Pilot 7: Acute Effects of Kalydeco in People with CF and the G551D Mutation

P.I.: Lucas Hoffman, MD, PhD
Associate Professor
Department of Pediatrics
Adjunct Associate Professor,
Department of Microbiology

In this study, we propose to test the hypothesis that the microbiota in respiratory samples from people with CF ill change within 1 week with Kalydeco treatment, a period during which sweat chloride values decreased by more than 50% on average (P. Singh, pers. comm.).

Aim: Define the CF sputum microbiome before and after beginning Kalydeco.
We will extract DNA from the sputum samples taken before and at days 2 and 7 after beginning treatment with Kalydeco from all 12 study subjects (36 total samples). A portion of each sample will be treated with a reagent that will exclude DNA from dead cells (propidium monoazide11), limiting the proposed analysis to only live cells (as time and funds allow, sample aliquots that were not PMA treated will also be sequenced and the results compared). These treated samples will then be studied using an existing analytical pipeline (Illumina HiSeq shotgun sequencing followed by phylogenetic analysis using the MetaPhlAn computational approach12), developed through an existing collaboration between the Hoffman and Miller laboratories, to define the identities and relative abundances of bacteria, and computational metagenomic analysis to define the content of specific gene families, such as adhesins or metabolic pathways that may be selected within the CF airway. Total and individual viable bacterial loads will be determined using quantitative PCR.