An extended PCR-RFLP assay for detection of parC, parE and gyrA mutations in fluoroquinolone-resistant Streptococcus pneumoniae.

Sir, 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 fluoroquinolones 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 corresponding 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 fluoroquinolones. S. pneumoniae CP1000 and 25 in vitroor 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 primers PnGyrA-F 5′-TTCACCGTCGCATTCTCTACGGA-3′ and PnGyrA-R 5′-CATCTACAAGCATGTAACGGTAGCTCCACCATTGAGCATACGGACCATGTC-3′. Primer PnGyrA-R was adjacent to the Glu-85 codon and differed by one base (underlined) from the gene sequence to generate a MboII recognition site. Mutations within parC were detected using HinfI (Ser-79 codon) and LweI (Asp-83 codon), those within gyrA by HinfI (Ser-81 codon) and MboII (Glu-85 codon) and that (Asp-435 codon) in parE with HinfI. Restricted DNA was analysed by electrophoresis in a 3% agarose gel. The 366 bp parC product from susceptible CP1000 contained two HinfI and one LweI recognition sites generating fragments of 183, 127 and 56 bp, and of 224 and 142 bp, respectively. Loss of a HinfI site in resistant isolates following mutations at the Ser-79 codon generated two 183 bp fragments that ran as a doublet. Mutations at the Asp-83 codon suppressed the LweI site and a 366 bp fragment was observed. The 290 bp parE product from the susceptible strain contained two HinfI sites generating 166, 87 and 37 bp fragments. Loss of one of the HinfI sites in the resistant isolates following mutations at the Asp-435 codon generated fragments of 203 and 87 bp. The 183 bp gyrA product from parental CP1000 contained a natural HinfI site (Ser-81 codon) and an artificially created MboII site generating fragments of 113 and 70 bp, and 141 and 42 bp, respectively. Loss of the HinfI site in resistant isolates due to mutations at the Ser-81 codon led to a 183 bp fragment. Mutations at the Glu-85 codon were associated with the loss of the MboII site leading also to a 183 bp fragment. Mutations resulting in amino acid changes at Ser-81 and/or Glu-85 in GyrA are often associated with decreased susceptibility to quinolones in S. pneumoniae.1 The mutated site in the Ser-81 codon is part of a naturally occurring HinfI restriction site; thus mutations are detected when HinfI fails to digest the PCR product, as analysed by electrophoresis in agarose gel.1 However, mutations within the Glu-85 codon do not generate any restriction site. To detect mutations in the corresponding codon, we introduced a base substitution near the mutated locus to create an artificial MboII cleavage site using the primer-specified restriction site modification method.3 The DNA fragment amplified from the wild-type gyrA gene had two naturally occurring HinfI restriction sites in the Ser-81 codon and an artificially created MboII cleavage site in the Glu-85 codon. Mutations in the Glu-85 codon can thus be detected by digesting the amplified DNA by MboII. This modified PCR-RFLP method has been used successfully to screen for mutations in the gyrA gene of Neisseria gonorrhoeae4 and Escherichia coli.5 The extended PCR-RFLP assay described here is simple, rapid and can be carried out in a diagnostic laboratory as a routine assay. However, this method has several limitations including the inability to assess the type of nucleotide substitution or to screen for mutations occurring at other positions in the gene. It has been shown that there is little correlation between specific mutations in type II topoisomerase genes and phenotypic susceptibility of the host to the most frequently used fluoroquinolones.6 Nevertheless, the present study provides data suggesting that this assay could be a useful screening tool for mutations and to facilitate epidemiological studies of decreased susceptibility to fluoroquinolones in clinical isolates of S. pneumoniae.