title: Limitations of plasmid complementation test for determination of quinolone resistance due to changes in the gyrase A protein and identification of conditional quinolone resistance locus.
abstract: Plasmid pJSW101 derived from pUC19 and carrying the wild-type gyrA gene was found to be unstable in HM72, a quinolone-resistant (QR) clinical isolate of Escherichia coli, and resulted in no change in quinolone MICs. MICs determined in the presence of ampicillin to ensure plasmid presence, however, resulted in complementation. HM72 was proved to have a gyrA mutation based on the DNA sequence of a 418-bp fragment of gyrA. DNA sequencing identified a common mutation encoding Leu-83 as the cause of QR. To identify loci other than gyrA and nfxB contributing to QR in KF111b, zgh-3075::Tn10 (67 min) in CAG12152 was transduced into KF111b. Sixteen percent of the transductants had a fourfold decrease in norfloxacin MIC, indicating the presence of a locus, nfxD, which contributes to QR. Outcross of nfxD from DH151 (gyrA nfxB nfxD zgh-3075::Tn10) resulted in 8% of the KF130 gyrA, 2% of the EN226-3 gyrA, and none of the KL16 (wild-type) transductants, with a four- to eightfold increase in norfloxacin MIC. In the presence of ampicillin, the resistance of a gyrA nfxD double mutant, DH161 nfxD gyrA (from EN226-3), was fully complemented by gyrA+. Thus, gyrA+ plasmid complementation tests for QR may be falsely negative with plasmid instability, a difficulty which may be circumvented by maintenance of plasmid selection. In addition, if nfxD-like mutations occur in gyrA clinical isolates, a positive test may overestimate the level of resistance attributable to gyrA alone.
authors.affiliation: Service de Bactériologie-Virologie-Hygiène, Hôpital Henri Mondor, Université de Paris XII, Créteil, France.
authors.forename: C J
authors.initials: CJ
authors.lastname: Soussy
authors.forename: J S
authors.initials: JS
authors.lastname: Wolfson
authors.forename: E Y
authors.initials: EY
authors.lastname: Ng
authors.forename: D C
authors.initials: DC
authors.lastname: Hooper
mesh_terms: D042462:4-Quinolones; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000494:pharmacology*; D001483:Base Sequence; D003001:Cloning, Molecular; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000187:drug effects / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D005188:False Negative Reactions; D005816:Genetic Complementation Test / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D014169:Transformation, Bacterial / Q000235:genetics
chemical_list: D042462:4-Quinolones; D000890:Anti-Infective Agents; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II