福氏志贺菌喹诺酮类耐药临床分离株gyrA和parC的基因突变

Mutations in the gyrA and parC genes of quinolone-resistant Shigella flexneri clinical isolates

目的检测福氏志贺菌喹诺酮类耐药临床分离株DNA旋转酶gyrA和拓扑异构酶ⅣparC基因的突变情况,探讨GyrA和ParC氨基酸改变与喹诺酮类耐药的相关性。方法根据药敏实验结果选取47株福氏志贺菌临床分离菌,对其gyrA、parC喹诺酮耐药决定区(QRDR)进行聚合酶链反应(PCR)扩增和测序分析,并采用SAS(V8.2)软件分析GyrA和ParC改变和喹诺酮类耐药性的关联程度。结果44株喹诺酮类耐药菌在gyrA、parC基因QRDR均发生有义突变,gyrA83位密码子突变(TCG→TTG)最为常见,存在于43株耐药菌。混合模型分析结果显示GyrA83位、ParC80位氨基酸改变与萘啶酸的耐药性密切相关(P<0.01,R2=0.999),GyrA83、87位氨基酸改变与环丙沙星的耐药性密切相关(P<0.05,R2=0.872)。结论GyrASer83→Leu突变是导致福氏志贺菌临床株对喹诺酮类耐药的关键突变,此单位点突变即可介导福氏志贺菌对萘啶酸的耐药,但对环丙沙星耐药需同时存在GyrA和/或ParC多个位点突变或其他耐药机制。

Objective To identify the mutations of DNA gyrase gyrA and Topoisomerase Ⅳ parC genes in quinolone-resistant Shigella flexneri clinical isolates and evaluate the relevance of amino acid changes in GyrA and ParC to quinolone resistance. Methods Based on the antimicrobial susceptibility testing, 47 S. flexneri clinical isolates with different quinolone susceptibility were selected and the fragments including the quinolone resistance-determining region （QRDR） in gyrA and parC were PCR amplified and sequenced. SAS （V 8. 2） software was used to analyze the correlation be tween quinolone resistance and changes in GyrA and ParC. Results Sense mutation（s） in gyrA and parC were commonly observed in all of 44 quinolone-resistant isolates, whereas no sense mutation was found in the 3 quinolone susceptible on.es. The most frequent mutation is at codon 83（TCG→TTG） of gyrA, which was observed in 43 quinolone-resistant isolates. The mixed model analysis indicated that the alterations in amino acid 83 of GyrA and amino acid 80 of ParC are close related to nalidixic acid resistance （P〈0.01, R^2 =0. 999） and alterations in amino acid 83,87 of GyrA are related to ciprofloxacin resistance （P〈0.05, R^2 =0. 872）. Conclusions The substitution Leu to Ser83 in GyrA apparently showed to be the pivotal mutation resulting in quinolone resistance in S. flexneri clinical isolates tested here. This single mutation could lead to resistance to nalidixic acid, while to obtain resistance to ciprofloxacin, the presence of additional mutation（s） in GyrA and/or ParC or other resistance mechanisms are required.