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Overexpression of Rob increases MIC to norfloxacin by upregulation of the MdfA efflux pump in Escherichia coli in vitro

Abstract number: 10.1111/j.1198-743X.2004.902_o280.x

Linde H.J.

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Objective:

In E. coli various efflux pump systems like AcrAB, AcrEF, EmrAB and MdfA are known to confer resistance to various classes of antimicrobials. Several transcriptional factors like MarA, Rob and SoxS are involved in their regulation. Here we report how overexpression of Rob affected the transcription of the acrAB, mdfA, emrAB and norE efflux pump systems, all known to confer resistance to fluoroquinolones.

Methods:

Rob was overexpressed from the arabinose-inducible plasmid pCC1 (Epicentre®) under control of the lac promoter in E. coli (pCC1-lacrob). MICs (Etest®, Solna, Sveden) to norfloxacin (NFX) and ciprofloxacin (CIP) and transcription levels of gapA, Rob, mdfA, acrAB, acrD, acrE, acrF, emrB, norE, tolC, marA, and soxS were determined by quantitative RT-PCR (LightCycler®) comparing induced vs. uninduced cultures (arabinose 0.1%).

Results:

Compared with uninduced control, maximum induction of pCC1-lacrob resulted in an increase of: MIC to NFX: threefold (0.064 vs. 0.023 mg/L), MIC to CIP: no change; transcription of Rob: 250-fold, mdfA 6.9-fold, acrAB 1.7-fold, emrB 0.6-fold, acrE 0.66-fold, acrF 0.69-fold, acrD 0.75-fold, norE 0.99-fold, marA 1.2-fold, soxS 0.66-fold, tolC 1.3-fold, and gapA 0.98-fold.

Conclusion:

MdfA is a recently recognised efflux pump system in E. coli with preference for norfloxacin (1,2). We demonstrate that increased transcription of rob from plasmid pCC1-lacrob resulted in increased transcription of mdfA but not acrAB and produces a correspondent phenotype with increased MIC to NFX but not CIP. Other efflux pump systems were only mildly affected (upregulation of acrAB, no effect for norE, downregulation of acrEF, emrB). The finding adds to the accumulating knowledge concerning the overlaps and parallels in regulation of efflux systems, and highlights their potential role in development of antimicrobial resistance.

References

1.Nishino, K. & Yamaguchi, A. (2001) Analysis of a complete library of putative drug transporter genes in Escherichia coli. J Bacteriol 183, 5803–5812.

2.Yang, S., Clayton, S.R. & Zechiedrich, E.L. (2003) Relative contributions of the AcrAB, MdfA and NorE efflux pumps to quinolone resistance in Escherichia coli. J Antimicrob Chemother 51, 545–556.

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Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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