A-048. Identification and Characterization of a Novel ABC Multidrug Efflux Pump, SmrA, in Stenotrophomonas maltophilia

A. Al-Hamad, J. Burnie, M. Upton;
Univ. of Manchester, Manchester, UNITED KINGDOM.

Background: Stenotrophomonas maltophilia is an emerging nosocomial pathogen that can cause difficult to treat infections and exhibits significant degrees of poorly understood multidrug resistance. Overexpression of efflux pump(s) is thought to be an important factor in this multiple resistance phenotype. We aimed to investigate the presence of a multidrug resistance encoding ATP-binding cassette (ABC)-type efflux pump in S. maltophilia. Methods: The SmrA sequence has been identified in the S. maltophilia genome based on ABC conserved motif detection in the C-terminal region of a polypeptide that is longer than 500 aa. This was then aligned with experimentally proven multidrug resistance encoding ABC transporters. The smrA gene was cloned and expressed in a hypersusceptible acrAB mutant E. coli strain. The same strain transformed with the lacZ gene alone was used as a control strain. MICs to several antimicrobial agents were measured. A norfloxacin efflux assay was performed using a fluorescence method with and without sodium O-vanadate, an ATPase inhibitor. Results: The SmrA protein sequence showed homologies with the sequences of N-MDR1, C-MDR1, LmrA, and VcaM of 39.5 %, 41.2 %, 42.2%, and 67.5%, respectively. Hydropathy analysis suggests the presence of an N-terminal hydrophobic domain with six putative transmembrane segments and a C-terminal hydrophilic ABC domain. Overexpression of smrA gene led to an 8-fold increase in the MIC of ciprofloxacin, norfloxacin, and tetracycline; other antimicrobial agents have shown a 2-fold increase in the MIC. Results of the efflux assay showed a more efficient extrusion of norfloxacin in cells harbouring the smrA gene. The efflux activity was inhibited by sodium O-vanadate in a concentration-dependent manner. Conclusion: SmrA is a member of the ABC multidrug efflux pump family that has six transmembrane helical regions followed by an ABC domain. Strains expressing SmrA showed elevated levels of resistance to a number of structurally dissimilar antimicrobial agents. The inhibition of the efflux activity by sodium O-vanadate provides evidence of the ATP-dependent nature of the SmrA.

160/A. Mechanisms of Antimicrobial Action

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