FIG. 5.

Oxacillin or vancomycin had no visible effect on S. aureus biofilms in PBS or bacterial media after incubation for 24 h. Polystyrene 96-well tissue culture wells were inoculated with, per ml, ∼10⁸ CFU of S. aureus ATCC 35556 (MIC of lysostaphin, 0.004 μg/ml; MIC of oxacillin, 0.125 μg/ml; and MIC of vancomycin, 1 μg/ml). Following 24 h of biofilm formation, wells were washed twice with PBS and then treated for 24 h in either PBS (A) (this panel shows one of the plates from the experiment reported in Fig. 4) or TSB plus 0.25% glucose bacterial medium (B) with either no added antibiotic (first column of wells in each plate) or serial twofold dilutions of lysostaphin (first two rows; 0.8 to 200 μg/ml), oxacillin (third and fourth rows; 1.6 μg/ml to 400 μg/ml), or vancomycin (bottom two rows; 3.2 to 800 μg/ml) as indicated on the figure. Following treatment, the wells were washed with PBS and then stained with safranin. The darkly staining wells indicate the presence of biofilm following treatment. Lysostaphin in PBS cleared the biofilm at 0.8 μg/ml (A), while lysostaphin in TSB plus 0.25% glucose cleared the biofilm at 12.5 μg/ml (B).