Quantitative Analysis Of The Gut Flora Imbalance In Liver Cirrhosis Patients

BackgroundThe human microbial community is huge, which widely distributes the skin, the oral cavity and gastrointestinal tract. Among them, the microbiota which lives in the gastrointestinal tract is referred to the intestinal flora. The gut flora is the most complex part in the human microbial community and plays an important role in human health. The weight of the intestinal flora with a normal adult can reach1-2kg, number as many as1014, including500-1000different species, mainly consisting of anaerobic bacteria, facultative anaerobic bacteria and aerobic bacteria etc. All the bacteria is different degree distribution in gut according to certain proportion and quantity. In normal circumstance, the intestinal microbiota keeps dynamic balance inside the human body and it is necessary and beneficial to maintain the human health. The intestinal microbiota participates in digestion nutrition, biosynthesis, biological barriers, and the immune functions promotion. Intestinal mucosa barrier system, consisting of normal intestinal flora, complete intestinal mucosa structure, mucin on the surface of mucosa, sIgA and gut immune cells, which prevents the gut bacteria and endotoxin produced by bacteria to shift outside. In a variety of pathological conditions, microflora imbalance is caused by the damage of the condition which is host to maintain the microbial balance. Flora imbalance includes proportion imbalance and bacteria translocation. The proportion imbalance refers to the dominant origin bacteria with growth suppression and a small number of bacterial with overgrowth, which induces the gut microflora imbalance. Bacterial translocation defines bacteria and bacterial toxin products (such as endotoxin) from the intestine into the mesenteric lymph nodes and other parenteral organs and parts, in most cases, endotoxemia is an important indicator of a reflection of bacterial translocation.At the same time, the intestine which is the biggest bacteria library and endotoxin pool in human is the main source of endogenous infections and endotoxemia. Recent years, increasing studies show that in some pathological conditions such as liver cirrhosis, severe hepatitis and serious burns etc, appearing gut flora imbalance, intestinal barrier function damage, bacteria translocation and enterogenous endotoxemia, which can influence patients life quality seriously.Liver cirrhosis is a common disease in China, the complications including bacterial infection, upper gastrointestinal bleeding and hepatic encephalopathy that usually induce the patients death. Intestinal flora and liver tie up closely not only in anatomical structure but also in functional status through the portal and the lymphatic system, which influence each other. One hand, the intestinal flora can provide vitamin K to promote the synthesis of liver clotting factors, and enhance the Kupffer cell number and function, strengthen body immunity; the other hand, the liver secretes bile acid to maintain the intestinal flora balance.Previous studies suggested that intestinal flora disorder is an important reason in the complications such as spontaneously peritonitis, enterogenous endotoxin and hepatic encephalopathy. Metagenomics study suggests that fecal microbial communities are distinct in cirrhotic patients compared with healthy individuals. The prevalence of potentially pathogenic bacteria, with the reduction of beneficial populations in cirrhotics may affect prognosis. Recently, increasing evidence suggests that gut flora is involved in the pathogenesis of liver cirrhosis and its complications in rat model. But there are few large-scale studies refer to human. In addition, the specific of bacteria in gut flora imbalance and the influence of microbiota disturbance to outcomes of patients are not completely clear.ObjectiveThe objective of our research is to quantitative analyze the change of fecal microbiota and endotoxemia in liver cirrhosis patients with different stage in comparison with healthy controls and to explore the relationship between gut flora disturbance and different classification and prognosis, which can provide the experimental evidence for the intestinal barrier function strengthen and new probiotics excavation in liver cirrhosis patients.Materials and methods1、 subjectsFecal and serum samples of patients with liver cirrhosis were collected from Guangzhou Southern hospital from September2010to Septembe2011and the control group gathered from healthy volunteers. Cirrhotic patients included in the standard reference of the Chinese Medical Association infectious diseases Parasitology, of Hepatology joint revised ((viral hepatitis prevention and treatment programs (September2000Xi’an)》 the diagnostic criteria for cirrhosis in hepatitis and in October2001Society of Hepatology of Chinese Medical Association developed 《the diagnostic criteria of alcoholic liver disease (draft)》 in alcoholic cirrhosis of the diagnostic criteria for diagnosis of patients with cirrhosis. The controls were composed of healthy people without acute or chronic liver disease, other gastrointestinal disease and regular check abnormality. All the subjects did not use any antibiotics or probiotics before4weeks at sampling.2、fecal bacterial cultureFresh feces in sterile boxes were immediately sent to the laboratory for culture. From this,0.5g of feces of each sample was mixed with4.5mL broth. After that, the mixture was serially diluted to10-2-10-7with broth.20μL of which with each degree concentration were applied to the different mediums correspondingly.10types of bacteria, including4aerobes (Enterobacter, Enterococcus, staphylococcus and yeast) and6anaerobes (Bacterioides, Bifidobacterium, Lactobacillus, Eubacterium, streptococcus and Clostridiurn), the former were cultured for24h under aerobes condition, while the latter for48h under anaerobes condition. The results were shown by lg colony forming unit (CFU) per gram feces each subject.3、fecal DNA extractionAll the steps of DNA extraction were followed TIANamp Stool DNA Kit (TIANGEN) protocol. The fecal DNA was detected the OD260/280value, concentration and stored at-20℃before analysis.4、specificity