Baseline characteristics
In this study, 162 patients were enrolled, 79 patients were randomly divided into group A, and 83 patients were randomly subjected into group B. 151 patients completed this trial, while 5 patients in group A and 6 patients in group B withdrew from this study (Fig. 1). Finally, a total of 755 stool samples were collected and subjected to analysis. The baseline characteristics of enrolled patients are presented in Table 1. No significant differences in age, gender, body mass index (BMI), smoking habits, alcohol consumption, marital status, education and exercise were found between the two groups (all P > 0.05). Before the eradication therapy, there was no significant difference in overall gastrointestinal symptoms between the two groups (Supplementary Table S1). Since the diet components had potential impact on the gut microbiota, we then assessed nutrient intake using a Food Frequency Questionnaire (FFQ) in these patients and found that there was no difference between the two groups (Supplementary Table S2). The eradication rates of group A and group B were 82.43% and 87.01% by ITT analysis. By PP analysis, the rates in group A and group B were 84.72% and 89.33%, respectively. No significant differences in eradication rates were observed (P > 0.05) (Table 2).
Table 1
Baseline characteristics of patients in the two groups.
Characteristics Group A (n = 74) Group B (n = 77) P |
Gender | | | 0.122 |
male | 44 (59.46%) | 55 (71.43%) | |
female | 30 (40.54%) | 22 (28.57%) | |
Agea | 45.32 ± 10.98 | 43.29 ± 11.30 | 0.263 |
BMIa | 23.30 ± 3.48 | 23.30 ± 3.65 | 0.996 |
Education | | | 0.904 |
primary | 15 (20.27%) | 12 (15.58%) | |
junior | 21 (28.38%) | 22 (28.57%) | |
senior | 20 (27.03%) | 22 (28.57%) | |
bachelor | 17 (22.97%) | 18 (23.38%) | |
postgraduate | 1 (1.35%) | 3 (3.90%) | |
Job | | | 0.607 |
Manual labor | 22 (29.73%) | 20 (25.97%) | |
Mental labor | 52 (70.27%) | 57 (74.03%) | |
Marital status | | | 0.219 |
unmarried | 9 (12.16%) | 15 (19.48%) | |
married | 65 (87.84%) | 62 (80.52%) | |
Smoking | 13 (17.57%) | 12 (15.58%) | 0.915 |
Alcohol | 17 (22.97%) | 14 (18.18%) | 0.676 |
Yogurt | 9 (12.16%) | 11 (14.29%) | 0.700 |
Exercise | 11 (14.86%) | 14 (18.18%) | 0.584 |
Group A: bismuth quadruple therapy; Group B: bismuth quadruple therapy + probiotics supplementation with Medilac-S; a Data are presented as mean ± standard deviation (SD). |
Table 2
Efficacy of H. pylori eradication in the two groups.
Analysis Group A (n = 74) Group B (n = 77) P |
PP | 61/72 (84.72%) | 67/75 (89.33%) | 0.226 |
ITT | 61/74 (82.43%) | 67/77 (87.01%) | 0.405 |
Group A: bismuth quadruple therapy; Group B: bismuth quadruple therapy + probiotics supplementation with Medilac-S; ITT, intention to treat; PP, per protocol; Data are presented as n (%). |
Alterations in gut microbial diversity after H. pylori eradication and probiotics supplementation
We first evaluated the alpha diversity in each group using four indices chao 1, observed OTUs, Shannon and faith_pd index. The alpha diversity indices were significantly decreased 2 weeks after treatment in both groups (P<0.01), which lasted for up to 4 weeks after treatment (P<0.05). There was a trend for restoration of microbiota with time. The alpha diversity almost returned to the baseline at week 8 (Figure 2A, Supplementary Figure S1). As for the comparison between the two groups, there were no significant differences in alpha diversity at baseline, week 2, week 4, week 6 and week 8 (P>0.01; Figure 2B and C, Supplementary Figure S2).
In order to examine the variability of microbial community between the two groups, we calculated beta diversity using PCoA on weighted Uniface distance. There were no significant differences in the beta diversity between the two groups before treatment (P=0.652; Figure 2D). However, there were significant differences in the beta diversity at week 2, week 4, week 6 and week 8 after treatment compared to the baseline based on all patients or each group alone (Figure 2E and F). There were no significant differences in beta diversity between the two groups at each time period (Figure 2F).
