Gut microbiota changes related to Helicobacter pylori eradication with vonoprazan containing triple therapy among adolescents: a prospective multicenter study

Currently, it is unclear whether treating Helicobacter pylori (H. pylori) infection is safe among adolescents. This study aimed to evaluate the safety of H. pylori eradication therapy by examining gut microbiota changes in adolescents 3 months after the therapy. H. pylori-infected adolescents were enrolled in this study. Their stool samples were collected at the following three time points: before treatment, 1–2 days after completion of treatment, and time of eradication successful judgment. We assessed the relative abundance, alpha-diversity, and beta-diversity of the gut microbiota and adverse events. The number of isolated Actinobacteria decreased immediately after eradication therapy in the 16 students included in the study, and it returned to pretreatment condition at the eradication judgment point. There was no change in the relative abundance at genus level. The alpha-diversity was lost immediately after eradication therapy; however, it recovered at the time of eradication judgment, and it was restored to pretreatment condition. Meanwhile, none of the participants experienced serious adverse events. H. pylori eradication therapy is safe for adolescents with respect to gut microbiota changes associated with H. pylori eradication therapy. Therefore, further long-term evaluations of gut microbiota changes following eradication therapy are warranted.


Results
We enrolled students with two positive test results for H. pylori between June 2017 and March 2019. The flow of patient enrolment is presented in Fig. 1. Among the 151 patients with an H. pylori infection, 31 provided consent and participated in this study. There were 29 students who provided stool samples before eradication, 26 immediately after eradication, and 19 at the time of eradication judgment. Of these 19 patients, 3 experienced primary treatment failure; therefore, 16 (n = 9, male, n = 7, female; median age: 15.6 [15.1-15.9] years) were considered eligible for this study. This study was conducted as a sub analysis of H. pylori infection screening of junior high school third-year students in Saga Prefecture. Considering this, we could not thoroughly examine the background of the students. These students submitted their stool samples at each of the three time points. The H. pylori eradication assessments were conducted on median day 92.5 (range 89-107 days) after the eradication therapy, and the stool samples were collected on the median day 99 (range 91.5-108.5 days). www.nature.com/scientificreports/ Relative abundance. Figure 2 shows the compositions and relative abundance of the gut microbiota at the phyla level. The proportion of Actinobacteria significantly decreased after eradication therapy compared with before eradication therapy (P = 0.006924). On the contrary, the proportion of Actinobacteria significantly increased at the time of eradication assessment compared with after eradication therapy (P = 0.047065), and no significant difference was observed between before eradication therapy and at the time of eradication assessment (P = 0.214554). The compositions and relative abundance of the gut microbiota at the class level are shown in Fig. 3. Compared with before eradication therapy, the proportion of Coriobacteriia significantly decreased after eradication therapy (P = 0.003564) and eradication assessment (P = 0.025209). The compositions and relative abundance of the gut microbiota at the family level are shown in Fig. 4. Compared with before eradication therapy, the proportion of Coriobacteriaceae significantly decreased after eradication therapy (P = 0.008656). Figure 5 shows the top seven genus compositions of the relative abundance of gut microbiota. In each of the seven genuses, no significant changes were observed in the comparison results before eradication therapy, after eradication therapy, and during eradication judgment.

Alpha-diversity.
We calculated the α-diversity according to the PD whole tree, Shannon index, observed OTUs, and Chao1 at the three time points. Phylogenetic richness (PD whole tree; P = 0.003), diversity of microbiota (Shannon index; P = 0.023), and microbial species richness (observed OTUs; P = 0.003, Chao1; P = 0.001) after eradication therapy were significantly lower than those before eradication therapy. However, there were no significant differences between the PD whole tree (P = 1.000), Shannon index (P = 1.000), observed OTUs (P = 1.000), and Chao1 (P = 1.000) at the time of eradication judgment and those before eradication therapy. Moreover, the PD whole tree (P = 0.063), Shannon index (P = 0.492), observed OTUs (P = 0.106), and Chao1 (P = 0.061) at eradication judgment point did not significantly differ from those after eradication therapy (shown in Fig. 6).
Adverse events. Among the 16 patients 1 (6.35%) presented with diarrhea and 2 (12.5%) presented with abdominal pain. None of the adverse events were serious. Therefore, therapy was not required, and treatment for the eradication of H. pylori was not interrupted.

