Commensal Oral Microbiota, Disease Severity, and Mortality in Fibrotic Lung Disease

Abstract

Rationale: Oral microbiota associate with diseases of the mouth and serve as a source of lung microbiota. However, the role of oral microbiota in lung disease is unknown. Objectives: To determine associations between oral microbiota and disease severity and death in idiopathic pulmonary fibrosis (IPF). Methods: We analyzed 16S rRNA gene and shotgun metagenomic sequencing data of buccal swabs from 511 patients with IPF in the multicenter CleanUP-IPF (Study of Clinical Efficacy of Antimicrobial Therapy Strategy Using Pragmatic Design in IPF) trial. Buccal swabs were collected from usual care and antimicrobial cohorts. Microbiome data were correlated with measures of disease severity using principal component analysis and linear regression models. Associations between the buccal microbiome and mortality were determined using Cox additive models, Kaplan-Meier analysis, and Cox proportional hazards models. Measurements and Main Results: Greater buccal microbial diversity associated with lower FVC at baseline (mean difference, -3.60; 95% confidence interval [CI], -5.92 to -1.29% predicted FVC per 1-unit increment). The buccal proportion of Streptococcus correlated positively with FVC (mean difference, 0.80; 95% CI, 0.16 to 1.43% predicted per 10% increase) (n = 490). Greater microbial diversity was associated with an increased risk of death (hazard ratio, 1.73; 95% CI, 1.03-2.90), whereas a greater proportion of Streptococcus was associated with a reduced risk of death (HR, 0.85; 95% CI, 0.73 to 0.99). The Streptococcus genus was mainly composed of Streptococcus mitis species. Conclusions: Increasing buccal microbial diversity predicts disease severity and death in IPF. The oral commensal S. mitis spp associates with preserved lung function and improved survival.

Keywords: buccal microbiome; disease progression; idiopathic pulmonary fibrosis.

Figure 1.

Streptococcus genus dominance of the buccal mucosa associates with preserved lung function and reduced disease severity in idiopathic pulmonary fibrosis (IPF). A total of 511 patients enrolled in the CleanUP-IPF (Study of Clinical Efficacy of Antimicrobial Therapy Strategy Using Pragmatic Design in IPF) trial provided buccal swabs for DNA extraction. Buccal microbial communities were characterized by 16 s rRNA sequencing. For initial evaluation, patients were stratified into groups based on percentage predicted FVC (ppFVC) tertiles. (A) Principal component analysis (PCA) and ordination of 16S rRNA gene data from patients with recorded baseline FVC (n = 490) demonstrates significant variation based on ppFVC tertiles by permutational multivariable ANOVA (PERMANOVA) (P = 0.0009). Data points are weighted by ppFVC with a range of minimum 22% to maximum 125% and colored by ppFVC tertile. (B) Biplot of genus ordination data demonstrating directionality of association between genera and FVC with the Streptococcus genus associating with higher FVC tertile clustering in the PCA. (C) Rank relative abundance of all clinical samples demonstrates domination of buccal communities by the genus Streptococcus, with other commonly reported buccal genera present. (D) Linear regression of Streptococcus genus and FVC (Spearmen regression line with 95% confidence intervals) with significant positive association. (E) Taxon domination is associated with higher FVC tertile cohort (pairwise Wilcoxon rank-sum with Benjamini Hochberg correction for multiple comparisons).

Figure 2.

Lower microbial diversity of the buccal mucosa associates with preserved lung function in idiopathic pulmonary fibrosis (IPF). A total of 511 patients enrolled in the CleanUP-IPF (Study of Clinical Efficacy of Antimicrobial Therapy Strategy Using Pragmatic Design in IPF) trial provided buccal swabs for DNA extraction. Buccal microbial communities were characterized by 16 s rRNA sequencing. A total of 490 patients had complete data available for analysis of associations between FVC and buccal microbial diversity. Microbial diversity was measured using the Shannon diversity index (SDI). Bacterial burden in swabs was calculated by droplet digital PCR of the 16S rRNA gene. (A) Strong negative correlation between Streptococcus genus relative abundance and microbial diversity, with increasing Streptococcus abundance associated with reduced SDI and higher percentage predicted FVC (ppFVC) (data points weighted by ppFVC with a range of minimum 22% to maximum 125% predicted) (Spearmen regression with line and 95% confidence intervals [CIs]). (B) Linear regression of SDI and ppFVC demonstrating a significant negative association (Spearmen regression with line and 95% CIs). (C) No association between bacterial burden in buccal swabs and ppFVC was observed (log₁₀ transformation of 16S rRNA copies per μl of buccal DNA) (Spearmen regression with line and 95% CIs).

Figure 3.

Key features of the buccal microbiome predict survival in idiopathic pulmonary fibrosis (IPF). Buccal swabs from 253 patients enrolled in the CleanUP-IPF (Study of Clinical Efficacy of Antimicrobial Therapy Strategy Using Pragmatic Design in IPF) trial and not exposed to long-term antimicrobials. Buccal microbial communities were characterized by 16S rRNA sequencing, and microbial diversity was calculated by Shannon diversity index (SDI). (A) Additive Cox model plot of SDI demonstrates a clear association between lower diversity and reduced relative death rate, with a threshold at approximately 1 unit SDI (dashed red line). (B) Kaplan-Meier plots of SDI stratified by diversity threshold of ⩾1 or <1. (C) Additive Cox model plot showing relationship between relative abundance of Streptococcus and relative death rate demonstrating a threshold abundance (approximately 70% relative abundance) of Streptococcus. (D) Kaplan-Meier plots of buccal Streptococcus abundance stratified at 70% and mortality. (E) Shotgun metagenomics allowing for species-level identification of microbial taxa, with Streptococcus mitis species dominating buccal communities.

Figure 4.

Clinical outcomes and the buccal microbiome in assigned antimicrobial treatment cohorts. Buccal swabs from patients enrolled in the CleanUP-IPF (Study of Clinical Efficacy of Antimicrobial Therapy Strategy Using Pragmatic Design in IPF) trial and exposed to either long-term cotrimoxazole (n = 120) or doxycycline (n = 116). Buccal microbial communities were characterized by 16S rRNA sequencing, and microbial diversity was calculated by Shannon diversity index (SDI). (A) All patients at baseline trial enrollment when stratified by assigned treatment groups (i.e., usual care, doxycycline, or cotrimoxazole), principal component analysis (PCA), and ordination demonstrate no difference in overall community composition. (B) No difference in SDI across assigned treatment groups at baseline. (C) No difference in Streptococcus relative abundance across all treatment groups at baseline. (D) Kaplan-Meier plot of diversity stratified by threshold of ⩾1 SDI in the cotrimoxazole cohort. (E) Kaplan-Meier plot of Streptococcus abundance stratified by threshold of ⩾70% relative abundance in the cotrimoxazole cohort. (F) Kaplan-Meier plot of diversity stratified by threshold of ⩾1 SDI in the doxycycline cohort. (G) Kaplan-Meier plot of Streptococcus abundance stratified by threshold of ⩾70% relative abundance in the doxycycline cohort.