Cai TW, Chen ZK, Lv ZY, Jiang WJ, Sun YY, Jin LX, Xia CM, Li QQ, Chen X. Effects of berberine-containing vs clarithromycin-containing quadruple therapy on intestinal microbiota during Helicobacter pylori eradication. World J Gastroenterol 2026; 32(43): 122243 [DOI: 10.3748/wjg.122243]
Corresponding Author of This Article
Xia Chen, MD, Chief Physician, Department of Gastroenterology, The First People’s Hospital of Wenling, Affiliated Wenling Hospital, Wenzhou Medical University, No. 333 Chuan’an South Road, Chengxi Street, Wenling 317500, Zhejiang Province, China. werla@sina.com
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Cai TW, Chen ZK, Lv ZY, Jiang WJ, Sun YY, Jin LX, Xia CM, Li QQ, Chen X. Effects of berberine-containing vs clarithromycin-containing quadruple therapy on intestinal microbiota during Helicobacter pylori eradication. World J Gastroenterol 2026; 32(43): 122243 [DOI: 10.3748/wjg.122243]
Teng-Wei Cai, Zi-Yan Lv, Wen-Jun Jiang, Ye-Yu Sun, Ling-Xiao Jin, Qian-Qian Li, Xia Chen, Department of Gastroenterology, The First People’s Hospital of Wenling, Affiliated Wenling Hospital, Wenzhou Medical University, Wenling 317500, Zhejiang Province, China
Zhi-Kai Chen, Chen-Mei Xia, Department of Gastroenterology, The First People’s Hospital of Wenling (Taizhou University Affiliated Wenling Hospital), School of Medicine, Taizhou University, Wenling 317500, Zhejiang Province, China
Author contributions: Cai TW and Chen ZK contributed equally to this work, as they are co-first authors; Cai TW and Chen ZK were involved in the study concept, design, and data collection; Jiang WJ and Chen X were involved in securing funding for the project; Lv ZY, Jiang WJ and Li QQ were involved in the interpretation of data; Sun YY, Jin LX and Xia CM were involved in the collection and analysis of data; Chen ZK, Lv ZY, Jiang WJ, Sun YY, Jin LX, Xia CM and Li QQ were involved in drafting the initial manuscript; Cai TW, Chen ZK, and Chen X were involved with the critical revision of the manuscript for important intellectual content and study supervision; all the authors have reviewed and approved the final manuscript.
AI contribution statement: Portions of this manuscript were edited using AI tools solely for language refinement. The authors carefully reviewed and verified all AI-assisted outputs and take full responsibility for the scientific content of the manuscript.
Supported by Zhejiang Provincial Science and Technology Program of Traditional Chinese Medicine, China, No. 2023ZL784; and Wenling Social Development Science and Technology Project, China, No. 2024S00303.
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board of the First People’s Hospital of Wenling, Affiliated Wenling Hospital, Wenzhou Medical University (No. KY-2024-1008-01).
Clinical trial registration statement: Retrospective trial registration submitted; registration number pending official review.
Informed consent statement: All study participants, or their legal guardians, provided informed written consent prior to study enrollment.
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
CONSORT 2010 statement: The authors have read the CONSORT 2010 Statement, and the manuscript was prepared and revised according to the CONSORT 2010 Statement.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at werla@sina.com.
Corresponding author: Xia Chen, MD, Chief Physician, Department of Gastroenterology, The First People’s Hospital of Wenling, Affiliated Wenling Hospital, Wenzhou Medical University, No. 333 Chuan’an South Road, Chengxi Street, Wenling 317500, Zhejiang Province, China. werla@sina.com
Received: April 14, 2026 Revised: June 15, 2026 Accepted: July 13, 2026 Published online: November 21, 2026 Processing time: 167 Days and 22.8 Hours
Abstract
BACKGROUND
Helicobacter pylori (H. pylori) eradication is crucial for managing H. pylori-related gastrointestinal diseases. Berberine, a natural alkaloid, has shown potential anti-H. pylori activity, but its effectiveness and feasibility remain unclear.
AIM
To evaluate the H. pylori eradication efficacy of berberine and its impact on intestinal flora, compared with clarithromycin-based therapy.
METHODS
A total of 41 subjects with positive 13C-urea breath test were randomly divided into group A (berberine group) and group B (clarithromycin group). All participants underwent 14-day quadruple therapy with omeprazole 20 mg once daily, amoxicillin 0.5 g three times daily and bismuth potassium citrate 1 g four times daily; group A substituted berberine 0.3 g three times daily, whereas group B substituted berberine with clarithromycin 0.5 g twice daily. Fecal samples were collected before (A1, B1) and after (A2, B2) treatment for 16S rRNA gene sequencing to analyze intestinal flora diversity, structure, and phenotypic characteristics. H. pylori eradication efficacy was evaluated by 13C-urea breath test 4 weeks post-treatment.
