Yuanyuan Zhao,1 Wenjing Le,1 Caroline A Genco,2 Peter A Rice,3 Xiaohong Su1 1Sexually Transmitted Disease Clinic, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, People’s Republic of China; 2Department of Immunology, Tufts University School of Medicine, Boston, MA, USA; 3Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USACorrespondence: Xiaohong Su, Sexually Transmitted Disease Clinic, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 12, Jiangwangmiao Road, Nanjing, 210042, Jiangsu Province, People’s Republic of China, Email suxh@ncstdlc.orgBackground: Since the first Chinese report of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone in 2016, additional FC428-like, penA 60.001 isolates have been identified in China.Objective: To document the rise in penA 60.001 isolates in Nanjing, China, and characterize their molecular and epidemiological features.Methods: N. gonorrhoeae minimum inhibitory concentrations (MICs, mg/L) for ceftriaxone, cefixime, penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, gentamicin and zoliflodacin were determined by agar dilution. MICs for ertapenem were measured by E-test. N. gonorrhoeae antimicrobial sequence typing (NG-STAR) of seven loci (penA, mtrR, porB, ponA, gyrA, parC and 23S rRNA) was analyzed together with N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST). Phylogenetic analysis was also performed using whole genomic sequencing (WGS).Results: Fourteen FC428-related penA 60.001 N. gonorrhoeae infections were identified out of 677 infections from 2017 to 2020, in Nanjing, representing an incremental yearly rise in the percentage of the city’s N. gonorrhoeae isolates that were FC428-related. Seven FC428-related N. gonorrhoeae infections were acquired in Nanjing, proper; four others in eastern Chinese cities and three from unknown locations. All FC428-related isolates were resistant to ceftriaxone, cefixime, ciprofloxacin, tetracycline and penicillin but susceptible to spectinomycin, gentamicin, ertapenem and zoliflodacin; three strains were resistant to azithromycin. penA 60.001 isolates displayed closely related MLST types and NG-STAR types but relatively distant NG-MAST types. WGS showed a phylogenetic analysis that intermingled with other international isolates.Conclusion: penA 60.001 N. gonorrhoeae isolates emerged in Nanjing, China, beginning in 2017, and have continued to rise.Keywords: Neisseria gonorrhoeae, penA 60.001, ceftriaxone-resistance, FC428, WGS phylogenetic analysis