Analysis And Pathogenesis Of Gut Microbiota In Ulcerative Colitis Patients And Their Healthy Spouses

Part 1Structure and diversity analysis of gut microbiota in patients with ulcerative colitis and their healthy spouses-Research based on 16S rRNA gene sequencing technologyObjective:Analyze the structure and diversity of intestinal microflora in UC patients and their healthy spouses byl6S rRNA gene sequencing technology,so as to reveal the differences in the structure and diversity of intestinal flora in UC patients and their healthy spouses.Methods:Feces and intestinal mucosa of 28 patients with early-onset,mild to moderate UC and their healthy spouses were collected.The total number of cases is 56,including 28 males and 28 females,28 from urban and 28 from country.DNA was extracted,amplified by PCR and followed by Illumina sequencing?25,000 tags,MiSeq platform?for the v3-v4 region sequencing of 16S rRNA.Bioinformation analysis was performed on the results of 16S rRNA sequencing.Results:1.Clustering OTU and species annotation:1129 OTU were generated from 112 samples.At the phylum level,the samples were mainly composed of Bacteroidetes,Firmicutes and Proteobacteria,in which 11 Baeteroides,Prevotella,Escherichia,Faecalibacterium,Roseburia,Lactococcus,Megamonas,Parabacteroides,Lactobacillus,Bacillus and Eubacterium were involved.2.The phylogenetic tree of intestinal microflora in UC patients:The intestinal microflora of UC patients could be roughly divided into 15 phyla,among which the most species were related to Firmicutes and Proteobacteria.3.Analysis of unique bacterial community shared by UC and HC:416 bacterial species of 1129 OTU were shared by the UC and HC groups in the feces and intestinal mucosa,while 224 bacterial species of OTU were unique to UC patients,among which 119 were unique to the intestinal mucosa of UC patients,42 were unique to the feces of UC patients and 63 are common.4.Comparison of microflora diversity of UC and HC:Alpha diversity of intestinal microflora was significantly different between UC and HC,and also between MAM and LAM.Compared with FH group,observed species index and chao index from FU group were significantly reduced.Compared with MU group,indexes of observed species,chao and ace from FU group were significantly decreased.Compared with MH group,indexes of observed species,chao and ace from FU group were also significantly reduced.Observed species index and ace index had no statistical difference between MH group and MU group.Beta diversity was different among groups.The diversity of FH was significantly less than MH,and the diversity of FU was significantly less than MU.5.Analysis and comparison of microflora from FU and FH:At the order level,the content of Puniceicoccales in FU was less than that in FH?P<0.01?.While the content of Aeromonadales,Alteromonadales,Bdellovibrionales and Coriobacteriales in FU were more than FH?P<0.05?.At the family level,the Acidaminococcaceae and Odoribacteraceae in FU were significantly lower than those in FH?P<0.05?,while Beijerinckiaceae and Eggerthellaceae were significantly increased?P<0.05?.On the genus level,compared with FH,Phascolarctobacterium and Odoribacter in FU were significantly reduced?P<0.05?,while Carnobacterium and Asticcacaulis were significantly increased?P<0.05?.6.Analysis and comparison of MU and MH microflora:At the order level,Acidaminoeoccales and Bacteroidales in MU were significantly decreased,while Bacillales,Enterobacteriales and Sphingomonadales were significantly increased.At the family level,Aminococcaceae and Odoribacteraceae in MU were significantly decreased,while Sphingomonadaceae were significantly increased.On the genus level,Peptostreptococcus was significantly increased,Odoribacter and Phascolarctobacterium were significantly decreased.At the family level,Acinetobacter baumannii and Acinetobacter junii in the intestinal mucosa of UC patients were significantly increased,P values were less than 0.05.7.Analysis and comparison between FU and MU microflora:The higher increased microflora in MAM than LAM of UC patients:Sphingomonadales?Sphingomonadaceae?Sphingobium,Enterobacteriales?Enterobacteriaceae?Escherichia,Bacillales?Bacillaceae)?Bacillus,Pseudomonadales?Pseudomonadaceae?Pseudomonas,Campylobacterale ? Campylobacteraceae?Campylobacter,Staphylococcaceae?Staphylococcus,Pseudomonadales?Moraxellacea?Acinetobacter?Acinetobacter_baumannii?Acinetobacter_junii?Acinetobacter_indicus_CIP₁10367?The higher increased microflora in LAM than MAM of UC patients:Prevotellaceae?Prevotella,Actinomycetales?Actinomycetaceae.8.Results of LEfSe analysis:The following categories of intestinal microflora in UC patients were significantly increased and had clinical significance:Proteobacteria)?Gammaproteobacteria Enterobacteriales?Enterobacteriaceae,Erysipelotrichia?Erysipelotrichales Erysipelothrichaceae,Actinobacteria?Actinomycetes,Streptococcaceae,Porphyromonadaceae,Clostridiume tertium,Clostridium_perfringens_ATCC₁3124.The following categories of intestinal microflora in UC patients were significantly decreased and had clinical