Abstract
BackgroundChronic lung infections are the primary cause of morbidity and mortality in Cystic Fibrosis (CF) patients. Recent molecular biological based studies have identified a surprisingly wide range of hitherto unreported bacterial species in the lungs of CF patients. The aim of this study was to determine whether the species present were active and, as such, worthy of further investigation as potential pathogens.MethodsTerminal Restriction Fragment Length Polymorphism (T-RFLP) profiles were generated from PCR products amplified from 16S rDNA and Reverse Transcription Terminal Restriction Fragment Length Polymorphism (RT-T-RFLP) profiles, a marker of metabolic activity, were generated from PCR products amplified from 16S rRNA, both extracted from the same CF sputum sample. To test the level of activity of these bacteria, T-RFLP profiles were compared to RT-T-RFLP profiles.ResultsSamples from 17 individuals were studied. Parallel analyses identified a total of 706 individual T-RF and RT-T-RF bands in this sample set. 323 bands were detected by T-RFLP and 383 bands were detected by RT-T-RFLP (statistically significant; P ≤ 0.001). For the group as a whole, 145 bands were detected in a T-RFLP profile alone, suggesting metabolically inactive bacteria. 205 bands were detected in an RT-T-RFLP profile alone and 178 bands were detected in both, suggesting a significant degree of metabolic activity. Although Pseudomonas aeruginosa was present and active in many patients, a low occurrence of other species traditionally considered to be key CF pathogens was detected. T-RFLP profiles obtained for induced sputum samples provided by healthy individuals without CF formed a separate cluster indicating a low level of similarity to those from CF patients.ConclusionThese results indicate that a high proportion of the bacterial species detected in the sputum from all of the CF patients in the study are active. The widespread activity of bacterial species in these samples emphasizes the potential importance of these previously unrecognized species within the CF lung.
Highlights
The Microbiological analysis of clinical specimens has relied traditionally on cultivation prior to identification
The banding positions generated through Terminal Restriction Fragment Length Polymorphism (T-RFLP) and RTT-RFLP analysis were compared for each sample. 178 bands were detected in both a T-RFLP profile and the corresponding RT-T-RFLP profile (356 bands in total), 145 bands were detected in a T-RFLP profile but were absent in the corresponding RT-T-RFLP profile, and 205 bands were detected in an RT-T-RFLP profile but were absent in the corresponding T-RFLP profile
It has been shown that many bacterial species not previously associated with Cystic Fibrosis (CF) lung infections, could be detected when molecular biological approaches were applied to the study of sputa [10,19]
Summary
The Microbiological analysis of clinical specimens has relied traditionally on cultivation prior to identification. The use of molecular biological approaches in clinical scenarios obviates the requirement for in vitro culture prior to analysis and so removes problems associated with microbial cultivation. These approaches are ideal for cases such as trauma where predicting the pathogen(s) responsible is challenging. Nucleic acids, extracted directly from samples, act as templates for PCR amplification of phylogenetically-informative ribosomal sequences using oligonucleotide primers "universal" for the Domain Bacteria. This means that no prior assumptions are made about the identity of species present. The aim of this study was to determine whether the species present were active and, as such, worthy of further investigation as potential pathogens
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