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A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids.

PloS one (2014-10-08)
Hugo Oliveira, Viruthachalam Thiagarajan, Maarten Walmagh, Sanna Sillankorva, Rob Lavigne, Maria Teresa Neves-Petersen, Leon D Kluskens, Joana Azeredo
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
DL-Malic acid, ReagentPlus®, ≥99%
Sigma-Aldrich
DL-Malic acid, ≥98% (capillary GC)
Supelco
Malic acid, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
DL-Malic acid, 99%
Sigma-Aldrich
Malic acid, meets USP/NF testing specifications
Supelco
DL-Malic acid, analytical standard
Sigma-Aldrich
DL-Malic acid, meets analytical specification of FCC, E296, 99-100.5% (alkalimetric)
USP
Malic acid, United States Pharmacopeia (USP) Reference Standard

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