New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate

Abstract

Plasmid-mediated Qnr and AAC(6')-Ib-cr have been recognized as new molecular mechanisms affecting fluoroquinolone (FQ) resistance. C316, an Escherichia coli strain demonstrating resistance to various FQs, was isolated in Japan. Resistance to FQs was augmented in an E. coli CSH2 transconjugant, but PCR failed to detect qnr genes, suggesting the presence of novel plasmid-mediated FQ resistance mechanisms. Susceptibility tests, DNA manipulation, and analyses of the gene and its product were performed to characterize the genetic determinant. A novel FQ-resistant gene, qepA, was identified in a plasmid, pHPA, of E. coli C316, and both qepA and rmtB genes were mediated by a probable transposable element flanked by two copies of IS26. Levels of resistance to norfloxacin, ciprofloxacin, and enrofloxacin were significantly elevated in E. coli transformants harboring qepA under AcrB-TolC-deficient conditions. QepA showed considerable similarities to transporters belonging to the 14-transmembrane-segment family of environmental actinomycetes. The effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on accumulation of norfloxacin was assayed in a qepA-harboring E. coli transformant. The intracellular accumulation of norfloxacin was decreased in a qepA-expressing E. coli transformant, but this phenomenon was canceled by CCCP. The augmented FQ resistance level acquired by the probable intergeneric transfer of a gene encoding a major facilitator superfamily-type efflux pump from some environmental microbes to E. coli was first identified. Surveillance of the qepA-harboring clinical isolates should be encouraged to minimize further dissemination of the kind of plasmid-dependent FQ resistance determinants among pathogenic microbes.

FIG. 1.

Organization of the element mediating qepA. qepA is located in a 10-kb region flanked by two copies of IS26, and this probable transposable element also contains rmtB, together with tnpR, bla_TEM-1, and several probable open reading frames. IRL, 5′-GGCACTGTTGCAAA-3′; IRR, 5′-TTTGCAACAGTGCC-3′.

FIG. 2.

Comparison of amino acid sequences of 14-TMS MFS-type efflux pumps with QepA. Fourteen portions of probable 14-TMS motifs and seven specific motifs, A, B, C, D1, E, F, and H, are shown by horizontal lines above the alignments. Asterisks and dots indicate identical and similar amino acid residues, respectively. Dashes represent gaps. Polaromonas, a putative efflux pump of the Polaromonas sp. strain JS666 chromosome (NCBI Protein Data Bank accession number ABE43318); N. farcinica, the putative arabinose efflux permease AraJ of the Nocardia farcinica chromosome (NCBI Protein Data Bank accession number BAD57397); S. globisporus, SgcB of Streptomyces globisporus (E/G/D accession number AY048670); S. clavuligerus, the putative efflux pump EpeA of the Streptomyces clavuligerus chromosome (E/G/D accession number AJ302083); SmfY, a Serratia marcescens chromosomal efflux pump protein (E/G/D accession number AB251607); SmvA, a Salmonella enterica chromosomal efflux pump protein (E/G/D accession number P37594); and EmrB, an Escherichia coli chromosomal efflux pump protein (E/G/D accession number AAC75733).

FIG. 3.

Predicted secondary structure of QepA. The secondary transmembrane structure of QepA (511 amino acid residues) was calculated and illustrated by the SOSUI system (http://bp.nuap.nagoya-u.ac.jp/sosui/). Fourteen probable α-helix TMS and seven specific motifs are indicated. Classes of amino acid residues are differentiated by four colors.

FIG. 4.

Phylogenetic tree of 14-TMS efflux pumps belonging to MFS transporters. Each amino acid sequence was subjected to analysis with reference to the following sources: LfrA, a Mycobacterium smegmatis proton antiporter efflux pump (E/G/D accession number U40487); Ptr, a “Streptomyces pristinaespiralis” pristinamycin compound and rifampin resistance protein (E/G/D accession number X84072); TcmA, a Streptomyces glaucescens tetracenomycin C resistance and export protein (E/G/D accession number M80674); EmrB, an E. coli multidrug efflux system protein (E/G/D accession number AAC75733); EmrY, an E. coli multidrug resistance protein (E/G/D accession number D78168); TetK, a Staphylococcus aureus tetracycline efflux protein (E/G/D accession number M16217); TetL, a Geobacillus stearothermophilus tetracycline efflux protein (NCBI Protein Data Bank accession number P07561); Mmr, a Streptomyces coelicolor methylenomycin A resistance protein (E/G/D accession number M18263); MmrB, a Bacillus subtilis methylenomycin A resistance protein (E/G/D accession number X66121); CmcT, an “Amycolatopsis lactamdurans” cephamycin export protein (NCBI Protein Data Bank accession number Q04733); LmrA, a Streptomyces lincolnensis lincomycin resistance protein (E/G/D accession number X59926); QacA, an S. aureus antiseptic resistance protein (E/G/D accession number X56628); SmfY, a Serratia marcescens multidrug efflux pump (E/G/D accession number AB251607); SmvA, a Salmonella enterica serovar Typhimurium methyl-viologen resistance protein (NCBI Protein Data Bank accession number P37594); U80063, a Streptomyces cinnamoneus proton-dependent transport protein (E/G/D accession number U80063); EpeA, a Streptomyces clavuligerus DHA2 subfamily multidrug transporter (E/G/D accession number AJ302083); Nocardia farcinica, N. farcinica strain IFM 10152, probable arabinose efflux permease AraJ (NCBI Protein Data Bank accession number BAD57397); Polaromonas sp., Polaromonas sp. strain JS666, a probable MFS efflux pump (NCBI Protein Data Bank accession number ABE43318); QepA, an E. coli plasmid-mediated FQ efflux pump (E/G/D accession number AB263754); Streptomyces coelicolor, a probable membrane transporter of S. coelicolor (NCBI Protein Data Bank accession number CAC37879); SgcB, a Streptomyces globisporus transmembrane efflux protein (E/G/D accession number AAL06672); and MonT, a Streptomyces cinnamonensis putative monensin transporter (NCBI Protein Data Bank accession number AAO65793). Blue, actimomycetes; brown, gram-positive cocci; green, gram-negative rods; purple, Mycobacterium smegmatis. The horizontal bar (0.1) indicates a 10% change in the amino acid residues calculated by the CLUSTAL W program.

FIG. 5.

Assay protocol and accumulation of norfloxacin in E. coli cells. The accumulations of norfloxacin in transformants that harbor a vacant plasmid (pSTV28), a qepA-carrying plasmid (pSTVqepA), and a disrupted qepA-carrying plasmid (pSTVΔqepA) are shown. *, statistical significance (P < 0.01) in the reduction of intracellular accumulation of norfloxacin that was seen in E. coli strains that produce QepA compared to QepA-nonproducing E. coli strains. The effect of CCCP on the accumulation of norfloxacin was also statistically significant in the QepA-producing strain. However, no statistical significance was seen in the norfloxacin levels of E. coli KAM32(pSTVqepA) cells between a sample taken at 10 min and a sample taken at 30 min in the absence of CCCP.