Characterization of four novel bacteriophages targeting multi-drug resistant Klebsiella pneumoniae strains of sequence type 147 and 307

Introduction: the development of alternative antimicrobial strategies is deemed to be a high priority to deal with the challenge caused by the spread of multidrug-resistant (MDR) bacteria in clinical settings. according to several international organizations, phages or components thereof are one of these possible options that could be useful to treat bacterial infections. among the drug-resistant bacteria, carbapenem resistant Klebsiella pneumoniae (CR-Kp) are particularly worrisome, given the extensive MDR profiles, their pandemic dissemination and primary role in healthcare associated and life-threatening infections. In this study we isolated and characterised four lytic bacteriophages targeting two major high-risk clones of CR-Kp circulating in hospital environments, i.e., those belonging to Sequence Type (ST) 307 and ST147. Materials and methods: wastewater samples collected from hospitals located in central Italy were screened for the presence of phages by using a previously characterized collection of K. pneumoniae clinical isolates as hosts and the top-agar overlay technique. host specificity and infection efficiency was assessed by spot test and efficiency of plating, respectively. Dynamic of bacterial infections was determined by the one-step growth curve method. Phages were visualized through transmission electron microscopy (TEM) and their genomes were obtained and analysed by a Next Generation Sequencing approach followed by bioinformatics analysis. Results: four bacteriophages, named GP-1, GP-2, GP-4 and GP-5, have been isolated, purified and produced at high titres. collectively, two phages were able to selectively lyse 12/14 K. pneumoniae strains of ST307, while the other two were active only against all the tested K. pneumoniae strains of ST147 (n=12). phages maintain an overall good stability to temperature and pH changes and were characterized by infection cycles having latency periods ranging from 10 to 50 minutes and burst sizes of 10-100 PFU. results from TEM analysis and genome sequencing demonstrated that the four phages were of different families and allowed to rule out the presence of antibiotic resistance genes, virulence factors or toxins. Discussion and Conclusions: Considering their strictly lytic nature and their high selectivity towards two of the major high-risk clones of K. pneumoniae, the isolated phages can be considered as good candidates for their evaluation in animal models as members of cocktails for applications to treat severe infections caused by CR-Kp strains.