Tongue-coating microbiome signatures associated with greasy tongue coating and radiological nodule progression risk in patients with pulmonary nodules: an integrated metagenomic and 16S rRNA sequencing study

Greasy tongue coating is a commonly observed tongue-coating phenotype in clinical practice, but its corresponding tongue-coating microbiological features and its relationship with the risk of radiological progression of persistent ground-glass pulmonary nodules remain unclear. This study integrated metagenomic sequencing and 16S rRNA sequencing to investigate tongue-coating microbiome characteristics, potential functional alterations, and their associations with subsequent radiological progression in patients with pulmonary nodules and greasy tongue coating. This ambispective observational study combined prospective tongue-image and tongue-coating sample collection at enrollment with retrospectively retrieved pre-enrollment chest CT data and post-enrollment CT follow-up. The clinical 16S cohort included 497 patients with persistent ground-glass pulmonary nodules, comprising 233 patients in the non-greasy tongue coating group and 264 patients in the greasy tongue coating group. This cohort was used to analyze the relationships among tongue-coating phenotype, CT imaging features, subsequent pulmonary nodule progression, and genus-level tongue-coating microbiome characteristics. The date of tongue-image and tongue-coating sample collection was defined as baseline for progression analysis, and only radiological progression identified during post-sampling follow-up was considered an outcome. Pre-enrollment CT data were used to confirm nodule persistence and characterize historical imaging features. An independent, non-overlapping metagenomic sequencing cohort recruited at the same institution included 150 participants, comprising 30 healthy controls, 60 patients with pulmonary nodules and non-greasy tongue coating, and 60 patients with pulmonary nodules and greasy tongue coating. This cohort was used to characterize species-level microbial differences and MetaCyc functional pathway features. Corresponding genus-level patterns were further assessed for cross-platform consistency in the 16S cohort. Compared with the non-greasy tongue coating group, the greasy tongue coating group had a larger average pulmonary nodule diameter and higher proportions of high-risk CT features and radiological progression of pulmonary nodules after sampling. 16S rRNA sequencing showed that the greasy tongue coating group had increased alpha diversity and a statistically significant but modest difference in overall microbial community composition compared with the non-greasy tongue coating group, together with enrichment of multiple anaerobe-related genera. Species-level metagenomic analysis identified six core differential species associated with greasy tongue coating: Campylobacter concisus , Campylobacter curvus , Selenomonas timonae , Treponema socranskii , Treponema vincentii , and Metamycoplasma salivarium . Metagenomic functional annotation indicated higher predicted abundances of MetaCyc pathways related to nitrate reduction, denitrification, menaquinol-8 biosynthesis, bacterial protein N-glycosylation, and thiamine diphosphate biosynthesis in the greasy tongue-coating group. In the clinical 16S cohort, Campylobacter and Selenomonas were significantly increased in the greasy tongue coating group, consistent with the metagenomic findings. Further clinical association analysis showed that Campylobacter abundance was weakly positively correlated with average nodule diameter and was higher in patients with documented radiological progression. Multivariable logistic regression showed that Campylobacter remained significantly associated with radiological progression status after adjustment for age, sex, average nodule diameter, nodule type, and high-risk CT features. Patients with pulmonary nodules and greasy tongue coating showed characteristic alterations in the tongue-coating microbiome, mainly manifested by enrichment of genera such as Campylobacter and Selenomonas, together with enrichment of predicted functional pathways related to anaerobic respiration, nitrate reduction, quinone-mediated electron transfer, and bacterial surface structure. Campylobacter may represent a candidate microbial feature associated with both the greasy tongue-coating microbiome phenotype and subsequent pulmonary nodule progression risk. However, these findings remain observational and exploratory and require further validation in independent multicenter cohorts and mechanistic studies.