Figure 4.

Bacterial survival in mouse blood and biofilm formation ability. (a) The ccs4 gene deletion had no effects on pneumococcal survival in mouse blood. Bacteria were incubated in heparinized mouse blood at 37°C for 1, 2, or 3 hours in a 5% CO₂ atmosphere. Survival rate was calculated by dividing the CFU value after the period of incubation by the CFU value of the original inoculum. Values are presented as the mean of 6 wells from one of 3 independent experiments. Vertical lines represent the mean + S.E. (b) Effect of ccs4 deletion on pneumococcal biofilm formation was assessed following incubation in THY at 37°C for 16 hours. Biofilm formed in 96-well microtiter plates was stained with Gram’s stain solution and absorbance was measured at 550 nm. The experiments were performed 3 times and data shown represent the mean of 6 wells from one of 3 independent experiments. Data are displayed as a box-whisker plot (min-[lower quartile–median-upper quartile]-max). Statistical differences between groups were analyzed using Mann-Whitney’s U-test. (c) Representative microscopic images of bacterial aggregation from WT strain-derived biofilm (left), and Δccs4 strain mutant-derived biofilm (right). Scale bars indicate 100 μm.