Changes in microbiota taxa after H. pylori eradication and probiotics supplementation
In order to identify the profiles of gut microbiome changes, we examined the microbiota taxonomic composition and relative abundance in the two groups at different taxonomic levels. At the phylum level, the relative abundance of Proteobacteria was significantly increased, while the abundance of Firmicutes, Bacterioidetes, Verrucomicrobia, and Actinobacteria was significantly decreased 2 weeks after treatment in both groups (Figure 3A). No significant difference in phyla was observed at week 4, week 6, and week 8 compared to the baseline (Figure 3A). Meanwhile, there was a decrease in the Bacterioidets:Firmicutes (B:F) ratio at week 2 after treatment which returned to the baseline from week 4 to week 8 (Figure 3B). At genus level, we observed an increased abundance of Clostridium, Klebsiella, Streptococcus, and Veillonella, while a decrease of Bacteroides, Faecalibacterium, Roseburia, Lachnospira, Phascolarctobacterium, Megamonas, Oscillospira, and Ruminococcus at 2 weeks after treatment compared to the baseline in both groups (Figure 3C and D).
We further used LEfSe to specifically identify the bacterial taxa in each group at different periods after eradication. In group A, several bacterial taxa were differentially abundant compared to the baseline with linear discriminant analysis (LDA) score>2 and P < 0.01. Klebsiella, Streptococcus, Veillonella, Fusobacterium, Morganella, and Prevotella were significantly enriched, while Bacteroides, Faecalibacterium, Roseburia, Lachnospira, Phascolarctobacterium, Bifidobacterium, and Butyricimonas were markedly decreased at week 2 (Supplementary Figure S3A). With the prolonged time periods, we observed that some of these bacterial taxa changes were transient, such as the increase of Prevotella, Fusobacterium, and Morganella, together with a decrease in Bacteroides, Faecalibacterium, Lachnospira, Bifidobacterium, and Butyricimonas at week 2 which recovered to the baseline by week 4. However, some other changes were longer lasting, such as the increase in relative abundance of Klebsiella, Streptococcus, and Veillonella, as well as a decrease of Roseburia and Oscillospira at week 2 which did not return to baseline by week 8 (Supplementary Figure S3A-D).
Similar in group B, Klebsiella, Streptococcus, and Veillonella were also significantly enriched, while Bacteroides, Faecalibacterium, Roseburia, Lachnospira, Phascolarctobacterium, Sutterella, Akkermansia, and Bifidobacterium were markedly decreased at week 2 (Supplementary Figure S3E). Differently, Enterococcus and Bacillus, main components of the probiotics, were markedly enriched at week 2 and week 4, normalized by week 6 and week 8, indicating the colonization of the probiotics during its supplementation (Supplementary Figure S3E-H). Furthermore, the Klebsiella and Streptococcus rapidly returned to normal by week 4 after probiotics supplementation (Supplementary Figure S3E-H).
To specify the distinct microbial taxa between the two groups at each time point, we further performed LEfSe to compare bacterial abundances. We observed that Enterococcus, Citrobacter, and Oscillospira were significantly enriched in group B, while Dialister, Anaerotruncus, and Megasphaera were mainly enriched in group A at week 2 (Figure 4A). At week 4, Enterococcus, Bacillus, and Lactobacillales were enriched in group B (Figure 4B), suggesting the successful colonization of probiotics after its supplementation. By week 6 and week 8, the enriched abundance of Enterococcus and Bacillus disappeared (Figure 4C and D). The above results suggested that H. pylori eradication could significantly disturb the composition of gut microbiota, enrich some detrimental bacteria taxa such as Klebsiella and Streptococcus, while decrease some beneficial taxa like Faecalibacterium, Roseburia, Lachnospira, Phascolarctobacterium, Bifidobacterium, and Butyricimonas. The effective colonization of probiotics could be observed during its supplementation, and the probiotics supplementation might rapidly decrease the enrichment of some detrimental bacteria taxa.
