Characteristics Of Gut Microbiota In Patients With Hepatitis B Virus Related Acute-on-chronic Liver Failure By Using Metagenomic Approach

The infection of chronic hepatitis B virus(HBV)is an important global public health problem that threat the health of people around the world.In China there was an estimated of 93 million HBV carriers.In the natural history of chronic hepatitis B(CHB),spontaneous acute exacerbation(AE)is not uncommon,with a cumulative incidence of 10-30%.While exacerbations can be mild hepatitis,some patients may develop to severe hepatitis(acute-on-chronic liver failure,ACLF).ACLF is associated with complications such as gastrointestinal bleeding,spontaneous bacterial peritonitis(SBP),hepatic encephalopathy and other manifestations of volume overload with high mortality.Although the encouraging progress have been made in the supportive treatment of organ failure,the treatment of ACLF is still a worldwide problem because of their unclear pathogenesis and high mortality.In recent years,great progress has been made in the pathogenesis of ACLF.The disordered in immune function and bacterial translocation which resulted from bacterial proliferation in small intestine together with the circulatory disturbance were considered as the key causes that led to ACLF.Once cirrhosis and ACLF have set in,the blood supply and immune status of intestinal microenvironment may be easily affected and the unbalance in the intestine microecology would even be caused.Spontaneous bacterial peritonitis(SBP),which is one of the common causes of deterioration of liver function,is a common and serious complication in ACLF patients.Gut microflora dysbiosis and bacterial translocation are considered as the main mechanism in SBP.In recent years,accumulating evidences indicated that the gut microbiota played a key role in chronic liver diseases and their various complications with development of techniques such as 16 S rDNA and metagenomics.However,changes in intestinal flora at various clinical settings of acute exacerbation in CHB were still unclear,especially in the gut microbiota that distinguished HBV-ACLF from CHB patients with non-ACLF.Our aim was to comprehensively analyzed the gut microbiota of CHB patients and to explore the characteristics of the intestinal flora community in HBV-ACLF patients.In the first part,our study aim was to investigate the characteristics of gut microbiota in HBV-ACLF patients with the severe chronic hepatitis B(S-CHB),mild to moderate chronic hepatitis B(M-CHB)and healthy were served as controls.The genomic DNA was extracted from stool samples and followed by high-throughput sequencing of the V3-V4 region of the 16 S r RNA gene.In the second part,we retrospectively analyzed the distribution characteristics of microorganisms in ascites from SBP patients and in vestigated the existence of bacteria in negative ascitic fluid samples by 16 S rDNA high-throughput sequencing in order to study the relationship between the intestinal flora disorder and the SBP,and to evaluate the efficacy of serum procalcitonin(PCT)and percentage of neutrophils(Neu%)in predicting SBP.The main results of this study were as follows:1.We studied the characteristics of the gut microbiota by high-throughput sequencing of 16 S rDNA in CHB patients and observed that the number of OTUs and community richness of intestinal microbiota in HBV-ACLF patients were significantly lower than those in M-CHB patients and healthy controls.The overall gut microbiota composition of patients with HBV-ACLF was significantly different from M-CHB group and healthy controls,as evidenced by their separation of the NMDS analysis.Their separation remained significantly even after the excluding patients of with cirrhosis,which suggested that the overall intestinal microbiota structure were significantly changed in HBV-ACLF patients regardless of concomitant cirrhosis.2.At genus-level,a wider range of gut microbiota community changes in HBV-ACLF patients were observed when it compared with those of M-CHB.We trained a random forest model in a training set of the HBV-ACLF and M-CHB patients using the profiles of genera.The microbiota composition can identify HBV-ACLF status and the genera were ranked in order of importance.The results suggested that microbiota composition may significantly distinguish between two disease status and there was different importance of the genera.3.Twenty-two genera,including Veillonella,Streptococcus,Fusobacterium and others,were significantly increased in HBV-ACLF patients and their abundance were significantly positively correlated with TB,PT,INR levels and MELD score;whereas another twenty-six genera,including Romboutsia,were enriched in M-CHB patients and their abundance were significantly negatively correlated with TB,INR,MELD scores.The correlations of genera abundance with clinical biomarkers suggested that the forty-eight characteristic genera were associated with the severity of the disease.4.We assessed the succession process associations between the abundances of genera included in the CHB signature described above and various clinical settings of acute exacerbation.The cumulative abundance of M-CHB-enriched genera(MEG)was the potential beneficial bacteria and that of ACLF-enriched genera(AEG)was the potential harmful bacteria.The succession process showed that MEG was significantly higher than AEG(P<0.05)both in HC and M-CHB group,MEG was similar to AEG(P>0.05)in S-CHB patiengs,and MEG was significantly lower than AEG(P<0.05)in ACLF patients.As the liver disease progressing,the cumulative abundance of MEG was significantly decreased and that of AEG was significantly increased.While ACLF ensues,there was a ratio inversion of the cumulative abundance of MEG to AEG.5.According to the clinical data of patients with SBP,the common bacteria in asc ites culture included Escherichia coli,Klebsiella pneumoniae,Streptococcus,staphylococcus et al..For patients with peritoneal irritation sign but negative cultured ascites,16 S rDNA high-throughput sequencing suggested that there might be one or more bacteria or bacterial DNA.Several types of bacterial genera from ascites were consistent with what were detected in intestinal flora of HBV-ACLF patients.6.In SBP patients with ascites polymorphnuclear(PMN)?250×106/L,serum PCT in patients with culture positive was significantly higher than that in patients with culture negative [(4.51(1.54,8.46)Vs 0.77(0.21,1.69),P<0.05] [median(quartile)].The diagnostic value of serum PCT and percentage of neutrophils in SBP patients was performed by ROC curve analysis and showed that the optimal cut-off values of PCT in the diagnosis of SBP1 group(culture positive and PMN?250×106/L),SBP2 group(culture positive and PMN<250×106/L),SBP3 group(culture negative and PMN?250×106/L)were: 0.795ng/ml,0.265ng/ml,0.405ng/ml respectively.The areas under curves values were 0.963,0.767 and 0.714,respectively.The sensitivities were 100.00%,90.00%,62.30%,respectively.The specificities were 92.70%,63.40%,80.50%,respectively.The optimal cut-off values of percentage of neutrophils in the diagnosis of SBP1,SBP2 and SPB3 were 68.45%,62.65%,65.00%,respectively.The areas under curves were 0.878,0.756,0.669,respectively.According to the cut-off values above,PCT and percentage of neutrophils in series in the diagnosis of SBP1,SBP2 and SPB3,the areas under curves were 0.976,0.865,0.706,respectively.Conclusion:This study,based upon the methods of high-throughput sequencing,preliminarily uncovered the disorders of intestinal flora at different stages of HBV-ACLF and CHB.As the increase severities of CHB,the gut microbiota turned into disorders progressively and structurally with a significant decrease in the whole flora abundance.The characteristics of intestinal flora in HBV-ACLF patients was significantly different from that in M-CHB patients,in which the balance of intestinal flora was destroyed and there was a proportional inversion between potential pathogen and the beneficial bacteria.Statistically,the genera of potential pathogen that increased in HBV-ACLF patients were partly consistent with what were detected from ascites in SBP patients.Conclusively,this study suggested that the abnormal increase in potential pathogen probably increased the infection risk and played an important role in the development of ACLF.In additon,it provided a supportive evidence that gut microbiota was a proper target for the diagnosis and treatment of ACLF.