1 +/−0.1% of cell lysis after 24 h of infection. P. ABT-263 cell line mosselii MFY161 exhibited a cytotoxic activity reaching 64.5 +/−0.1% of lysis and the cytotoxic activity of P. aeruginosa PAO1 was higher with 85.6 +/−0.2% of lysis. Enumeration of P. mosselii ATCC BAA-99 (5 × 108 CFU.mL-1), P. mosselii MFY161 (4.8 × 108 CFU.mL-1) and P. aeruginosa PAO1 (4.9 × 108 CFU.mL-1), at the end of the infection period showed that higher cytotoxicity was not due to bacterial overgrowth. Figure 1 Cytotoxic effects of P. mosselii ATCC BAA-99, P. mosselii

MFY161 and P. aeruginosa PAO1 on Caco-2/TC7 cells. Cytotoxicity was determined by LDH release assay after 24 h of infection. Results were calculated as the mean values (+/−SEM) of three independent experiments. *** P < 0.001 versus uninfected Caco-2/TC7 cells, 3 Methyladenine ∆∆∆ P < 0.001 versus P. aeruginosa PAO1, ∆∆ P < 0.01 versus P. aeruginosa PAO1, •• P < 0.01 versus P. mosselii ATCC BAA-99. Bacterial invasion assay The capacity of P. mosselii ATCC BAA-99 and

MFY161 to enter Caco-2/TC7 cells has been investigated using the gentamicin exclusion test Linsitinib purchase (Figure 2). The results show that the two P. mosselii strains studied can have an invasive behavior with 0.5 +/−0.2 × 105 and 0.2 +/−0.2 × 105 CFU.mL-1 detected intracellularly for P. mosselii ATCC BAA-99 and MFY161, respectively. The invasive capacity of P. aeruginosa PAO1 was significantly higher with 1.4 +/−0.1 × 105 CFU.mL-1 that entered Caco-2/TC7 cells. Figure 2 Invasive P-type ATPase capacity of P. mosselii ATCC BAA-99, P. mosselii MFY161 and P. aeruginosa PAO1. 4 h after infection of Caco-2/TC7 cells with the bacteria, extracellular germs were killed by gentamicin. Cells were lysed and the intracellular bacteria were enumerated by plating onto nutrient agar medium. Results were calculated as the mean values (+/−SEM) of three independent experiments. * P < 0.05 versus

P. mosselii ATCC BAA-99 and P. mosselii MFY161, NS not significant between P. mosselii ATCC BAA-99 and P. mosselii MFY161. Quantification of IL-6, IL-8 and HBD-2 secretion The bacterial proinflammatory effect of P. mosselii ATCC BAA-99, P. mosselii MFY161 and P. aeruginosa PAO1 was assessed by measuring IL-6 and IL-8 secretion in Caco-2/TC7 after 24 h of infection. The results show that the two strains of P. mosselii studied did not induce significant stimulation of IL-6 (Figure 3A) and IL-8 (Figure 3B) secretion in Caco-2/TC7 compared to uninfected cells. On the contrary, the infection of Caco-2/TC7 cells with P. aeruginosa PAO1 led to a major secretion of IL-8 with 92 +/−13 pg.mL-1 (Figure 3B). Figure 3 Proinflammatory effects of P. mosselii ATCC BAA-99, P. mosselii MFY161 and P. aeruginosa PAO1 on Caco-2/TC7 cells. IL-6 and IL-8 cytokines, and HBD-2 were measured in Caco-2/TC7 cells supernatant after 24 h of infection. Results were calculated as the mean values (+/−SEM) of three independent experiments. *** P < 0.001 versus uninfected Caco-2/TC7 cells, ** P < 0.