Analysis Of The Gastric Microbiota Of H.pylori-infected Patients With Non-atrophic Gastritis,Intestinal Metaplasia And Gastric Cancer

BackgroundThe stomach used to be regarded as a sterile environment until H.pylori was first isolated in 1983.Decades of research have convinced the fact that there are many non-H.pylori bacteria in the stomach.Recently,with the development of detection methods,especially the high-throughput sequencing,our knowledge about the gastric microbiota has come to a new stage.The relatively normal gastric microbiota and the impact of H.pylori on the gastric microbiota have been studied.Gastric ·cancer is a multi-factorial disease that is involved with H.pylori infection,environment and heredity.Correa et al.proposed a cascade from chronic non-atrophic gastritis,by way of atrophic gastritis and intestinal metaplasia,to dysplasia,which was mainly induced by H,pylori infection.The Correa’s cascade is widely accepted as the process through which non-cardia gastric cancer develops.Current research suggested that with the development of Correa’s cascade,the role of H.pylori in carcinogenesis is becoming weaker,which means the H.pylori is more like a trigger.Lots of bacteria cannot live in the stomach because of the gastric acid.And the stomach with atrophy and intestinal metaplasia is hypochlorhydric,which may lead to many non-H.pylori to overgrowth.Prior studies suggested that some non-H.pylori bacteria may participate in gastric carcinogenesis.However,only a few studies have focused on this,and the results were different.AimThis study is aimed to analyze whether the hypochlorhydric micro-environment of the stomach will causes non-H.pylori bacteria to overgrowth,and whether the non-H.pylori bacteria have association with gastric cancer.MethodsWe collected 22 patients(40 gastric mucosal samples),including 10 patients with non-atrophic gastritis(10 gastric antral mucosal samples and 9 corpus samples),6 patients with intestinal metaplasia(4 antral mucosal samples and 6 corpus samples),7 patients with gastric cancer(6 gastric tumor samples and 5 peri-tumorial samples).These samples were collected by sterile biopsy forceps under the gastroduodenoscopy and analyzed by the high-throughput sequencing technologies on the Illumina MiSeq platform.The status of H.pylori infection was confirmed by RUT,histopathology and sequencing.ResultsProteobacteria,Firmicutes,Bacteroidetes,Actinobacteria,Fusobacteria and unclassified-k-norank-d-Bacteria were the predominant bacteria on the phylum level and the abundance of Proteobacteria was more than 70%.By analyzing the microbial difference in these H.pylori-infected patients,we found that the proportion of H.pylori was highest in samples from patients with intestinal metaplasia,and lowest in samples from patients with gastric cancer.No matter the samples of gastric tumor or peri-tumorial tissues were compared with samples from the other two groups with different histopathology,only the changes of the unclassified-k-norank-d-Bacteria had significance,and that the proportion of the bacteria was decreased from non-atrophic gastritis to intestinal metaplasia and to gastric cancer group.No bacterium was found to be increased from non-atrophic gastritis to intestinal metaplasia and to gastric cancer group.Except for H.pylori,no bacterium was increased in intestinal metaplasia group compared with non-atrophic gastritis.In addition,the bacterial differences between samples of gastric tumor and peri-tumorial tissue were no statistical significance.The richness of gastric microbiota was increased from non-atrophic gastritis to intestinal metaplasia and to gastric cancer group,and the diversity of intestinal metaplasia group was the lowest.However,the diversity of samples of gastric tumor was higher than that of non-atrophic gastritis group,but almost no diversity difference was observed between peri-tumorial tissues and samples from patients with non-atrophic gastritis.Additionally,the bacterial richness and diversity of samples of gastric tumor were higher than that of peri-tumorial tissues.NMDS analysis showed that there was a clear separation between samples of gastric cancer group and non-atrophic gastritis group,and samples of intestinal metaplasia group were overlapped with them.ConclusionsCompared with that of H.pylori-infected patients with non-atrophic gastritis and gastric cancer,bacteria didn’t overgrowth in stomach of patients with intestinal metaplasia.Maybe H.pylori is still the most important carcinogenic factor for H.pylori-infected patients.And some bacteria may secondary overgrowth in gastric tumor.In the future,studying the gastric microbiota of H.pylori-negative patients and patients with intraepithelial neoplasia or early gastric canceris is meaningful for analyzing the association between the gastric microbiota and gastric cancer.More advanced technologies,like metagenomic sequencing,should be applied for functional analysis of the gastric microbiota.SignificanceThis study firstly analyzed the gastric bacteria in patients with non-atrophic gastritis,intestinal metaplasia and gastric cancer in China by using high-throughput sequencing.The status of H.pylori infection was confirmed by RUT,histopathology and sequencing in this study to reduce the false-negative rate.Moreover,this study is important for revealing the role of H.pylori and other bacteria in gastric carcinogenesis in patients with H.pylori infection.