Bactericidal Action and in Vitro Immunomodulatory Effects of Ozone on Methicillin-Resistant Staphylococcus Aureus

Methicillin-resistant Staphylococcus aureus (MRSA) poses a major challenge in clinical settings due to limited susceptibility to conventional antibiotics. Ozone (O₃), a naturally occurring molecule composed of three oxygen atoms, exhibits strong antimicrobial properties against a wide spectrum of microorganisms, including both Gram-positive and Gram-negative bacteria. The antibacterial efficacy of O₃ against pathogenic bacteria has been demonstrated both in vivo and in vitro, although the mechanisms of action remain poorly understood. Notably, O₃ has proven effective against MRSA, underscoring the potential of O₃ as a valuable adjunctive therapy for managing resistant bacterial infections.

This study aimed to confirm the antibacterial effects of oxygen-O₃ (O₂-O₃) therapy on MRSA strains and to investigate the associated immunomodulatory and antioxidant properties in human peripheral blood mononuclear cells (PBMCs) treated in vitro with MRSA.

PBMCs from healthy donors were initially exposed to heat-killed or O₃-treated MRSA strains at 37 °C for 2 hours, then treated with 40 μg/mL O₃ for an additional 24 hours to simulate O₃ therapy. Subsequently, cytokine release, antioxidant and pro-oxidant gene expression, reactive oxygen species production, and redox-related biomarker levels were assessed.

Our findings demonstrated that O₃, at various concentrations (20-60 μg/mL) within the therapeutic range (20-80 μg/mL), exerts a potent antibacterial effect against MRSA, resulting in complete loss of bacterial colonies, in the absence of antioxidants in the medium. Using a multidisciplinary approach encompassing biochemical, molecular, and cell biology techniques, we observed that O₃ exerts immunomodulatory and antioxidant effects on PBMCs exposed to O₃ and stimulated with MRSA as early as 1 hour after stimulation, with these effects persisting for up to 48-72 hours, regardless of whether the bacteria were inactivated by O₃ or heat.

We successfully demonstrated that O₃ exerts an early antibacterial effect, followed by antioxidant activity and immunomodulatory properties, as confirmed by multiple integrated analyses. These findings highlight the potential of O₃ as a promising strategy to combat antibiotic resistance and support therapeutic protocols.