
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.09.12.612745
preprint
1
Article
Context-Seq: CRISPR-Cas9 Targeted Nanopore Sequencing for Transmission Dynamics of Antimicrobial Resistance
Fuhrmeister Erica R. http://orcid.org/0000-0001-5264-7533

Kim Sooyeol
Mairal Shruteek A.
McCormack Caroline
Chieng Benard
Swarthout Jenna M.
Paulos Abigail Harvey
Njenga Sammy M.
Pickering Amy J. http://orcid.org/0000-0001-6193-2221

12 9 2024
2024.09.12.612745https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
http://biorxiv.org/lookup/doi/10.1101/2024.09.12.612745
nihpp-2024.09.12.612745.pdf
Abstract

Antimicrobial resistance (AMR) aligns with a One Health framework in that resistant bacteria and antibiotic resistance genes (ARGs) can be transmitted between humans, animals, and the environment. However, there is a critical need to more precisely understand how and to what extent AMR is exchanged between animals and humans. Metagenomic sequencing has low detection for rare targets such as ARGs, while whole genome sequencing of isolates is burdensome and misses exchange between uncultured bacterial species. We developed a novel, targeted sequencing assay using CRISPR-Cas9 to selectively sequence ARGs and their genomic context with long-read sequencing. Using this method, termed Context-Seq, we investigated overlapping AMR elements containing the ARGs bla CTX-M and bla TEM between adults, children, poultry, and dogs in animal-owning households in Nairobi, Kenya. We identified 22 genetically distinct clusters (> 80%ID over ≥ 3000 bp) containing bla TEM and one cluster containing bla CTX-M that were shared within and between households. Half of the clusters were shared between humans and animals, while the other half were shared only between animals (poultry-poultry, dog-dog, and dog-poultry). We identified potentially pathogenic hosts of ARGs including Escherichia coli, Klebsiella pneumonia , and Haemophilus influenzae across sample types. Context-Seq complements conventional methods to obtain an additional view of bacterial and mammalian hosts in the proliferation of AMR.
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