Programme Hospitalier Recherche Clinique, Institut Pasteur, Inserm, French Public Health Agency.

No abstract

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).

b Mutation-dependent overproduction of intrinsic ␤-lactamase AmpC is considered the main cause of resistance of clinical strains of Pseudomonas aeruginosa to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the ␤-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient P. aeruginosa 4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on ␤-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gramnegative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that P. aeruginosa is able to adapt to efficacious ␤-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic ␤-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are >1.5% in the clinical setting. Pseudomonas aeruginosa is a well-known cause of acute and chronic infections in fragile patients. One of the most remarkable traits of this opportunistic pathogen is its ability to evolve and become resistant to many antibiotics through a variety of mutational and transferable mechanisms (reviewed in reference 1). Some mechanisms tend to prevent the interaction of drugs with their cognate cellular targets, while others result in drug inactivation (1). Like several other Gram-negative species, P. aeruginosa harbors a chromosomal drug-inducible gene, bla AmpC , encoding a wide-spectrum class C ␤-lactamase (2). This enzyme contributes to the natural resistance of the microorganism toward labile and inducing molecules, such as aminopenicillins, first-and secondgeneration cephalosporins (3). More importantly, when overproduced as a result of mutations altering the peptidoglycan recycling process, AmpC becomes a major cause of resistance to widely used antipseudomonal penicillins (ticarcillin and piperacillin), monobactams (aztreonam), and third-generation (ceftazidime) and fourth-generation (cefepime) cephalosporins (4-7). The so-called "derepressed mutants" are common in the clinical setting and account for a large proportion of strains resistant to ceftazidime and cefepime in various studies (8-11)....

MexXY is an inducible efflux system that contributes to the natural resistance of Pseudomonas aeruginosa to antibiotics. Experiments involving real-time PCR after reverse transcription in reference strain PAO1 showed concentration-dependent induction of gene mexY by various ribosome inhibitors (e.g., chloramphenicol, tetracycline, macrolides, and aminoglycosides) but not by antibiotics acting on other cellular targets (e.g., ␤-lactams, fluoroquinolones). Confirming a functional link between the efflux system and the translational machinery, ribosome protection by plasmid-encoded proteins TetO and ErmBP increased the resistance of a ⌬mexAB-oprM mutant of PAO1 to tetracycline and erythromycin, respectively, as well as the concentrations of both drugs required to induce mexY. Furthermore, spontaneous mutations resulting in specific resistance to dihydrostreptomycin or spectinomycin also raised the minimal drug concentration for mexXY induction in strain PAO1. While strongly upregulated in a PAO1 mutant defective in gene mexZ (which codes for a putative repressor of operon mexXY), gene mexY remained inducible by agents such as tetracycline, chloramphenicol, and spectinomycin, suggesting additional regulatory loci for mexXY. Altogether, these data demonstrate physiological interplays between MexXY and the ribosome and are suggestive of an alternative function for MexXY beyond antibiotic efflux.

We recently identified a hypervirulent strain of Pseudomonas aeruginosa, differing significantly from the classical strains in that it lacks the type 3 secretion system (T3SS), a major determinant of P. aeruginosa virulence. This new strain secretes a novel toxin, called ExlA, which induces plasma membrane rupture in host cells. For this study, we collected 18 other exlA-positive T3SS-negative strains, analyzed their main virulence factors and tested their toxicity in various models. Phylogenetic analysis revealed two groups. The strains were isolated on five continents from patients with various pathologies or in the environment. Their proteolytic activity and their motion abilities were highly different, as well as their capacity to infect epithelial, endothelial, fibroblastic and immune cells, which correlated directly with ExlA secretion levels. In contrast, their toxicity towards human erythrocytes was limited. Some strains were hypervirulent in a mouse pneumonia model and others on chicory leaves. We conclude that (i) exlA-positive strains can colonize different habitats and may induce various infection types, (ii) the strains secreting significant amounts of ExlA are cytotoxic for most cell types but are poorly hemolytic, (iii) toxicity in planta does not correlate with ExlA secretion.

