Impact of supplementing Lactobacillus fermentum MN–LF23 on the eradication of Helicobacter pylori with 14–day standard quadruple therapy: A randomized, double–blind, placebo–controlled trial

Impact of supplementing Lactobacillus fermentum MN–LF23 on the eradication of Helicobacter pylori with 14–day standard quadruple therapy: A randomized, double–blind, placebo–controlled trial | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impact of supplementing Lactobacillus fermentum MN–LF23 on the eradication of Helicobacter pylori with 14–day standard quadruple therapy: A randomized, double–blind, placebo–controlled trial Yuyang Zhao, Xiaokang Niu, Yong Zhang, Liang Zhao, Liwei Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5403420/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Jul, 2025 Read the published version in Nutrition Journal → Version 1 posted 7 You are reading this latest preprint version Abstract Background: The effect of probiotics on Helicobacter pylori (Hp) infection demonstrates considerable heterogeneity. This study aims to elucidate the role of Lactobacillus fermentum MN–LF23 (MN–LF23) in Hp–infected populations. Methods: A total of 94 adult patients with confirmed Hp infection were enrolled in this study and randomly allocated to the placebo or MN–LF23 group. Patients initially received either placebo or probiotics along with standard quadruple therapy for 2 weeks, followed by continued administration of either placebo or probiotics for an additional 4 weeks. The eradication of Hp, serum levels of inflammatory factors, and alterations in gastrointestinal symptoms were assessed at weeks 0, 2, and 6, while fecal samples were collected for metagenomic sequencing. Results: The results showed no significant difference ( P = 1) in the eradication rate between the placebo group (85.11%) and the probiotic group (82.98%). Following treatment, the incidence of constipation, dyspepsia, and Gastrointestinal Symptom Rating Scale (GSRS) scores in the probiotic group were markedly lower ( P < 0.05) compared to those observed in the placebo group. Throughout the treatment process, there were no significant differences in TNF–α and IL–1β levels between the two groups. Compared to the placebo group, the probiotic group exhibited a significant increase in beneficial bacteria such as Lactobacillus fermentum , Lactobacillus plantarum , Bifidobacterium longum , Coprococcus caltus , and Clostridium butyricum. Conclusion: MN–LF23 supplementation did not improve the eradication rate of standard quadruple therapy. However, it significantly reduced the overall GSRS score, improved digestive and constipation symptoms, and promoted the proliferation of beneficial bacteria in the intestine. Helicobacter pylori Lactobacillus fermentum standard quadruple therapy gut microbiota metagenomic sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hp is a microaerophilic Gram–negative bacterium that widely colonizes the gastric antral mucosa. Hp is estimated to have infected more than half of the world’s population 1 , with an infection rate in the Chinese mainland of approximately 44.2% 2 . In 1994, the International Agency for Research on Cancer (IARC) under the World Health Organization (WHO) classified Hp as a Class I carcinogen 3 . The presence of Hp infection is closely associated with an increased risk of gastric diseases, including chronic gastritis, peptic ulcers, and gastric cancer 4 . Eradicating Hp can effectively reduce the risk of developing these diseases 5 . Triple and quadruple therapy with antibiotics are widely recognized as first–line treatment options internationally 6 . However, due to the widespread use of antibiotics, there has been a steady increase in Hp resistance over the years, leading to a rapid decline in the eradication rate of antibiotic therapy 7, 8 . Additionally, antibiotic therapy can result in side effects such as gastrointestinal dysfunction and an imbalance in gut microbiota in patients 9, 10 . Therefore, it is imperative to explore and develop new treatment plans. In recent years, probiotics have demonstrated significant potential in combating Hp infection. Studies have indicated that probiotics can enhance the eradication rate of antibiotic therapy and effectively mitigate the side effects associated with such treatment. Ismail et al. found that supplementing Lactobacillus reuteri for 4 weeks following a standard 2–week triple therapy regimen resulted in a substantial increase in the Hp eradication rate (91.1 vs. 68.9%; P = 0.007) and an improvement in drug treatment side effects compared to the placebo group 11 . Additionally, Viazis et al. discovered that the addition of four mixed probiotics to a 10–day non–bismuth quadruple Hp eradication regimen enhanced eradication rates and reduced side effects 12 . However, some studies have indicated that supplementation with Lactobacillus plantarum and Pediococcus acidilactici acidophilus in antibiotic treatment regimens does not improve eradication rates or reduce side effects 13 . Therefore, the impact of probiotics on the eradication rate and side effects of antibiotic treatment can be considered heterogeneous. We previously demonstrated that MN–LF23, isolated from dairy products, effectively reduced the abundance of Hp in mice infected with the bacteria. MN–LF23 has also been shown to regulate the immune response, decrease gastric inflammation, and improve the composition of the gastric antral mucosal microbiota 14 . However, its impact on human populations infected with Hp remains unknown. Therefore, we conducted a randomized, double–blind, placebo–controlled experiment to assess the impact of MN–LF23 on antibiotic therapy for eradicating Hp. We used metagenomic sequencing technology to analyze changes in the gastrointestinal microbiota before and after treatment. This study provides new insights for developing innovative strategies against Hp. Materials and methods Participants From July to October 2023, 94 subjects with Hp infection were consecutively recruited at the First Medical Center of Chinese PLA General Hospital in Beijing. The inclusion criteria were as follows: individuals aged 18–65 years with positive results from the 13 C–Urea Breath Test ( 13 C–UBT) within 2 weeks before enrollment. The exclusion criteria included a history of gastrointestinal surgery, previous treatment for Hp infection, use of antibiotics, probiotic products, proton pump inhibitors, H 2 receptor antagonists, bismuth agents, laxatives, antidiarrheals, or certain antibacterial traditional Chinese medicines within the past 4 weeks; current pregnancy, lactation, or preparation for pregnancy; presence of mental disorders, immune system disorders, cardiovascular diseases, lung diseases, liver diseases, kidney diseases, malignant tumors, or coagulation disorders; contraindications or allergies to amoxicillin, clarithromycin, bismuth agents or proton pump inhibitors; and participation in other clinical trials simultaneously. This study was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR220059817) and was approved by the Medical Ethics Committee of The First Medical Center of Chinese PLA General Hospital (approval number: S2022–060–02). This study was conducted in accordance with the principles of good clinical practice and the Declaration of Helsinki. All participants voluntarily signed a written informed consent form after fully understanding the potential benefits and risks of participating in the trial. Study design The doctor developed an eradication therapy based on clinical medication guidelines and the patient’s past medication history. The eradication regimen utilized in this study was a 14–day standard quadruple therapy consisting of omeprazole (20 mg) and colloidal bismuth pectin (200 mg) taken before meals, as well as amoxicillin 1 g and clarithromycin (500 mg) taken after meals twice a day. All medications were to be taken during morning and evening meal times. This is a prospective, randomized, double–blind, placebo–controlled trial. Following the prescription of the aforementioned drugs, the minimum randomization method was employed. The subjects were randomly assigned to either the placebo or probiotics group for treatment based on stratified factors, such as sex, age, and initial DOB value. We used probiotics in the form of a single MN–LF23 freeze–dried powder, with each bag containing 1 × 10 10 colony–forming units (CFU) of live cells. The placebo consisted of maltodextrin and had the same weight, appearance, and taste as the probiotic powder but lacked active ingredients. During the 14–day treatment period, both groups consumed one bag of probiotic powder or placebo 2 h after taking their medication. Within 4 weeks after the end of the medication period, both groups were instructed to take one bag of probiotic powder or placebo after breakfast and dinner. The participants are not allowed to consume any other probiotic products during the treatment period. The adherence of subjects to antibiotics and probiotic powders was assessed via daily online questionnaires. Study evaluations This study was conducted in four stages: the baseline screening period (weeks − 2–0, V0), a 2–week emptying period (weeks 0–2, V1), a 2–week probiotic and drug administration period (weeks 2–4, V2), and a 4–week probiotic administration period (weeks 4–8, V3). During the V0 phase, subjects underwent the 13 C–UBT. Baseline population data on age, sex, height, weight, body mass index (BMI), smoking habits, alcohol consumption, and allergy history were collected. Blood safety index tests were performed on the subjects at the end of phases V1 and V3. The Hp eradication rate was evaluated by the 13 C–UBT at the end of phases V2 and V3. Experimental data were collected throughout the entire intervention period. A comprehensive timeline of the study is outlined in Fig. 1 a. Assessment of gastrointestinal symptoms and adverse effects The