Multiplex PCR for detection of plasmid-mediated quinolone resistance genes in Enterobacteriaceae consecutively isolated from blood cultures
Abstract number: P1467
Cano M.E., Calvo J., Agüero J., Martínez-Martínez L.
Objectives: Plasmid-mediated quinolone resistance (PMQR) caused by qnr, qepA and/or aac(6')-Ib-cr genes have often been investigated in enterobacteria with concrete resistance phenotypes or in particular species. In this study, the presence of PMQR genes in enterobacteria consecutively isolated from blood cultures was evaluated using a multiplex PCR.
Methods: Three hundred and forty-two Enterobacteriaceae (1/patient) consecutively isolated from blood cultures (January 2007-March 2008) were studied. Identification and susceptibility testing were performed with the MicroScan WalkAway system (Dade Behring). Isolates containing PMQR genes were typed by REP-PCR and the MICs of nalidixic acid (NAL), ciprofloxacin (CIP) and levofloxacin (LEV) against them were determined by Etest. qnrA, qnrB, qnrS, qepA and aac(6')-Ib were detected by a multiplex PCR (degenerated primers for amplifying 20 qnrB variants were used). Positive reactions were confirmed by sequencing (both strands) of amplicons obtained with other primers amplifying the entire, or almost entire, gene.
Results: We tested 180 E. coli, 44 K. pneumoniae, 31 E. cloacae, 24 S. marcescens, 17 P. mirabilis, 13 K. oxytoca, 10 C. freundii, 8 E. aerogenes and 15 other species. The multiplex PCR correctly detected PMQR genes of positive controls, individually and in all possible combinations. PMQR genes were overall detected in 24 (7%) isolates. qnrB, qnrS and aac(6')-Ib-cr were detected in five (1.5%), 13 (3.8%) and seven (2%) isolates, respectively. One E. coli contained both qnrS1 and aac(6')-Ib-cr. No isolates carried qnrA or qepA. Four of 5 qnrB were new alleles detected in 4 clonally unrelated C. freundii; the remaining case corresponded to qnrB1 in an E. cloacae. qnrS genes corresponded to 12 qnrS1 (3 in 3 clones of E. coli, 8 in 3 clones of E. cloacae, 1 isolate of K. pneumoniae) and 1 qnrS2 (in a K. pneumoniae strain clonally unrelated to the qnrS1-positive strain). aac(6')-Ib-cr was detected in 6 E. coli (4 clones) and 1 E. aerogenes. In the 24 isolates with PMQR genes, resistance (CLSI breakpoints) to NAL, CIP and LEV were 75%, 46% and 42%, respectively.
Conclusion: PMQR genes were successfully detected with the multiplex PCR developed in this study. qnrS variants were the more common PMQR genes detected, followed by aac(6')-Ib-cr and qnrB alleles. A significant proportion of strains with these genes are susceptible to both nalidixic acid and fluoroquinolones.
|Session name:||19th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Helsinki, Finland, 16 - 19 May 2009|
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