# Resistance of Pseudomonas aeruginosa and Staphylococcus aureus to the airway epithelium oxidative response assessed by a cell-free in vitro assay

## TL;DR

This study examines how Pseudomonas aeruginosa and Staphylococcus aureus resist oxidative stress from the airway epithelium using a cell-free in vitro assay.

## Contribution

A cell-free in vitro assay was characterized to assess bacterial resistance to oxidative stress in airway epithelium.

## Key findings

- H2O2 and OSCN- were steadily produced for up to 25 hours in the in vitro assay.
- Staphylococcus aureus showed resistance to oxidative stress regardless of antibiotic resistance phenotype.
- Pseudomonas aeruginosa survival rates varied depending on inoculum concentration.

## Abstract

The antibacterial oxidative response, which relies on the production of hydrogen peroxide (H2O2) and hypothiocyanite (OSCN-), is a major line of defense protecting the human airway epithelium (HAE) from lesions when infected. The in vitro studies of the oxidative responses are performed mainly by one-shot H2O2 exposure that does not recapitulate the complex H2O2/LPO/SCN- system releasing the reactive oxygen species in airway secretions. A cell-free in vitro assay mimicking this system has been described but was not fully characterized. Here, we comprehensively characterized the hourly H2O2/OSCN- concentrations produced within this in vitro assay and assessed the resistance of Pseudomonas aeruginosa and Staphylococcus aureus clinical strains to the HAE oxidative response. We found that H2O2/OSCN- were steadily produced from 7h and up to 25h, but OSCN- was detoxified in 15 minutes by bacteria upon exposure. Preliminary tests on PA14 showed survival rates at 1-hour post-exposure (hpe) to H2O2 of roughly 50% for 105 and 107 colony-forming unit (CFU)/mL inocula, while 102 and 104 CFU/mL inocula were cleared after one hpe. Thirteen clinical strains were then exposed, highlighting that conversely to P. aeruginosa, S. aureus showed resistance to oxidative stress independently of its antibiotic resistance phenotype. Our results demonstrated how this in vitro assay can be helpful in assessing whether pathogens can resist the antibacterial oxidative HAE response. We anticipate these findings as a starting point for more sophisticated in vitro models that could serve as high-throughput screening for molecules targeting the bacterial antioxidant response.