Figure 3.

Pneumococci after intranasal challenge of mice and treatment with a low dose of azithromycin. a) Animals (n = 5) were intranasally challenged with 1 × 10⁶ CFU of D39 pneumococci and after 30 min treated intraperitoneally with either a sub-inhibitory 1.128 μg/g BW azithromycin (open circle) or saline (closed circle). The antibiotic dose was chosen to be approximately 10x below the MIC in the serum. Mice were sampled at 6, 12, 24 or 48 hours after infection and bacterial counts were determined for lungs (blue), spleen (green) and blood (red). The limit of detection is shown as dotted line for the spleen and lung (green) and blood (red) samples. b) Signs of disease in mice were assessed and scored as 0 Normal, 1 Hunched, 2 Pilo-erection, 3 Loss of activity, 4 Lethargic, which was the severity limit of the experiment (filled circles: saline, open circles: azithromycin). c) Concentrations of azithromycin were determined by a bioassay of five homogenised spleens (green) and serum (red) of azithromycin-treated mice (values in Table S3). d) Effect of azithromycin treatment (same dose as panel a) on numbers of bacteria in the blood (red) and the spleen (green) after a separate intravenous challenge control experiment (n = 5; open circle azithromycin; closed circle saline). Statistical significance was determined by ANOVA (**, p<0.01; ***, p<0.001; ****, p<0.0001; ns not significant, p>0.05). e) 3D reconstruction of confocal image of CD169+ macrophages in the spleen at 24 h of a saline-treated mouse (CD169, red; bacteria, green; nuclei, blue). The scale bar represents 15µm.