Use of bacteriophage endolysin EL188 and outer membrane permeabilizers against Pseudomonas aeruginosa

Abstract

Aims: To select and evaluate an appropriate outer membrane (OM) permeabilizer to use in combination with the highly muralytic bacteriophage endolysin EL188 to inactivate (multi‐resistant) Pseudomonas aeruginosa. Methods and Results: We tested the combination of endolysin EL188 and several OM permeabilizing compounds on three selected Ps. aeruginosa strains with varying antibiotic resistance. We analysed OM permeabilization using the hydrophobic probe N‐phenylnaphtylamine and a recombinant fusion protein of a peptidoglycan binding domain and green fluorescent protein on the one hand and cell lysis assays on the other hand. Antibacterial assays showed that incubation of 106Ps. aeruginosa cells ml−1 in presence of 10 mmol l−1 ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA) and 50 μg ml−1 endolysin EL188 led to a strain‐dependent inactivation between 3·01 ± 0·17 and 4·27 ± 0·11 log units in 30 min. Increasing the EL188 concentration to 250 μg ml−1 further increased the inactivation of the most antibiotic resistant strain Br667 (4·07 ± 0·09 log units). Conclusions: Ethylene diamine tetraacetic acid disodium salt dihydrate was selected as the most suitable component to combine with EL188 in order to reduce Ps. aeruginosa with up to 4 log units in a time interval of 30 min. Significance and Impact of the Study: This in vitro study demonstrates that the application range of bacteriophage encoded endolysins as ‘enzybiotics’ must not be limited to gram‐positive pathogens.

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…Permeabilizers can also enable the use of new antimicrobial compounds that are not very effective because of their low ability to enter the cell. For instance, bacteriophage endolysin EL188, which was considered to be limited to Gram-positive pathogens, inhibited P. aeruginosa when combined with certain outer membrane permeabilizers, in particular EDTA (25).…”

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…EDTA), or high hydrostatic pressure in combination with an endolysin have been studied. 21,22,91,92 Although these measures have shown that chemical and/or physical disruption of the outer membrane allows the endolysin to gain access to the protected peptidoglycan, they may not lend themselves well to human therapies. These methods have not always been successful, with some Gram-negative strains remaining insensitive to lysis by endolysins after membrane disruption.…”

Bacteriophages and Their Derivatives as Biotherapeutic Agents in Disease Prevention and Treatment

“…Thus, antibacterial activity has only been shown to be possible after treatment of the outer membrane of Gram-negative cells with EDTA [116] or by the fusion of hydrophobic amino acids to the endolysin, which enables the movement of the endolysin across the outer membrane. The latter approach has been recently developed in endolysins by the group of Lavigne in Belgium [117] and was based on earlier observations where the action of lysozyme against E. coli was enhanced by the fusion of a hydrophobic pentapeptide onto the C-terminus [118]. The advantage of the use of lysins over other antibacterials (or antibiotics) is their specificity for one bacterial pathogen without disturbing other nonpathogenic bacterial flora.…”