Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium

Zhang, Xinglin; Paganelli, Fernanda L.; Bierschenk, Damiën; Kuipers, Annemarie; Bonten, Marc J. M.; Willems, Rob J. L.; Schaik, Willem van

doi:10.1371/journal.pgen.1002804

Cited by 95 publications

(181 citation statements)

References 61 publications

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“…To gather more insight into the correlation between catabolic pathway utilization and tolerance to bactericidal antibiotics, we created a genome-wide transposon mutant library in the E. faecalis ⌬sodA mutant and screened it for variants with increased tolerance of vancomycin in the presence of glucose as the energy source. Recently, construction of a high-density transposon mutant library has been established for E. faecium using a new transposon delivery plasmid named pZXL5 (22). We therefore tested if this plasmid could also be a useful tool for transposon mutagenesis in E. faecalis.…”

Section: Resultsmentioning

confidence: 99%

“…For transposon mutagenesis in the E. faecalis ⌬sodA JH2-2 and OG1RF strains, we used the pZXL5 transposon delivery plasmid (GenBank accession number JQ088279) ( Table 1) and a method previously described (22). Briefly, after electroporation, gentamicin-resistant transformants were grown overnight in BHI broth supplemented with 300 g/ml gentamicin and 10 g/ml chloramphenicol at the permissive temperature of 28°C.…”

Section: Methodsmentioning

confidence: 99%

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Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci

et al. 2015

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Enterococci are naturally tolerant to typically bactericidal cell wall-active antibiotics, meaning that their growth is inhibited but they are not killed even when exposed to a high concentration of the drug. The molecular reasons for this extraordinary tolerance are still incompletely understood. Previous work showed that resistance to killing collapsed specifically in mutants affected in superoxide dismutase (Sod) activity, arguing that bactericidal antibiotic treatment led to induction of a superoxide burst. In the present work, we show that loss of antibiotic tolerance in ⌬sodA mutants of pathogenic enterococci is dependent on the energy source present during antibiotic treatment. Hexoses induce greater killing than the pentose ribose, and no killing was observed with glycerol as the energy source. These results point to glycolytic reactions as crucial for antibiotic-mediated killing of ⌬sodA mutants. A transposon mutant library was constructed in Enterococcus faecalis ⌬sodA mutants and screened for restored tolerance of vancomycin. Partially restored tolerance was observed in mutants with transposon integrations into intergenic regions upstream of regulators implicated in arginine catabolism. In these mutants, the arginine deiminase operon was highly upregulated. A model for the action of cell wall-active antibiotics in tolerant and nontolerant bacteria is proposed. IMPORTANCEAntibiotic tolerance is a serious clinical concern, since tolerant bacteria have considerably increased abilities to resist killing by bactericidal drugs. Using enterococci as models for highly antibiotic-tolerant pathogens, we showed that tolerance of these bacteria is linked to their superoxide dismutase (Sod), arguing that bactericidal antibiotics induce generation of reactive oxygen species inside cells. Wild-type strains are tolerant because they detoxify these deleterious molecules by the activity of Sod, whereas Sod-deficient strains are killed. This study showed that killing depends on the energy source present during treatment and that an increase in arginine catabolism partially restored tolerance of the Sod mutants. These results are used to propose a mode-of-action model of cell wall-active antibiotics in tolerant and nontolerant bacteria. Even though enterococci are considered low-virulence pathogens, treatment of enterococcal infections is often difficult and lengthy. International guidelines for treatment of infective enterococcal endocarditis recommend 4 to 6 weeks of administration of penicillin or ampicillin plus an aminoglycoside, with a risk of acute renal failure due to the nephrotoxicity of the latter antibiotics (1-3). Enterococci, which are lactic acid bacteria and part of the human commensal flora, are naturally highly tolerant to antibiotics considered to be bactericidal such as penicillins and glycopeptides (4, 5). The mechanisms that allow these organisms to escape the lethal action of those drugs are still incompletely understood. On the basis of previous studies, it was suggested that the tolerance...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci

et al. 2015

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“…We are currently investigating the differential effect of class A penicillin binding protein (PBP) inactivation on susceptibility to daptomycin and antimicrobial peptides and whether ceftaroline and/or ampicillin exert their effects on VRE through these targets (15). Additional evidence by others suggests that low-molecular-weight PBPs, such as ddcP, which influences D-alanine-D-alanine carboxypeptidase activity (16), warrant study as well, particularly with the possible tangential effects on vancomycin MIC by ceftaroline.…”

