i Culture of bacteria from bronchial secretions in respiratory patients has low sensitivity and does not allow for complete assessment of microbial diversity across different bronchial compartments. In addition, a significant number of clinical studies are based on sputum samples, and it is not known to what extent they describe the real diversity of the mucosa. In order to identify previously unrecognized lower airway bacteria and to investigate the complexity and distribution of microbiota in patients with chronic obstructive pulmonary disease (COPD), we performed PCR amplification and pyrosequencing of the 16S rRNA gene in patients not showing signs or symptoms of infection. Four types of respiratory samples (sputum, bronchial aspirate, bronchoalveolar lavage, and bronchial mucosa) were taken from each individual, obtaining on average >1,000 16S rRNA sequences per sample. The total number of genera per patient was >100, showing a high diversity, with Streptococcus, Prevotella, Moraxella, Haemophilus, Acinetobacter, Fusobacterium, and Neisseria being the most commonly identified. Sputum samples showed significantly lower diversity than the other three sample types. Lower-bronchial-tree samples, i.e., bronchoalveolar lavage and bronchial mucosa, showed a very similar bacterial compositions in contrast to sputum and bronchial aspirate samples. Thus, sputum and bronchial aspirate samples are upper bronchial tree samples that are not representative of the lower bronchial mucosa flora, and bronchoalveolar lavage samples showed the results closest to those for the bronchial mucosa. Our data confirm that the bronchial tree is not sterile in COPD patients and support the existence a different microbiota in the upper and lower compartments.
Background The respiratory microbiome is altered in COPD patients but its relationship with core components of the disease, such as the severity of airflow limitation, the frequency of exacerbations or the circulating levels of eosinophils, is unclear. Methods Cross-sectional study comprising 72 clinically stable COPD patients (mean age 68 [SD 7.9] years; FEV1 48.7 [SD 20.1]% of reference) who provided spontaneous sputum samples for 16S rRNA gene amplification and sequencing. The microbiome composition was analysed with QIIME. Results We observed that: (1) more severe airflow limitation was associated with reduced relative abundance (RA) of Treponema and an increase in Pseudomonas ; (2) patients with ≥2 exacerbations the previous year showed a significantly different bacterial community with respect to non-exacerbators ( p = 0.014), with changes in 13 genera, including an increase of Pseudomonas, and finally, (3) peripheral eosinophils levels ≥2% were associated with more diverse microbiome [Chao1 224.51 (74.88) vs 277.39 (78.92) p = 0.006; Shannon 3.94 (1.05) vs 4.54 (1.06) p = 0.020], and a significant increase in the RAs of 20 genera. Conclusion The respiratory microbiome in clinically stable COPD patients varies significantly according to the severity of airflow limitation, previous history of exacerbations and circulating eosinophils levels. Electronic supplementary material The online version of this article (10.1186/s12890-019-0867-x) contains supplementary material, which is available to authorized users.
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