To compare the characteristics of tongue coating flora in healthy people, patients with chronic hepatitis B (CHB) and patients with CHB complicated with non-alcoholic fatty liver disease (NAFLD), and to explore the characteristics of tongue coating flora in patients with CHB complicated with NAFLD and its comorbidity mechanism.

From January 2024 to December 2024, 42 with CHB complicated with NAFLD patients with CHB complicated with NAFLD(CHB complicated with NAFLD group) and 34 patients with simple CHB (simple CHB group) were subjected in our department, and 28 healthy individuals were selected and served as control group. Baseline clinical data were collected from all the participants, and intergroup differences were analyzed. After tongue coating samples were harvested for 16S ribosomal RNA (16S rRNA) sequencing using the PacBio Sequel sequencing platform. After sequence quality control, denoising, and amplicon sequence variant (ASV) clustering, the microbial composition, α diversity, and β diversity were analyzed in the 3 sample groups. Linear discriminant analysis effect size (LEfSe) was used to screen for intergroup differential marker species, by linear discriminant analysis (LDA) score >2 and P<0.05, and Kyoto encyclopedia of genes and genomes (KEGG) and MetaCyc functional prediction analyses were also performed.

Significant differences were observed among the 3 groups in terms of age, gender, body mass index (BMI), alanine aminotransferase (ALT), fasting plasma glucose (FPG) and free fatty acids (FFA) (P<0.05). There were totally 48 236 ASVs obtained, with CHB complicated with NAFLD group having the highest number of unique ASVs. Compared to the healthy control group, the CHB complicated with NAFLD group exhibited an increasing trend in species richness, community heterogeneity, and distribution uniformity of the tongue coating microbiota. At the phylum level, the proportions of Firmicutes (32%) and Proteobacteria (26%) were increased, while the proportion of Bacteroidota (27%) was decreased in the CHB complicated with NAFLD group than the healthy control group. In comparison to the CHB-only group, the proportion of Bacteroidota was increased. At the genus level, the proportions of Veillonella (18%) and Neisseria (18%) were increased, while that of Prevotella_7 (15%) was decreased in the CHB complicated with NAFLD group than the healthy control group, and that of Neisseria and Prevotella_7 was increased in the CHB complicated with NAFLD group when compared with the CHB-only group. LEfSe analysis revealed that Actinobacteriota, Neisseria_elongata, and Streptococcus_oralis were potential marker species in the CHB complicated with NAFLD group. Both KEGG and MetaCyc functional prediction analyses indicated that the differentially expressed functions of the tongue coating microbiota among the 3 groups were primarily enriched in pathways such as cofactor and vitamin metabolism, carbohydrate metabolism, and lipid metabolism.

CHB complicated with NAFLD patients exhibit characteristic changes in their tongue coating microbiota. The flora of tongue coating may affect the glucose and lipid metabolism of CHB patients through the "Oral-gut-liver axis", and promote the process and progress of NAFLD.