CHARACTERIZATION OF FLUORO QUINOLONE-RESISTANT MUTANTS OF ESCHERICHIA-COLI SELECTED IN-VITRO

Wild-type mutants highly resistant to fluoroquinolones were selected i n vitro from a quinolone-susceptible Escherichia coli isolate by stepw ise exposure to increasing concentrations of nalidixic acid and ciprof loxacin (CIP) either in liquid medium or on solid medium. Mutant R17 w as selected by serial passage in liquid medium; the MIC of CIP for mut ant R17 was 256 mu g/ml. On solid medium, consecutive mutants MI, MII, MIII, MIVa, and MIVb were selected in four steps. The frequencies of mutations were between 10(-9) and 10(-11), and the MICs of CIP ranged from 0.5 mu g/ml (for mutant MI) to 256 mu g/ml (for mutant MlVb). Fro m the results of a dominance test with the gyrB(+) plasmid (pBP547), n o gyrB mutations were detectable. In the first step, mutant h II, a mu tation from a Ser to a Leu residue at position 83 (a Ser-83-->Leu muta tion), was detected in the quinolone resistance-determining region of the gyrA gene. In addition, the second-step mutation was associated wi th a reduced uptake of CIP and an altered outer membrane protein profi le. The third mutation was identified as an Asp-87-->Gly mutation in t he quinolone resistance-determining region of the gyrA gene. Concomita ntly, a slight increase in the doubling time was detected. For two dif ferent four-step mutants, mutants MIVa and MIVb, the MICs of only some quinolones, including CIP, increased. The accumulation of CIP in the mutants was comparable to that in their parent MIII. The doubling time of mutant MIVa was similar to that of mutant MIII, but differed by a factor of 3 from that of the very slow growing mutant MIM,. In contras t, a clinical isolate off. coli (isolate 205096) described previously (P. Heisig, H. Schedletzky, and H. Falkenstein-Paul, Antimicrob. Agent s Chemother, 37:696-701, 1993) which has the same double mutation in g yrA, had a doubling time comparable to that of the wild-type isolate.