Helicobacter pylori ORIGINAL RESEARCH Gut: first published as 10.1136/gutjnl-2019-319826 on 23 January 2020. Downloaded from Gastric microbes associated with gastric inflammation, atrophy and intestinal metaplasia 1 year after Helicobacter pylori eradication Joseph J Y Sung, Olabisi Oluwabukola Coker, Eagle Chu, Chun Ho Szeto, Simson Tsz Yat Luk, Harry Cheuk Hay Lau, Jun Yu ► Additional material is ABSTRact published online only. To view Objective Helicobacter pylori is associated with gastric Significance of this study please visit the journal online (http:// dx. doi. org/ 10. 1136/ inflammation, precancerous gastric atrophy (GA) and What is already known on this subject? gutjnl- 2019- 319826). intestinal metaplasia (IM). We aimed to identify microbes that are associated with progressive inflammation, GA ► Helicobacter pylori infection is associated with and IM 1 year after H. pylori eradication. gastric inflammation, precancerous gastric Correspondence to Design A total of 587 H. pylori–positive patients were atrophy and intestinal metaplasia. Professor Joseph J Y Sung, ► Less than 3% of H. pylori–infected individuals Department of Medicine and randomised to receive H. pylori eradication therapy (295 Therapeutics, Chinese University patients) or placebo (292 patients). Bacterial taxonomy develop gastric cancer, and about 20% of of Hong Kong, New Territories, was analysed on 404 gastric biopsy samples comprising individuals with chronic gastritis are H. pylori Hong Kong; 102 pairs before and after 1 year H. pylori eradication negative; other organisms may induce gastric jjysung@ cuhk. edu. hk and 100 pairs before and after 1 year placebo by 16S inflammation and gastric carcinogenesis. Received 9 September 2019 rRNA sequencing. What are the new findings? Revised 3 December 2019 Results Analysis of microbial sequences confirmed ► Microbial co- occurrence was reduced and Accepted 14 December 2019 the eradication of H. pylori in treated group after 1 year. characterised by a distinct cluster of oral Principal component analysis revealed distinct microbial bacteria 1 year after eradication of H. pylori, clusters reflected by increase in bacterial diversity while gastric microbial ecology remained (p<0.00001) after H. pylori eradication. While microbial largely unchanged 1 year after placebo interactions remained largely unchanged after placebo treatment. treatment, microbial co-occurrence was less in treated ► One year after H. pylori eradication, http://gut.bmj.com/ group. Acinetobacter lwoffii, Streptococcus anginosus Acinetobacter lwoffii, Streptococcus anginosus and Ralstonia were enriched while Roseburia and and Ralstonia were enriched while Roseburia Sphingomonas were depleted in patients with persistent and Sphingomonas were depleted in patients inflammation 1year after H. pylori eradication. A distinct with persistent inflammation. cluster of oral bacteria comprising Peptostreptococcus, ► A distinct cluster of oral bacteria comprising Streptococcus, Parvimonas, Prevotella, Rothia and Peptostreptococcus, Streptococcus, Parvimonas, Granulicatella were associated with emergence and Prevotella, Rothia and Granulicatella were on September 27, 2021 by guest. Protected copyright. persistence of GA and IM. Probiotic Faecalibacterium associated with emergence and persistence praustznii was depleted in subjects who developed GA of atrophy and intestinal metaplasia, while following H. pylori eradication. Functional pathways probiotic Faecalibacterium praustznii was including amino acid metabolism and inositol phosphate depleted in subjects who developed atrophy metabolism were enriched while folate biosynthesis and 1 year after H. pylori eradication. NOD-lik e receptor signalling decreased in atrophy/IM- ► Amino acid metabolism and inositol phosphate associated gastric microbiota. metabolism were enriched while folate Conclusion This study identified that gastric microbes biosynthesis and NOD- like receptor signalling contribute to the progression of gastric carcinogenesis decreased in subjects with emerged intestinal after H. pylori eradication. metaplasia following H. pylori eradication. How might it impact on clinical practice in the foreseeable future? © Author(s) (or their INTRODUCTION ► This study demonstrated the contribution employer(s)) 2020. Re- use Independent epidemiological studies have permitted under CC BY-­NC. No of gastric microbes in the development and commercial re-use . See rights confirmed that infection with Helicobacter pylori perpetuation of precancerous gastric lesions and permissions. Published is the most important acquired aetiological agent after H. pylori eradication. by BMJ. 1 for gastric cancer, a global leading cause of cancer- ► The identified microbes that associated with related deaths. The