The Intestinal Microecology Research Of Acute On Chronic Liver Failure Patients By The Approach Of Metagenomics

Liver failure is a serious liver damage which caused by many factors with high mortality.Acute on chronic liver failure(ACLF)is the most common type in China,on the basis of chronic liver disease with mid and short-term mortality as high as 50%～900/%.There is a closely relationship between liver and gut.Significant changes appeared in the intestinal microenvironment,which can lead to the imbalance of intestinal microecology.The abnormal intestinal microecology in turn lead to liver damage.The alteration of gut microbes in ACLF is unknown.More and more attention were paid in the role of imbalanced intestinal mieroecological.The development of high-throughput sequencing technologies greatly promoted the progress of ecology field,which provide a powerful tool to reveal the relationship of microecology and disease.Object:To clarify the change of the microbial phylogenetic structure and functional genes in ACLF intestinal flora,reveal the profile of antibiotic genes of intestinal microbial flora,and investigate the relationship between ACLF and the intestinal flora,we use the high-throughput sequencing technologies to study intestinal microbial flora in patients with ACLF.Methods:28 ACLF patients were enrolled and 28 controls were selected.Their gut microorganisms were extracted.High-throughput sequencing methods with bioinformatics analysis were used to reveal the variation of phylogenesis,functional genomics and antibiotic resistance gene in acute on chronic liver failure patients.Result:1.Using Hiseq2000 sequencer,we got an average of 4 GB data per samples of 28 ACLF patients,111 GB in total.Through assembling andannotating,a non-redundant gene set from intestinal microflora of ACLF patients were constructed,here we called ACLF gene set.Combined the ACLF gene set with liver cirrhosis gene set.we got a non-redundant gene set of liver disease.Here we called L gene set.Among the L gene set,220,759 genes were new,account for 37%of ACLF gene set,and 7.6%of L gene set.28 healthy controls were selected from metagenomic database of intestinal microflora in our lab.Two groups were matched with age,gender and BMI.Compared with control group,we found that the gene richness of ACLF patients were significant decreased.2.Significant changes in phylogenetic structure of intestinal flora were found in ACLF patients.Most of the different species were enriched in control group.Faecalibacterium_prausnitzii,and species from Ruminococcaceae,which played important roles in human nutrition and anti-inflammatory,were significantly decreased in ACLF group.Veillonella andStreptococcus,which were from oral,were significantly enriched in ACLF group.Through the enterotype analysis,we found that there are three kinds of enterotype,driven by threegenus,including Bacteroides,Prevotella and Veillonella respectively.67303 gene markers specific to ACLF were found.Then gene markers were clustered,and 27 clusters were found.Most of the MGS enriched in ACLF group were collaborative with each other,while antagonistic actionwas found betweenMGS from two groups.MGS enriched in ACLF group were positive correlated with prothrombin time,international normalized ratio,copies of HBV-DNA and CTP score,and negative correlated with serum globulin.Using MGS,diagnostic model were constructed with high sensitivity and specificity.Combination of MGS could improve the diagnostic value.Functional genes of gut microbes in ACLF patients were significantly changed.The most enriched orthologues in patients group were transcription,translation,replication and repair,which implied that the proportion of gut microbes which were in the state of proliferation were increased significantly.3.Most of the antibiotic resistance(AR)genes in gut microbiome were from Proteobacteria,which were not agreed with the species composition of gut microbe.Tetq is the mostabundant antibiotic resistance genotype in our study.Macrolides,lincosamides and streptogramins was the most common resistance antibioticin our study,and enriched in ACLF patients.The abundance AR gene,AR gene number and AR genotype is significantly higher in ACLF patients than in healthy controls.Conclusion:We first revealed the imbalance of intestinal structure and function genes in patients with acute on chronic liver failure,revealed the profile of antibiotic genes and the kind of antibiotics types for AR in intestinal microbial flora.Through the study of MGS,we found that the imbalance of intestinal flora were significantly associated with the illness,and could be used in diagnosis.This provided a new insight of clinical diagnosis,and provide a theoretical basis for interventional treatment of microecology.