The E. coli Selleck ATM Kinase Inhibitor strain CFT073 and the culture medium supplemented with 1% (v/v) glucose were used as positive and negative controls, respectively. Assays were performed in quintuplicate and repeated at least 4 times. The cut-off optical density (ODc) was defined as three standard deviations above the mean OD of the negative control (culture medium), and strains were classified

as non-adherent (OD ≤ ODc), weakly adherent (ODc < OD ≤ 2 × ODc), moderately adherent (2 × ODc < OD ≤ 4 × ODc), or strongly adherent (OD > 4 × ODc). The A-1210477 ultrastructural analysis of biofilm was performed by a Field Emission Scanning Electron Microscope (FESEM) (Zeiss, Germany). Briefly, adjusted inocula (200 μl, 0.5 McF) of each strain diluted with 1.8 ml of fresh LB supplemented with 1% (v/v) glucose were added to 24-well plates with round

glass coverslips (1 cm diameter) put into each well and incubated at 37°C for 24 h. The content of each well was removed and the round coverslips were washed with PBS (1%) twice. Biofilms grown on coverslips were fixed with 2,5% glutaraldehyde in Na-cacodylate 0,1 M (pH 7.4) buffer solution (AppliChem, Germany) for 2 h at room temperature. Following three washing steps with the same buffer solution, samples were dehydrated through graded ethanol (30°, 50°, MCC950 datasheet 70°, 85°, 95°, 100°) and dried with hexamethyldisilazane (Alfa Aesar, USA) for 1 h30′. Samples were air dried overnight and coated by sputtering with a gold target [19]. Results and discussion Diversity among clonal groups of E. coli phylogroup D Isolates belonging to the three analysed STs exhibited inter and intraclonal variability regarding the VF profile and the ability Inositol monophosphatase 1 to form biofilm. On the basis of their virulence scores, all ST69 (n = 13/13; median = 14/range = 9-15) and all ST393 (n = 11/11; median = 14/range = 8-15), and only sporadic ST405 (n = 2/11; median = 6/range = 2-14) isolates were classified as ExPEC (Table 2). While most ST69 and ST393 carried pap alleles (papA, papC, papEF, papG II), iha, kpsMTII-K5 and ompT, ST405

isolates frequently contained fyuA, malX and traT, suggesting the presence of different genomic islands among E. coli phylogroup D isolates. Table 2 Virulence gene profiles of phylogenetic group D E . coli clonal groups Virulence genesa N° of isolates (%) P valuea   ST69 (n = 13) ST393 (n = 11) ST405 (n = 11) ST69 vs ST393 ST69 vs ST405 ST393 vs ST405 Adhesins           fimH 13 (100%) 11 (92%) 9 (82%) 0.480 0.199 0.590 papA 11 (85%) 8 (67%) 0 (0%) 0.378 0.000 0.001 papC 12 (92%) 10 (83%) 0 (0%) 0.593 0.000 0.000 papEF 12 (92%) 9 (75%) 2(18%) 0.322 0.001 0.012 papG allele I 0 (0%) 1 (8%) 0 (0%) 0.480 – 1.000 papG allele II 9 (69%) 10 (83%) 0 (0%) 0.645 0.001 0.000 papG allele III 9 (69%) 2 (17%) 1 (9%) 0.015 0.005 1.000 bmaE 2 (15%) 0 (0%) 0 (0%) 0.480 0.482 – gafD 2 (15%) 0 (0%) 0 (0%) 0.480 0.482 – iha 10 (77%) 10 (83%) 2 (18%) 1.