Abnormal fecal microbiota community and functions in patients with hepatitis B liver cirrhosis as revealed by a metagenomic approach

Abstract

Background: Assessment and characterization of human colon microbiota is now a major research area in human diseases, including in patients with hepatitis B liver cirrhosis (HBLC).

Methods: We recruited 120 patients with HBLC and 120 healthy controls. The fecal microbial community and functions in the two groups were analyzed using high-throughput Solexa sequencing of the complete metagenomic DNA and bioinformatics methods.

Results: Community and metabolism-wide changes of the fecal microbiota in 20 HBLC patients and 20 healthy controls were observed and compared. A negative correlation was observed between the Child-Turcotte-Pugh scores and Bacteroidetes (P < 0.01), whereas a positive correlation was observed between the scores and Enterobacteriaceae and Veillonella (P < 0.01). Analysis of the additional 200 fecal microbiota samples demonstrated that these intestinal microbial markers might be useful for distinguishing liver cirrhosis microbiota samples from normal ones. The functional diversity was significantly reduced in the fecal microbiota of cirrhotic patients compared with in the controls. At the module or pathway levels, the fecal microbiota of the HBLC patients showed enrichment in the metabolism of glutathione, gluconeogenesis, branched-chain amino acid, nitrogen, and lipid (P < 0.05), whereas there was a decrease in the level of aromatic amino acid, bile acid and cell cycle related metabolism (P < 0.05).

Conclusions: Extensive differences in the microbiota community and metabolic potential were detected in the fecal microbiota of cirrhotic patients. The intestinal microbial community may act as an independent organ to regulate the body's metabolic balance, which may affect the prognosis for HBLC patients.

Figure 1

Composition of fecal microbiota from HBLC patients and healthy individuals. Data are represented as the average percentage of each individual profile. Fecal microbiota community analysis at the phylum, class, order, family levels demonstrated that the fecal microbiota from the HBLC patients contained a significant absence of Bacteroidetes (4% compared with 53% in the microbiota from the controls) and enrichment of Proteobacteria (43% compared with 4% in the microbiota from the controls), which included most of the pathogens. The microbiota in the HBLC patients showed a significant enrichment of Gammaproteobac (42%), Negativicutes (21%), and Bacilli (20%) at the class level, Enterobacteriales (41%), Selenomonadales(20%), and Lactobacillales (19%) at the order level, and Enterobacteriaceae (39%), Veillonellaceae (19%), and Streptococcaceae (18%) at the family level.

Figure 2

Comparison of healthy and HBLC microbial structure. (A) Bacterial species abundance differentiates HBLC patients and healthy individuals. The first five principal components (P value in Tracy-Widom test < 0.05 and contribution > 3%) were examined: PC1 = 33.38%, PC2 = 21.81%, PC3 = 12.74%, PC4 = 10.31%, and PC5 = 6.17%. The first two components (PC1 and PC2) are plotted. (B) Species annotation of the significantly differential genes. Blue bars represent gene coverage of the significantly differential species, red bars represent gene coverage of the significantly differential species in the HBLC samples, and green bars represent gene coverage of the significantly differential species in the control samples. (C) Bacterial groups quantified using real-time qPCR. Blue bars represent the control samples, and red bars represent the HBLC samples. The Student t test was used to evaluate the statistical difference between the two groups. *P < 0.05; ** P < 0.01.

Figure 3

Function and metabolism analysis of fecal microbiota from HBLC patients and control samples. Blue bars represent the control samples, and red bars represent the HBLC samples. The Student t test was used to evaluate the statistical difference between the two groups. *P < 0.05; ** P < 0.01 (A) Genes annotated using the eggNOG database. The significantly differential genes were annotated as: E, amino acid transport and metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; and W, extracellular structures. (B) Genes annotated using the KEGG database. The significantly differential genes were annotated with pathways: Cellular, cell growth and death; Environmental, membrane transport; Environmental, signal transduction; Metabolism, biosynthesis of other secondary metabolites; Metabolism, energy metabolism; Metabolism, enzyme families; Metabolism, metabolism of other amino acids; and Metabolism, xenobiotics biodegradation and metabolism. (C) Relative abundance of genes annotated as BSH related to primary and secondary bile acid biosynthesis [KEGG: K01442]. Green bars represent the control samples, and red bars represent the HBLC samples. The Student t test was used to evaluate statistical difference between the two groups, P = 0.013.