大肠杆菌acrR插入序列对acrAB表达水平及多重耐药性的影响

Effect of insertion within acrR gene on acrAB expression and multiple resistance in an Escherichia coli isolate

为研究大肠杆菌(E.coli)分离株ED28的acrR基因中777bp插入片段对外排泵AcrAB表达水平及多重耐药性的影响,本研究将野生型acrR通过互补实验导入ED28中得到C-ED28。通过药物的最低抑菌浓度(MIC)测定、有机溶剂耐受性试验和实时定量PCR(qPCR)等方法检测菌株中AcrAB外排泵的活性。通过PCR方法检测ED28和C-ED28喹诺酮耐药决定区(QRDR)的氨基酸突变及携带的质粒介导喹诺酮类耐药(PMQR)基因和超广谱β-内酰胺酶(ESBLs)耐药基因。结果显示,临床分离菌株ED28对12种抗生素呈多重耐药表型,而互补菌株C-ED28恢复了对多种药物的敏感性。当能量抑制剂(CCCP)存在时,喹诺酮类和β-内酰胺类药物对ED28的MIC普遍降低,而对C-ED28的MIC保持不变。ED28对染料溴化乙锭(EB)的MIC为512μg/mL,对正己烷和环己烷耐受;而C-ED28对染料EB的敏感性增强,MIC为16μg/mL,对正己烷耐受,对环己烷不耐受。qPCR结果显示,基因acrA和acrB在C-ED28中的表达水平低于亲本菌株ED28,而基因marA在C-ED28中的表达水平高于ED28。靶位突变检测和耐药基因检测结果显示,ED28存在gyrA(Ser83Leu和Asp87Asn)和parC(Ser80Ile)双突变,携带aac(6')-Ib-cr、blaCTX-M-27和blaTEM-13个质粒编码耐药基因;而C-ED28仅存在gyrA(Asp87Asn)一个突变位点,并丢失aac(6')-Ib-cr、blaTEM-1两个基因。

To investigate effect of a 777bp fragment insertion within acrR gene on multiple resistance in an Escherichia coli isolate, the E.coli C-ED28 was constructed from wild type E.coli ED28 by the genetic complementation test and the activity of effiux pump AcrAB-TolC in C-ED28 was detected by minimal inhibition concentration （MIC）, organic solvent tolerance （OST） and real time PCR. In addition, PCR method was used to detect the mutations in quinolone resistance determining region （QRDR）, the presence of plasmid mediated quinolone resistance （PMQR） and extended spectrum β-1actamases （ESBLs）. The results showed that the C-ED28 with acrR mutation restored susceptibility to all tested antibiotics. The MIC of ED28 to fluoroquinolones and β-Lactamases were decreased in the presence of CCCP, however, it was no change in C-ED28. ED28 was able to effectively extrude ethidium bromide and grow in the presence of cyclohexane, but not C-ED28. Complementation with wild type acrR decreased the expression of acrA and acrB, and increased the expression of marA, compared with that of the parental strain ED28. Double mutations were detected within QRDR of gyrA （SerS3Leu and Asp87Asn） and parC （Senile） in the ED28 carrying three plasmid borne resistant genes aac（6＇）-Fo-cr, blacrx-M-27, blaTEM-1 While only one mutation gyrA （Asp87Asn） was detected in C-ED28, and of two plasmid borne resistant genes, aac（6＇）-Po-cr and blare1 were lost.