DEVELOPMENT OF A RAPID ASSAY FOR DETECTING GYRA MUTATIONS IN ESCHERICHIA-COLI AND DETERMINATION OF INCIDENCE OF GYRA MUTATIONS IN CLINICAL STRAINS ISOLATED FROM PATIENTS WITH COMPLICATED URINARY-TRACT INFECTIONS

The MICs of ofloxacin for 743 strains of Escherichia coli isolated fro m 1988 to 1994 were determined by testing, The strains were from patie nts with urinary tract infections complicated by functional or anatomi cal disorders of the urinary tract, Those determined to be ofloxacin r esistant (MIG, greater than or equal to 12.5 mu g/ml) comprised 3 of 3 95 strains (1.3%) from the 1988 to 1990 group, 2 of 166 strains (1.2%) from the 1991 to 1992 group, and 7 of 182 strains (3.8%) from the 199 3 to 1994 group, The incidence of resistant strains increased signific antly during this period, The percentage of isolates with moderately d ecreased susceptibilities to ofloxacin (MIG, 0.39 to 3.13 mu g/ml) als o rose during the same period, To determine the incidence of gyrA muta tions in urinary-tract-derived strains of E, coli, we developed a simp le and rapid assay based on PCR amplification of the region of the gyr A gene containing the mutation sites followed by digestion of the PCR product with a restriction enzyme, Using this assay, we examined all 1 82 strains isolated in 1993 and 1994 for the presence of mutations at Ser-83 and Asp-87 in the gyrA gene, Of these strains, 33 (18.1%) had m utations in the gyrA gene, The incidences of mutations at Ser-83, at A sp-87, and at both codons were 10.4 (19 strains), 4.4 (8 strains), and 3.3% (6 strains), respectively, To determine the correlation of the m utations in the gyrA gene with susceptibilities to quinolones (nalidix ic acid, ofloxacin, norfloxacin, and ciprofloxacin), we further examin ed 116 strains for which the MICs of ofloxacin were greater than or eq ual to 0.2 mu g/ml that were chosen from the isolates in the 1988 to 1 992 group, The MICs of nalidixic acid for the strains without mutation s at either Ser-83 or Asp-87 were less than or equal to 25 mu g/ml, wh ereas those for the strains with single mutations or double mutations were from 50 to >800 mu g/ml. For the fluoroquinolones, significant di fferences in the distributions of the MICs were observed among the str ains without mutations, with single mutations, and with double mutatio ns, The accumulation of mutations in the gvrA gene was associated with an increase in fluoroquinolone resistance, Ofloxacin MICs for the maj ority of the strains with single and double mutations were 0.39 to 3.1 3 and 6.25 to 100 mu g/ml, respectively, This study demonstrates a chr onological increase in the percentage of not only highly fluoroquinolo ne-resistant strains, corresponding to those with double mutations in the gyrA gene, but also strains with moderately decreased susceptibili ties to fluoroquinolones, corresponding to those with single mutations , This increase in the incidence of strains with a single mutation in the gyrA gene portends a further increase in the incidence of strains with clinically significant resistance to fluoroquinolones.