Molecular cloning and characterization of SmrA, a novel ABC multidrug efflux pump from Stenotrophomonas maltophilia
Molecular cloning and characterization of SmrA, a novel ABC multidrug efflux pump from Stenotrophomonas maltophilia
复制标题
DOI：
10.1093/jac/dkp271
复制
发表时间：
2009-10-01
影响因子：
5.2
通讯作者：
Burnie, James
中科院分区：
文献类型：
--
作者：
Al-Hamad, Arif;Upton, Mathew;Burnie, James
目的：嗜麦芽寡养单胞菌是一种新兴的医院病原体，可引起难以治疗的感染，并表现出严重程度的多药耐药性（MDR），而人们对此知之甚少。本研究的目的是鉴定和表征嗜麦芽糖链球菌中的多药 ATP 结合盒 (ABC) 外排泵。方法：基于 ABC 转运蛋白保守基序的检测以及与实验证明的 MDR ABC 转运蛋白的比对，在嗜麦芽糖链球菌基因组中鉴定了 SmrA。 smrA 基因在高敏感性 acrAB 突变型大肠杆菌菌株 SM1411 中克隆并表达。使用 Stokes 圆盘扩散和肉汤微量稀释 MIC 方法测试了对几种抗菌药物的耐药性。使用荧光方法进行诺氟沙星累积和外排测定，使用和不使用外排泵抑制剂 O-钒酸钠和利血平。 结果：大肠杆菌中 smrA 的克隆和表达赋予了对结构不相关化合物（包括氟喹诺酮类、四环素、阿霉素和多种染料）增强的耐药性。此外，大肠杆菌中smrA的表达减少了诺氟沙星的摄取并增强了其外排，这些特征可以被ABC外排泵抑制剂抑制。结论：SmrA是ABC多药外排泵家族的成员。这些发现值得进一步研究该分子在嗜麦芽糖链球菌分离株中的作用，以估计该系统对抗菌素耐药性的潜在影响。
Objectives: Stenotrophomonas maltophilia is an emerging nosocomial pathogen that can cause difficult-to-treat infections and exhibits significant degrees of poorly understood multidrug resistance (MDR). The aim of this study was to identify and characterize a multidrug ATP-binding cassette (ABC) efflux pump in S. maltophilia.Methods: SmrA was identified in the S. maltophilia genome based on the detection of ABC transporter conserved motifs and alignment with experimentally proven MDR ABC transporters. The smrA gene was cloned and expressed in the hypersusceptible acrAB mutant Escherichia coli strain SM1411. The resistance to several antimicrobial agents was tested using Stokes' disc diffusion and broth microdilution MIC methods. Norfloxacin accumulation and efflux assays were performed using a fluorescence method with and without the efflux pump inhibitors sodium O-vanadate and reserpine.Results: Cloning and expression of smrA in Escherichia coli conferred increased resistance to structurally unrelated compounds, including fluoroquinolones, tetracycline, doxorubicin and multiple dyes. Moreover, the expression of smrA in E coli reduced norfloxacin uptake and enhanced its efflux, features that could be inhibited by the ABC efflux pump inhibitors.Conclusions: SmrA is a member of the ABC multidrug efflux pump family. The findings warrant further study of the role of this molecule in S. maltophilia isolates, to estimate the potential impact of this system in antimicrobial resistance.