Plasmid-encoded multidrug efflux pump conferring resistance to olaquindox in Escherichia coli - PubMed (original) (raw)

Plasmid-encoded multidrug efflux pump conferring resistance to olaquindox in Escherichia coli

Lars Hestbjerg Hansen et al. Antimicrob Agents Chemother. 2004 Sep.

Abstract

We report here the first gene-encoded resistance mechanism to the swine growth enhancer olaquindox. The genetic elements involved in resistance to olaquindox were subcloned and sequenced from a conjugative plasmid isolated from Escherichia coli. The subcloned fragment contained two open reading frames, oqxA and oqxB, that are homologous to several resistance-nodulation-cell-division family efflux systems from different species. The putative protein sequences were aligned to both experimentally verified and putative efflux pumps. We show that oqxA and oqxB are expressed in E. coli. Plasmids containing the oqxAB genes yielded high (>128 microg/ml) resistance to olaquindox in E. coli, whereas strains containing the control plasmid showed low resistance to the drug (8 microg/ml). The oqxAB-encoded pump also conferred high (>64 microg/ml) resistance to chloramphenicol. We demonstrate that the subcloned fragment conferred H(+)-dependent ethidium efflux abilities to E. coli strain N43. In addition, we show that the efflux system is dependent on the host TolC outer membrane protein when expressed in E. coli.

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Figures

FIG. 1.

FIG. 1.

The 6-kb ApaLI insert from pOLA52 revealed three ORFs, and their locations are indicated in the diagram. The oqxA and oqxB genes are situated on the plus strand, ORF3 is located on the minus strand. Some selected restriction sites are indicated.

FIG. 2.

FIG. 2.

(A) Comparison of the putative protein sequence of OqxA and the entire sequences of the E. coli AcrA, X. axonopodis MexE, and P. aeruginosa MexX proteins. (B) Comparison of the putative protein sequence of OqxB and the entire sequences of the E. coli AcrB, X. axonopodis MexF, and P. aeruginosa MexY proteins. Black letters on a white background indicate different amino acid residues. Shaded black letters indicate similar residues. White letters on a black background indicate identical residues.

FIG. 2.

FIG. 2.

(A) Comparison of the putative protein sequence of OqxA and the entire sequences of the E. coli AcrA, X. axonopodis MexE, and P. aeruginosa MexX proteins. (B) Comparison of the putative protein sequence of OqxB and the entire sequences of the E. coli AcrB, X. axonopodis MexF, and P. aeruginosa MexY proteins. Black letters on a white background indicate different amino acid residues. Shaded black letters indicate similar residues. White letters on a black background indicate identical residues.

FIG. 2.

FIG. 2.

(A) Comparison of the putative protein sequence of OqxA and the entire sequences of the E. coli AcrA, X. axonopodis MexE, and P. aeruginosa MexX proteins. (B) Comparison of the putative protein sequence of OqxB and the entire sequences of the E. coli AcrB, X. axonopodis MexF, and P. aeruginosa MexY proteins. Black letters on a white background indicate different amino acid residues. Shaded black letters indicate similar residues. White letters on a black background indicate identical residues.

FIG. 3.

FIG. 3.

RT-PCR analysis of oqxAB operon and agarose gel electrophoresis of RT-PCR products. Products were visualized in a 1.2% agarose gel. Lanes 1 and 6, 100-bp marker; lanes 2 and 3, RT-PCRs with N43 (lane 2) or N43/pOLA52 (lane 3) as a template; lanes 4 and 5, control PCRs on templates of total RNA from N43 (lane 4) or N43/pOLA52 (lane 5).

FIG. 4.

FIG. 4.

(Left panel) Uptake of ethidium in E. coli N43 cells containing either pLOW2 (○) or pLOW2::oqxAB (▵). Cells were exposed to ethidium bromide at 0 min. CCCP was added to the cells after 9 min (point indicated by arrow). The fluorescence of cells (shown as relative fluorescence units [RFU]), caused by the presence of ethidium, was measured continuously during the assay. (Right panel) Ethidium efflux. Starved cells of N43 cells containing either pLOW2 (○) or pLOW2::oqxAB (▵) were loaded with ethidium bromide for 1 h prior to the start of the assay. At 5 min after assay start, glucose and thiamine were added to energize the cells (indicated by arrow). The fluorescence of cells (shown as relative fluorescence units [RFU]), caused by the presence of ethidium, was measured continuously during the assay.

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