RESULTS
The H. pylori eradication rates were 80.95% in group A and 75.00% in group B, with no statistical difference (χ2 = 0.212, P = 0.719). Alpha diversity analysis showed that clarithromycin treatment (group B) significantly decreased ACE, Chao1, Sobs, and Shannon indices (all P < 0.05), while berberine treatment (group A) had no significant effect. Beta diversity and enterotype analysis revealed that clarithromycin induced significant changes in intestinal flora structure and enterotype distribution, whereas berberine maintained relatively stable flora structure. Neutral community model analysis indicated that berberine enhanced deterministic processes more significantly than clarithromycin in regulating flora assembly. Differential species analysis identified that Blautia was significantly enriched in group A post-treatment, and Dorea longicatena significantly enriched in group B.
CONCLUSION
Berberine exhibits comparable H. pylori eradication efficacy to clarithromycin, and has a milder impact on intestinal flora, maintaining flora homeostasis and reducing the risk of antibiotic resistance.
Core Tip: This study compares berberine and clarithromycin-based therapy for Helicobacter pylori (H. pylori) eradication. The two regimens have comparable H. pylori eradication efficacy (80.95% vs 75.00%). Berberine causes milder intestinal flora disturbance, maintains flora homeostasis, enriches beneficial bacteria, and reduces antibiotic resistance risk, serving as a promising alternative for H. pylori eradication, especially in antibiotic-resistant populations.
Citation: Cai TW, Chen ZK, Lv ZY, Jiang WJ, Sun YY, Jin LX, Xia CM, Li QQ, Chen X. Effects of berberine-containing vs clarithromycin-containing quadruple therapy on intestinal microbiota during Helicobacter pylori eradication. World J Gastroenterol 2026; 32(43): 122243
Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic bacterium that colonizes the human gastric mucosa, and affects approximately half of the global population[1,2]. As a class I carcinogen, it is the primary etiology of chronic gastritis, peptic ulcers, and gastric cancer, imposing a heavy burden on global public health[3]. Eradication of H. pylori infection is the cornerstone of managing H. pylori-related diseases. However, conventional antibiotic-based regimens face increasing challenges, including antibiotic resistance, adverse gastrointestinal reactions, and high recurrence rates, which has prompted the search for alternative or complementary antibiotic agents[4-6].
Clarithromycin is an antibiotic that is currently used in first-line therapy for H. pylori eradication. However, the global prevalence of H. pylori antibiotic resistance has increased dramatically in recent years, becoming a major barrier to successful eradication. In particular, clarithromycin resistance rates have increased in many regions, often exceeding 30%, which significantly reduces the efficacy of clarithromycin-containing regimens[7-9]. This increase in antibiotic resistance, coupled with adverse gastrointestinal reactions and intestinal flora disturbance caused by long-term or combined antibiotic use, has become a critical clinical challenge in H. pylori eradication[10]. In this context, berberine, a natural isoquinoline alkaloid derived from Phellodendron chinense and Coptis chinensis, has emerged as a promising candidate for H. pylori eradication because of its inherent anti-H. pylori activity and milder effects on intestinal microecology, addressing the key drawbacks of conventional antibiotic therapies[11]. Multiple randomized controlled trials have verified the clinical efficacy of berberine-containing regimens. Combined administration of berberine can achieve eradication rates no lower than those of antibiotic-based regimens while reducing adverse reactions[12,13].
This study aimed to evaluate the effectiveness and feasibility of using berberine as an alternative or complementary agent for H. pylori eradication, with clarithromycin used as a control. We hypothesized that berberine would yield short-term H. pylori eradication rates comparable to those of clarithromycin, but with milder intestinal flora disturbance and fewer adverse reactions. By analyzing intestinal flora and eradication outcomes, this study provides experimental evidence for optimizing H. pylori eradication regimens with antibiotic-sparing or complementary strategies.
MATERIALS AND METHODS
Study participants and sample collection
This study was designed as a pilot exploratory trial. Patients who presented to the First People’s Hospital of Wenling with upper gastrointestinal discomfort were enrolled in this study. The inclusion criteria were as follows: (1) Aged 18-70 years (male or female); (2) Positive results of the 13C-urea breath test; (3) No use of antibiotics, bismuth, H2 receptor antagonists, or proton pump inhibitors within the previous 4 weeks; and (4) Willingness to receive H. pylori eradication therapy and comply with followup assessments. Patients were excluded if they had severe cardiac, hepatic, or renal dysfunction, were pregnant or lactating, had a history of esophageal, gastric, or intestinal surgery; were unable to provide informed consent because of psychiatric disorders or severe neurosis; were concurrently using nonsteroidal anti-inflammatory drugs; had a history of alcohol abuse; or had a penicillin allergy. Fecal samples (approximately 0.5 g) were collected from each participant before treatment and at the 4-week follow-up, immediately frozen at -80 °C, and transported on dry ice to the laboratory for subsequent analysis. The study was performed in accordance with the Declaration of Helsinki. Ethical approval was obtained from the Institutional Review Board of the First People’s Hospital of Wenling, Affiliated Wenling Hospital, Wenzhou Medical University (No. KY-2024-1008-01). Written informed consent was obtained from all participants.
