Abstract
Background: The global rise in multidrug-resistant (MDR) Gram-negative bacteria (GNB) poses an urgent challenge for infection control and antibiotic stewardship. Among these, Klebsiella pneumoniae is a major cause of hospital-acquired infections and is listed as a critical priority pathogen by the World Health Organization. Peru reports an exceptionally high prevalence of MDR K. pneumoniae, underscoring the need for innovative antimicrobial approaches. Methods: Here, we describe the isolation and characterization of lytic Klebsiella bacteriophage from sewage samples collected from the Huatanay River (Cusco, Peru) in 2023. Phages were isolated using the reference strain MDR K. pneumoniae ATCC BAA-2814. Then, they were screened against 50 clinical MDR K. pneumoniae strains. Results: The phage H33IIK demonstrated effective antibacterial capability, showing strict host specificity for K. pneumoniae, thermal stability, moderate pH tolerance, and high burst size. Whole-genome sequencing analysis classified it within the class Caudoviricetes, family Ackermannviridae, and genus Taipeivirus. The genomic analysis confirmed the absence of lysogeny-related, antimicrobial resistance, and virulence genes, supporting its suitability and safety for potential biotechnological applications. Conclusions: These findings highlight phage H33IIK as a lytic agent effective against MDR K. pneumoniae. It could contribute to the development of phage-based approaches to combat MDR GNB.