Purpose: This study compares the pathogenicity of biofilm-forming and planktonic L. monocytogenes cells as to their virulence genes expression, adhesion, invasion and cytotoxicity to mammalian cells.
Methods: Firstly, the biofilm-forming capability of over 100 L. monocytogenes strains, including the food- and clinical-isolated cultures, was screened using crystal violate staining method. Secondly, 6 strains representing high and medium biofilm-forming groups were selected for further in vitro pathogenicity study. Two types of human intestinal epithelial cell lines, Caco-2 and HCT-8, were used for in vitro adhesion and invasion experiment. A B cell hybridoma, Ped-2E9, was applied to test the cytotoxicity of L. monocytogenes using flow cytometry. Then, reverse transcriptional PCR (qRT-PCR) was used to test the expression of genes related to flagellum biosynthesis (flaA), pathogenesis (lap and inlA) and quorum sensing (luxS).
Results: In general, L. monocytogenes food isolates showed higher biofilm forming phenotype than the clinical isolates. The results of in vitro experiments using Caco-2, HCT-8 and Ped-2E9 cells suggest that biofilm-forming L. monocytogenes cells grown in minimal nutrient medium had significantly decreased adhesion, invasion and cytotoxicity capabilities compared to the planktonic cells grown in rich or poor nutrient media. Furthermore, qRT-PCR results indicated that flaA gene was over expressed in the biofilm-forming cells compared to the planktonic cells. While luxS and virulence genes (lap and inlA) were underexpressed during biofilm formation and overexpressed in planktonic cells.
Significance: These results reveal that the virulence properties of L. monocytogenes diminish when the pathogen switches from planktonic cells to biofilm-forming cells.