Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture SequencingTools 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, Posch, Andreas E. and Weinberger, Johannes (2021) Culture-Free Detection of Antibiotic Resistance Markers from Native Patient Samples by Hybridization Capture Sequencing. Microorganisms. Preview Culture-Free Detection of Antibiotic Resistance Markers from - Ferreira et al..pdf - Published Version Download (941kB) | Preview
Official URL: https://doi.org/ 10.3390/microorganisms9081672
AbstractThe 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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