Molecular analysis of diversity within the genus Pseudomonas in the lungs of cystic fibrosis patients

The genus Pseudomonas contains species that can act as human pathogens and are important in many infections such as those occurring in the lungs of many cystic fibrosis (CF) patients. Current methodologies that rely on the cultivation of bacteria are too cumbersome and often lack sufficient discriminatory power to define the diversity of Pseudomonas spp. As a result, molecular-based approaches have many advantages when attempting to differentiate between species of this genus. This study assessed the ability of terminal restriction fragment length polymorphism (T-RFLP) profiling of the 16S–23S rRNA ITS1 gene region to differentiate species of the genus Pseudomonas. Before application to clinical samples, this approach was validated on a panel of 10 different Pseudomonas spp. T-RFLP profiling of the 16S–23S rRNA ITS1 gene region amplified from these strains differentiated all Pseudomonas spp. tested. The presence of Pseudomonas spp. in CF sputum was assessed through the detection of this ITS1 gene region as amplified from DNA extracted from 40 samples of CF sputum. The ITS1 gene region was detected in 75% of these samples, including 5 from which no Pseudomonas spp. had been identified using culture-based methods. In silico analysis showed that all sequences amplified had a high homology with the ITS1 region of Pseudomonas aeruginosa. T-RFLP gave data that were consistent with the band being generated from P. aeruginosa for each patient. No correlation between Pseudomonas diversity and severity of lung disease was observed in this group of CF patients. This study has however shown that molecular analysis of the ITS1 region is effective in resolving diversity within the genus Pseudomonas. The wider use of this application is discussed.