participants were requested to recall their gastrointestinal symptom history over a 2–week period at the conclusion of the V1, V2, and V3 phases. The participants were also asked to complete the GSRS in the form of a questionnaire, which consisted of 15 questions across five levels: diarrhea, indigestion, constipation, abdominal pain, and reflux 15 . A 4–point Likert scale (absent, mild, moderate, severe) was used for scoring. Any other adverse reactions occurring outside the gastrointestinal tract were documented and assessed. Blood and stool samples Serum and fecal samples were collected from subjects at the conclusion of phases V1, V2, and V3. The blood collector used a blood collection vessel to obtain blood from the subjects, followed by immediate centrifugation of the collected serum and storage at -80°C. The participants were instructed to use disposable sterile fecal collection tubes for collecting fecal samples at the visit site, with immediate storage at -80°C. Serum indicator detection Enzyme–linked immunosorbent assay (ELISA) was used to detect variations in serum levels of tumor necrosis factor alpha (TNF–α) and interleukin1–β (IL–1β) during treatment, according to the instructions of the test kit (Jiangsu Meimian Industrial Co., Ltd, Yancheng, China). DNA extraction and metagenomic sequencing Fecal samples were subjected to whole metagenome shotgun sequencing on the Illumina NovaSeq PE150 platform at Majorbio Bio Pharm Technology Co., Ltd. (Shanghai, China). Metagenomic sequencing was conducted as previously described 16 . Briefly, total genomic DNA was extracted from human feces using an E.Z.N.A.® Soil DNA Kit (Omega Biotek, Norcross, GA, U.S.) according to the manufacturer’s instructions. The concentration and purity of total genomic DNA were assessed using a TBS 380 micro fluorometer (Turner Bio Systems, USA) and a NanoDrop2000 ultra micro spectrophotometer (Thermo Fisher Scientific, USA), respectively. Raw sequencing reads underwent quality control processing using fastp (0.23.0) 17 , which involved removing low–quality reads (> 45 bases with a quality score 5 ‘N’ bases), as well as low complexity and adapter–containing reads; the remaining reads were trimmed at the tails for low quality (< Q20) or ‘N’ bases. Human genomic reads were eliminated by mapping to the reference human genome (GRCh38) using Bowtie2 (2.4.4) 18 . The gut microbiota composition was quantified using MetaPhlAn4 (4.0.2) algorithms 19 . The alpha diversity of the gut microbiota was determined by calculating the Shannon and Simpson indices. The beta diversity based on principal coordinate analysis (PCoA) was estimated by calculating the Bray–Curtis distance. LEfSe was used to analyze differences in gut microbiota composition between groups. Statistical analysis Statistical analysis was performed using SPSS 28 software. For continuous variables, a normal or approximately normal distribution is described by mean ± standard deviation, while frequency (%) is used to describe the comparison of multiple groups of categorical variables. One–way analysis of variance was used for variables with a normal distribution or those that conformed to a normal distribution after log conversion, whereas Kruskal–Wallis rank sum test was used for non–normally distributed variables. The comparison of categorical variables between groups was conducted using the chi–square test or Fisher’s exact probability method. A P value < 0.05 was taken to indicate statistical significance. Results Baseline characteristics of patients Figure 1 b illustrates the process of subject enrollment and follow–up. Out of the 130 screened subjects, 94 eligible individuals were randomly assigned to receive placebo (n = 47) or probiotic (n = 47) intervention. Four participants from each group withdrew from the trial during the intervention process, and ITT analysis was employed to assess changes in relevant indicators throughout the intervention period. The baseline demographic data of the participants are supplemented in Table 1 . Table 1 Demographic and clinical characteristics of the patients included in the study Placebo (n = 47) Probiotic (n = 47) P value Age (y), mean (SD) 43.85 (12.02) 43.43 (11.63) 0.862 Male, n (%) 15 (31.91) 15 (31.91) 1 Female, n (%) 32 (68.09) 32 (69.09) 1 Height (cm), mean (SD) 165.57 (6.96) 165.38 (8.47) 0.905 Weight (kg), mean (SD) 66.18 (11.53) 65.78 (10.59) 0.860 BMI (kg/m2), mean (SD) 24.07 (3.45) 24.04 (3.34) 0.962 Smoker, n (%) 6 (12.77) 8 (17.02) 0.773 Drinker, n (%) 8 (17.02) 7 (14.89) 1 Baseline DOB, mean (SD) 35.65 (24.04) 33.56 (20.57) 0.653 13 C–UBT results The primary outcome measure of this study was Hp eradication, as indicated by a negative 13 C–UBT result. The eradication rates for the placebo and probiotics groups were 85.11% (40/47) and 82.98% (39/47), respectively, with no significant differences between the groups. The changes in DOB values during the intervention period are illustrated in Fig. 2 . Both groups showed significantly lower DOB values in stages V2 and V3 compared to stage V1 ( P < 0.001), with no significant difference observed between the groups during this period. GSRS scores The changes in the total GSRS score and scores for the five subscales (constipation, indigestion, reflux, abdominal pain, diarrhea) before and after treatment in both groups are depicted in Fig. 3 . The probiotic group exhibited a significantly lower total GSRS score at the V3 stage than at the V1 and V2 stages ( P < 0.01) (Fig. 3 a). In terms of constipation, the V2 score of the probiotic group was significantly lower than that of the V1 group ( P < 0.05), while the V3 score was significantly lower than that of the V1 group ( P < 0.001), with the V3 probiotic group score also being significantly lower than that of the placebo group ( P < 0.05) (Fig. 3 b). For indigestion, the V3 score of the probiotic group was significantly lower than that of both the V1 and V2 stages ( P < 0.01), and it was also significantly lower than that of the placebo group at this stage ( P < 0.05) (Fig. 3 c). Regarding reflux, the placebo group had a significantly lower score at stage V3 than at stage V2 ( P < 0.05) (Fig. 3 d). No significant trend was detected in the scale for abdominal pain and diarrhea (Figs. 4 e, f). Blood index analysis Figure 4 illustrates the variations in the concentration of pro–inflammatory cytokines in the serum of the subjects throughout the treatment period. Both groups exhibited a significant decrease in serum TNF–α levels after treatment compared to baseline (Fig. 4 a). However, no significant trend was observed in the serum IL–1β levels for either group during the treatment process (Fig. 4 b). Additionally, there were no notable differences in TNF–α and IL–1β levels between the probiotic and placebo groups during the same period. Gut microbiota analysis The changes in gut microbiota composition and diversity throughout the treatment period are illustrated in Fig. 5 . Compared to V1, both groups exhibited changes in the composition and abundance of gut microbiota at V2, which subsequently returned to baseline levels by V3. The composition of gut microbiota was similar between the two groups during the same period (Figs. 5 a–c). Throughout the treatment process, the trends in alpha and beta diversity were similar in both groups. No significant differences were observed in α and β diversity between the two groups during this period (Figs. 5 f–h). Relative to V1, there was a notable decrease in alpha diversity in both groups at V2, whereas beta diversity exhibited significant variations. However, both the alpha and beta diversity reverted to baseline levels by V3 (Figs. 5 d, e, i, j). LEfSe was used to analyze differences in gut microbiota levels between the two groups during the intervention process (Figs. 6 ). During the 2–week intervention, the probiotic group exhibited a significant enrichment of beneficial bacteria, such as Lactobacillus fermentum and Lactobacillus plantarum . In contrast, the placebo group exhibited a notable enrichment of harmful bacteria, including Fusobacterium nucleatum , Prevotella bivia , and Fusobacterium polymorphum . At 6 weeks of intervention, the probiotic group continued to show significant enrichment of beneficial bacteria, such as Bifidobacterium longum , Coprococcus caltus , Lactobacillus fermentum , Lactobacillus plantarum , and Clostridium butyricum . Discussion In this study, we assessed the impact of MN–LF23 supplementation on the eradication rate and side effects of 14–day standard quadruple therapy for Hp. Overall, our findings indicate that while supplementing with MN–LF23 did not lead to a significant improvement in the eradication rate of Hp, it did demonstrate effectiveness in reducing patients’ indigestion and constipation symptoms, as well as lowering GSRS scores. It was also found to have a positive effect on gut microbiota composition. The Hp eradication rate is influenced by various factors, including antibiotic resistance, past history of Hp treatment, smoking, and alcohol consumption. A previous meta–analysis revealed that the resistance rates of Hp to metronidazole (51.1 to 83.3%), clarithromycin (21.1 to 37.5%), and levofloxacin (17.8 to 42.7%) significantly increased from 2013 to 2023, whereas the resistance rates to tetracycline (1.4 to 0.2%) and furazolidone (4 to 0%) has decreased year by year 20 . Research has indicated that a history of Hp treatment, smoking, and alcohol consumption is correlated with the failure of Hp eradication 21–23 . In the current study, both the placebo and probiotic groups demonstrated eradication rates exceeding 80%, which may be attributed to the fact that the subjects had no history of Hp treatment, as well as low rates of smoking and alcohol consumption. Our results showed that MN–LF23 did not enhance the Hp eradication rate in quadruple therapy, which is consistent with previous findings. Supplementation of