Fluoroquinolone resistance-conferring gyrA variants alter the fitness cost and potentiate the resistance of the zoliflodacin resistance mutation gyrB ^D

Neisseria gonorrhoeae is a major public health concern due to its high global prevalence and rapid evolution of antibiotic resistance. A first-in-class topoisomerase inhibitor, zoliflodacin (a spiropyrimidinetrione), recently received FDA approval for treatment of gonorrhea, but its potential for cross-resistance with another topoisomerase inhibitor, the fluoroquinolone antibiotic ciprofloxacin, remains poorly understood. Here, we investigated how genetic diversity in the fluoroquinolone target gyrA influences the resistance and fitness effects of the zoliflodacin resistance mutation gyrB ^D429N . We constructed an isogenic panel of N. gonorrhoeae to determine how the resistance and fitness effects of the gyrB ^D429N mutation are modulated by the most common ciprofloxacin resistance-associated variants in gyrA . In the presence of gyrB ^D429N , the zoliflodacin minimum inhibitory concentration (MIC) was two- to fourfold higher in strains that also contained ciprofloxacin resistance-associated gyrA alleles, and the gyrB ^D429N mutation reciprocally increased ciprofloxacin MICs of these strains three- to sixfold. The fitness cost of the gyrB ^D429N mutation varied from modest to severe across gyrA backgrounds, with the largest cost in ciprofloxacin-resistant gyrA ^91F/95G and gyrA ^91F/95N backgrounds and comparatively minimal cost in the ciprofloxacin-resistant gyrA ^91F/95A background. These results demonstrate the capacity for epistatic interactions among resistance-associated gyrA and gyrB mutations, underscoring the need for genomic surveillance to monitor high-risk combinations of resistance determinants as new therapies are deployed.