FIG 3.

Synergistic hemolysis is partially dependent on extracellular pH and can be overridden by its modulation. (A) The pH of monomicrobial (SA or CA) and polymicrobial (CA+SA) cultures was measured after 16 h of incubation at 37°C. (B) Assays were conducted similarly as in panel A, except that the pH of TSB-g was buffered as indicated. Activation of staphylococcal agr was assessed using a GFP reporter assay. (C) Aliquots from each culture were taken at the endpoint to measure microbial counts by plating on selective media. Blue bars indicate C. albicans counts, and yellow bars indicate S. aureus counts. Solid color bars depict counts from monomicrobial (mono) cultures, while hatched bars depict counts from polymicrobial (poly) cultures. (D) Buffered (pH 7.0) spent CA and SA culture supernatants or fresh media were added to S. aureus(pDB22) initially grown at pH 5.5. Fluorescence (488 nm) was captured kinetically to assess agr activation. All data are representative of three independent repeats and expressed as the means ± SEM. Data were assessed for significance using one-way ANOVA and Dunnet’s posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001.