Preclinical safety assessment of Koomba kaat 1 and Biyabeda mokiny 1 phages for respiratory application against Staphylococcus aureus

Therapeutic bacteriophages are promising alternatives to antibiotics for treating antimicrobial-resistant bacterial infections. For pulmonary infections, direct respiratory delivery offers therapeutic advantages; however, preclinical evaluation of respiratory safety remains underdeveloped. Bacteriophages exhibit genomic and biological diversity, and safety cannot be assumed for individual candidates. Here, we evaluated the respiratory safety of lytic Staphylococcus aureus bacteriophages, Koomba kaat 1 and Biyabeda mokiny 1, using human and murine preclinical models. Differentiated primary airway epithelial cells derived from six healthy paediatric donors were exposed apically to purified phage (1 x 109 PFU/mL) for 24 hours. Barrier integrity, epithelial morphology, mucus production, cytotoxicity, and interleukin-8 release were assessed. Safety was further investigated in adult C57BL/6J mice receiving intranasal phage administration (1 x 109 PFU) twice daily (14 days). Clinical observations, body weight, organ pathology, blood biochemistry, bronchoalveolar lavage cellularity, protein concentration, and inflammatory mediators were evaluated. Neither phage altered epithelial morphology, barrier integrity, mucus production, cytotoxicity, nor inflammatory responses in differentiated cultures. Repeated intranasal administration was well tolerated in vivo, with no adverse clinical signs, weight loss, macroscopic pathology, or treatment-associated changes in pulmonary cellularity or tissue histopathology. Differences in blood biochemistry and inflammatory mediators were small and not accompanied by epithelial injury or pulmonary inflammation. Collectively, these findings demonstrate that Koomba kaat 1 and Biyabeda mokiny 1 exhibit favourable safety profiles following repeated airway administration. This study establishes a comprehensive framework for the preclinical respiratory safety assessment of bacteriophages and provides support for the clinical development of inhaled phage for S. aureus respiratory infections.