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Background Gepotidacin (GEP) is a novel triazaacenaphthylene bacterial type II topoisomerase inhibitor targeting both bacterial DNA gyrase and topoisomerase IV by a different mechanism from fluoroquinolone antibiotics. Although in vitro frequency of resistance to GEP in Neisseria gonorrhoeae (NG) is low, during a phase 2 trial, clinical resistance to gepotidacin in NG emerged in a subset of fluoroquinolone-resistant NG isolates that contained a pre-existing ParC D86N mutation by introduction of a new GyrA A92T mutation. The objective of this study was to evaluate the role of GyrA A92T & Parc D86N mutations in resistance to GEP. Methods We utilized the high frequency of natural transformation to introduce GyrA A92T and ParC D86N mutations, individually and in combination, into NG isolates either with GyrA S91F D95G mutations or with wild type (WT) GyrA by selection on ciprofloxacin (CIP) or GEP to generate isogenic strains for susceptibility evaluation. Results Results are summarized in enclosed table. Overall, GyrA A92T and ParC D86N mutations alone did not confer a significant (>4-fold) increase in GEP MIC; whereas together they gave >16-fold increases in GEP MIC. Importantly, quinolone target mutations (GyrA S91F D95G and ParC D86N) together showed no significant effect on the GEP MIC; while they gave >1000-fold increase in CIP MIC. As expected, GyrA A92T and ParC D86N mutations alone or together in WT GyrA background had no significant effect on CIP susceptibility. Susceptibility of isogenic NG strains to gepotidacin and ciprofloxacin Conclusion Our results indicated that unlike fluoroquinolones that primarily target DNA gyrase in NG, there is no obvious primary target for GEP, supporting well-balanced dual targeting of DNA gyrase and topoisomerase IV by GEP in NG. Though, the pre-existing ParC D86N mutation is a potential risk marker for clinical resistance development, as this mutation compromises dual targeting of GEP, our studies provide mechanistic insight for appropriate clinical dose selection to potentially suppress further resistance development in this subset of clinical isolates. Disclosures Pan Chan, PhD, GlaxoSmithKline (Employee, Shareholder) Karen Ingraham, MS, GlaxoSmithKline (Employee, Shareholder) Sharon Min, MS, GlaxoSmithKline (Employee, Shareholder) Nicole Scangarella-Oman, MS, GlaxoSmithKline plc. (Employee, Shareholder) Steve Rittenhouse, PhD, GlaxoSmithKline (Employee, Shareholder) Jianzhong Huang, PhD, GlaxoSmithKline (Employee, Shareholder)