Gastric microbiome analysis in Helicobacter pylori-negative NSAID-induced ulcer: A prospective multicenter pilot study

Objective: to characterize the gastric mucosa-associated microbiome (MAM) in H. pylori-negative gastric ulcers. Methods: 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 16S rRNA sequencing and QIIME 2. Results: Clinical characteristics were comparable among groups, with no significant differences in demographics, H. pylori eradication status, comorbidities, or NSAID use. The gastric MAM differed significantly in patients with Case 2, featuring distinct ß-diversity at antrum with homogeneous microbial composition compared with the other groups. Taxonomic analyses consistently demonstrated enrichment of phylum Bacillota A 368345, Lachnospiraceae and genera including Phocaeicola A, Anaerobutyricum, and Bacteroides H 857956, with depletion of Streptococcus in Case 2, findings that remained robust after adjustment for age, sex, and atrophy. 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). Conclusions: In this pilot study, distinct gastric microbiome alterations are observed in H. pylori-negative idiopathic gastric ulcers at antrum, suggesting potential roles in pathogenesis and opportunities for microbiome-targeted interventions.