parE and gyrA mutations in fluoroquinolone-resistant Streptococcus pneumoniae

In Gram-positive cocci, fluoroquinolone resistance is associated with mutational alterations in DNA gyrase and/or topoisomerase IV or with active efflux of the drugs. In Streptococcus pneumoniae, fluoroquinolone resistance is mainly due to substitutions in ParC (Ser-79→Phe or Tyr, Asp-83→Asn), ParE (Asp-435→Asn) or GyrA (Ser-81→Phe or Tyr, Glu-85→Lys or Gly).1,2 Therefore, detection of these substitutions is important for the understanding of the mechanisms of resistance to fluoro-quinolones and for epidemiological studies of transmission and spread of resistant strains. A PCR-RFLP assay has been developed by Pan et al.1 for detection of point mutations within the parC (Ser-79 codon) and gyrA (Ser-81 codon) genes of S. pneumoniae. Detection included amplification of the quinolone resistance-determining regions (QRDR) of the corres-ponding gene and digestion of the PCR product by restriction enzymes. In this study, we report an extension of this assay to detect mutations within the Ser-79 and Asp-83 codons of parC, the Asp-435 codon of parE and the Ser-81 and Glu-85 codons of gyrA genes in S. pneumoniae which lead to decreased susceptibility to fluoro-quinolones. S. pneumoniae CP1000 and 25 in vitro- or in vivo-generated resistant mutant derivatives, which were studied for mutations in the QRDR of the parC, parE and gyrA genes by sequencing of the PCR products, were used to validate the assay. A 366 bp parC and a 290 bp parE region encompassing the QRDR were amplified with already described primers.2 Similarly, a 183 bp gyrA region containing the QRDR was amplified using primer