Plasmid-mediated quinolone resistance determinant qepA1 and extended-spectrum β-lactamase gene blaCTX-M-14 co-located on the same plasmid in two Escherichia coli strains from China

With the common use of fluoroquinolones (FQs) and β-lactam antibiotics in both human and animal diseases, increasing numbers of bacterial clinical isolates are being reported with resistance to these agents. 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, the qepA gene and the oqxAB gene (Strahilevitz et al., 2009). The PMQR determinants can confer only low-level resistance to quinolones; however, resistant mutants are likely to occur among PMQR gene- carrying isolates (Cattoir & Nordmann, 2009). Enzymes of the CTX-M group have become the dominant extended-spectrum β-lactamases (ESBLs) reported worldwide (Livermore et al., 2007). The association of multiple antibiotic resistance genes on mobile genetic elements such as plasmids has been an important mechanism of the dissemination of multidrug resistance, and a strong association of PMQR determinents with ESBLs or AmpC-type β-lactamases has been reported (Strahilevitz et al., 2009). These two kinds of resistance genes are often found to be co-located on the same plasmid (Strahilevitz et al., 2009); however, there is a paucity of data with regard to the coexistence of qepA and genes encoding ESBLs in Escherichia coli strains. In this study, we looked at the dissemination mechanism of qepA and the coexistence of qepA and genes encoding ESBLs in E. coli strains of animal origin.

E. coli isolates GDP5 and GDS466, which were recovered from faeces of a diseased dog in an animal hospital in Guangzhou, China, and from a liver sample of a diseased piglet on a farm in Guangdong, respectively, were both isolated in 2010. 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 Clinical and Laboratory Standards Institute (CLSI, 2008). The genes bla_CTX-M, bla_SHV, qepA, qnrA, qnrB, qnrS, aac(6′)-Ib, gyrA, parC and oqxAB were detected and sequenced by specific PCRs using primers described previously (Briñas et al., 2003; Liu et al., 2011; Yamane et al., 2008; Yue et al., 2008). The primers used for rmtB were as follows: rmtB-F (5′-atg aac atc aac gat gcc ct-3′) and rmtB-R (5′-cct tct gat tgg ctt atc ca-3′).

Both of the E. coli strains GDS466 and GDP5 exhibited an ESBL phenotype. The MICs of some antibiotics for the two isolates are listed in Table 1. Strain GDS466 was resistant to all 15 tested antibiotics, whereas GDP5 was resistant to 8 of the 15 tested antibiotics (Table 1). Sequence analysis of quinolone resistance-determining regions revealed that amino acid changes were detected in both GyrA (S83L and D87N) and ParC (S80I) proteins in E. coli GDS466. However, no mutation was detected in GDP5, which was susceptible to nalidixic acid, ciprofloxacin and enrofloxacin. The genes qepA1, rmtB, oqxAB, aac(6′)-Ib and bla_CTX-M-14 were all detected in E. coli GDS466 and GDP5, and an additional qnrS1 gene was also harboured in GDP5 (Table 1).

Conjugation experiments were performed to determine whether these genes were located on plasmids and whether these PMQR genes were associated with the detected bla_CTX-M-14, using E. coli C600 (streptomycin-resistant) as the recipient. Both of the transconjugants GDS466T and GDP5T, obtained from GDS466 and GDP5, respectively, were found to harbour qepA1, bla_CTX-M-14, rmtB and aac(6′)-Ib, simultaneously, without any amino acid changes in GyrA or ParC proteins (Table 1). Also, as shown in Table 1, GDS466T and GDP5T both showed multidrug-resistant phenotypes.

The MICs of ciprofloxacin, enrofloxacin and nalidixic acid for GDS466T were 16-, 16- and 2-fold higher than those for the recipient, respectively, which indicated that qepA1 contributes to the decrease of susceptibility to hydrophilic FQs rather than to hydrophobic FQs. The MICs of amikacin, gentamicin and chloramphenicol showed >256-, >256- and 8-fold increases, respectively. For the β-lactam antibiotics, GDS466T showed 128-, 1032- and 4096-fold increases in the MICs of ampicillin, cefotaxime and ceftriaxone, respectively, when compared with the recipient.

For GDP5T, the MICs of ampicillin, cefotaxime and ceftriaxone were 128-, 4096- and 8192-fold higher than those for the recipient. The MICs of enrofloxacin and ciprofloxacin for this transconjugant showed 16–32-fold increases compared to those of the recipient. This was similar to those of GDS466T. Like GDS466T, GDP5T also showed a >256-fold increase in both of the MICs of amikacin and gentamicin, due to the transfer of the gene rmtB.

Southern blot hybridization was performed with digoxigenin-labelled probes specific for qepA1 and bla_CTX-M-14. The results shown in Fig. 1 confirmed the transfer of the plasmids and revealed the coexistence of qepA1 and bla_CTX-M-14 on the same plasmid of ~70 kb (pGDS466 and pGDP5, respectively) in strains GDS466 and GDP5. Notably, the rmtB gene might also be co-located on plasmids pGDS466 and pGDP5, according to the results of the analysis of these two plasmids and the association of qepA and rmtB reported previously (Liu et al., 2008). Plasmids pGDS466 and pGDP5 were both detected to harbour aac(6′)-Ib rather than aac(6′)-Ib-cr which often occurred in isolates harbouring aac(6′)-Ib during persistent exposure to FQs. Replicon typing of plasmids was performed using the method described by Carattoli et al. (2005) and both the plasmids carrying the qepA and bla_CTX-M-14 genes belonged to the IncFII group, which was confirmed by Southern blot hybridization with a digoxigenin-labelled probe specific for FII (Fig. 1). This was consistent with the results of Strahilevitz et al. (2009). However, these two plasmids showed very different plasmid restriction patterns (data not shown), indicating that the two plasmids were not derived from one identical plasmid through horizontal spread. The results suggest that the two E. coli strains are reservoirs of plasmids that contain multidrug-resistance genes.

To our knowledge, this is the first report of the coexistence of qepA1 and bla_CTX-M-14 on the same plasmid in an E. coli strain. The co-location of these resistance determinants on the same plasmid may be an important mechanism of dissemination of multidrug resistance and the dissemination could be promoted by use of the various antimicrobial agents. This may be worrisome and be a threat to public health. With the increasing numbers of bacterial clinical isolates that are resistant to FQs and cephalosporins, there is a great need to obtain more detailed knowledge on the association of FQ resistance and resistance to extended-spectrum β-lactams.