| As a superorganism, human’s physical health is affected not only by external environment and autologous genes, but also by alien microbial. Microbiome sojourn with human, regarded as a virtual organ, carries out various metabolic functions. Most bacteria are dwells in the gut, and the gut microbiome is hourned as human’s second genome.The colon pH of normal individuals is6.5～7.5, and the intestinal microbiome composition tends to approximate, as shown by Bacteroides spp. accounting for50%, whereas Enterobacter and Enterococcus spp. less than1%. As a credible bacteria barrier, the obligateanaerobes play an important role in maintaining the intestinal microbiome composition balance. On the one hand, the intestinal obligateanaerobes tightly adhere to the enterocyte to prevent potential pathogenic bacteria integrating with enterocyte; on the other hand, intestinal obligateanaerobes inhibit potential pathogenic bacteria adhering to enterocyte through striving for nutriment followed by producing acid metabolite and reducing intestinal pH.As reported by World Health Organism, about600,000people died related to chronic HBV infection every year in the world. By2010, China has120million people were infected with HBV. Afer invade into liver, HBV can reproduce in the liver cells and further induce adaptive immune response, which will produce two effects:killing liver cells and clearing HBV, leading to liver cirrhosis over several years. China has a high serum HBV DNA positive rate of76.7%among patients with liver cirrhosis.The main clinical syndromes of cirrhosis are portal hypertension, liver function impairment and resisitance reduction in human. Portal hypertension will lead to gastrointestinal stasis, edema, stomach and intestine slowdown, intestinal permeability increase, then the intestinal pH changes and intestinal microbes cryptic forms. The impaired liver function is a crucial reason of the reduced secretion of bile and bile acids, as well as the decreased ability for liver detoxification. Taken together, all these confounding factors are responsible for the changes in intestinal micro-environment and microbial community imbalance. Microbes in the human gut undergo selective pressure from liver cirrhosis, yet its specific circumstances and molecular mechanisms in the intestinal microbial community remain obscure, highlighting the importance of a metagenomic analysis to understand microbial communities under the influence of liver cirrhosis.Metagenomics is a study of the metagenome, extracted directly from environmental samples, processed with high-throughput sequencing and bioinformation analysis. Both Traditional microbiology and microbe genomics are based on microbe cloning products and various microbial diversities are missed due to the cultured limitations. At present, more than99%bacteria can’t be cultured and detected in lab. Because of its ability to disclosure the species diversity hidden in micro-environment previously, metagenomics, as a revolution for understanding the whole living world, provide a wider view for us to observe microbial world. Collecting and extracting valuable information from enormous and complicated data undoubtedly represent a significant challenge for researchers.Here we present the first metagenomic research on the intestinal bacterial communities from patients with hepatitis B liver cirrhosis. Thirty-six faecal samples (sixteen patients with hepatitis B cirrhosis and twenty normal individuals, respectively from Gastroenterology dept of302Hospital of PLA and the patients’ family members),40～60years old, with a body mass index (BMI)=18.5～24.9kg m-2, and without food preferences, were selected for this study. Patients had positive serum HBV DNA, liver diffuse fibrosis and false lobules formation found by histology and no complications, other gastrointestinal diseases or metabolic diseases. The Child-Pugh scoring system was used to assess the prognosis of cirrhosis. All healthy subjects had normal liver biochemistry tests without evidence of hepatic or other diseases. None of the subjects had received antibiotics, probiotics, steroids or other hormones (including oral, intramuscular or intravenous injection) for at least one week before sampling. Patients and normal individuals were each asked to provide a frozen fresh stool sample, and microbiota metagenomes were extracted immediately. Metagenomes were subjected to high-throughput Solexa sequencing and bioinformation analysis, including rarefaction analysis, principal component analysis, species annotation, eggNOG function annotation, KEGG pathway annotation of significant genes, and so on.Our study suggested that the faecal microbiota of patients with hepatitis B cirrhosis contains a marked absence of Bacteroides(P<0.05) and enrichment of Veillonella (P<0.05) and Enterobacteriaceae (P<0.05). Functional and metabolic analysis shoewed that patients with cirrhosis had a significant enrichment of genes annotated to amino acid transport and metabolism, inorganicions transport and metabolism, secondary metabolite transport and metabolism, energy metabolism, xenobiotic metabolism (P<0.01), as well as a significant absence of genes annotated to cell wall/membrane biogenesis, signal transduction mechanism, reproduction, recombination and repair (P<0.05). Bile acid metabolism analysis showed that patients with liver cirrhosis had a significant absence of BSHs (P=0.013).Primary bile acid, produced in liver, flowed into intestine with bile, and broke down to secondary bile acid in the role of BSH. Bacteroides produce BSH, and BSH-mediated dissociation role provides carbon, nitrogen, sulfur source and energy for Bacteroides. So on reduces, the other one weakens.Sodium cholate, due to its molecular structure characteristics, can aggregate to form micelles when reaches a certain concentration and Veillonella spp. can hydrolyze conjugated bile salts and promote the bile stranded, which will lead to bile duct obstruction and aggravate liver cirrhosis.The disordered intestinal microbial community structure in patients implied the decreased intestinal wall resistance and changed intestinal micro-environment, which is conducive to bacteria growth due to the reduced secretion of antibodies (such as IgA), lysozyme, and mucus and destructed acid-base balance under the influence of liver cirrhosis. Additionally, the reduced intestinal blood perfusion, mesenteric ischemia and decreased bowel movements caused by liver cirrhosis give rise to a significant increase of opportunistic pathogens such as gram-negative aerobic and facultative anaerobic Enterobacteriaceae and anaerobic bacilli Veillonella. The significant enrichment of Veillonella and Enterobacteriaceae, which usually isolated from jejuna separator of healthy adults, in colon from patients with liver cirrhosis might be the result of bacteria translocation.Lipopolysaccharide (LPS), part of the outer membrane of gram-negative bacteria Veillonella and Enterobacteriaceae, is the prototypical form of endotoxin and its ability to cause disease has been confirmed. Gram-negative bacteria release endotoxin during active cell growth, autolysis, or external lysis mediated by complement and lysozyme, and phagocytic digestion of bacterial cells. Immune dysfunction, inhibited Kupffer cells function and the formation of collateral circulation caused by liver cirrhosis are highly responsible for the decreased ability of endotoxin clearance, and high concentrations of intestinal endotoxin enter into systemic circulation. LPS binds to the lipid-binding protein (LBP) in human serum, and triggers increased secretion of some pro-inflammatory cytokines (such as TNF, IL-1and IL-6), contributing to the pathophysiology of liver fibrosis and cirrhosis. Also, excess endotoxin will inhibit the intestinal epithelial synthesis, so the intestinal cell barrier is damaged, which will further make the intestinal microbes imbalanced. The inhibition of intestinal epithelial synthesis caused by excess endotoxin will also make the carrier anchor point changed, leading to the defection of amino acid and carbohydrate transportion.Patients with hepatitis B liver cirrhosis had a significant enrichment of genes annotated to the function of transport and metabolism, and absence of genes annotated to cell circle. Intestinal microhabitat of patients changed under the influence of portal hypertension, arrested liver function and host resistance declining, creating an inverse environment to the intestinal microbiota development, and promoting the formation of more extensive metabolic pathway.Here we present the first metagenomic research on the intestinal bacterial communities from patients with hepatitis B liver cirrhosis.Indeed, intestinal microbiome acts as a virtual system in human and regulates the body’s metabolic balance by gene adjustment and microbes restructuring, which are important in the disease prognosis, and this is currently under investigation. |
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