Genomic and phenotypic characterization of Klebsiella pneumoniae phage KP Ø1: a novel lytic Slopekvirus targeting uropathogenic multidrug-resistant Klebsiella pneumoniae

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The rise of multidrug-resistant (MDR) uropathogenic gram-negative bacteria (GNB) necessitates the development of alternative therapeutic strategies. This study aimed to isolate, phenotypically characterize, and perform whole-genome sequencing of the bacteriophage demonstrating the broadest host range against MDR uropathogens. Fifty MDR GNB isolates were screened for lytic phages. The most promising candidate, Klebsiella pneumoniae phage KP Ø1, was characterized using plaque assay, Transmission Electron Microscopy (TEM), and pH/thermal stability testing. Genomic characterization was performed via whole-genome sequencing (WGS), with functional annotation and lifestyle prediction using PhaBOX and PhageScope software. Klebsiella pneumoniae was the most prevalent MDR uropathogen. Klebsiella pneumoniae phage KP Ø1 exhibited a 50% host range and high lytic titer (10⁸ PFU/mL). TEM revealed an icosahedral head and short contractile tail. Genomic characterization by WGS revealed that Klebsiella pneumoniae phage KP Ø1 possesses a 174,591 bp double-stranded deoxyribonucleic acid (dsDNA) genome containing 274 predicted open reading frames (ORFs). No lysogeny-related genes, toxins, or antibiotic resistance markers were detected, confirming its strictly lytic nature and supporting its potential as a candidate for phage therapy applications. The phage remained stable (10⁸ PFU/mL) across temperatures of - 20 °C to 50 °C; supporting its suitability for long-term biobanking and suggesting potential activity at physiological temperature, and across a pH range of 7-9. Klebsiella pneumoniae phage KP Ø1 is a novel, obligately lytic Slopekvirus whose genomic architecture, stability profile, and absence of lysogeny-associated, virulence, and antimicrobial resistance genes ( AMR) collectively support its candidacy for further preclinical evaluation as a phage therapy agent against uropathogenic MDR Klebsiella pneumoniae.

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