Isolation and characterization of an antibiofilm bacteriophage against Klebsiella michiganensis and evaluation of its therapeutic potential in an insect larval gut colonization model

Bacteriophages (phages) have therapeutic potential against AMR bacteria. Recently, Klebsiella michiganensis (a member of the Klebsiella oxytoca complex) was discovered. This pathogen is associated with nosocomial infection and sepsis and has the ability to neutralize the β-lactam class of antibiotics. This study focused on detailed characterization of the phage vB_KleM_kp20 (kp20) and evaluation of its in vitro and in vivo efficacy against K. michiganensis. Phage kp20 was isolated from sewage water, purified, and characterized via standard methods, including adsorption assay, one-step growth curve, host range determination, pH/temperature stability, in vitro lysis kinetics, transmission electron microscopy (TEM), and genome sequencing. The therapeutic potential of kp20 was also evaluated through in vitro antibiofilm activity and in vivo decolonization in a mosquito larval model (Aedes albopictus). kp20 showed narrow host specificity, a rapid adsorption rate (> 60% adsorption within 10 min), a short latency period (~ 10 min), a large burst size (~ 100 PFU per infected cell), and strong in vitro antibacterial activity at a low MOI (0.1). Phage kp20 retained infectivity over a range of temperatures (4–40 °C) and pH values (6–10). In the in vitro antibiofilm assay, a 39% reduction in biomass was observed at an MOI of 1. Transmission electron micrography revealed a myovirus morphology, and genetic analyses revealed an ~ 174 kb dsDNA genome (GenBank PP993148) harboring at least 267 CDSs and 2 tRNA genes. No AMR/toxin- or integration-related genes were detected. Analyses of the proteomic tree revealed the affinity of kp20 for the genus Slopekvirus under the Straboviridae family of viruses. Compared with no treatment, treatment with kp20 significantly reduced survival (70% vs. 40%; P < 0.001) and growth (2.1 ± 0.38 mm vs. 1.4 ± 0.17 mm; p < 0.0001) in the mosquito larval gut colonization model. The results of our study suggest promising therapeutic potential for phage kp20. Furthermore, we demonstrated that the mosquito larval gut colonization model could be utilized as a convenient pre-murine model to evaluate phage efficacy in vivo.