T-RFLP – a representative tool to study pulmonary microbial communities

Background: It has become apparent that lungs are constantly exposed to complex microbial communities. Changes in such communities may play critical role in the initiation of lung diseases. The aim of this study was to characterize the normal microbiota of lungs and COPD-induced alterations in lower respiratory tract.

Methods: Conventional culture-based methods for the analysis of pulmonary flora miss bacteria that are difficult or impossible to culture. Therefore we applied PCR-based tools – terminal restriction fragment length polymorphism (T-RFLP) and sequencing. T-RFLP generates fragments that differ in length due to the variation in the position of the first specific restriction endonuclease site. The fragment of 16S RNA gene was chosen to identify bacteria. Total DNA, isolated from clinical bronchoalveolar lavage (BAL) samples and mice lung sections was used for the analysis.

Results: Generated terminal restriction fragment (T-RF) patterns revealed both similarities and differences inside the same group of patients. The frequency at which the T-RF bands were detected in each sample was determined. Universal peaks demonstrated the presence of 1 to 3 major bacterial pathogens. Unique or rarely observed peaks reflect individual bacteria present in different samples. The mean species richness was 13.2 (±4.4). The genera Haemophillus and Streptococcus seem to play the leading role in the colonization process. As for the mice lungs tissue samples, the reproducibility of number, position and intensity of peaks by different animals were observed. This data support the idea, that lower airways are the site of stable bacterial colonization.

Discussion: We detected various species in the lower respiratory tract of healthy persons and patients with COPD and mice lungs sections. A better understanding of the resident microbial communities and their dynamics should shed the light on the pathogenesis, diagnosis and treatment of pulmonary disorders.