Phage training improves the bactericidal ability against carbapenem-resistant Klebsiella pneumoniae

Abstract:

[Objective] This study aims to improve the bactericidal ability of phages against carbapenem-resistant Klebsiella pneumoniae, a major pathogen in clinical practice, and reduce host resistance to phages by developing a novel method of phage training. [Methods] Phages were isolated from municipal wastewater by the double-layer agar plate method with clinical carbapenem-resistant K. pneumoniae strain Kp2092 as the host, and their host ranges were analyzed. The morphological and genetic characteristics of a phage strain with strong lysis ability and a broad host range were analyzed by transmission electron microscopy and whole-genome sequencing. Phage training was performed by phage-host co-culture for generations, and the phages before and after training were compared in terms of biological properties such as bactericidal activity, optimal multiplicity of infection, one-step growth curve, and stability under different stress conditions. [Results] A total of 9 phage strains were isolated, in which P55anc was a short-tailed phage with the strongest lysis ability and the broadest host range. The genome (40 301 bp) of P55anc included 51 coding sequences, of which 27 showed possible functions involving nucleic acid metabolism, virion morphogenesis, DNA packaging, and host lysis. Three evolved phages of P55anc were obtained after 9 days of phage training. These evolved phages showed significantly enhanced bactericidal activities, manifested by the increased burst size, broadened host range, and reduced host resistance. Moreover, the evolved phages maintained stable when being exposed to heat, ultraviolet, and serum treatments. [Conclusion] Phage training by phage-host co-culture can be employed to obtain evolved phages with enhanced bactericidal effects. Furthermore, the evolved phages reduced the host resistance and remained stable under different stress conditions.

Key words:bacterial drug resistance;phage therapy;phage training;co-evolution