Therefore, the diarrhea-isolated EAEC strain 340-1 and the prototype selleck chemicals EAEC strain 042 were chosen in order to continue the mixed infection assays employing quantitative analyses. As verified in the preliminary tests, the preinfection of HeLa cells with EACF strain 205 increased the bacterial adherence when Selleck LY2603618 followed by coinfection with EAEC strains 340-1 or 042 (Figure 2A). In contrast, preinfection with control-isolated C. freundii strain 047 did not cause any increment of bacterial adhesion. Figure
2 Mixed infection assays. A- Qualitative assay. Aggregative C. freundii (EACF) strain 205 improves bacterial adhesion when in combination with typical EAEC strains. B- Quantitative mixed infection assay. Adherence to HeLa cells
displayed by EACF 205 and EAEC strains in mixed infections assays was quantified using the counting of colony-forming units (CFU), and was compared with adhesion displayed by the monocultures. EAEC strains showed antagonistic behaviors when in presence of EACF 205. a denotes P < 0.05 for comparison of 2 groups; b and c P < 0.001. Statistical analyses: independent-sample T test. MK-0457 concentration To exclude the possibility that the increased adhesion was an unspecific synergic effect triggered by any pair of aggregative strains, coinfection assays were performed with several pairs of EAEC strains (EAEC 340-1 and EAEC 042; EAEC 205-1 and EAEC 042; EAEC 340-1 and EAEC 205-1). No increment in bacterial adhesion was observed using any strain combination. In order to determine what species accounted for the increased adhesion, quantitative mixed infection assays were DCLK1 conducted and the colony forming units (CFU) were counted (Figure 2B). Assays showed that EAEC strains 340-1 and 042
displayed antagonistic behaviors when HeLa cells were preinfected with EACF strain 205. Regarding EAEC 340-1, preinfection with EACF 205 induced a 10-fold increase in the adherence of strain 340-1 when compared with the single infection (P < 0.001). By contrast, preinfection with EACF 205 decreased adhesion of the EAEC strain 042 at 43.5% (P < 0.05). The overall increased adhesion displayed by coinfection of EACF 205 plus EAEC 042 was supported by the 2.8-fold increased adherence of the EACF 205 (P < 0.001). Search for biochemical signaling The role of inter-specific chemical signals in the increase of bacterial adherence was evaluated using permeable inserts that allow the division of culture-plate wells into two diffusion chambers. Thus, DMEM media were pre-conditioned inoculating the upper chamber with bacterial cultures, and then HeLa cells, in the lower chamber, were infected in order to test the bacterial adherence. Media pre-conditioned by EACF 205 or by EAEC strains did not induce changes in the adhesion developed by EAEC 340-1, EAEC 042 or EACF 205. Such results indicated that the increase in adherence was not triggered by chemical signaling.