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Journal of Clinical Microbiology, August 2003, p. 3548-3558, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3548-3558.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Bacterial Diversity in Cases of Lung Infection in Cystic Fibrosis Patients: 16S Ribosomal DNA (rDNA) Length Heterogeneity PCR and 16S rDNA Terminal Restriction Fragment Length Polymorphism Profiling

G. B. Rogers,1 C. A. Hart,2 J. R. Mason,1 M. Hughes,3 M. J. Walshaw,4 and K. D. Bruce1*

Department of Life Sciences, King's College London, London SE1 9NN,1 Department of Medical Microbiology and Genitourinary Medicine, University of Liverpool, Liverpool L69 3GA,2 Regional Adult Cystic Fibrosis Unit, The Cardiothoracic Centre, Liverpool L14 3PE, United Kingdom,4 Silicon Genetics, Redwood City, California 940633

Received 18 December 2002/ Returned for modification 13 February 2003/ Accepted 28 April 2003

The leading cause of morbidity and mortality in cystic fibrosis (CF) patients stems from repeated bacterial respiratory infections. Many bacterial species have been cultured from CF specimens and so are associated with lung disease. Despite this, much remains to be determined. In the present study, we characterized without prior cultivation the total bacterial community present in specimens taken from adult CF patients, extracting DNA directly from 14 bronchoscopy or sputum samples. Bacterial 16S ribosomal DNA (rRNA) gene PCR products were amplified from extracted nucleic acids, with analyses by terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), and sequencing of individual cloned PCR products to characterize these communities. Using the same loading of PCR products, 12 distinct T-RFLP profiles were identified that had between 3 and 32 T-RFLP bands. Nine distinct LH-PCR profiles were identified containing between one and four bands. T-RFLP bands were detected in certain samples at positions that corresponded to pathogens cultured from CF samples, e.g., Burkholderia cepacia and Haemophilus influenzae. In every sample studied, one T-RFLP band was identified that corresponded to that produced by Pseudomonas aeruginosa. A total of 103 16S rRNA gene clones were examined from five patients. P. aeruginosa was the most commonly identified species (59% of clones). Stenotrophomonas species were also common, with eight other (typically anaerobic) bacterial species identified within the remaining 17 clones. In conclusion, T-RFLP analysis coupled with 16S rRNA gene sequencing is a powerful means of analyzing the composition and diversity of the bacterial community in specimens sampled from CF patients.

* Corresponding author. Mailing address: Department of Life Sciences, Franklin-Wilkins Bldg., King's College London, 150 Stamford St., London SE1 9NN, United Kingdom. Phone: (44) 020-7836-5454. Fax: (44) 020-7848-4500. E-mail: kenneth.bruce{at}kcl.ac.uk.

Journal of Clinical Microbiology, August 2003, p. 3548-3558, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3548-3558.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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