Analysis of the airway microbiota of healthy individuals and patients with chronic obstructive pulmonary disease by T-RFLP and clone sequencing

Abstract

Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease that affects a large number of patients and has significant impact. One hallmark of the disease is the presence of bacteria in the lower airways.

Objective: The aim of this study was to analyze the detailed structure of microbial communities found in the lungs of healthy individuals and patients with COPD. Nine COPD patients as compared and 9 healthy individuals underwent flexible bronchoscopy and BAL was performed. Bacterial nucleic acids were subjected to terminal restriction fragment (TRF) length polymorphism and clone library analysis. Overall, we identified 326 T-RFLP band, 159 in patients and 167 in healthy controls. The results of the TRF analysis correlated partly with the data obtained from clone sequencing. Although the results of the sequencing showed high diversity, the genera Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus constituted the major part of the core microbiome found in both groups. A TRF band possibly representing Pseudomonas sp. monoinfection was associated with a reduction of the microbial diversity. Non-cultural methods reveal the complexity of the pulmonary microbiome in healthy individuals and in patients with COPD. Alterations of the microbiome in pulmonary diseases are correlated with disease.

Figure 1. T-RFLP profiles revealed highly interindividual variation of detected bands.

Band sizes were correlated with bacterial general, keeping in mind that T-FRLP only allows a prediction but not a determination of the microbial taxa.

Figure 2. Biplot of the principal component analysis of the bacterial communities from bronchoalveolar lavages samples of the chronic obstructive pulmonary disease patients and healthy individuals.

The terminal restriction fragment lengths (in base pairs) corresponding to the correlated bacterial genera are specified in the brackets.

Figure 3. Sample richness (a) and Shannon diversity index (b) based on fragment lengths.

Terminal restriction fragments shorter than 50 bp were excluded from the analysis. Empty squares correspond to healthy individuals, filled squares to COPD patients.

Figure 4. A phylogenetic tree obtained from sequencing analysis illustrates the standard microbiota present in healthy lungs containing 75 leafs.

Different genera are denoted with different colors with one exception: the family Sphingomonadaceae represents the taxon of higher rank. Bacteria marked with green circles were detected only in healthy individuals, but not in COPD patients.

Figure 5. A phylogenetic tree obtained from sequencing analysis illustrates the microbial community of lungs of patients with stable COPD and contains 76 leafs.

Different genera are denoted with different colors with two exceptions: the families Rhodocyclaceae and Lachnospiraceae represent the taxa of higher rank. Bacteria marked with red circles were detected only in COPD patients, whereas other microbes are the part of a core lung microbiome.