The OD was determined at 570nm and the results were expressed as CAT (U)/BBU [16]. 2.4. Vero Cell Cytotoxicity AssayThe cytotoxicity of culture supernatants was evaluated by Vero cells assay. Vero cells were grown at 37��C in Eagle’s minimal essential medium (MEM) supplemented with 10% (vol/vol) fetal calf serum, 100mg/liter penicillin, 200mg/liter streptomycin, and 2.2g/liter selleck chemicals llc NaHCO3 in an atmosphere of 5% CO2. The supernatant of each strain and culture conditions was centrifuged at 17,228��g, 10min at 4��C, filtered with 0.22��m membrane and 50��L of each one was inoculated in 96-well-plates containing 4 �� 104 freshly trypsinized Vero cells and incubated 48h at 37��C in a 5% CO2 atmosphere. The cell monolayers were fixed with 10% (v/v) formaldehyde and then stained with 0.2% (w/v) CV in PBS [6, 7].

The cytotoxic effects were evaluated after 24h by light microscopy. Then, for each sample, images from three randomly selected positions were obtained and analyzed using an Olympus Fluoview FV 1000. For image analysis, three investigators (N.A.V., I.A., and M.G.P.) evaluated the images independently in a blinded retrospective manner. Results are expressed as damage percentage (%) with respect to controls��SD LPS. E. coli EDL 933 (strain N�� 3) was used as positive control, and a strain Stx positive without cytotoxic effect was used as negative control (E. coli serotype O15:H21) [23].2.5. Statistical AnalysisAll experiments were performed in triplicate, and numerical data are presented as means with error bars representing standard deviations.

The data were statistically analyzed by using ANOVA followed by the Student-Newman-Keuls test for multiple comparisons. The differences between means were assessed with a *P versus TSB < 0.01 and #P versus thioglycollate medium < 0.01 being considered statistically significant. 3. Results3.1. Influence of Different Culture Conditions on Oxidant Metabolites and Antioxidant Defenses in BiofilmsA quantitative analysis of biofilm formation indicated that the three STEC strains showed ��weak biofilm producer�� (according to the scale described in Materials and Methods) biofilm formation in TSB (Figure 1(a)). When we studied the effect of adding 0.5% glucose to TSB on the production of biofilms, a significant increase in biofilm production ��moderate biofilm producer�� was seen in E. coli O157:H7 (strain N�� 1) (BBU = 1.

31 �� 0.02 to 3.23 �� 0.07) and a smaller but still significant increase in both E. coli O111 (strain N�� 2) (BBU = 1.52 �� 0.06 to 1.82 �� 0.06) and E. coli EDL 933 (strain N�� 3) (BBU = 1.50 �� 0.07 to 1.93 �� 0.05) (*Pversus TSB < 0.01). Biofilm formation was increased Cilengitide similarly with the addition of mannose to TSB (data not shown). Figure 1Quantification of biofilm formation of STEC strains by crystal violet (CV) staining expressed in biofilm biomass units (BBU): (a) in TSB; with addition of 0.