| ObjectiveThis study was to analyze the changes of intestinal microflora in cirrhotic patients and cirrhotic patients with diabetes mellitus,and to compare the difference of the flora between the two groups by high-throughput sequencing.On this basis,we further discussed the relationship between blood glucose and intestinal microflora in cirrhotic patients with diabetes mellitus,and the relationship between the intestinal microflora and the Child-Pugh score of cirrhosis.MethodsWe selected thirty-five inpatients in department of Gastroenterology of Tianjin Third Central Hospital from 2015 November to 2016 April,and the patients were divided into two groups.One group included 20 patients with liver cirrhosis,the other group had 15 cirrhotic patients with diabetes mellitus.Fresh fecal samples were collected from the two groups and quickly stored at-80 ?.The DNA was extracted from the fecal through DNA extraction kit.The extracted DNA was purified and then subjected to agarose gel electrophoresis.The purified DNA was amplified by PCR and the quality and purity of the amplified product was detected.The V3-V6 region of the 16 S rDNA of the fecal flora of two group was sequenced by the Roche454 high-throughput sequencing platform.The results were analyzed according to the sequencing results.To compare and analysize the differences and similiraty of intestinal flora between the two groups of patients with cirrhosis and cirrhosis with diabetes mellitus.The patients of the two groups were classified by pathogens,and compared the differences according to different pathogens,and compared the difference of intestinal flora for the same pathogens.Furethermore,to analysize intestinal flora associated with fasting blood glucose among the patients with cirrhosis and diabetes,and analisize the correlation of intestinal flora between Child-Pugh score among patients with cirrhosis.Results1.There were no statistically significant differences in age,sex and Child-Pugh score between 20 cirrhotic patients and 15 patients with cirrhosis and diabetes mellitus.The levels of fasting blood glucose in cirrhosis with diabetes mellitus were significantly higher than those in cirrhosis Group(P = 0.004).2.The number of valid sequences in the liver cirrhosis group was 162499,the average number of 8125 sequences in each sample;the total number of valid sequences in 15 cases of liver cirrhosis with diabetes mellitus group was 199388,the average of each sample was 13293 sequences.A total of 7,159 OTUs were obtained from the two groups by using Uclust software with a similarity of 97% as a cutoff shreshold,a total of 7159 OTUs,an average of 333 OTUs per sample in cirrhosis,at the same time a total of 6653 OTUs in the cirrhotic patients with diabetes mellitus,the average number of OTUs was 477,and there was no significant difference between the two groups.3.The analysis of sample diversity showed that there was no significant difference in chao1 index,Simpson index and shannon index between cirrhosis and cirrhosis with diabetes mellitus.The complexity analysis showed that the sparse curve of each sample tended to flat with the increase of sequencing volume gentle.The principal component analysis showed that 35 samples could not be distinguished by PC1-PC2-PC3 coordinate system,which showed that there was no significant difference in cluster and had similarity of intestinal flora structure between the two groups.4.From the analysis of the sample community structure,the species of cirrhosis and cirrhosis with diabetes mellitus were similar in the five classification levels,but there were still significant differences in the distribution of the species.At the level of the phylum,the ratio of Proteobacteria in intestinal microflora of cirrhotic patients with diabetes increased significantly(8.8%vs2.4%,P = 0.001),and the ratio of Bacteroidetes / Firmicutes was also slightly larger than that of cirrhosis(3.3VS2.1,P=0.063).At the level of the class,the level of Gammaproteobacteria and Betaproteobacteria in the intestinal flora of the patients with cirrhosis and diabetes mellitus were significantly higher than those in the cirrhosis group(5.5%vs1.6%,P = 0.001;1.5%vs0.5%,P <0.001).At the level of the order,Erysipelotrichales and Desulfovibrionales in the intestinal microflora of cirrhosis patients had obvious advantage compare to cirrhosis patients with diabetes mellitus(0.6%vs0.2%,P = 0.016;0.2%vs0.1%,P = 0.01),but the percentage of Burkholderiales and Enterobacteriales in the intestinal flora of patients with cirrhosis and diabetes mellitus increased obviously(1.5%vs0.6%,P <0.001;6.5%vs2.6%,P = 0.001),the percentage of Pasteurellale