Phage ZH4 rescues murine mastitis infected with hypervirulent multidrug-resistant Klebsiella pneumoniae through pathogen elimination and mammary barrier restoration

pfu/gland achieved complete bacterial clearance in mammary tissue within 72 h, significantly outperforming cefotaxime sodium, which failed to reduce bacterial burdens. We found that ZH4 therapy resolved histopathological damage, downregulated key proinflammatory cytokines (IL-6, IL-1β, TNF-α) and toll-like receptors (TLR2 and TLR4), and crucially restored blood-milk barrier integrity, reducing fluorescein isothiocyanate-labeled BSA leakage into alveoli by >80% versus antibiotic-treated mice. Immunohistochemistry and titer quantification confirmed ZH4's self-limiting replication kinetics: phage density peaked at 24 h after treatment and declined to undetectable levels by 120 h after treatment, aligning with host bacterial load. No adverse effects were observed. These findings establish phage ZH4 as a safe and highly effective therapeutic against intractable MDR K. pneumoniae mastitis, uniquely combining rapid pathogen eradication with structural barrier preservation and offering a transformative alternative to failing antibiotics for sustainable dairy production.