The CF sputum metagenomic response to inhaled tobramycin

Fellow: Maria T Nelson, PhD

Mentor:  Lucas Hoffman, MD, PhD
Professor, Pediatrics

Cystic Fibrosis (CF) respiratory disease is characterized by chronic, polymicrobial infections that require frequent treatment with antibiotics. These infections are considerable contributors to reduced quality and length of life in people with CF, yet neither the microbial determinants of respiratory disease severity nor of response to therapy are well understood. CF lung infections have historically been characterized using standard culture techniques, which identify a small number of classic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. Recently, culture-free sequencing methods have shown CF sputum to harbor complex polymicrobial communities, often comprising multiple species not easily detected using classic laboratory methods. These newer techniques have suggested the CF “microbiota” (the taxonomic membership of the community) to be relatively stable across time, even through times of clinical stability, respiratory exacerbation and antibiotic therapy. By comparison, little is known about how the “metagenome” (the complement of microbial genes in the community, representing aggregate functional capacity, including antibiotic resistance mechanisms) changes with therapy. The mechanisms underlying the resilience of CF respiratory infections in the face of potent antibiotic treatments are not clear; for example, even bacteria such as P. aeruginosa and S. aureus, which are effectively killed in vitro by commonly-used antibiotics, persist during treatment with the same drugs. How the polymicrobial nature of CF infections contribute to this resilience of standard pathogens, and how the respiratory microbiome responds as a whole to antibiotics, has not been defined. This project aims to study these questions with a focus on tobramycin, an antibiotic used both acutely and as maintenance therapy for people chronically infected with P. aeruginosa. Tobramycin is the most commonly prescribed antibiotic in CF, and maintenance treatment with inhaled tobramycin has been shown to reduce CF pulmonary exacerbations and mortality in the long term. However, studies have shown that two key short-term effects of tobramycin, reduction in P. aeruginosa viable counts and improvement in respiratory symptoms, diminish with each successive course of the drug and some individuals do not respond at all. We hypothesize that features of pre-treatment sputum microbiomes (both microbiota and metagenome) will correlate with subsequent microbiological responses to tobramycin (defined by sputum abundances of traditional pathogens, including P. aeruginosa and S. aureus). To address this hypothesis, this study will utilize longitudinal, weekly sputa from individuals during a standard, month-long cycle of inhaled tobramycin. The sputa were collected concomitantly with clinical microbiological culture results, respiratory function measures and subjective symptoms scores to allow for comparison of metagenomic findings with standard microbiological and clinical measures; because this maintenance treatment is intended to stabilize, rather than improve, clinical symptoms in the short term, this project will focus on the metagenomic correlates of traditional microbiological response. Aim 1 will use next generation sequencing to define the sputum microbiota before and after antibiotic therapy, allowing for identification of taxonomic factors associated with microbiological response as defined above. Aim 2 will analyze the sputum metagenome to identify the functional components of sputum microbial communities that are associated with microbiological response to therapy. The results of this analysis will be used to identify candidate mechanisms and predictors of antibiotic effect on CF pathogens, toward the ultimate goal of developing more effective therapies for CF respiratory infections.