From gene detection to resistome ecology: targeted hybrid capture in AMR surveillance

Antimicrobial resistance (AMR) surveillance requires approaches capable of capturing the full resistome beyond culture-based and targeted molecular diagnostics. Here, we compared a commercial real-time PCR assay for carbapenemase detection with a targeted hybrid capture next-generation sequencing (xHYB) approach for comprehensive antimicrobial resistance gene (ARG) profiling in rectal swab samples. The xHYB panel showed high concordance with PCR for bla KPC and bla NDM , with overall agreement varying by gene target. Sequencing demonstrated higher analytical sensitivity, detecting additional ARGs not identified by PCR, particularly among OXA -like variants. Sequencing read counts were inversely correlated with PCR cycle threshold values, supporting a semi-quantitative relationship between methods, while no association was observed with total DNA concentration. Beyond single-gene detection, resistome analysis revealed significant compositional differences between carbapenemase-positive and -negative samples. Positive samples displayed a broader multidrug resistance profile, including β-lactamases, aminoglycoside-modifying enzymes, tetracycline, sulfonamide, macrolide, trimethoprim resistance genes, and multidrug efflux systems. Comparative analyses across resistance databases showed variability at gene and peptide-marker levels, whereas gene-family-level profiles were more consistent. Overall, targeted xHYB sequencing enables sensitive detection of clinically relevant ARGs and reveals structured resistome patterns associated with carbapenemase carriage, supporting its integration into AMR surveillance strategies. The xHYB panel showed high concordance with PCR for bla KPC and bla NDM , with overall agreement varying by gene target. Sequencing demonstrated higher analytical sensitivity, detecting additional ARGs not identified by PCR, particularly among OXA -like variants. Sequencing read counts were inversely correlated with PCR cycle threshold values, supporting a semi-quantitative relationship between methods, while no association was observed with total DNA concentration. IMPORTANCE Antimicrobial resistance is a growing global health threat driven by the spread of resistance genes among bacteria, particularly in the human gut, which acts as a major reservoir. This study focuses on intestinal antimicrobial resistance genes from human rectal swabs, highlighting that the presence of carbapenemases was associated not only with single resistance genes but with a broader and more complex multidrug resistance profile. This indicates that conventional diagnostic methods may underestimate the true resistance potential of microbial communities. By providing a more comprehensive view of resistance gene diversity, this approach can improve surveillance and help identify high-risk resistance patterns earlier. These findings support the use of sequencing-based methods to complement existing diagnostics and strengthen monitoring of antimicrobial resistance in clinical and public health settings.