Liver cirrhosis occurs as a consequence of many chronic liver diseases that are prevalent worldwide. Here we characterize the gut microbiome in liver cirrhosis by comparing 98 patients and 83 healthy control individuals. We build a reference gene set for the cohort containing 2.69 million genes, 36.1% of which are novel. Quantitative metagenomics reveals 75,245 genes that differ in abundance between the patients and healthy individuals (false discovery rate < 0.0001) and can be grouped into 66 clusters representing cognate bacterial species; 28 are enriched in patients and 38 in control individuals. Most (54%) of the patient-enriched, taxonomically assigned species are of buccal origin, suggesting an invasion of the gut from the mouth in liver cirrhosis. Biomarkers specific to liver cirrhosis at gene and function levels are revealed by a comparison with those for type 2 diabetes and inflammatory bowel disease. On the basis of only 15 biomarkers, a highly accurate patient discrimination index is created and validated on an independent cohort. Thus microbiota-targeted biomarkers may be a powerful tool for diagnosis of different diseases.
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This work was supported by the National Program on Key Basic Research Project (2013CB531401), the National Natural Science Foundation of China (81301475 and 81330011), the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (81121002), the Technology Group Project for Infectious Disease Control of Zhejiang Province (2009R50041) and the Metagenopolis grant ANR-11-DPBS-0001. We thank Q. Cao, K. Su, J. Shao and A. Ghozlane for help with data computation, and H. Zhang, H. Lu, Q. Bao, J. Ge, J. Jiang, Z. Ren and M. Ye for assistance with sample collection. We are thankful to the MetaHIT consortium for generating the gut gene set and the Human Microbiome Project for generating the reference genomes from human gut microbes.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Venn diagram comparing the current major human microbiome gene set and the results of a principal component analysis of biomarkers distributed between patients with liver cirrhosis and healthy controls.
a, Venn diagram of the four currently available major human microbiome gene sets. The total gene number in each gene set and the overlapping areas are indicated. b, Venn diagram of the three major human gut gene sets (LC, liver cirrhosis gene set; T2D, type 2 diabetes gene set; MetaHIT, MetaHIT gene set; HMP, HMP gene set). c, Visualization of the principal component analysis results for the liver-cirrhosis-associated genes that differed significantly in the discovery cohort (FDR < 0.0001, Wilcoxon rank-sum test adjusted for multiple testing). The principal component analysis is built here using these genes in the validation cohort (25 patients with liver cirrhosis in red, 31 healthy controls in green).
Extended Data Figure 2 Phylogenetic abundance at the phylum, genus and species levels from liver cirrhosis and healthy control samples.
a, Phylogenetic abundance variation box plot at the phylum level and the 30 most abundant phylotypes at the genus and species levels in the healthy controls are shown. Red, green, blue, turquoise and purple represent Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria and other phyla, respectively. The colour of each genus and species corresponds with the colour of its respective phylum. b, Phylogenetic abundance variation box plot at the phylum level and the 30 most abundant phylotypes at the genus and species levels in the liver cirrhosis are shown (see Methods for the calculations). The boxes represent the interquartile range, from the first and third quartiles, and the inside line represents the median. The whiskers denote the lowest and highest values within an interquartile range of 1.53 from the first and third quartiles. The circles represent outliers beyond the whiskers.
Extended Data Figure 3 MGS enriched in healthy Chinese individuals (n = 114) are present in Danish individuals (n = 292).
Presence and abundance of 50 ‘tracer’ genes for each species; genes are in rows; abundance is indicated by colour gradient (white, not detected; red, most abundant). Individuals, ordered by increasing gene count, are in columns. Significance of correlation of species abundance (computed as mean abundance of the tracer genes) and gene count (q value, FDR adjusted) is given. Species in the Chinese cohort that were identical to those previously found, as correlated with the gene diversity in the Danish cohort27, are highlighted in red. Left, the Chinese healthy cohort. Right, the Danish obesity cohort.
Top left, healthy individuals have more gut microbial genes than patients with liver cirrhosis. Gene count was computed after downsizing the mapped reads to a level of 6.2 million (ref. 27). The significance of the difference was computed using a Student’s t-test. Bottom, abundance of patient-enriched species (n = 28) in patients with liver cirrhosis (n = 98) and healthy controls (n = 83). The relative abundance of each patient-enriched species was computed as a sum of the abundances of all the genes assigned to it divided by the sum of the abundances of all gut microbial genes in each patient, which is equal to 1 in the normalized data set. Bar length indicates the relative abundance of a given species depicted by a different colour. Patients were ordered by the total patient-enriched species abundance; LPA and HPA quartiles (n = 24) are separated by red vertical lines. Top right, oral species are frequent in patients with liver cirrhosis. MGS enriched in healthy controls are largely not assigned to a species level, while those enriched in patients with liver cirrhosis are largely assigned to a species level and are mostly of oral origin (see Methods for species assignment).
