Abstract
Lytic bacteriophages and phage-encoded endolysins (peptidoglycan hydrolases) provide a source for the development of novel antimicrobial strategies. In the present study, we focus on the closely related (96 % DNA sequence identity) environmental myoviruses vB_KpnM_KP15 (KP15) and vB_KpnM_KP27 (KP27) infecting multidrug-resistant Klebsiella pneumoniae and Klebsiella oxytoca strains. Their genome organisation and evolutionary relationship are compared to Enterobacter phage phiEap-3 and Klebsiella phages Matisse and Miro. Due to the shared and distinct evolutionary history of these phages, we propose to create a new phage genus “Kp15virus” within the Tevenvirinae subfamily. In silico genome analysis reveals two unique putative homing endonucleases of KP27 phage, probably involved in unrevealed mechanism of DNA modification and resistance to restriction digestion, resulting in a broader host spectrum. Additionally, we identified in KP15 and KP27 a complete set of lysis genes, containing holin, antiholin, spanin and endolysin. By turbidimetric assays on permeabilized Gram-negative strains, we verified the ability of the KP27 endolysin to destroy the bacterial peptidoglycan. We confirmed high stability, absence of toxicity on a human epithelial cell line and the enzymatic specificity of endolysin, which was found to possess endopeptidase activity, cleaving the peptide stem between l-alanine and d-glutamic acid.
Highlights
IntroductionLytic bacteriophages (viruses that attack and lyse bacteria) are ubiquitous in nature
As an addition to existing annotation, the regulatory sequence positions were localized using MEME/MAST and ARNold software. Despite this high level of identity, the genomes differ significantly: 53 and 118 rho-independent terminators have been predicted for phage KP15 and KP27 respectively (Tables S2 and S3, Online Resource 1)
There is an increasing number of Klebsiella phages propagating on especially extended spectrum betalactamase (ESBL) and carbapenemase (KPC) producing isolates (Drulis-Kawa et al 2011; Kęsik-Szeloch et al 2013; Mijalis et al 2015; Provasek et al 2015; Wangkahad et al 2015; Jamal et al 2015), which enables analysis of homology and particular properties of this specific clade of phages
Summary
Lytic bacteriophages (viruses that attack and lyse bacteria) are ubiquitous in nature. As natural predators, they control the bacterial population and have a large impact on bacterial ecosystems (Weinbauer and Rassoulzadegan 2004). Particular attention has been drawn to phages and endolysins which demonstrate activity on highly virulent and multidrug-resistant pathogens including Klebsiella pneumoniae. This bacterium is associated with nosocomial infections and commonly found in natural environments, such as water reservoirs, soil, sewage and on plant surface (Bagley 1985). Noteworthy is the occurrence of different Klebsiella spp. capable of expressing virulence factors including serum resistance, capsular polysaccharides, pili and
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