Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs

Abstract

The mutations in the quinolone resistance-determining region of the gyrA and gyrB genes from 27 clinical isolates of Escherichia coli with a range of MICs of ciprofloxacin from 0.007 to 128 ,ug/ml and of nalidixic acid from 2 to >2,000 ,ug/ml were determined by DNA sequencing. All 15 isolates with ciprofloxacin MICs of .1 ,ug/ml showed a change in Ser-83 to Leu of GyrA protein, whereas in clinical isolates with a MIC of .8 ,ug/ml (11 strains), a double change in Ser-83 and Asp-87 was found. All isolates with a MIC of nalidixic acid of .128 ,ug/ml showed a mutation at amino acid codon Ser-83. Only 1 of the 27 clinical isolates of E. coli analyzed showed a change in Lys-447 of the B subunit of DNA gyrase. A change in Ser-83 is sufficient to generate a high level of resistance to nalidixic acid, whereas a second mutation at Asp-87 in the A subunit of DNA gyrase may play a complementary role in developing the strain's high levels of ciprofloxacin resistance.New fluoroquinolones are broad-spectrum antibacterial agents which inhibit DNA gyrase activity (26). DNA gyrase contains two subunits of GyrA and two subunits of GyrB (13,19). Gyrase A mediates DNA strand breakage and reunion with the Tyr residue at position 122 forming a transient phosphotyrosine linkage with a broken DNA strand. The mechanisms of quinolone resistance essentially fall into two classes: (i) mutations in gyrA (1,2,5,6,8,17,28,30) or gyrB genes (27, 29) or (ii) reduced levels of quinolone accumulation in the cells (3, 4, 9-12). The mutations in the gyrA gene involved in the resistance are clustered in a region between nucleotides 199 (Ala-67) and 318 (Gln-106), which contains nucleotide 247 (Ser-83), the most frequently changed in spontaneus gyrA mutations. In the gyrB gene, two quinolone resistance-determining sites (amino acids 426 and 447) have been found (27,29). The mutation at amino acid codon Asp-426 confers resistance to nalidixic acid and the new fluoroquinolones, whereas the change of Lys-447 results in resistance to nalidixic acid and increased susceptibility to the fluoroquinolones (29). Recently, Heisig et al. (8) identified mutations in the gyrA gene of Escherichia coli 205096, a highly fluoroquinolone-resistant strain (MIC of ciprofloxacin, 128 ,ug/ml). From 1989 to 1993, the level of resistance to ciprofloxacin in our hospital increased from 0 to 16% in E. coli isolated from urine samples. To further assess the importance of these gyrA and gyrB mutations in the acquisition of high-level quinolone resistance, we examined by DNA sequencing 27 clinical isolates of E. coli with a range of MICs of ciprofloxacin from 0.007 to 128 ,ug/ml and of nalidixic acid from 2 to >2,000 ,ug/ml.The clinical isolates of E. coli were obtained from urine samples from outpatients, who had not previously received quinolones,

The Impact of Mutations in Topoisomerase Genes and the Plasmid-Mediated Quinolone Resistance (PMQR) Determinants on the Resistance to Fluoroquinolones in Klebsiella pneumoniae

“…The MIC of fluoroquinolones for isolates with two mutations in gyrA gene was higher than isolates with only one mutation that is similar to other studies' results (25)(26)(27)(28). Sequence analysis of QRDR in parC gene revealed two mutations with amino acid changes which have been reported by several previous studies (19,29).…”

Susceptibility patterns of enteroaggregative Escherichia coli associated with traveller's diarrhoea: emergence of quinolone resistance

“…This con®rms previous ®ndings that high-level resistance to cipro¯oxacin in E. coli is related to the presence of concomitant mutations in the gyrA and parC genes [23,24].…”

“…Ampli®cation of the quinolone-resistant determining regions (QRDR) of the gyrA and parC genes by PCR PCR was performed as described elsewhere [23,24]. The PCR reaction was performed in a DNA Thermal Cycler 480 (Perkin-Elmer Cetus, Emeryville, CA, USA).…”

Identification of strain harboring both aac(6')-Ib and aac(6')-Ib-cr variant simultaneously in Escherichia coli and Klebsiella pneumoniae

“…In E. coli, several mutations have been identified in the gyrA gene. Among these, mutations at Ser-83 and Asp-87 have been found with a higher frequency in quinolone resistant E. coli clinical isolates than in susceptible isolates (19). The genes for multidrug efflux pump oqxAB, which is active on fluoroquinolones, were detected in human clinical isolates on a plasmid in E. coli and on the chromosome of K. pneumoniae (20).…”