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Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae

Metagenomic 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...

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Autores principales: Peto, Leon, Fawcett, Nicola J., Crook, Derrick W., Peto, Tim E. A., Llewelyn, Martin J., Walker, A. Sarah
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839868/
https://www.ncbi.nlm.nih.gov/pubmed/31703058
http://dx.doi.org/10.1371/journal.pone.0222831
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author Peto, Leon
Fawcett, Nicola J.
Crook, Derrick W.
Peto, Tim E. A.
Llewelyn, Martin J.
Walker, A. Sarah
author_facet Peto, Leon
Fawcett, Nicola J.
Crook, Derrick W.
Peto, Tim E. A.
Llewelyn, Martin J.
Walker, A. Sarah
author_sort Peto, Leon
collection PubMed
description Metagenomic 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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spelling pubmed-68398682019-11-15 Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae Peto, Leon Fawcett, Nicola J. Crook, Derrick W. Peto, Tim E. A. Llewelyn, Martin J. Walker, A. Sarah PLoS One Research Article Metagenomic 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. Public Library of Science 2019-11-08 /pmc/articles/PMC6839868/ /pubmed/31703058 http://dx.doi.org/10.1371/journal.pone.0222831 Text en © 2019 Peto et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Peto, Leon
Fawcett, Nicola J.
Crook, Derrick W.
Peto, Tim E. A.
Llewelyn, Martin J.
Walker, A. Sarah
Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae
title Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae
title_full Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae
title_fullStr Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae
title_full_unstemmed Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae
title_short Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae
title_sort selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant enterobacteriaceae
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839868/
https://www.ncbi.nlm.nih.gov/pubmed/31703058
http://dx.doi.org/10.1371/journal.pone.0222831
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