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Antimicrobial Agents and Chemotherapy, July 2004, p. 2415-2423, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2415-2423.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Efflux Pump-Mediated Intrinsic Drug Resistance in Mycobacterium smegmatis

Xian-Zhi Li,{dagger} Li Zhang, and Hiroshi Nikaido*

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202

Received 8 October 2003/ Returned for modification 30 January 2004/ Accepted 21 March 2004

The Mycobacterium smegmatis genome contains many genes encoding putative drug efflux pumps. Yet with the exception of lfrA, it is not clear whether these genes contribute to the intrinsic drug resistance of this organism. We showed first by reverse transcription (RT)-PCR that several of these genes, including lfrA as well as the homologues of Mycobacterium tuberculosis Rv1145, Rv1146, Rv1877, Rv2846c (efpA), and Rv3065 (mmr and emrE), were expressed at detectable levels in the strain mc2155. Null mutants each carrying an in-frame deletion of these genes were then constructed in M. smegmatis. The deletions of the lfrA gene or mmr homologue rendered the mutant more susceptible to multiple drugs such as fluoroquinolones, ethidium bromide, and acriflavine (two- to eightfold decrease in MICs). The deletion of the efpA homologue also produced increased susceptibility to these agents but unexpectedly also resulted in decreased susceptibility to rifamycins, isoniazid, and chloramphenicol (two- to fourfold increase in MICs). Deletion of the Rv1877 homologue produced some increased susceptibility to ethidium bromide, acriflavine, and erythromycin. The upstream region of lfrA contained a gene encoding a putative TetR family transcriptional repressor, dubbed LfrR. The deletion of lfrR elevated the expression of lfrA and produced higher resistance to multiple drugs. Multidrug-resistant single-step mutants, independent of LfrA and attributed to a yet-unidentified drug efflux pump (here called LfrX), were selected in vitro and showed decreased accumulation of norfloxacin, ethidium bromide, and acriflavine in intact cells. Finally, use of isogenic ß-lactamase-deficient strains showed the contribution of LfrA and LfrX to resistance to certain ß-lactams in M. smegmatis.

* Corresponding author. Mailing address: Department of Molecular and Cell Biology, 426 Barker Hall, University of California, Berkeley, CA 94720-3202. Phone: (510) 642-2027. Fax: (510) 643-6334. E-mail: nhiroshi{at}uclink4.berkeley.edu.

{dagger} Present address: Human Safety Division, Veterinary Drugs Directorate, Health Canada, Ottawa, Ontario, Canada K1A 0K9

Antimicrobial Agents and Chemotherapy, July 2004, p. 2415-2423, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2415-2423.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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