Lactobacillus plantarum and Pediococcus acidilactici in both triple therapy and non–bismuth quadruple therapy regimens did not enhance the Hp eradication rate [placebo 95% (95% confidence interval [CI]: 89 to 98%) vs. probiotic 97% (95% CI: 92 to 99%), P = 0.721] 13 . While other studies have shown that Saccharomyces boulardii CNCM I–745 24 and Lactobacillus gasseri OLL2716 25 can significantly improve the eradication rate of antibiotic therapy. The comparative study design revealed significant differences in the baseline characteristics, including history of Hp treatment, average age, and location. There were also variations in probiotic use regimens, such as the duration of use, dosage, and type of intake and antibiotic used. The subjects recruited for this study had no history of Hp treatment, which may have contributed to the higher antibiotic efficacy and may have weakened the role of probiotics in the study’s findings. Multiple studies have demonstrated the efficacy of probiotics in alleviating the adverse effects of antibiotic treatment 12, 26 . Following a 2 week standard triple therapy regimen, subjects were instructed to continue taking Lactobacillus rhamnosus DSM17648 or placebo for an additional 4 weeks. The probiotics group exhibited significant reductions in symptoms such as indigestion, constipation, abdominal pain, and overall GSRS scores post–treatment 11 . Furthermore, this study revealed that MN–LF23 effectively improved the symptoms of indigestion and constipation. Hp infection can induce the expression of pro–inflammatory cytokines, such as TNF–α 27 and IL–1β 28 , in gastric mucosal cells, leading to gastric inflammation. However, Hp infection does not necessarily cause elevated serum proinflammatory cytokine levels. A previous meta–analysis revealed that serum TNF–α levels were significantly higher in patients with Hp infection than in uninfected individuals (15 studies, 1224 subjects; standardized mean difference [SMD] 0.88; 95% CI: 0.46, 1.29; P ≤ 0.0001). However, there was no significant impact on serum IL–1β levels (6 studies, 463 subjects, SMD: 0.10; 95% CI: -0.39, 0.59; P = 0.682) 29 . Our research findings agree with the conclusions of this meta–analysis. Following the eradication of Hp, the serum TNF–α levels of the subjects were significantly reduced, whereas there was no significant effect on IL–1β levels. During the same period, there was no significant difference in serum inflammatory cytokine levels between the probiotic and placebo groups, which may be attributed to the initial eradication of Hp. The high efficacy of standard quadruple therapy weakened the role of probiotics. Given the rapid development of DNA sequencing technology, the close relationship between changes in the gut microbiota and various gastrointestinal diseases has been widely confirmed 30–33 . Metagenomic sequencing technology can identify species and even strains at the species level, with a higher depth of species identification 34 . However, most studies on the impact of Hp eradication on gut microbiota are based on 16S rRNA sequencing analysis 9, 35, 36 , which has limited effectiveness in identifying microbial species. The administration of antibiotics can have a significant impact on the composition of the gastrointestinal microbiota in individuals 37 . The findings of this study revealed that the alpha diversity of the gut microbiota in both the placebo and probiotic groups experienced a notable decrease at 2 weeks but returned to baseline levels by 6 weeks. Additionally, both groups exhibited significant changes in beta diversity at 2 weeks, which were restored by 6 weeks. However, no substantial difference in diversity was observed between the two groups during this timeframe. These results are consistent with previous research conducted by Tang et al., which also found no significant variance in diversity between a probiotic group (supplemented with Enterococcus faecalis and Bacillus subtilis ) and a placebo group 38 . However, other studies have also demonstrated that supplementation with inactivated Lactobacillus reuteri significantly enhances antibiotic–induced disruption of gut microbiota diversity 39 . In this study, although there was no discernible difference in the overall structure of the gut microbiota between the probiotic and placebo groups, the probiotic group exhibited a significant enrichment of beneficial bacteria such as Lactobacillus fermentum , Lactobacillus plantarum , Bifidobacterium longum , Coprococcus callus , and Clostridium butyricum , compared to the placebo group. These bacteria contribute to gut microbiota health. Specifically, Lactobacillus fermentum 40 , Lactobacillus plantarum 41 , and Bifidobacterium longum 42 have been shown to be beneficial in improving IBD, whereas Coprococcus caltus 43 and Clostridium butyricum 44 can produce butyric acid, which is advantageous for intestinal health. This suggests that MN–LF23 plays a positive role in mitigating antibiotic–induced disruption of the gastrointestinal microbiota and may explain why the probiotic group experienced significantly lower levels of indigestion, constipation, and GSRS scores compared to the placebo group. In this study, we used the independently developed MN–LF23, which has a comprehensive preliminary experimental basis from screening to animal experiments. The changes in the gastrointestinal microbiota of patients during the treatment process and the role of MN–LF23 were subjected to in–depth analysis using metagenomic sequencing technology. We also assessed the impact of MN–LF23 on the efficacy and adverse reactions of antibiotics at the end of treatment (V2). There are still some limitations to this study. First, the subjects included in the study were middle–aged and elderly. Second, the tracking period was relatively short and can be followed for another 4 weeks after V3 to fully detect dynamic changes in the gut microbiota. Furthermore, the gastric mucosa of the subjects was not collected; therefore, the impact of probiotics on the patients’ gastric microbiota remains unknown. Conclusions In conclusion, our findings suggest that supplementation with MN–LF23 does not improve the eradication rate of the 14–day standard quadruple therapy. However, MN–LF23 was effective in alleviating the side effects associated with antibiotic treatment, including reducing indigestion, constipation, and GSRS scores, as well as regulating the gut microbiota balance. Abbreviations Hp : Helicobacter Pylori MN–LF23 : Lactobacillus Fermentum MN–LF23 GSRS : Gastrointestinal Symptom Rating Scale IARC : International Agency for Research on Cancer WHO : World Health Organization 13 C–UBT : 13 C–Urea Breath Test CFU : Colony–forming Units BMI : Body Mass Index ELISA : Enzyme–linked Immunosorbent Assay TNF – α : Tumor Necrosis Factor Alpha IL – 1β : Interleukin1–β PCoA : Principal Coordinate Analysis ITT : Intention–to–treat Declarations Ethics approval and consent to participate This study was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR220059817) and was approved by the Medical Ethics Committee of The First Medical Center of Chinese PLA General Hospital (approval number: S2022–060–02). This study was conducted in accordance with the principles of good clinical practice and the Declaration of Helsinki. All participants voluntarily signed a written informed consent form after fully understanding the potential benefits and risks of participating in the trial. Consent for publication All study subjects gave their written informed consent that their data could be used for publications in anonymized form. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research was funded by the National Key R&D Program of China (No. 2022YFF1100100). Authors’ contributions Conceptualization, LZ, YM, XZ and RW; Methodology, YZ, YZ, LZ, JH, and RW; Formal analysis and investigation, YZ, XN, LZ and QZ; Writing–original draft preparation, YZ; Writing–review and editing, FW, XZ and RW; Supervision, YZ, YM, FW and XZ; Resources, YM, FW, XZ and RW. All authors read and approved the final manuscript. 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Overlapping cytokines in H. pylori infection and gastric cancer: A tandem meta-analysis [J]. Front Immunol, 2023, 14: 1125658. Halfvarson J, Brislawn CJ, Lamendella R, et al. Dynamics of the human gut microbiome in inflammatory bowel disease [J]. Nat Microbiol, 2017, 2: 17004. Cao C, Yue S, Lu A, et al. Host-Gut Microbiota Metabolic Interactions and Their Role in Precision Diagnosis and Treatment of Gastrointestinal Cancers [J]. Pharmacol Res, 2024, 207: 107321. Shan Y, Lee M, Chang EB. The Gut Microbiome and Inflammatory Bowel Diseases [J]. Annu Rev Med, 2022, 73: 455-68. De Vos WM, Tilg H, Van Hul M, et al. Gut microbiome and health: mechanistic insights [J]. Gut, 2022, 71(5): 1020-32. Mannion A, Sheh A, Shen Z, et al. Shotgun Metagenomics of Gastric Biopsies Reveals Compositional and Functional Microbiome Shifts in High- and Low-Gastric-Cancer-Risk Populations from Colombia, South America [J]. Gut Microbes, 2023, 15(1): 2186677. Chen L, Xu W, Lee A, et al. The impact of Helicobacter pylori infection, eradication therapy and probiotic supplementation on gut microenvironment homeostasis: An open-label, randomized clinical trial [J]. EBioMedicine, 2018, 35: 87-96. Hu Y, Xu X, Ouyang YB, et al. Altered Gut Microbiota and Short-Chain Fatty Acids After Vonoprazan-Amoxicillin Dual Therapy for Helicobacter pylori Eradication [J]. Front Cell Infect Microbiol, 2022, 12: 881968. Wang L, Yao H, Tong T, et al. Dynamic changes in antibiotic resistance genes and gut microbiota after Helicobacter pylori eradication therapies [J]. Helicobacter, 2022, 27(2): e12871. Tang B, Tang L, Huang C, et al. The Effect of Probiotics Supplementation on Gut Microbiota After Helicobacter pylori Eradication: A Multicenter Randomized Controlled Trial [J]. Infect Dis Ther, 2021, 10(1): 317-33. Yang C, Liang L, Lv P, et al. Effects of non-viable Lactobacillus reuteri combining with 14-day standard triple therapy on Helicobacter pylori eradication: A randomized double-blind placebo-controlled trial [J]. Helicobacter, 2021, 26(6): e12856. Jang YJ, Kim WK, Han DH, et al. Lactobacillus fermentum species ameliorate dextran sulfate sodium-induced colitis by regulating the immune response and altering gut microbiota [J]. Gut Microbes, 2019, 10(6): 696-711. Liu Y, Liu G, Fang J. Progress on the mechanisms of Lactobacillus plantarum to improve intestinal barrier function in ulcerative colitis [J]. J Nutr Biochem, 2024, 124: 109505. Yao S, Zhao Z, Wang W, et al. Bifidobacterium Longum : Protection against Inflammatory Bowel Disease [J]. J Immunol Res, 2021, 2021: 8030297. Sheridan PO, Louis P, Tsompanidou E, et al. Distribution, organization and expression of genes concerned with anaerobic lactate utilization in human intestinal bacteria [J]. Microb Genom, 2022, 8(1). Stoeva MK, Garcia-So J, Justice N, et al. Butyrate-producing human gut symbiont, Clostridium butyricum , and its role in health and disease [J]. Gut Microbes, 2021, 13(1): 1-28. