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ISEcp1-mediated transposition of qnrB5 gene in Escherichia coli

Abstract number: P2013

Cattoir V., Poirel L., Silva J., Espinal P., Soussy C., Nordmann P.

Objectives: Plasmid-mediated quinolone resistance due to Qnr determinants is increasingly reported worldwide in enterobacterial isolates being of the QnrA, QnrB and QnrS types. The qnrA-and qnrB-like genes are often identified in sul1-type integrons (ISCR1-based mobilisation). The aim of this study was (i) to investigate the genetic environment of a qnrB gene identified in an Escherichia coli isolate recovered from Colombia, and (ii) to experimentally evaluate the mobility of that structure in E. coli.

Methods: Cloning experiments were performed with EcoRI-restricted whole-cell DNA of E. coli clinical isolate R4525 and the genetic environment surrounding the qnrB gene was determined by primer walking. The transcription start site was determined by 5'RACE analysis. The ISEcp1-mediated mobilisation of the qnrB5 gene was attempted from E. coli R4525 to recA (-) E. coli recipient strain using self-conjugative and IS-free plasmid pOX38-Gen as a target for transposition events.

Results: The qnrB5 gene was located at the right-hand extremity of the ISEcp1 element, forming a 2,739-bp long transposon flanked by a 5-bp duplication of the target site (ATCAA) and inserted inside orf1 of Tn1721. As opposed to what has been reported for b-lactamase genes of the CTX-M type, the qnrB5 gene was located in an opposite orientation, relative to the transposase ORF of ISEcp1. Therefore, ISEcp1 did not provide promoter sequences for its expression. ISEcp1B-mediated transposition of qnrB5 into E. coli was successful in vitro and was found to occur at a frequency of ca. 10–6 per donor by a mechanism similar to one-ended transposition. The promoter sequences able to enhance the qnrB5 expression were determined, being made of a -35 box (TTGACG) and a -10 box (TACCAT) separated by a 17-bp sequence, with the transcription start site located 30 bp upstream of the codon start. This allowed us to consider the QnrB5 protein to be 214 amino-acid long thus shorter than previously hypothesised.

Conclusion: These findings show that ISEcp1 is at the origin of acquisition of the qnrB5 gene in E. coli. ISEcp1-like elements have previously shown to mobilize b-lactamase genes (blaCTX-M) and rmtC genes (resistance to aminoglycosides).

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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