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目的:探讨铜绿假单胞菌(Pseudomonas aeruginosa,Pa)的耐药机制及其耐药性与外排泵表达水平关系。方法:PCR法对30株铜绿假单胞菌进行MexB基因检测;采用实时荧光定量PCR的方法检测MexAB-OprM的mRNA相对表达水平;对多重耐药铜绿假单胞菌采用琼脂对倍稀释法进行外排泵抑制剂的抑制试验,观察抗菌药物的MIC变化。结果:联合外排泵抑制剂后,庆大霉素(GEN)和环丙沙星(CIP)MIC值与之前相比大部分有2个折点的变化,有4株MPa的MIC值有4个折点以上的变化,但总体耐药率差异无统计学意义(P>0.05);30株多重耐药Pa株中,均存在外排泵Mex-AB-OprM的高表达,多重耐药菌组外排泵表达量达敏感组的36.5倍。当外排泵抑制剂CCCP浓度为7.5μg/ml时,庆大霉素(GEN)、环丙沙星(CIP)、四环素(TET)MIC值不变或增加的菌株(≥1MIC组)与MIC值减小的菌株(≤1/2MIC组)的外排泵相对表达量之间有着显著性的差异(P<0.05),而头孢噻肟(CTX)MIC值不变或增加的菌株(≥1MIC组)与MIC值减小的菌株(≤1/2MIC组)的外排泵相对表达量之间无显著性的差异(P>0.05)。结论:外排泵MexAB-OprM在多重耐药铜绿假单胞菌临床分离株中起着十分重要的作用,MexAB-OprM的高表达与多重耐药Pa菌庆大霉素、环丙沙星的耐药性有着密切关系,可能起着基础性耐药作用,但其对头孢噻肟的耐药性影响较小。其高表达还与庆大霉素、环丙沙星、四环素的耐药有着直接的量的关系,具体机制有待进一步的研究。
Objective: To investigate the relationship between drug resistance and efflux pump expression of Pseudomonas aeruginosa (Pa). Methods: MexB gene was detected in 30 strains of Pseudomonas aeruginosa by PCR. The mRNA expression of MexAB-OprM was detected by real-time fluorescence quantitative PCR. The multi-resistant Pseudomonas aeruginosa was amplified by agar dilution Inhibition of efflux pump inhibitors test to observe the MIC of antibacterial drugs. RESULTS: After combination of efflux pump inhibitors, MIC of gentamicin (GEN) and ciprofloxacin (CIP) had two breakpoints most of the time, 4 of which had MIC of 4 (P> 0.05). There was a high expression of Mex-AB-OprM in efflux pump in 30 multi-drug resistant Pa strains, and multi-drug resistant bacteria Group of efflux pump expression of 36.5 times the sensitive group. When the CCCP concentration of efflux pump inhibitor was 7.5μg / ml, the MIC values of GEN, ciprofloxacin (CIP) and tetracycline (TET) were the same as MIC There was a significant difference (P <0.05) between the relative expression levels of efflux pump in the strains with reduced value (≤1 / 2MIC group), while those with constant or increased MIC values in cefotaxime (≥1 MIC There was no significant difference (P> 0.05) between the relative expression of efflux pump in the group with MIC value (≤1 / 2MIC group). Conclusion: The efflux pump MexAB-OprM plays a very important role in clinical isolates of multi-drug resistant Pseudomonas aeruginosa. The high expression of MexAB-OprM is associated with multi-drug resistant Pa bacteria gentamicin, ciprofloxacin Drug resistance is closely related, may play a fundamental role in resistance, but its resistance to cefotaxime less. Its high expression with gentamicin, ciprofloxacin, tetracycline resistance has a direct quantitative relationship, the specific mechanism needs further study.