Supplementary Material

Klebsiella pneumoniae is an important pathogen responsible for bovine mastitis and milk contamination, causing serious consequences for the dairy industry. Owing to the growing antibiotic resistance of bacteria, phage therapy has regained attention, but there is limited study on the antibacterial properties of Klebsiella phages in milk. First, we evaluated the antibacterial effect of the K5-specific bacteriophage in milk at 4°C and 38°C at different multiplicities of infection (MOI=10, 1 and 0.1). Phage treatment groups achieved significant bacterial load reductions under 4°C, with decreases ranging from 1.13 log10 CFU/mL to 1.89 log10 CFU/mL compared with control group after 24 h incubation. At 38°C, phage treatment groups demonstrated reductions in viable bacterial counts ranging from 0.04 log10 CFU/mL to 0.21 log10 CFU/mL compared with control group. Phage P1011 exhibited significant antibacterial activity initially, but the bacteria eventually regained growth at 38°C, suggesting the emergence of phage resistant strains. Subsequently, we collected phage resistant strains using the double-layer agar method. Through whole-genome sequencing, gene knockout and complementation, along with plaque formation assay, we investigated the mechanisms of phage resistance. Both strains acquired phage resistance through mutations in receptor synthesis-related genes. Specifically, strain B16R1 resistance was related to a wcaJ gene mutation, which disrupts capsular polysaccharide receptor synthesis, while strain B16R17 resistance arose from an interrupted OmpC gene that abolished expression of its outer membrane protein receptor. Strain B16R1 exhibited a rough colony appearance but strain B16R17 retained smooth colony appearance. The acquisition of phage resistance in these strains was accompanied by compensatory costs, including decreased virulence and antibiotic susceptibility change, which underscores the potential advantages of phage therapy. Besides, we found that receptor expression levels affected plating efficiency, plaque morphology, size and clarity. In summary, we explored the antibacterial efficacy of Klebsiella phages in the dairy industry and elucidated the mechanisms of phage resistance in K. pneumoniae. These findings lay the groundwork for the use of Klebsiella phages in the dairy production and their long-term therapeutic efficacy.