淋病奈瑟氏菌中国流行株gyrA和parC基因与喹诺酮类耐药性的相关性

Correlation of in vitro susceptibilities to quinolones of naturally occurring quinolone-resistant Neisseria gonorrhoeae strains isolated with changes in gyrA and parC in China

目的 调查中国地区淋病奈瑟氏菌 (NG)对喹诺酮类的耐药性状况 ,探讨高水平耐药的基因突变株对喹诺酮类耐药机制的作用 .方法 对 9a来临床分离保存淋球菌流行株进行了体外环丙沙星 (CIP)药敏实验 .筛选出 76株高水平CIP耐药株 ,通过 PCR扩增了其中的 1 8株 NG的 gyr A和par C喹诺酮耐药决定区基因 ,并对扩增子直接测序 .通过N he I酶切、脉冲电场凝胶电泳 (PFEG)分析了这些菌株的遗传关系 .结果 环丙沙星的耐药检出率有逐年增高的趋势 ,MIC≥ 1 .0 mg· L- 1的菌株由 1 993年的 5 .1 %上升到 2 0 0 1年的 2 1 .4% .对照组中的 1 8株敏感菌只有两株发生了 gyr A的单一突变 ,未发现 par C变异 ;而耐药组中 89% (1 6 )菌株的gyr A发生了 Ser- 91向 Phe的单一突变株或 (和 ) par C的双突变 .88.9% (1 5 )的菌株的 gyr A发生了 Ser- 91向 Phe和 Asp-95向 Gly的突变 .在发生 par C突变中 72 %属于 Asp- 86向Asn突变 .PFEG分析发现 1 0株具有完全一致的 gyr A和par C突变模式 .同时也发现来自不同地区的菌株的 PFEG指纹图是不同的 .结论 研究表明 ,在中国 NG流行株对喹诺酮类耐药率呈增长趋势 ;NG流行株对喹诺酮产生耐药性与gyr A和 par

AIM To investigate the status of Gonococcal fluoroquinolone resistance and analyse the contribution of mechanisms of quinolone resistance in N. gonorrhoeae . METHODS The 594 N. gonorrhoeae strains isolated in China from 1993 to 2001, were examined susceptible to ciprofloxacin. Seventy six strains were highly resistant, 18 N. gonorrhoeae strains were investigated for the frequency and patterns of mutations within the gyrA and parC genes. To identify mutations in the gyrA and parC genes of the gonococcal mutants, the quinolone resistance determining regions of the gyrA and parC genes were amplified by polymerase chain reaction (PCR) and the PCR products were directly sequenced. The strains were analyzed for genetic relationship by pulsed field gel electrophoresis (PFGE). RESULTS There was a trend of increasing detection rate of resistance strains every year, and the strains (MIC≥1 mg·L -1 ) increased from 5.1% to 21.4% during 1993 to 2001.In control group, only 2 strains exhibited single mutation of gyrA, not parC mutation. In resistance group, alterations at Ser 91 and Asp 95 in gyrA and a single or double alteration in parC were identified in 16 strains (89%). Ser 91 to Phe and Asp 95 to Gly alterations in gyrA were detected in 15 strains (88.9%). The most common parC alteration, Asp 86 to Asn, was found in 13 strains (72%). The strains were analyzed for genetic relationship by PFGE. The analysis showed 10 strains with the same mutation pattern in the gyrA and parC genes, originating from different geographical areas over nine years, had the same PFGE patterns after Nhe I digestion. CONCLUSION The results from this study suggest that resistance strains would be increasing in China; double gyrA mutations plus a parC mutation (s) play an important role in the development of high level fluoroquinolone resistance in N. gonorrhoeae .