Constitutive overexpression of the active efflux system MexXY/OprM is a major cause of resistance to aminoglycosides, fluoroquinolones, and cefepime in clinical strains of Pseudomonas aeruginosa. Upregulation of this pump often results from mutations occurring in mexZ, the local repressor gene of the mexXY operon. In this study, analysis of MexXY-overproducing mutants selected in 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 intact mexZ 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 that mexXY overexpression was independent of mexZ and the PA5471 gene, which is required for drug-dependent induction of mexXY. Furthermore, the transcript levels of the oprD gene, which encodes the carbapenem-selective porin OprD, were found to be reduced drastically in PAOW2. Wholegenome 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 of P. aeruginosa to polycationic peptides such as colistin. The multidrug resistance phenotype was suppressed in PAOW2 by deletion of the parS and parRS genes and conferred to PAO1Bes by chromosomal insertion of the mutated parRS 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 operon arnBCADTEF-ugd, responsible for resistance to polycationic agents. Exposure of wild-type PAO1Bes to different polycationic peptides, including colistin, was shown to result in increased mexY and repressed oprD 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 ⌬parRS 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).

bConstitutive 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 mexXY operon.

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.

The Iccare web server, http://genopole.toulouse.inra.fr/bioinfo/Iccare, provides a simple yet efficient tool for crude EST (expressed sequence tag) annotation specifically dedicated to comparative mapping approaches. Iccare uses all the EST and mRNA sequences from public databases for an organism of interest (query species) and compares them to all the transcripts of one reference organism (Homo sapiens or Arabidopsis thaliana). The results are displayed according to the location of the genes on the chromosomes of the reference organism. Gene structure information and sequence similarities are combined in a graphical representation in order to pinpoint the nature of the transcript query sequence. The user can subsequently design primers or probes for the purpose of physical or genetic mapping. In addition to the query organisms already available in Iccare, users can perform a tailor-made search with their own sequences against the animal or plant reference organism genes.

Resistance mechanisms of Pseudomonas aeruginosa to ceftolozane/tazobactam (C/T) were assessed on a collection of 420 nonredundant strains non-susceptible to ceftazidime (MIC > 8 μg/ml) and/or imipenem (> 4 μg/ml), collected by 36 French hospital laboratories over a one month period (GERPA study). Rates of C/T resistance (MIC > 4/4 μg/ml) were equal to 10% in this population (42/420 strains), and 23.2% among the isolates resistant to both ceftazidime and imipenem (26/112). A first group of 21 strains (50%) was found to harbor various extended-spectrum β-lactamases (1 OXA-14; 2 OXA-19; 1 OXA-35; 1 GES-9; 3 PER-1), carbapenemases (2 GES-5; 1 IMP-8; 8 VIM-2), or both (1 VIM-2/OXA-35; 1 VIM-4/SHV-2a). All the strains of this group belonged to widely distributed epidemic clones (ST111, ST175, CC235, ST244, ST348, ST654), and were highly resistant to almost all the antibiotics tested except colistin. A second group was composed of 16 (38%) isolates moderately resistant to C/T (MIC from 8/4 to 16/4 μg/ml), of which 7 were related to international clones (ST111, ST253, CC274, ST352, ST386). As demonstrated by targeted mass spectrometry, cloxacillin-based inhibition tests and gene blaPDC deletion experiments, this resistance phenotype was correlated to an extremely high production of cephalosporinase PDC. In part accounting for this strong PDC upregulation, genomic analyses revealed the presence of mutations in regulator AmpR (D135N/G in 6 strains) and enzymes of the peptidoglycan recycling pathway such as AmpD, PBP4, and Mpl (9 strains). Finally, all the 5 (12%) remaining C/T resistant strains (group 3) appeared to encode PDC variants with mutations known to improve the hydrolytic activity of the β-lactamase to ceftazidime and C/T (F147L, ΔL223-Y226, E247K, N373I). Collectively, our results highlight the importance of both intrinsic and transferable mechanisms in C/T resistant P. aeruginosa. Which mutational events lead some clinical strains to massively produce the natural cephalosporinase PDC remains incompletely understood.