Quinolone Resistance Detection by PCR-RFLP and Multiplex-PCR among Extended- Spectrum β- Lactamase Producing Enterobacteriaceae

Abstract

Quinolone resistance limits the therapeutic potential for the Extended-Spectrum β- lactamase (ESBL) producing Enterobacteriaceae. The aim of this study was to investigate the most common mechanisms of quinolones resistance in ESBL- producing Enterobacteriaceae using both phenotypic and genotypic methods. Out of 1766 clinical isolates collected between October 2012 and September 2013, 219 Enterobacteriaceae clinical isolates were ESBL producers, nalidixic acid and ciprofloxacin resistant were identified by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDITOF/ MS) and the Minimal Inhibitory Concentration (MIC) values of ciprofloxacin and nalidixic acid wasdetected before and after addition of phenylalanine-arginine β-naphthylamide (PaβN) efflux pump inhibitor. Thirty three isolates were selected for screening of the plasmid-mediated fluoroquinolone resistance (PMQR) genes; (qnr, aac(6’)-Ib-cr and qepA) and the efflux pump genes (oqxAB genes) by multiplex PCR. Whereas GyrA and ParC genes mutations were detected by PCR-RFLP assay. The PaβN changed the MIC values of 28 isolates. The GyrA gene mutation was detected in 24/33 (72.7%), while the par C gene mutation was detected in 3/33 (9.1%). Qnr-genes were detected in 13/33 (39.4%), aac(6')-Ib gene was detected in 24/33 (72.7%). qepA gene was detected in only one Klebsiella pneumoniae isolate. Finally the oqxA gene was detected in 16/33 (48.5%) of the studied isolates. The present study indicated that the PaβN was an effective phenotypic screening method for quinolones resistance efflux pump; moreover PCR-RFLP offered simple and rapid method for detection of ciprofloxacin-resistance that could be useful for clinical diagnosis and epidemiological studies.