生物膜铜绿假单胞菌多药外排泵的表达研究

Expression of Multi-drug Efflux Pump Gene in Pseudomonas aeruginosa Biofilms

目的研究铜绿假单胞菌在浮游态、生物膜态及经亚胺培南、环丙沙星诱导后,多药外排泵MexAB-OprM及其阻遏蛋白MexR基因表达情况。方法用铜绿假单胞菌PAO1在eppendorf管内建立生物膜;用1/2 MIC亚胺培南、环丙沙星对浮游菌诱导4 h;4 MIC亚胺培南2、MIC环丙沙星对生物膜菌诱导12 h,分别于生物膜形成的第35、天,用实时荧光定量PCR的方法,测定铜绿假单胞菌外排泵MexAB-OprM及MexR的基因表达。结果铜绿假单胞菌由浮游菌变为生物膜菌后,其外排泵MexAB-OprM的表达水平明显降低,其相对表达量最高相差近10倍,其差异有统计学意义(P<0.05);铜绿假单胞菌由浮游菌变为生物膜菌后,阻遏蛋白MexR表达差异无统计学意义;在亚胺培南及环丙沙星诱导后,浮游菌和生物膜菌其基因MexAB-OprM、MexR表达差异均无统计学意义。结论铜绿假单胞菌由浮游菌转变为生物膜菌后,外排泵MexAB-OprM表达明显减少,MexR基因表达无影响;亚胺培南、环丙沙星对浮游菌及生物膜菌的MexAB-OprM、MexR均无诱导作用。

OBJECTIVE To investigate the expression of MexAB-OprM and its repressor protein-MexR in planktonic and biofilm Pseudomonas aeruginosa（PAE）.METHODS P.aeruginosa PAO1 was used in this study.PAO1 biofilm were built up in eppendorf tube in vitro.The planktonic PAE were exposed to 1/2 MIC of imipenem and ciprofloxacin for 4 hours.The biofilm PAE was exposed to 4 MIC imipenem and 2 MIC ciprofloxacin for 12 hours,the expressions of multiple drugs efflux pumps MexAB-OprM and MexR were detected by real-time quantitative PCR at the day 3 and 5 after biofilm formation.RESULTS Expression of efflux pump MexAB-OprM gene was significantly decreased（P〈0.05）with the formation of biofilm as compared to the plantonic one,reaching its lowest expression nearly 10 folds decrease.There was no difference between planktonic and bioiflms in the expression of repressor protein MexR.No significant difference was found in the expression level of MexAB-OprM and MexR with the induction of imipenem and ciprofloxacin between planktonic and biofilms PAE.CONCLUSION Formation of PAE biofilm can decrease the expression of efflux pump MexAB-OprM gene with no effect on MexR gene.Imipenem and ciprofcoxacin showed no effect on the expression of MexAB-OprM and MexR gene in either planktonic or biofilm PAE.