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ABSTRACTMultidrug-resistant mutants of Pseudomonas aeruginosathat overproduce the active efflux system MexEF-OprN (called nfxCmutants) have rarely been characterized in the hospital setting. Screening of 221 clinical strains exhibiting a reduced susceptibility to ciprofloxacin (a substrate of MexEF-OprN) and imipenem (a substrate of the negatively coregulated porin OprD) led to the identification of 43 (19.5%) nfxCmutants. Subsequent analysis of 22 nonredundant mutants showed that, in contrast to their in vitro-selected counterparts, only 3 of them (13.6%) harbored a disrupted mexSgene, which codes for the oxidoreductase MexS, whose inactivation is known to activate the mexEF-oprNoperon through a LysR-type regulator, MexT. Nine (40.9%) of the clinical nfxCmutants contained single amino acid mutations in MexS, and these were associated with moderate effects on resistance and virulence factor production in 8/9 strains. Finally, the remaining 10 (45.5%) nfxCmutants did not display mutations in any of the regulators known to control mexEF-oprNexpression (the mexS, mexT, mvaT, and ampRgenes), confirming that other loci are responsible for pump upregulation in patients. Collectively, these data demonstrate that nfxCmutants are probably more frequent in the hospital than previously thought and have genetic and phenotypic features somewhat different from those of in vitro-selected mutants.

ABSTRACTThe development of novel antimicrobial agents is urgently required to curb the widespread emergence of multidrug-resistant bacteria like colistin-resistant Pseudomonas aeruginosa. We previously synthesized a series of amphiphilic neamine derivatives active against bacterial membranes, among which 3′,6-di-O-[(2″-naphthyl)propyl]neamine (3′,6-di2NP), 3′,6-di-O-[(2″-naphthyl)butyl]neamine (3′,6-di2NB), and 3′,6-di-O-nonylneamine (3′,6-diNn) showed high levels of activity and low levels of cytotoxicity (L. Zimmermann et al., J. Med. Chem. 56:7691–7705, 2013). We have now further characterized the activity of these derivatives against colistin-resistant P. aeruginosaand studied their mode of action; specifically, we characterized their ability to interact with lipopolysaccharide (LPS) and to alter the bacterial outer membrane (OM). The three amphiphilic neamine derivatives were active against clinical colistin-resistant strains (MICs, about 2 to 8 μg/ml), The most active one (3′,6-diNn) was bactericidal at its MIC and inhibited biofilm formation at 2-fold its MIC. They cooperatively bound to LPSs, increasing the outer membrane permeability. Grafting long and linear alkyl chains (nonyl) optimized binding to LPS and outer membrane permeabilization. The effects of amphiphilic neamine derivatives on LPS micelles suggest changes in the cross-bridging of lipopolysaccharides and disordering in the hydrophobic core of the micelles. The molecular shape of the 3′,6-dialkyl neamine derivatives induced by the nature of the grafted hydrophobic moieties (naphthylalkyl instead of alkyl) and the flexibility of the hydrophobic moiety are critical for their fluidifying effect and their ability to displace cations bridging LPS. Results from this work could be exploited for the development of new amphiphilic neamine derivatives active against colistin-resistant P. 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.

ABSTRACTAn NDM-1 carbapenemase-producing Pseudomonas aeruginosaisolate was recovered from a patient hospitalized in France after a previous hospitalization in Serbia. Genetic studies revealed that the blaNDM-1gene was surrounded by insertion sequence ISAba125and a truncated bleomycin resistance gene. This blaNDM-1region was a part of the variable region of a new complex class 1 integron bearing IS common region 1 (ISCR1). The presence of ISPa7upstream of this integron suggests insertion in a chromosomally located Tn402-like structure.

ABSTRACTConstitutive 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 mexXYoperon. In this study, analysis of MexXY-overproducing mutants selected in vitrofrom 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 mexZgene 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 mexXYoverexpression was independent of mexZand the PA5471 gene, which is required for drug-dependent induction of mexXY. Furthermore, the transcript levels of the oprDgene, 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 of P. aeruginosato polycationic peptides such as colistin. The multidrug resistance phenotype was suppressed in PAOW2 by deletion of the parSand parRSgenes and conferred to PAO1Bes by chromosomal insertion of the mutated parRSlocus 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 mexYand repressed oprDexpression via ParRS, independent of PA5471. In agreement with these results, colistin antagonized activity of the MexXY/OprM substrates in PAO1Bes but not in a ΔparRSderivative. 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).

ABSTRACTSince their initial description 2 decades ago, MexCD-OprJ-overproducing efflux mutants of Pseudomonas aeruginosa(also called nfxBmutants) have rarely been described in the clinical setting. Screening of 110 nonreplicate clinical isolates showing moderate resistance to ciprofloxacin (MIC from 0.5 μg/ml to 4 μg/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 nfxBmutants 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. aeruginosastrains with low-level resistance to ciprofloxacin, MexCD-OprJ-overproducing mutants may be isolated after single therapy with fluoroquinolones and may be pathogenic.

