Comparison of the respiratory microbiome in healthy nonsmokers and smokers

Abstract

Rationale: Results from 16S rDNA-encoding gene sequence-based, culture-independent techniques have led to conflicting conclusions about the composition of the lower respiratory tract microbiome.

Objectives: To compare the microbiome of the upper and lower respiratory tract in healthy HIV-uninfected nonsmokers and smokers in a multicenter cohort.

Methods: Participants were nonsmokers and smokers without significant comorbidities. Oral washes and bronchoscopic alveolar lavages were collected in a standardized manner. Sequence analysis of bacterial 16S rRNA-encoding genes was performed, and the neutral model in community ecology was used to identify bacteria that were the most plausible members of a lung microbiome.

Measurements and main results: Sixty-four participants were enrolled. Most bacteria identified in the lung were also in the mouth, but specific bacteria such as Enterobacteriaceae, Haemophilus, Methylobacterium, and Ralstonia species were disproportionally represented in the lungs compared with values predicted by the neutral model. Tropheryma was also in the lung, but not the mouth. Mouth communities differed between nonsmokers and smokers in species such as Porphyromonas, Neisseria, and Gemella, but lung bacterial populations did not.

Conclusions: This study is the largest to examine composition of the lower respiratory tract microbiome in healthy individuals and the first to use the neutral model to compare the lung to the mouth. Specific bacteria appear in significantly higher abundance in the lungs than would be expected if they originated from the mouth, demonstrating that the lung microbiome does not derive entirely from the mouth. The mouth microbiome differs in nonsmokers and smokers, but lung communities were not significantly altered by smoking.

Figure 1.

Neutral model for nonsmokers (A, B, C) and smokers (D, E, F) from 16S molecular surveys with variable regions 1 through 3. The solid line is the model prediction and dashed lines represent 95% confidence intervals. Green points are the outlying operational taxonomic units (OTUs) for which the observed frequency is greater than the model prediction and dark golden points are the outlying OTUs for which the observed frequency is less than the model prediction. Mean abundance (B and E) and detection frequency (C and F) of the outlying OTUs that are the strongest candidates for members of a lung microbiome in nonsmokers and smokers, respectively. Error bars in all histograms represent SE (*P value < 0.05, **P value < 0.01).

Figure 2.

Neutral model for nonsmokers (A, B, C) and smokers (D, E, F) from the 16S molecular surveys with variable regions 3 through 5. The solid line is the model prediction, and dashed lines represent 95% confidence intervals. Green points are the outlying operational taxonomic units (OTUs) for which the observed frequency is greater than the model prediction, and dark golden points are the outlying OTUs for which the observed frequency is less than the model prediction. Mean abundance (B and E) and detection frequency (C and F) of the outlying OTUs that are the strongest candidates for members of a lung microbiome in nonsmokers and smokers, respectively. Error bars in all histograms represent SE (*P value < 0.05, **P value < 0.01).

Figure 3.

Relative abundance of bacterial operational taxonomic units (OTUs) observed in the bronchoalveolar lavage and oral wash samples from nonsmokers and smokers. These bacterial genera represent those populations that averaged more than 1% of the relative abundance across all samples when sequencing variable regions 1 through 3 (A) and variable regions 3 through 5 (B). Some genus names appear twice because multiple OTUs had the same consensus taxonomy.

Figure 4.

Relative abundance of bacterial genera observed in the oral wash samples from nonsmokers and smokers. These bacterial genera represent those populations that averaged more than 1% of the relative abundance across all samples when sequencing variable regions 1 through 3 (A) and variable regions 3 through 5 (B). Some genus names appear twice because multiple operational taxonomic units (OTUs) had the same consensus taxonomy.