Featured Gut Microbiomes Associated With the Progression of Chronic Hepatitis B Disease

Abstract

Dysbiosis of gut microbiota during the progression of HBV-related liver disease is not well understood, as there are very few reports that discuss the featured bacterial taxa in different stages. The aim of this study was to reveal the featured bacterial species whose abundances are directly associated with HBV disease progression, that is, progression from healthy subjects to, chronic HBV infection, chronic hepatitis B to liver cirrhosis. Approximately 400 fecal samples were collected, and 97 samples were subjected to 16S rRNA gene sequencing after age and BMI matching. Compared with the healthy individuals, significant gut microbiota alterations were associated with the progression of liver disease. LEfSe results showed that the HBV infected patients had higher Fusobacteria, Veillonella, and Haemophilus abundance while the healthy individuals had higher levels of Prevotella and Phascolarctobacterium. Indicator analysis revealed that 57 OTUs changed as the disease progressed, and their combination produced an AUC value of 90% (95% CI: 86-94%) between the LC and non-LC groups. In addition, the abundances of OTU51 (Dialister succinatiphilus) and OTU50 (Alistipes onderdonkii) decreased as the disease progressed, and these results were further verified by qPCR. The LC patients had the higher bacterial network complexity, which was accompanied with a lower abundance of potential beneficial bacterial taxa, such as Dialister and Alistipes, while they had a higher abundance of pathogenic species within Actinobacteria. The compositional and network changes in the gut microbiota in varied CHB stages, suggest the potential contributions of gut microbiota in CHB disease progression.

Keywords: cooccurrence network; gut dysbiosis; hepatitis B virus; liver cirrhosis; random forest.

FIGURE 1

Ordination analyses of clinical parameters and gut bacterial community grouped by disease progression. (A) Non-metric multidimensional scaling (NMDS) based on the Bray–Curtis distance showing the overall distribution pattern of clinical parameters. (B) db-RDA analyses reflecting differences in gut microbiota structures fitted with significantly correlated clinical properties. The letter with arrows indicated the total variance explanation degree. (C) Non-metric multidimensional scaling (NMDS) plot based on Bray–Curtis distances calculated using OTU compositions. (D) NMDS plot based on Bray–Curtis distances calculated using genus compositions. Adonis R² (effect size) between groups of different category subjects was calculated with 999 permutations and shown in the lower part of the figure. HBVI, chronic HBV infection; CHB, chronic hepatitis B; and LC, liver cirrhosis. OTU, operational taxonomic unit. The color scheme for Healthy, HBVI, CHB and LC in the graph are red, green, blue, and purple.

FIGURE 2

Comparisons of bacterial diversity among healthy and the HBV infection progression. (A) The Venn graph of percentage of shared and unique OTUs. (B) The observed species number comparisons. (C) The diversity indexes (Shannon, J, Simpson and Chao1) comparisons. Letters indicate the ANOVA groupings. The color scheme for Healthy, HBVI, CHB and LC in the graph are red, green, blue, and purple.

FIGURE 3

The bacterial composition of fecal samples from patients in different taxa levels. (A) Phyla distribution. (B) Class distribution. (C) Comparisons of top 40 genus taxa among four disease stages by heatmap. The color scheme for Healthy, HBVI, CHB and LC in the graph are red, green, blue, and purple.

FIGURE 4

Indicator species significantly (q < 0.1) associated with the disease progression. The bar indicates the relative abundance of each indicator species, while the size of each circle represents the indicator value (association strength) of a specific species with the different group: 0–0.25, not characteristic; 0.25–0.5, weakly characteristic; 0.5–0.75, characteristic; and 0.75–1.0, strongly characteristic. The color scheme for Healthy, HBVI, CHB and LC in the graph are red, green, blue, and purple.

FIGURE 5

The indicator species’ distribution and correlation with clinics along with the disease progression. (A) The correlation graph between clinics and indicator species, * denotes significance p < 0.05 and ** denotes p < 0.01. (B) The ROC curve based on the indicator OTU to predict its potential to distinguish healthy and non-healthy, HBVI and non-HBVI, CHB and non-CHB, LC and non-LC. (C) The distribution of those indicator species with higher abundance (>0.1%) that significantly correlated with clinics. * Denotes significance p < 0.05 and ** denotes p < 0.01. The color scheme for Healthy, HBVI, CHB and LC in the graph are red, green, blue, and purple.

FIGURE 6

The network analysis revealing the co-occurrence patterns between bacterial taxa for each sample group. (A) Healthy group network. (B) HBVI group network. (C) CHB group network. (D) LC group network. The nodes were colored according to the phyla assigned by the taxa. A connection represents a strong (Spearman’s correlation coefficient R² > 0.6) and significant (p < 0.05) correlation. Edges weighted according to the correlation coefficient and node size weighted according to the relative abundance of microbial taxa.