DNA extraction and 16S rRNA gene amplification
Total microbial genomic DNA was extracted from fecal samples using the FastPure Stool DNA Isolation Kit (MJYH, Shanghai, China). The quality and concentration of DNA were determined by 1.0% agarose gel electrophoresis and a NanoDrop2000 spectrophotometer (Thermo Scientific, United States), and DNA samples were kept at -80 °C prior to further use. The hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified with the primer pairs 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) using a T100 Thermal Cycler polymerase chain reaction (PCR) thermocycler (BIO-RAD, United States). The PCR reaction mixture contained 10 μL 2 × Pro Taq, 0.8 μL of each primer (5 μM), 10 ng of template DNA, and double distilled water to a final volume of 20 μL. The PCR amplification cycling conditions were set as follows: Initial denaturation at 95 °C for 3 minutes, followed by 27 cycles of denaturing at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds and extension at 72 °C for 45 seconds, a single extension at 72 °C for 10 minutes, and a final hold at 4 °C. The PCR product was extracted from 2% agarose gel and purified using the PCR Clean-Up Kit (YuHua, Shanghai, China) according to the manufacturer’s instructions, and quantified using a Qubit 4.0 fluorometer (Thermo Fisher Scientific, United States).
16S rRNA gene sequencing and bioinformatics analysis
Exploratory intestinal microbiota analyses based on 16S rRNA gene sequencing were performed to characterize shifts in gut flora before and after treatment. Purified amplicons were pooled in equimolar amounts and paired-end sequenced on an Illumina Nextseq2000 platform (Illumina, San Diego, CA, United States) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China), and the raw sequencing reads were deposited into the NCBI Sequence Read Archive database. After demultiplexing, the resulting sequences were quality filtered with fastp (0.23.4) and merged with FLASH (v1.2.11). Then the high-quality sequences were de-noised using the DADA2 plugin in the Qiime2 (version 2024) pipeline with recommended parameters, which obtains single nucleotide resolution based on error profiles within samples; DADA2 denoised sequences are usually referred to as amplicon sequence variants (ASVs). Taxonomic assignment of ASVs was performed using the Naive bayes consensus taxonomy classifier implemented in Qiime2 and the SILVA 16S rRNA database (v138), and BugBase phenotypic prediction analysis was conducted. P values were used to identify taxa with significantly different relative abundance (P < 0.05), and multiple testing correction was performed for all differential species comparisons using the Benjamini-Hochberg false discovery rate (FDR) approach.
Treatment regimens and eradication evaluation
Eligible patients were allocated into two groups according to their outpatient visit days: Patients who attended on Tuesdays, Thursdays or Saturdays were assigned to the berberine group (group A), while those presenting on Mondays, Wednesdays or Fridays were assigned to the clarithromycin group (group B). For the convenience of subsequent analysis, the samples collected from group A before treatment were designated A1, and those collected at the 4-week follow-up after treatment were designated A2; similarly, the samples collected from group B before treatment were designated B1, and those collected at the 4-week follow-up after treatment were designated B2. Patients in group A (n = 21) were given berberine (Northeast Pharmaceutical Group Shenyang No.1 Pharmaceutical Co., Ltd., No. H21022453, 0.1 g/tablet) at 0.3 g per dose three times daily, amoxicillin (Ausmech Pharmaceutical Factory, HC20130014, 0.5 g/capsule) at 0.5 g per dose three times daily; omeprazole (Zhejiang Jinhua Conba Bio pharmaceutical Co., Ltd., No. H19991118, 10 mg/capsule) at 20 mg per dose once daily and bismuth potassium citrate (Livzon Group Livzon Pharmaceutical Factory, No. H10900086, 1 g/bag) at 1 g per dose four times daily for H. pylori eradication treatment for 14 days. The dosage regimen of berberine was selected in accordance with widely adopted dosing protocols from previous clinical studies focusing on berberine against H. pylori, which demonstrated reliable antibacterial and immunomodulatory effects without obvious adverse reactions[12,14]. Patients in group B (n = 20) were given clarithromycin (Jiangsu Hengrui Pharmaceutical Co., Ltd., No. H20031041, 0.5 g/tablet) at 0.5 g per dose, twice daily to replace berberine, while the other three medications remained identical. During treatment, all patients received health education, including information on the etiology, clinical manifestations, and disease progression of their condition, and were advised of the importance of complying with medical advice; they were also instructed to avoid smoking, alcohol, tea, spicy and other irritating foods, and drugs that cause gastrointestinal damage. All patients were followed-up on day 7 of treatment to inquire about medication intake by telephone. Adverse reactions, such as dizziness, nausea, vomiting, and rash, were closely observed. At 4 weeks after the completion of the 14-day regular eradication treatment course, patients returned to the hospital for a 13C-breath test to confirm the eradication effect; a negative breath test result was defined as successful H. pylori eradication, and a positive result was defined as treatment failure.
