In Vitro Characterization of Antilisterial Activity by Bacteriophage Endolysin PlyP100

Introduction: The Gram-positive pathogen Listeria monocytogenes is highly problematic in the manufacture and processing of ready-to-eat foods due to its environmental persistence and its ability to grow under refrigerated storage. Special care must be taken to prevent listerial contamination during the production of fresh cheeses, as their delicate flavor and texture are sensitive to disruption by many of the antimicrobial processes and additives commonly used for other foods. Bacteriophage-derived cell wall hydrolytic enzymes, known as endolysins, comprise one possible intervention and may not suffer from the high strain-specificity of their parent phages.

Purpose: To address limitations of current antilisterial processes for fresh cheeses, by assessing antimicrobial efficacy of a Listeria-specific endolysin in vitro.

Methods: The L. monocytogenes phage P100 endolysin, PlyP100, was recombinantly expressed in Escherichia coli BL21(DE3), purified, and applied to listerial cultures suspended in phosphate buffer at a range of pH levels, temperatures, and salt-concentrations. Turbidity reduction assays were conducted over 30 min periods with optical densities of samples monitored via microplate spectrophotometer.

Results: Turbidity reduction assays confirm that PlyP100 exhibits optimal cell destruction at pH 8, mesophilic temperatures, and 100-250 mM NaCl. All listerial strains subjected to PlyP100, including 18 strains of L. monocytogenes and 5 strains of related species, were susceptible to enzymatic degradation, while all other gram-positive organisms tested (n=20) were insensitive except for Bacillus subtilis, Clostridium difficile, Brevibacterium linens, and Lactobacillus plantarum. Furthermore, we confirmed that sensitivity to PlyP100 is dependent upon the growth phase of target cultures.

Significance: This study demonstrates the potential of bacteriophage endolysins in targeting foodborne pathogens and highlights factors to consider for their study and application in foods, including target specificity and environmental tolerance.