Discussion
The present study revealed changes in the gut microbiota before and after H. pylori eradication therapy using 16S rRNA gene/DNA/amplicon sequencing with next-generation sequencing and amplicon analysis in Japanese adolescents. Two important clinical suggestions were obtained. First, change in the gut microbiota during eradication therapy of H. pylori with vonoprazan fumarate containing triple therapy in adolescents led to dysbiosis immediately after eradication therapy; however, it returned to pretreatment conditions 3 months after the eradication therapy (shown in Figs. 2 and 6). Second, the eradication therapy of H. pylori with vonoprazan fumarate containing triple therapy was found safe for children from the perspective of adverse events. Yap TW et al. reported that the eradication of H. pylori caused perturbation of the gut microbiota, and it may indirectly affect the health of the patients 30 . By contrast, Gotoda T et al. reported that although H. pylori eradication therapy caused short-term dysbiosis, microbial diversity was restored in healthy Japanese adolescents 31 . To the best of our knowledge, this was the first study to evaluate the changes in the gut microbiota before and after H. pylori eradication therapy in adolescents. Moreover, α-diversity analysis revealed that species microbial richness and evenness were recovered to pretreatment levels at 2 months after eradication therapy. The proportion of Actinobacteria also significantly decreased immediately after eradication therapy, and it recovered similarly 2 months after eradication. Our results were considerably similar to those of a previous study (shown in Figs. 2 and 6) 31 . Cornejo-Pareja et al. observed a significant decrease in the number of Actinobacteria after the administration of omeprazole, clarithromycin, and amoxicillin in patients with H. pylori infection 32 . Our results also showed a decrease in Actinobacteria, which was due to a decrease in Coriobacteriia and Coriobacteriaceae at the class and family levels, respectively. We assumed that this occurred due to the effect of drug susceptibility to   www.nature.com/scientificreports/ the antibiotics used in the eradication therapy, but we are unsure of the exact etiology. In the present study, the safety of eradication therapy among adolescents may be recommended with respect to gut microbiota because the dysbiosis of gut microbiota recovered after several months to pretreatment levels. Liou 34 . However, information related to the relationship between H. pylori infection and intestinal bacteria included in the gut microbiota is still limited. Owing to the presence of differences in gut microbiota between H. pylori-infected and non-infected children, it cannot be denied that H. pylori infection affects children. From this perspective as well, H. pylori eradication in children appears to be necessary. To confirm the long-term safety of H. pylori eradication, future studies must be conducted to directly compare gut microbiota between the children who have remained healthy for a long time after H. pylori eradication therapy and healthy children without H. pylori infection.
The adverse events observed in our study included diarrhea and abdominal pain. However, these were not serious adverse events. The safety of eradication therapy of H. pylori with triple therapy containing vonoprazan fumarate for adolescents remains unclear; however, reports have shown no major problems in short-term evaluations 9,12,16 . In the present study, the number of cases was limited, and only adverse events during oral administration of antibacterial drugs and P-CAB were evaluated. Therefore, future studies with a larger sample size are warranted.
In Japan, the screening and treatment of H. pylori infection among adolescents are performed as a primary preventive measure against gastric cancer. By contrast, the clinical practice guidelines do not recommend screening and treatment for asymptomatic H. pylori-infected children to prevent gastric cancer 38,39 . However, these guidelines are for children and adolescents living in Europe and North America, and they may not be applicable   Figs. 2 and 6). However, it returned to pretreatment condition after 3 months. This may be one factor for the safety of H. pylori eradication treatment.
To prevent gastric cancer, H. pylori infection must be eradicated at a young age 6 . However, from the viewpoint of gastric cancer prevention, it is controversial whether the ideal period of H. pylori eradication is childhood or adulthood. It is certain that gastric mucosal atrophy associated with H. pylori infection is a high risk of welldifferentiated gastric cancer 41 . Furthermore, gastric mucosal atrophy is known to occur since childhood [42][43][44][45] , and a small proportion of gastric cancers that occur in childhood 46 . Therefore, it seems unsafe to conclude that eradication in adulthood is absolutely sufficient to prevent gastric cancer. Although the risk of H. pylori reinfection after eradication therapy must also be considered, the reinfection rate is considered to be extremely low [47][48][49] . Moreover, the safety of H. pylori eradication therapy for adolescents must be further validated 7,50 . This study had several limitations. First, the sample size was relatively small. Second, this study did not include placebo treatments for comparison. Third, the altered intestinal microbiota could be evaluated; the functionality of the bacteria itself and how it affected humans were also not evaluated. Intestinal microbiota analysis could not be performed by further analysis methods, such as UPGMA, LEfSe, and KEGG pathway. Fourth, because diet was important for microbiota evaluation, it was not possible to grasp the meal contents of each participant. However, because they were Japanese adolescents of almost the same age living in a single prefecture, it was presumed that there would be no major dietary differences. www.nature.com/scientificreports/ H. pylori eradication therapy may be safe for adolescents with respect to gut microbiota. Therefore, further long-term evaluations of the changes in gut microbiota after eradication therapy must be performed. If the safety of H. pylori eradication therapy in adolescents is validated, it will promote screening and treatment among adolescents to prevent gastric cancer.

