Altered oral microbiota in chronic hepatitis B patients with different tongue coatings

Abstract

Aim: To elucidate tongue coating microbiota and metabolic differences in chronic hepatitis B (CHB) patients with yellow or white tongue coatings.

Methods: Tongue coating samples were collected from 53 CHB patients (28 CHB yellow tongue coating patients and 25 CHB white tongue coating patients) and 22 healthy controls. Microbial DNA was extracted from the tongue samples, and the bacterial 16S ribosomal RNA gene V3 region was amplified from all samples and sequenced with the Ion Torrent PGM™ sequencing platform according to the standard protocols. The metabolites in the tongue coatings were evaluated using a liquid chromatography-mass spectrometry (LC-MS) platform. Statistical analyses were then performed.

Results: The relative compositions of the tongue coating microbiotas and metabolites in the CHB patients were significantly different from those of the healthy controls, but the tongue coating microbiota abundances and diversity levels were not significantly different. Compared with the CHB white tongue coating patients, the CHB yellow tongue coating patients had higher hepatitis B viral DNA (HBV-DNA) titers (median 21210 vs 500, respectively, P = 0.03) and a significantly lower level of Bacteroidetes (20.14% vs 27.93%, respectively, P = 0.013) and higher level of Proteobacteria (25.99% vs 18.17%, respectively, P = 0.045) in the microbial compositions at the phylum level. The inferred metagenomic pathways enriched in the CHB yellow tongue coating patients were mainly those involved in amino acid metabolism, which was consistent with the metabolic disorder. The abundances of bacteria from Bacteroidales at the order level were higher in the CHB white tongue coating patients (19.2% vs 27.22%, respectively, P = 0.011), whereas Neisseriales were enriched in the yellow tongue coating patients (21.85% vs 13.83%, respectively, P = 0.029). At the family level, the abundance of Neisseriaceae in the yellow tongue patients was positively correlated with the HBV-DNA level but negatively correlated with the S-adenosyl-L-methionine level.

Conclusion: This research illustrates specific clinical features and bacterial structures in CHB patients with different tongue coatings, which facilitates understanding of the traditional tongue diagnosis.

Keywords: 16S rRNA gene sequencing; Chronic hepatitis B; Metabolomics; Microbiota; Tongue diagnosis.

Figure 1

Overview of the tongue coating microbiota among groups. A: Detailed characteristics of alpha diversity; B: PLS-DA discriminant analysis plot; C: Venn diagram based on OTUs. PLS-DA: Partial least squares-discriminant analysis; OTUs: Operational taxonomic units.

Figure 2

Taxonomic classification of the tongue coating microbiota at the phylum level. A: Taxonomic classification of the tongue-coating microbiota at the phylum level; B: Metastats analysis of differences in the relative proportions difference at the phylum level. ^aP < 0.05, ^bP < 0.01.

Figure 3

The most differentially abundant taxa in the chronic hepatitis B yellow and white tongue coating patients. A: Comparison of the relative abundance at the bacterial family and genus levels, the marks of significance from left to right in parentheses are the CHB yellow tongue coating patients vs the healthy controls, the CHB yellow tongue coating patients vs the CHB white tongue coating patients and the CHB white tongue coating patients vs the healthy controls. ^aP < 0.05, ^bP < 0.01, “−” indicates no significant difference based on the Metastats analysis; B: Taxonomic cladogram of the LEfSe analysis of the 16S sequences. (Red) healthy-control enriched taxa; (Green) CHB white tongue coating patients-enriched taxa; and (Blue) CHB yellow tongue coating patients-enriched taxa. C: LDA scores of enriched taxa in the healthy controls (Red), CHB white tongue coating patients (Green) and CHB yellow tongue coating patients (Blue). LDA: Linear discriminant analysis.

Figure 4

Linear discriminant analysis scores of the enriched microbial functions in the healthy-controls (Red), chronic hepatitis B white tongue coating patients (Green) and chronic hepatitis B yellow tongue coating patients (Blue). LDA: Linear discriminant analysis.

Figure 5

Associations of the tongue coating microbiotas with metabolites and clinical indices. Correlations were detected, and the indices with Spearman’s correlation coefficients > 0.3 were visualized in Cytoscape. Triangles represent clinical indicators, arrows represent metabolites and dots represent oral microbial families. If the correlations are negative, the connecting line is blue; if the correlation is positive, the connecting line is red. The line thickness represents the size of the correlation coefficient.