bs_bs_banner doi:10.1111/jgh.14736 GASTROENTEROLOGY Short-term and long-term impacts of Helicobacter pylori eradication with reverse hybrid therapy on the gut microbiota Ping-I Hsu,* Chao-Yu Pan,† John Y Kao,‡ Feng-Woei Tsay,* Nan-Jing Peng,§ Sung-Shuo Kao,* Yan-Hua Chen,* Tzung-Jiun Tsai,* Deng-Chyang Wu¶ and Kuo-Wang Tsai**,†† *Division of Gastroenterology and Hepatology, Department of Internal Medicine, Kaohsiung Veterans General Hospital and National Yang-Ming University, Kaohsiung, Taiwan†Institute of Biomedical InformaticsAcademia Sinica, National Yang-Ming University and Institute of Biomedical Sciences, Taipei, Taiwan‡Department of Internal Medicine, Division of Gastroenterology, Michigan Medicine, University of Michigan, Ann Arbor, Michigan USA§Department of Nuclear Medicine, Kaohsiung Veterans General Hospital and National Yang-Ming University, Kaohsiung, Taiwan¶Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan**Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan††Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan Key words Abstract dysbiosis, erm(B), Helicobacter pylori, microbiome, treatment. Background and Aims: Anti-Helicobacter pylori therapy may lead to the growth of path- ogenic or antibiotic-resistant bacteria in the gut. The study aimed to investigate the short- Accepted for publication 13 May 2019. term and long-term impacts of H. pylori eradication with reverse hybrid therapy on the components and macrolide resistance of the gut microbiota. Correspondence Methods: Helicobacter pylori-related gastritis patients were administered a 14-day reverse Dr Kuo-Wang Tsai, Department of Medical hybrid therapy. Fecal samples were collected before treatment and at the end of week 2, Education and Research, Kaohsiung Veterans week 8, and week 48. The V3–V4 region of the bacterial 16S rRNA gene in fecal specimens General Hospital, 386 Ta Chung 1st Road, was amplified by polymerase chain reaction and sequenced on Illumina MiSeq platform. Kaohsiung 813, Taiwan. Additionally, amplification of erm(B) gene (encoding erythromycin resistance methylase) Email: [email protected] was performed. Dr Chao-Yu Pan, Institute of Biomedical Infor- Results: Reverse hybrid therapy resulted in decreased relative abundances of Firmicutes matics, National Yang-Ming University and In- (from 62.0% to 30.7%; P < 0.001) and Actinobacteria (from 3.4% to 0.6%; 0.032) at stitute of Biomedical Sciences, 2 Linong Street, the end of therapy. In contrast, the relative abundance of Proteobacteria increased from Beitou District. Taipei 112, Taiwan. 10.2% to 49.1% (0.002). These microbiota alterations did not persist but returned to the ini- Email: [email protected] tial levels at week 8 and week 48. The amount of erm(B) gene in fecal specimens was com- parable with the pretreatment level at week 2 but increased at week 8 (0.025) and then Declaration of conflict of interest: All the authors disclose no conflicts of interest. returned to the pretreatment level by week 48. Conclusions: Helicobacter pylori eradication with reverse hybrid therapy can lead to short-term gut dysbiosis. The amount of erm(B) gene in the stool increased transiently after treatment and returned to the pretreatment level at 1-year post-treatment. Introduction dysbiosis. Antibiotic treatment is known to alter the composition of the normal human gut microbiota. Adverse events such as Human gut microbiota influence essential biological functions of pseudomembranous colitis associated with Clostridium difficile in- the hosts including energy metabolism, immune modulation, and 12,13 1,2 fection have been reported after anti-H. pylori therapy. In ad- host defense against pathobionts. Numerous studies have reported that abnormal alterations of the gut microbiota (a.k.a., dition, gastric acid suppression by PPI is a known risk factor for fi 14,15 dysbiosis) may promote the development illness including C. dif cile-associated diarrhea in hospitalized patients. An- colorectal cancer, inflammatory bowel disease, obesity, type 2 other concern with the administration of antibiotics is the selection diabetes mellitus, asthma, rheumatoid disorders, and neurodegen- of antibiotic-resistant strains. It is known that gut microbial erative diseases.2–4 macrolide resistance is mediated by erythromycin resistance 16 Helicobacter pylori infection is the major cause of chronic methylases encoded by erm genes. The erm genes have been gastritis, gastric ulcer, duodenal ulcer, gastric adenocarcinoma, found in different genera of bacteria and erm(B) has the largest 16 and gastric mucosa-associated lymphoid tissue lymphoma.5,6 host range. Highly macrolide-resistant enterococci have been