FIG 2.

Eradication of pneumococcal colonization with DoC using a model of human pharyngeal colonization. Polarized human pharyngeal Detroit 562 cells were immobilized with 2% paraformaldehyde and then infected with S. pneumoniae strain GA47281 (∼5.15 × 10⁸ CFU/ml). Infected cells were incubated for 4 h, and planktonic cells were removed. (A and B) These S. pneumoniae-colonized human pharyngeal cells were left untreated (control) or (A) treated with different dosages of deoxycholic acid (DoC) and incubated for an additional 2 h period or (B) treated with DoC (0.5 mg/ml) and incubated for the indicated time. At the end of the incubation, the cultures were serially diluted and plated onto blood agar plates to obtain the density (CFU/ml). In panels A and B, error bars represent the standard errors of the means calculated using data from at least three independent experiments. *, P < 0.05 compared to the untreated control. LOD, limit of detection. (C and D) S. pneumoniae-colonized human pharyngeal cells (infected as described above) were left untreated or treated with DoC (0.5 mg/ml) and incubated for 2 h. (C and D) Preparations were fixed, and S. pneumoniae was stained with (C) an anti-S19-Alexa-555 labeled antibody (red) and the DNA was stained with TOPRO3 (green) or with (D) an anti-S4-Alexa-488 labeled antibody (green); cell membranes were labeled with WGA (red), and DNA was labeled with DAPI (blue). Preparations were analyzed with a confocal microscope. Panels show z-projections of z-stacks obtained from xy optical sections. Lower bottom of panel C shows a 3D digital reconstruction. The merge of the channels is shown in each panel. Arrows point out extracellular S. pneumoniae bacteria.