Whole-Genome Sequencing and Antibiotic Resistance Profiling of Helicobacter pylori Isolates from a Tertiary Hospital in Southern Thailand

<b>Background</b>: <i>Helicobacter pylori</i> is associated with a wide range of gastroduodenal diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. Eradication efforts are challenged by increasing antimicrobial resistance rates, particularly in Southeast Asia. We sequenced the whole genomes of clinical <i>H. pylori</i> isolates from Southern Thailand to elucidate their resistance profiles, virulence determinants, and evolutionary relationships. <b>Methods</b>: Three clinical <i>H. pylori</i> isolates (004, 117, and 189) were subjected to whole-genome sequencing, phenotypic antimicrobial susceptibility testing, and comparative genomic analyses. <b>Results</b>: All strains exhibited high-level resistance to metronidazole. Additionally, <i>H. pylori</i> 117 was resistant to both amoxicillin and levofloxacin, classifying it as multidrug-resistant. Genomic analysis revealed mutations in <i>rdxA</i>, <i>frxA</i>, and <i>rpoB</i>, as well as in penicillin-binding protein genes (<i>pbp2</i> and <i>pbp3</i>), supporting the phenotypic findings. While all isolates harboured clarithromycin resistance mutations (A2142G and A2143G in the 23S rRNA gene), they were phenotypically susceptible, highlighting a potential discordance that requires further investigation. Virulence gene profiling identified 115-118 conserved genes per strain, including <i>cagA</i>, <i>vacA</i>, <i>oipA</i>, <i>babA</i>, and flagellar, urease, and lipopolysaccharide biosynthesis genes. Phylogenetic analysis using core-genome single-nucleotide polymorphisms demonstrated that these strains formed a distinct Southern Thai monophyletic clade, suggesting localised clonal expansion driven by regional selective pressures. <b>Conclusions</b>: Region-specific surveillance strategies and treatment guidelines are urgently needed in Thailand. The combination of high-risk virulence genes and rising antimicrobial resistance in <i>H. pylori</i> strains necessitates tailored therapeutic approaches, the integration of genomic surveillance into clinical diagnostics, and expanded studies linking genotype to clinical outcomes in diverse populations.