Section: Discussionmentioning

confidence: 99%

“…Daptomycin-nonsusceptible enterococci are different from their susceptible counterparts in sensing cell wall stress (via liaFSR) (15,16,18), phospholipid composition (via cls) (20)(21)(22), and increased proclivity to septation (via ezrA) (6). While we did not perform analysis of phospholipid content with ceftaroline therapy, we noticed changes in septation and cell wall thickness induced by ceftaroline.…”

Section: Discussionmentioning

confidence: 99%

Ceftaroline Restores Daptomycin Activity against Daptomycin-Nonsusceptible Vancomycin-Resistant Enterococcus faecium

Sakoulas

Rose

Nonejuie

et al. 2014

Antimicrob Agents Chemother

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c Daptomycin-nonsusceptible vancomycin-resistant Enterococcus faecium (VRE) strains are a formidable emerging threat to patients with comorbidities, leaving few therapeutic options in cases of severe invasive infections. Using a previously characterized isogenic pair of VRE strains from the same patient differing in their daptomycin susceptibilities (Etest MICs of 0.38 mg/liter and 10 mg/liter), we examined the effect of ceftaroline, ceftriaxone, and ampicillin on membrane fluidity and susceptibility of VRE to surface binding and killing by daptomycin and human cathelicidin antimicrobial peptide LL37. Synergy was noted in vitro between daptomycin, ampicillin, and ceftaroline for the daptomycin-susceptible VRE strain, but only ceftaroline showed synergy against the daptomycin-nonsusceptible VRE strain (ϳ2 log 10 CFU reduction at 24 h). Ceftaroline cotreatment increased daptomycin surface binding with an associated increase in membrane fluidity and an increase in the net negative surface charge of the bacteria as evidenced by increased poly-L-lysine binding. Consistent with the observed biophysical changes, ceftaroline resulted in increased binding and killing of daptomycin-nonsusceptible VRE by human cathelicidin LL37. Using a pair of daptomycinsusceptible/nonsusceptible VRE strains, we noted that VRE is ceftaroline resistant, yet ceftaroline confers significant effects on growth rate as well as biophysical changes on the cell surface of VRE that can potentiate the activity of daptomycin and innate cationic host defense peptides, such as cathelicidin. Although limited to just 2 strains, these finding suggest that additional in vivo and in vitro studies need to be done to explore the possibility of using ceftaroline as adjunctive anti-VRE therapy. Loss of susceptibility to daptomycin is an increasing concern among vancomycin-resistant Enterococcus faecium (VRE) (1). When faced with invasive infections by daptomycin-nonsusceptible VRE, clinicians have limited therapeutic options. Of great concern are the lack of a bactericidal agent, antibiotic-associated side effects such as linezolid-induced thrombocytopenia and quinupristin-dalfopristin (QD)-associated myalgias, and drug-drug interactions such as microsomal P450 effects of QD and serotonin syndrome concerns with linezolid and concomitant serotonin reuptake inhibitors. Therefore, a great need exists for infectiousdisease physicians practicing in tertiary medical centers with patients at high risk for VRE infections, e.g., bone marrow and liver transplant recipients, to develop innovative pharmacotherapies to treat such patients (2, 3). The clinical dilemma faced by physicians treating these patients is further compounded by the facts that novel therapeutics targeting VRE are lacking and that single or combination antibiotics with in vitro activity against VRE have not been clinically validated by appropriate trials (4).Our group has previously shown a perhaps counterintuitive effect of ampicillin in converting daptomycin from a bacteriostatic to a bactericidal an...

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“…6B), and both proteins were found to increase bacterial resistance to antimicrobial compounds. 35,56,57 While the cellular localization of AhyD remains undefined, VirJ is known to be anchored to the periplasmic surface in the cytoplasmic membrane of the gram-negative bacterium P. aeruginosa. VirJ exhibits broad substrate specificity and can hydrolyse the Ala-, Gly-, and Lys- moieties of artificial substrates.…”

Section: Homeostasis and Utilization Of Aa-pg In The Membranementioning

confidence: 99%

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances

Fields

Roy

2017

RNA Biology

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ABSTRACTtRNA-dependent addition of amino acids to lipids on the outer surface of the bacterial membrane results in decreased effectiveness of antimicrobials such as cationic antimicrobial peptides (CAMPs) that target the membrane, and increased virulence of several pathogenic species. After a brief introduction to CAMPs and the various bacterial resistance mechanisms used to counteract these compounds, this review focuses on recent advances in tRNA-dependent pathways for lipid modification in bacteria. Phenotypes associated with amino acid lipid modifications and regulation of their expression will also be discussed.

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Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium

Cited by 95 publications

References 61 publications

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci

Ceftaroline Restores Daptomycin Activity against Daptomycin-Nonsusceptible Vancomycin-Resistant Enterococcus faecium

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances

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