Characterization of fluoroquinolone-resistant mutants of escherichia coli selected in vitro.

Wild-type mutants highly resistant to fluoroquinolones were selected in vitro from a quinolone-susceptible Escherichia coli isolate by stepwise exposure to increasing concentrations of nalidixic acid and ciprofloxacin (CIP) either in liquid medium or on solid medium. Mutant R17 was selected by serial passage in liquid medium; the MIC of CIP for mutant R17 was 256 micrograms/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 microgram/ml (for mutant MI) to 256 micrograms/ml (for mutant MIVb). From the results of a dominance test with the gyrB+ plasmid (pBP547), no gyrB mutations were detectable. In the first step, mutant MI, a mutation from a Ser to a Leu residue at position 83 (a Ser-83-->Leu mutation), was detected in the quinolone resistance-determining region of the gyrA gene. In addition, the second-step mutation was associated with a reduced uptake of CIP and an altered outer membrane protein profile. The third mutation was identified as an Asp-87-->Gly mutation in the quinolone resistance-determining region of the gyrA gene. Concomitantly, a slight increase in the doubling time was detected. For two different 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 MIVb. In contrast, a clinical isolate of E.coli (isolate 205096) described previously (P. Heisig, H. Schedletzky, and H. Falkenstein-Paul, Antimicrob. Agents Chemother. 37:696-701, 1993) which has the same double mutation in gyrA had a doubling time comparable to that of the wild-type isolate.