Control of Listeria monocytogenes in food industry by a combination treatment of natural aromatic compound with Listeria-specific bacteriophage cocktail

Most Listeria monocytogenes found in the food industry are listeriosis-causing pathogens and possess the ability to form biofilms on food and food contact materials (FCMs). This study aims to evaluate the efficacy of the combination treatment of natural aromatic compounds (thymol, eugenol, carvacrol, and citral) with a Listeria-specific phage cocktail in mitigating the threat posed by L. monocytogenes in the food industry. In vitro combination treatment of 1 minimal inhibitory concentration (MIC) of natural aromatic compound with phage cocktail at multiplicity of infection (MOI) 100 reduced more than 4 log CFU/mL of L. monocytogenes planktonic cells and inhibited biofilm formation. In addition, the expression of virulence-related genes (flaA, mota, hlyA, prfA, and actA) and the stress response (siga) gene were significantly downregulated. The combination of natural aromatic compound with phage cocktail reduced the biofilm cell population on contaminated celery by more than 2 log CFU/g and by more than 2 log CFU/cm2 on already-formed biofilm on FCMs, but it was less effective on chicken meat, with an approximate reduction of only 1 log CFU/g. The antibiofilm activity toward preformed L. monocytogenes biofilms was also observed using field-emission scanning electron microscopy (FESEM) and confocal laser scanning microscopy (CLSM). COMSTAT analysis of the structural change of biofilms revealed that major biofilm structure parameters (biovolume, thickness, diffusion distance, and microcolonies at substratum) were reduced after treatment. Our findings suggest that the combination of natural aromatic compounds with a phage cocktail has enormous potential as an antimicrobial and antibiofilm agent for controlling L. monocytogenes in the food industry.