Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates

Objectives: To develop a rapid and reliable single-tube-based PCR technique for detecting simul-taneously the plasmid-mediated quinolone resistance qnrA, qnrB and qnrS genes. Methods: After multiple alignments, primers were designed to detect known qnr variants (six for qnrA-, six for qnrB- and two for qnrS-like genes). They were used for screening a collection of 64 expanded-spectrum b-lactamase (ESBL)-producing enterobacterial isolates from Kuwait, collected from 2002 to 2004, as ESBL genes have been often associated with qnr genes. Sequencing was performed to ident-ify qnr and associated ESBL genes. Results: In optimized conditions, all positive controls (used separately or mixed) confirmed the speci-ficity of the PCR primers. Out of 64 isolates, only 3 isolates were positive for a qnrB-like gene (4.7%), whereas no qnrA-like and qnrS-like gene was detected. A qnrB2 gene was detected in an Enterobacter cloacae K34 (SHV-121) isolate, whereas qnrB1-like (termed qnrB7) and qnrB6-like (termed qnrB8) genes were identified from E. cloacae K37 (SHV-121) and Citrobacter freundii K70 (VEB-1b1) isolates, respectively. Conclusions: We report here a fast and reliable technique for rapid screening of qnr-positive strains to be used for epidemiological surveys. A low prevalence of Qnr determinants among ESBL-producing Enterobacteriaceae was identified in the study with Kuwaiti isolates