Lactobacillus acidophilus ATCC 4356-derived metabolites inhibit biofilm formation and motility in Listeria monocytogenes

Listeria monocytogenes is a major foodborne pathogen whose persistence in food-processing environments is strongly associated with its ability to survive under a wide range of environmental conditions and form biofilms on different surfaces. Metabolites produced by beneficial lactic acid bacteria may represent a natural source of antimicrobial and antibiofilm compounds capable of affecting bacterial behavior even at subinhibitory concentrations. This study investigated the effects of the cell-free supernatant of the probiotic strain Lactobacillus acidophilus ATCC 4356 ( La -CFS) on growth, biofilm formation, motility, and the expression of selected quorum sensing- and motility-associated genes in L. monocytogenes . The antibacterial activity and minimum inhibitory concentration (MIC) of La -CFS were determined against multiple L. monocytogenes strains. Extracellular signaling activity was evaluated using the AI-2-responsive Vibrio harveyi BB170 bioluminescence reporter assay, while the effects of sub-MIC concentrations on biofilm formation and swimming and swarming motility were assessed using phenotypic assays. The expression of luxS , agrC , flaA , and motA was analyzed by RT-qPCR. La -CFS inhibited the growth of all tested L. monocytogenes strains, whereas sub-MIC concentrations induced a transient delay in bacterial growth without significantly affecting the final optical density. Cell-free supernatants from the evaluated L. monocytogenes strains induced bioluminescence in the V. harveyi BB170 reporter assay, indicating extracellular signaling activity detectable by an AI-2-responsive system. At sub-MIC concentrations, La -CFS significantly reduced biofilm formation and impaired swimming and swarming motility in a strain- and concentration-dependent manner. Transcriptional analysis of L. monocytogenes CLIST 3833 exposed to La -CFS at 0.5 × MIC for 24 h revealed significant downregulation of the motility-associated genes flaA and motA (p < 0.05), whereas the expression of luxS and agrC was not significantly altered. These findings demonstrate that metabolites present in the cell-free supernatant of L. acidophilus ATCC 4356 can impair biofilm formation and motility in L. monocytogenes , supporting further investigation of La -CFS as a potential source of bioactive compounds for controlling the persistence of this pathogen in food-processing environments.