Probiotics modulate gastrointestinal microbiota after Helicobacter pylori eradication: A multicenter randomized double-blind placebo-controlled trial

Abstract

Background: Helicobacter pylori (H. pylori) eradication has been reported to cause short-term disruption of gut microbiota. It is acknowledged that probiotics supplementation mitigates side effects induced by H. pylori eradication, yet its role on alleviating dysbiosis of microbiota is obscure.

Objectives: To evaluate the impact of probiotics on gastrointestinal microbiota after eradication therapy.

Methods: This was a multicenter, double-blinded, randomized trial done at seven centers in China. A total of 276 treatment-naïve H. pylori-positive patients were randomly assigned to receive 14-day bismuth-containing quadruple therapy (esomeprazole, bismuth, amoxicillin, furazolidone) combined with probiotics (Bifidobacterium Tetragenous viable Bacteria Tablets) (n=140) or placebo (n=136) for 28 days. Saliva, gastric mucosa and fecal samples were collected before and after therapy for 16S rRNA gene sequencing.

Results: The incidence of gastrointestinal adverse events was lower in probiotics group compared to placebo group (23.6% vs 37.7%, p=0.016), while there was no significant difference in eradication rate. We found dramatic perturbations of gut microbiota immediately following eradication, with the predominance of Proteobacteria in replacement of commensal Firmicutes and Bacteroidetes, and gradually restored after two weeks. The reduction of gut Bacteroidetes caused by eradication drugs was neutralized with probiotics supplementation. The gastric microbiota was completely reconstituted with H. pylori depleted and other taxa flourished. Of note, patients treated with probiotics showed smaller fluctuations of gastric microbiota compared to those with placebo. We also observed changes of saliva microbiota after H. pylori eradication, illustrated by the overgrowth of Neisseria and depletion of Streptococcus. The expansion of some pathogenic genera, including Porphyromonas, Leptotrichia, in the mouth was suppressed by probiotics.

Conclusion: This study not only demonstrated the beneficial effect of probiotics implementation on side events during H. pylori eradication but also provided a comprehensive profile of microbiome alterations along gastrointestinal tract that modulated by probiotics.

Keywords: 16S rRNA gene sequence; gastric microbiota; gut microbiota; helicobacter pylori eradication; probiotics; saliva microbiota.

Figure 1

Flow diagram showing the study design. BQT, bismuth quadruple therapy; ITT, intention-to-treat; PP, per protocol; D, day; ¹³C-UBT, ¹³C-Urea Breath Test.

Figure 2

Comparison of diversity and composition of gut microbiota after eradication therapy. (A, B) Alpha diversity, representing the richness of microbiota, was significantly reduced immediately following eradication treatment (T2) and then gradually returned to baseline from T3 to T5. (C) Principal coordinate analysis (PCoA) based on Bray-Curtis distances revealed the overall microbiota composition differed between T2 and other time points. (D) Relative proportions of the top 10 bacterial phyla. (E) Relative proportions of the top 20 bacterial genera. A, probiotics group; B, placebo group; T1, pretreatment; T2, cessation of quadruple therapy; T3, 2 weeks after quadruple therapy; T4, 8 weeks after quadruple therapy; T5, 24 weeks after quadruple therapy; *, p<0.05; **, p<0.01, ***, p<0.001.

Figure 3

Dynamic alterations of genus-level gut microbiota after different periods of H. pylori eradication. 14 most abundant genera that differed between T1 and T2 were identified, and their alterations from T2 to T5 were calculated based on their abundance relative to T1. Value that lower than zero represents reduced relative abundance compared to baseline, whereas higher than zero represents increased relative abundance. *, p<0.05; **, p<0.01; ***, p<0.001; star without a line, group compared to T1; star with a line, comparison between (A, B). (A) probiotics group; (B) placebo group; T1, pretreatment; T2, cessation of quadruple therapy; T3, 2 weeks after quadruple therapy; T4, 8 weeks after quadruple therapy; T5, 24 weeks after quadruple therapy.

Figure 4

Co-occurrence network reveals the transformation of gut microbial pattern and their interconnection. (A) Heatmap shows the relative abundance of genera in each module. An obvious transformation of module_9 to module_1 was observed at T2, which was reverted from T3 to T5. (B) Significant co-occurrence and co-exclusion relationships among the modules. Each node represents a genus colored by module and its size is proportional to the relative abundance. Edge color indicates the positive (red) and negative (blue) association. The width of each line exhibits the correlation strength among genus.

Figure 5

Longitudinal changes of gastric microbial diversity and composition after H. pylori eradication. (A, B) Box and whisker plots of the alpha diversity indices for richness and diversity of the bacterial communities on the ASV level in patients treated with either probiotics or placebo at T1 and T4, respectively. (C) Beta diversity analysis is represented by principal coordinate analysis based on Bray-Curtis distance. The red samples (T1) are deviated from the blue samples (T4). Relative abundance of the top 10 phyla (D) and the top 20 genera (E) in gastric mucosa. ***, p<0.001. A, probiotics group; B, placebo group; T1, pretreatment; T4, 8 weeks after quadruple therapy; T1, pretreatment; T4, 8 weeks after quadruple therapy.

Figure 6

Alterations of distinct gastric genera after H. pylori eradication. The most abundant genera that differed between T1 and T4 were identified. The difference was illustrated based on the relative abundance of T4 compared to T1. Values above zero represent that the genera increased after therapy, whereas those below zero represent the decreased genera. *p<0.05; **p<0.01; ***p<0.001; star without a line, comparison between T1 and T4; star with a line, comparison between (A, B); (A) probiotics group; (B) placebo group.

Figure 7

Effect of probiotics on saliva microbiota in patients following H. pylori eradication. (A) Compositional changes of the top 20 most abundant saliva genera. (B) Alterations of differential genera after H. pylori eradication. Values above zero represented the increased relative abundance at T4 relative to baseline, while those below zero represented decreased abundance. *p<0.05; **p<0.01; star without a line, comparison between T1 and T4; star with a line, comparison between (A, B); (A) probiotics group; (B) placebo group.

Figure 8

There were dramatical changes in gut microbiota immediately after H. pylori eradication, which were swiftly restored after 8 weeks. These perturbations were partially counteracted by probiotics. Meanwhile, alterations of oral and gastric microbiota were also observed. Probiotics mitigated the fluctuations of gastric microbiota as well as promoted the enrichment of beneficial bacteria in the mouth.