In vitro selection of fluoroquinolone-resistant Neisseria gonorrhoeae harboring alterations in DNA gyrase and topoisomerase IV.

Abstract

We attempted to select increasingly fluoroquinolone-resistant strains of Neisseria gonorrhoeae in vitro and to assess whether selected mutants harbored alterations in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV, which were analogous to those in fluoroquinolone-resistant clinical isolates. A fluoroquinolone-susceptible strain was exposed to norfloxacin in vitro. Selected mutants were sequentially exposed to norfloxacin, and this procedure was repeated. For 11 mutants, minimum inhibitory concentrations (MICs) of antimicrobial agents were determined, and mutations in the region corresponding to the quinolone resistance-determining region (QRDR) of the Escherichia coli gyrA gene and the analogous region of the parC gene were analyzed. Mutants obtained in one step exhibited significantly increased MICs of norfloxacin, ofloxacin and ciprofloxacin and had a single amino acid change in GyrA. Two-step mutants exhibited significantly higher norfloxacin MICs. Three of four two-step selected strains had single amino acid changes in both GyrA and ParC. Three-step mutants exhibited further increases in fluoroquinolone MICs and were assigned to the ciprofloxacin-resistant category. Two had a double amino acid change in GyrA, and one had a double GyrA change and a single amino acid change in ParC. We selected fluoroquinolone-resistant strains that carried GyrA and ParC alterations analogous to those in clinical isolates. The serial accumulation of changes in the QRDR of GyrA and the analogous region of ParC was associated with a stepwise increase in fluoroquinolone resistance, although the development of additional alterations in other regions of GyrA and ParC or other mechanisms of fluoroquinolone resistance also might contribute to the enhancement in fluoroquinolone resistance. The clinical emergence of fluoroquinolone-resistant strains may be due to in-vivo stepwise selection of strains with genetic alterations in GyrA and ParC, as observed here in the in-vitro selection of fluoroquinolone-resistant mutants.