Analysis Of Bacteria Of The Human Gastric Microbiome In Patients With Functional Dysplasia And In Controls

OBJECTIVE To characterize the gastric microbiome in patients with FunctionalDysplasia and in controls. Comparison of the gastric mucosa associated microbiotadiversity between the two groups.METHODS Patients with FD (group FD,n=30)were recruited according to the RomeⅢ criteria; all subjects have normal gastric mucosal morphology in gastroscopicexamination and with H. Pylori negative under these test (Histological results, Rapidurease test and C13-urea breath tests).Two Biopsies from gastric antrum and corpuswere recovered form all subjects who undergoing upper gastrointestinal endoscopy,The Samples were collected in sterile containers and stored in refrigerator at-80℃.Total genomic DNA from each samples was extracted with the QIAamp DNA Minikit, DNA amplification of16S Rrna were performed. After purified from1.5%agarose gel, the products were mixed and prepared for one sequencing libraries, Thepurified amplicon mixtures were sequenced on Illumina Miseq platform usingpair-end method. Pairs of reads from the original16s gene fragments are merged byusing FLASH. Then Sequences were processed with the QIIME software package. weuse operational taxonomic units (OTUs) at97%identity through to making OTU table.In order to compute α Divesity, we rarefied the sample-by-OUT table and calculatethree metrics: Chao1metric, Observed Species metric and shannon index. We useunweighted unifrac to do Principal Coordinate Analysis (PCoA) and Unweighted PairGroup Method with Arithmetic mean (UPGMA) Clustering，which interpret theaverages distance matrix between the two compared groups.RESULTS28,262and30,261sequences per sample were obtained form FD andHC group, binned into a mean of271±72and293±88operational taxonomic units(OUTs)in the two group,respectively. The Rarefaction,chao1,shannon index in FDand HC group were71±72and293±88，290±89and319±108,4.57±1.19and5.03±1.29,respectively. No significant difference was observed between the two groups(p>0.05).The gastric bacterial community in FD group was dominated by15phyla, the dominant pyla were Proteobacteria(79.13%)，Bacteroidetes(5.91%)，Firmicutes(5.42%)，Actinobacteria(5.41%). while,the reads in HC groups resulting indetection of17phylum and predominated by Proteobacteria(72.40%),Bacteroidetes(8.53%), Actinobacteria(7.23%), Firmicutes(6.34%), and Crenarchaeota(1.71%).At the phylum level,the relative abundance of proteobacteria, Fusobacteriawere increased in the FD group,and Nitrospirae were increased in control group.Themain prevalent genus in FD sample set was Halomonas (45.361%), Shewanella(12.442%), Acinetobacter (3.336%), Corynebacterium (1.973%), Streptococcus(1.661%). In the HC subject, the Halomonas(41.484%) dominated, followed bymembers of the Shewanella(10.121%), Acinetobacter (2.446%), Corynebacterium(2.422%), Bacteroides（2.258%），Helicobacter（1.754%），Rubrobacter（1.575%）.Among these phylotypes,Halomonas and Shewanella identity to isolates from marinelsource. At the genus level,FD had higher representation in5genera(Halomonas,Shewanella, Sphingomonas, Burkholderia and Parapedobacter). On the contrary,theproportions of Propionibacterium, Rubrobacter and Roseburia was lower in comparedto controls.In beta diversity analysis, Principal coordinate analysis (PCoA) plots usingweighted or unweighted UniFrac distances clustered samples by groups(FD and HC),whereas we did not observe distinct clustering due to their individual variablility.UPGMA dendrogram Results show the close clustering of gastric body and antrumsamples from the same subject, as evidenced by the short branch lengths in thedendrograms.CONCLUSION The human gastric environment in FD and healthy containsdistinctive microbial communities,including some member of the marine phylotypeswere previously uncharacterized. Despite of some phylum/genera were significantlydiferent between the FD and healty subjects, no considerable community compositiondifferences were observed among the two groups.