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 07 Jul, 2025 Read the published version in Nutrition Journal → Version 1 posted Editorial decision: Revision requested 14 Mar, 2025 Reviews received at journal 05 Mar, 2025 Reviewers agreed at journal 28 Feb, 2025 Reviewers invited by journal 23 Nov, 2024 Editor assigned by journal 13 Nov, 2024 Submission checks completed at journal 07 Nov, 2024 First submitted to journal 06 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5403420","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381912261,"identity":"07599f3a-7d8e-452f-ba96-0fc17432db6d","order_by":0,"name":"Yuyang Zhao","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yuyang","middleName":"","lastName":"Zhao","suffix":""},{"id":381912262,"identity":"9d4a1fa0-9204-4e02-beda-af165cca922a","order_by":1,"name":"Xiaokang Niu","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaokang","middleName":"","lastName":"Niu","suffix":""},{"id":381912263,"identity":"c676f11f-6b46-4983-8994-a351be7776f5","order_by":2,"name":"Yong Zhang","email":"","orcid":"","institution":"The First Medical Center of Chinese PLA General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Zhang","suffix":""},{"id":381912264,"identity":"fb449576-9e27-456e-95b1-1e081dd78db6","order_by":3,"name":"Liang Zhao","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Zhao","suffix":""},{"id":381912265,"identity":"909af752-6ea8-471b-a21c-a8385c42fa5b","order_by":4,"name":"Liwei Zhang","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Liwei","middleName":"","lastName":"Zhang","suffix":""},{"id":381912266,"identity":"ce7f3bf9-f1c4-4afd-acac-77ab11945182","order_by":5,"name":"Jingjing He","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"He","suffix":""},{"id":381912267,"identity":"75ed9c6a-a68e-452d-88ee-ec4d81d2e8d9","order_by":6,"name":"Qi Zhang","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Zhang","suffix":""},{"id":381912268,"identity":"a3be7d67-7934-40a8-bd80-89f6cec15b23","order_by":7,"name":"Yuejian Mao","email":"","orcid":"","institution":"Inner Mongolia Mengniu Dairy (Group) Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Yuejian","middleName":"","lastName":"Mao","suffix":""},{"id":381912269,"identity":"d3e5bb36-bd20-4566-af32-97e8d08a1b6c","order_by":8,"name":"Fuqing Wang","email":"","orcid":"","institution":"Tibet Tianhong Science and Technology Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Fuqing","middleName":"","lastName":"Wang","suffix":""},{"id":381912270,"identity":"df4b6592-1e6a-4aa3-aedb-231e884cbc22","order_by":9,"name":"Xiaohui Zhao","email":"","orcid":"","institution":"Inner Mongolia Mengniu Dairy (Group) Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Zhao","suffix":""},{"id":381912271,"identity":"1952e111-4d19-4ecc-994a-e117b43a2402","order_by":10,"name":"Ran Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBADOQjFRoIWY9K1JDYQrcXg+NnDrytq7qT3t58xYPhQdpiBf3YDAS1n8tIszxx7ljvjTI4B44xzhxkk7hzAr8XsQI6ZYQPb4dwNDDkGzLxthxkMJBIIaDn/Bqjl3+F0A/43Bsx/idJyI8f4YWPb4QQDCaAtjMRosb/xxoyxse+w4YwbzwoO9pxL55G4QUCLZH+O8ceGb4fl+fuTNz74UWYtxz+DgBYgYJOAsQ4AMQ9B9UDA/IEYVaNgFIyCUTCCAQD0jkV20kxo2AAAAABJRU5ErkJggg==","orcid":"","institution":"China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Ran","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-11-06 14:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5403420/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5403420/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12937-025-01124-6","type":"published","date":"2025-07-07T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70924563,"identity":"e85af208-c91e-4624-9f1b-252d4ae9f81a","added_by":"auto","created_at":"2024-12-09 09:04:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":105187,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of patients throughout the study. a: Timeline of the study. b: Flow diagram of the study. ITT: Intention–to–treat.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5403420/v1/9976f6b0d6cb0b07f24545b6.png"},{"id":70923833,"identity":"d71e7072-0995-4e66-b4b3-2c43a78f319e","added_by":"auto","created_at":"2024-12-09 08:56:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42210,"visible":true,"origin":"","legend":"\u003cp\u003eChange in DOB values of the two groups during treatment. DOB: \u003csup\u003e13\u003c/sup\u003eC–UBT detection value, DOB \u0026gt; 4 indicates a positive Hp infection, whereas anything less indicates a negative result; *\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5403420/v1/b0f0cecd1880a9bc63588713.png"},{"id":70923832,"identity":"bc240a83-15a0-4e4a-a53c-e41b28d896c9","added_by":"auto","created_at":"2024-12-09 08:56:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":167142,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the GSRS total score and the scores of its five subscales. a: GSRS total score. b: Constipation symptom score. c: Indigestion symptom score. d: Reflux symptom score. e: Abdominal symptom score. f: Diarrhea symptom score. *\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5403420/v1/38915f04f8d1c99008004eba.png"},{"id":70923827,"identity":"4213645f-4f4e-4f37-9efe-43a8fa4238e1","added_by":"auto","created_at":"2024-12-09 08:56:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90187,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the serum levels of pro–inflammatory cytokines before and after treatment. a: TNF–α concentration changes. b: IL–1β concentration changes. *\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5403420/v1/e056a33ce7db30f717de934f.png"},{"id":70923830,"identity":"42f693fa-a8e6-4b08-ad33-edcc9b8a458e","added_by":"auto","created_at":"2024-12-09 08:56:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":298600,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in gut microbiota composition and alpha and beta diversity during treatment. a–c: Changes in gut microbiota at the phylum, genus, and species levels. d and e: Changes in alpha diversity of gut microbiota between the two groups. f–h: Comparison of beta diversity between the two groups of subjects during the same period. i and j: Changes in beta diversity in the placebo group and probiotic group. Means followed by different letters indicate significant differences, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5403420/v1/a3f581262feb81515d15b919.png"},{"id":70923831,"identity":"85a2f09b-329d-4993-8058-d63db699b735","added_by":"auto","created_at":"2024-12-09 08:56:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":116140,"visible":true,"origin":"","legend":"\u003cp\u003eThe species level differences in gut microbiota between the two groups during treatment. a: Two–week intervention. b: Six–week intervention.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5403420/v1/6c1813d503f27e89be122464.png"},{"id":86699358,"identity":"c93c4749-647e-485d-b0be-edeb0993332f","added_by":"auto","created_at":"2025-07-14 16:08:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1544364,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5403420/v1/976b3646-2a6c-436c-a4d2-ab3463b8d9de.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of supplementing Lactobacillus fermentum MN–LF23 on the eradication of Helicobacter pylori with 14–day standard quadruple therapy: A randomized, double–blind, placebo–controlled trial","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHp is a microaerophilic Gram\u0026ndash;negative bacterium that widely colonizes the gastric antral mucosa. Hp is estimated to have infected more than half of the world\u0026rsquo;s population\u003csup\u003e1\u003c/sup\u003e, with an infection rate in the Chinese mainland of approximately 44.2%\u003csup\u003e2\u003c/sup\u003e. In 1994, the International Agency for Research on Cancer (IARC) under the World Health Organization (WHO) classified Hp as a Class I carcinogen\u003csup\u003e3\u003c/sup\u003e. The presence of Hp infection is closely associated with an increased risk of gastric diseases, including chronic gastritis, peptic ulcers, and gastric cancer\u003csup\u003e4\u003c/sup\u003e. Eradicating Hp can effectively reduce the risk of developing these diseases\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTriple and quadruple therapy with antibiotics are widely recognized as first\u0026ndash;line treatment options internationally\u003csup\u003e6\u003c/sup\u003e. However, due to the widespread use of antibiotics, there has been a steady increase in Hp resistance over the years, leading to a rapid decline in the eradication rate of antibiotic therapy\u003csup\u003e7, 8\u003c/sup\u003e. Additionally, antibiotic therapy can result in side effects such as gastrointestinal dysfunction and an imbalance in gut microbiota in patients\u003csup\u003e9, 10\u003c/sup\u003e. Therefore, it is imperative to explore and develop new treatment plans.