某三级医院四环素耐药淋病奈瑟菌临床分离株的耐药机制及分子特征

Tetracycline-resistant Neisseria gonorrhoeae strains isolated in a Grade Ⅲ hospital in eastern China:molecular characteristics and genetic diversity

目的对中国东部温州地区淋病奈瑟菌流行菌株进行分型,并对四环素耐药的相关机制进行研究。方法从淋病患者中分离出77株淋病奈瑟菌,采用E-test法进行青霉素、四环素、环丙沙星、大观霉素、头孢曲松和阿奇霉素的最小抑菌浓度(MIC)测定。PCR和DNA测序用于检测四环素抗性相关基因,包括Tet-M基因、mtrR启动子区和mtrR编码区。多抗原序列分型(NG-MAST)和多位点序列分型(MLST)分别用于确定所有临床分离株和四环素耐药分离株的分子特征。结果在77株淋病奈瑟菌中,74株(96.10%)、27株(35.06%)、70株(90.91%)和15株(19.48%)分别对青霉素、四环素、环丙沙星和阿奇霉素耐药。所有测试的分离株对大观霉素和头孢曲松敏感。19株耐四环素菌株均存在Tet-M基因,其中17株mtrR启动区域发生1个碱基A的缺失突变,3株mtrR编码区域发生G45D突变。19株四环素耐药淋病奈瑟菌共测得11个不同的NG-MAST基因型,其中ST14781、ST1766和ST1866各占15.79%(均为3株),2个ST型(10.52%,2/19)未在NG-MAST数据库中报道过。19株四环素耐药淋病奈瑟菌共测序得到12个不同的MLST基因型别,其中ST10899占26.32%(5株),ST1600占15.79%(3株)。结论淋病奈瑟菌对四环素的耐药可能与mtrR启动区域和Tet-M基因的突变相关。NG-MAST基因分型结果显示温州地区淋球菌临床分离物表现出分子分型多样化。应采取措施监测温州地区具有高抗药性的NG-MASTST1766和MLSTST1600淋病菌克隆。

Objective To analyze the genotypes of Neisseria gonorrhoeae(N.gonorrhoeae)epidemic strains in Wenzhou,eastern China,and to study the mechanism of tetracycline resistance in these strains.Methods A total of 77 N.gonorrhoeae strains were isolated from patients with gonorrhea.Antimicrobial susceptibility of these strains to penicillin,tetracycline,ciprofloxacin,spectinomycin,ceftriaxone and azithromycin was analyzed using E-test.PCR and DNA sequencing were used to detect the genes associated with tetracycline resistance,such as Tet-M,mtrR promoter region and mtrR coding region.N.gonorrhoeae multi-antigen sequence typing(NG-MAST)and multilocus sequence typing(MLST)were used to determine the molecular characteristics of all clinical isolates and tetracycline-resistant isolates,respectively.Results Among the 77 N.gonorrhoeae isolates,74(96.10%),27(35.06%),70(90.91%)and 15(19.48%)were resistant to penicillin,tetracycline,ciprofloxacin and azithromycin,respectively.All tested isolates were susceptible to spectinomycin and ceftriaxone.Nineteen isolates were resistant to tetracycline and all of them carried Tet-M gene.Among them,17 had one deletion mutation of base A in mtrR promoter region and three had G45D mutation in mtrR coding region.NG-MAST classified the 19 tetracycline-resistant isolates into 11 different sequence types(ST).ST14781,ST1766 and ST1866 each accounted for 15.79%(three strains).Two ST(10.52%,2/19)found in the present study had not been reported previously in the NG-MAST database.MLST showed the 19 tetracycline-resistant isolates belonged to 12 different STs,in which ST10899 accounted for 26.32%(five strains)and ST1600 accounted for 15.79%(three strains).Conclusions Mutations in mtrR promoter region and carrying Tet-M gene were associated with tetracycline resistance in N.gonorrhoeae.Clinical strains isolated in Wenzhou showed considerable molecular diversity.Measures should be implemented to monitor the spread of NG-MAST ST1766 and MLST ST1600 N.gonorrhoeae clones with high resistance to tetracycline in Wenzhou.