Moreover, it was shown that resistance to the tested antibiotics

decreased #CRT0066101 randurls[1|1|,|CHEM1|]# in the presence of efflux inhibitors in the studied strains, demonstrating that these inhibitors have a broad range of activity that is not specific to a given genotype. In conclusion, the methodology used in this study demonstrates that porin MspA plays an important role in the entrance of quaternary ammonium compounds and antibiotics into the cell. Whether its absence is the main cause for decreased permeability, or that its absence has resulted in altered lipid structure of the outer membrane that is less permeable remains to be elucidated. The same methodology used to assess permeability also assessed the activity of the main efflux pump LfrA of the wild-type strain and of LfrA and LfrR depleted mutants and correlated the degree of activity with low-level resistance to several antimicrobial drugs. The methodology used and the results obtained in this work will be used in future studies as a working

model for the evaluation Z-DEVD-FMK of influx and efflux of substrates by multidrug resistant M. tuberculosis clinical isolates and, therefore, determine the cause for the multidrug resistant phenotype beyond simple mutation of relevant targets. Methods Materials EtBr, glucose, phosphate buffered solution (PBS), chlorpromazine, thioridazine, verapamil, amikacin, ciprofloxacin, ethambutol, erythromycin, rifampicin and streptomycin were purchased from Sigma Aldrich Química Oxymatrine SA (Madrid, Spain). Clarithromycin was obtained from Abbott Laboratories (Abbott Park, IL, USA). Middlebrook 7H9 broth and OADC supplement were purchased from Difco (Detroit, MI, USA). All solutions were prepared on the day of the experiment. Bacteria The M. smegmatis strains used in this work are described in Table 1. M. smegmatis strains SMR5, MN01 and ML10 were kindly provided by Michael

Niederweis (Department of Microbiology, University of Alabama at Birmingham, Birmingham, U.S.A); strains XZL1675 and XZL1720 were kindly provided by Hiroshi Nikaido (Department of Molecular and Cell Biology, University of California, Berkeley, California, U.S.A). Mycobacteria were grown at 37°C in Middlebrook 7H9 broth or Middlebrook 7H11 solid medium, supplemented with 10% (v/v) of OADC. Determination of Minimum Inhibitory Concentrations The determination of MICs of EtBr, the efflux inhibitors chlorpromazine, thioridazine and verapamil and of antibiotics studied alone and in the presence of an efflux inhibitor, was performed by the broth microdilution method according to the CLSI guidelines [33]. Briefly, mycobacterial strains were grown at 37°C in Middlebrook 7H9 broth supplemented with 10% OADC until an optical density (O.D.) of 0.8 at a wavelength of 600 nm.