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Emergence of multidrug-resistant Neisseria gonorrhoeae resulting from new genetic mutations is a serious threat to controlling gonorrhea.To determine 1) antimicrobial susceptibilities and the corresponding genetic mutations and 2) the role of MtrRCDE efflux system in gonococcal resistance to fluoroquinolones.Antimicrobial susceptibility testing and sequence analysis of gyrA, parC, and mtrR loci of 131 N. gonorrhoeae isolates from Japan.The proportion of N. gonorrhoeae strains resistant and intermediate-resistant to antimicrobials was 25.2% and 48.9% for ciprofloxacin, 25.2% and 30.5% for ofloxacin, 12.2% and 53.4% for penicillin; and 17.6% and 51.1% for tetracycline, respectively. Strains were categorized into 22 mutation profiles, with GyrA-S91F/ParC-D86N/MtrR-G45D being the most predominant profile. The frequency of mutation in gyrA, parC, mtrR, and the mtrR promoter was 71%, 47.3%, 77.1%, and 23.7%, respectively. Seventy-one percent of strains carried mutations in both gyrA and mtrR.This study reports simultaneous mutations in fluoroquinolone target enzymes and the MtrRCDE efflux system as a fluoroquinolone-resistant mechanism in N. gonorrhoeae.