Development and <i>in-vivo</i> efficacy of Leicester-UTI1 phage cocktail that targets ESBL-associated <i>Klebsiella</i> from urinary tract infections

ABSTRACT Uropathogenic Klebsiella pneumoniae strains are of major concern in the context of antimicrobial resistance, which has sparked interest in phage therapy as an alternative or complement to antibiotics. To address this, we developed and evaluated the Leicester-UTI1 phage cocktail, composed of six obligate lytic phages. Leicester-UTI1 phage cocktail demonstrated an in vitro efficacy of 80% against a panel of 30 diverse extended-spectrum β-lactamase (ESBL) and non-ESBL Klebsiella clinical isolates, as well as the reference K. pneumoniae Top52 strain. To assess in vivo activity, catheterized female C57BL/6J mice were infected transurethrally with K. pneumoniae Top52 and treated with a single dose of the purified Leicester-UTI1. Over a 7-day observation period modeling an acute urinary tract infection (UTI), the phage treatment significantly reduced or eradicated the K. pneumoniae infection in the urine, bladder, and kidneys, with effects observable as early as 4 h post-treatment. Phage recovery data indicated active replication in the urine, with no evidence of systemic dissemination, as no phage were detected in the spleen or blood. No emergence of phage resistance was observed. Phage treatment also reduced bladder inflammation, decreased pro-inflammatory IL-6, and increased anti-inflammatory IL-10 protein levels, indicating a positive immunomodulatory effect and enhanced infection resolution. Overall, the Leicester-UTI1 phage cocktail is a promising therapeutic agent for the treatment of Klebsiella pneumoniae UTI. IMPORTANCE The emergence of antibiotic resistance, coupled with a slow development of new antimicrobials, has renewed the interest in phage therapy, including for the treatment of urinary tract infections (UTIs). Despite the growing number of animal studies evaluating phage therapy, UTIs remained relatively understudied in this context. Here, we established a robust acute UTI murine model and used it to assess the efficacy of our optimized Leicester-UTI1 phage cocktail targeting extended-spectrum β-lactamase-associated Klebsiella . We found that a single transurethral administration of Leicester-UTI1 was sufficient to consistently eradicate bacterial infection in the urine, bladder, and kidneys. Further investigation also demonstrated that the phage exhibited immunomodulatory effects that supported infection resolution. Overall, our study provides strong evidence for the therapeutic potential of phages against UTI pathogens and highlights their promise as an alternative approach to antibiotics.