RESULTS
Baseline characteristics and H. pylori eradication efficacy
A total of 41 subjects with positive 13C-urea breath tests were enrolled in this study and randomly divided into two groups: Group A (berberine group) and group B (clarithromycin group). The baseline characteristics of the two groups were comparable with no statistically significant differences. The mean age of the subjects in group A was 43.24 ± 9.32 years, while the mean age in group B was 47.45 ± 11.77 years (P = 0.367). In terms of gender distribution, there were 11 males and 10 females in group A, and 10 males and 10 females in group B (χ2 = 0.879, P = 1.000). A follow-up 13C-urea breath test was performed 4 weeks after the completion of H. pylori eradication treatment. The eradication rate was 80.95% (4 positive cases) in group A and 75.00% (5 positive cases) in group B, with no statistically significant difference between the two groups (χ2 = 0.212, P = 0.719).
Alpha diversity analysis of intestinal flora
After 16S rRNA gene sequencing, sequence denoising methods (DADA2) were used to optimize the data, and representative ASV representative sequences and abundance information were obtained. Rarefaction curve analysis revealed that both the observed species (Sobs) and Shannon index rarefaction curves tended to plateau, indicating that the sequencing data volume was reasonable and could reflect the community structure characteristics of the samples (Figure 1A and B). The coverage index values were all between 0.997 and 1.0, with no significant difference between groups before and after treatment, suggesting sufficient sequencing depth, few missing rare species, and no bias in subsequent analyses (Figure 1C).
Figure 1 Rarefaction curves and comparison of alpha diversity indices of intestinal microbiota.
A: Rarefaction curves based on observed species (Sobs), X-axis: Randomly sampled sequencing data volume. Y-axis: Observed species number or diversity index; B: Rarefaction curves based on Shannon index, X-axis: Randomly sampled sequencing data volume. Y-axis: Observed species number or diversity index; C: Coverage index curves, X-axis: Randomly sampled sequencing data volume. Y-axis: Observed species number or diversity index; D-I: Comparisons of alpha diversity indices including ACE, Simpson, Shannon, Sobs, Chao1, and coverage among groups. Groups with significant differences are labeled as follows: 0.01 < aP < 0.05. bP ≤ 0.01. The X-axis represents group names, and the Y-axis represents the index value of each group. Group A (berberine group): A1, before treatment; A2, after treatment. Group B (clarithromycin group): B1, before treatment; B2, after treatment. ASV: Amplicon sequence variant.
Before treatment, there were no statistically significant differences in the alpha diversity indices between the two groups (A1 vs B1). After treatment, the alpha diversity indices (ACE, Chao1, Sobs, and Shannon) decreased in both groups, indicating a reduction in the number of gastrointestinal flora species (Figure 1D-I). Interestingly, there was no statistically significant difference in the alpha diversity indices between A2 (after treatment in group A) and A1 (before treatment in group A). In contrast, compared with those in B1 (before treatment), the ACE, Chao1, Sobs, and Shannon indices in B2 (after treatment in group B) significantly decreased, whereas the Simpson index significantly increased.
Beta diversity analysis and enterotype classification
Community distance heatmaps at the genus level clearly revealed differences in the intestinal microbiota (Figure 2A). Venn and QIIME analyses revealed distinct clustering of microbial community structures was observed among the four groups (Figure 2B). Principal co-ordinates analysis (PCoA), non-metric multidimensional scaling (NMDS), and principal components analysis (PCA) at the ASV level indicated that H. pylori eradication treatment altered the flora structure. There were no statistically significant differences in the matrix distances of PCA, PCoA, or NMDS before and after treatment in group A (Figure 2C-E), suggesting that berberine treatment had no significant effect on the flora structure. In contrast, the matrix distances before and after treatment in group B significantly differed (Figure 2F-H), indicating significant changes in the flora after clarithromycin treatment.
Figure 2 Beta diversity analysis of intestinal microbiota in berberine group and clarithromycin group before and after treatment.
A: Heatmap of microbial community composition at the genus level. The X-axis represents sample names, and the Y-axis represents species names. A color gradient is used to indicate the relative proportion of each species, with the corresponding values shown on the right side of the figure; B: Venn map at the genus level. Different colors represent different groups. The overlapping regions indicate species shared by multiple groups, while the non-overlapping regions represent unique species specific to that group. The numbers indicate the corresponding number of species; C-E: Non-metric multidimensional scaling (NMDS), principal co-ordinates analysis (PCoA), and principal components analysis (PCA) plots based on amplicon sequence variant (ASV) level of berberine group; F-H: NMDS, PCoA, and PCA plots based on ASV level of clarithromycin group. The horizontal and vertical axes represent the two selected principal coordinate components, and the percentages indicate the contribution of each principal coordinate component to the differences in sample composition. The scales of the horizontal and vertical axes are relative distances and have no actual physical meaning. Points of different colors or shapes represent samples from different groups. The closer two sample points are, the more similar their species composition. Group A (berberine group): A1, before treatment; A2, after treatment. Group B (clarithromycin group): B1, before treatment; B2, after treatment. NMDS: Non-metric multidimensional scaling; PC: Principal component.
