gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae

Abstract

Fluoroquinolone resistance (FQ-R) in clinical isolates of Enterobacteriaceae species has been reported with increasing frequency in recent years. Two mechanisms of FQ-R have been identified in gram-negative organisms: mutations in DNA gyrase and reduced intracellular drug accumulation. A single point mutation in gyrA has been shown to reduce susceptibility to fluoroquinolones. To determine the extent of gyrA mutations associated with FQ-R in enteric bacteria, one set of oligonucleotide primers was selected from conserved sequences in the flanking regions of the quinolone resistance-determining regions (QRDR) of Escherichia coli and Klebsiella pneumoniae. This set of primers was used to amplify and sequence the QRDRs from 8 Enterobacteriaceae type strains and 60 fluoroquinolone-resistant clinical isolates of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, E. coli, K. pneumoniae, Klebsiella oxytoca, Providencia stuartii, and Serratia marcescens. Although similarity of the nucleotide sequences of seven species ranged from 80.8 to 93.3%, when compared with that of E. coli, the amino acid sequences of the gyrA QRDR were highly conserved. Conservative amino acid substitutions were detected in the QRDRs of the susceptible type strains of C. freundii, E. aerogenes, K. oxytoca (Ser-83 to Thr), and P. stuartii (Asp-87 to Glu). Strains with ciprofloxacin MICs of >2 microg/ml expressed amino acid substitutions primarily at the Gly-81, Ser-83, or Asp-87 position. Fluoroquinolone MICs varied significantly for strains exhibiting identical gyrA mutations, indicating that alterations outside gyrA contribute to resistance. The type and position of amino acid alterations also differed among these six genera. High-level FQ-R frequently was associated with single gyrA mutations in all species of Enterobacteriaceae in this study except E. coli.

FIG. 1

PCR amplification of the gyrA QRDR sequences from type strains of Enterobacteriaceae species. (A) Amplification of the predicted 626-bp fragment (including primers) from the 5′ end of the gyrA gene from various species of the Enterobacteriaceae. In the left-most lane are molecular size markers (100-bp DNA ladder). (B) Schematic diagram of the gyrA region amplified by synthetic oligonucleotide primers gyrA6 and gyrA631R (arrows), including the 120-bp QRDR (heavy line) encoding amino acids 67 to 106 of the E. coli GyrA protein (21).

FIG. 2

DNA sequence similarities of the gyrA QRDRs from eight species of Enterobacteriaceae. Dots indicate nucleotide positions identical to the corresponding E. coli gyrA sequence. Nucleotide positions conserved in all sequences are designated by asterisks. The Ser-83 and Asp-87 codons, in which mutations frequently associated with fluoroquinolone resistance are found, are indicated by the solid bars above the sequence. Numbers refer to the nucleotide positions in the E. coli gyrA sequence (21).

FIG. 3

Alignment of deduced amino acid sequences of the QRDRs of Enterobacteriaceae type strains. Amino acid differences are noted, and dots indicate amino acids identical to the corresponding E. coli sequence. Amino acid positions conserved in all sequences are designated by asterisks. Numbers refer to the amino acid positions in the E. coli GyrA sequence (21).