Kinetic Parameters of Efflux of Penicillins by the Multidrug Efflux Transporter AcrAB-TolC of Escherichia coli
Kinetic Parameters of Efflux of Penicillins by the Multidrug Efflux Transporter AcrAB-TolC of Escherichia coli
复制标题
DOI：
10.1128/aac.01714-09
复制
发表时间：
2010-05-01
影响因子：
4.9
通讯作者：
Nikaido, Hiroshi
中科院分区：
文献类型：
--
作者：
Lim, Siew Ping;Nikaido, Hiroshi
众所周知，多药外排转运体AcrAB-TolC能排出多种抗生素，包括β-内酰胺类抗生素。然而，定量了解耐药性所需的外排过程的动力学常数是无法获得的，直到最近通过将外排与周质β-内酰胺酶对药物的水解相结合来确定某些头孢菌素外排的动力学常数。在本研究中，我们将这种方法扩展到广泛的青霉素类药物的研究中，通过将外排和水解与OXA-7青霉素酶相结合，从氨苄青霉素和青霉素V到尿素青霉素类和异恶唑青霉素类。我们发现青霉素类药物对AcrB有更强的表观亲和力，最大外排速率也高于头孢菌素类药物。所有青霉素类药物对出口均表现出较强的正协同动力学。根据外排和水解动力学从理论上预测的最低抑菌浓度与观察到的最低抑菌浓度非常相似(异恶唑青霉素除外)，因此得到的动力学常数得到了验证。然而，令人惊讶的是，例如氯唑西林在AcrB基因缺失后在大肠杆菌中的MIC下降了512倍，其外排动力学与氨苯西林非常相似，相同处理下其MIC仅下降2倍。对这一现象的分析表明，AcrB突变体的MIC大幅下降主要是由于药物的低渗透性，亲本和AcrB菌株之间的MIC比较是衡量AcrB泵出药物能力的非常差的衡量标准。
The multidrug efflux transporter AcrAB-TolC is known to pump out a diverse range of antibiotics, including beta-lactams. However, the kinetic constants of the efflux process, needed for the quantitative understanding of resistance, were not available until those accompanying the efflux of some cephalosporins were recently determined by combining efflux with the hydrolysis of drugs by the periplasmic beta-lactamase. In the present study we extended this approach to the study of a wide range of penicillins, from ampicillin and penicillin V to ureidopenicillins and isoxazolylpenicillins, by combining efflux with hydrolysis with the OXA-7 penicillinase. We found that the penicillins had a much stronger apparent affinity to AcrB and higher maximum rates of efflux than the cephalosporins. All penicillins showed strong positive cooperativity kinetics for export. The kinetic constants obtained were validated, as the MICs theoretically predicted on the basis of efflux and hydrolysis kinetics were remarkably similar to the observed MICs (except for the isoxazolylpenicillins). Surprisingly, however, the efflux kinetics of cloxacillin, for example, whose MIC decreased 512-fold in Escherichia coli upon the genetic deletion of the acrB gene, were quite similar to those of ampicillin, whose MIC decreased only 2-fold with the same treatment. Analysis of this phenomenon showed that the extensive decrease in the MIC for the acrB mutant is primarily due to the low permeation of the drug and that comparison of the MICs between the parent and the acrB strains is a very poor measure of the ability of AcrB to pump a drug out.