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Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture Sequencing

The increasing incidence of antimicrobial resistance (AMR) is a major global challenge. Routine techniques for molecular AMR marker detection are largely based on low-plex PCR and detect dozens to hundreds of AMR markers. To allow for comprehensive and sensitive profiling of AMR markers, we develope...

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Autores principales: Ferreira, Ines, Lepuschitz, Sarah, Beisken, Stephan, Fiume, Giuseppe, Mrazek, Katharina, Frank, Bernhard J. H., Huber, Silke, Knoll, Miriam A., von Haeseler, Arndt, Materna, Arne, Hofstaetter, Jochen G., Posch, Andreas E., Weinberger, Johannes
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8398375/
https://www.ncbi.nlm.nih.gov/pubmed/34442751
http://dx.doi.org/10.3390/microorganisms9081672
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author Ferreira, Ines
Lepuschitz, Sarah
Beisken, Stephan
Fiume, Giuseppe
Mrazek, Katharina
Frank, Bernhard J. H.
Huber, Silke
Knoll, Miriam A.
von Haeseler, Arndt
Materna, Arne
Hofstaetter, Jochen G.
Posch, Andreas E.
Weinberger, Johannes
author_facet Ferreira, Ines
Lepuschitz, Sarah
Beisken, Stephan
Fiume, Giuseppe
Mrazek, Katharina
Frank, Bernhard J. H.
Huber, Silke
Knoll, Miriam A.
von Haeseler, Arndt
Materna, Arne
Hofstaetter, Jochen G.
Posch, Andreas E.
Weinberger, Johannes
author_sort Ferreira, Ines
collection PubMed
description The increasing incidence of antimicrobial resistance (AMR) is a major global challenge. Routine techniques for molecular AMR marker detection are largely based on low-plex PCR and detect dozens to hundreds of AMR markers. To allow for comprehensive and sensitive profiling of AMR markers, we developed a capture-based next generation sequencing (NGS) workflow featuring a novel AMR marker panel based on the curated AMR database ARESdb. Our primary objective was to compare the sensitivity of target enrichment-based AMR marker detection to metagenomics sequencing. Therefore, we determined the limit of detection (LOD) in synovial fluid and urine samples across four key pathogens. We further demonstrated proof-of-concept for AMR marker profiling from septic samples using a selection of urine samples with confirmed monoinfection. The results showed that the capture-based workflow is more sensitive and requires lower sequencing depth compared with metagenomics sequencing, allowing for comprehensive AMR marker detection with an LOD of 1000 CFU/mL. Combining the ARESdb AMR panel with 16S rRNA gene sequencing allowed for the culture-free detection of bacterial taxa and AMR markers directly from septic patient samples at an average sensitivity of 99%. Summarizing, the newly developed ARESdb AMR panel may serve as a valuable tool for comprehensive and sensitive AMR marker detection.
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spelling pubmed-83983752021-08-29 Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture Sequencing Ferreira, Ines Lepuschitz, Sarah Beisken, Stephan Fiume, Giuseppe Mrazek, Katharina Frank, Bernhard J. H. Huber, Silke Knoll, Miriam A. von Haeseler, Arndt Materna, Arne Hofstaetter, Jochen G. Posch, Andreas E. Weinberger, Johannes Microorganisms Article The increasing incidence of antimicrobial resistance (AMR) is a major global challenge. Routine techniques for molecular AMR marker detection are largely based on low-plex PCR and detect dozens to hundreds of AMR markers. To allow for comprehensive and sensitive profiling of AMR markers, we developed a capture-based next generation sequencing (NGS) workflow featuring a novel AMR marker panel based on the curated AMR database ARESdb. Our primary objective was to compare the sensitivity of target enrichment-based AMR marker detection to metagenomics sequencing. Therefore, we determined the limit of detection (LOD) in synovial fluid and urine samples across four key pathogens. We further demonstrated proof-of-concept for AMR marker profiling from septic samples using a selection of urine samples with confirmed monoinfection. The results showed that the capture-based workflow is more sensitive and requires lower sequencing depth compared with metagenomics sequencing, allowing for comprehensive AMR marker detection with an LOD of 1000 CFU/mL. Combining the ARESdb AMR panel with 16S rRNA gene sequencing allowed for the culture-free detection of bacterial taxa and AMR markers directly from septic patient samples at an average sensitivity of 99%. Summarizing, the newly developed ARESdb AMR panel may serve as a valuable tool for comprehensive and sensitive AMR marker detection. MDPI 2021-08-05 /pmc/articles/PMC8398375/ /pubmed/34442751 http://dx.doi.org/10.3390/microorganisms9081672 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ferreira, Ines
Lepuschitz, Sarah
Beisken, Stephan
Fiume, Giuseppe
Mrazek, Katharina
Frank, Bernhard J. H.
Huber, Silke
Knoll, Miriam A.
von Haeseler, Arndt
Materna, Arne
Hofstaetter, Jochen G.
Posch, Andreas E.
Weinberger, Johannes
Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture Sequencing
title Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture Sequencing
title_full Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture Sequencing
title_fullStr Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture Sequencing
title_full_unstemmed Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture Sequencing
title_short Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture Sequencing
title_sort culture-free detection of antibiotic resistance markers from native patient samples by hybridization capture sequencing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8398375/
https://www.ncbi.nlm.nih.gov/pubmed/34442751
http://dx.doi.org/10.3390/microorganisms9081672
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