Antibacterial efficacy of a K5-specific Klebsiella phage in milk and investigation of phage resistance mechanisms.

Klebsiella pneumoniae is an important pathogen causing bovine mastitis and milk contamination, with severe consequences for the dairy industry. Due to increasing bacterial antibiotic resistance, phage therapy has regained attention; however, studies on the antibacterial properties of Klebsiella phages in milk are limited. We evaluated the antibacterial efficacy of the K5-specific phage in milk at 4°C and 38°C with different multiplicities of infection (MOI = 10, 1, and 0.1). At 4°C, phage treatment groups achieved significant bacterial load reductions, ranging from 1.13 to 1.89 log cfu/mL, compared with the control group after 24 h incubation. At 38°C, phage treatment groups showed reductions in viable bacterial counts, ranging from 0.04 to 0.21 log cfu/mL, compared with the control group. Phage P1011 initially exhibited significant antibacterial activity, but bacterial regrowth occurred at 38°C, indicating the emergence of phage-resistant strains. Subsequently, we isolated phage-resistant strains using the double-layer agar plate method. Using whole-genome sequencing, gene knockout, complementation, and plaque formation assays, we investigated phage resistance mechanisms. Both strains developed phage resistance through mutations in genes related to receptor synthesis. Specifically, the resistance of strain B16R1 resulted from a wcaJ gene mutation that disrupts capsular polysaccharide receptor synthesis, whereas resistance of strain B16R17 arose from an OmpC gene mutation that abolished outer membrane protein expression. Strain B16R1 displayed a rough colony morphology, whereas strain B16R17 retained a smooth colony morphology. Phage resistance in these strains was associated with fitness costs, including reduced virulence and altered antibiotic susceptibility, highlighting the potential benefits of phage therapy. Additionally, we found that receptor expression levels influenced plating efficiency, plaque morphology, size, and clarity. In summary, we evaluated the antibacterial efficacy of a K5-specific Klebsiella phage in milk and elucidated phage resistance mechanisms in K. pneumoniae. These findings provide a foundation for using Klebsiella phages in dairy production and ensuring their long-term therapeutic efficacy.