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

Yunge Liu,Yimin Zhang,Xin Luo,Lixian Zhu,Pengcheng Dong,Jina Han,Lina Wu

doi:10.3389/fmicb.2020.617473

Abstract

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.

Highlights

Listeria monocytogenes (L. monocytogenes) is a Gram-positive food-borne pathogen
All compounds inhibited the growth of Lm 54004, and the minimum inhibitory concentration (MIC) for cinnamaldehyde, eugenol, resveratrol, thymoquinone, and sodium hypochlorite were 640 μg/ml, 1,280 μg/ml, 400 μg/ ml, 50 μg/ml and 1560 ppm, respectively
Our results showed that the growth of planktonic bacteria was totally inhibited by compounds at MIC – 4 × MIC, while 1/32 × MIC – 1/4 × MIC of five compounds all exhibited no obvious impacts on the concentration of Lm 54004 at stationary phase (Figure 1)

Summary

Introduction

Listeria monocytogenes (L. monocytogenes) is a Gram-positive food-borne pathogen. It is causes listeriosis with a high mortality rate (20–30%) among immunocompromised individuals (e.g., pregnant women, neonates, and the elderly; Gandra et al, 2019; Liu et al, 2020b). The biofilm formation of L. monocytogenes is the main cause for its persistence and stress resistance in food processing environments (GarcíaGonzalo and Pagán, 2015). Microorganisms in biofilms can enhance their resistance to antimicrobial agents, and are more difficult to eradicate compared with planktonic cells (Jolivet-Gougeon and Bonnaure-Mallet, 2014; Chen et al, 2019). L. monocytogenes cells in biofilms often cause recurrent contamination in food products, which enhances the food safety risks and leads to potential human health threats

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