Andrographolide Inhibits Biofilm and Virulence in Listeria monocytogenes as a Quorum-Sensing Inhibitor.

Listeria monocytogenes is a major foodborne pathogen that can cause listeriosis in humans and animals. Andrographolide is known as a natural antibiotic and exhibits good antibacterial activity. We aimed to investigate the effect of andrographolide on two quorum-sensing (QS) systems, LuxS/AI-2 and Agr/AIP of L. monocytogenes, as well as QS-controlled phenotypes in this study. Our results showed that neither luxS expression nor AI-2 production was affected by andrographolide. Nevertheless, andrographolide significantly reduced the expression levels of the agr genes and the activity of the agr promoter P2. Results from the crystal violet staining method, confocal laser scanning microscopy (CLSM), and field emission scanning electron microscopy (FE-SEM) demonstrated that andrographolide remarkably inhibited the biofilm-forming ability of L. monocytogenes 10403S. The preformed biofilms were eradicated when exposed to andrographolide, and reduced surviving cells were also observed in treated biofilms. L. monocytogenes treated with andrographolide exhibited decreased ability to secrete LLO and adhere to and invade Caco-2 cells. Therefore, andrographolide is a potential QS inhibitor by targeting the Agr QS system to reduce biofilm formation and virulence of L. monocytogenes.

Benzalkonium Chloride Adaptation Increases Expression of the Agr System, Biofilm Formation, and Virulence in Listeria monocytogenes.

Benzalkonium chloride (BC) is widely used for disinfection in food industry. However, prolonged exposure to BC may lead to the emergence of BC adapted strains of Listeria monocytogenes, an important foodborne pathogen. Until now, two communication systems, the LuxS/AI-2 system and the Agr system, have been identified in L. monocytogenes. This study aimed to investigate the role of communication systems in BC adaptation and the effect of BC adaptation on two communication systems and the communication-controlled behaviors in L. monocytogenes. Results demonstrated that the Agr system rather than the LuxS system plays an important role in BC adaptation of L. monocytogenes. Neither luxS expression nor AI-2 production was affected by BC adaptation. On the other hand, the expression of the agr operon and the activity of the agr promoter were significantly increased after BC adaptation. BC adaptation enhanced biofilm formation of L. monocytogenes. However, swarming motility was reduced by BC adaptation. Data from qRT-PCR showed that flagella-mediated motility-related genes (flaA, motA, and motB) were downregulated in BC adapted strains. BC adaptation increased the ability of L. monocytogenes to adhere to and invade Caco-2 cells but did not affect the hemolytic activity. Compared with the wild-type strains, the expression levels of virulence genes prfA, plcA, mpl, actA, and plcB increased more than 2-fold in BC adapted strains; however, lower than 2-fold changes in the expression of hemolysis-associated gene hly were observed. Our study suggests that BC adaptation could increase the expression of the Agr system and enhance biofilm formation, invasion, and virulence of L. monocytogenes, which brings about threats to food safety and public health. Therefore, effective measures should be taken to avoid the emergence of BC adapted strains of L. monocytogenes.

Phenyllactic acid application to control Listeria monocytogenes biofilms and its growth in milk and spiced beef.

Listeria monocytogenes, as a food-associated pathogen, is able to develop biofilms on different surfaces of food contact, which seriously threatens food safety. Phenyllactic acid (PLA) exhibits excellent inhibitory effects on many bacterial strains including L. monocytogenes. Our study aimed to investigate effects of PLA on L. monocytogenes biofilms and its growth in milk and on spiced beef. Biofilm biomass was measured by the microplate method and biofilm structure was observed by electron microscopy. Growth of L. monocytogenes in food samples was determined by colony counting. Results from the agar dilution method demonstrated that L. monocytogenes 10403S had a PLA minimum inhibitory concentration (MIC) value of 6 mg/ml. Sub-inhibitory concentrations of PLA could inhibit biofilm formation by reducing the secretion of exopolysaccharides and extracellular proteins in L. monocytogenes. PLA at concentrations above 1/2MIC could destroy mature biofilms of L. monocytogenes by decreasing the exopolysaccharides and extracellular proteins in the biofilm framework. Both swimming and swarming motilities of L. monocytogenes were inhibited by PLA. The hemolytic activity of L. monocytogenes was inactivated by PLA. However, the capacity to attach and invade Caco-2 cells was not affected by PLA. The results displayed that PLA had no effect on the expression of genes associated with motility, but reduced the expression level of the hly gene encoding Listeria hemolysin. When added to ultra-high temperature (UHT) whole and pasteurized milk, PLA at 3 mg/ml inhibited L. monocytogenes growth through 14 days of storage at 4 °C. PLA at concentrations ≥3 mg/ml significantly reduced L. monocytogenes counts on spiced beef samples during storage. PLA has potential as an alternative antimicrobial to control L. monocytogenes contamination and its biofilms in food industry.

Role of the VirSR-VirAB system in biofilm formation of Listeria monocytogenes EGD-e.

The ability of Listeria monocytogenes, an important foodborne pathogen, to form biofilms in food processing environments leads to increased opportunity for contamination of food products, which is a major concern for food safety. In this study, the role of a complex system composed of the VirSR two-component signal transduction system (TCS) and the ATP-binding cassette (ABC) transporter VirAB in biofilm formation of L. monocytogenes EGD-e was investigated. Biofilm formation was measured using the microplate assay with crystal violet staining, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), and attachment and swarming motility were compared between strain EGD-e and its isogenic deletion mutants. Additionally, the relative expression levels of genes associated with the early steps of biofilm development in the wild-type and mutant strains were also determined by RT-qPCR. Results from microplate assay, CLSM and SEM showed that VirR is not required for biofilm formation in L. monocytogenes EGD-e. A central finding of this study is that both VirAB and VirS are essential for biofilm formation and they could function as a whole in biofilm formation of L. monocytogenes EGD-e. The results also demonstrated that both VirAB and VirS are involved in attachment, but they are not associated with swarming motility. Results from RT-qPCR showed that flaA, motA and motB were downregulated in the mutant strains ΔvirAB and ΔvirS, which could be the possible reason for reduced attachment and biofilm formation in these mutants. This study provides a better understanding of the mechanisms involved in biofilm formation of L. monocytogenes, leading to improved processes to control this biofilm-forming foodborne pathogen.