HvgA enables GBS crossing of the BBBs. (a and b) Groups of 15–21 d old BALB/c mice (n > 8) were inoculated orally with 109 (a) or 5 × 108 CFUs (b) WT ST-17 or ΔhvgA mutant strain and brain invasion was assessed 12-h after inoculation by CFUs enumeration. (c and d) Groups of 3–4-wk-old BALB/c mice (n > 8) were infected by repeated i.v. injections of 5 × 106 CFUs every 12 h and sacrificed at 48 h, and bacteria enumerated in blood (c) and brain (d). Data shown are the results of three independent experiments. (e) Real-time imaging of CNS infection. Groups of BALB/c mice (n = 4) were infected i.v. with 108 CFUs every 12 h. Bioluminescence was assessed every 12 h (the images shown correspond to the 36 h after injection time point). (f–h) Histopathological examination (Gram staining) of CNS tissue samples from WT-ST-17 infected mice reveals Gram-positive cocci in the meninges (f), in the choroid plexuses (g), in brain microvessel endothelial cells and the surrounding brain parenchyma (h). (i–k) Immunofluorescence staining of brain sections obtained from animals infected with the WT ST-17 in d, showing infection of meninges (i), submeningeal space (j), and brain microvessel endothelial cells (k). Bars: (f–h) 25 µm; (i–5) 20 µm. All experiments were repeated at least three times. NS, not significant. Asterisks indicate significant differences as assessed by the Mann-Whitney test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
