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Antimicrobial Agents and Chemotherapy, July 2008, p. 2503-2511, Vol. 52, No. 7
0066-4804/08/$08.00+0     doi:10.1128/AAC.00298-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of a Novel Multidrug Efflux Pump of Mycobacterium tuberculosis ^,

Department of Microbiology, University of Alabama at Birmingham, 609 Bevill Biomedical Research Building, 845 19th Street South, Birmingham, Alabama 35294,¹ Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, D-91058 Erlangen, Germany²

Received 3 March 2008/ Returned for modification 28 March 2008/ Accepted 24 April 2008

The impermeability of the outer membrane in combination with drug efflux are major determinants of the natural drug resistance of mycobacteria. β-Lactams are the most widely used antibiotics for treatment of bacterial infections. However, it is unknown how β-lactams enter Mycobacterium tuberculosis and whether efflux pumps exist that can export these drugs out of the cell. To identify the molecular mechanisms of M. tuberculosis resistance to β-lactams, a library of 7,500 transposon mutants was generated in the model organism Mycobacterium bovis BCG. Thirty-three unique insertion sites were determined that conferred medium or high-level (2,000 µg/ml) resistance to ampicillin. Three mutants in sulfolipid synthesis or transport were highly resistant to ampicillin, indicating an indirect effect of the lipid composition on the outer membrane permeability of M. bovis BCG to ampicillin. Mutants with insertions in genes encoding surface molecules such as PPE proteins or lipoarabinomannan were also completely resistant to ampicillin, thus suggesting a lack of transport across the outer membrane. Insertion of the transposon in front of bcg0231 increased transcription of the gene and concomitantly the resistance of M. bovis BCG to ampicillin, streptomycin, and chloramphenicol by 32- to 64-fold. Resistance to vancomycin and tetracycline was increased four- to eightfold. Bcg0231 and Rv0194 are almost identical ATP-binding cassette transporters. Expression of rv0194 significantly reduced accumulation of ethidium bromide and conferred multidrug resistance to Mycobacterium smegmatis. Both effects were abrogated in the presence of the efflux pump inhibitor reserpine. These results demonstrate that Rv0194 is a novel multidrug efflux pump of M. tuberculosis.

Published ahead of print on 5 May 2008.

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