As a control, bacteria were grown in Panobinostat clinical trial an equal volume of cell culturing medium. The plate was incubated at 5% CO2 and 37°C and the GW4869 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 AMN-107 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 Glycogen branching enzyme (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.