泛耐药铜绿假单胞菌体外耐药模型构建及耐药机制的研究

目的 通过多种抗菌药物体外诱导构建泛耐药铜绿假单胞菌(PDRPA)体外模型,检测其相关的耐药机制;探讨联合抗菌药物体外诱导对铜绿假单胞菌( PAE)耐药性的影响.方法 采用梯度浓度琼脂平皿传代诱导法,将4株细菌接种于梯度浓度的药物平皿,直至对该药产生耐药,诱导前后测定细菌的敏感性,采用E-test法对诱导前的敏感株、诱导的泛耐药株以及临床分离的泛耐药菌株进行MBL检测；采用Real-time PCR技术,对诱导前的4株敏感PAE和诱导后的4株PDRPA以及4株临床分离的PDRPA进行主动外排系统的检测,包括:MexAB-OprM、MexCD-OprJ、MexEF-OprN、MexXY-OprM.结果 经诱导后4株敏感PAE对抗菌药物的敏感性下降,MIC明显升高,按照CLSI耐药折点4株菌株全部转变为泛耐药菌株,4株临床分离的敏感PAE株诱导前后MBL检测均为阴性;临床分离的4株PDRPA株MBL检测有2株为阳性,诱导前的4株敏感PAE,4个外排系统均无高表达；相对于诱导前,经过诱导成为泛耐药菌株之后,4株耐药诱导株均存在MexA-OprM外排系统高表达,有1株存在MexCD-OprJ和1株MexEF-OprN高表达,全部菌株均无MexXY-OprM高表达；在临床分离的PDRPA中,有1株所有4个主动外排系统均高表达,有1株只有MexAB-OprM高表达,另外3个外排系统无高表达,另外2株4个外排系统均无高表达.结论 在体外抗菌药物的压力下,敏感的PAE可以经诱导变为PDRPA,耐药性稳定；体外诱导的PDRPA非产金属β-内酰胺酶,其耐药性的产生可能与主动外排泵有关,尤其是mexAB-OprM.

Construction of in vitro resistance model of pan-resistant Pseudomonas aeruginosaand investigation of its resistance mechanism

OBJECTIVE To establish in vitro model of pan-resistant Pseudomonas aeruginosa(P.aeruginosa) by in vitro induction of multiple antibiotics and detect its resistance mechanism;to discuss the effect of in vitro induction of combined antibiotics on the drug resistance of P.aeruginosa.METHODS By gradient plate method,4 isolates were inoculated on the agar plates carrying drug of gradient concentrations until the emergence of resistance to this drug.The bacterial susceptibility before and after the induction was determined;the MBL detection was performed using E-test method in the susceptible isolates before induction,the induced pan-resistant isolates,and pan-resistant clinical isolates.The detection of active efflux pumps system of the 4 susceptible isolates before induction,the 4 pan-resistant P.aeruginosa isolates after induction,and 4 clinical isolates was performed by real-time PCR.The four efflux pumps included MexAB-OprM,MexCD-OprJ,MexEF-OprN and MexXY-OprM.RESULTS The susceptibility of 4 susceptible P.aeruginosa isolates to 6 antibiotics after induction decreased,and the value of MIC for 6 antibiotics increased markedly.According to the breaking points of CLSI,all of the 4 strains became pan-resistant.MBL detections were all negative for 4 clinical susceptible P.aeruginosa isolates before and after induction;2 of 4 clinical pan-resistant P.aeruginosa isolates were positive for MBLdetection,and the other 2 strains were negative.Of the 4 susceptibleP.aeruginosa isolates before induction,there were no high expression of 4 efflux pumps;compared with that before the induction,there were high expression of mexA-OprM in 4 induced resistant isolates,high expression of mexCD-OprJ in 1 isolate,high expression of mexEF-OprN in 1 isolate,but high expression of mexXY-OprM were found in none of the isolates.Of all the clinical isolates of pan-resistantP.aeruginosa,there were high expression of 4 efflux pumps in 1 isolate,mexAB-OprM only in 1 isolate without high expression of other 3 efflux pumps,and no high expression of 4 efflux pumps in the other 2 isolates. CONCLUSION Under the pressure of in vitro antibiotics,the drug resistance of pan-resistantP.aeruginosa resulted from the induction of susceptible isolates is stable;pan-resistantP.aeruginosa induced in vitro do not produce metallo-beta-lactamases,therefore the emergence of drug resistance maybe related to active efflux pumps,in particular MexAB-OprM.