JAC Advance Access originally published online on April 8, 2009
Journal of Antimicrobial Chemotherapy 2009 63(6):1128-1134; doi:10.1093/jac/dkp111
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Original research |
Mutational analysis of quinolone resistance in the plasmid-encoded pentapeptide repeat proteins QnrA, QnrB and QnrS
1 Department of Microbiology, University of Seville, Seville, Spain 2 Service of Microbiology, University Hospital Virgen Macarena, Seville, Spain 3 Service of Microbiology, University Hospital Marqués de Valdecilla, Santander, Spain 4 Department of Molecular Biology, University of Cantabria, Santander, Spain
Received 16 January 2009; returned 9 February 2009; revised 4 March 2009; accepted 4 March 2009
* Corresponding author. Tel: +34-954-55-28-63; Fax: +34-954-37-74-13; E-mail: jmrodriguez{at}us.es
Objectives: Pentapeptide repeat proteins (PRPs) QnrA, QnrB and QnrS confer reduced susceptibility to quinolones. This study presents an in vitro analysis of the genetic evolution of quinolone resistance mediated by changes in the coding sequences and promoter regions of qnrA1, qnrS1 and qnrB1 genes.
Methods: A random mutagenesis technique was used to predict the evolutionary potential of these PRPs against nalidixic acid and fluoroquinolones. After comparing the amino acid sequences of these and other PRPs protecting bacteria from quinolone activity, several conserved positions were found. The role of these residues in their effect against quinolones was evaluated by site-directed mutagenesis.
Results: Three different phenotypes (similar resistance, higher resistance or lower resistance to quinolones) were obtained in the random mutagenesis assays when compared with wild-type phenotypes. Only one mutant increased quinolone resistance: QnrS1 containing D185Y substitution (4-fold for ciprofloxacin). Using site-directed mutagenesis, residues G56, C72, C92, G96, F114, C115, S116, A117 and L159, according to the sequence of QnrA1, were analysed and despite the wide amino acid variability of the PRPs, most conserved residues analysed were critical to QnrA1, QnrB1 and QnrS1.
Conclusions: Amino acid sequences of PRPs QnrA1, QnrB1 and QnrS1 could be already optimized for quinolone resistance. One or several changes appear to be insufficient to obtain variants producing fluoroquinolone clinical resistance (MIC > 1 mg/L). Critical residues for quinolone resistance in PRPs were described. Interestingly, different effects were observed for QnrA1, QnrB1 and QnrS1 with the same substitution in several positions.
Keywords: fluoroquinolone resistance , qnr , PRPs
These authors contributed equally to this work.
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