As a control, bacteria were grown in AZD1480 cell line an equal volume of cell culturing medium. The plate was incubated at 5% CO2 and 37°C and the absorbance was measured in a microplate reader (Multiska Ascent, Thermo labsystems, Helsingfors, Finland) at 620 nm every 30 min for 6 h. The absorbance of PMN cells only was measured and subtracted from the absorbance of the co-incubated samples (bacteria + PMN). The relative growth inhibition (delta OD620) was calculated as absorbance of bacteria-(absorbance of bacteria + PMN).

The viability of the PMN was > 80% as determined by trypan blue exclusion test 6 h after bacterial stimulation. Transwell PMN migration assay A498 cells were seeded onto a inverted 3 μm pore size transwell insert (Falcon, BD Biosciences Pharmingen, San Diego, USA) for 3 h (at 5% CO2 and 37°C) to facilitate cell settling. After 3 h the inserts were placed in 6-well plates with fresh medium and the cells were cultured on the inserts for 2 weeks at 5% CO2 and 37°C. Medium was changed every second day. The cells were pre-stimulated

with the bacteria (MOI 10) for 4 h by adding the different Omipalisib order check details strains to the bottom wells. The PMN were prepared as described above and 106 PMN were added to the top well after the pre-stimulation. PMN cells were collected from the bottom well after 1 and 3 h and counted in a cell counter (TC10™ automated cell counter, Bio-Rad). Measurement of epithelial cytokine production An enzyme-linked immunosorbent assay (ELISA) was performed to measure the cytokine production of A498 cells stimulated with different

bacterial strains for 3 and 6 h. The cytokines IL-6 and IL-8 were measured using human IL-8 and IL-6 kits DOK2 (ELISA MAX™ Deluxe Sets, BioLegend, San Diego, CA, USA). Statistical analysis The variables were normally distributed and differences between groups were evaluated with the unpaired Student’s t-test or one-way ANOVA followed by Bonferroni test. Differences were considered statistically significant when p < 0.05. Data were presented as mean ± standard error of the mean (SEM), n = number of independent experiments. Results Selection and characterization of the UPEC strains The renal epithelial (A498) cells were stimulated with the different bacterial isolates for 6 h and the cell viability was assessed. Bacterial isolates that decreased the cell viability (> 20%) were not suitable for the in vitro infection study design and were excluded. Two ESBL-producing (2/8; 25%) and five non-ESBL-producing (5/11; 45%) isolates were excluded based on this criteria. Six ESBL-producing and six non-ESBL producing isolates remained for investigation. The characteristics of the different isolates included in the study are summarized in Table 1. All ESBL-producing isolates belonged to either the CTX-M-14 or CTX-M-15 enzyme type. The phylogenetic analysis showed that 50% of the susceptible strains belonged to the B2, 33% to the B1 and 17% to the D group.