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Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae
Version 2 2023-06-07, 08:29
Version 1 2023-06-07, 06:46
journal contribution
posted on 2023-06-07, 08:29 authored by Leon Peto, Nicola J Fawcett, Derrick W Crook, Tim E A Peto, Martin LlewelynMartin Llewelyn, A Sarah WalkerMetagenomic sequencing of fecal DNA can usefully characterise an individual’s intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer feces were spiked with carbapenemase-producing Enterobacteriaceae and incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a fecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goal
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Publication status
- Published
File Version
- Published version
Journal
PLoS MedicineISSN
1549-1277Publisher
Public Library of ScienceExternal DOI
Issue
11Volume
14Page range
1-14Department affiliated with
- Global Health and Infection Publications
Full text available
- No
Peer reviewed?
- Yes
Legacy Posted Date
2019-09-24First Open Access (FOA) Date
2019-11-26First Compliant Deposit (FCD) Date
2019-09-24Usage metrics
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