Statistical analysis for figures.

<div><p>The antibacterial oxidative response, which relies on the production of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) and hypothiocyanite (OSCN<sup>-</sup>), is a major line of defense protecting the human airway epithelium (HAE) from lesions when infected. The <i>in vitro</i> studies of the oxidative responses are performed mainly by one-shot H<sub>2</sub>O<sub>2</sub> exposure that does not recapitulate the complex H<sub>2</sub>O<sub>2</sub>/LPO/SCN<sup>-</sup> system releasing the reactive oxygen species in airway secretions. A cell-free <i>in vitro</i> assay mimicking this system has been described but was not fully characterized. Here, we comprehensively characterized the hourly H<sub>2</sub>O<sub>2</sub>/OSCN<sup>-</sup> concentrations produced within this <i>in vitro</i> assay and assessed the resistance of <i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i> clinical strains to the HAE oxidative response. We found that H<sub>2</sub>O<sub>2</sub>/OSCN<sup>-</sup> were steadily produced from 7h and up to 25h, but OSCN<sup>-</sup> was detoxified in 15 minutes by bacteria upon exposure. Preliminary tests on PA14 showed survival rates at 1-hour post-exposure (hpe) to H<sub>2</sub>O<sub>2</sub> of roughly 50% for 10<sup>5</sup> and 10<sup>7</sup> colony-forming unit (CFU)/mL inocula, while 10<sup>2</sup> and 10<sup>4</sup> CFU/mL inocula were cleared after one hpe. Thirteen clinical strains were then exposed, highlighting that conversely to <i>P</i>. <i>aeruginosa</i>, <i>S</i>. <i>aureus</i> showed resistance to oxidative stress independently of its antibiotic resistance phenotype. Our results demonstrated how this <i>in vitro</i> 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 <i>in vitro</i> models that could serve as high-throughput screening for molecules targeting the bacterial antioxidant response.</p></div>