Cells were incubated for 2 h at 37°C to allow for adhesion and internalisation of the bacteria and then washed twice with DMEM to remove unbound bacteria.
For the adhesion assay, #ITF2357 clinical trial randurls[1|1|,|CHEM1|]# cells were analysed using osmotic shock in pure water and extensively pipetted to achieve full release of cell-associated bacteria. For the invasion assay, infected cells were further incubated for 1 h in culture medium containing 200 mg/L gentamicin to kill extracellular bacteria but not internalised bacteria. Cells were then washed twice in DMEM and analysed as described above to release internalised bacteria. For both the adhesion and invasion assays, viable bacteria in cell lysates were enumerated by plate counting on agar. The number of adherent bacteria was calculated by subtracting the number of internalised bacteria from the number of total cell-associated bacteria. The results were expressed as the mean ± standard deviation of the percentage of recovered
internalised or adherent bacteria with respect to inoculated bacteria, derived from four independent experiments performed in duplicate. Statistical analysis The statistical analyses were based on the use of one-way ANOVA followed by the a posteriori Dunnett’s test. Correlation analysis was performed using Spearman’s rank correlation coefficient. The level of statistical significance was set at 0.05. The tests were carried out with SPSS for Windows version 12.0 software. Results Susceptibility Selleck GDC 0449 to antibiotics of bacterial strains cultured in BHI We first examined the influence of the experimental conditions on the MIC values of tested strains. The oxacillin, gentamicin, vancomycin, clindamycin, linezolid, moxifloxacin and rifampin MICs were determined using CLSI recommendations
and compared to those obtained with BHI inoculated with 5 × 105 CFU/mL (Table 3). MICs in BHI were of the same magnitude as those obtained in Mueller-Hinton, therefore we used BHI medium for the rest of this study. Table 3 MICs of antibiotics tested in BHI medium against 6 selected S. aureus strains MIC (mg/L) in Celecoxib BHI mediuma Antibiotic 8325-4 DU5883 ST2008 1028 ST2008 0563 HT2000 0594 HT2001 0390 Oxacilllin 0.25 0.25 0.25 0.25 0.25 0.25 Gentamicin 1 1 1 1 1 1 Vancomycin 2 2 1 1 2 2 Clindamycin 0.15 > 128b 0.30 0.30 0.30 0.30 Linezolid 1 1 1 1 1 1 Rifampicin 0.006 0.006 0.006 0.006 0.006 0.006 Moxifloxacin 0.12 0.12 0.12 0.12 0.12 0.12 aMICs were determined with a standard microdilution method recommended by the CSLI in BHI medium inoculated with 5 × 105 CFU/mL bDU5883 strain is resistant to clindamycin as it harbours the ermB gene  Effect of antibiotics on S. aureus adhesion to fibronectin-coated microplates We determined the influence of antibiotics on the adhesion of S. aureus to fibronectin using a fibronectin-coated microplate adhesion assay.