Gastric microbiome analysis in Helicobacter pylori-negative gastric ulcer: a prospective multicenter pilot study

To characterize the gastric mucosa-associated microbiome (MAM) in H. pylori -negative gastric ulcers. Between August 2022 and January 2024, 38 H. pylori -negative participants were enrolled: control ( n = 11; NSAID use without ulcer), Case 1 ( n = 21; ≥ 1-month NSAID use with active ulcer), and Case 2 ( n = 6; idiopathic ulcer; no NSAID use with active ulcer). All participants discontinued antisecretory medications ≥ 4 weeks before sampling. Mucosal biopsies from the antrum, body, duodenal bulb, and second portion, and hypopharyngeal swabs were analysed using 16 S rRNA sequencing and QIIME 2. Clinical characteristics were generally comparable among groups. The gastric MAM showed significant differences in antral β-diversity in Case 2, although significant PERMDISP results indicated that differences in within-group dispersion may have contributed to this pattern. The age- and sex-adjusted ANCOM-BC2 analysis identified multiple taxa-level differences in Case 2; however, the specific taxa identified were not fully consistent across analytical approaches. Gastrotyping of the antral MAM suggested a two-cluster solution along a compositional gradient, characterized by relative predominance of Phocaeicola A (Cluster 1) or Streptococcus (Cluster 2), with ulcer patients (Cases 1 and 2) showing a significantly higher representation of Cluster 1 ( p = 0.044). In this pilot study, alterations in the antral gastric microbiome were associated with H. pylori - negative idiopathic gastric ulcers. Although exploratory, these findings support further investigation of the gastric microbiome in the pathophysiology of idiopathic gastric ulcer and require validation in larger independent cohorts.