Previously, we reported potent activity of a novel spiropyrimidinetrione, zoliflodacin, against N. gonorrhoeae isolates from symptomatic men in Nanjing, China, collected in 2013. Here, we investigated trends of susceptibilities of zoliflodacin in 986 isolates collected from men between 2014 and 2018. N. gonorrhoeae isolates were tested for susceptibility to zoliflodacin and seven other antibiotics. Mutations in gyrA, gyrB, parC, parE and mtrR genes were determined by PCR and sequencing. The

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... of zoliflodacin ranged from ≤0.002 to 0.25 mg/L; the overall MIC50s and MIC90s were 0.06 mg/L and 0.125mg/L in 2018, increasing two-fold from 2014. However, the percent of isolates with lower zoliflodacin MICs declined in each year sequentially while the percent with higher MICs increased yearly (P≤0.00001). All isolates were susceptible to spectinomycin but resistant to ciprofloxacin (MIC ≥1 mg/L); 21.2% (209/986) were resistant to azithromycin (≥1 mg/L), 43.4% (428/986) were penicillinase-producing (PPNG), 26.9% (265/986) tetracycline-resistant (TRNG) and 19.4% (191/986) were multi-drug resistant (MDR) isolates. Among 202 isolates tested, all were quinolone resistant with double or triple mutations in gyrA; One hundred ninety three (193/202; 95.5%) also had mutations in parC There were no D429N/A and/or K450T mutations in GyrB identified in the 143 isolates with higher zoliflodacin MICs; a S467N mutation in GyrB was identified in one isolate. We report that zoliflodacin continues to have excellent in vitro activity against clinical gonococcal isolates, including those with high-level resistance to ciprofloxacin, azithromycin and extended spectrum cephalosporins.