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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Veröffentlicht in:Microorganisms (Basel) 2021-08, Vol.9 (8), p.1672, Article 1672
Hauptverfasser: 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
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Sprache:eng
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Zusammenfassung: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.
ISSN:2076-2607
2076-2607
DOI:10.3390/microorganisms9081672