significance:Acidaminococcales?Acidaminococcaceae,Odoribacteraceae?Odoribacter?Odoribacter_splanchnicus,Odoribacteraceae?Butyricimonas?Butyricimonas_virosa,Phascolarctobacterium?Phascolarctobacterium faecium,Bacteroides_caccae,Bacteroides_cellulosilyticus_DSM₁4838,Bacteroides_stercoris_ATCC₄3183,Selenomonadaceae.Conclusions:1.The increased abundance of Proteobacteria and decrease of firmicutes may be biomarkers for the diagnosis and prognosis of UC patients.2.Alpha diversity of intestinal microflora is significantly different between UC and HC,and between MAM and LAM.The richness of FU microflora is significantly lower than that of FH,MU and MH groups.There is no statistical difference in the abundance between MH group and MU group.The microflora diversity of FH is significantly less than MH,and the microflora diversity of FU is significantly less than MU.3,Compared with FH and MH group,the following microflora in FU and MU of UC patients are reduce simultaneously:Acidaminococcaceae,Odoribacteraceae,and Phascolarctobacterium,suggesting that the above microflora have the potential to be used as biomarkers to predict the disorder of UC enterobacteria.4.This work successfully identifies some microflora from UC patients,such as:Eggerthellaceae,Carnobacterium,Asticcacaulis,Sphingomonadacea,Serratia,Corynebacterium,Acinetobacter_baumannii,Acinetobacter_junii,Acinetobacter_indicus_CIP₁10367.There is no report about the relationship between the above microflora and UC.It provides a new direction and target for the future study of the role and mechanism of intestinal flora in the pathogenesis of UC.5.The intestinal microflora changed significantly in UC patients is analyzed by LEfSe and we find that the change of above microflora may be the core microflora related to the occurrence and development of UC.KEGG,eggNOG and MetaCyc metabolic pathways were used to explore the key functions of the core microflora and the possible pathogenic mechanism.Part ?:Function analysis of gut microbiota in patients with ulcerative colitis and their healthy spouses-Research based on metagenomicsObjective:Analyze the functional differences of intestinal microflora between patients with UC and their healthy partners by macromolecomic sequencing technolgy and speculate the possible pathogenesis of UC.Methods:In 28 cases of UC patients and their healthy spouses,the feces of 11 pairs and the mucosa of 21 pairs of UC patients and their healthy spouses were randomly selected,samples above were sequenced using Illumina HiSeq sequencing platform.MGS results were analyzed by bioinformatics.Results:1.Annotation analysis of COGs database:In FU,65?17.6%?genes showed high abundance?15 involved in metabolism,23 involved in information processing and 27 involved in cellular processes and signaling?,while 305?82.4%?genes showed low abundance?79 involved in metabolism,108 involved in information processing and 117 involved in participate in cellular processes and signaling?.In FM,only 44?2.2%?genes showed high abundance?2 involved in metabolism,13 involved in information processing and 29 involved in cellular processes and signaling?,while 1946?97.8%?genes showed low abundance?874 involved in metabolism,543 involved in information storage and processing and 529 involved in cellular processes and signaling?.2.Annotation analysis of KEGG metabolic pathways:the abundance 10 KEGG metabolic pathways were significant different in FU.The functions of macrogene in the Glucagon signaling pathway,Caprolactam degradation,Steroid biosynthesis,Ribosome and other metabolic pathways of FU were suppresssed.While the Flagellar assembly,Riboflavin metabolism and Calcium signaling pathway might be activated.The relative abundance of 110?69.2%?pathways in FM was significantly reduced,and that of 48?30.8%?pathways was higher than normal.3.Annotation analysis of MetaCyc Pathway:The abundance of 13 metabolic pathways in FU were significant different.The relative abundance of pwy-6788,pwy-3821,pwy-7459 and pwy66-428 were decreased,while the relative abundance of pwy-7226,pwy-7227,pwy-2301 and pwy-5427 were increased.There were 265 abnormal pathways in FM,among which 51 pathways showed higher abundance than the healthy control and 214 pathways showed lower abundance than normal.Conclusions:1.UC patients show obviously structure and function disorder of intestinal microflora,and the diversity of intestinal microflora in UC patients decreases not only at the level of species but also at the level of macro genome.2.Compared with healthy spouses,the inflammatory response,oxidative stress,flagellum assembly and other metabolic pathways of intestinal microflora function in UC patients is significantly enriched in.Metabolic pathways involved in carbohydrate,amino acid,cofactor synthesis and nucleotide for nutrient transport and intake are signifieantly down-regulated.3.Intestinal microorganism structure may be involved in the occurrence and development of UC through increasing of peptidoglycan and lipopolysaccharide,and the possible pathogenesis of intestinal microorganism in UC 1s proposed based on the above results.