To further characterize the changes in intestinal microbiota composition induced by different treatments, exploratory analysis of intestinal microbiota enterotypes was performed at the genus level on the basis of the Jensen-Shannon divergence distance (Figure 3A). Samples from 41 subjects collected before and after treatment were clustered into different enterotypes. The peak Calinski-Harabasz index were observed at k = 9, confirming that 9 clusters represented the statistically optimal grouping for the present cohort (Figure 3B). Before treatment (A1), the dominant enterotypes in group A were enterotypes 1, 2, and 3, accounting for 19.05%, 19.05%, and 33.33%, respectively. After treatment (A2), the community structure remained relatively stable, with enterotypes 1 and 3 still being the main types (19.05% each). In contrast, the enterotype distribution in group B changed more significantly: Samples before treatment (B1) were dominated mainly by enterotypes 2 (25.00%) and 3 (20.00%), whereas samples after treatment (B2) were clustered mainly into enterotypes 3, 4, and 8 (20.00% each) (Figure 3C).
Figure 3 Composition and enterotype analysis of intestinal microbiota based on Jensen-Shannon divergence.
A: Enterotype distribution in each group. Enterotype of specific clinical samples is reflected by the structure of the dominant flora in each sample. The top right corner indicates the sample groups; different colors represent different types; the circles represent the range within the confidence interval; B: Optimal cluster number for enterotype classification selected by Calinski-Harabasz (CH) index values. The X-axis represents the number of clusters (k value), and the Y-axis denotes the magnitude of the CH index; C: Relative abundances of signature genera for each enterotype. The percentage bar chart shows the proportion of different types in each group of samples. The bar plot presents the typing composition of samples in each group, with different colors representing different types; D and E: Microbial composition at the phylum level and genus level. This figure shows the composition and proportion of the top abundant phylum/genus in all samples, with other low-abundance phylum/genus classified as others. It mainly illustrates the changes in dominant species composition across different samples/groups. The X-axis/Y-axis represents sample names, and the Y-axis/X-axis represents the relative proportion of each species in the sample. Bars of different colors represent different species, and the length of the bar indicates the relative proportion of the species. Group A (berberine group): A1, before treatment; A2, after treatment. Group B (clarithromycin group): B1, before treatment; B2, after treatment. CH: Calinski-Harabasz; BBR: Berberine; CLA: Clarithromycin.
Enterotype characterized by signature genera with high relative abundances: Enterotype 1 was enriched in Blautia (18.31%), Faecalibacterium (11.01%), and Romboutsia (6.84%); Enterotype 2 was dominated by Blautia (14.82%), Bacteroides (6.97%), and Escherichia (6.09%); Enterotype 3 was marked by Blautia (29.41%), Anaerobutyricum (9.80%), and Romboutsia (6.55%); Enterotype 4 was featured with Blautia (33.90%), Romboutsia (11.28%), and Escherichia (8.16%); Enterotype 8 was characterized by Blautia (18.44%),Romboutsia (10.41%), and Ruminococcus (10.22%). Overall, these results indicated that berberine intervention had a mild effect on the enterotype structure of the intestinal microbiota, while clarithromycin treatment induced significant structural changes in the microbial community. The taxonomic composition of each group at the phylum and genus levels is shown in Figure 3D and E.
Neutral community model analysis of microbial community assembly
To clarify the effects of treatment regimens A and B on intestinal microbiota assembly, the neutral community model (NCM) was used to analyze the relative importance of neutral processes, where the R2 value represents the overall goodness of fit of the model. For group A, the proportion of microbiota variation explained by stochastic processes decreased sharply from 21.33% before treatment to 2.40% after treatment.
For group B, the contribution rate of stochastic factors before treatment (24.01%) was slightly greater than that in group A before treatment (21.33%), and decreased to 10.83% after treatment. These results suggest that regimen B also enhanced deterministic processes, but the enhancement was much lower than that of regimen A (the decreases in R2 values were 88.75% and 54.89%, respectively). Notably, 10.83% of the microbiota variation in group B after treatment was still attributed to stochastic processes.
Species typing based on the detection rate and identification of core species
To further identify the core taxa in the microbial community, all the species were divided into three categories on the basis of their detection rates: Transient species (low abundance), intermediate species (medium abundance), and persistent species (high abundance) (Figure 4A). The results revealed that persistent species accounted for 60%-70% of the species in both groups, whereas transient species accounted for only 2%-3%. The high proportion of persistent species indicates that the microbial community is insensitive to the environment, which is consistent with the results of the NCM, in which deterministic processes dominate community assembly. However, we found that the proportion of intermediate species in group B decreased from 34.15% to 27.47%, which was greater than the change in group A before and after treatment, suggesting that some medium-abundance flora potentially sensitive to antibiotics were affected by the antibiotics.
