance (AMR)nanopore sequencingDNA methylationstrain-resolved metagenomicsplasmid host prediction |
| spellingShingle | Bram Bloemen Bram Bloemen Mathieu Gand Moniek Ringenier Bert Bogaerts Kevin Vanneste Kathleen Marchal Kathleen Marchal Nancy H. C. Roosens Jeroen Dewulf Filip Boyen Sigrid C. J. De Keersmaecker Overcoming challenges in metagenomic AMR surveillance with nanopore sequencing: a case study on fluoroquinolone resistance Frontiers in Microbiology metagenomic sequencing antimicrobial resistance (AMR) nanopore sequencing DNA methylation strain-resolved metagenomics plasmid host prediction |
| title | Overcoming challenges in metagenomic AMR surveillance with nanopore sequencing: a case study on fluoroquinolone resistance |
| title_full | Overcoming challenges in metagenomic AMR surveillance with nanopore sequencing: a case study on fluoroquinolone resistance |
| title_fullStr | Overcoming challenges in metagenomic AMR surveillance with nanopore sequencing: a case study on fluoroquinolone resistance |
| title_full_unstemmed | Overcoming challenges in metagenomic AMR surveillance with nanopore sequencing: a case study on fluoroquinolone resistance |
| title_short | Overcoming challenges in metagenomic AMR surveillance with nanopore sequencing: a case study on fluoroquinolone resistance |
| title_sort | overcoming challenges in metagenomic amr surveillance with nanopore sequencing a case study on fluoroquinolone resistance |
| topic | metagenomic sequencing antimicrobial resistance (AMR) nanopore sequencing DNA methylation strain-resolved metagenomics plasmid host prediction |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1614301/full |
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