Multiple mutations lead to MexXY / OprM - dependent aminoglycoside resistance in 1 clinical strains of Pseudomonas aeruginosa 2 3 4 5

24 25 Constitutive overproduction of the pump MexXY/OprM (XY+/M) is recognized as a major 26 cause of resistance to aminoglycosides, fluoroquinolones, and zwitterionic cephalosporins in 27 P. aeruginosa. In this study, 57 clonally-unrelated strains recovered from non-cystic fibrosis 28 patients were analyzed to characterize the mutations resulting in upregulation of the mexXY 29 operon. Forty four (77.2%) of the strains, classified as agrZ mutants, were found to harbor 30 mutations inactivating the local repressor gene (mexZ) of mexXY operon (n=33; 57.9%) or 31 introducing amino acid substitutions in its product, MexZ (n=11; 19.3%). These sequence 32 variations which mapped in the dimerization domain, the DNA binding domain, or the rest of 33 the MexZ structure, mostly affected amino acid positions conserved in TetR-like regulators. 34 The 13 remaining XY+/M strains (22.8%) contained intact mexZ genes encoding wild-type 35 MexZ proteins. Eight (14.0%) of these isolates, classified as agrW1 mutants, overexpressed 36 gene PA5471 which codes for the MexZ antirepressor AmrZ, with 5 strains exhibiting growth 37 defects at 37C and 44C consistent with mutations impairing ribosome activity. Interestingly, 38 one agrW1 mutant appeared to harbor a 7-bp deletion in the coding sequence of leader 39 peptide PA5471.1 involved in ribosome-dependent, translational attenuation of PA5471 40 expression. Finally DNA sequencing and complementation experiments revealed that 5 41 (8.8%) strains, classified as agrW2 mutants, harbored single amino acid variations in the 42 sensor histidine kinase of ParRS, a two component system known to positively control mexXY 43 expression. Collectively, these results demonstrate that clinical strains of P. aeruginosa 44 exploit different regulatory circuitries to mutationally overproduce the MexXY/OprM pump 45 and become multidrug resistant, which accounts for the high prevalence of XY+/M mutants in 46 the clinical setting. 47