Characterization of plasmid-mediated quinolone resistance genes in enterobacteriaceae clinical isolates in Thailand

Abstract

Quinolone resistance is mainly caused by chromosomal mutations in the quinolone resistance-determining region (QRDR) of DNA gyrase and topoisomerase IV. Recently, plasmid-mediated quinolone resistance (PMQR) including target protection mechanism encoded by qnr, efflux pump encoded by qepA and fluoroquinolone-modifying enzyme encoded by aac(6’)-Ib-cr has been described. This study characterized genes encoding PMQR and investigated the prevalence of quinolone and extended-spectrum cephalosporin resistance in Enterobactericeae isolates. A total of 671 Enterobacteriaceae isolated from patients during 2009-2011 were included in this study. The resistance rates to nalidixic, norfloxacin, ciprofloxacin, cefoxitin, ceftazidime, cefotaxime and ceftriaxone were 47.10%, 36.22%, 37.71%, 19.68%, 46.20%, 35.77% and 44.11%, respectively. The PMQR genes were detected in 20.57% (138 isolates).Of the 671 Enterobacteriaceae isolates, 86(12.82%), 49 (7.30%) and 3 (0.45%) carried qnr, aac(6’)-Ib-cr and qepA, respectively. The qnrS gene (8.05%) was the most common qnr found in Enterobacteriaceae isolates followed by qnrA (2.38%) and qnrD (2.38%) genes, respectively. The qnrB and qnrC genes were not detected in any isolates. Of the138 PMQR-positive isolates, 60.14% (83/138) were ESBL producers, 3.62% (5/138) were AmpC producers and 6.52% (9/138) were both ESBL and AmpC producers. The bla CTX-M gene was the most common ESBL determinant (31.16%, 43/138). The aac(6’)-Ib-cr-carrying isolates were commonly associated with ESBL genes (87.76%, 43/49) and qnr gene were found to be co-harboured with ESBL genes in 43.02% (37/86). All 3 qepA-positive isolates harboured ESBL genes (blaCTX-M and/or bla TEM). Mutations in quinolone target were investigated in 23 PMQR representative isolates. Three isolates which were highly resistant to ciprofloxacin (MICs of >256 µg/ml) had amino acid substitutions at S83L, D87N in GyrA and S80I in ParC, whereas no mutation was found in 20 isolates with reduced susceptibility and intermediate resistance to ciprofloxacin. All 3 ciprofloxacin-resistant isolates harbored the qepA gene and had efflux pump activity. The qnrA, qnrS, blaCIT, blaCTX-M, blaTEM and blaVEB genes were successfully transferred to E. coli UB1637AzR by conjugation method. The qnr, ESBL and ampC genes were found to be co-transferred. Our study showed high rates of quinolone and extended spectrum-cephalosporin resistance in Enterobacteriaceae. The qnr genes were the most prevalent PMQR determinant and were able to be transferred with ESBL and ampC genes. This is the first report of the presence of qepA and qnrD genes in Enterobacteriaceae isolated from Thailand.