耐喹诺酮类大肠埃希菌耐药机制的研究

Mechanisms of Quinolones Resistance in Escherichia coli

目的探讨耐喹诺酮类大肠埃希菌的耐药机制。方法收集天津市第一中心医院2004年3月-2005年10月临床分离的大肠埃希菌,并随机选取40株作为研究对象,通过K-B药片试纸法和微量稀释法测40株目的菌株的耐药性;PCR扩增耐药株的gyrA QRDR区和parC基因,进行PCR-SSCP分析;同时,PCR扩增marOR基因,在耐药株中随机选取进行测序,检测marOR基因突变情况。结果37株对耐喹诺酮类菌株均出现了gyrA基因突变,除常见氨基酸改变外,还发现Asp87→Asn、Ala84→Pro;36株耐喹诺酮菌株发生了parC基因突变,只对萘啶酸耐药,对环丙沙星中介、氧氟沙星、加替沙星敏感的ECO24未出现parC基因突变;只对氟喹诺酮类耐药的ECO11未出现marOR区突变;存在多重耐药的ECO5 marOR区发现6处突变,且1 879 bp处的突变改变了marOR的终止密码子。结论gyrA和parC基因突变引起大肠埃希菌产生耐药,gyrA基因突变是产生对氟喹诺酮类耐药的主要原因,parC基因突变可引起菌株对氟喹诺酮类药物的高水平耐药,marOR的多位点突变在多重耐药机制中有一定的作用。

OBJECTIVE To study the mechanisms of quinolones resistance in Escherichia coli, METHODS Forty E. coli clinical isolates were randomly collected from clinical specimens at the Tianjin First Central Hospital from Mar 2004 to Dec 2005. Then we detected the susceptibility to antibiotics in 40 clinical isolates of E, coli by MICs and K-B disk diffusion method. In order to investigate the mutations in the target genes, we amplified the QRDR of gyrA and parC by PCR. Later we analyzed the PCR products by single strand conformation polymorphism analysis（SSCP）. In the meantime, the PCR products of marOR region were sequenced to detect the possible gene changes which contributed to the increasing expression of MarA and then lead to the Mar phenotype. RESULTS The alterations in gyrA were found in all quinolones-resistante strains, Asp87→→Asn and Ala84→Pro were found besides the common amino acid alteration. The alterations in parC were found in thirty-six strains resistant to quinolones, There were no parC alterations in ECO24 which was nalidixic acid-resistant and ofloxacin/gatifloxacinsusceptible. ECO11 Which was resistant to quinolones only had no gene changes in marOR region. Six gene changes in marOR region were found in ECO5 which was resistant to mutiple antibodies. The alteration in 1879 bp changed the terminator. CONCLUSIONS The alterations in gyrA and parC are responsible for the resistant phenotypes in E. coll. That is, the alterations in the gyrA are primarily responsible for resistance to quinolones, and the alterations in the parC may play a complemental role in enhancing resistance to fluoroquinolones. Moreover, the randomly collected strains resistant to quinolones, have found some mutations in marOR. It may be play certain roles in multiple antibiotic resistance of E. coll.