Exploring bacteriophages as potential tools for combating multi drug-resistant Klebsiella pneumoniae: Isolation, characterization, genomic insights, and in vitro evaluation.

Bacteriophages are promising alternatives to antibiotics for combating multidrug-resistant (MDR) pathogens such as Klebsiella pneumoniae, a major cause of hospital-acquired infections. In this study, two K. pneumoniae-specific phages, vB_Kpn_RAH1 and vB_Kpn_RAH2, were isolated from sewage using an MDR clinical isolate (KP26) as the host. Their morphological, physicochemical, and genomic characteristics were analyzed. Both phages exhibited Siphovirus-like morphology, and the genome sizes of RAH1 and RAH2 were 46,784 bp and 59,380 bp, respectively. The phages were stable at 25-37 °C and pH 7-9 but were sensitive to high temperatures (80-90 °C), extreme pH, and prolonged UV exposure. Killing-curve assays confirmed strong bactericidal activity, with MOI 0.1 producing the most sustained bacterial reduction (90-97 %), while higher MOIs showed reduced long-term efficacy. The phage cocktail exhibited superior and prolonged inhibition of K. pneumoniae, outperforming RAH1 and RAH2 individually. One-step growth analysis revealed burst sizes of approximately 400 virions for RAH1 and 250 for RAH2. Host-range analysis indicated narrow specificity for both phages, whereas their cocktail displayed a broader lytic spectrum, lysing 9 of 15 clinical isolates. The absence of lysis in non-Klebsiella species emphasizes the high specificity of the phages, supporting a targeted therapeutic approach while minimizing unintended effects on the host microbiota. Genomic analysis confirmed that RAH1 and RAH2 are novel species lacking antimicrobial resistance and virulence genes. RAH2 is strictly lytic, whereas RAH1 is temperate. The strictly lytic nature and favorable genomic features of RAH2 support its potential as a candidate for therapeutic applications.