DETECTION OF MUTATIONS IN THE GYRA AND PARC GENES IN QUINOLONE-RESISTANT CLINICAL ISOLATES OF ENTEROBACTER-CLOACAE

We have determined partial sequences of the gyrA and parC genes of Ent erobacter cloacae type strain including the regions analogous to the q uinolone resistance-determining region of the Escherichia coli gyrA ge ne. The deduced 65- and 49-amino acid sequences of the determined regi ons of the E. cloacae gyrA and parC genes were identical to the corres ponding regions of the E. coli GyrA and ParC proteins, respectively. W e examined 40 clinical strains of E. cloacae isolated from patients wi th urinary tract infection for susceptibilities to nalidixic acid and ciprofloxacin. Based on the nalidixic acid and ciprofloxacin MICs, the se isolates were divided into 19 quinolone-susceptible strains (MICs o f nalidixic acid, 3.13-25 mg/L; MICs of ciprofloxacin, less than or eq ual to 0.025 mg/L) and 21 quinolone-resistant strains (MICs of nalidix ic acid, 400 to >800 mg/L; MICs of ciprofloxacin, 0.39-100 mg/L). We a nalysed five quinolone-susceptible and 21 quinolone-resistant strains for alterations in GyrA and ParC. The five quinolone-susceptible strai ns had amino acid sequences in GyrA and ParC identical to those of typ e strain. Of the 21 quinolone-resistant isolates, three (MICs of nalid ixic acid, 400 to >800 mg/L; MICs of ciprofloxacin, 0.39-3.13 mg/L) ha d a single amino acid change at the position equivalent to Ser-83 in t he E. coli GyrA protein and no alterations in ParC; one (MIG of nalidi xic acid, >800 mg/L; MIC of ciprofloxacin, 3.13 mg/L) had a single ami no acid change at Ser-83 in GyrA and a single amino acid change at the position equivalent to Glu-84 in the E. coli ParC protein; two (MIG o f nalidixic acid, >800 mg/L; MIC of ciprofloxacin, 25 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and no alterations in ParC; and 15 (MICs of nalidixic acid, >800 mg/L; MICs of ciprofloxaci n, 25-100 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and a single amino acid change at Ser-80 or Glu-84 in ParC. This study suggests, that in clinical isolates of E. cloacae, DNA gyrase is a primary target of quinolones, that only a single amino acid change at Ser-83 in GyrA is sufficient to generate high-level resistance to n alidixic acid and to decrease susceptibility to ciprofloxacin, and tha t the accumulation of amino acid changes in GyrA and the simultaneous presence of the ParC alterations play a central role in developing hig h-level resistance to ciprofloxacin.