Development of a septicaemic murine model allowing selection of virulent mutants of Streptococcus pneumoniae

Genetic construction of virulence deficient mutant is a strategy to analyse virulence genes of Streptococcus pneumoniae and was used to virulence fact ors as capsule, pneumolysin, autolysin and PspA. We perform a model allowin g the in vivo positive selection of virulent S. pneumoniae mutants. Mice wh ich are the most susceptible animals to pneumococcal infection, offer the b est model for screening virulent S. pneumoniae. Indeed after intraperitonea l injection of bacterial mix which was composed to a lot of avirulent bacte ria (6 log(10) CFU per mouse) (V1015 strain, DL50 = 7.05) and Pew virulent pneumococci (1 to 2 log(10) CFU per mouse) (P4241 strain, DL50 < 1), mice c leared all avirulent bacteria but not virulent pneumococci. Thus, mice dead in 3 to 4 days with septicaemia and positive hemoculture contained only vi rulent strain. This model was validated by in vivo selection of a virulent mutant (V1042, DL50 = 4.1) which was obtained after transformation of aviru lent strain V1015 with the genomic fragment of virulent strain P4241. Our m odel of screening was the only one allowing detection of virulent S. pneumo niae mutants. This new genetic strategy which consisted in gene addition an d used mouse as selection agent, could be used to discover new virulence ge nes required to in vivo bacterial development.