Study On PCR Of Detecting Six Escherichia Coli Plasmid-Mediated Quinolone Resistance Genes And Its Molecular Epidemiology

We used K-B(Kirby-Bauer) method to detect 256 Escherichia coli isolates collected at large scale pig farms, hospitals and wild animal zoos in 5 provinces in China from 2006 to 2008. The results showed that the resisrance rate to NA,NOR,OFX,ENR,CIP and LEV in 256 E.coli strains were 50.00%,48.83%,48.05%,47.66%,44.14% and 40.63 %. The E.coli isolates collected from pig farms, hospitals resistance to NOR, NA remarkably and the resisrance rate of isolates collected from wild animals is 12.50%.To investigate the prevalence of six plasmid-mediated quinolone resistance determinants genes:qnrA, qnrB, qnrS, aac(6')-Ib-cr, qepA and qnrD in 256 Escherichia coli isolates collected at large scale pig farms, hospitals and wild animal zoos in 5 provinces in China from 2006 to 2008.We established multi-PCR method for qnrB, qnrS, qnrD and conventional PCR method for qnrA, aac(6')-Ib-cr and qepA.The multiplex PCR amplification system were optimized including find out the best concentration of Mg2+. dNTPs,Taq enzyme and primers, the best annealing temperature and the best cycles. The best method of preparing template was also choosed and optimized, and 60% glycerin in the template makes Taq enzyme more stabilization. Specificity, sensibility test of the multi-PCR were carried out after we choosed the best modality of its combination.We used multi-PCR method to detect plasmid-mediated quinolone resistance determinants genes(qnrB, qnrS, qnrD) from bactaria successfully. The best parameters of amplification system is showed as follow:10×PCR Buffer 2.5μl, MgCl2 (25 mmol/L) 3μl, dNTPs (2.5 mmol/L) 3.5μl, template 2.5μl (McFarland No 1.0), Taq enzyme (2.5 U/μl) 0.35μl, forward and reverse primers each (25 mmol/L):qnrB (0.9μl),qnrS (0.6μl),qnrD (0.5μl), and ensure the total volume was 25μl by adding ddH2O. The reaction was performed with 5 min initial denaturation at 94℃.The PCR program comprised 30 cycles (1min at 94℃, 1min at 55℃,1min at 72℃). A final elongation was done in 10 min at 72℃followed the last cycle. Results showed that the multi-PCR method was specific, had a very high sensibility (3.0×104CFU/mL) and repeatability.The multi-PCR method was sensitive and cost less time.We usd multiple PCR to detect PMQR gene:qnrB, qnrS and qnrD and using conventional PCR to detect qnrA, aac (6)-Ib-cr and qepA.The detected plasmid-mediated quinolone resistance determinants were aac(6')-Ib-cr (59.38%),qnrD (30.47%), qnrA (30.47%), qepA (25.00%), qnrS (20.70%), qnrB (19.53%). The fluoroquinolones resistance genetype of aac(6')-Ib-cr (69.29%) and qnrD(43.57%) were detected frequently among swine isolates. The resistance genetype of aac(6')-Ib-cr (54.35%) and qnrS (37.00 %) were prevalent among human isolates remarkably.qnrB(8.33%), qepA(16.67%) and aac(6')-Ib-cr(20.83%) were detected in wildlife isolates.To our knowledge this is the first investigate of 6 PMQR genes of different origin E.coli strains.The fluoroquinolones resistance genetype of qnrD were prevalent among swine isolates remarkably.