Comprehensive characterization of gastrointestinal microbiota dysbiosis in patients with refractory Helicobacter pylori infection.

While repeated Helicobacter pylori eradication attempts are increasingly common due to antimicrobial resistance, the compounding effects of multiple antibiotic courses on gastrointestinal microbiota remain unquantified. We enrolled 32 treatment-naïve patients and 84 patients with refractory H. pylori infections, defined as failure of at least two prior eradication attempts. Antibiotic susceptibility testing was conducted for refractory H. pylori isolates to assess resistance patterns. Gastric mucosal biopsies and fecal samples were obtained for 16S rRNA gene sequencing. Antimicrobial susceptibility testing revealed resistance rates of 96.55% to metronidazole, 77.59% to clarithromycin, 56.90% to levofloxacin, 5.17% to rifampicin, 1.72% to tetracycline, and 0% to amoxicillin and furazolidone. The rates of dual drug and multidrug resistance were 27.59% and 55.17%, respectively. Refractory patients exhibited significant gastric microbiota restructuring, marked by reduced alpha diversity. The refractory group had higher concentrations of pathogens like Pseudomonas and Burkholderia and lower relative abundances of beneficial bacteria, including Bifidobacterium, Blautia, and Roseburia. Levofloxacin-resistant individuals exhibited an amplified abundance of pathogenic Veillonella. The gastric microbial diversity was higher in clarithromycin-resistant patients, with the enrichment of Micromonospora. Compared with the single-resistant group, pathogens, such as Veillonella and Peptostreptococcus, were increased in the multidrug-resistant group. We also observed perturbations of gut microbiota in refractory H. pylori-infected patients, with the predominance of Streptococcus and depletion of Bacteroides. Our findings establish that refractory H. pylori infection induces a disruption of gastrointestinal microbiota, highlighting the importance of optimizing first-line regimens to prevent retreatment cycles and the potential benefit of microbiota-modulating strategies in refractory H. pylori management.IMPORTANCEPrevious research has demonstrated that H. pylori eradication therapies can transiently alter gut microbiota. However, the long-term consequences of repeated antibiotic treatments in refractory infections remain unexplored. In this study, we link failed eradication attempts to persistent gastrointestinal dysbiosis, characterized by increased Pseudomonas and antibiotic-resistant Veillonella, alongside depletion of beneficial bacteria. In addition, we also demonstrate distinct gastric microbiota structure in patients with different antibiotic resistance patterns. Our findings showed distinct microbial dysbiosis after repeated eradication attempts, highlighting the need to explore microbiota-modulating approaches in future clinical trials.