Study On The Genotyping Of Neisseria Gonorrhoeae With Resistance To Azithromycin By Neisseria Gonorrhoeae Multiantigen Sequence Typing And Genetic Polymorphisms In PenA, PorB And MtrR Genes Of Neisseria Gonorrhoeae With Reduced Susceptibility To Ceftriaxo

Neisseria gonorrhoeae (NG) is the causative pathogen of gonorrhea, which causes both symptomatic and asymptomatic infections. Increasing antimicrobial resistance in NG is a major challenge for prevention and control of gonorrhea. Azithromycin is usually used for the treatment of urethritis and cervicitis in syndromic management, so azithromycin-resistant NG strains should be monitored in China. Although the recommended first-line treatment for gonorrhea in China is ceftriaxone or spectinomycin, in recent years, the emergence and spread of NG strains with reduced susceptibility to ceftriaxone (Cef) has been reported in China. It’s essential to monitor the susceptibility to ceftriaxone of NG strains and launch research on the resistance mechanism of NG strains resistant or non-susceptible to ceftriaxone. NG multiantigen sequence typing (NG-MAST) is a discriminatory genotyping method and easy for performing. In the current study, NG-MAST method was used to investigate the genotypes of azithromycin-resistant NG isolates and Cef isolates. The current study was divided into three parts as follow:Chapter1. Resistance to azithromycin of NG isolates from Nanjing and ChongqingWe aimed to investigate azithromycin susceptibility in NG from Nanjing and Chongqing. NG isolates (n=318) were cultured from Nanjing and Chongqing between2008and2009. Minimum inhibitory concentration (MIC) of azithromycin, ceftriaxone, spectinomycin, tetracycline, and ciprofloxacin was determined using the agar dilution method. Azithromycin-resistant isolates were defined as having an MIC value equal to or greater than that of strain WHO P, which is internationally recognized as azithromycin-resistant (MIC=2mg/L). Seventeen (5.3%) isolates showed resistance to azithromycin. A relatively high prevalence of azithromycin-resistant NG strains implies that azithromycin should not be recommended for the treatment of gonococcal urethritis or cervicitis in Nanjing and Chongqing.Chapter2. Genotyping of azithromycin-resistant NG isolates by NG-MAST methodOur objective was to investigate the genotypes of azithromycin-resistant NG isolates from Nanjing and Chongqing. Seventeen azithromycin-resistant NG isolates identified in Chapter1were genotyped by NG-MAST method and analyzed using Mega software to construct the phylogenetic tree. Among these isolates (n=17),11sequence types were identified by NG-MAST method, of which5were novel. The most common sequence type (ST) was ST3356, represented by6isolates. ST1866was represented by2isolates, which were isolated from patients with an unknown relationship, and both isolates were highly resistant to azithromycin, i.e., displayed an MIC of>64mg/L. In the phylogenetic analysis, the isolates of4novel STs and1known ST clustered closely to the ST3356isolates and, accordingly, these may all have a common ancestor.Chapter3. Genetic polymorphisms in penA, porB and mtrR genes of Cef1isolatesOur aim was to investigate the correlation of different polymorphisms in the penA, porB, and mtrR genes of Cef isolates. Seventeen Cef isolates and4susceptible NG isolates were characterized by NG-MAST method and sequencing of penA, porB, and mtrR alleles. Among the21NG isolates,9mutation patterns were observed in penicillin-binding protein2(PBP2), encoded by penA gene, of which2were novel. The most common mutation pattern is XVIII, represented by8Cef isolates. And mutation pattern XIII was represented by4Cef isolates. None of the21NG isolates contained a mosaic PBP2(pattern X). Eighteen of the21NG isolates had a porBlb gene, encoding porin protein PorBlb. Substitutions of I218M/M257T/G259V were present in PorBlb of4Cef isolates, and another257substitution (M257R) were present in PorBlb of6Cef isolates. Substitution of A39T was observed in MtrR, encoded by mtrR gene, of3Cef isolates. Substitutions of G45D or H105Y were observed in MtrR, encoded by mtrR gene, of both Cef and susceptible isolates. All17Cef isolates were assigned13NG-MAST sequence types, of which3were novel. In conclusion, these mutations in penA, porB and mtrR genes may play a role for the reduced susceptibility to ceftriaxone in NG...