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

Purpose: We attempted to select increasingly fluoroquinolone-resistant stra ins of Neisseria gonorrhoeae in vitro and to assess whether selected mutant s harbored alterations in the GyrA subunit of DNA gyrase and the ParC subun it of DNA topoisomerase TV, which were analogous to those in fluoroquinolon e-resistant clinical isolates. Materials and Methods: A fluoroquinolone-susceptible strain was exposed to norfloxacin in vitro. Selected mutants were sequentially exposed to norflox acin, and this procedure was repeated. For 11 mutants, minimum inhibitory c oncentrations (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 p arC gene were analyzed. Results: Mutants obtained in one step exhibited significantly increased MIC s of norfloxacin, ofloxacin and ciprofloxacin and had a single amino acid c hange 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 f luoroquinolone MICs and were assigned to the ciprofloxacin-resistant catego ry. Two had a double amino acid change in GyrA, and one had a double GyrA c hange and a single amino acid change in ParC. Conclusion: We selected fluoroquinolone-resistant strains that carried GyrA and ParC alterations analogous to those in clinical isolates. The serial a ccumulation of changes in the QRDR of GyrA and the analogous region of ParC was associated with a stepwise increase in fluoroquinolone resistance, alt hough the development of additional alterations in other regions of GyrA an d ParC or other mechanisms of fluoroquinolone resistance also might contrib ute to the enhancement in fluoroquinolone resistance. The clinical emergenc e of fluoroquinolone-resistant strains may be due to in-vivo stepwise selec tion of strains with genetic alterations in GyrA and ParC, as observed here in the in-vitro selection of fluoroquinolone-resistant mutants.