Supplementary Material

The emergence of multidrug-resistant <i>Klebsiella pneumoniae</i> (MDR <i>K. pneumoniae</i>) 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, demonstrating potent activity against prevalent MDR <i>K. pneumoniae</i> strains from mastitic cows in Shanghai farms. Morphological characterization revealed that ZH4 belonged to the<i> </i><i>Podoviridae</i> family, while 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 exhibited exceptional stability across wide temperature, pH, and fetal bovine serum (FBS) concentration ranges. <i>In vitro</i>, ZH4 reduced MDR<i> K. pneumoniae</i> loads by &gt;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. 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 FITC-BSA leakage into alveoli by &gt;80% versus antibiotic-treated mice. Immunohistochemistry and titer quantification confirmed ZH4's self-limiting replication kinetics: phage density peaked at 24 h post-treatment and declined to undetectable levels by 120 h post-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<i> K. pneumoniae</i> mastitis, uniquely combining rapid pathogen eradication with structural barrier preservation—offering a transformative alternative to failing antibiotics for sustainable dairy production.