Helicobacter pylori whole genome enrichment and sequencing for resistome and virulome characterization in gastric biopsies

Antimicrobial resistance in Helicobacter pylori (H. pylori) increasingly compromises empirical eradication therapy, yet current comprehensive susceptibility testing is limited by slow culture methods and molecular assays targeting only a limited set of resistance loci. We evaluated a hybrid-capture whole genome sequencing (WGS) workflow for direct analysis of native and formalin-fixed paraffin-embedded (FFPE) gastric biopsies, including challenging samples with low bacterial burden and highly fragmented DNA. Using the Qiagen QIAseq xHYB H. pylori capture probe panel, we sequenced 72 FFPE samples previously genotyped by PCR and Sanger sequencing and 32 native gastric biopsies processed for culture-based phenotyping. The next-generation sequencing (NGS) assay enabled genome-wide interrogation of resistance determinants to clarithromycin, levofloxacin, metronidazole, amoxicillin, tetracycline, and rifamycin in a single test and produced interpretable results in 92.9% of the specimens. Genotypic data for clarithromycin and levofloxacin resistance showed high concordance with legacy molecular assays and phenotypic susceptibility. Notably, WGS also detected low-frequency allelic variants that were missed by traditional methods. Genome-wide data also supported multilocus sequence typing (MLST) and profiling of virulence factors directly from biopsy material, enabling culture-independent resistance profiling and comprehensive strain characterization for surveillance and individualized management. This study lays the groundwork for integrating WGS-based H. pylori resistance testing into precision diagnostics in pathology. The ability to profile resistance at the genome level can significantly improve antimicrobial stewardship, guide targeted therapy, and support more precise clinical decision-making.