Isolation and characterization of bacteriophages against IMP-6-producingKlebsiella pneumoniaeisolated from clinical settings in Japan

Abstract

AbstractCarbapenemase-producingEnterobacteriaceae(CPE) are one of the most detrimental species of antibiotic-resistant bacteria worldwide. Phage therapy has emerged as an effective strategy for the treatment of infections caused by CPE pathogens. In west Japan, the increasing occurrence ofKlebsiella pneumoniaeharboring the pKPI-6 plasmid, which encodesblaIMP-6, is a growing concern. To manage such major antimicrobial-resistant pathogens, we isolated 29 novel phages from sewage in Japan, targeting 31 strains ofK. pneumoniaeand one strain ofEscherichia coliharboring the pKPI-6 plasmid. Electron microscopy analysis indicated that of the 29 isolated phages, 21 (72.4%), 5 (17.2%), and 3 (10.3%) belonged toMyoviridae, Siphoviridae, andPodoviridae, respectively. Host range analysis revealed that 20Myoviridaemembers in isolated phages infected 25–26 strains ofK. pneumoniae, indicating that most of the isolated phages have a broad host range. TheK. pneumoniaeKp21 can only be infected by phage øKp_21, while Kp22 can be infected by more than 20 phages. We applied a phage cocktail, which consists of 10 phages, against Kp21 and Kp22 and found that the phage cocktail delayed the emergence of phage-resistant bacteria for Kp21 strain but not for the Kp22 strain. Furthermore, phage-resistant Kp21 (Kp21r) became prone to be infected from other bacteriophages as a “trade-off” of resistance to phage øKp_21. Our proposed phage set has an adequate number of phages to combat theK. pneumoniaestrain isolated in Japan. Notably, our work demonstrates how a suitable phage cocktail diminishes the occurrence of phage-resistant bacteria.ImportanceKlebsiella pneumoniaeharboring the plasmid carryingblaIMP-6is becoming an increasingly hazardous species in Japan. We collected and characterized 29 novel bacteriophages that infectK. pneumoniaecarrying the pKPI-6 plasmid, isolated in clinical settings of west Japan. Our phages showed broad host ranges. We applied a phage cocktail treatment constructed from 10 phages against two host strains, Kp21 and Kp22, which show different phage susceptibility patterns each other. Although the phage cocktail delayed phage-resistant Kp21 emergence, the emergence of phage-resistant Kp22 could not be delayed. Moreover, phage-resistant Kp21 became sensitive to other phages, which did not originally infect wild-type Kp21. Our study demonstrates how a suitable phage cocktail can diminish the occurrence of phage-resistant bacteria.