Dose-and regimen-dependent therapeutic efficacy of the lytic Klebsiella phage TRK61 in a Galleria mellonella infection model.

Multidrug-resistant (MDR) Klebsiella infections pose a significant challenge and necessitate alternative antimicrobial approaches. Bacteriophage therapy represents a promising option; however, therapeutic outcomes depend on phage dose, dosing strategy, and host-pathogen interactions. In this study, we isolated and characterized a lytic Klebsiella phage, vB_KpnS_TRK61 (TRK61), from municipal wastewater. Electron microscopy revealed that TRK61 has an icosahedral head and a long, flexible tail, consistent with members of the family Demerecviridae. Host range analysis demonstrated lytic activity against clinical isolates of Klebsiella pneumoniae and K. oxytoca, as well as K. quasipneumoniae ATCC 700,603 and K. michiganensis ATCC 43,086. Genome sequencing revealed a 113,941 bp double-stranded DNA genome encoding 147 predicted proteins and 24 tRNA genes, with no identifiable antibiotic resistance, toxin, or virulence-associated genes. One-step growth experiments indicated a latent period of approximately 30 min and a burst size of ~ 50 plaque-forming units per infected cell. The in vivo efficacy of TRK61 was evaluated using the Galleria mellonella infection model at multiplicities of infection (MOIs) of 10, 100, and 1,000 under single- and double-dose regimens. Phage-mediated protection was strongly influenced by both MOI and dosing frequency. Moderate MOIs combined with repeated dosing produced the highest survival rates, whereas excessively high MOIs occasionally reduced efficacy. Overall, TRK61 shows strong lytic activity and in vivo efficacy, supporting its potential as a therapeutic or biocontrol agent against MDR Klebsiella.