Topographical continuity of bacterial populations in the healthy human respiratory tract

Abstract

Rationale: Defining the biogeography of bacterial populations in human body habitats is a high priority for understanding microbial-host relationships in health and disease. The healthy lung was traditionally considered sterile, but this notion has been challenged by emerging molecular approaches that enable comprehensive examination of microbial communities. However, studies of the lung are challenging due to difficulties in working with low biomass samples.

Objectives: Our goal was to use molecular methods to define the bacterial microbiota present in the lungs of healthy individuals and assess its relationship to upper airway populations.

Methods: We sampled respiratory flora intensively at multiple sites in six healthy individuals. The upper tract was sampled by oral wash and oro-/nasopharyngeal swabs. Two bronchoscopes were used to collect samples up to the glottis, followed by serial bronchoalveolar lavage and lower airway protected brush. Bacterial abundance and composition were analyzed by 16S rDNA Q-PCR and deep sequencing.

Measurements and main results: Bacterial communities from the lung displayed composition indistinguishable from the upper airways, but were 2 to 4 logs lower in biomass. Lung-specific sequences were rare and not shared among individuals. There was no unique lung microbiome.

Conclusions: In contrast to other organ systems, the respiratory tract harbors a homogenous microbiota that decreases in biomass from upper to lower tract. The healthy lung does not contain a consistent distinct microbiome, but instead contains low levels of bacterial sequences largely indistinguishable from upper respiratory flora. These findings establish baseline data for healthy subjects and sampling approaches for sequence-based analysis of diseases.

Figure 1.

The two-bronchoscope procedure used to sample the respiratory tract. The samples collected are shown in blue lettering. (A) Sampling with the first bronchoscope. After nebulized oropharyngeal anesthesia, bronchoscope #1 was introduced transorally and advanced to the glottis, where local anesthesia was administered and visible secretions aspirated. The bronchoscope was removed and a sample obtained from the scope tip with a flocked swab (Scope #1 Tip) and from the channel by rinsing with lavage saline (Scope #1 Post-wash). (B) Sampling with the second bronchoscope. Bronchoscope #2 was introduced transorally, passed through the vocal cords without aspiration, and wedged in a right middle lobe segmental bronchus. Fifty milliliters of saline were instilled and then aspirated (BAL-A 1st Return), after which 100 ml of additional saline were instilled in the same location, without loss of wedge position, and aspirated (BAL-A 2nd Return). The bronchoscope was then repositioned and wedged in an immediately adjacent segmental bronchus, where 150 ml of saline were instilled and then aspirated (BAL-B). The bronchoscope was then repositioned in the left mainstem bronchus, and a protected specimen brush extended and used to sample the left lower lobe bronchial mucosa (PSB).

Figure 2.

Quantification of bacterial 16S rDNA gene copies in the airway samples and controls. The subject studied is indicated along the x-axis by Subject ID. The 10 types of samples studied are summarized in the key to the right. Along the bottom is shown selected demographic information (S indicates smoker, NS nonsmoker). The y-axis shows the relative number of 16S gene copies detected using the Q-PCR assay. Error bars are shown as the standard error of the mean. All 16S copy numbers are normalized to the fractions collected (key at right) and are represented as either per ml of liquid sample (wash/BAL) or per whole swab/brush eluate. LOQ = lower limit of quantification, 725 16S rDNA copies/ml.

Figure 3.

Proportions of bacterial taxa in each sample type inferred from 16S rDNA pyrosequence data. Each column corresponds to an individual respiratory tract or control sample. The type of sample in each group is indicated in the boxes at the bottom of each group of columns. Each row corresponds to a specific bacterial family. OTUs were collected into families, so that some rows harbor multiple OTUs. OTUs that were not assigned at the family level are omitted. Rows were subjected to hierarchical clustering to emphasize families that show similar abundance patterns. The proportional representation (relative abundance) of each family is represented by the color code (key to the right). The absolute number of 16S copies determined from quantitative PCR of genomic DNA extracts are shown along the bottom. # indicates copies per ml; ^ indicates copies in the total volume eluted from the swab or brush.