Katy Jeannot

Induction of the MexXY Efflux Pump in Pseudomonas aeruginosa Is Dependent on Drug-Ribosome Interaction

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., beta-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 DeltamexAB-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.

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

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. 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. 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 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).

Pseudomonas aeruginosa May Accumulate Drug Resistance Mechanisms without Losing Its Ability To Cause Bloodstream Infections

ABSTRACT In 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. aeruginosa strains 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. aeruginosa may 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. aeruginosa may remain fully pathogenic.

Cumulative Effects of Several Nonenzymatic Mechanisms on the Resistance of Pseudomonas aeruginosa to Aminoglycosides

ABSTRACT 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 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. aeruginosa may gradually develop high resistance to these antibiotics via intrinsic (i.e., nonenzymatic) mechanisms, as in cystic fibrosis patients.

Resistance and Virulence of Pseudomonas aeruginosa Clinical Strains Overproducing the MexCD-OprJ Efflux Pump

ABSTRACT 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 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 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.

Resistance to polymyxins in Gram-negative organisms

Polymyxins have recently been re-introduced into the therapeutic arsenal to combat infections caused by multidrug-resistant Gram-negative bacteria. However, the emergence of strains resistant to these last-resort drugs is becoming a critical issue in a growing number of countries. Both intrinsic and transferable mechanisms of polymyxin resistance have been characterised. These mechanisms as well as the epidemiological data regarding four relevant bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa) are considered in this review. A special focus is made on plasmid-mediated resistance and the spread of mcr genes.

Complexity of resistance mechanisms to imipenem in intensive care unit strains of Pseudomonas aeruginosa

BACKGROUND Pseudomonas aeruginosa can become resistant to carbapenems by both intrinsic (mutation-driven) and transferable (β-lactamase-based) mechanisms. Knowledge of the prevalence of these various mechanisms is important in intensive care units (ICUs) in order to define optimal prevention and therapeutic strategies. METHODS A total of 109 imipenem-non-susceptible (MIC >4 mg/L) strains of P. aeruginosa were collected in June 2010 from the ICUs of 26 French public hospitals. Their resistance mechanisms were characterized by phenotypic, enzymatic, western blotting and molecular methods. RESULTS Single or associated imipenem resistance mechanisms were identified among the 109 strains. Seven isolates (6.4%) were found to produce a metallo-β-lactamase (one VIM-1, four VIM-2, one VIM-4 and one IMP-29). Porin OprD was lost in 94 (86.2%) strains as a result of mutations or gene disruption by various insertion sequences (ISPa1635, ISPa1328, IS911, ISPs1, IS51, IS222 and ISPa41). Thirteen other strains were shown to be regulatory mutants in which down-regulation of oprD was coupled with overexpressed efflux pumps CzcCBA (n = 1), MexXY (n = 9) and MexEF-OprN (n = 3). The lack of OprD was due to disruption of the oprD promoter by ISPsy2 in one strain and alteration of the porin signal sequence in another. CONCLUSIONS Imipenem resistance in ICU P. aeruginosa strains may result from multiple mechanisms involving metallo-β-lactamase gene acquisition and genetic events (mutations and ISs) inactivating oprD, turning down its expression while increasing efflux activities or preventing insertion of porin OprD in the outer membrane. This diversity of mechanisms allows P. aeruginosa, more than any other nosocomial pathogen, to rapidly adapt to carbapenems in ICUs.

Multiple mutations lead to MexXY/OprM-dependent aminoglycoside resistance in clinical strains of Pseudomonas aeruginosa

ABSTRACT Constitutive 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. Forty-four (77.2%) of the strains, classified as agrZ mutants were found to harbor mutations inactivating the local repressor gene (mexZ) of the mexXY operon (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 mexZ genes encoding wild-type MexZ proteins. Eight (14.0%) of these isolates, classified as agrW1 mutants, 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 agrW1 mutant 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 agrW2 mutants, harbored single amino acid variations in the sensor histidine kinase of ParRS, a two-component system known to positively control mexXY expression. Collectively, these results demonstrate that clinical strains of P. aeruginosa exploit 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.

Genetic and Biochemical Characterization of OXA-405, an OXA-48-Type Extended-Spectrum β-Lactamase without Significant Carbapenemase Activity

ABSTRACT The epidemiology of carbapenemases worldwide is showing that OXA-48 variants are becoming the predominant carbapenemase type in Enterobacteriaceae in many countries. However, not all OXA-48 variants possess significant activity toward carbapenems (e.g., OXA-163). Two Serratia marcescens isolates with resistance either to carbapenems or to extended-spectrum cephalosporins were successively recovered from the same patient. A genomic comparison using pulsed-field gel electrophoresis and automated Rep-PCR typing identified a 97.8% similarity between the two isolates. Both strains were resistant to penicillins and first-generation cephalosporins. The first isolate was susceptible to expanded-spectrum cephalosporins, was resistant to carbapenems, and had a significant carbapenemase activity (positive Carba NP test) related to the expression of OXA-48. The second isolate was resistant to expanded-spectrum cephalosporins, was susceptible to carbapenems, and did not express a significant imipenemase activity, (negative for the Carba NP test) despite possessing a blaOXA-48-type gene. Sequencing identified a novel OXA-48-type β-lactamase, OXA-405, with a four-amino-acid deletion compared to OXA-48. The blaOXA-405 gene was located on a ca. 46-kb plasmid identical to the prototype IncL/M blaOXA-48-carrying plasmid except for a ca. 16.4-kb deletion in the tra operon, leading to the suppression of self-conjugation properties. Biochemical analysis showed that OXA-405 has clavulanic acid-inhibited activity toward expanded-spectrum activity without significant imipenemase activity. This is the first identification of a successive switch of catalytic activity in OXA-48-like β-lactamases, suggesting their plasticity. Therefore, this report suggests that the first-line screening of carbapenemase producers in Enterobacteriaceae may be based on the biochemical detection of carbapenemase activity in clinical settings.

Antibiotic Resistance Determinants in a Pseudomonas putida Strain Isolated from a Hospital

Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267) kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts.