(A) Penicillin resistance was generated via de novo mutation in T4-derived pneumococcal cells by continuous passaging of planktonic cells in liquid culture containing gradually increasing subinhibitory concentrations of penicillin.
(B) Penicillin MICs of WT pneumococcal strain T4 and its derivative experimentally evolved pneumococcal populations. Data are represented as mean MIC ± SD (n = 3).
(C) Blood titers at 24 h post-infection with WT T4 or a derivative strain.
(D) Mouse survival followed 10 days after intranasal challenge with experimentally evolved versus WT pneumococci. Seven-week-old BALB/c mice were intranasally infected with 106 CFUs of T4 or an experimentally evolved derivative population (n = 10 mice/groups). p values of 0.05 or less as determined by Mann-Whitney U test were considered significant.