碳青霉烯类抗生素诱导铜绿假单胞菌外膜蛋白及外排系统突变的机制研究  

A study on the mechanisms of carbapenems-induced gene mutations of outer membrane proteins and efflux systems in Pseudomonas aeruginosa

作　　者：曾章锐[1] 王卫萍[1] 王莹[1] 邵海枫[1] 

机构地区：[1]南京军区南京总医院临床中心实验科微生物室,南京210002

出　　处：《临床检验杂志》2013年第6期450-454,共5页Chinese Journal of Clinical Laboratory Science

摘　　要：目的探讨临床分离的因外膜蛋白突变导致的铜绿假单胞菌(Pa)碳青霉烯类耐药机制是否与药物诱导相关。方法在M-H平板上用亚胺培南(IPM)和美罗培南(MEM)药敏纸片分别诱导3株Pa(Pa标、Pa1和Pa2)。琼脂稀释法测定诱导前后Pa对IPM、MEM、头孢他啶和头孢吡肟4种药物的最低抑菌浓度(MIC)。改良三维试验检测诱导后产碳青霉烯酶变化。SDS-PAGE观察Pa外膜蛋白OprD的改变。PCR扩增oprD基因并进行序列分析。实时荧光定量PCR检测Pa外排系统表达情况。并对诱导耐药菌株进行稳定性分析。结果诱导第5天出现对IPM和MEM耐药(MIC≥8μg/mL),且MEM诱导株对2种药物敏感性降低速度明显快于IPM诱导株。诱导株未检出耐药酶,外膜蛋白OprD出现缺失或者减少。oprD基因测序发现Pa标(I)、Pa标(M)和Pa1(M)分别在831(TGG→TGA)、1 017(TGG→TGA)和1 014(TGG→TAG)有碱基突变产生终止密码子,Pa2(I)在1 206处有新的碱基插入导致移码突变。外排基因以MexA过度表达为主,同时伴有其他外排系统的过度表达。稳定性分析发现5株诱导株的耐药表型稳定,另1株外排系统表达量呈现持续升高,导致MIC值的右移。结论在碳青霉烯类药物环境中,外膜蛋白OprD表达减少和基因的突变以及外排系统的过度表达是引起诱导株对IPM和MEM耐药的主要原因。此型耐药株不仅耐药表型稳定,甚至可能由于某种机制导致脱离药物后MIC值仍持续升高。Objective To investigate the relationship between carbapenems resistance and outer membrane proteins gene mutation in clinical isolates of carbapenems-induced Pseudomonas aeruginosa （Pa） in vitro. Methods Imipenems （IPM） and meropenems （MEM） susceptibility test discs were used to induce 3 sensitive strains of Pa on Muller-Hinton agar. Agar dilution method was used to determine the minimal inhibitory concentrations （MIC） of 4 antibiotics （IPM, MEM, cefepime and eeftazidime）for all the 3 strains of Pa before and after induction. Carbapenemases were detected by the modified three-dimensional test. SDS-PAGE, PCR, DNA sequencing and real-time reverse transcriptase PCR were used to analyze the mechanisms of earbapenems resistance in Pa. Results The imipenem- and meropenem-induced strains presented resistance （ MIC≥ 8 μg/mL） in the 5 day after induction, and the decrease of susceptibility of IPM-induced strains to the 2 drugs was faster than the MEM-induced strains. The strains induced by the three-dimensional test did not produce carbapenemase. All of the induced strains exhibited loss or reduction of outer membrane proteins OprD which was demonstrated by SDS-PAGE. By oprD sequencing it was observed that base mutations leaded to presence of terminator codons （ TGG→TGA at 831 in Pa ATCC 27853 （I）, 1 017 in Pa ATCC 27853 （M） , and 1 014 in Pal （M） and base insertions leaded to frameshift mutations at 1 206 point. In the expression of efflux system genes mexA overexpression was prevalent with the presence of overexpression of other genes. In the stability analysis stable phenotype of resistance presented in 5 induced strains, but continuous rise of expression of efflux pumps was exhibited in a strain, which leaded to right shift of the MIC. Conclusion In the carbapenems environment, decreased expression of OprD, mutations of genes and overexpression of efflux systems should be the main mechanisms leading to the resistance of the induced strains to imipenems and meropenems. The ea

关 键 词：碳青霉烯类 铜绿假单胞菌 外膜蛋白 OPRD 外排系统 

分 类 号：R446.5[医药卫生—诊断学]