Antimicrobial potential of a novel K5-specific phage and its recombinant strains against Klebsiella pneumoniae in milk

The nutrient-rich composition of milk creates an optimal environment for bacterial proliferation, making the inhibition of microbial growth essential for maintaining dairy product quality and ensuring consumer safety. Klebsiella pneumoniae is an important contaminant of milk and a leading cause of bovine mastitis. Although the increasingly serious antibiotic resistance has led to a renewed interest in phage therapy, research on antimicrobial potential of Klebsiella phages in milk remains scarce. The K5 serotype of K. pneumoniae is a major concern due to its high virulence and prevalence in dairy farming operations. Despite its clinical and economic importance, the availability of phages specifically targeting this serotype remains substantially limited. Here, we successfully isolated and sequenced 2 K1-specific Klebsiella phages, P284 and P287, and one K5-specific Klebsiella phage P252. We identified the receptor-binding proteins with depolymerization activity in these phages. The phage library against K5 K. pneumoniae was enriched by phage genome modification. Specifically, we replaced the receptor-binding protein of K1-specific phage P284 with that of K5-specific phage P252, resulting in the generation of recombinant phages T and F, which exhibit specific lytic activity against K5 K. pneumoniae. Compared with phage P252, recombinant phages T and F exhibited better and more prolonged antibacterial potential in planktonic assay. In addition, all these K5-specific phages could significantly inhibit bacterial growth and reduce bacterial populations in milk at 4°C and 38°C. In summary, this study provided K5-specific phages with potential application in managing K. pneumoniae contamination and infection in the dairy industry.