CF Research Translation Center and Research Development Program
Seattle Children’s Research Institute
University of Washington
B. Wayne Hughes Building (Building Cure)
1920 Terry Ave
Seattle, WA 98101
CFF RDP Pilot
Diagnostic Utility of mRNA for Characterizing Heteroresistant P. aeruginosa Population Response to Antibiotic Treatment
PI: Dustin Long, MD
Assistant Professor, Anesthesiology and Pain Medicine
Funding Period: 2024 – 2026
Abstract: Chronic respiratory infections remain a leading cause of morbidity and mortality among people with cystic fibrosis (CF), and studies show infection persists even after CFTR modulator treatment. Laboratory-determined minimum inhibitory concentration (MIC)-determined antimicrobial susceptibilities for the chronic pathogen Pseudomonas aeruginosa are poor predictors of treatment response and clinical outcomes in CF. Several problems including resistance heterogeneity of bacterial populations, condition-dependent resistance phenotypes, the instability of resistance traits in culture, and the diversity of antibiotic resistance genotypes all likely contribute to the inability of current approaches to guide individualized treatment of infection.
Rather than identifying specific culture phenotypes or resistance genes, we propose that measurement of bacterial mRNA transcripts after in vitro antibiotic challenge can enable sensitive, specific, and genotype-independent assessments of antibiotic efficacy that better predict response to treatment in vivo. In this pilot proposal, we will focus on susceptibility of P. aeruginosa to tobramycin and ciprofloxacin. In Aim 1, we will identify P. aeruginosa mRNA sequences for which rapid reductions in transcript abundance after antibiotic challenge are associated with the extent of antibiotic-induced cell killing. Using RNA-Seq of antibiotic susceptible wild-type P. aeruginosa and highly resistant mutant derivatives, we will identify and develop RT-qPCR assays for such candidate transcripts. In Aim 2, we will use RT-qPCR to validate these candidate mRNA markers of P. aeruginosa cell survival under CF-like conditions that pose challenges to characterization by standard lab measures.
This pilot will demonstrate the feasibility of using quantitative, culture-independent molecular measurements of P. aeruginosa mRNA to test antibiotic efficacy in complex conditions that pose challenges to current testing strategies in CF.