Figure 4 Species typing based on detection rate and differential species analysis of intestinal microbiota.
A: Microorganisms are classified according to environmental sensitivity and categorized into transient, intermediate, and persistent taxa based on their prevalence. This figure shows the proportion of average relative abundance and the proportion of detected number of transient, intermediate, and persistent species in the samples. The X-axis/Y-axis represents percentage, corresponding to average relative abundance or detected number; B-D: The differences in the average relative abundance of the same species among different groups, with annotations indicating the significance of differences, directly presenting the variation in average relative abundance of the same species across groups. The X-axis represents the species names at different taxonomic levels; the Y-axis represents the percentage value of the abundance of a given species in the sample; different colors represent different groups. The P value is shown on the far right. 0.01 < aP < 0.05. bP ≤ 0.01. Group A (berberine group): A1, before treatment; A2, after treatment. Group B (clarithromycin group): B1, before treatment; B2, after treatment. ES: Effect size; FC: Fold change.
Differential species analysis between groups
Differential species analysis was performed to further explore the changes and impacts of key species. Specifically, 10 differential species (FDR-adjusted P < 0.05) were identified in the berberine treatment group, among which the abundances of Bifidobacterium catenulatum (B. catenulatum) and Thomasclavelia ramosa were significantly increased, while the abundances of Collinsella aerofaciens (C. aerofaciens) and Bifidobacterium adolescentis (B. adolescentis) were significantly decreased. Among these differential species, B. adolescentis, Butyricimonas virosa, and Cloacibacillus evryensis were transient species, and the remaining species were intermediate species (Figure 4B).
In the clarithromycin treatment group, 10 differential species (FDR-adjusted P < 0.05) were also identified, among which the abundances of B. catenulatum, Mediterraneibacter faecis, Dorea longicatena, C. aerofaciens, Faecalibacillus intestinalis, and Gemmiger formicilis were significantly decreased, while the abundance of Thomasclavelia ramosa was significantly increased. All differential flora in the clarithromycin group were intermediate species (Figure 4C). Notably, 9 differential species (FDR-adjusted P < 0.05) were identified between group A and group B after treatment. Among them, Turicibacter bilis and Clostridium scindens were significantly enriched in the clarithromycin group, while 7 species, including Phocaeicola plebeius and Bilophila wadsworthia were significantly enriched in the berberine group (Figure 4D). Among the differential species after treatment, Bacteroides timonensis, Holdemania filiformis, Anaerococcus vaginalis, and Massiliimalia massiliensis were all transient species.
Phenotypic prediction of intestinal flora
Phenotypic functional prediction of bacteria in the two groups before and after treatment was performed based on BugBase (Figure 5A). There was a significant intergroup difference in the Forms Biofilms phenotype (P < 0.05) (Figure 5B), whereas no significant intragroup changes before and after treatment were observed under either therapeutic regimen (A1 vs A2, B1 vs B2, all FDR-adjusted P > 0.05) (Figure 5C). Biofilm formation was mainly mediated by Bifidobacterium (Figure 5D). Furthermore, the results of other phenotypic functions are presented in Supplementary Figure 1. After clarithromycin treatment (group B2), the Longicatena taxa were significantly enriched in the contains mobile elements phenotypic category (Supplementary Figure 1). In addition, key flora in the stress tolerant phenotypic category completely disappeared after antibiotic treatment (Supplementary Figure 1).
Figure 5 Phenotypic prediction of intestinal microbiota.
A: Variations in composition of phenotypes. The X-axis represents sample names, and the Y-axis represents relative abundance. The color gradient of the blocks shows the phenotypic categories; B: Kruskal-Wallis H test on phenotype. The X-axis represents the phenotypic categories, and the Y-axis shows the percentage value of the relative abundance of each phenotype in the samples. Different colors correspond to different groups. The rightmost column displays P values, aP < 0.05; C: Kruskal-Wallis H test for forms biofilms phenotype. The X-axis represents phenotypic categories, and the Y-axis indicates the percentage of relative abundance for each phenotype in samples. Different colors represent different groups; D: Prediction of species contribution to forms biofilms phenotype. This figure displays the dominant species composition of forms biofilms phenotypes, and reflects the matching relationship between species and forms biofilms phenotypes. The X-axis shows group names, different colors in the legend represent distinct species, the Y-axis represents the relative abundance values of each species under the target phenotype. The top 5 genera are shown. Group A (berberine group): A1, before treatment; A2, after treatment. Group B (clarithromycin group): B1, before treatment; B2, after treatment. BBR: Berberine; CLA: Clarithromycin.
