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.

Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis

“…This and previous studies show that the sequence-based molecular approach to clinical microbiology provides microbiological perspective that can significantly enhance the standard clinical culture-based view (16)(17)(18)(19). The use of sequences for identification in this study is a substantial improvement over electrophoretic patterns for the identification of microbes, because there is no ambiguity in identity.…”

The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection

“…Other studies place diversity higher, with an average of 5.4 bacterial species per chronic wound [36]. Such degrees of species diversity are also observed in the CF lung, resulting from the diverse microenvironments that arise [97][98][99][100]. Conversely, Kirketerp-Moller and colleagues (2008) [5] struggled to identify multiple species during a venous leg ulcer study, highlighting the diversity and complexity of different types of chronic infection.…”

Analysis of the Bacterial Communities Present in Lungs of Patients with Cystic Fibrosis from American and British Centers

“…Conventional diagnostic microbiology focuses on the detection of a limited group of species, including those listed above (16). However, more recent studies have indicated that the range of bacterial species harbored in sputa from adult CF patients is much wider (28,29,31). These findings emerged through the application of culture-independent analytical strategies that detect bacterial species signatures in nucleic acids extracted directly from clinical samples.…”