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

The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae as a major cause of bovine mastitis poses a critical threat to global dairy production, with conventional antibiotics increasingly failing to control infections. To address this crisis, a lytic phage vB_KpnP_ZH4 (ZH4) was isolated from dairy farm sewage and demonstrated to possess potent activity against prevalent MDR K. pneumoniae strains from mastitic cows in Shanghai farms. Morphological characterization revealed that ZH4 belonged to the Podoviridae family, whereas genomic analysis confirmed the absence of virulence, resistance, or lysogeny genes in its 36.2-kb genome. Phage ZH4 demonstrated strong lytic ability, evidenced by a short latency period (20 min) and high burst size (89 pfu/cell) in one-step growth analysis, and it exhibited exceptional stability across wide temperature, pH, and fetal bovine serum concentration ranges. In vitro, ZH4 reduced MDR K. pneumoniae loads by >3 log in raw milk within 24 h and blocked intracellular infection in bovine mammary epithelial (MAC-T) cells without cytotoxicity. In a murine mastitis model challenged with a hypervirulent MDR isolate (ZH-KP-4), a single intramammary ZH4 dose of 5 × 10⁶ 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.