Cell cytotoxicity and viability assays A549 cells (cultured in either 24- or 96-well plates) were infected with K. pneumoniae strains (MOI 500:1

or 1000:1, 5 h). Lactate dehydrogenase (LDH) release was measured using a commercial kit (CytoTox 96, Promega). see more Per cent cytotoxicity was calculated as: (OD490 sample – OD490 medium)/(OD490 max – OD490 medium)*100. OD490 max was obtained with the provided lysis positive control. Measure of formazan production from reduction of MTS tetrazolium by metabolically active cells was performed using cells cultured in 96-well plates. Formazan production (% viability) was measured using a kit (CellTiter 96 AQueous One, Promega) and calculated as: OD490 sample/OD490 max*100. OD490 max was obtained from a monolayer of non-infected cells. Ethidium bromide is taken up by host cells when GSK2118436 cytoplasmic membrane integrity is lost, staining nuclei red when visualised by fluorescence microscopy. Cells were cultured on coverslips in 24-well plates and infected as described above (MOI 500:1, 5 h). 15 min before the end of the infection, culture medium was removed

and wells were washed with 1 ml PBS. Cells were stained for 10 min with 250 μl of 6 TM ethidium bromide prepared in PBS, washed three times with 1 ml PBS, fixed with 3.7% paraformaldehyde in PBS, and mounted for immunofluorescence analysis as described above. Cytotoxicity (red nuclei) was quantified by counting a minimum of 100 cells in three ACP-196 cost independent experiments. Mouse pneumonia model Overnight-grown bacteria were subcultured and grown to exponential phase. Bacteria were

centrifuged (2500 × g, 20 min, 22°C), resuspended in PBS and adjusted to 5 × 106 colony-forming units (c.f.u.)/ml. Five to seven-week-old female C57BI/6j mice were anaesthetized by i.p. injection with a mixture containing ketamine (100 mg/ml) and xylazine (10 mg/ml). 20 μl of bacterial suspension were inoculated intranasally in 4 × 5 μl aliquots. 48 or 72 h post-infection the mice were sacrificed by cervical DNA Methyltransferas inhibitor dislocation and trachea, spleen and liver were dissected, weighed and homogenized in 1 ml PBS. Serial dilutions of the homogenates in PBS were plated on LB agar to determine c.f.u. per gram of tissue. Statistics Statistical analyses were performed with Prism4 for PC (GraphPad Software) using the analysis of variance (ANOVA) or the two-sample t test or, when the requirements were not met, by the Mann-Whitney U test. P < 0.05 was considered statistically significant. Results K. pneumoniae induces a cytotoxic effect in lung epithelial cells A549 lung epithelial cells were infected with K. pneumoniae 52145 (52145), a highly capsulated strain (339 μg per 105 c.f.u.) for 5 h with different MOIs and the host actin cytoskeleton was stained.