Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Jacob Vieira; Tara Gallagher; Hui-Yu Sui; Sirus Jesudasen; Katrine Whiteson; George A. O'Toole; Kurt Hanselmann; Peggy S. Lai

doi:10.3791/62888

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

2.2K views

⸱

DOI:

10.3791/62888

⸱

August 3rd, 2021

Jacob Vieira¹ , Tara Gallagher² , Hui-Yu Sui¹ , Sirus Jesudasen³ , Katrine Whiteson² , George A. O'Toole⁴ , Kurt Hanselmann⁵ , Peggy S. Lai¹

¹Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, ²Department of Molecular Biology & Biochemistry, University of California, Irvine, ³Department of Medicine, Massachusetts General Hospital, ⁴Department of Microbiology & Immunology, Geisel School of Medicine at Dartmouth, ⁵Swiss i-research and teaching institute

Summary

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Abstract

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

Introduction

Cystic fibrosis (CF) is a genetic disease characterized by an inability to clear thick mucus from the lungs, leading to repeated infections and progressive lung function decline that often results in the need for lung transplantation or death. The airway microbiome of people with cystic fibrosis (pwCF) appears to track disease activity¹, with a reduction in microbial diversity associated with adverse long-term outcomes²^,³. In clinical studies of pwCF, supplemental oxygen therapy has been associated with more advanced disease⁴^,⁵,....