Extended Data Figure 5 The distribution of eggNOG orthologue group and KEGG functional categories for liver-cirrhosis-related markers.
a, Comparison between the liver-cirrhosis-enriched and control-enriched eggNOG orthologue group markers for 24 eggNOG orthologue group functional categories shown by number. b, Comparison between the liver-cirrhosis-enriched and control-enriched eggNOG orthologue group markers for 24 eggNOG orthologue group functional categories shown by percentage. c, Comparison between the liver-cirrhosis-enriched and control-enriched KEGG orthologue group markers for each KEGG functional category shown by number. d, Comparison between the liver-cirrhosis-enriched and control-enriched KEGG orthologue group markers for each KEGG functional category shown by percentage.
a, Venn diagram showing a gene marker comparison of case-enriched gene markers from the liver cirrhosis, T2D and IBD studies. b, Venn diagram showing a gene marker comparison of control-enriched gene markers from the liver cirrhosis, T2D and IBD studies. c, The length of the bar (y axis) represents the number of genes; the P value in the related range is shown on the x axis. The pink and light green bars show genes involved in type 2 diabetes and liver cirrhosis, respectively. Inset, the log P value of the gene markers between the two studies.
Extended Data Figure 7 The distribution of eggNOG functional categories for case-enriched and control-enriched gene markers in liver cirrhosis only, T2D only and the liver cirrhosis/T2D groups.
a, Comparison of the eggNOG orthologue group functional categories for case-enriched gene markers shown by number. b, Comparison of the eggNOG orthologue group functional categories for case-enriched gene markers shown by percentage. c, Comparison of the eggNOG orthologue group functional categories for the control-enriched gene markers shown by number. d, Comparison of the eggNOG orthologue group functional categories for the control-enriched gene markers shown by percentage.
Extended Data Figure 8 The distribution of the KEGG functional categories for case-enriched and control-enriched gene markers in liver cirrhosis only, T2D only or the liver cirrhosis/T2D group.
a, Comparison of the KEGG pathway categories for the case-enriched gene markers shown by number. b, Comparison of the KEGG pathway categories for the case-enriched gene markers shown by percentage. c, Comparison of the KEGG pathway categories for the control-enriched gene markers shown by number. d, Comparison of the KEGG pathway categories for the control-enriched gene markers shown by percentage.
Extended Data Figure 9 Estimating the optimum number of markers and establishing the taxonomic assignment of MGS.
a, Comparison of the case-enriched gene markers. b, Comparison of the control-enriched gene markers. c, The mRMR method was used to identify the liver-cirrhosis-associated markers. Sequential subsets were generated at five-marker intervals. For each subset, the error rate was estimated using a leave-one-out cross-validation of a linear discrimination classifier. The optimum (highest value of the Matthews correlation coefficient) subset contains 15 gene markers. d, The study included a discovery and a validation phase. Volunteers for both phases were recruited in the same hospital. Both direct read mapping and de novo assembly were performed for each sample. A taxonomy profiling table was established for taxonomy analysis. A novel gut gene set was established, and annotated. Identification of the MGS, finding markers and validating markers is also shown. e, MGS enriched in Chinese patients with liver cirrhosis and healthy individuals. Species-level assignment was deduced from the best BlastN hits of genes from a given MGS at thresholds of the average of more than 95% identity and more than 90% overlap with genes from a sequenced genome. For MGS where these thresholds were not reached, an assignment was attributed at the lowest taxonomy level where at least 80% of the genes had the same best hit BlastP taxonomy; in all cases these criteria held true at higher taxonomic levels. f, Taxonomic assignments of 58 species related to gut gene richness in a Danish cohort27.
Extended Data Figure 10 Phylogenetic abundance of healthy controls in the discovery stage in the liver cirrhosis and T2D studies.
The relative abundance of top bacterial phylotypes at the phylum, genus and species levels, respectively, in the liver cirrhosis study (top three panels) and in the T2D study (bottom three panels).
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Qin, N., Yang, F., Li, A. et al. Alterations of the human gut microbiome in liver cirrhosis. Nature 513, 59–64 (2014). https://doi.org/10.1038/nature13568
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