of primersWe use the fecal sample from healthy controls for regular PCR amplification. Each PCR mixture(25uL) were composed of12.5uL Premix Taq,1uL forward and reverse primers, respectively, luL DNA template and9.5uL ddH2O. The amplification program consisted of one cycle of94℃for5min;30cycles of94℃for30s, annealing for30s(temperature in table2), and72℃for60s; and finally one cycle of72℃for10min. The PCR products were subjected to gel electrophoresis in1.5%agarose with1000bp DNA Ladder for the standard, followed by ethidium bromide staining.5^real-time PCREach standard was serially diluted to101-107as DNA template to make a standard curve by real-time PCR.20uJ amplification reaction was performed with10uL of SYBR Green Master Mix,1uL forward and reverse primers, respectively,5uL DNA template and3uL ddH2O. Amplifications were performed under the following profiles:one cycle at95℃for5minutes,45cycles of denaturation at95℃for10seconds, annealing at corresponding tempreture(Escherichia coli, Lactobacillus, Bifidobacterium58℃, Bacteroides, Clostridium, enterococcus,52℃) for20seconds, and72℃extension for30seconds, fluorescence was measured at this stage. The specificity of PCR products was evaluated by melt curve. The copy number of target DNA was determined by comparison with serially diluting standards running on the same plate. Bacterial quantity was expressed as lg copy numbers per gram of stool.6、metagenomicsWe designed universal primers regard to the V4-V6region of bacterial16S rRNA for conventional PCR reaction. Each PCR mixture(25uL) were composed of10uL Premix Taq,0.5uL forward and reverse primers(universal primer), respectively,5uL DNA template and9uL ddH2O. The amplification program consisted of one cycle of94℃for2min;25cycles of94℃for30s,59℃annealing for30s, and72℃for45s; and finally one cycle of72℃for5min. The PCR product was the illumina HiSeq2000sequencing platform sequencing to get the data to cluster analysis.7、serum endotoxin detectionThe blood samples were treated. All the steps of endotoxin test were followed the kit protocol.8、statistical analysisThe analysis was conducted in SPSS version13.0. The data of the bacteria cultures was expressed as Mean±SD and the rest of the data as M (median);The Mann-Whitney U test and Kruskal-Wallis H tests were used to evaluate the difference between two groups and among three, respectively. Correlation between variables was evaluated by Spearman Rank correlation. The comparison of rate was evaluated by chi-square test, rates were compared using chi-square test.We took results with a level of significance<5%to be statistically significant.Results1、Traditional bacteria cultureTo compare with healthy controls, culture results showed that the lever of Escherichia coli(Z=-2.030, P=0.042), staphylococcus(Z=-1.974, P=0.048)and yeast (Z=-2.413, P=0.016)was significantly enriched, Bacteroides(Z=-2.198, P=0.028), Lactobacillus(Z=-2.044, P=0.041)and Clostridium(Z=-2.566, P=0.010)were significantly reduced in patient group.2、Real-time quantitative PCRTo compare with healthy controls, qPCR results suggested that the amount of Escherichia coli(Z=-3.963, P=0.000)and Enterococcus(Z=-6.762, P=0.000)were statistically increased, Lactobacillus(Z=-7.093, P=0.000), Bifidobacterium(Z=-5.186, P=0.000), Bacteroides(Z=-6.058, P=0.000) and Clostridium(Z=-4.268, P=0.000)were statistically decreased.3、MetagenomicsCompared with the control group, the intestinal flora of cirrhotic patients had a significant difference, that was correlated with liver function classification. Also they had a characteristics of the genus, Veillonellaceae was significantly increased, Verrucomicrobia, Bacteroides, Porphyromonadaceae, Acidaminococcus and Desulfovibrionaceae was significantly reduced.4^Quantitative analysis of the gut flora in patients with different liver classification The lever of Escherichia coli(x2=13.321, P=0.001), Enterococcus(x2=7.403, P=0.025), Bacteroides(x2=13.425, P=0.001), Clostridium(x2=12.277, P=0.002), Lactobacillus(x2=49.314, P=0.000)and Bifidobacterium(x2=6.760, P=0.034) were significant differences between the different classification of liver function in patients.5^Quantitative analysis of relationship between flora imbalance and prognosis in patientsEscherichia coli had a positive correlation with Child-classification (R=0.241, P=0.002), Child-Turcotte-Pugh (CTP) score(R=0.235, P=0.003), model for end-stage liver disease (MELD) score(R=0.231, P=0.010). While Lactobacillus and Clostridium were correlated negatively with Child-classification(former R=-0.276, P=0.000; latter R=-0.532, P=0.000), Child-Turcotte-Pugh (CTP) score(former R=-0.280, P=0.000; latter R=-0.420, P=0.000), model for end-stage liver disease (MELD) score(former R=-0.227, P=0.000; latter R=-0.435, P=0.011).6、Quantitative analysis of correlation between endotoxemia and disease severity, prognosisThe lever of endotoxin(Z=-7.593, P=0.000) were significantly increased in patients with liver cirrhosis in comparison with controls. Endotoxin(x2=36.168, P=0.000) were statistically different in cirrhotic patients with varied liver function, which greatly increased while the liver function deterioration. Endotoxin had a positive correlation with Child-classification(R=0.44, P=0.000), CTP score(R=0.30, P=0.000) and MELD score(R=0.37, P=0.000).Conclusion1、 The intestinal bacteria disorder with different degrees is prevalent in patients with liver cirrhosis, increased of Escherichia coli and reduction of Bacteroides, Lactobacillus and Clostridium is consistent in the bacterial culture and qPCR.2、The increased of Escherichia coli and decreased of Lactobacillus and Clostridium have a positive correlation with the severity and bad prognosis of the disease in the cirrhotic patients.3、Clostridium are likely to play a positive role in the prognosis of patients with liver cirrhosis and may become a new probiotic, which needs our further more study to confirm.4、The results of traditional culture and qPCR are consistent. Metagenomics reflect the diversity of the intestinal flora more intuitively and completely, that can find more subtle flora imbalance.5、The prevalence of endotoxemia in cirrhosis patients may accelerate disease progression and affect the prognosis.