Lytic bacteriophages against Klebsiella pneumoniae and their impact on bacterial virulence

The use of lytic bacteriophages as therapeutic agents for the treatment of human infections is regaining interest, especially due to the spread of antibiotic-resistant bacteria. Beyond directly killing prokaryotic cells, phages also exert a selective pressure that can promote the selection of lysis-resistant cells during treatments. This phenomenon typically arises from modifications of phage receptors, often located on bacterial surface, which are involved in early virus-bacterium interactions. Here, we aimed to i) obtain in vitro phage-resistant derivatives, ii) characterize the genetic mechanisms underlying this phenotype and iii) evaluate changes in interactions with the host immune system. The vB_KpM_GP-7 phage was isolated from wastewater using the BO-FR-1 Klebsiella pneumoniae strain as host. This strain is a member of the K. pneumoniae ST258 clade II clonal lineage, previously obtained by infection at high multiplicity with the φBO1E phage of the KKBO-1 clinical strain. Three independent vB_KpM_GP-7 resistant derivatives have been characterized by genome sequencing and by evaluating their virulence on human macrophages and susceptibility to human serum. The vB_KpM_GP-7 resistant strains were all characterized by different alterations of genes involved in the lipopolysaccharide biosynthesis pathway, suggesting that components of this macromolecule are the receptors used by this phage to start an infective cycle. Phage resistant derivatives were all more susceptible than the parental clinical strain to the action of complementactive human serum and to macrophage killing, even if differences were observed among the tested strains. These results underscore that selective pressure exerted by phages could lead to decreased virulence in K. pneumoniae.