Materials and methods
Study design and participants. The present study performed a sub analysis of H. pylori infection screening as part of the junior high school health screening system among third-year students in Saga Prefecture 9 . Using local governmental grants, a screening and treatment program for eradicating H. pylori infection among third-grade junior high students was started in Saga Prefecture in 2016. The students underwent urinary anti-H. pylori antibody tests (RAPIRAN; Otsuka Pharmaceutical Co., Ltd. Tokyo, Japan), followed by H. pylori stool antigen tests (TESTMATE RAPID RYLORI ANTIGEN; Wakamoto Pharmaceutical Co., Ltd. Tokyo, Japan). To eradicate H. pylori infection, those who tested positive on both tests received triple therapy comprising 20 mg vonoprazan fumarate (Takeda Pharmaceutical Co., Ltd. Tokyo, Japan), 750 mg amoxicillin, and 200 mg clarithromycin twice a day for 7 days. No probiotics were added to the eradication treatment nor were they used during the subsequent follow-up period. Then, 8-12 weeks after the eradication therapy, 13 C-urea breath test ( 13 C-UBT) was conducted at one of the cooperating medical institutions to assess for treatment efficacy. Breath samples were obtained 4 h after a meal and 20 min after the ingestion of 100 mg 13 C-urea (UBIT tablet, 100 mg). An infrared spectrometer (POC ONE; Otsuka Electronics Co., Ltd., Hirakata, Japan) was used for the test (negative ≤ 2.5%).
Students were excluded from receiving eradication therapy if they were allergic to any study drugs, had impaired liver or kidney function, were receiving colchicine, had infectious mononucleosis (or suspected www.nature.com/scientificreports/ infection with Epstein-Barr virus), or weighed < 30 kg. Students who had taken antibiotics or antacids (PPI, P-CAB, and histamine H2-receptor antagonist) within a year were excluded because of their effects on gut microbiota. The stool samples were collected prior to treatment (Before), 1-2 days after completion of treatment (After), and after 8-12 weeks of eradication therapy when it was determined that H. pylori had been eradicated (Judgment). At the time of judgment of eradication therapy, students with unsuccessful treatment outcomes were excluded to rule out the effect of the presence of H. pylori on the gut microbiota 34 . The outcome of H. pylori eradication therapy was confirmed using the 13 C-UBT. The stools were immediately stored at − 20 °C until DNA extraction. Adverse events associated with the eradication therapy were collected through a self-report questionnaire during the eradication treatment and by interview with the attending physician during the follow-up period from the eradication treatment at 13 C-UBT for eradication judgment.
The institutional review board of Saga University Hospital (approval numbers: 2017-03-01, approval date: June 5, 2017) approved the present study. As a sub analysis of the previous studies 13 , we examined the gut microbiota changes 3 months after therapy related to H. pylori eradication with vonoprazan fumarate containing triple therapy among adolescents. All methods were carried out in accordance with the relevant guidelines and regulations or the guidelines of the Declaration of Helsinki. Written informed consent was obtained from all participants and their parents or guardians. 16 s rDNA sequence analysis. The 16S rRNA gene/DNA/amplicon sequencing results with next-generation sequencing were analyzed as follows: low-quality sequences were removed, chimeras were checked, operational taxonomic units (OTUs) were constructed, and taxonomy was assigned using CD-HIT-OTU, Quantitative Insights Into Microbial Ecology pipeline (http://qiime .org/). OTUs were established by clustering with a 97% identity threshold and were completed using an RDP classifier with the Green genes database. The observed OTUs, Chao1 (microbial species richness), and Shannon indices (microbial evenness) and PD whole tree (phylogenetic richness) were calculated to determine the alpha (α)-diversity of the microbiota in the samples. The beta (β)-diversity of our samples was calculated using the default beta-diversity metrics of the weighted and unweighted UniFrac distance. To compare the differences in the overall bacterial gut microbiota structure, a principal coordinates analysis was performed to reduce the dimensionality of the resulting distance. Statistical analysis. The raw data were expressed as percentage, mean, and standard deviation whenever applicable. For skewed data, the Mann-Whitney U test and Wilcoxon signed-rank test were used to compare variables between and within groups, respectively. The comparison of α-diversity and group distance for β-diversity was performed using the Monte Carlo two-sample test, followed by the false discovery rate and Bonferroni correction. A P value < 0.05 was considered statistically significant. Statistical analyses of the microbiota were performed using Python. Other statistical analyses were performed using R (R Core Team 2018]. R: A language and environment for statistical computing; R Foundation for Statistical Computing, Vienna, Austria; URL: https ://www.R-proje ct.org/).

Study endpoint.
The study endpoint was the change in the intestinal microbiota during H. pylori eradication with vonoprazan-containing triple therapy. In particular, the relative abundance, α-diversity, and β-diversity of the gut microbiota were compared before eradication, immediately after eradication, and during assessment of treatment efficacy. In addition, the adverse effects of H. pylori eradication with vonoprazan-containing triple therapy in children were evaluated. www.nature.com/scientificreports/