CONTRIBUTION OF MUTATIONS IN GYRA AND PARC GENES TO FLUOROQUINOLONE RESISTANCE OF MUTANTS OF STREPTOCOCCUS-PNEUMONIAE OBTAINED IN-VIVO AND IN-VITRO

We have analyzed by gene amplification and sequencing mutations in the quinolone resistance-determining regions of the gyrA, gyrB, and parC genes of fluoroquinolone-resistant Streptococcus pneumoniae mutants ob tained during therapy or in vitro, Mutations leading to substitutions in ParC were detected in the two mutants obtained in vivo, BM4203-R (s ubstitution of a histidine for an aspartate at position 84 [Asp-84-->H is]; Staphylococcus aureus coordinates) and BM4204-R (Ser-80-->Phe), a nd in two mutants obtained in vitro (Ser-80-->Tyr). An additional muta nt obtained in vitro, BM4205-R3, displayed a higher level of fluoroqui nolone resistance and had a mutation in gyrA leading to a Ser-84-->Phe change, We could not detect any mutation in the three remaining mutan ts obtained in vitro, Total DNA from BM4203-R, BM4204-R, and BM4205-R3 was used to transform S. pneumoniae CP1000 by selection on fluoroquin olones. For the parC mutants, transformants with phenotypes indistingu ishable from those of the donors were obtained at frequencies (5 x 10( -3) to 8 x 10(-3)) compatible with monogenic transformation, By contra st, transformants were obtained at a low frequency (4 x 10(-5)), compa tible with the transformation of two independent genes, for the gyrA m utant. Resistant transformants of CP1000 were also obtained with an am plified fragment of parC from BM4203-R and BM4204-R but not with a gyr A fragment from BM4205-R3. All transformants had mutations identical t o those in the donors, These data strongly suggest that ParC is the pr imary target for fluoroquinolones in S. pneumoniae and that BM4205-R3 is resistant to higher levels of the drugs following the acquisition o f two mutations, including one in gyrA.