Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008 Lisboa, Portugal.
BMC Microbiol. 2011 Feb 18;11:35. doi: 10.1186/1471-2180-11-35.
Active efflux systems and reduced cell-wall permeability are considered to be the main causes of mycobacterial intrinsic resistance to many antimicrobials. In this study, we have compared the Mycobacterium smegmatis wild-type strain mc2155 with knockout mutants for porins MspA (the main porin of M. smegmatis) and MspC, the efflux pump LfrA (the main efflux pump system of M. smegmatis) and its repressor LfrR for their ability to transport ethidium bromide (EtBr) on a real-time basis. This information was then correlated with minimum inhibitory concentrations (MICs) of several antibiotics in the presence or absence of the efflux inhibitors chlorpromazine, thioridazine and verapamil.
In the absence of porins MspA and MspC, accumulation of ethidium bromide decreased and the cells became more resistant to several antibiotics, whereas the knockout mutant for the LfrA pump showed increased accumulation of EtBr and increased susceptibility to EtBr, rifampicin, ethambutol and ciprofloxacin. Moreover, the efflux inhibitors caused a reduction of the MICs of streptomycin, rifampicin, amikacin, ciprofloxacin, clarithromycin and erythromycin in most of the strains tested.
The methodology used in this study demonstrated that porin MspA plays an important role in the influx of quaternary ammonium compounds and antibiotics and that efflux via the LfrA pump is involved in low-level resistance to several antimicrobial drugs in M. smegmatis. The results obtained with this non-pathogenic mycobacterium will be used in future studies as a model for the evaluation of the activity of the same efflux inhibitors on the susceptibility of multidrug resistant strains of Mycobacterium tuberculosis to isoniazid and rifampicin.
主动外排系统和细胞壁通透性降低被认为是分枝杆菌对许多抗菌药物固有耐药的主要原因。在这项研究中，我们比较了耻垢分枝杆菌野生型菌株 mc2155 与缺失突变体 mspA（耻垢分枝杆菌的主要孔蛋白）和 mspC、外排泵 lfrA（耻垢分枝杆菌的主要外排泵系统）及其抑制剂 lfrR，研究它们在实时条件下摄取溴化乙锭（EtBr）的能力。然后将这些信息与几种抗生素的最低抑菌浓度（MIC）进行相关分析，这些抗生素要么存在外排抑制剂氯丙嗪、硫利达嗪和维拉帕米，要么不存在。
在缺乏 mspA 和 mspC 孔蛋白的情况下，溴化乙锭的积累减少，细胞对几种抗生素的耐药性增加，而 lfrA 泵的缺失突变体表现出 EtBr 积累增加和对 EtBr、利福平、乙胺丁醇和环丙沙星的敏感性增加。此外，外排抑制剂导致所测试的大多数菌株中链霉素、利福平、阿米卡星、环丙沙星、克拉霉素和红霉素的 MIC 降低。
本研究中使用的方法学表明，孔蛋白 MspA 在季铵化合物和抗生素的流入中起重要作用，并且通过 LfrA 泵的外排参与了几种抗菌药物对耻垢分枝杆菌的低水平耐药性。从这种非致病性分枝杆菌中获得的结果将在未来的研究中用作评估相同外排抑制剂对异烟肼和利福平耐药的结核分枝杆菌多药耐药株敏感性的模型。