\u003c/p\u003e \u003cp\u003eIn recent years, probiotics have demonstrated significant potential in combating Hp infection. Studies have indicated that probiotics can enhance the eradication rate of antibiotic therapy and effectively mitigate the side effects associated with such treatment. Ismail et al. found that supplementing \u003cem\u003eLactobacillus reuteri\u003c/em\u003e for 4 weeks following a standard 2\u0026ndash;week triple therapy regimen resulted in a substantial increase in the Hp eradication rate (91.1 vs. 68.9%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007) and an improvement in drug treatment side effects compared to the placebo group\u003csup\u003e11\u003c/sup\u003e. Additionally, Viazis et al. discovered that the addition of four mixed probiotics to a 10\u0026ndash;day non\u0026ndash;bismuth quadruple Hp eradication regimen enhanced eradication rates and reduced side effects\u003csup\u003e12\u003c/sup\u003e. However, some studies have indicated that supplementation with \u003cem\u003eLactobacillus plantarum\u003c/em\u003e and \u003cem\u003ePediococcus acidilactici acidophilus\u003c/em\u003e in antibiotic treatment regimens does not improve eradication rates or reduce side effects\u003csup\u003e13\u003c/sup\u003e. Therefore, the impact of probiotics on the eradication rate and side effects of antibiotic treatment can be considered heterogeneous.\u003c/p\u003e \u003cp\u003eWe previously demonstrated that MN\u0026ndash;LF23, isolated from dairy products, effectively reduced the abundance of Hp in mice infected with the bacteria. MN\u0026ndash;LF23 has also been shown to regulate the immune response, decrease gastric inflammation, and improve the composition of the gastric antral mucosal microbiota\u003csup\u003e14\u003c/sup\u003e. However, its impact on human populations infected with Hp remains unknown.\u003c/p\u003e \u003cp\u003eTherefore, we conducted a randomized, double\u0026ndash;blind, placebo\u0026ndash;controlled experiment to assess the impact of MN\u0026ndash;LF23 on antibiotic therapy for eradicating Hp. We used metagenomic sequencing technology to analyze changes in the gastrointestinal microbiota before and after treatment. This study provides new insights for developing innovative strategies against Hp.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants\u003c/h2\u003e \u003cp\u003eFrom July to October 2023, 94 subjects with Hp infection were consecutively recruited at the First Medical Center of Chinese PLA General Hospital in Beijing. The inclusion criteria were as follows: individuals aged 18\u0026ndash;65 years with positive results from the \u003csup\u003e13\u003c/sup\u003eC\u0026ndash;Urea Breath Test (\u003csup\u003e13\u003c/sup\u003eC\u0026ndash;UBT) within 2 weeks before enrollment. The exclusion criteria included a history of gastrointestinal surgery, previous treatment for Hp infection, use of antibiotics, probiotic products, proton pump inhibitors, H\u003csub\u003e2\u003c/sub\u003e receptor antagonists, bismuth agents, laxatives, antidiarrheals, or certain antibacterial traditional Chinese medicines within the past 4 weeks; current pregnancy, lactation, or preparation for pregnancy; presence of mental disorders, immune system disorders, cardiovascular diseases, lung diseases, liver diseases, kidney diseases, malignant tumors, or coagulation disorders; contraindications or allergies to amoxicillin, clarithromycin, bismuth agents or proton pump inhibitors; and participation in other clinical trials simultaneously.\u003c/p\u003e \u003cp\u003e This study was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR220059817) and was approved by the Medical Ethics Committee of The First Medical Center of Chinese PLA General Hospital (approval number: S2022\u0026ndash;060\u0026ndash;02). This study was conducted in accordance with the principles of good clinical practice and the Declaration of Helsinki. All participants voluntarily signed a written informed consent form after fully understanding the potential benefits and risks of participating in the trial.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy design\u003c/h3\u003e\n\u003cp\u003e The doctor developed an eradication therapy based on clinical medication guidelines and the patient\u0026rsquo;s past medication history. The eradication regimen utilized in this study was a 14\u0026ndash;day standard quadruple therapy consisting of omeprazole (20 mg) and colloidal bismuth pectin (200 mg) taken before meals, as well as amoxicillin 1 g and clarithromycin (500 mg) taken after meals twice a day. All medications were to be taken during morning and evening meal times.\u003c/p\u003e \u003cp\u003eThis is a prospective, randomized, double\u0026ndash;blind, placebo\u0026ndash;controlled trial. Following the prescription of the aforementioned drugs, the minimum randomization method was employed. The subjects were randomly assigned to either the placebo or probiotics group for treatment based on stratified factors, such as sex, age, and initial DOB value.\u003c/p\u003e \u003cp\u003eWe used probiotics in the form of a single MN\u0026ndash;LF23 freeze\u0026ndash;dried powder, with each bag containing 1 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e colony\u0026ndash;forming units (CFU) of live cells. The placebo consisted of maltodextrin and had the same weight, appearance, and taste as the probiotic powder but lacked active ingredients. During the 14\u0026ndash;day treatment period, both groups consumed one bag of probiotic powder or placebo 2 h after taking their medication. Within 4 weeks after the end of the medication period, both groups were instructed to take one bag of probiotic powder or placebo after breakfast and dinner.\u003c/p\u003e \u003cp\u003eThe participants are not allowed to consume any other probiotic products during the treatment period. The adherence of subjects to antibiotics and probiotic powders was assessed via daily online questionnaires.\u003c/p\u003e\n\u003ch3\u003eStudy evaluations\u003c/h3\u003e\n\u003cp\u003eThis study was conducted in four stages: the baseline screening period (weeks \u0026minus;\u0026thinsp;2\u0026ndash;0, V0), a 2\u0026ndash;week emptying period (weeks 0\u0026ndash;2, V1), a 2\u0026ndash;week probiotic and drug administration period (weeks 2\u0026ndash;4, V2), and a 4\u0026ndash;week probiotic administration period (weeks 4\u0026ndash;8, V3).\u003c/p\u003e \u003cp\u003eDuring the V0 phase, subjects underwent the \u003csup\u003e13\u003c/sup\u003eC\u0026ndash;UBT. Baseline population data on age, sex, height, weight, body mass index (BMI), smoking habits, alcohol consumption, and allergy history were collected.\u003c/p\u003e \u003cp\u003eBlood safety index tests were performed on the subjects at the end of phases V1 and V3. The Hp eradication rate was evaluated by the \u003csup\u003e13\u003c/sup\u003eC\u0026ndash;UBT at the end of phases V2 and V3. Experimental data were collected throughout the entire intervention period.\u003c/p\u003e \u003cp\u003eA comprehensive timeline of the study is outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAssessment of gastrointestinal symptoms and adverse effects\u003c/h3\u003e\n\u003cp\u003eThe participants were requested to recall their gastrointestinal symptom history over a 2\u0026ndash;week period at the conclusion of the V1, V2, and V3 phases. The participants were also asked to complete the GSRS in the form of a questionnaire, which consisted of 15 questions across five levels: diarrhea, indigestion, constipation, abdominal pain, and reflux\u003csup\u003e15\u003c/sup\u003e. A 4\u0026ndash;point Likert scale (absent, mild, moderate, severe) was used for scoring. Any other adverse reactions occurring outside the gastrointestinal tract were documented and assessed.\u003c/p\u003e\n\u003ch3\u003eBlood and stool samples\u003c/h3\u003e\n\u003cp\u003eSerum and fecal samples were collected from subjects at the conclusion of phases V1, V2, and V3. The blood collector used a blood collection vessel to obtain blood from the subjects, followed by immediate centrifugation of the collected serum and storage at -80\u0026deg;C. The participants were instructed to use disposable sterile fecal collection tubes for collecting fecal samples at the visit site, with immediate storage at -80\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSerum indicator detection\u003c/h2\u003e \u003cp\u003e Enzyme\u0026ndash;linked immunosorbent assay (ELISA) was used to detect variations in serum levels of tumor necrosis factor alpha (TNF\u0026ndash;α) and interleukin1\u0026ndash;β (IL\u0026ndash;1β) during treatment, according to the instructions of the test kit (Jiangsu Meimian Industrial Co., Ltd, Yancheng, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA extraction and metagenomic sequencing\u003c/h3\u003e\n\u003cp\u003eFecal samples were subjected to whole metagenome shotgun sequencing on the Illumina NovaSeq PE150 platform at Majorbio Bio Pharm Technology Co., Ltd. (Shanghai, China). Metagenomic sequencing was conducted as previously described\u003csup\u003e16\u003c/sup\u003e. Briefly, total genomic DNA was extracted from human feces using an E.Z.N.A.