An in vitro exposure platform for investigating bacterial and epithelial cell responses to aerosolized phage challenge.
Mathura Thirugnanasampanthar1,2, Michelle Feng1,3, Fereshteh Bayat1,4, Rod G Rhem5, Myrna B Dolovich1,5,6 & Zeinab Hosseinidoust1,2,4,7
1McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4L8, Canada
2Department of Chemical Engineering
3Department of Electrical Engineering
4School of Biomedical Engineering
5St. Joseph’s Healthcare, Firestone Research Aerosol Laboratory, 50 Charlton Avenue East, Hamilton, ON, L8N 4A6, Canada
6Faculty of Health Sciences, Department of Medicine
7Michael DeGroote Institute for Infectious Disease Research
Pseudomonas aeruginosa (Pa) is a bacterial pathogen responsible for chronic respiratory infections in cystic fibrosis (CF) patients. Bacteriophages (also known as phages) are viruses which infect bacteria; phage therapy is the use of these natural predators to treat infections. Investigations with aerosolized phage are limited and primarily focus on proof-of-concept demonstrations with animal models. In vitro models are needed to conduct comprehensive, mechanistic research. We report the development of an in vitro platform for evaluating the response of Pa bacterial cells and Calu-3 human airway epithelial cells to aerosolized Pseudomonas phage vB_Pae-TbilisiM32. Phage aerosols were generated using a single jet Blaustein atomizer. Transwells containing Pa biofilms and air-liquid interface cultures of Calu-3 were separately exposed to aerosolized phage within a cascade impactor. Assays performed 24 hours later were used to determine the effect of airflow and phage treatments on Pa and Calu-3 cells. XTT assay and Live/Dead staining were used to assess the metabolic activity and viability of bacterial cells, respectively. Significant reductions in metabolic activity and viability of Pa cells were observed after phage exposure, indicating phage-mediated killing of bacterial cells has occurred. Fluorescein isothiocyanate-conjugated dextran (FITC-dextran) assay and Live/Dead staining were used to assess the barrier integrity and viability of Calu-3 cells, respectively. The results show exposure to high-velocity airflow and phage aerosols does not affect barrier integrity or viability of Calu-3 cells. In summary, our platform can be used to assess bacterial and epithelial cell responses to aerosolized phage treatment.
An aerosol exposure platform was developed and used to demonstrate the effects of phage aerosol exposure on bacterial and epithelial cells. Phage exposure had negligible effects on the viability and barrier integrity of epithelial cells; in contrast significant reductions in the metabolic activity and viability of 24-hour Pa biofilms were observed.