Eradication of H. pylori not only can prevent the recurrence of identified after anti-H. pylori therapy with clarithromycin- peptic ulcers but also may decrease the incidence of gastric containing regimen, and an increase in erm(B) levels in entero- 22adenocarcinoma.7,8 However, most H. pylori eradication regi- cocci has been reported following the treatment.17 Therefore, a mens contain antibiotics and proton pump inhibitor (PPI),9–11 comprehensive investigation of the effects of H. pylori eradication which may deplete gut resident commensal microbes resulting in treatment on the growth of pathogenic or antibiotic-resistant 1968 Journal of Gastroenterology and Hepatology 34 (2019) 1968–1976 © 2019 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd P-I Hsu et al. Gut microbiota change in H. pylori eradication bacteria is quite important before recommending global criteria were excluded from this study: (i) previous eradication anti-H. pylori therapy for cancer prevention in asymptomatic therapy, (ii) allergy to any antibiotic of our study, (iii) previous subjects. gastrectomy, (iv) the coexistence of severe concomitant illness Previous studies18 showed that standard triple therapy contain- (e.g. decompensated cirrhosis, uremia, congestive heart failure, ing amoxicillin and clarithromycin led to a reduction of the chronic obstructive pulmonary disease, and cancer), (v) pregnancy relative abundance of Firmicutes phylum. In contrast, bismuth or lactating women, (vi) the use of antibiotics within the previous quadruple therapy containing metronidazole and tetracycline 8 weeks, and (vii) taking PPI or histamine-2 receptor antagonist resulted in a dramatic decrease in the relative abundance of within previous 8 weeks. This trial was approved by the Institu- Bacteroidetes.19 Hybrid therapy is a recommend anti-H. pylori tional Review Board of the Kaohsiung Veterans General Hospital treatment in areas of either high or low clarithromycin resistance (VGHKS15-CT2-10). in the Taiwan H. pylori Consensus Report.20 and also in the American College of Gastroenterology guideline and the Sample collection procedures. The eligible subjects re- Bangkok H. pylori Consensus Report.21,22 Our group has recently ceived a 14-day reverse hybrid therapy consisting of pantoprazole shown that a modified hybrid therapy, called reverse hybrid ther- 40 mg plus amoxicillin 1 g twice daily for 14 days and apy consisting of a PPI and amoxicillin for 14 days and clarithromycin 500 mg plus metronidazole 500 mg twice daily clarithromycin and metronidazole in the initial 7 days, is a more for the initial 7 days. Patients were asked to return in 2 weeks to simplified hybrid therapy regimen.23 It achieves a higher eradica- check drug adherence and adverse events. They underwent a urea tion rate than standard triple therapy with similar tolerability and breath test to assess post-treatment H. pylori status at week 8.25 Fe- at a lower cost.24 Additionally, reverse hybrid therapy has compa- cal samples for gut microbiota analysis were collected the morning rable efficacy as bismuth quadruple therapy in the treatment of of day 1 before anti-H. pylori therapy and at the end of week 2, H. pylori infection and was also found to have fewer side effects.25 week 8, and week 48. Patients collected fecal samples at home Because the impact of reverse hybrid therapy on the gut micro- and stored them at 4 °C. The samples were sent to our laboratory biota is unknown, the aims of our study are (i) to clarify the short- within 6 h after collection and were immediately stored at term and long-term impacts of reverse hybrid therapy on the com- À70 °C until DNA extraction. ponents of the gut microbes and (ii) to examine the short-term and long-term impacts of reverse hybrid therapy on the amount of erm(B) gene in the fecal microbiota. 16S rRNA gene amplification and sequencing by MiSeq. Bacterial DNA in fecal samples was extracted with Bac- terial DNA Extraction Kits (Topgen Biotechnology Co. Ltd, Methods Kaohsiung, Taiwan). The 16S rRNA gene was amplified in a 50- μL reaction containing bacterial DNA (5 ng/μL), hotStart Taq Study population. Helicobacter pylori-infected adult pa- (Qiagen, Hilden, Germany), and polymerase chain reaction tients (age ≥ 20 years) with gastritis documented by (PCR) primers (Rd1-16S-V3-V4-Forward/Rd2-16S-V3-V4-Re- esophagogastroduodenoscopy were recruited. H. pylori infection verse) as previously described.19 The PCR conditions used were was confirmed by at least two positive test results (e.g. rapid ure- 95 °C for 3 min, 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and ase test, histology, and culture). Subjects with any of the following 72 °C for