chronic inflammation induced To cite: Sung JJY, Coker OO, progression of gastric inflammation, atrophy or Chu E, et al. Gut Epub ahead by H. pylori induces several histopathological intestinal metaplasia are potential therapeutic of print: [please include Day changes in the gastric epithelium and maintains a targets in gastric cancer prevention. Month Year]. doi:10.1136/ constant production of a cascade of cytokines which gutjnl-2019-319826 in turn attracts immune cells that generate oxidative Sung JJY, et al. Gut 2020;0:1–10. doi:10.1136/gutjnl-2019-319826 1 Helicobacter pylori radicals with the potential to damage host DNA.2 The mech- Histological assessment anism employed by H. pylori in promoting the emergence of Severity of gastric inflammation, GA and IM was graded Gut: first published as 10.1136/gutjnl-2019-319826 on 23 January 2020. Downloaded from pre- neoplastic gastric lesions (atrophy and intestinal metaplasia) according to the updated Sydney classification by a pathologist is predominantly chronic inflammation. Treatment regimens who was unaware of the treatment condition. Progression and targeted at H. pylori eradication have been demonstrated to be regression were defined as increase or decrease, respectively, of effective in preventing the progression of pathological changes inflammation, GA or IM scores after 1 year. in the gastric mucosa.3 4 However, despite the reported effec- tiveness of eradication therapy, some patients do continue to Sample sequencing develop pre- neoplastic gastric lesions including gastric atrophy Purification of DNA for 16S rRNA gene sequencing was (GA) and intestinal metaplasia (IM).3 Notably, less than 3% of performed on a total of 404 gastric biopsy samples, comprising H. pylori–infected individuals develop gastric cancer,5 and about 100 pairs before and after 1 year of placebo treatment, and 102 20% of individuals with chronic gastritis are H. pylori negative, pairs before and after eradication of H. pylori by OAC treatment suggesting that other organisms may induce gastric inflammation (online supplementary table 1). Gastric biopsies tissues were and even gastric carcinogenesis.6 digested using lysozyme and mutanolysin enzymes (Sigma, Hong It is possible that, while H. pylori initiate the inflamma- Kong), followed by bead beating and DNA purification with tory process in the stomach, other gastric microbes with pro- QIAamp DNA Mini Kit. Illumina Primer pair 515 f, 5′-GTGC- inflammatory potential play an important role in maintaining the CAGCMGCCGCGGTAA-3′ and 806 r, 5′- GGACTACHVGG- progression of inflammation and dysplastic changes leading to GTWTCTAAT-3′ targeted across the V4 hypervariable regions development of gastric cancer. The loss of acid- secreting parietal of the 16S rRNA gene was used for sequencing on Illumina cells induced by chronic H. pylori infection in the gastric epithe- MiSeq platform. Invitrogen SequelPrep Normalization Plate Kit lium may provide a favourable environment for colonisation was used for Library clean-up and normalisation. by non-H. pylori microbes to induce further pro- carcinogenic process. Moreover, the ‘point of no return’ in the cascade of Quality control and annotation of sequences events leading to gastric cancer is reportedly associated with Mothur software9 suite was used for sequence curation and patients with IM and dysplasia, independent of H. pylori status.2 analysis. Briefly, Needleman- Wunsch alignment algorithm was The relevance of non- H. pylori gastric microbes in the devel- used to merge paired- end reads into contigs with default param- opment of gastric cancer was previously demonstrated by using eters. This was followed by alignment against SILVA 16S rRNA transgenic INS- GAS mice, overexpressing gastrin. Antibiotic- sequence database (V.123) with nearest alignment space termina- treated INS- GAS mice colonised with H. pylori had delayed tion (NAST) algorithm.10 Contigs that mapped within V4 region onset of gastric cancer compared with control mice infected were trimmed and merged with sequences having a difference with H. pylori without antibiotic treatment,7 suggesting that of most two nucleotide bases, followed by chimeric sequences gastric microbes could enhance the effects of H. pylori in gastric screening using de novo Uchime.11 Greengenes database (V.13.8) carcinogenesis. Single- nucleotide polymorphisms in host genes was used for taxonomy assignment. Reads assigned as non- bacterial or unknown kingdoms were discarded. Average neigh- that influence bacterial pattern recognition receptors in gastric http://gut.bmj.com/ cancer patients have also been reported,8 underscoring the bour clustering algorithm was the used to cluster the resulting active role of bacteria–host cross- talk in gastric tumourigenesis.