A Thermostable Salmonella Phage Endolysin, Lys68, with Broad Bactericidal Properties against Gram-Negative Pathogens in Presence of Weak Acids

Hugo Oliveira,Viruthachalam Thiagarajan,Rob Lavigne,Sanna Sillankorva,Leon D Kluskens,Joana Azeredo,Maria Teresa Neves-Petersen,Maarten Walmagh,Kornelius Zeth

doi:10.1371/journal.pone.0108376

Hugo Oliveira, Viruthachalam Thiagarajan + Show 7 more

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https://doi.org/10.1371/journal.pone.0108376

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Abstract

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.

Highlights

Gram-negative bacterial pathogens are common causes of foodborne (e.g. Salmonella, Escherichia coli O157:H7, Shigella) and hospital-acquired (e.g. Pseudomonas, Acinetobacter) infectious diseases [1,2]
In an era in which the threat of antibiotic and multi-resistant bacteria is increasing and solutions are becoming scarce, it is important to search for alternative antimicrobials
One promising alternative approach to prevent or destroy pathogenic bacteria is the use of bacterial cell wall hydrolases

Summary

Introduction

Gram-negative bacterial pathogens are common causes of foodborne (e.g. Salmonella, Escherichia coli O157:H7, Shigella) and hospital-acquired (e.g. Pseudomonas, Acinetobacter) infectious diseases [1,2]. One promising alternative approach to prevent or destroy pathogenic bacteria is the use of bacterial cell wall hydrolases. These enzymes cause bacteriolysis by degrading the peptidoglycan (PG) layer, known as murein, the major component of the bacterial cell wall and responsible for the mechanical integrity. Among these bacterial cell wall hydrolases, an increased interest has been given to bacteriophage (phage) endolysins. Endolysins are specialized PG-degrading enzymes, part of a universal lytic cassette system encoded by all double stranded DNA phages and expressed during the terminal stage of the reproduction cycle [3]

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