Efflux Unbalance in Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

Abstract

Retrospective analysis of 189 nonredundant strains of Pseudomonas aeruginosa sequentially recovered from the sputum samples of 46 cystic fibrosis (CF) patients over a 10-year period (1998 to 2007) revealed that 53 out of 189 (28%) samples were hypersusceptible to the ␤-lactam antibiotic ticarcillin (MIC < 4 g/ml) (phenotype dubbed Tic hs ). As evidenced by trans-complementation and gene inactivation experiments, the mutational upregulation of the efflux system MexXY was responsible for various degrees of resistance to aminoglycosides in a selection of 11 genotypically distinct strains (gentamicin MICs from 2 to 64 g/ml). By demonstrating for the first time that the MexXY pump may evolve in CF strains, we found that a mutation leading to an F1018L change in the resistance-nodulation-cell division (RND) transporter MexY was able to increase pump-promoted resistance to aminoglycosides, cefepime, and fluoroquinolones twofold. The inactivation of the mexB gene (which codes for the RND transporter MexB) in the 11 selected strains showed that the Tic hs phenotype was due to a mutational or functional loss of function of MexAB-OprM, the multidrug efflux system known to contribute to the natural resistance of P. aeruginosa to ␤-lactams (e.g., ticarcillin and aztreonam), fluoroquinolones, tetracycline, and novobiocin. Two of the selected strains synthesized abnormally low amounts of the MexB protein, and 3 of 11 strains expressed truncated MexB (n ‫؍‬ 2) or MexA (n ‫؍‬ 1) polypeptide as a result of mutations in the corresponding genes, while 7 of 11 strains produced wild-type though nonfunctional MexAB-OprM pumps at levels similar to or even higher than that of reference strain PAO1. Overall, our data indicate that while MexXY is necessary for P. aeruginosa to adapt to the hostile environment of the CF lung, the MexAB-OprM pump is dispensable and tends to be lost or inactivated in subpopulations of P. aeruginosa.

MexXY efflux pump overexpression and aminoglycoside resistance in cystic fibrosis isolates of Pseudomonas aeruginosa from chronic infections

“…The range of overexpression of mexY varied from 6.76-to 73-fold. It has been suggested that MexXY expression contributes not only to the antibiotic resistance of CF P. aeruginosa isolates but also to its adaptive strategy in the CF lung environment (Smith et al 2006;Vettoretti et al 2009). Widespread expression of MexXY pump in the CF isolates we tested does suggest that this pump may have a role to play that goes beyond their antibiotic resistance.…”

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…In some cases, these mutations cause amino acid changes that make a pump more efficient at extruding the antibiotics out of the cell. For instance, Vettoretti et al (252) described an F1018L substitution in MexY, an RND transporter of P. aeruginosa, which increased resistance to aminoglycosides, fluoroquinolones, and the ␤-lactam cefepime, mediated by the MexXY efflux pump. Notably, this mutation was present in clinical isolates from cystic fibrosis (CF) patients.…”

Oxidative Stress Induction of the MexXY Multidrug Efflux Genes and Promotion of Aminoglycoside Resistance Development in Pseudomonas aeruginosa

“…Upregulation of mexXY by antimicrobials or fmt/ folD mutations is dependent upon a gene, PA5471, encoding a conserved hypothetical protein whose expression is also promoted by ribosome-disrupting antimicrobials (34) and by fmt/ folD mutations (6). Despite this primary link to translation disruption, the MexXY-OprM efflux system is a significant determinant of resistance to antimicrobials in clinical isolates, particularly aminoglycosides (19,40,52) but also ␤-lactams (3,19,23,37,51). Indeed, while it is uncommon as a mechanism of aminoglycoside resistance in most clinical strains of P. aeruginosa, MexXY-OprM is the predominant mechanism of resistance to these agents in cystic fibrosis (CF) isolates (19,40,52).…”