Epidemiology Of Neisseria Gonorrhoeae Antimicrobial Susceptibility And The Emerging FC428 Clone And Analysis Of The Impact Of Bla_TEM Gene And Vector Variations On Penicillin Resistance

Neisseria gonorrhoeae specifically infects human and causes sexually transmitted disease gonorrhoea.The lack of effective treatment can lead to serious complications.The current clinical first-line therapy for the treatment of gonorrhea is ceftriaxone monotherapy or ceftriaxone plus azithromycin dual therapy.However,the increasing antimicrobial resistance is a serious threat to the future treatment of gonorrhea,which has attracted continuous global attention.The purpose of this study is to monitor the molecular epidemiology and resistance mechanisms of gonococcal antibiotic resistance to provide experimental evidence for the spread and impact of gonococcal resistance and antibacterial management.379 clinical isolates of Neisseria gonorrhoeae were collected from seven hospitals in Hangzhou,China during the period of 2015-2017.The agar dilution method was used to analyze the antimicrobial susceptibility,and the Neisseria gonorrhoeae multiple antigen sequence（NG-MAST）analysis was used to determine the sequencing type and perform isolates genetic relationship analysis.The resistance determinants were determined by PCR and sequencing.A total of 17%of the isolates showed reduced susceptibility to ceftriaxone,including 35 isolates（6%）resistant to ceftriaxone,and resistance to ceftriaxone increased from 2%（3/127）in 2015 increased to 5%（5/107）in2017;a total of 20%showed resistance to azithromycin,of which 55 strains（10%）were of high level resistance to azithromycin.In 2019,70 isolates were collected from a single hospital in Hangzhou.21（30%）isolates were resistant to ceftriaxone,and 10%isolates showed high level resistance to ceftriaxone,which all contained pen A 60.001allele and were characterized as the emerging FC428 clone-related isolates by whole-genome sequencing.This study found that bla _TEM-135 alleles were highly abundant in penicillinase-producing N.gonorrhoeae（PPNG）isolates in Hangzhou,and were commonly identified in Asian type plasmids.The agar dilution method was used to analyze the penicillin susceptibility,and the results showed that the PPNG isolates of Asian plasmid/bla _TEM-135 had a much higher minimum inhibitory concentration（MIC）for penicillin than other isolates.Quantitative PCR(detection of plasmid copy number and bla _TEM gene expression),western blotting analysis（to detect TEM level and TEM stability）and in vivoβ-lactamase activity analysis were performed for epresentative isolates to clarify the penicillin MIC differences.The results showed that the Asian plasmid/bla _TEM-135 isolates had the highestβ-lactamase activity due to the higher expression of bla _TEM genes in Asian-type plasmids than African-type plasmids and the higher protein stability of TEM-135 than TEM-1.In summary,this study showed that the resistance of Neisseria gonorrhoeae to ceftriaxone is rapidly increasing,and the emergence of multi-drug resistantisolates poses a major threat to the current monotherapy of ceftriaxone and the possible implementation of ceftriaxone plus azithromycin dual therapy in Hangzhou and other parts of China.