Rapid formulation of a genetically diverse phage cocktail targeting uropathogenic Escherichia coli infections using the UTI89 model

Urinary tract infections are commonly caused by uropathogenic Escherichia coli (UPEC). Due to the emergence of multidrug-resistant UPEC, rendering antibiotic treatment ineffective, phage combination-based therapy has been proposed as a potential alternative. Here, we present a formulation of a genetically diverse phage-derived cocktail that is rapidly customized for UPEC using E. coli UTI89 as a model strain. Through our rapid selection and combination of four phages against UPEC strain UTI89 (SR01, SR02, SR04, and Zappy) from our library, the combination of two lytic phages, SR02 and SR04, exhibits the strongest suppression of bacterial growth for at least 16 h, with no emergence of phage resistance observed in vitro. Phage SR02 undergoes subcellular activity for 25 min, producing approximately 106 progeny particles per cell, while SR04 completes its replication cycle in 20 min, generating around 564 progeny particles per cell. These two novel phages are genetically diverse, and their cocktail exhibited potent suppression of bacterial growth, independent of multiplicities of infection (MOIs), significantly reducing the viable bacterial counts after treatment in vitro. The phage cocktail has low immunogenicity and does not induce any proinflammatory gene responses in human bladder uroepithelial cells. Moreover, the cocktail effectively eradicates the invading UPEC strain UTI89 in the uroepithelial cells at a comparable level to that of phage SR04 alone, likely releasing some immunostimulatory agents that, in turn, trigger upregulation of MIP-3 and IL-8 genes. Altogether, this study offers an alternative pipeline for rapidly formulating genetically diverse phage-derived cocktails, which is specifically customized for targeted bacteria.