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Context-Seq: CRISPR-Cas9 Targeted Nanopore Sequencing for Transmission Dynamics of Antimicrobial Resistance
Erica R Fuhrmeister1,2, Sooyeol Kim2, Shruteek A Mairal3
1Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Seattle, Washington, USA.
Abstract:
Precisely understanding how and to what extent antimicrobial resistance (AMR) is exchanged between animals and humans is needed to inform control strategies. Metagenomic sequencing has low detection for rare targets such as antibiotic resistance genes, while whole genome sequencing of isolates misses exchange between uncultured bacterial species. We introduce Context-Seq, CRISPR-Cas9 targeted sequencing of ARGs and their genomic context with long-reads. Using Context-Seq, we investigate genetically similar AMR elements containing the ARGs bla CTX-M and bla TEM between adults, children, poultry, and dogs in Nairobi, Kenya. We identify genetically distinct clusters containing bla TEM and bla CTX-M that are shared between animals and humans within and between households. We also uncover potentially pathogenic hosts of ARGs including Escherichia coli, Klebsiella pneumonia, and Haemophilus influenzae in this study context. Context-Seq complements conventional methods to obtain an additional view of bacterial and mammalian hosts in the proliferation of AMR.
Insights
Antimicrobial resistance (AMR) spreads between people and animals. A new method, Context-Seq, tracked antibiotic resistance genes (ARGs) like blaCTX-M and blaTEM, revealing shared AMR in households.
Area of Science:
- Microbiology
- Genomics
- One Health
Background:
- Antimicrobial resistance (AMR) is a significant public health threat, necessitating understanding of transmission pathways within the One Health framework.
- Current sequencing methods have limitations in detecting rare antibiotic resistance genes (ARGs) and tracking their genomic context or exchange between uncultured species.
Purpose of the Study:
- To develop and validate a novel targeted sequencing assay, Context-Seq, for precise detection and characterization of ARGs and their surrounding genetic elements.
- To investigate the exchange of specific AMR elements, blaCTX-M and blaTEM, between humans, poultry, and dogs in animal-owning households in Nairobi, Kenya.
Main Methods:
- Developed Context-Seq, a CRISPR-Cas9-based targeted sequencing assay utilizing long-read technology for selective amplification and sequencing of ARGs and their genomic context.
- Applied Context-Seq to samples from human and animal populations within animal-owning households to identify and analyze shared AMR clusters.
Main Results:
- Identified 22 distinct genetic clusters of blaTEM and one cluster of blaCTX-M shared within and between households.
- Found that half of the identified AMR clusters were transmitted between humans and animals, while the other half were exclusively shared among animals.
- Detected potentially pathogenic bacterial hosts, including Escherichia coli and Klebsiella pneumoniae, carrying ARGs across various sample types.
Conclusions:
- Context-Seq provides a powerful complementary approach to conventional methods for studying AMR proliferation and host dynamics.
- The findings highlight significant inter-species transmission of AMR within households, emphasizing the interconnectedness of human, animal, and environmental health.
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