全耐药铜绿假单胞菌中多药外排泵mRNA表达的定量RT-PCR检测

目的分析4种重要的药物外排泵系统在临床分离的全耐药铜绿假单胞菌与全敏感铜绿假单胞菌中表达的差异。方法用SYBR G reenⅠ实时定量RT-PCR方法检测临床分离鉴定的全耐药和全敏感铜绿假单胞菌中4种外排泵M exAB-OprM、M exCD-OprJ、M exEF-OprN、M exXY-OprM的mRNA。结果全耐药菌株较全敏感菌株多药外排泵M exAB、M exXY、M exCD、M ex-EF的mRNA表达明显增高(P<0.01)。以敏感菌株拷贝平均数×4判定明显高表达,则全耐药菌M exAB高表达率为18.9%;M exXY高表达率为13.5%;M exCD高表达率为56.8%;M exEF高表达率为35.1%。37株全耐药菌有1种外排泵高表达的8株;2种外排泵高表达的10株;3种外排泵高表达的4株;4种外排泵同时高表达的2株,4种外排泵表达均未增高的有13株。结论药物外排泵系统在临床全耐药铜绿假单胞菌耐药机制中起着重要作用,研究结果提示可能还存在其他耐药机制或存在外排泵功能与其表达量存在差异的情况。

Measurement for mRNA levels of efflux pumps in clinical stains of Pseudomonas aeruginosa by real-time RT-PCR

Objective To evaluate the role of multi-drug efflux pumps system involved in the resistance of pan-time pseudomonas aeruginosas to antibiotics.Methods Established SYBR-Green I real-time RT-PCR method was employed to quantify the mRNA levels of MexAB-OprM,MexCD-OprJ,MexEF-OprN,MexXY-OpM and the differences of mRNA levels between pan-resistant P.aeruginosas and susceptible p.aeruginosas were compared.Results The mRNA levels of MexAB-OprM,MexCD-OprJ,MexEF-OprN,MexXY-OprM in pan-resistant P.aeruginosas were higher than those in susceptible P.aeruginosas(P<0.01).The percentage of overexpression was 18.9% in MexAB-OprM,13.5% in MexCD-OprJ,56.8% in MexEF-OprN and 35.1% in MexXY-OprM respectively when the cutoff value was defined as four times of the mean value of susceptible P.aeruginosas.Conclusion The efflux pump system may play a key role in the resistance of pan-resistant Pseudomonas aeruginosas to antibiotics.