FIG 3.

Altered virulence and growth characteristics of D39 stocks from different laboratories. (A) Hemolytic activity of D39 stocks. Bacterial lysates diluted to a total protein concentration of 5 μg were incubated with sheep red blood cells, and blood cell lysis was then determined by the absorbance at 540 nm. Data are means ± standard deviations (n = 5 biological replicates per group). (B) Pneumolysin concentration expressed as nanograms per 10⁷ CFU. Bacterial cell pellets were lysed and used for the detection of pneumolysin protein by ELISAs. Data are means ± standard deviations (n = 3 biological replicates per group). (C) Growth of D39 stocks in brain heart infusion broth. The optical density at 500 nm was measured every 15 min for 12 h. Only strain H1 demonstrated significantly different growth characteristics compared to NCTC7466 (P < 0.01 between 315 and 615 min by two-way analysis of variance [ANOVA] with Dunnett’s multiple-comparison test). Data are pooled from three independent experiments, for each of which the mean from two technical replicates per strain was taken. (D) The pneumococcal cell density at 10 h was determined by serial dilution onto blood agar (n = 4 biological replicates per group). P values in panels A, B, and D were determined by one-way ANOVA with Dunnett’s multiple-comparison test versus NCTC7466.