| Background: Gastric ulcer(Gastric ulcer,GU)is a kind of peptic ulcer,there are 5%-10% of the people in the world have gastric ulcer.As an important part of the digestive system,there are a lot of special microbial communities in stomach.However,the structure and function of the microbial community in the stomach,especially related to human stomach disease,is not clear.A small number of studies have focused on the relationship between the structure and function of the microbial community and malignant gastric diseases(gastric cancer).The characteristics of microbial community in patients with gastric ulcer and the differences between health and health people is not clear.Therefore,this study focus on the microbial community and use 16 S r DNA high-throughput sequencing and bioinformatics methods to analyze the gastric mucosa flora characteristics and differences in patients and health people in Changchun.After that we analyze the construction of gastric ulcer is remarkable specific bacterial composition profiles,and screening significant diseases related to key bacteria for further molecular evolution research and related correlation analysis.This study will lay the foundation for the relationship between the structure of gastric flora and the gastric ulcer,and provide a new way for the clinical treatment of gastric ulcer.Methods: Gastric mucosal biopsy samples were collected from patients with gastric ulcer and health volunteers,and the total DNA was extracted.The 16 S gene V3-V4 variable region of r DNA was amplified by PCR.The products were sequenced by Illumina Mi Seq high-throughput sequencing instrument.The high quality sequence of each sample is obtained by preprocessing the data.Furthermore,the OTU distribution,the OTU analysis and the taxonomic status identification were used to examine the distribution of OTU at different levels.The ?-diversity of the bacteria was evaluated by species rarefaction curve,species accumulation curves,rank aboundance curve.According to the composition and distribution of samples in each taxonomic level,we compare the differences between the two groups.By analysing the principal component and non metric multidimensional scaling analysis,we evaluate the ?-diversity of bacterial community structure.According to the abundance distribution of dominant genera in different samples,the association network of dominant microbial groups was established.We isolated H.pylori from patients with gastric antrum gastric ulcer,PCR amplify cag A gene 3'-terminal sequences to draw phylogenetic tree,and make the correlation analysis.Results: In this study,10 cases of gastric antrum mucosa samples were collected from health people,and 9 cases of gastric antrum mucosa samples were collected from patients with gastric ulcer.The results showed that all the samples had obvious bands in the 500 bp region,and the purity is enough for further study.After the data processing,the OTU analysis showed that the number of OTU in the health group was significantly higher than that in the gastric ulcer group.There were 1172 OTU in the two groups,and the health group had exclusive 413 OTU,and the gastric ulcer group had exclusive 92 OTU.The results showed that the sequence and the depth of sequencing were sufficient to cover the species of bacteria in the stomach,and the richness and evenness of the two groups were good.Comparing the health people and patients with gastric ulcer and gastric mucosa flora diversity,we found that in the same number of sequence extraction cases,health groups observed species richness,Chao1 index and Shannon diversity index were higher than the disease group.More than 250 genera of bacteria from the 30 phyla were identified by OTU in the level of the taxonomy.The dominant bacteria in all the antral mucosa samples were as follows: Proteobacteria,Bacteroidetes,Actinobacteria and Firmicutes bacteria.The gastric ulcer group compared with health group of bacterial abundance have significant differences as follows: Bacteroidetes and Firmicutes,Proteobacteria and Acidobacteria,Fusobactera,Actinobacteria,Tenericutes,Chloroflexi,Synergistetes.In the genus level,the relative abundance of H.pylori in gastric ulcer group is significantly higher than that of health group,while in Lactococcus,Bacteroides,Actinomyces and other 19 genera,relative abundance was significantly lower than that of health group.The beta diversity analysis of the bacterial community structure showed that there were significant differences in the microbial community structure between the health group and the gastric ulcer group.According to the analysis of the network,the dominant bacteria in the stomach were divided into two typical groups,and the link of the two groups was H.pylori.9 strains of H.pylori were isolated and identified from the gastric antrum tissues.The isolation rate was 100%.The results of cag A amplification showed that 9 strains had obvious bands around 800 bp.The cag A gene 3'-terminal variable sequence polymorphism analysis showed that JLU-1 and JLU-9 were EPIYA-ABBD,JLU-4 was EPIYA-AB,and the others were EPIYA-ABD.Molecular phylogenetic analysis showed that all 9 strains were of the East-Asian-type.After correlation analyzing,we found that the number of EPIYA site is positively correlated with the relative abundance of H.pylori in gastric mucosa and the severity of gastric ulcer.Conclusion: 1.Based on 16 S r DNA high-throughput sequencing of 9 gastric ulcer patients and 10 health people gastric mucosal biopsy samples,we found that: there are a large number of Proteobacteria,Bacteroidetes,Actinobacteria and Firmicutes bacteria;At the phylum level,Proteobacteria were dominant(accounting for 80%),and the genus level were mainly Helicobacter(accounting for 50%);Compared with health people,the distribution uniformity of bacteria was poor and the diversity was significantly reduced;The abundance of Proteobacteria,Bacteroidetes,Actinobacteria etc.9 phyla and Helicobacter,Lactococcus,Actinomyces etc.20 genera are significantly different between health people and gastric ulcer patients.2.All 9 H.pylori strains isolated from gastric ulcer patients in Changchun area were East-Asian-type according to the phylogenetic tree based on cag A gene.3.For the first time,we found that the repeatability of the carboxyl terminal EPIYA-D sites,of Cag A protein in H.pylori clinical isolates from patients with gastric ulcer in Changchun,may be related to the colonization of H.pylori in the stomach. |
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