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 g/ ml) resistance to olaquindox in E. coli, whereas strains containing the control plasmid showed low resistance to the drug (8 g/ml). The oqxAB-encoded pump also conferred high (>64 g/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.Transfer of multiresistance plasmids is an increasing threat to human health. In recent years, the use of antibiotics as growth enhancers has been the subject of intense debate due to the possibility for selection of drug resistance as a consequence of this practice. The quinoxaline-di-N-oxide olaquindox (OQX) has been a widely used growth enhancer in pigs (3). Its antibiotic activity is due to inhibition of DNA synthesis (19). Curiously, for many years, no genetically encoded resistance to this drug had been isolated. Therefore, it has been considered a relatively safe antibiotic. However, Sørensen et al. (17) recently isolated a conjugative plasmid conferring strong resistance to OQX. In short, an OQX-resistant strain of Escherichia coli was isolated from swine manure. Plasmid pOLA52, containing the genetic determinant for OQX resistance (MIC ϭ 128 g/ml), was subsequently transferred by conjugation from this strain to E. coli CSH26. The size of the plasmid was ca. 52 kb. It also confers resistance to ampicillin (AMP; MIC Ͼ 32 g/ml) and chloramphenicol (CHL; MIC ϭ 64 g/ml). This raises some concern that the use of OQX could select for the proliferation of a conjugative plasmid, which also carries resistance determinants for two therapeutic antibiotics, AMP (-lactams) and CHL.We describe here the isolation, subcloning, and sequencing of the plasmid-borne resistance determinant for OQX. Furthermore, a mode of action for this resistance mechanism is proposed and validated. MATERIALS AND METHODSBacterial strains and media. E. coli CSH26 Rif r /pOLA52 (17) was used as the source of pOLA52. E. coli DH5␣ (8) was used as cloning strain prior to sequencing of the OQX resistance determinants. E. coli N43 and E. coli N43tolC::Tn10 (5) were used as host strains in the resistance tests. All E. coli strains were grown in Luria-Bertani (LB) broth (15) containing the appropriate antibiotics at 37°C.Antibiotics were added to both liquid and soli...
A conjugative plasmid, pOLA52, conferring resistance to the antibiotic growth promoter olaquindox has been isolated from Escherichia coli from swine manure. It also confers resistance to ampicillin and chloramphenicol and has a high frequency of transfer between strains of E. coli. Plasmid-borne olaquindox resistance has not been demonstrated before.The synthetic chemotherapeutic agent olaquindox has found wide use as a growth promoter in pig farming. It is active against coliform bacteria (3), where it inhibits DNA synthesis (10). Resistance has been defined with a breakpoint of 64 g/ml (3) or 50 g/ml (7). Until 2000, the compound has been allowed in concentrations of up to 100 mg/kg in feed for pigs younger than 4 months.Since the introduction of the compound in the 1980s, there has been some concern about whether resistance would arise and, if so, whether this resistance would be transferable and whether it would be linked to other resistance determinants (2, 5, 7). A survey study in Denmark has demonstrated the presence of a small fraction of olaquindox-resistant coliform bacteria in farm animals (3). Other studies have demonstrated increased resistance to olaquindox on pig farms using olaquindox. There was a slight correlation between resistance to olaquindox and resistance to chloramphenicol or ampicillin (5, 7). The aim of the present study was to find out if plasmid-bound olaquindox resistance exists.Isolation of a resistant bacterium. Swine manure from a farm using olaquindox as a feed additive was tested for the presence of bacteria able to grow on Gould S1 medium (4) with 100 g of olaquindox per ml. Diluted manure corresponding to 10 l of undiluted manure gave rise to three uniform colonies that did not show the fluorescence characteristic of fluorescent pseudomonads. One of the colonies was restreaked several times on Levine EMB plates (GIBCO Products for Microbiology: Technical Manual and Catalog; GIBCO Laboratories, Madison, Wis.) and Luria-Bertani (LB) agar plates (9), both containing 100 g of olaquindox per ml. Olaquindox (98% pure; ICN, Costa Mesa, Calif.) was added as a 10-mg/ml stock solution in 2.5 M NaOH; HCl was added to the final medium to counteract the high pH. All incubations were done at 37°C. The isolate was identified as Escherichia coli by use of the API 20E system (BioMérieux, Marcy l'Etoile, France).Olaquindox resistance was tested with an agar dilution test, while resistance to an array of other antibiotics was tested with Sensititre plates (1). The isolate was found to be resistant to ampicillin (MIC, Ͼ32 g/ml), kanamycin (MIC, Ͼ64 g/ml), chloramphenicol (MIC, Ͼ64 g/ml), nitrofurantoin (MIC, 128 g/ml), streptomycin (MIC, 128 g/ml), olaquindox (MIC, 128 g/ml), sulfamethoxazole (MIC, Ͼ512 g/ml), trimethoprim (MIC, Ͼ32 g/ml), and carbadox (MIC, Ͼ128 g/ml). The strain was sensitive to apramycin (MIC, 4 g/ml), ciprofloxacin (MIC, 0.125 g/ml), colistin (MIC, 1 g/ml), gentamicin (MIC, 0.5 g/ml), nalidixic acid (MIC, 16 g/ml), and tetracycline (MIC, 1 g/ml). In addition, the isolate did...
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