Inhibition of Listeria monocytogenes biofilm formation by phenyllactic acid in pasteurized milk is associated with suppression of the Agr system

The biofilm-forming propensity of Listeria monocytogenes on food-contact surfaces presents critical challenges to food safety. Phenyllactic acid (PLA), a microbial-derived organic acid compound, exhibits potent biofilm inhibition against this pathogen, yet its mechanistic basis remains undefined. This study investigated PLA's effects on the Agr system, which promotes L. monocytogenes biofilm formation, to explore its potential as a biofilm control agent. Quantitative biofilm analysis through crystal violet-based microplate assays demonstrated decreased biofilm biomass under PLA treatment. Quantitative real time PCR (RT-qPCR) revealed marked downregulation of the agr system components, paralleled by diminished β-galactosidase activity in agr promoter-reporter constructs. These evidences suggested that PLA can inhibit L. monocytogenes biofilm formation by suppressing the Agr system in medium. Microscale thermophoresis analysis excluded direct molecular interactions between PLA and AgrC/AgrA proteins. Our data demonstrated that signal molecule autoinducing peptide (AIP) produced by AgrBD in L. monocytogenes was a cyclic hexapeptide whose biosynthesis was inhibited by PLA. Notably, PLA maintained its biofilm-inhibitory capacity by suppressing the Agr system in pasteurized milk. This preserved activity across distinct experimental environments highlights the application potential of PLA as a food-safe antimicrobial agent.