title: A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids.
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.
authors.affiliation: Centre of Biological Engineering, University of Minho, Braga, Portugal.
authors.forename: Hugo
authors.initials: H
authors.lastname: Oliveira
authors.affiliation: School of Chemistry, Bharathidasan University, Tiruchirappalli, India.
authors.forename: Viruthachalam
authors.initials: V
authors.lastname: Thiagarajan
authors.affiliation: Laboratory of Gene Technology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Walmagh
authors.affiliation: Centre of Biological Engineering, University of Minho, Braga, Portugal.
authors.forename: Sanna
authors.initials: S
authors.lastname: Sillankorva
authors.affiliation: Laboratory of Gene Technology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Rob
authors.initials: R
authors.lastname: Lavigne
authors.affiliation: Nanomedicine Department, International Iberian Nanotechnology Laboratory, Braga, Portugal; Medical Faculty, Aalborg University, Aalborg, Denmark.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Neves-Petersen
authors.affiliation: Centre of Biological Engineering, University of Minho, Braga, Portugal.
authors.forename: Leon D
authors.initials: LD
authors.lastname: Kluskens
authors.affiliation: Centre of Biological Engineering, University of Minho, Braga, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Azeredo
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D019343:Citric Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D004795:Enzyme Stability; D006090:Gram-Negative Bacteria / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008293:Malates / Q000494:pharmacology; D012483:Salmonella Phages / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D000900:Anti-Bacterial Agents; D008293:Malates; D019343:Citric Acid; C030298:malic acid; D010450:Endopeptidases; C001143:endolysin