A new pathway involved in cross-resistance to antibiotics in Pseudomonas aeruginosa
Abstract number: P769
Objectives: Constitutive overproduction of the multidrug efflux system MexXY/OprM provides P. aeruginosa with a moderate resistance to fluoroquinolones, aminoglycosides, and cefepime. In mutants named agrZ, MexXY/OprM upregulation results from inactivation of gene mexZ, the product of which represses the meXY operon. However, other efflux mutants (agrW) have been reported among clinical strains, that exhibit an intact mexZ gene. This study investigates a subclass of this latter type of mutants, dubbed agrW2.
Methods: Spontaneous MexXY/OprM overproducing mutants from reference strain PAO1 were selected on amikacin in vitro. Their drug susceptibility profiles were determined with the conventional agar dilution method according to standard guidelines. Search for mexZ mutations was performed by DNA sequencing and BLAST alignement analysis. Transript levels of genes mexY, mexZ, oprD, PA5470, and PA5471 together with production of transporter MexY were assessed by quantitative Real Time PCR (RT-qPCR) and immunoblotting, respectively. The point mutation in a selected agrW2 mutant was identified by whole genome sequencing (6.3 Mbp) by using an Illumina Genome Analyser.
Results: Analysis of spontaneous amikacin resistant clones from strain PAO1 led to identification of a new type of MexXY/OprM overproducing mutants (agrW2). Unlike previously reported agrW mutants, the agrW2 mutants exhibited increased resistance to carbapenems (imipenem 8 mg/mL) in addition to cefepime (16 mg/mL), ciprofloxacin (1 mg/mL), and aminoglycosides (amikacin 1632 mg/mL). Upregulation of MexY gene (1517 fold) and downregulation of porin OprD gene (0.060.07 fold) were in full agreement with this new resistance phenotype. Gene deletion experiments demonstrated that neither repressor gene mexZ nor the PA5471-PA5470 locus known as essential for mexXY drug induction were involved in MexXY/OprM derepression in the agrW2 mutants. Whole genome sequence analysis of a selected agrW2 clone revealed a single nucleotide change (G®A) inactivating a yet uncharacterized signal transduction system. Alteration of this system was recognized in clinical multidrug resistant strains.
Conclusion: This study provides new insight into the complex regulation of the MexXY/OprM pump. Importantly, it shows that aminoglycosides are able to select porin-deficient mutants of P. aeruginosa cross-resistant to carbapenems.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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