淋球菌对氟喹诺酮类药物敏感性及其耐药机制的研究(英文)

Study on fluoroquinolone susceptibility and resistance mechanism in clinical isolates of Neisseria gonorrhoeae

目的了解长沙地区淋球菌流行株对氟喹诺酮类药物的耐药现状,探讨淋球菌对氟喹诺酮类药物耐药的分子机制。方法纸片扩散法检测126株淋球菌对4种氟喹诺酮类药物的敏感性。E-test法定量检测其中63株淋球菌环丙沙星的最小抑菌浓度(MIC)。对淋球菌喹诺酮类药物作用靶位编码基因gyrA和parC的喹诺酮耐药决定区(QRDR)进行PCR扩增和直接测序分析。结果淋球菌对氧氟沙星、氟罗沙星、洛美沙星、依诺沙星耐药率分别为70.6%、81.7%、72.2%和82.5%。环丙沙星MIC为0.004～12.0μg/mL,耐药率65.1%。对环丙沙星敏感的菌株gyrA和parC基因均未发生突变,而中介或耐药菌株均出现gyrA突变或同时伴有parC突变,且所有parC突变均发生在gyrA突变基础上。突变位点包括gyrA上Ser91→Tyr,Ser91→Phe,Asp95→Gly,Asp95→Ala,Asp95→Asn以及parC上Gly85→Cys、Asp86→Asn、Ser87→Arg、Ser87→Ile、Ser87→Asn、Ser88→Pro、Glu91→Lys、Glu91→Gly。其中以gyrASer91→Phe频率最高。结论长沙地区淋球菌流行株对氟喹诺酮类药物耐药已十分严重。淋球菌对氟喹诺酮类药物耐药与gyrA和parC基因突变密切相关;GyrASer91→Phe是形成该耐药性的关键突变。多位点突变具有协同作用,可使耐药性进一步增强。

[Objeetive] To evaluate the situation of Fluroquinolone resistance in Neisseria Gonorrhoeae in Changsha, P.R.China and to investigate the molecular mechanisms of Fluoroquinolone resistance in clinical isolates of Neisseria Gonorrhoeae. [Methods] The susceptibilities of 126 clinical isolates of N.gonorrhoeae to 4 Fluorcquinolones were tested by disc diffusion method. The minimum inhibitory concentration （MIC） of Ciprofloxacin was determined by E-test method in 63 strains. The fragments including quinolone resistance-determining region （QR- DR） were amplified by PCR in gyrA and parC genes, and their relative fragments were directly sequenced. [Resuits] The percentages of resistance to Ofloxacin, Fleroxacin, Lomefloxacin, and Enoxacin were 70.6%, 81.7%, 72.2% and 82.5% respectively. Range of ciprofloxacin MIC was 0.004-12.0 μg/mL in 63 strains. Percentage of resistance was 65.1%. Ciprofloxacin-susceptible strains contained no mutation in gyrA and parC genes,while intermediate or resistant strains contained mutations in gyrA gene, and some contained mutations in parC gene simultaneously. All parC gene mutations were found on the basis of mutations in gyrA gene. These amino substitutions included Sergl→Tyr, Ser91→Phe, Asp95→Gly, Asp95→Ala, Asp95→Asn in GyrA and Gly85→Cys, Asp86→Asn, Ser87→Arg, Ser87→Ile, Ser87→Asn, Ser88→Pro, Glu91→Lys, Glu91→Gly in ParC. Of all the mutations in gyrA gene,the percentage of substitution of Ser91→Phe was the highest. [Conclusions] Fluoroquinolone resistanee in N. gonorrhoeae in Changsha, P.R.China is very severe at present. Fluoroquinolone resistance in N.gonorrhoeae has close relationship with mutations in gyrA and parC genes. Substitution of Ser91→Phe in gyrA gene is the pivotal mutation resulting in fluoroquinolone resistance in N.gonorrhoeae. The eooperation of multi-point mutations makes the resistance stronger.