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. 2005 Jun 1;6(1):49.
doi: 10.1186/1465-9921-6-49.

Bacterial activity in cystic fibrosis lung infections

Geraint B Rogers  1 , Mary P Carroll, David J Serisier, Peter M Hockey, Valia Kehagia, Graeme R Jones, Kenneth D Bruce
Affiliations

Bacterial activity in cystic fibrosis lung infections

Geraint B Rogers et al. Respir Res. .

Abstract

Background: Chronic 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.

Methods: Terminal 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.

Results: Samples 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 < or = 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.

Conclusion: These 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.

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Figures

Figure 1
Figure 1
Electrophoretic gel images generated by T-RFLP and RT-T-RFLP. This figure shows the profiles generated from five sputum samples within the sample set. By a process of automated comparison of band positions with those in marker lanes allows their length to be determined and direct comparisons to be made between lanes.
Figure 2
Figure 2
Identification of individual bands within regions of corresponding T-RFLP and RT-T-RFLP profiles. This figure shows regions of profiles as analysed using Phoretix 1D Advanced v.5.10 (Nonlinear Dynamics, Newcastle upon Tyne, UK). In each case, the region of electrophoretic profile is shown (below) next to a trace of relative band intensity. The manual confirmation of correct band identification minimises the inclusion of erroneous peaks.
Figure 3
Figure 3
Dendrogram constructed using T-RFLP and RT-T-RFLP profiles generated from the sample set. A dendrogram was constructed using the results of Hierarchical Cluster Analysis (HCA), using Dice measure, of the T-RFLP and RT-T-RFLP profile data. HCA results in the formation of clusters in which profiles are iteratively joined in a descending order of similarity.
Figure 4
Figure 4
Dendrogram constructed using T-RFLP profiles generated from sputum samples obtained from CF patients and healthy individuals. A dendrogram was constructed using the results of Hierarchical Cluster Analysis (HCA), using Dice measure, of the T-RFLP profile data.
See this image and copyright information in PMC

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