摘要目的 了解深圳地区淋球菌临床分离株对阿奇霉素的耐药水平、分子机制及流行特征.方法 收集2011-2015年深圳市淋球菌临床分离株,采用琼脂稀释法和E-test法筛选出阿奇霉素耐药株(AZ-R, MIC≥1 μg/ml),将MIC≥2 μg/ml的耐药菌株以及随机抽取(SPSS17.0软件)的一定数量的敏感(MIC≤0.25 μg/ml)菌株(AZ-S)作为实验菌株进行耐药基因23S rRNA、mtrR、erm基因分析及淋球菌多抗原序列分型(NG-MAST).结果 共收集到788株淋球菌,AZ-R菌株有148株,耐药率为18.8%(148/788).高水平阿奇霉素耐药菌株(AZ-HLR,21株)中有18株23S rRNA基因发生错义突变,均为4个等位基因的A2059G突变.中等水平阿奇霉素耐药菌株(AZ-MLR,29株)中有12株23S rRNA基因发生错义突变,其中主要为C2611T在4个等位基因中的全部突变(10株).对mtrR启动子和编码区的突变分析,仅发现G45D/Y105H突变在AZ-HLR组中所占比例高于AZ-MLR组(χ2=12.702,P=0.000)和AZ-S组(χ2=4.462,P=0.035).发现1株菌株携带有ermB基因(MIC值为2 μg/ml).NG-MAST分型,共得到了81个ST型别,其中大部分ST型别由单一菌株构成,AZ-R组8个成簇的ST型别中ST1866、ST3356分别被国内城市南京、重庆、广州报道过,Neighbor-joining建树未观察到耐药株单独成簇的情况.结论 目前深圳地区已不适用于单独采用阿奇霉素治疗淋病.淋球菌对阿奇霉素的高度耐药和中等水平耐药主要由23S rRNA 上A2059G和C2611T突变分别介导.多地报道出现的ST1866和ST3356有助于我们更好地对阿奇霉素耐药淋球菌菌株的流行特征进行监测和分析.

abstractsObjective To investigate the prevalence, molecular mechanism and genetic characteristics of azithromycin-resistant Neisseria gonorrhoeae (N.gonorrhoeae) strains isolated in Shenzhen.Methods N.gonorrhoeae strains were collected in Shenzhen from 2011 to 2015.Agar dilution method and E-test were used to detect the minimum inhibitory concentrations (MIC) of these strains to azithromycin.All azithromycin-resistant (AZM-R) strains (MIC≥2 μg/ml) and some azithromycin-sensitive strains (MIC≤0.25 μg/ml) which were randomly selected as the control group were screened for mutations in 23S rRNA, mtrR and erm genes and genotyped by using N.gonorrhoeae multi-antigen sequence typing (NG-MAST).Results A total of 788 N.gonorrhoeae strains were collected, 148 (18.8%) of which were AZM-R strains (MIC≥1 μg/ml).Eighteen out of 21 high-level AZM-R (AZM-HLR) strains had A2143G mutations in the four copies of the 23S rRNA gene.Twelve out of 29 middle-level AZM-R (AZ-MLR) strains had missense mutations, among which C2611T mutations in the four copies of the 23S rRNA were detected in 10 strains.Incidence of G45D/Y105H mutation in AZM-HLR strains was higher than that in AZM-MLR (χ2=12.702, P=0.000) or AZ-S (χ2=4.462, P=0.035) strains according to the analysis of the promoter and coding region of mtrR gene.PCR analysis revealed that only one strain carried ermB gene (MIC=2 μg/ml).The 788 N.gonorrhoeae strains were typed into 81 sequence types (STs) by NG-MAST, most of which were represented by one strain only.STs of ST3356 and ST1866 that were identified in the AZ-R strains in the current study had been noted in a previous report of emerging AZM-R N.gonorrhoeae strains in Nanjing, Chongqing and Guangzhou.Neighbor-joining (NJ) phylogenetic tree showed that the resistant strains did not form a separate cluster.Conclusion Currently, it is not suitable to use azithromycin as a monotherapy for gonorrhea in Shenzhen.Mutations of A2059G and C2611T in 23S rRNA of N.gonorrhoeae were respectively responsible for high-level and middle-level resistance to azithromycin.Repeated emergence of ST1866 and ST3356 will help us monitor and analyze the epidemiological characteristics of N.gonorrhoeae strains resistant to azithromycin in Shenzhen.