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 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.

Automated summary

The newly developed ARESdb AMR panel allows for comprehensive and sensitive detection of antimicrobial resistance markers. Compared to metagenomics sequencing, the capture-based workflow has higher sensitivity and lower sequencing depth requirements, with a limit of detection for specific pathogens in synovial fluid and urine samples of 1000 CFU/mL. It also enables culture-free detection of bacterial taxa and AMR markers with an average sensitivity of 99% in septic patient samples.