\u0026reg; Soil DNA Kit (Omega Biotek, Norcross, GA, U.S.) according to the manufacturer\u0026rsquo;s instructions. The concentration and purity of total genomic DNA were assessed using a TBS 380 micro fluorometer (Turner Bio Systems, USA) and a NanoDrop2000 ultra micro spectrophotometer (Thermo Fisher Scientific, USA), respectively. Raw sequencing reads underwent quality control processing using fastp (0.23.0)\u003csup\u003e17\u003c/sup\u003e, which involved removing low\u0026ndash;quality reads (\u0026gt;\u0026thinsp;45 bases with a quality score\u0026thinsp;\u0026lt;\u0026thinsp;20 or \u0026gt;\u0026thinsp;5 \u0026lsquo;N\u0026rsquo; bases), as well as low complexity and adapter\u0026ndash;containing reads; the remaining reads were trimmed at the tails for low quality (\u0026lt;\u0026thinsp;Q20) or \u0026lsquo;N\u0026rsquo; bases. Human genomic reads were eliminated by mapping to the reference human genome (GRCh38) using Bowtie2 (2.4.4)\u003csup\u003e18\u003c/sup\u003e. The gut microbiota composition was quantified using MetaPhlAn4 (4.0.2) algorithms\u003csup\u003e19\u003c/sup\u003e. The alpha diversity of the gut microbiota was determined by calculating the Shannon and Simpson indices. The beta diversity based on principal coordinate analysis (PCoA) was estimated by calculating the Bray\u0026ndash;Curtis distance. LEfSe was used to analyze differences in gut microbiota composition between groups.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS 28 software. For continuous variables, a normal or approximately normal distribution is described by mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, while frequency (%) is used to describe the comparison of multiple groups of categorical variables. One\u0026ndash;way analysis of variance was used for variables with a normal distribution or those that conformed to a normal distribution after log conversion, whereas Kruskal\u0026ndash;Wallis rank sum test was used for non\u0026ndash;normally distributed variables. The comparison of categorical variables between groups was conducted using the chi\u0026ndash;square test or Fisher\u0026rsquo;s exact probability method. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was taken to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBaseline characteristics of patients\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb illustrates the process of subject enrollment and follow\u0026ndash;up. Out of the 130 screened subjects, 94 eligible individuals were randomly assigned to receive placebo (n\u0026thinsp;=\u0026thinsp;47) or probiotic (n\u0026thinsp;=\u0026thinsp;47) intervention. Four participants from each group withdrew from the trial during the intervention process, and ITT analysis was employed to assess changes in relevant indicators throughout the intervention period. The baseline demographic data of the participants are supplemented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and clinical characteristics of the patients included in the study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlacebo\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;47)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProbiotic\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;47)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (y), mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43.85 (12.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43.43 (11.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.862\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15 (31.91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15 (31.91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32 (68.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32 (69.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeight (cm), mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e165.57 (6.96)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e165.38 (8.47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.905\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight (kg), mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e66.18 (11.53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e65.78 (10.59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.860\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (kg/m2), mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24.07 (3.45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24.04 (3.34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.962\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoker, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6 (12.77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8 (17.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.773\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrinker, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8 (17.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7 (14.89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaseline DOB, mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e35.65 (24.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33.56 (20.57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.653\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC\u0026ndash;UBT results\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe primary outcome measure of this study was Hp eradication, as indicated by a negative\u003c/p\u003e \u003cp\u003e \u003csup\u003e13\u003c/sup\u003eC\u0026ndash;UBT result. The eradication rates for the placebo and probiotics groups were 85.11% (40/47) and 82.98% (39/47), respectively, with no significant differences between the groups. The changes in DOB values during the intervention period are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Both groups showed significantly lower DOB values in stages V2 and V3 compared to stage V1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with no significant difference observed between the groups during this period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGSRS scores\u003c/h2\u003e \u003cp\u003eThe changes in the total GSRS score and scores for the five subscales (constipation, indigestion, reflux, abdominal pain, diarrhea) before and after treatment in both groups are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The probiotic group exhibited a significantly lower total GSRS score at the V3 stage than at the V1 and V2 stages (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In terms of constipation, the V2 score of the probiotic group was significantly lower than that of the V1 group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the V3 score was significantly lower than that of the V1 group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with the V3 probiotic group score also being significantly lower than that of the placebo group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). For indigestion, the V3 score of the probiotic group was significantly lower than that of both the V1 and V2 stages (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and it was also significantly lower than that of the placebo group at this stage (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Regarding reflux, the placebo group had a significantly lower score at stage V3 than at stage V2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). No significant trend was detected in the scale for abdominal pain and diarrhea (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBlood index analysis\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the variations in the concentration of pro\u0026ndash;inflammatory cytokines in the serum of the subjects throughout the treatment period. Both groups exhibited a significant decrease in serum TNF\u0026ndash;α levels after treatment compared to baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). However, no significant trend was observed in the serum IL\u0026ndash;1β levels for either group during the treatment process (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Additionally, there were no notable differences in TNF\u0026ndash;α and IL\u0026ndash;1β levels between the probiotic and placebo groups during the same period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGut microbiota analysis\u003c/h2\u003e \u003cp\u003eThe changes in gut microbiota composition and diversity throughout the treatment period are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Compared to V1, both groups exhibited changes in the composition and abundance of gut microbiota at V2, which subsequently returned to baseline levels by V3. The composition of gut microbiota was similar between the two groups during the same period (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026ndash;c). Throughout the treatment process, the trends in alpha and beta diversity were similar in both groups. No significant differences were observed in α and β diversity between the two groups during this period (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef\u0026ndash;h). Relative to V1, there was a notable decrease in alpha diversity in both groups at V2, whereas beta diversity exhibited significant variations. However, both the alpha and beta diversity reverted to baseline levels by V3 (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, e, i, j).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLEfSe was used to analyze differences in gut microbiota levels between the two groups during the intervention process (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring the 2\u0026ndash;week intervention, the probiotic group exhibited a significant enrichment of beneficial bacteria, such as \u003cem\u003eLactobacillus fermentum\u003c/em\u003e and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e. In contrast, the placebo group exhibited a notable enrichment of harmful bacteria, including \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e, \u003cem\u003ePrevotella bivia\u003c/em\u003e, and \u003cem\u003eFusobacterium polymorphum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAt 6 weeks of intervention, the probiotic group continued to show significant enrichment of beneficial bacteria, such as \u003cem\u003eBifidobacterium longum\u003c/em\u003e, \u003cem\u003eCoprococcus caltus\u003c/em\u003e, \u003cem\u003eLactobacillus fermentum\u003c/em\u003e, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, and \u003cem\u003eClostridium butyricum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we assessed the impact of MN\u0026ndash;LF23 supplementation on the eradication rate and side effects of 14\u0026ndash;day standard quadruple therapy for Hp. Overall, our findings indicate that while supplementing with MN\u0026ndash;LF23 did not lead to a significant improvement in the eradication rate of Hp, it did demonstrate effectiveness in reducing patients\u0026rsquo; indigestion and constipation symptoms, as well as lowering GSRS scores. It was also found to have a positive effect on gut microbiota composition.