临床分离铜绿假单胞菌环丙沙星体外诱导相关耐药基因分析

The muLti-resistant mechanism of clinic Pseudomonas aeruginosa induced by ciprofloxacin in vitro

目的利用环丙沙星诱导全敏感铜绿假单胞菌临床分离株耐药后,检测前后突变频率的变化,分析耐药率的变化及其耐药机制的研究。方法收集临床分离的全敏感铜绿假单胞菌28株,采用低浓度环丙沙星体外诱导方法诱导其对环丙沙星耐药,用E-test检测抗菌药物的MIC值变化,检测诱导前后突变频率,采用PCR方法检测gyrA、parC和oprD并测序分析,采用qRT-PCR方法检测外排泵mexAB-oprM、mexCD-oprJ和mexXY-oprM诱导前后的mRNA表达水平。结果诱导后有24株菌发生了环丙沙星耐药、亚胺培南、左氧氟沙星、头孢吡肟及头孢噻肟耐药率明显上升,其中有6株细菌转变为多重耐药菌,诱导后突变频率明显高于诱导前(t=-2.57,P=0.01),有6株耐药菌发生了gyrA Thr83(ACC)→Ile(ATC)替换,诱导后有15株菌外排泵mexAB-oprMmRNA高表达,17株菌外排泵mexCD-oprJ高表达,5株菌外排泵mexXY-oprM高表达,6株菌株oprD低表达,且皆发生了多位点核苷酸突变。结论环丙沙星可诱导铜绿假单胞菌多重耐药,并产生oprD广泛有意突变和外排泵高表达对亚胺培南、环丙沙星等抗菌药物耐药,菌株突变频率诱导前后发生明显变化。

Objective Using the ciprofloxacin to induce the pan-susceptible pseudomonas aeruginosa isolated from clinc into resistant isolates, we detected the change of mutation frequency before and after induction, and analysed the change of the resistant rate and study the resistant mechanism. Methods A total of twenty-eight pan- susceptible pseudomonas aeruginosa isolate were collected from clinic, the vitro induce methods were used to change them into resistant to ciprofloxacin, and the E-test method were used to determinate the MIC of these strains to several antibiotics. We detected the mutation frequency of the strains before or after induction. The gene gyrA, parC and oprD were detected by PCR. PCR products were purified and sequenced. The efflux pump mexAB-oprM, mexCD-oprJ and mexXY-oprM mRNA expression were detected by qRT-PCR before or after induction. ResuLts The resistant rate of twenty-four strains to ciprofloxacin, imipenem, levofloxacin, meropenem, cefepime and cefotaxime were distinctly increased. There were six strains changed into muLti-drug resistant bacteria. The mutation frequency after induction was obviously higher than before. There were six strains which the Thr83（ACC） ofgyrA was substitute for lle（ATC）. After induction, there were fifteen strains high expression of mexAB-oprM, seventeen strains high expression of mexCD- opt J, five strains high expression of mexXY-oprM. There were six strains low expression of oprD, and all oprD gene mutations were wide and diverse. Conclusions Ciprofloxacin can induce pseudomonas aeruginosa into the multi- drug resistant strains. Most of them were resuLted from the wide oprD gene mutations and high expression of efflux pump. The mutation frequency was increased obviously after induction.