Discovery of potent endolysins against Pseudomonas aeruginosa and other gram-negative ESKAPE pathogens by an agar-based screening method.

Antibiotic resistance is a growing concern, and there is an urgent need for alternative treatments. A proposed solution includes endolysins. Naturally occurring endolysins have limited activity against Gram-negative bacteria due to the presence of an outer membrane. Previous agar-based screening methods did not consider this and screened for activity on autoclaved cell debris. In this study, we used a soft-agar-based screening method to identify endolysins targeting Pseudomonas aeruginosa. This method enabled us to screen for activity of endolysins on live Pseudomonas aeruginosa, taking the outer membrane into account. The application of this randomized forward screening method led to the discovery of three highly bactericidal endolysins, L0779, L0782, and L0799. These endolysins displayed a broad spectrum of activity against other Gram-negative bacterial species. We report minimal inhibitory concentrations (MICs) for endolysins acting on multiple Gram-negative ESKAPE species, with MICs for L0782 on Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii ranging from 4 to 16 µg/mL. In summary, our screening method enables rapid identification of endolysins with broad-spectrum activity against Gram-negative bacteria. These show potential for further development as alternative treatments for antibiotic-resistant infections.