\u003c/p\u003e \u003cp\u003eThe Hp eradication rate is influenced by various factors, including antibiotic resistance, past history of Hp treatment, smoking, and alcohol consumption. A previous meta\u0026ndash;analysis revealed that the resistance rates of Hp to metronidazole (51.1 to 83.3%), clarithromycin (21.1 to 37.5%), and levofloxacin (17.8 to 42.7%) significantly increased from 2013 to 2023, whereas the resistance rates to tetracycline (1.4 to 0.2%) and furazolidone (4 to 0%) has decreased year by year\u003csup\u003e20\u003c/sup\u003e. Research has indicated that a history of Hp treatment, smoking, and alcohol consumption is correlated with the failure of Hp eradication\u003csup\u003e21\u0026ndash;23\u003c/sup\u003e. In the current study, both the placebo and probiotic groups demonstrated eradication rates exceeding 80%, which may be attributed to the fact that the subjects had no history of Hp treatment, as well as low rates of smoking and alcohol consumption.\u003c/p\u003e \u003cp\u003eOur results showed that MN\u0026ndash;LF23 did not enhance the Hp eradication rate in quadruple therapy, which is consistent with previous findings. Supplementation of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e and \u003cem\u003ePediococcus acidilactici\u003c/em\u003e in both triple therapy and non\u0026ndash;bismuth quadruple therapy regimens did not enhance the Hp eradication rate [placebo 95% (95% confidence interval [CI]: 89 to 98%) vs. probiotic 97% (95% CI: 92 to 99%), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.721]\u003csup\u003e13\u003c/sup\u003e. While other studies have shown that \u003cem\u003eSaccharomyces boulardii\u003c/em\u003e CNCM I\u0026ndash;745\u003csup\u003e24\u003c/sup\u003e and \u003cem\u003eLactobacillus gasseri\u003c/em\u003e OLL2716\u003csup\u003e25\u003c/sup\u003e can significantly improve the eradication rate of antibiotic therapy. The comparative study design revealed significant differences in the baseline characteristics, including history of Hp treatment, average age, and location. There were also variations in probiotic use regimens, such as the duration of use, dosage, and type of intake and antibiotic used. The subjects recruited for this study had no history of Hp treatment, which may have contributed to the higher antibiotic efficacy and may have weakened the role of probiotics in the study\u0026rsquo;s findings.\u003c/p\u003e \u003cp\u003eMultiple studies have demonstrated the efficacy of probiotics in alleviating the adverse effects of antibiotic treatment\u003csup\u003e12, 26\u003c/sup\u003e. Following a 2 week standard triple therapy regimen, subjects were instructed to continue taking \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e DSM17648 or placebo for an additional 4 weeks. The probiotics group exhibited significant reductions in symptoms such as indigestion, constipation, abdominal pain, and overall GSRS scores post\u0026ndash;treatment\u003csup\u003e11\u003c/sup\u003e. Furthermore, this study revealed that MN\u0026ndash;LF23 effectively improved the symptoms of indigestion and constipation.\u003c/p\u003e \u003cp\u003eHp infection can induce the expression of pro\u0026ndash;inflammatory cytokines, such as TNF\u0026ndash;α\u003csup\u003e27\u003c/sup\u003e and IL\u0026ndash;1β\u003csup\u003e28\u003c/sup\u003e, in gastric mucosal cells, leading to gastric inflammation. However, Hp infection does not necessarily cause elevated serum proinflammatory cytokine levels. A previous meta\u0026ndash;analysis revealed that serum TNF\u0026ndash;α levels were significantly higher in patients with Hp infection than in uninfected individuals (15 studies, 1224 subjects; standardized mean difference [SMD] 0.88; 95% CI: 0.46, 1.29; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001). However, there was no significant impact on serum IL\u0026ndash;1β levels (6 studies, 463 subjects, SMD: 0.10; 95% CI: -0.39, 0.59; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.682)\u003csup\u003e29\u003c/sup\u003e. Our research findings agree with the conclusions of this meta\u0026ndash;analysis. Following the eradication of Hp, the serum TNF\u0026ndash;α levels of the subjects were significantly reduced, whereas there was no significant effect on IL\u0026ndash;1β levels. During the same period, there was no significant difference in serum inflammatory cytokine levels between the probiotic and placebo groups, which may be attributed to the initial eradication of Hp. The high efficacy of standard quadruple therapy weakened the role of probiotics.\u003c/p\u003e \u003cp\u003eGiven the rapid development of DNA sequencing technology, the close relationship between changes in the gut microbiota and various gastrointestinal diseases has been widely confirmed\u003csup\u003e30\u0026ndash;33\u003c/sup\u003e. Metagenomic sequencing technology can identify species and even strains at the species level, with a higher depth of species identification\u003csup\u003e34\u003c/sup\u003e. However, most studies on the impact of Hp eradication on gut microbiota are based on 16S rRNA sequencing analysis\u003csup\u003e9, 35, 36\u003c/sup\u003e, which has limited effectiveness in identifying microbial species.\u003c/p\u003e \u003cp\u003eThe administration of antibiotics can have a significant impact on the composition of the gastrointestinal microbiota in individuals\u003csup\u003e37\u003c/sup\u003e. The findings of this study revealed that the alpha diversity of the gut microbiota in both the placebo and probiotic groups experienced a notable decrease at 2 weeks but returned to baseline levels by 6 weeks. Additionally, both groups exhibited significant changes in beta diversity at 2 weeks, which were restored by 6 weeks. However, no substantial difference in diversity was observed between the two groups during this timeframe. These results are consistent with previous research conducted by Tang et al., which also found no significant variance in diversity between a probiotic group (supplemented with \u003cem\u003eEnterococcus faecalis\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e) and a placebo group\u003csup\u003e38\u003c/sup\u003e. However, other studies have also demonstrated that supplementation with inactivated \u003cem\u003eLactobacillus reuteri\u003c/em\u003e significantly enhances antibiotic\u0026ndash;induced disruption of gut microbiota diversity\u003csup\u003e39\u003c/sup\u003e. In this study, although there was no discernible difference in the overall structure of the gut microbiota between the probiotic and placebo groups, the probiotic group exhibited a significant enrichment of beneficial bacteria such as \u003cem\u003eLactobacillus fermentum\u003c/em\u003e, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eBifidobacterium longum\u003c/em\u003e, \u003cem\u003eCoprococcus callus\u003c/em\u003e, and \u003cem\u003eClostridium butyricum\u003c/em\u003e, compared to the placebo group. These bacteria contribute to gut microbiota health. Specifically, \u003cem\u003eLactobacillus fermentum\u003c/em\u003e\u003csup\u003e40\u003c/sup\u003e, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e\u003csup\u003e41\u003c/sup\u003e, and \u003cem\u003eBifidobacterium longum\u003c/em\u003e\u003csup\u003e42\u003c/sup\u003e have been shown to be beneficial in improving IBD, whereas \u003cem\u003eCoprococcus caltus\u003c/em\u003e\u003csup\u003e43\u003c/sup\u003e and \u003cem\u003eClostridium butyricum\u003c/em\u003e\u003csup\u003e44\u003c/sup\u003e can produce butyric acid, which is advantageous for intestinal health. This suggests that MN\u0026ndash;LF23 plays a positive role in mitigating antibiotic\u0026ndash;induced disruption of the gastrointestinal microbiota and may explain why the probiotic group experienced significantly lower levels of indigestion, constipation, and GSRS scores compared to the placebo group.\u003c/p\u003e \u003cp\u003eIn this study, we used the independently developed MN\u0026ndash;LF23, which has a comprehensive preliminary experimental basis from screening to animal experiments. The changes in the gastrointestinal microbiota of patients during the treatment process and the role of MN\u0026ndash;LF23 were subjected to in\u0026ndash;depth analysis using metagenomic sequencing technology. We also assessed the impact of MN\u0026ndash;LF23 on the efficacy and adverse reactions of antibiotics at the end of treatment (V2).\u003c/p\u003e \u003cp\u003eThere are still some limitations to this study. First, the subjects included in the study were middle\u0026ndash;aged and elderly. Second, the tracking period was relatively short and can be followed for another 4 weeks after V3 to fully detect dynamic changes in the gut microbiota. Furthermore, the gastric mucosa of the subjects was not collected; therefore, the impact of probiotics on the patients\u0026rsquo; gastric microbiota remains unknown.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our findings suggest that supplementation with MN\u0026ndash;LF23 does not improve the eradication rate of the 14\u0026ndash;day standard quadruple therapy. However, MN\u0026ndash;LF23 was effective in alleviating the side effects associated with antibiotic treatment, including reducing indigestion, constipation, and GSRS scores, as well as regulating the gut microbiota balance.