Abstract Title: Antimicrobial Effects of Inhaled Nitric Oxide Therapy on Pseudomonas Aeruginosa Lung Colonization in a Swine Model of Independent Lung Ventilation.

Background: Pseudomonas Aeruginosa is the most common cause of gram-negative nosocomial pneumonia, and up to 26% of ventilator-associated pneumonia globally. Recent findings indicate that inhaled nitric oxide (iNO) at 300 parts-per-million (ppm) effectively reduces lung bacterial colonization of Klebsiella pneumonia and Pseudomonas aeruginosa (P. aeruginosa). Unlike inhaled antibiotics, iNO300 therapy might enhance the immune response in non-ventilated lung regions due to consolidated parenchyma.

Methods: Twelve swine were kept under general anesthesia and mechanical ventilation for 72 hours. All pigs were challenged with P. aeruginosa via bronchoscopy and twice via nebulization at 24 and 48 hours. The control group (n=6) received standard ventilation through a single-lumen endotracheal tube. The iNO300 group (n=6) was ventilated using a double-lumen endotracheal tube (DLT), with one lung receiving iNO300 and the other standard ventilation. Lung isolation was confirmed through respiratory mechanics, bronchoscopy, and lung imaging. iNO300 was administered for 30 minutes every 4 hours, to right (n=3) and left (n=3) lungs in different animals. After 72 hours, lung tissue samples were collected. Measurements of Colony Forming Units (CFUs)/gram of P. aeruginosa in each lung lobe, and pathohistological scoring of infection and inflammation (0=none/minimal, 1=focal/mild, 2=moderate, 3=diffuse/severe), were obtained. Percent methemoglobin (MetHb), mean arterial pressure, and PaO2/FiO2 were measured during treatments.

Results: Lung tissue samples demonstrated that animals treated with iNO300 had statistically lower bacterial loads in both lungs compared to the control group (2.3±1.0 vs. 4.4±0.6 Log10 CFU/g, p-value=0.045). In the iNO group there was no difference between the treated and non-treated lungs (2.3±1.0 vs 2.3±1.4 Log10 CFU/g, p-value=0.98).

Compared to controls, iNO300-treated swine showed less severe signs of acute bronchopneumonia (0.59±0.91, vs 1.91±0.72, p-value <0.001), interstitial inflammation (0.54±0.63 vs 1.19±0.69, p-value <0.001) and airway inflammation (0.67±0.7 vs 1.46±0.69, p-value <0.001) in both lungs. In the iNO group there was no difference in acute bronchopneumonia (p-value=0.71) or interstitial inflammation (p-value=0.76) between treated and non-treated lungs.

Baseline MetHb levels were measured (1.73±1.02%), and levels peaked 30 minutes (3.67±1.82%) during iNO treatments but returned to baseline within 30 minutes post-treatment (1.13±0.76%). Mean arterial pressure (p-value=0.97) and PaO2/FiO2 (p-value=0.22) remained stable during treatments

Conclusion: In a swine model of P. aeruginosa pneumonia, iNO300 delivered to only one lung reduces bacterial load and lung tissue inflammation in both treated and untreated lungs, suggesting antimicrobial mechanisms beyond direct contact. The transient rise in MetHb levels and stable hemodynamic parameters indicate iNO300 does not adversely affect cardiovascular health.