DISCUSSION
H. pylori is an opportunistic pathogen that colonizes the human gastric mucosa, and its eradication is a key measure for preventing peptic ulcers and gastric cancer[15]. Commonly used clinical antibiotics (such as clarithromycin) are prone to disturbing the intestinal microecology, by contrast, berberine, a natural plant extract, possesses both anti-H. pylori activity and microecological regulatory potential[16]. In this study, we compared differences in intestinal flora diversity, community structure, species composition, and phenotypic characteristics before and after H. pylori eradication between the berberine group (group A) and the clarithromycin group (group B). We further deeply explored the potential effects of the two treatment regimens on H. pylori eradication, microecological safety, and clinical application value were explored in depth to provide an experimental basis and theoretical support for the optimization of H. pylori eradication regimens.
In this study, the H. pylori eradication rates were comparable between the berberine group and the clarithromycin group, with no significant difference in therapeutic effectiveness (P > 0.05). Berberine is commonly administered at 300 mg thrice daily or 500 mg twice daily in H. pylori eradication regimens, with no significant difference in eradication efficacy between the two dosing schedules, and they yield comparable eradication rates to bismuth-containing quadruple antibiotic therapy (Table 1)[14,17,18]. Results of alpha and beta diversity analyses indicate that berberine exerts a mild regulatory effect on the intestinal flora without causing significant disturbance. This phenomenon is closely associated with the multitarget mechanism through which berberine exerts anti-H. pylori effects. Berberine binds to the sulfonic groups of urease active sites to inhibit urease activity and abolish bacterial acid resistance[12,19]. It changes bacterial morphology, triggers phosphatidylserine externalization to raise membrane permeability, downregulates flagellar genes flaA and flaB to weaken bacterial motility and gastric colonization[20-23], and induces severe oxidative stress while destroying membrane integrity to cause leakage of intracellular substances and suppress bacterial survival[24]. Besides, it downregulates key virulence factors to reduce the pathogenicity of H. pylori[16]. For mucosal immune regulation, berberine inhibits the IRF8-interferon (IFN)-γ signaling axis to cut excessive IFN-γ secretion[25], balances pro-inflammatory cytokines [interleukin (IL)-1β, transforming growth factor-β, IL-6] and anti-inflammatory IL-10 to alleviate gastric mucosal inflammation, and activates IL-4-STAT6 pathway to promote anti-inflammatory macrophage polarization[26,27]. It balances intestinal regulatory T cell/T helper 17 subsets, upregulates tight junction proteins to repair intestinal barrier and relieve flora disorder[26]. Moreover, stable intestinal microbiota modulated by berberine can restrain H. pylori adhesion via gut-gastric axis and lower gastrointestinal adverse reactions without decreasing eradication efficiency[28,29].
In contrast, significant alterations of alpha and beta diversity in the clarithromycin group (group B, B2 vs B1) demonstrate that the clarithromycin treatment (group B) induced a profound remodeling of the microbial community. This observation is probably attributable to its antibacterial properties. While H. pylori is eradicated, clarithromycin non-selectively inhibits a variety of susceptible intestinal flora, especially intermittent flora, leading to a simplified flora structure and reduced diversity[30,31]. Such alterations may lead to a decreased abundance of beneficial bacteria (e.g., butyrate-producing bacteria), an increased release of pro-inflammatory cytokines, reduced gastric mucosal defense capacity, and enhanced H. pylori recolonization potential[32,33]. Additionally, analysis based on the NCM indicated that both treatments increased the dominant role of deterministic processes in flora assembly, which dominated microbiota changes through nonrandom mechanisms (e.g., targeted elimination of specific taxa and alteration of intestinal niches for microbiota selection). However, the finding that 10.83% of the variation in post-treatment flora in group B was attributed to stochastic processes suggests that the regulatory effect of clarithromycin on intestinal flora is limited; this may result in a long-term failure of the intestinal flora to restore homeostasis in some patients, thereby increasing the risk of H. pylori recurrence and intestinal flora-related disorders (e.g., irritable bowel syndrome)[34].
The characteristics of the differential species further clarify the distinct effects of the two treatment regimens on H. pylori eradication and their differences in microecological safety, which are closely correlated with the stability of H. pylori eradication efficacy and recurrence risk. A total of 10 differential species were identified in the berberine group (group A) before and after treatment. Among these differential species, B. adolescentis, Butyricimonas virosa, and Cloacibacillus evryensis were transient species, and the remaining species were intermediate species. As a beneficial intestinal bacterium, B. catenulatum can produce short-chain fatty acids, inhibit the release of proinflammatory factors, repair intestinal mucosal barriers, and suppress H. pylori adhesion and colonization through the gut-gastric axis, thereby indirectly consolidating eradication efficacy[35]. The decrease in C. aerofaciens abundance can reduce intestinal endotoxin release and alleviate mucosal inflammation, further facilitating H. pylori eradication[36]. Notably, the Blautia genus was significantly enriched after treatment in group A. As a core beneficial intestinal bacterium, Blautia not only produces butyrate to maintain intestinal barrier integrity but also regulates immunity and inhibits H. pylori-related inflammatory responses[37,38]. These findings suggest that berberine plays a dual role, exerting both bactericidal effects and probiotic-protective activity. All differential flora in the clarithromycin group were intermediate species. As important components of the intestinal flora, intermediate species regulate intestinal metabolism, barrier function, and inflammatory responses. Previous studies have confirmed that a reduced abundance of beneficial intestinal bacteria constitutes an important risk factor for H. pylori recurrence following eradication[30]. The decrease in beneficial bacteria (e.g., B. catenulatum) in the clarithromycin group may weaken the indirect inhibitory effect of the intestinal tract. Additionally, a total of 9 differential species were identified between group A and group B after treatment, among which Turicibacter bilis and Clostridium scindens were significantly enriched in group B. These two bacterial species are associated with intestinal inflammation and flora dysbiosis, and their increased abundance may further aggravate intestinal mucosal damage[39]. In contrast, 7 species were significantly enriched in group A, further highlighting the ability of berberine to regulate intestinal flora and protect microecological stability[40,41].
