Inhibition of Biofilm Formation and Related Gene Expression of Listeria monocytogenes in Response to Four Natural Antimicrobial Compounds and Sodium Hypochlorite

The aim of this study was to assess the efficacy of four natural antimicrobial compounds (cinnamaldehyde, eugenol, resveratrol and thymoquinone) plus a control chemical disinfectant (sodium hypochlorite) in inhibiting biofilm formation by Listeria monocytogenes CMCC54004 (Lm 54004) at a minimum inhibitory concentration (MIC) and sub-MICs. Crystal violet staining assay and microscopic examination were employed to investigate anti-biofilm effects of the evaluated compounds, and a real-time PCR assay was used to investigate the expression of critical genes by Lm 54004 biofilm. The results showed that five antimicrobial compounds inhibited Lm 54004 biofilm formation in a dose dependent way. Specifically, cinnamaldehyde and resveratrol showed better anti-biofilm effects at 1/4 × MIC, while sodium hypochlorite exhibited the lowest inhibitory rates. A swimming assay confirmed that natural compounds at sub-MICs suppressed Lm 54004 motility to a low degree. Supporting these findings, expression analysis showed that all four natural compounds at 1/4 × MIC significantly down-regulated quorum sensing genes (agrA, agrC, and agrD) rather than suppressing the motility- and flagella-associated genes (degU, motB, and flaA). This study revealed that sub-MICs of natural antimicrobial compounds reduced biofilm formation by suppressing the quorum sensing system rather than by inhibiting flagella formation.