Real-time PCR for fast detection of plasmid-mediated qnr genes in extended spectrum beta-lactamase producing Enterobacteriaceae

Aim of the study. - To develop a fast and reliable real time PCR technique for detecting plasmid-mediated quinolone resistance genes gnrA, gnrB and gnrS. Methods. - A real-time PCR assay using SYBR Greenland Roche LightCycler (R) was developed to detect qnr genes. Detection of qnr genes was based on comparison of melting temperature differences with a positive control of each qnr genes. This assay was performed to study 138 isolates collected from diagnostic and screening samples in the Champagne-Ardenne region in 2004 (France). Results. - In optimized conditions, the three positive controls tested alone and with isolates confirmed the specificity of the PCR primers. Each PCR assay was able to test 30 strains in 60 min for 1 qnr gene. Out of 138 isolates screened, 3.6% isolates were positive for a gnrAI, 1.5% for qnrSI and no gnrB-like gene. Prevalence of qnr determinants was 5% and reached 9.5% in clinical isolates. Conclusion. - Real-time PCR is a fast and reliable technique for screening of qnr-positive strains. This study shows a relatively high prevalence of qnr determinants (5%) among ESBL-producing Enterobacteriaceae. (C) 2009 Elsevier Masson SAS. All rights reserved.