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHp\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e \u003cem\u003e\u0026nbsp;Helicobacter Pylori\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMN\u0026ndash;LF23\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003e\u003cem\u003eLactobacillus Fermentum\u0026nbsp;\u003c/em\u003eMN\u0026ndash;LF23\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGSRS\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eGastrointestinal Symptom Rating Scale\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIARC\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e International Agency for Research on Cancer\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWHO\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eWorld Health Organization\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003csup\u003e13\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eC\u0026ndash;UBT\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC\u0026ndash;Urea Breath Test\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCFU\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eColony\u0026ndash;forming Units\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBMI\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eBody Mass Index\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eELISA\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Enzyme\u0026ndash;linked Immunosorbent Assay\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTNF\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026alpha;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eTumor Necrosis Factor Alpha\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIL\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026ndash;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e1\u0026beta;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Interleukin1\u0026ndash;\u0026beta;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePCoA\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Principal Coordinate Analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eITT\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Intention\u0026ndash;to\u0026ndash;treat\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR220059817) and was approved by the Medical Ethics Committee of The First Medical Center of Chinese PLA General Hospital (approval number:\u0026nbsp;S2022\u0026ndash;060\u0026ndash;02). This study was conducted in accordance with the principles of good clinical practice and the Declaration of Helsinki. All participants voluntarily signed a written informed consent form after fully understanding the potential benefits and risks of participating in the trial.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll study subjects gave their written informed consent that their data could be used for publications in anonymized form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Key R\u0026amp;D Program of China (No. 2022YFF1100100).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, LZ, YM, XZ and RW; Methodology, YZ, YZ, LZ, JH, and RW; Formal analysis and investigation, YZ, XN, LZ and QZ; Writing\u0026ndash;original draft preparation, YZ; Writing\u0026ndash;review and editing, FW, XZ and RW; Supervision, YZ, YM, FW and XZ; Resources, YM, FW, XZ and RW. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank LetPub Ltd. for its linguistic assistance during the preparation of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHooi JKY, Lai WY, Ng WK, et al. Global Prevalence of \u003cem\u003eHelicobacter pylori\u003c/em\u003e Infection: Systematic Review and Meta-Analysis [J]. Gastroenterology, 2017, 153(2): 420-9.\u003c/li\u003e\n\u003cli\u003eRen S, Cai P, Liu Y, et al. Prevalence of \u003cem\u003eHelicobacter pylori\u003c/em\u003e infection in China: A systematic review and meta-analysis [J]. 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Effects of non-viable \u003cem\u003eLactobacillus reuteri \u003c/em\u003ecombining with 14-day standard triple therapy on \u003cem\u003eHelicobacter pylori\u003c/em\u003e eradication: A randomized double-blind placebo-controlled trial [J]. Helicobacter, 2021, 26(6): e12856.\u003c/li\u003e\n\u003cli\u003eJang YJ, Kim WK, Han DH, et al. \u003cem\u003eLactobacillus fermentum\u003c/em\u003e species ameliorate dextran sulfate sodium-induced colitis by regulating the immune response and altering gut microbiota [J]. Gut Microbes, 2019, 10(6): 696-711.\u003c/li\u003e\n\u003cli\u003eLiu Y, Liu G, Fang J. Progress on the mechanisms of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e to improve intestinal barrier function in ulcerative colitis [J]. J Nutr Biochem, 2024, 124: 109505.\u003c/li\u003e\n\u003cli\u003eYao S, Zhao Z, Wang W, et al. \u003cem\u003eBifidobacterium Longum\u003c/em\u003e: Protection against Inflammatory Bowel Disease [J]. J Immunol Res, 2021, 2021: 8030297.\u003c/li\u003e\n\u003cli\u003eSheridan PO, Louis P, Tsompanidou E, et al. Distribution, organization and expression of genes concerned with anaerobic lactate utilization in human intestinal bacteria [J]. Microb Genom, 2022, 8(1).\u003c/li\u003e\n\u003cli\u003eStoeva MK, Garcia-So J, Justice N, et al. Butyrate-producing human gut symbiont, \u003cem\u003eClostridium butyricum\u003c/em\u003e, and its role in health and disease [J]. Gut Microbes, 2021, 13(1): 1-28.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nutrition-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nutj","sideBox":"Learn more about [Nutrition Journal](http://nutritionj.biomedcentral.com/)","snPcode":"12937","submissionUrl":"https://submission.nature.com/new-submission/12937/3","title":"Nutrition Journal","twitterHandle":"@NutrJournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Helicobacter pylori, Lactobacillus fermentum, standard quadruple therapy, gut microbiota, metagenomic sequencing","lastPublishedDoi":"10.21203/rs.3.rs-5403420/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5403420/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The effect of probiotics on\u003cem\u003eHelicobacter pylori\u003c/em\u003e (Hp) infection demonstrates considerable heterogeneity. This study aims to elucidate the role of \u003cem\u003eLactobacillus fermentum \u003c/em\u003eMN–LF23 (MN–LF23) in Hp–infected populations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eA total of 94 adult patients with confirmed Hp infection were enrolled in this study and randomly allocated to the placebo or MN–LF23 group. Patients initially received either placebo or probiotics along with standard quadruple therapy for 2 weeks, followed by continued administration of either placebo or probiotics for an additional 4 weeks. The eradication of Hp, serum levels of inflammatory factors, and alterations in gastrointestinal symptoms were assessed at weeks 0, 2, and 6, while fecal samples were collected for metagenomic sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The results showed no significant difference (\u003cem\u003eP \u003c/em\u003e= 1) in the eradication rate between the placebo group (85.11%) and the probiotic group (82.98%). Following treatment, the incidence of constipation, dyspepsia, and Gastrointestinal Symptom Rating Scale (GSRS) scores in the probiotic group were markedly lower (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05) compared to those observed in the placebo group. Throughout the treatment process, there were no significant differences in TNF–α and IL–1β levels between the two groups. Compared to the placebo group, the probiotic group exhibited a significant increase in beneficial bacteria such as\u003cem\u003e Lactobacillus fermentum\u003c/em\u003e, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eBifidobacterium longum\u003c/em\u003e, \u003cem\u003eCoprococcus caltus\u003c/em\u003e, and \u003cem\u003eClostridium butyricum.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e MN–LF23 supplementation did not improve the eradication rate of standard quadruple therapy. However, it significantly reduced the overall GSRS score, improved digestive and constipation symptoms, and promoted the proliferation of beneficial bacteria in the intestine.\u003c/p\u003e","manuscriptTitle":"Impact of supplementing Lactobacillus fermentum MN–LF23 on the eradication of Helicobacter pylori with 14–day standard quadruple therapy: A randomized, double–blind, placebo–controlled trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-09 08:56:10","doi":"10.21203/rs.3.rs-5403420/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-14T09:16:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-05T10:58:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316385030684411093902658624021309857016","date":"2025-02-28T10:30:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-23T15:26:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-13T22:16:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-07T06:24:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Nutrition Journal","date":"2024-11-06T13:55:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nutrition-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nutj","sideBox":"Learn more about [Nutrition Journal](http://nutritionj.biomedcentral.com/)","snPcode":"12937","submissionUrl":"https://submission.nature.com/new-submission/12937/3","title":"Nutrition Journal","twitterHandle":"@NutrJournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b60c3987-ba2d-439b-8a5c-3ff42292f584","owner":[],"postedDate":"December 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-14T16:01:26+00:00","versionOfRecord":{"articleIdentity":"rs-5403420","link":"https://doi.org/10.1186/s12937-025-01124-6","journal":{"identity":"nutrition-journal","isVorOnly":false,"title":"Nutrition Journal"},"publishedOn":"2025-07-07 15:57:28","publishedOnDateReadable":"July 7th, 2025"},"versionCreatedAt":"2024-12-09 08:56:10","video":"","vorDoi":"10.1186/s12937-025-01124-6","vorDoiUrl":"https://doi.org/10.1186/s12937-025-01124-6","workflowStages":[]},"version":"v1","identity":"rs-5403420","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5403420","identity":"rs-5403420","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}