铜绿假单胞菌对环丙沙星的抗药性和耐药性机制研究  

Resistance and tolerance of Pseudomonas aeruginosa to ciprofloxacin

作　　者：高超[1,2] 胡明[2] 白华[2] 齐静[2] 朱小玲[2] 刘昌彬[3] 单虎[1] 秦晓冰[1] 刘玉庆[1,2] 高培基[3] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]山东省农业科学院畜牧兽医研究所,山东省畜禽疫病防治与繁育重点实验室,济南250100 [3]山东大学微生物技术国家重点实验室,济南250100

出　　处：《山东大学学报（医学版）》2011年第6期38-45,共8页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助项目(30770044);山东省中青年科学家科研奖励基金资助项目(2006BS02008);中国博士后科学基金资助项目(20080440451)

摘　　要：目的研究铜绿假单胞菌对环丙沙星的抗药性(resistance)和耐药性(tolerance)机制,并予以区分。方法分离养殖场和医院铜绿假单胞菌33株,用微量肉汤稀释法测定铜绿假单胞菌临床分离株对喹诺酮类抗生素的最低抑菌浓度(MIC),筛选喹诺酮抗药菌5株;体外连续培养诱导铜绿假单胞菌标准菌株P.aeruginosa ATCC27853,得到对环丙沙星和头孢噻肟的高抗药性菌株PA34。检测及分析铜绿假单胞菌喹诺酮耐药决定区(QRDR)的基因突变;荧光比色法检测不同抗药性表型的铜绿假单胞菌外排泵活性;荧光定量RT-PCR法检测铜绿假单胞菌外排泵、跨膜转运蛋白及喹诺酮靶酶编码基因的mRNA表达水平。结果 临床分离的5株喹诺酮抗药表型的铜绿假单胞菌,其外排泵活性均显著高于标准菌株(P<0.01),外排泵、喹诺酮靶位酶编码基因的mRNA的表达量上调、跨膜转运基因表达量下调,DNA旋转酶A亚基gyrA基因均发生点突变Thr-83→Ile。PA34和这5株抗药性菌株表现一致,但其MIC远高于这5株抗药性菌株,主要由于PA34除了gyrA基因发生点突变Thr-83→Ile外,parC基因发生多点突变Glu-84→Gln、Gln-91→Lys,且跨膜转运基因表达量极低。结论 在喹诺酮类抗生素选择压力下,铜绿假单胞菌能发展多种可能的机制以消除抗生素对细菌代谢的损伤,对类似gyrA、parC基因突变称为抗药性,其他生理性适应机制称为耐药性,其抗药性突变和耐药性调节共同决定了抗药性表型,但对其加以区分并分别研究,更容易集中研究单纯的抗药性突变机制,采取相应的用药措施。Objective To study and discriminate the resistance and tolerance of P. aeruginosa to ciprofloxacin. Methotis MICs of fluoroquinolones against 33 strains of P. aeruginosa, isolated from farm and hospital together with P. aeruginosa ATCC 27853 and its high resistant mutant strain PA34 consecutively induced by ciprofloxacin and cefo-tarqme in vitro, were determined with broth microdilution method. Among the clinic strains, and five multi-drug resistant strains were selected for the following study. The resistance-determining region （QRDR） was sequenced to determine the mutations for fluoroquinolone resistance. The activities of the efflux pump in different resistant phenotypes of P. aeruginosa were detected by fluorescent colorimetric method, also mRNA expression of the efflux pump, the transmembrane transporter protein and fluoroquinolones target enzymes were detected by the fluorescence quantitative PCR method. Result Compared with that of ATCC 27853, the activities of the efflux pumps of the five clinic resistant strains of P. aeruginosa remarkably increased（ P 〈0.01 ）, and efflux pumps and fluoroquinolones target enzymes were over-expressed while mRNA expression of the trans-membrane transporter protein comparatively declined. There was a point-mutation in gyrA at Thr-83→Ⅱe for each of the five resistance phenotypic strains. However, their MICs were far below that of the induced super high-esistant mutant strain PA34 in which the gyrA mutation occurred at Thr-83→Ⅱe, parC mutations occurred at Glu-84→Ghi,Ghi-91→Lys and mRNA expression of the transmembrane transporter protein declined in the extreme. Conclusion To resist fluoroquinolones, P. aeruginosa can develop any possible phenotype, which can be genitically disciiminated as resistance （gyrA, parC mutation） or tolerance （adaptive expression of efflux pumps protein, the tran-smembrane transporter protein and fluoroquinolones target enzymes）. The new evolutionary taxonomy facilitates the focus on the simplex study of resistance muta

关 键 词：铜绿假单胞菌 喹诺酮 耐药性 抗药性 

分 类 号：Q931[生物学—微生物学]