Figure 4.

Effects of oritavancin on spx expression and oxacillin susceptibility of USA300 in mature biofilms. (a) The effect of oritavancin on spx expression was detected with spx promoter-GFP reporter assay: the USA300-Pspx strain was cultivated to mid-log phase (4 h, OD₆₀₀ ∼0.6), then oritavancin was added to a final concentration of 0.7 μM. The cell densities and GFP fluorescence intensities were monitored at different time points during cultivation. no antibiotic treated culture was used as a control. The spx expression is indicated with the intensity of GFP fluorescence normalized with OD₆₀₀ value. (b) Biofilms of the MRSA USA300 strain TCH1516 were grown in fluorodishes at 37°C for 24 h. Afterwards, the planktonic cells were removed and fresh TSB containing 0.1% DMSO (control), 32 mg/L oxacillin, 3.1 μM oritavancin, or 32 mg/L oxacillin plus 3.1 μM oritavancin was added and incubated at 37°C for another 24 h. The mature biofilms were stained with SYTO9 and propidium iodide (PI) and observed under a Leica TCS SP8 CLSM with a 63× 1.4-NA oil-immersion objective. The viable cells were stained with green fluorescence while the dead cells with red fluorescence. Images of three-dimensional biofilm structure were constructed using IMARIS 9.0 software and the thicknesses of the biofilms were shown. The fluorescence intensities of SYTO9 and PI were determined by using Image J software, and ratio of PI/Total (PI + SYTO9) were calculated.