Differences in floral phenotype and function provide important insights into the clinical application of the two treatment regimens, particularly regarding the long-term safety and efficacy stability of H. pylori eradication. The phenotype of forms biofilms was markedly reduced in both groups after eradication treatment, with Bifidobacterium identified as the primary contributor to this phenotype. As demonstrated in the above taxonomic analysis, B. adolescentis was markedly depleted while B. catenulatum was significantly enriched after berberine-based treatment. In contrast, B. catenulatum exhibited a remarkable reduction in the clarithromycin group. These two species may be closely associated with alterations in the forms biofilms phenotype. Both B. adolescentis and B. catenulatum can synthesize extracellular polysaccharides to construct biofilm structures on the intestinal mucosal surface, and their biofilms exert protective effects by reinforcing intestinal barrier function and inhibiting the adhesion of harmful microbes[42]. Therefore, shifts in their relative abundance jointly drive the dynamic changes of the forms biofilms phenotype. Longicatena genus was enriched in group B after treatment under the contains mobile elements phenotypic category. As a first-line antibiotic for H. pylori eradication, the widespread use of clarithromycin has led to a gradual increase in H. pylori clarithromycin resistance rates[7]. The enrichment of mobile genetic elements (e.g., plasmids and transposons) in the intestinal flora may accelerate the dissemination of H. pylori resistance genes, increasing the difficulty of subsequent H. pylori eradication and representing a major limitation of clarithromycin-based regimens[43]. Additionally, key stress-responsive flora (the stress tolerant phenotype) in group B completely disappeared after treatment. The disappearance of stress-responsive flora reduces the resistance of the intestinal microecosystem to H. pylori colonization, increasing the likelihood of H. pylori reinfection.
Notably, this study has several limitations. Although the H. pylori eradication rates of the two groups were comparable, the correlation between changes in the bacterial flora and the stability of eradication efficacy (e.g., the recurrence rate) was not further analyzed. No long-term follow-up was conducted, so the long-term effects of the two regimens on intestinal flora and H. pylori recurrence cannot be verified. Furthermore, berberine is documented to inhibit H. pylori urease activity, which may induce false-negative 13C-urea breath test results and underestimate residual bacterial colonization. Hence, histopathological detection with specific H. pylori staining is preferred for efficacy verification in future trials. Additionally, the sample size was small, which may affect the generalizability of the results.
CONCLUSION
In conclusion, clarithromycin significantly disturbed the intestinal flora, leading to decreased diversity, structural remodeling, and the extensive inhibition of susceptible flora. It may also increase the enrichment of mobile genetic elements and the risk of H. pylori antibiotic resistance, which is not conducive to intestinal microecological balance or long-term prognosis after H. pylori eradication. The therapeutic effectiveness of berberine and clarithromycin is comparable for short-term H. pylori eradication. Notably, berberine has mild regulatory effects on the intestinal flora, modulates differential species, increases the abundance of beneficial bacteria (e.g., Blautia and B. catenulatum), and maintains flora homeostasis. It maximizes the protection of the intestinal microecological balance while ensuring equivalent eradication efficacy, providing important experimental evidence and theoretical support for optimizing clinical H. pylori eradication regimens and balancing therapeutic efficacy with microecological protection.
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Footnotes
Peer review: Externally peer reviewed.
Peer-review model: Single blind
Specialty type: Gastroenterology and hepatology
Country of origin: China
Peer-review report’s classification
Scientific quality: Grade A, Grade B, Grade B, Grade B, Grade C
Novelty: Grade A, Grade B, Grade B, Grade B, Grade B
Creativity or innovation: Grade B, Grade B, Grade B, Grade C, Grade C
Scientific significance: Grade A, Grade B, Grade B, Grade B, Grade C
P-Reviewer: Ansari S, Assistant Professor, PhD, United Arab Emirates; Baryshnikova NV, Associate Professor, MD, PhD, Russia; Sitkin S, Associate Professor, Head, MD, PhD, Russia S-Editor: Fan M L-Editor: A P-Editor: Lei YY
