Effect of Cycled Antibiotic Therapy on the Cystic Fibrosis Sputum Microbiome

Abstract

Cystic fibrosis (CF) is a genetic disease resulting in chronic, complex, polymicrobial respiratory infections, ultimately contributing to lung destruction and death. Despite extensive study, primarily using culture methods, the microbial determinants of clinical status and therapeutic response have yet to be completely elucidated. The most common antibiotic used to treat people with CF is inhaled tobramycin, administered as maintenance therapy for chronic Pseudomonas aeruginosa lung infections. While the effects of inhaled tobramycin on P. aeruginosa abundance and lung function diminish with continued therapy, this maintenance treatment improves long-term CF morbidity and mortality, underscoring how little is known about why antibiotics work in CF infections, their effects on complex CF sputum microbiomes, and how to improve these treatments. Metagenomic sequencing is a promising approach for identifying and characterizing organisms and their functional characteristics in complex, polymicrobial infections, such as airway infections in people with CF. These analyses are often hampered, however, by overwhelming quantities of human DNA, yielding only a small proportion of microbial reads for analysis. Additionally, many abundant microbes in respiratory samples can produce large quantities of extracellular bacterial DNA originating either from biofilms or dead cells. We first describe a method for simultaneously depleting DNA from intact human cells and extracellular DNA (human and bacterial) in sputum, using selective lysis of eukaryotic cells and endonuclease digestion. We show that this method increases microbial sequencing depth and, consequently, both the number of taxa detected and coverage of individual genes such as those involved in antibiotic resistance, underscoring the substantial impact of DNA from sources other than live bacteria in microbiological analyses of complex, chronic infection specimens. We then collected sputum from 30 individuals with CF at standardized time points before, during and after a month-long course of maintenance inhaled tobramycin. We used culture, quantitative PCR and metagenomic sequencing to define the dynamic effects of inhaled tobramycin on sputum microbiomes, including abundance changes in both clinically targeted and untargeted bacteria, and in functional gene categories. CF sputum microbiota changed most markedly by one week of therapy and plateaued thereafter. This shift was largely driven by changes in non-dominant taxa. Genetically conferred functional capacities (metagenomes) of subjects’ sputum communities changed little with antibiotic perturbation, despite taxonomic shifts, suggesting functional redundancy within the CF sputum microbiome. What changes did occur were primarily driven by non-dominant taxa. These results highlight the importance of monitoring the broad microbiological effects of antibiotics and other treatments as we work towards accurately defining and improving the clinical impact of these therapies.