Mutations in the gyrA gene of a highly fluoroquinolone-resistant clinical isolate of Escherichia coli.
We have determined the DNA sequence of the gyrA gene of the fluoroquinolone-resistant Escherichia coli isolate 205096 (MIC of ciprofloxacin, 128 micrograms/ml), which was recently demonstrated to be a gyrA mutant (P. Heisig and B. Wiedemann, Antimicrob. Agents Chemother. 35:2031-2036, 1991). Compared with the gyrA+ gene of E. coli K-12, 55 nucleotide changes were found. Three of these resulted in amino acid exchanges: Ser-83-->Leu, Asp-87-->Gly, and Asp-678-->Glu. A 0.7-kb DNA fragment containing two of these mutations (Ser-83-->Leu and Asp-87-->Gly) was isolated and fused in frame to the residual 3' coding region of gyrA+ in a plasmid to yield a chimeric gyrA gene (gyrA#). After introduction into E. coli 205096, this gyrA# gene does not increase the fluoroquinolone susceptibility of the resulting heterodiploid strain in a dominance test, while the gyrA+ gene does. The ciprofloxacin concentration necessary to inhibit by 90% (IC90) the supercoiling activity of gyrase isolated from E. coli 205096 is above 2,000 micrograms/ml. An identical result was found for gyrase reconstituted in vitro from the gyrB+ gene product and the chimeric gyrA# gene product. This is more than a 4,000-fold increase compared with the IC90 determined for gyrase from E. coli K-12 (gyrA+) (IC90, 0.5 microgram of ciprofloxacin per ml). No indications for the involvement of the gyrB gene or for alterations in quinolone permeation were found.
Heisig
P
P
Abteilung Pharmazeutische Mikrobiologie, Universität Bonn, Germany.
Schedletzky
H
H
Falkenstein-Paul
H
H
D000890:Anti-Infective Agents / Q000494:pharmacology*; D001483:Base Sequence; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D004927:Escherichia coli Infections / Q000382:microbiology; D024841:Fluoroquinolones; D005784:Gene Amplification; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000187:drug effects* / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D014162:Transfection
D000890:Anti-Infective Agents; D004278:DNA, Superhelical; D024841:Fluoroquinolones; D004250:DNA Topoisomerases, Type II