Stepwise evolution and clonal enrichment of gepotidacin resistance in <i>Neisseria gonorrhoeae</i>

Although gepotidacin is a promising oral candidate for treating multidrug-resistant <i>Neisseria gonorrhoeae</i>, its resistance mechanisms and clinical implications remain poorly understood. In this study, we collected 989 clinical <i>N. gonorrhoeae</i> isolates from 33 hospitals in Shanghai, China (2022-2024). Antimicrobial susceptibility testing showed that gepotidacin exhibited high <i>in vitro</i> activity with MIC₅₀ and MIC₉₀ of 0.5 and 1 μg/mL, respectively. Further investigation identified that elevated gepotidacin MICs were significantly associated with substitutions at GyrA position 92/95 and ParC position 86/87, including GyrA A92P (odds ratio [OR], 4.25; 95% confidence interval [CI], 2.61-6.94) and D95Y (OR, 4.61; 95% CI, 2.82-7.54), as well as ParC D86N (OR, 3.12; 95% CI, 1.99-4.90) and S87N (OR, 5.92; 95% CI, 3.63-9.64). Substitution combinations analyses revealed that GyrA D95A plus ParC D86N, GyrA A92P/D95Y plus ParC S87N, GyrA D95A plus ParC S87N, and GyrA D95G plus ParC D86N were significantly associated with elevated gepotidacin MICs. Notably, these high-risk substitutions were enriched in international clones ST7363 and ST8123. <i>In vitro</i> induction experiments demonstrated a stepwise resistance trajectory: from initial diverse QRDR mutations to an intermediate state with GyrA A92P/D95Y, followed by ParC D86N and GyrA A92T/D95A, culminating in high-level resistance, with MICs all reaching 64 μg/mL. We also found that the strain with preexisting GyrA D95A and ParC D86N more readily acquired A92T and developed high-level resistance under gepotidacin exposure. Our study highlights key mutational patterns and clonal backgrounds that promote gepotidacin resistance, emphasizing the need for optimized dosing strategies as well as targeted molecular surveillance to preserve its effectiveness.