A Two-Component Regulatory System Interconnects Resistance to Polymyxins, Aminoglycosides, Fluoroquinolones, and β-Lactams in Pseudomonas aeruginosa

Constitutive overexpression of the active efflux system MexXY/OprM is a major cause of resistance to aminoglycosides, fluoroquinolones, and cefepime in clinical strains ofPseudomonas aeruginosa . Upregulation of this pump often results from mutations occurring inmexZ , the local repressor gene of themexXY operon. In this study, analysis of MexXY-overproducing mutants selectedin vitro from reference strain PAO1Bes on amikacin (at a concentration 1.5-fold higher than the MIC) led to identification of a new class of mutants harboring an intactmexZ gene and exhibiting increased resistance to colistin and imipenem in addition to aminoglycosides, fluoroquinolones, and cefepime. Reverse transcription-quantitative PCR (RT-qPCR) experiments on a selected clone named PAOW2 demonstrated thatmexXY overexpression was independent ofmexZ and the PA5471 gene, which is required for drug-dependent induction ofmexXY . Furthermore, the transcript levels of theoprD gene, which encodes the carbapenem-selective porin OprD, were found to be reduced drastically in PAOW2. Whole-genome sequencing revealed a single mutation resulting in an M59I substitution in the ParR protein, the response regulator of the ParRS two-component regulatory system (with ParS being the sensor kinase), which is required for adaptive resistance ofP. aeruginosa to polycationic peptides such as colistin. The multidrug resistance phenotype was suppressed in PAOW2 by deletion of theparS andparRS genes and conferred to PAO1Bes by chromosomal insertion of the mutatedparRS locus from PAOW2. As shown by transcriptomic analysis, only a very small number of genes were expressed differentially between PAOW2 and PAO1Bes, including the lipopolysaccharide (LPS) modification operonarnBCADTEF-ugd , responsible for resistance to polycationic agents. Exposure of wild-type PAO1Bes to different polycationic peptides, including colistin, was shown to result in increasedmexY and repressedoprD expression via ParRS, independent of PA5471. In agreement with these results, colistin antagonized activity of the MexXY/OprM substrates in PAO1Bes but not in a ΔparRS derivative. Finally, screening of clinical strains exhibiting the PAOW2 resistance phenotype allowed the identification of additional alterations in ParRS. Collectively, our data indicate that ParRS may promote either induced or constitutive multidrug resistance to four different classes of antibiotics through the activation of three distinct mechanisms (efflux, porin loss, and LPS modification).