Expanding resistance databases: integrating efflux pump genes for accurate genomic prediction in Helicobacter pylori

Helicobacter pylori remain a major global cause of gastric disease, and increasing antibiotic resistance threatens treatment outcomes. Despite wider use of whole‑genome sequencing for resistance prediction, genomic results do not always match phenotypic susceptibility patterns. This study used an exploratory genomic approach to characterize known antimicrobial resistance genes (ARGs) and the broader diversity of efflux pump genes in Iranian clinical isolates. Gastric biopsies from gastritis patients were cultured, and antibiotic susceptibility was determined following CLSI guidelines. Genomic DNA from 30 isolates was sequenced using Illumina NovaSeq technology, assembled in CLC Genomics Workbench, and screened for ARGs using ABRicate, ResFinder, RGI/CARD, and AMRFinderPlus. EP genes were identified using Prokka and BLAST, and allelic diversity was assessed by sequence comparison. Phenotypic testing showed that 49% of isolates were resistant to at least one antibiotic, and 27.3% were multidrug resistant. Genomes carried on average 86% of the 46 EP genes. Several EP genes ( czcB , hefH , ybhR , ybhS ) were present in nearly all isolates, while ceuE appeared in 24 isolates and was duplicated in four genomes. Thirty-five EP genes formed the core genome, including yeeO , which was highly enriched in Iranian strains (93.3% vs. 0.1% globally). Eleven EP genes were accessory, and 43% of EP alleles were novel. This study reveals extensive efflux pump gene diversity in Iranian H. pylori isolates and suggests a possible role in unexplained resistance. Including efflux‑related loci in genomic analyses may improve resistance prediction and clarify non‑traditional resistance mechanisms.