Plasmid-mediated quinolone resistance determinants oqxAB and aac(6')-Ib-cr and extended-spectrum  -lactamase gene blaCTX-M-24 co-located on the same plasmid in one Escherichia coli strain from China

Sir, With the common use of fluoroquinolones in both human and animal diseases, fluoroquinolone resistance in veterinary clinics has become an important public health problem since the discovery of horizontally transmissible elements, such as plasmids. To date, a number of plasmid-mediated quinolone resistance (PMQR) determinants have been described: the qnr genes (A, B, S, C and D); the aac(6′)-Ib-cr gene; and the qepA gene.1 However, OqxAB, a plasmid-encoded multidrug efflux pump that confers reduced susceptibility to multiple agents, including fluoroquinolones,2 was not recognized as a PMQR determinant until recently. The PMQR determinants can confer only low-level resistance to quinolones; however, they can facilitate the selection of resistant mutants upon exposure to ciprofloxacin.3 With the development of both quinolone resistance and β-lactam resistance, many studies on the association of PMQR genes and extended-spectrum β-lactamases (ESBLs) have been reported. There is a paucity of data with regard to the association of oqxAB and other resistance genes, although other PMQR genes were often found to be co-carried with genes encoding ESBLs or AmpC-type β-lactamases on the same plasmid.1 In this study, we investigated the dissemination mechanism of oqxAB and the coexistence of oqxAB and genes encoding ESBLs in an Escherichia coli strain. E. coli strain a6 was isolated from a liver sample of diseased chicken from a farm in Guangdong Province, China. Susceptibility to 15 antibiotics was measured by agar dilution methods and an ESBL production test was also performed according to the guidelines provided by the CLSI (2008). The genes blaCTX-M-9 group, blaTEM, qepA, qnrA, qnrB, qnrS, aac(6′)-Ib-cr, gyrA and parC were detected and sequenced by specific PCRs using previously described primers.4–7 The primers used for oqxA and oqxB were as follows: oqxA-F (5′-CTTGCACTTAGTTAAGCGCC-3′) and oqxA-R (5′-GAGGTTTTGATAGTGGAGGTAGG-3′) for oqxA; and oqxB-F (5′-GCGGTGCTGTCGATTTTA-3′) and oqxB-R (5′-TACCGGAACCCATCTCGAT -3′) for oqxB. E. coli strain a6 showed a multidrug resistance phenotype and was positive for the ESBL production test. The high MICs of nalidixic acid, ciprofloxacin, olaquindox, chloramphenicol, ampicillin and cefotaxime were >512, 512, 128, 256, 512 and >256 mg/L, respectively. Amino acid changes were detected in both GyrA (Ser83Leu Asp87Tyr) and ParC (Ser80Ile) proteins. The genes qepA1, oqxAB and aac(6′)-Ib-cr were all detected in E. coli a6 harbouring the genes encoding CTX-M-24 and TEM-1b β-lactamases. A conjugation experiment was performed to investigate whether these genes were located on plasmids and whether the transfer of these genes contributed to the reduced susceptibility of the recipient E. coli towards antibiotics, by using E. coli C600 (streptomycin resistant) as the recipient. The transconjugants showed a multidrug resistance phenotype that included resistance to nalidixic acid, ampicillin, cefotaxime, trimethoprim/sulfamethoxazole, tetracycline and ceftiofur. Transconjugants showed 8-, 4- and 8-fold increases in the MICs of nalidixic acid, ciprofloxacin and enrofloxacin, respectively, when compared with the recipient strain. With regard to the MICs of olaquindox and chloramphenicol, they both showed 8-fold increases. For the β-lactam antibiotics, the transconjugants showed 128-, 2048-, 32-, 256- and 4-fold increases in the MICs of ampicillin, cefotaxime, ceftazidime, ceftiofur and cefoxitin, respectively, when compared with the recipient. The transconjugant strain a6T was detected to harbour oqxAB, blaCTX-M-24, blaTEM-1b and aac(6′)-Ib-cr simultaneously, while qepA and any amino acid changes in GyrA or ParC proteins were not detected. Southern blot hybridization was performed with digoxigenin-labelled probes specific for oqxB, blaCTX-M-24 and aac(6′)-Ib-cr. Notably, the results shown in Figure 1 revealed the coexistence of oqxAB, blaCTX-M-24 and aac(6′)-Ib-cr on the same plasmid of ∼54 kb in E. coli a6 and its transconjugant, a6T. Analysis of the genetic environment of the blaCTX-M-24 gene showed that the sequence upstream of it contained the ISEcp1 element; the orf477 sequence and the virulence gene iroN, a urovirulence factor in E. coli,8 were located downstream of the β-lactamase gene. According to their plasmid incompatibility group, plasmids were classified using the method described by Carattoli,9 and the conjugative plasmid harbouring blaCTX-M-24 in this study was found to belong to the IncF group, which was consistent with the results of Carattoli.9 The result suggests that E. coli a6 is a reservoir of plasmids that contain multidrug resistance genes. Panel I shows electrophoresis of the plasmids of E. coli a6 (lane 1) and its transconjugant strain (lane 2); lane M is the plasmid profile of E. coli standard V517. Panels II, III and IV show the results of Southern blot analysis of uncut plasmids hybridized with oqxAB, blaCTX-M-24 and aac(6′)-Ib-cr, respectively. To our knowledge, this is the first description of the coexistence of the oqxAB, aac(6′)-Ib-cr and blaCTX-M-24 resistance genes and the virulence gene iroN on the same plasmid in one E. coli strain. The association of these resistance determinants is worrisome, because it may facilitate the selection of high-level multidrug-resistant strains in some communities and this process may be promoted by the co-selection of various antimicrobial agents under subinhibitory concentrations. Moreover, the coexistence of iroN with these resistance genes may raise the concern that antibiotic use will co-select for virulence determinants and pose a potential threat to public health. There is a great need to obtain more detailed knowledge on the association of various resistance genes in E. coli, and more studies should be carried out in this field. This work was supported by grants U0631006 and 30800836 from the National Natural Science Foundation of China. None to declare.