Biofilms have been shown to become increasingly resistant to repeated doses of antibiotics or nonspecific oxidizing biocides [16], but the chronic myelocytic leukemia basis for this apparent acquired resistance is currently unknown. We previously described a model system for examining the oxidative stress generated in a biofilm [13] but further studies are still necessary to determine the consequences of the imbalance between the production of oxidants and the levels of antioxidant defenses in the biofilms. The objective of the present study was to determine the relationships among STEC biofilm formation, cellular stress, and release of Stx under different culture conditions. To our knowledge, it is the first study that has attempted to correlate this biofilm formation with the disturbance in the prooxidant-antioxidant balance and its effect on the production and release of Stx.

The study shows that the alteration of biofilm environment can be suitable for release of Stx and it could contribute to the understanding of the pathogenesis of infection by this pathogen.2. Material and Methods2.1. Bacterial Strains and Culture ConditionsThe biofilm formation of E. coli O157:H7 (strain N�� 1-Stx1 and Stx2-) and E. coli O111:H-(strain N�� 2-Stx1-) clinical isolates (these strains produce Shiga-toxin and they were associated with HUS) and the reference strain E. coli EDL 933 (strain N�� 3-Stx1 and Stx2-) were studied. Clinical isolates were kindly provided by the Microbiology Laboratory of the Pediatric Hospital of C��rdoba, Provincia de C��rdoba, Argentina [14, 17].

Stock cultures were preserved at ?80��C using glycerol 15% (v/v) as the cryoprotectant, and the E. coli strains were grown in tryptic soy broth (TSB) at 37��C for 18h. 2.2. Quantification of Biofilm Assay Using a Microtiter Plate Assay The assay for the biofilm formation used in this study was adapted from the method of O’Toole and Kolter [18], which is based on the ability of bacteria to form biofilm on solid surfaces and it uses crystal violet (CV) to stain biofilms. In brief, 200��L of dilution 1/10 of overnight culture in TSB was added in each well of flat-bottomed microtiter plates (96-well, Greiner Bio-One, Germany), at 37��C for 24h without shaking. After incubation, the supernatant was separated and the flat-bottomed microtiter plate was washed twice with phosphate buffer solution (PBS) pH 7.2.

Diverse culture conditions were assayed using TSB alone, with added glucose (0.5%) or mannose (0.5%) [13, 19]. The influence of the reduction conditions was assayed in thioglycollate broth, and the microaerobic conditions were also studied [13]. TSB or thioglycollate was used as negative controls to obtain a background value. H2O2 is considered a AV-951 major endogenous source of oxidative stress [20]. Influence of oxidative stress induced by exogenous application of H2O2 (Merck) was assayed in concentrations ranging from 2.