Multiple Mutations Lead to MexXY-OprM-Dependent Aminoglycoside Resistance in Clinical Strains of Pseudomonas aeruginosa

ABSTRACTConstitutive overproduction of the pump MexXY-OprM is recognized as a major cause of resistance to aminoglycosides, fluoroquinolones, and zwitterionic cephalosporins in Pseudomonas aeruginosa. In this study, 57 clonally unrelated strains recovered from non-cystic fibrosis patients were analyzed to characterize the mutations resulting in upregulation of the mexXYoperon. Forty-four (77.2%) of the strains, classified as agrZmutants were found to harbor mutations inactivating the local repressor gene (mexZ) of the mexXYoperon (n= 33; 57.9%) or introducing amino acid substitutions in its product, MexZ (n= 11; 19.3%). These sequence variations, which mapped in the dimerization domain, the DNA binding domain, or the rest of the MexZ structure, mostly affected amino acid positions conserved in TetR-like regulators. The 13 remaining MexXY-OprM strains (22.8%) contained intact mexZgenes encoding wild-type MexZ proteins. Eight (14.0%) of these isolates, classified as agrW1mutants, overexpressed the gene PA5471, which codes for the MexZ antirepressor AmrZ, with 5 strains exhibiting growth defects at 37°C and 44°C, consistent with mutations impairing ribosome activity. Interestingly, one agrW1mutant appeared to harbor a 7-bp deletion in the coding sequence of the leader peptide, PA5471.1, involved in ribosome-dependent, translational attenuation of PA5471 expression. Finally, DNA sequencing and complementation experiments revealed that 5 (8.8%) strains, classified as agrW2mutants, harbored single amino acid variations in the sensor histidine kinase of ParRS, a two-component system known to positively control mexXYexpression. Collectively, these results demonstrate that clinical strains of P. aeruginosaexploit different regulatory circuitries to mutationally overproduce the MexXY-OprM pump and become multidrug resistant, which accounts for the high prevalence of MexXY-OprM mutants in the clinical setting.