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Antimicrobial Agents and Chemotherapy, November 2000, p. 3079-3086, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Cloning and Characterization of SmeDEF, a Novel Multidrug Efflux Pump from Stenotrophomonas maltophilia

Ana Alonso and José L. Martínez*

Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049-Madrid, Spain

Received 20 March 2000/Returned for modification 16 July 2000/Accepted 18 August 2000

Stenotrophomonas maltophilia is a nosocomial bacterial pathogen intrinsically resistant to several antibiotics. The mechanisms involved in this intrinsic multiresistance phenotype are poorly understood. A library of chromosomal DNA from a spontaneous multidrug-resistant S. maltophilia D457R mutant (A. Alonso and J. L. Martinez, Antimicrob. Agents Chemother. 41:1140-1142, 1997) was screened for complementation of erythromycin susceptibility on an antibiotic-hypersusceptible Escherichia coli Delta acrAB strain. Cloning and further analysis revealed that a 6-kbp region constituting a transcriptional unit was capable of complementing the antibiotic-susceptible phenotype of an E. coli Delta acrAB strain. We identified three open reading frames, smeD, smeE and smeF, which code for members of the membrane fusion protein, resistance nodulation division, and outer membrane factor families, respectively. Drug susceptibility assays indicated that the SmeDEF system cloned in E. coli mediates resistance to a wide range of antibiotics. Ethidium bromide and norfloxacin accumulation experiments in the presence and in the absence of carbonyl cyanide m-chlorophenylhydrazone showed that this system constitutes a drug efflux pump dependent on the membrane proton motive force. The presence of high levels of smeDEF mRNA in the multiresistant D457R mutant was consistent with the high levels of SmeF (formerly Omp54) observed in the same strain. In contrast, transcription levels of smeDEF in the D457 strain were tiny, which correlates with the low levels of SmeF observed for this strain. Also, for both the D457 and D457R strains, we observed growth phase-dependent regulation in which the highest level of transcription corresponded to early exponential phase, with transcription decreasing throughout the growth curve to undetectable levels at 24 h.

* Corresponding author. Mailing address: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049-Madrid, Spain. Phone: 34-91-5854551. Fax: 34-91-5854506. E-mail: jmtnez{at}cnb.uam.es.

Antimicrobial Agents and Chemotherapy, November 2000, p. 3079-3086, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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