Dermal fibroblast cells interactions with single and triple bacterial-species biofilms.

Polymicrobial biofilm leads to wound healing delay. We set up an in vitro co-culture model of single- and triple-species biofilms of Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis with dermal fibroblast to assess the fibroblast response against to the different biofilms. Scratch and viability assays and biofilm cell quantifications were performed by WST-1, CLSM and plating method, respectively. Quorum sensing-related gene expression levels in P. aeruginosa and E. faecalis were analysed by reverse-transcriptase PCR. The immune responses of cells against S. aureus, P. aeruginosa and E. faecalis biofilms were measured by cytokine and matrix metalloproteinase analyzes. The influence of biofilm soluble factors on fibroblasts was also determined. After 24 h, triple-species biofilm cells caused the removal of the fibroblasts from the surfaces indicating the negative synergistic effect of three species. After co-cultures, twenty-five cytokines were significantly increased in fibroblast cells compared to control. Compared to other strains, the most important cytokine, chemokine and growth factors increased was observed in P. aeruginosa co-cultures with fibroblast. While the expressions of fsrB and gelE genes were significantly upregulated in E. faecalis biofilm cells cultured with fibroblast cells, no significant difference was observed in P. aeruginosa. The wound healing and cell growth of fibroblasts were disrupted more aggressively in the presence of P. aeruginosa and triple-species biofilm cells. P. aeruginosa generally induced a stronger immune response in the fibroblasts than E. faecalis and S. aureus.