Predictive functional pathways of microbial community after H. pylori eradication and probiotics supplementation
To investigate the potential role of gut microbiome, we tried to identify the functional variations in the microbial communities using PICRUSt analysis with KEGG database to predict microbiota associated functional pathways. We observed differential predicted functions between samples before and after H. pylori treatment. At week 2 in group A, pathways involved in starch degradation, pyruvate fermentation, glycolysis, glucarate degradation were decreased, whereas those involved in fatty acid oxidation, sucrose degradation, formaldehyde assimilation were increased at week 2 (Supplementary Figure S4A). Consistent with the tendency of microbial changes, we observed some changes of the predicted pathways were also transient, while some other changes such as an increase of sucrose degradation and fatty acid oxidation as well as the decrease of starch degradation and glycolysis were longer lasting until week 8 (Supplementary Figure S4A-D). In group B, pathways involved in propanediol degradation, fatty acid oxidation, pyruvate_dehydrogenase, hexitol_fermentation sucrose_degradation, methylphosphonate_degradation were increased, whereas those involved in glycogen_degradation, peptidoglycan_biosynthesis, L_histidine_biosynthesis, coenzyme_A_biosynthesis, formaldehyde_oxidation were decreased at week 2 (Supplementary Figure S4E). Similarly, most of these changes seemed to be transient, while some others were lasting until week 8 (Supplementary Figure S4E-H).
To further compare the differential functional pathways between the two groups, we conducted LEfSe analysis to identify the discriminating functional pathways. Interestingly, the distinct pathways were only identified between the two groups at week 4. We predicted 9 different microbiota-associated functional pathways between group A and group B. Four pathways with increased abundance in group B were predicted, including superpathway of thiamin diphosphate biosynthesis, sulfate_reduction, coenzyme_A_biosynthesis, and N10 formyl tetrahydrofolate biosynthesis. In contrast, the abundances of 5 pathways, which were associated with superpathway_of_lipopolysaccharide_biosynthesis, polymyxin_resistance, starch degradation, CDP_diacylglycerol_biosynthesis, and mannan_degradation were significantly decreased compared with group A (Figure 5A).
Furthermore, we investigated whether the differential microbial taxa between group A and group B were related to the predicted pathways to depict how specific taxa was involved in the functional pathways. The Enterococcus was significantly enriched in group B after probiotics supplementation, and we found Enterococcus was related to five of the nine predicted pathways at week 4. Specifically, Enterococcus was positively associated with superpathway of thiamin diphosphate biosynthesis, N10 formyl tetrahydrofolate biosynthesis, and coenzyme_A_biosynthesis, while negatively related to superpathway of lipopolysaccharide biosynthesis and polymyxin_resistance (Figure 5B-F). In terms of the Collinsella, which was significantly decreased in group B, was positively related to starch degradation and CDP_diacylglycerol_biosynthesis, and mannan_degradation (Figure 5G-I). Taken together, we hypothesized that the altered gut microbiota after eradication and probiotics supplementation might contribute to the various metabolic or clinical features through these microbiota related pathways.
Gut microbiome correlated with patients’ features
Since various factors such as diet, age, alcohol, smoke, BMI might have impact on gut microbiota, we sought to investigate the relationship of gut microbiome and these factors after eradication. We incorporated the following variables into analysis: age, gender, BMI, alcohol, smoking, exercise. We found that age, alcohol and BMI were closely correlated with the microbial community diversity. We further identified specific bacterial taxa in group A and group B by dividing patients into different subgroups based on these factors. The analysis showed that Streptococcus, Dorea, Selenomonas, and Mogibacterium were significantly increased, while Paraprevotella and Turicibacter decreased in elder patients in group A (Figure 6A). However, Ruminococcus was significantly increased with a decrease of Megasphaera in elder patients in group B (Figure 6A). Compared to non-drinkers, Gemmiger and Collinsella were increase after eradication, while Sporosarcina, Alloscardovia, and Moryella were enriched after probiotics supplementation (Figure 6B). In terms of BMI, we found that Prevotella, Bilophila, Dialister, and Eikenella were markedly enriched after eradication, while Megasphaera, Blautia, and Faecalibacterium were increased after probiotics supplementation in obesity patients (Figure 6C). These results indicated that age, BMI and alcohol had an impact on gut microbiota after H. pylori eradication, and probiotics supplementation might partially eliminate the adverse effect in patients with these specific features.