主动外排系统介导的鲍曼不动杆菌耐药机制研究

发布时间：2018-12-20 09:31

【摘要】： [目的]了解临床分离54株鲍曼不动杆菌的耐药现状及对外排泵底物的耐药分析；探讨主动外排泵基因adeB、adeJ、abeM的在鲍曼不动杆菌中的分布及其mRNA表达水平与该菌耐药性的关系。 [方法]收集昆明医学院第一附属医院临床分离鲍曼不动杆菌54株,采用K-B法检测其对21种抗菌药物的药敏情况；用PCR方法扩增鲍曼不动杆菌外排泵编码基因adeB、adeJ、abeM的片段；荧光RT-PCR法检测外排系统基因mRNA的表达水平,采用分子克隆方法制作绝对定量标准品进行荧光定量,并分析外排系统基因mRNA的表达水平与该菌耐药性的关系。 [结果] 鲍曼不动杆菌的耐药检测及外排泵底物的药敏结果： 1、16种常用抗菌药物药敏结果显示：庆大霉素(GEN)、甲氧苄啶／磺胺甲恶唑(SXT)、哌拉西林(PIP)、头孢曲松(CRO)、四环素(TCY)、头孢他啶(CAZ)、头孢噻肟(CTX)、妥布霉素(TOB)、阿米卡星(AMK)的耐药率高达70%左右,而对亚胺培南(IMP)、美洛培南(MEM)、头孢哌酮／舒巴坦(CSL),仍保持较高的敏感性,其耐药率均低于15%。54株鲍曼不动杆菌耐5类抗菌药物菌株15株(27.8%)、耐4类药菌株15株(27.8%)、耐2类药菌株9株(16.7%)、耐1类药菌株9(16.7%)、敏感株6株(11.1%)。 2、54株鲍曼不动杆菌对三种外排泵底物共10种抗菌药物的耐药率依次从高到低为林可霉素(LIN)100.0%、利福平(RIF)98.2%、氯霉素(CHL)96.4%、庆大霉素(GEN)75.0%、四环素(TET)71.4%、红霉素(ERY)64.3%、阿米卡星(AMK)53.6%、妥布霉素(TOB)53.6%、环丙沙星(CIP)50.0%、左旋氧氟沙星(LVX)41.1%。外排蛋白编码基因adeB、adeJ、abeM的PCR扩增及RT-PCR定量其mRNA的表达水平与该菌耐药性分析结果： 1.adeB基因在54株鲍曼不动杆菌中49株(90.7%)检测到,未检测到adeB基因的5株菌(9.3%)中,有4株是敏感株,1株为耐1类抗菌药物株。多重耐药株adeB基因mRNA的表达水平与敏感株、非多重耐药株均有显著性差异(P0.05)；非多重耐药株与敏感株也有显著性差异(P0.05)。外排泵AdeB的底物：庆大霉素、四环素、环丙沙星、左氧氟沙星,妥布霉素和阿米卡星在本次研究中,其mRNA表达水平耐药株与敏感株之间有显著性差异(P0.05)。 2、ade J基因本次研究显示54株均检测到。其mRNA表达水平耐药株与敏感株之间无显著性差异(P0.05)。外排泵底物药物的耐药株与敏感株之间也无显著性差异(P0.05)。 3、abeM基因在54株中均检测到。其mRNA表达水平耐药菌株与敏感株有显著性差异(P0.05)。外排泵AbeM的底物中环丙沙星、左氧氟沙星耐药株与敏感株abeM基因mRNA表达水平有显著性差异(P＜0.001)。[结论]鲍曼不动杆菌耐药形式严峻；其临床分离株普遍存在外排泵编码基因adeB、adeJ和abeM;主动外排泵AdeABC和AbeM可能是鲍曼不动杆菌发生耐药的机制之一。
[Abstract]:[objective] to investigate the current situation of drug resistance of 54 strains of Acinetobacter baumannii and analysis of drug resistance of external pump substrates. To investigate the distribution of active efflux pump gene adeB,adeJ,abeM in Acinetobacter baumannii and the relationship between mRNA expression level and drug resistance of Acinetobacter baumannii. [methods] 54 strains of Acinetobacter baumannii isolated from the first affiliated Hospital of Kunming Medical College were collected and their susceptibility to 21 antimicrobial agents was detected by K-B method. The adeB,adeJ,abeM fragment of acinetobacter baumannii efflux pump coding gene was amplified by PCR. Fluorescence RT-PCR method was used to detect the expression level of efflux system gene mRNA, and molecular cloning method was used to make the absolute quantitative standard product for fluorescence quantification. The relationship between the expression level of efflux system gene mRNA and the drug resistance of the bacteria was analyzed. [results] the detection of drug resistance of Acinetobacter baumannii and the drug sensitivity of efflux pump substrates: 1. The results of 16 common antimicrobial agents showed that: gentamicin (GEN), trimethoprim / sulfamethoxazole (SXT), Piperacillin (PIP), ceftriaxone (CRO), tetracycline (TCY), ceftazidime (CAZ), cefotaxime (CTX), tobramycin (TOB), (AMK) resistance to imipenem (IMP), The sensitivity of (MEM), cefoperazone / sulbactam (CSL), was higher than 15.54 strains of Acinetobacter baumannii and 15 strains (27.8%) of Acinetobacter baumannii resistance. 15 strains (27.8%) were resistant to 4 kinds of drugs, 9 strains (16.7%) were resistant to 2 drugs, 9 (16.7%) were resistant to class 1, 6 strains (11.1%) were sensitive. (2) the drug resistance rates of Acinetobacter baumannii strains to three efflux pump substrates were lincomycin (LIN) 100.0g, rifampicin (RIF) 98.2i, chloramphenicol (CHL) 96.4B from high to low. Gentamycin (GEN) 75.0, tetracycline (TET) 71.4, erythromycin (ERY) 64.3, amicacin (AMK) 53.6, tobramycin (TOB) 53.6, ciprofloxacin (CIP) 50.0. Levofloxacin (LVX) 41.1. The results of PCR amplification and RT-PCR quantitative analysis of mRNA expression and drug resistance of efflux protein encoding gene adeB,adeJ,abeM showed that 1.adeB gene was detected in 49 strains (90.7%) of 54 Acinetobacter baumannii strains. Of the 5 strains (9.3%) with no adeB gene detected, 4 strains were sensitive and 1 strain was resistant to class 1 antimicrobial agents. The expression level of adeB gene mRNA in multidrug resistant strain was significantly higher than that in sensitive strain and non-multidrug resistant strain (P0.05), and there was also significant difference between non-multidrug resistant strain and sensitive strain (P0.05). In this study, the mRNA expression levels of gentamicin, tetracycline, ciprofloxacin, levofloxacin, tobramycin and amikacin were significantly different from those of sensitive strains (P0.05). 2Ade J gene was detected in 54 strains. There was no significant difference in mRNA expression between resistant and sensitive strains (P0.05). There was also no significant difference between the drug resistant strains and the sensitive strains of the efflux pump substrates (P0.05). The gene was detected in 54 strains. There was significant difference in mRNA expression between resistant strains and sensitive strains (P0.05). The mRNA expression of ciprofloxacin, levofloxacin resistant and sensitive AbeM substrates in efflux pump AbeM was significantly different (P < 0.001). [conclusion] the drug resistance of Acinetobacter baumannii is severe and the gene adeB,adeJ and abeM; active efflux pump AdeABC and AbeM may be one of the mechanisms of drug resistance of Acinetobacter baumannii.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378.99

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