ABSTRACTIn this study, we systematically investigated the resistance mechanisms to β-lactams, aminoglycosides, and fluoroquinolones of 120 bacteremic strains of Pseudomonas aeruginosa. Pulsed-field gel electrophoresis genotyping showed that 97 of these strains were represented by a single isolate, 10 by 2 and 1 by 3 clonally related isolates, respectively. Seventy-five percent (90 out of 120) of the bacteremic P. aeruginosastrains displayed a significant resistance to one or more of the tested antimicrobials (up to 11 for 1 strain). These strains were found to harbor a great diversity of resistance mechanisms (up to 7 in 1 strain), leading to various levels of drug resistance. Interestingly, 11 and 36% of the isolates appeared to overproduce the MexAB-OprM and MexXY-OprM efflux systems, respectively. Altogether, our results show that P. aeruginosamay accumulate intrinsic (overproduction of cephalosporinase AmpC, increased drug efflux, fluoroquinolone target mutations, and deficient production of porin OprD) and exogenous (production of secondary β-lactamases and aminoglycoside-modifying enzymes) resistance mechanisms without losing its ability to generate severe bloodstream infections. Consequently, clinicians should be aware that multidrug-resistant P. aeruginosamay remain fully pathogenic.

ABSTRACTScreening of a Tn5-Hg insertional library (12,000 clones) constructed in wild-type Pseudomonas aeruginosastrain 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 β-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. aeruginosamay gradually develop high resistance to these antibiotics via intrinsic (i.e., nonenzymatic) mechanisms, as in cystic fibrosis patients.

Resistance mechanisms of Pseudomonas aeruginosato ceftolozane/tazobactam (C/T) were assessed on a collection of 420 nonredundant strains nonsusceptible to ceftazidime (MIC > 8 μg/ml) and/or imipenem (>4 μg/ml), collected by 36 French hospital laboratories over a one-month period (the 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% (26/112) of the isolates were resistant to both ceftazidime and imipenem.

The intrinsic resistance of Pseudomonas aeruginosato polymyxins in part relies on the addition of 4-amino-4-deoxy-l-arabinose (Ara4N) molecules to the lipid A of lipopolysaccharide (LPS), through induction of operon arnBCADTEF-ugd(arn) expression. As demonstrated previously, at least three two-component regulatory systems (PmrAB, ParRS, and CprRS) are able to upregulate this operon when bacteria are exposed to colistin. In the present study, gene deletion experiments with the bioluminescent strain PAO1::luxshowed that ParRS is a key element in the tolerance of P. aeruginosato this last-resort antibiotic (i.

Emergence of resistance to polymyxins in Pseudomonas aeruginosais mainly due to mutations in two-component systems that promote the addition of 4-amino-4-deoxy-l-arabinose to the lipopolysaccharide (LPS) through upregulation of operon arnBCADTEF-ugd(arn) expression. Here, we demonstrate that mutations occurring in different domains of histidine kinase PmrB or in response regulator PmrA result in coresistance to aminoglycosides and colistin.

ABSTRACTResistance of clinical strains of Pseudomonas aeruginosato aminoglycosides can result from production of transferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and/or mutational derepression of intrinsic multidrug efflux pump MexXY(OprM). We report here the characterization of a new type of mutant that is 4- to 8-fold more resistant to 2-deoxystreptamine derivatives (e.g., gentamicin, amikacin, and tobramycin) than the wild-type strain PAO1. The genetic alterations of three in vitromutants were mapped on fusA1and found to result in single amino acid substitutions in domains II, III, and V of elongation factor G (EF-G1A), a key component of translational machinery. Transfer of the mutated fusA1alleles into PAO1 reproduced the resistance phenotype. Interestingly, fusA1mutants with other amino acid changes in domains G, IV, and V of EF-G1A were identified among clinical strains with decreased susceptibility to aminoglycosides. Allelic-exchange experiments confirmed the relevance of these latter mutations and of three other previously reported alterations located in domains G and IV. Pump MexXY(OprM) partly contributed to the resistance conferred by the mutated EF-G1A variants and had additive effects on aminoglycoside MICs when mutationally upregulated. Altogether, our data demonstrate that cystic fibrosis (CF) and non-CF strains of P. aeruginosacan acquire a therapeutically significant resistance to important aminoglycosides via a new mechanism involving mutations in elongation factor EF-G1A.

ABSTRACTThe multidrug efflux system MexEF-OprN is produced at low levels in wild-type strains of Pseudomonas aeruginosa. However, in so-called nfxCmutants, mutational alteration of the gene mexSresults in constitutive overexpression of the pump, along with increased resistance of the bacterium to chloramphenicol, fluoroquinolones, and trimethoprim. In this study, analysis of in vitro-selected chloramphenicol-resistant clones of strain PA14 led to the identification of a new class of MexEF-OprN-overproducing mutants (called nfxC2) exhibiting alterations in an as-yet-uncharacterized gene, PA14_38040 (homolog of PA2047 in strain PAO1). This gene is predicted to encode an AraC-like transcriptional regulator and was called cmrA(for chloramphenicol resistance activator). In nfxC2mutants, the mutated CmrA increases its proper gene expression and upregulates the operon mexEF-oprNthrough MexS and MexT, resulting in a multidrug resistance phenotype without significant loss in bacterial virulence. Transcriptomic experiments demonstrated that CmrA positively regulates a small set of 11 genes, including PA14_38020 (homolog of PA2048), which is required for the MexS/T-dependent activation of mexEF-oprN. PA2048 codes for a protein sharing conserved domains with the quinol monooxygenase YgiN from Escherichia coli. Interestingly, exposure of strain PA14 to toxic electrophilic molecules (glyoxal, methylglyoxal, and cinnamaldehyde) strongly activates the CmrA pathway and upregulates MexEF-OprN and, thus, increases the resistance of P. aeruginosato the pump substrates. A picture emerges in which MexEF-OprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.