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

Abstract

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.

FIG. 1.

LH-PCR (700IR-labeled) profiles of 16S rRNA gene products amplified from single bacterial strains and separated on a LI-COR IR² automated sequencer. (a) S. maltophilia; (b) P. aeruginosa, (c) E. coli; (d) H. influenzae; (e) A. pleuropneumoniae; (f) B. cepacia; (g) K. pneumoniae; (h) S. aureus. The x axis on each graph corresponds to the signal intensity, and the y axis on each graph corresponds to the retardation factor.

FIG. 2.

CfoI-generated T-RFLP profiles formed from 16S rRNA gene PCR products (700IR labeled) amplified from single bacterial strains and separated on a LI-COR IR² automated sequencer. (a) S. maltophilia; (b) P. aeruginosa; (c) E. coli; (d) K. pneumoniae; (e) A. pleuropneumoniae; (f) H. influenzae; (g) S. aureus; (h) B. cepacia. The x axis on each graph corresponds to the signal intensity, and the y axis on each graph corresponds to the retardation factor.

FIG. 3.

LH-PCR product (700IR-labeled) profiles generated from DNA extracted directly from a sputum sample separated on a LI-COR IR² automated sequencer. Lanes 1 and 12, microSTEP 15a ladder (sizes in bases). Lanes 2 to 6 were loaded with equal quantities of PCR product amplified from five separate DNA extractions from a single sputum sample; lanes 7 to 11 were loaded with equal quantities of PCR product amplified in five separate reactions with a template from a single DNA extraction.

FIG. 4.

LH-PCR product (700IR-labeled) profiles generated from DNA extracted directly from bronchoscopy and sputum samples separated on a LI-COR IR² automated sequencer. Lanes 1 and 16, microSTEP 15a ladder (sizes in bases); lane 2, individual 1, sputum sample; lane 3, individual 2, sputum sample; lane 4, individual 3, bronchoscopy sample; lane 5, individual 4, bronchoscopy sample; lane 6, individual 5, sputum sample; lane 7, individual 6, bronchoscopy sample; lane 8, individual 7, sputum sample; lane 9, individual 8, sputum sample; lane 10, individual 9, bronchoscopy sample; lane 11, individual 10, sputum sample; lane 12, individual 11, sputum sample; lane 13, individual 12, sputum sample; lane 14, individual 13, sputum sample; lane 15, individual 14, sputum sample.

FIG. 5.

CfoI-generated T-RFLP profiles formed from 16S rRNA gene PCR products (700IR labeled) amplified from DNA extracted directly from a sputum sample separated on a LI-COR IR² automated sequencer. Lane 1 and 12, microSTEP 15a ladder (sizes in bases). Lanes 2 to 6 were loaded with equal quantities of CfoI-digested T-RFLP PCR products amplified from five separate DNA extractions from a single sputum sample; lanes 7 to 11 were loaded with equal quantities of CfoI-digested T-RFLP PCR products amplified in five separate reactions with a template from a single DNA extraction.

FIG. 6.

CfoI-generated T-RFLP profiles formed from 16S rRNA gene PCR products (700IR labeled) amplified from DNA extracted directly from bronchoscopy and sputum samples separated on a LI-COR IR² automated sequencer. Lanes 1 and 16, microSTEP 15a ladder (sizes in bases); lane 2, individual 1, sputum sample; lane 3, individual 2, sputum sample; lane 4, individual 3, bronchoscopy sample; lane 5, individual 4, bronchoscopy sample; lane 6, individual 5, sputum sample; lane 7, individual 6, bronchoscopy sample; lane 8, individual 7, sputum sample; lane 9, individual 8, sputum sample; lane 10, individual 9, bronchoscopy sample; lane 11, individual 10, sputum sample; lane 12, individual 11, sputum sample; lane 13, individual 12, sputum sample; lane 14, individual 13, sputum sample; lane 15, individual 14, sputum sample. Labeled locations within these profiles correspond to position 1, band size 155 bases (P. aeruginosa); position 2, band size 102 bases (P. oris); position 3, band size 373 (C. murliniae); and position 4, band size 215 (A. defectiva).