in the intestinal flora of the patients with cirrhosis and diabetes was little higher than that in the cirrhosis patients(0.6%vs0.3%,P=0.075).At the level of the family,the Porphyromonadaceae,Enterobacteriaceae,Veillonellaceae and Alcaligenaceae in the intestinal flora of cirrhosis patients with diabetes mellitus had obvious advantages(4.1%vs2.2%,P=0.005;6.5%vs1.3%,P=0.001;3.8%vs2.6%,P=0.019;1.5%vs0.6%,P < 0.001).The percentage of Clostridiaceae,Lachnospiraceae,Erysipelotrichaceae and Desulfovibrionaceae in the intestinal flora of patients with cirrhosis increased apparently(0.3%vs0.2%,P=0.028;14.8%vs9.9%,P=0.016;0.5%vs0.2%,P=0.016;0.2%vs0.1%,P=0.016),the percentage of Bacteroidaceae in the intestinal flora of the patients with cirrhosis and diabetes was little higher than that in the cirrhosis patients(51.6%vs41.9%,P=0.07).In the genus level,the populations of Butyricimonas,Blautia,Coprococcus,Dorea,Faecalibacterium and Bifidobacterium were significantly enriched in patients with liver cirrhosis(0.3% vs0.1%,P=0.011;0.5% vs0.2%,P=0.017;0.6% vs0.2%,P=0.012;0.4% vs0.2%,P=0.022;6.4% vs2.2%,P=0.001;0.7% vs0%,P=0.001,),while the proportions of Parabacteroides,Sutterella,Roseburia and Klebsiella were larger than the proportions in cirrhosis gut flora(4.3% vs2.2%,P=0.002;1.5% vs0.5%,P<0.001;1.6%vs0.1%,P<0.001;0.7% vs0%,P<0.001),the percentage of Lachnospira in the intestinal flora of the patients with cirrhosis was little higher than that in the cirrhosis patients and diabetes(3.3%vs1.6%,P=0.069).5.Through the Kruskal-Wallis H test our subject found that between the the cirrhosis group and cirrhosis with diabetes group of patients with different pathogens there were no significant difference in the five levels of phylum,class,order,family and genus.6.We compared the intestinal microflora in patients with cirrhosis and cirrhosis complicated with diabetes mellitus with the same etiology.The results showed that in the etiology group of hepatitis B,the ratios of Proteobacteria,Betaproteobacteria,Burkholderiales,Veillonellaceae,Parabacteroides,Roseburia,Veillonella and Sutterella in hepatitis B cirrhosis patients were significantly less than that in patients with hepatitis B cirrhosis with diabetes mellitus(2.8%vs7.7%,P=0.007;0.5%vs2.2%,P=0.001;0.5%vs2.2%,P=0.001;2.7%vs6.8%,P=0.003;0.5%vs2.2%,P=0.001;2.7%vs5.2%,P=0.036;0.1%vs1.5%,P<0.001;0.2%vs2.8%,P=0.011;0.5%vs2.2%,P=0.001),while Streptococcus,Clostridium,Faecalibacterium and Streptococcus had advantages in the intestinal tract of patients with hepatitis B cirrhosis(0.7%vs0.1%,P=0.021;0.4%vs0.2%,P=0.027;8.1%vs2.6%,P=0.044;0.7%vs0.1%,P=0.02).In autoimmune pathogens,the proportions of the Bacteroidetes,Proteobacteria,Gammaproteobacteria,Enterobacteriales,Streptococcus,Enterobacteriaceae,Roseburia,Veillonella and Streptococcus in the autoimmune cirrhosis patients with diabetes mellitus were more than autoimmune cirrhosis patients(80.2%vs52.3%,P=0.044;8.6%vs2.5%,P=0.002;8.0%vs1.8%,P<0.001;7.9%vs1.4%,P < 0.001;0.9%vs0.2%,P=0.015;7.9%vs1.4%,P < 0.001;1.0%vs0.2%,P=0.015;1.2%vs0.9%,P=0.012;0.9%vs0.2%,P=0.015),whereas autoimmune cirrhosis patients had more Coprococcus in their intestinal(0.5%vs0.1%,P=0.001).The proportions of the Erysipelotrichales,Lachnospiraceae,Butyricimonas?Dorea in cryptogenic cirrhosis were higher than that in cryptogenic patients with diabetes mellitus(0.3%vs0.2%,P=0.026;15.7%10.8%,P=0.024;0.8%vs0.1%,P=0.004;0.2%vs0.1%,P=0.001).There was no significant difference between other same etiologies in the two groups.7.In cirrhosis with diabetes mellitus patients,Gammaproteobacteria and fasting blood glucose were positively correlated,Enterobacteriaceae,Veillonellaceae and Klebsiella had approximately positive correlation with fasting blood glucose.8.No intestinal flora associated with the Child-Pugh score was found in the intestinal flora of patients with cirrhosis complicated with diabetes mellitus.9.In the intestinal flora of patients with liver cirrhosis,the proportion of Roseburia,Parabacteroides and Porphyromonadaceae were neg atively correlated with Child-Pugh score,Veillonella and Child-Pugh score were positively correlated.ConclusionCirrhosis and cirrhosis patients with diabetes have similar intestinal flora structure but there are specificities between them,There are some bacteria in the intestinal flora associated with cirrhosis with diabetes mellitus and the bacteria associated with the condition of patients with liver cirrhosis,which provide a theoretical basis for the clinical treatment of cirrhosis and cirrhosis with diabetes by regulating the flora structure. |
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