Novel molecular methods for the analysis of cystic fibrosis (CF) sputum

Project Leader

Joseph Schwartzman, M.D.
Director, Microbiology Professor of Pathology
Geisel School of Medicine at Dartmouth

The goal of this project is to utilize RNA-based technologies to detect and provide quantitative information on a broad array of microbes that form the complex microbiome of the CF lung. Colonization and infection of the lower respiratory tract with microorganisms in CF and in other chronic pulmonary diseases is associated with progressive lung destruction and pulmonary failure. We will compare traditional culture based methods, the current standard technique for CF microbiological diagnostics, to DNA sequencing of ribosomal rDNA elements to detect the spectrum of organisms present, and the NanoString nCounter technology to detect and quantify the RNA of the microbiome in CF respiratory specimens. Based on previous literature and studies at Dartmouth we will design sets of probes to detect the complex mix of bacteria and fungi that have been shown to comprise the flora of the CF lung. We will initially validate that these probe sets identify the ribosomal RNA of the microbiota, compared to 16S (bacterial) and ITS1 (fungal) DNA sequence profiles, and to standard cultures. The ribosome-associated RNAs are likely to represent not only the relative abundance of organisms within the specimens, but to reflect their metabolic activity, and provide additional data to inform the importance of particular organisms in the progressive damage seen with clinical exacerbations of pulmonary disease. We will test this by comparison of specimens from patients during exacerbations and over the course of treatment that will be collected with the aid of our clinical colleagues. Our long term goal is to develop an RNA-based detection methodology to improve the diagnosis and management of individuals with CF.

Laboratory Personnel

Joseph Schwartzman, M.D.
Director, Microbiology Professor of Pathology
Geisel School of Medicine at Dartmouth

Deborah Hogan, Ph.D.
Associate Professor of Microbiology and Immunology
Study design and sample acquisition

Isabella Martin, M.D., Clinical Microbiology Fellow
Microscopic and culture-based analysis of sputum

Sven Willger, Ph.D., Post doctoral fellow, Hogan Lab
Development of probes for characterization of fungi

Laura Filkins, Graduate student, O'Toole Lab
Development of probes for the characterization of bacteria

George O'Toole, Professor of Microbiology and Immunology
Development of probes for the characterization of bacteria

Emily Dolben, Research Assistant
Sample analysis