Retrospective analysis of 189 non redundant strains of Pseudomonas aeruginosa sequentially recovered from the sputums of 46 cystic fibrosis (CF) patients over a 10 year period (1998-2007) revealed that 53/189 (28%) were hypersusceptible to ss-lactam antibiotic ticarcillin (MIC = 4 microg/mL, phenotype dubbed Tic (HS)). As evidenced by trans complementation and gene inactivation experiments, 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 microg/mL). Demonstrating for the first time that the MexXY pump may evolve in CF strains, we found that a mutation leading to a F1018L change in RND transporter MexY was able to increase pump-promoted resistance to aminoglycosides, cefepime and fluoroquinones 2-fold. Inactivation of gene mexB (which codes for RND transporter MexB) in the 11 selected strains showed that the Tic (HS) phenotype was due to mutational or functional loss-of-function of MexAB-OprM, the multidrug efflux system known to contribute to the natural resistance of P. aeruginosa to ss-lactams (e.g., ticarcillin, aztreonam), fluoroquinolones, tetracycline, and novobiocin. Two of the selected strains synthetized abnormally low amounts of protein MexB, 3/11 expressed truncated MexB (n = 2) or MexA (n = 1) polypeptides as a result of mutations in corresponding genes, while 7/11 produced wild-type though non functional 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 CF lung, the MexAB-OprM pump is dispensable and tends to be lost or inactivated in subpopulations of P. aeruginosa.