Molecular cloning and characterization of SmrA, a novel ABC multidrug efflux pump from Stenotrophomonas maltophilia

Abstract

Stenotrophomonas maltophilia is an emerging nosocomial pathogen that can cause difficult-to-treat infections and exhibits significant degrees of poorly understood multidrug resistance (MDR). The aim of this study was to identify and characterize a multidrug ATP-binding cassette (ABC) efflux pump in S. maltophilia. SmrA was identified in the S. maltophilia genome based on the detection of ABC transporter conserved motifs and alignment with experimentally proven MDR ABC transporters. The smrA gene was cloned and expressed in the hypersusceptible acrAB mutant Escherichia coli strain SM1411. The resistance to several antimicrobial agents was tested using Stokes' disc diffusion and broth microdilution MIC methods. Norfloxacin accumulation and efflux assays were performed using a fluorescence method with and without the efflux pump inhibitors sodium O-vanadate and reserpine. Cloning and expression of smrA in Escherichia coli conferred increased resistance to structurally unrelated compounds, including fluoroquinolones, tetracycline, doxorubicin and multiple dyes. Moreover, the expression of smrA in E. coli reduced norfloxacin uptake and enhanced its efflux, features that could be inhibited by the ABC efflux pump inhibitors. SmrA is a member of the ABC multidrug efflux pump family. The findings warrant further study of the role of this molecule in S. maltophilia isolates, to estimate the potential impact of this system in antimicrobial resistance.