A phage cocktail with bactericidal and virulence-attenuating effects against dairy-associated hypervirulent Klebsiella pneumoniae

Klebsiella pneumoniae is an opportunistic pathogen associated with bovine mastitis and dairy contamination, threatening animal health and food safety. This study addresses iuc3 -positive, multidrug-resistant, hypervirulent K. pneumoniae (MDR-hvKP) from dairy sources. Two virulent phages, vB-KP80 and vB-KPT2, were isolated from dairy farm wastewater and characterized as members of distinct genera. They mediate specific lysis by targeting different surface receptors: vB-KP80 recognizes capsular polysaccharide encoded by genes ( wzc/wcaJ ), whereas vB-KPT2 utilizes lipopolysaccharide and outer membrane proteins encoded and regulated by genes ( rfaH/fhuA ). The phage cocktail combining both showed superior bactericidal activity over individual phages in pasteurized milk and a murine mastitis model, significantly reducing bacterial loads and alleviating tissue inflammation. Critically, the cocktail exerted receptor-directed evolutionary pressure, promoting resistant mutants with capsule deficiencies from mutations at multiple loci. These mutants displayed marked virulence attenuation, including reduced biofilm formation, impaired host cell adhesion, lost serum resistance, and increased phagocytosis susceptibility. Thus, the cocktail provides dual benefits: direct killing of MDR-hvKP and indirect protection by evolving less virulent resistant populations. This eco-friendly, antibacterial–antivirulence strategy shows promise for controlling bovine mastitis and enhancing dairy product safety. • Two novel lytic phages targeting distinct bacterial receptors were isolated and characterized. • Novel phage cocktail effectively controls MDR-hvKP in milk and bovine mastitis. • The cocktail's multi-receptor targeting imposed evolutionary pressure, selecting for resistant mutants with attenuated virulence in vivo. • Phage therapy offers a green, evolution-guided strategy to enhance dairy safety.