Isolation of Phage Lysins That Effectively Kill Pseudomonas aeruginosa in Mouse Models of Lung and Skin Infection

Abstract

Multidrug resistance (MDR) is rapidly increasing in prevalence among isolates of the opportunistic pathogen Pseudomonas aeruginosa, leaving few treatment options. Phage lysins are cell wall hydrolases that have a demonstrated therapeutic potential against Gram-positive pathogens; however, the outer membrane of Gram-negative bacteria prevents most lysins from reaching the peptidoglycan, making them less effective as therapeutics. Nevertheless, a few lysins from Gram-negative bacterial phage can penetrate the bacterial outer membrane with the aid of an amphipathic tail found in the molecule’s termini. In this work, we took a phylogenetic approach to systematically identify those lysins from P. aeruginosa phage that would be most effective therapeutically. We isolated and performed preliminary characterization of 16 lysins and chose 2 lysins, PlyPa03 and PlyPa91, which exhibited >5-log killing activity against P. aeruginosa and other Gram-negative pathogens (particularly Klebsiella and Enterobacter). These lysins showed rapid killing kinetics and were active in the presence of high concentrations of salt and urea and under pH conditions ranging from 5.0 to 10.0. Activity was not inhibited in the presence of the pulmonary surfactant beractant (Survanta). While neither enzyme was active in 100% human serum, PlyPa91 retained activity in low serum concentrations. The lysins were effective in the treatment of a P. aeruginosa skin infection in a mouse model, and PlyPa91 protected mice in a lung infection model, making these lysins potential drug candidates for Gram-negative bacterial infections of the skin or respiratory mucosa.

Lysins breaking down the walls of Gram-negative bacteria, no longer a no-go

“…A genome-based screening approach using the C-terminal amphipathic helix of PlyF307 as query successfully yielded a new prophage lysin with intrinsic antibacterial activity [15]. In addition, genome-wide homology searches and phylogenetic analyses starting from the same lysin led to the identification of more lysins with intrinsic antibacterial activity [16 ]. An exception to this rule are lysins from extremophilic Thermus bacteriophages.…”

“…Moreover, in a mouse model of P. aeruginosa lung infection, PlyPa91 rescued 70% of mice when treated with a subsequent intranasal and intratracheal instillation, while only 20% of mice treated survived when treated with two intranasal instillations. These differences highlight the key importance of the route of delivery [16 ]. Recently, LysECD7 was used to eliminate Klebsiella pneumonia biofilms formed into diffusion chambers implanted into rats.…”

Gram-Negative Bacterial Lysins

“…Using an in silico approach to search P. aeruginosa phage genomes, the Fischetti laboratory has identified 16 P. aeruginosa specific lysins that had phylogenic similarities to A. baumannii lysin PlyF307 [22,51]. Of the 16 identified lysins, two lysins have shown promise as drug candidates: PlyPa03 and PlyPa91.…”

In vitro and in vivo efficacy of combinations of colistin and different endolysins against clinical strains of multi-drug resistant pathogens

“…Mouse skin infection model. A superficial skin wound infection by tape stripping in mouse was done as previously described 59,60 , with some modifications. Female BALB/c mice (6-8 weeks old) were anaesthetized with an injection of ketamine (500 μg/mouse) and medetomidine (15 μg/mouse).…”