Screening of a Tn5-Hg insertional library (12,000 clones) constructed in wild-type Pseudomonas aeruginosa strain PAO1 identified four genes (namely, galU, nuoG, mexZ, and rplY) whose disruption individually led to increased resistance to aminoglycosides (means of twofold). Inactivation of these genes was associated with (i) impaired outer membrane uptake, (ii) reduced active transport, (iii) increased MexXY-OprM-mediated active efflux, and (iv) alteration of target of aminoglycosides, respectively. In addition, suppression of the gene rplY, which codes for ribosomal protein L25, was found to result in both moderate upregulation of the efflux system MexXY-OprM and hypersusceptibility to ␤-lactam antibiotics. Construction of double, triple, and quadruple mutants demonstrated cumulative effects of the different mechanisms on aminoglycoside resistance, with MICs increasing from 16-to 64-fold in the quadruple mutant compared to the wild-type strain PAO1. Altogether, these results illustrate how P. aeruginosa may gradually develop high resistance to these antibiotics via intrinsic (i.e., nonenzymatic) mechanisms, as in cystic fibrosis patients.

Since their initial description 2 decades ago, MexCD-OprJ-overproducing efflux mutants of Pseudomonas aeruginosa (also called nfxB mutants) have rarely been described in the clinical setting. Screening of 110 nonreplicate clinical isolates showing moderate resistance to ciprofloxacin (MIC from 0.5 g/ml to 4 g/ml) yielded only four mutants (3.6%) of that type harboring various alterations in the repressor gene nfxB. MexCD-OprJ upregulation correlated with an increased resistance to ciprofloxacin, cefepime, and chloramphenicol in most of the clinical strains, concomitant with a higher susceptibility to ticarcillin, aztreonam, imipenem, and aminoglycosides. Evidence was obtained that this increased susceptibility to aminoglycosides results from the impaired activity of efflux pump MexXY-OprM. Furthermore, MexCD-OprJ upregulation was found to impair bacterial growth and to have a strain-specific, variable impact on rhamnolipid, elastase, phospholipase C, and pyocyanin production. Review of patient files indicated that the four nfxB mutants were responsible for confirmed cases of infection and emerged during long-term therapy with ciprofloxacin. Taken together, these data show that, while rather infrequent among P. aeruginosa strains with low-level resistance to ciprofloxacin, MexCD-OprJ-overproducing mutants may be isolated after single therapy with fluoroquinolones and may be pathogenic.Ten intrinsic multidrug efflux systems belonging to the RND (resistance nodulation cell division) family have been characterized so far in Pseudomonas aeruginosa (58). Of all these pumps, only MexAB-OprM, MexXY-OprM, MexCD-OprJ, and MexEF-OprN have been reported to provide significant resistance to antibiotics when stably overproduced upon mutations (see reference 53 for a recent review). In contrast to MexAB-OprM and MexXY-OprM, the MexCD-OprJ system does not contribute to the natural resistance of the pathogen to antimicrobials (54). However, alteration of the nfxB gene, whose product strongly represses the mexCD-oprJ operon, leads to a dramatic increase in MexCD-OprJ production and significant cross-resistance to fluoroquinolones, macrolides, and zwitterionic cephems such as cefpirome and cefepime (15,35,46,51,54). In addition, most but not all of the so-called "nfxB mutants" appear to be more susceptible than wild-type strains to aminoglycosides and other ␤-lactams (e.g., carbenicillin, aztreonam, and imipenem) (15,24,25,45,54). They also may exhibit variable levels of resistance to tetracycline and chloramphenicol, thus making the NfxB phenotype difficult to identify among clinical strains. Such changes in the NfxB phenotype have been attributed to variations in the levels of MexCD-OprJ production (45). More recently, the increased susceptibility of in vitro-selected nfxB mutants to ␤-lactams (except imipenem) was attributed to the decreased expression of system MexAB-OprM and/or deficient drug induction of intrinsic ␤-lactamase AmpC (13, 47).Since the first description of these mutants by Hirai et al. (15) 2 decades ago, very ...