001) (Figures 5A and 5C). In contrast, the mixed biofilm developed by EACF 205 and EAEC 17-2 (traA-negative strain) URMC-099 (OD 0.431 ± 0.084) did not display a statistically significant increase when compared with the EAEC 17-2 single biofilm (OD 0.383 ± 0.079) (P = 0.237) (Figures 5A and 5C). Figure 5 Biofilm formation on glass coverslips. A- Micrographs showing the upper-facing side of the glass coverslips. Selleck NSC 683864 biofilms formed by EACF 205 or by EAEC strains were compared with mixed biofilms produced by cocultures of EACF 205 and EAEC strains. EAEC genotype

denotes the specific combination of EAEC markers hosted by E. coli strains. Enhanced biofilms were formed by the coculture of EACF 205 and traA-positive EAEC strains. B- Micrographs showing the down-facing side of the glass coverslips. Enhanced biofilms formed by the coculture of EACF 205 and traA-positive EAEC strains indicating an active processes rather than a mere fate following the bacterial settling. C- Quantitative assays. a, b, c, d and e denote P < 0.001 for comparison of 2 groups; f P < 0.05. Statistical analyses: independent-sample T test. Zinc effect on single and mixed biofilms Single and mixed biofilm assays were performed in order to evaluate the impact of zinc, and consequently the role of

putative F pili, on biofilm formation (Figure 5C). Zinc at a concentration of 0.25 mM (12-fold lower GSK458 than zinc MIC – minimum inhibitory concentration) reduced the single

biofilm formation by EAEC strain 205-1 by 23% (P = 0.038) (Figure 5C). In the case of EAEC strains 340-1 and 17-2 no reduction in single biofilms was noted. In contrast, the single biofilm formed by EACF 205 displayed a 3-fold increase when zinc was present (P < 0.001) (Figure 5C). Focusing on the traA-positive EAEC strains, these results indicate that putative F pili assume variable relevance in the formation of single biofilms. The impact of zinc on mixed biofilm developed by cocultures of EACF 205 and EAEC strains was also evaluated. Zinc significantly reduced (P < 0.001) EACF-205 mixed biofilms formed by EAEC 205-1 (59%) or by EAEC 340-1 (45%) which displayed Pazopanib in vitro in these conditions similar levels to those reached by EACF 205 single biofilms (Figure 5C). As expected, zinc treatment did not impact the mixed biofilm produced by EACF 205 and EAEC 17-2 (traA-negative strain) endorsing the conclusion that this biofilm was formed in the absence of putative F pili. Taken together, these results indicated that putative F pili engaged EAEC strains in mixed biofilm formation when EACF was present. SEM analyses of biofilms SEM micrographs showed that EACF-205 biofilms occurred in the absence of any extracellular appendage (Figure 1E). By contrast, biofilms formed by EAEC strains 340-1 or 205-1 were mediated by thick pili that emanated from bacteria and regularly attached to the abiotic surface (Figure 6A).