Abstract
Aim: To identify microorganisms unrecognized through culture in the lower airway of patients with chronic pulmonary disease through amplification and pyrosequencing of specific genes for microbial diversity assessment
Method: To avoid culture-related bias, 1) DNA extraction from respiratory samples (sputum, bronchial aspirate (BAS), bronchoalveolar lavage (BAL) and bronchial mucosa); 2) 16S rRNA amplification and purification with modified primers which include a specific 8 nucleotides code per sample; 3) Pyrosequencing of amplified products by Titanium/GS-FLX System (454-Roche; 4) Obtention of >500 sequences per sample; and 6) Comparison of the results obtained with the diferent samples using bronchial mucosa as gold standard.
Results: Sputum, BAS, BAL and bronchial mucosa samples from 6 patients were studied. Over 500 sequences of gen 16S were identified in 17 out of 24 samples. Total number of bacterial genera per patient were over 100. Analysis of coincidence obtained from bronchial mucosa showed low coincidence with BAS and sputum, and maximal coincidence with BAL, that showed a range of 55-74 bacterial genera. Streptococcus, Prevotella, Moraxella, Haemophilus, Acinetobacter, Fusobacterium and Neisseria were the most often identified genders, accounting for up to 60% of the overall.
Conclusions: Pyrosequencing is applicable to samples of bronchial secretions and is able to demonstrate a microbial diversity over 600 microorganisms. Sputum and BAS samples were non-representative of bronchial mucosa, but BAL that mostrates over fifty bacterial genera, shows the closest results with bronchial mucosa.
Sponsorship by Fundacio Parc Tauli.
- © 2011 ERS
