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. 2017 Aug;9(8):1165-1178.
doi: 10.15252/emmm.201707661.

Synergistic Antibacterial Effect of Silver and Ebselen Against Multidrug-Resistant Gram-negative Bacterial Infections

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Free PMC article

Synergistic Antibacterial Effect of Silver and Ebselen Against Multidrug-Resistant Gram-negative Bacterial Infections

Lili Zou et al. EMBO Mol Med. .
Free PMC article

Abstract

Multidrug-resistant (MDR) Gram-negative bacteria account for a majority of fatal infections, and development of new antibiotic principles and drugs is therefore of outstanding importance. Here, we report that five most clinically difficult-to-treat MDR Gram-negative bacteria are highly sensitive to a synergistic combination of silver and ebselen. In contrast, silver has no synergistic toxicity with ebselen on mammalian cells. The silver and ebselen combination causes a rapid depletion of glutathione and inhibition of the thioredoxin system in bacteria. Silver ions were identified as strong inhibitors of Escherichia coli thioredoxin and thioredoxin reductase, which are required for ribonucleotide reductase and DNA synthesis and defense against oxidative stress. The bactericidal efficacy of silver and ebselen was further verified in the treatment of mild and acute MDR E. coli peritonitis in mice. These results demonstrate that thiol-dependent redox systems in bacteria can be targeted in the design of new antibacterial drugs. The silver and ebselen combination offers a proof of concept in targeting essential bacterial systems and might be developed for novel efficient treatments against MDR Gram-negative bacterial infections.

Keywords: ebselen; multidrug‐resistant Gram‐negative bacteria; silver; synergistic antibacterial effect; thiol‐dependent redox system.

Figures

Figure 1
Figure 1. Effects of silver with ebselen in combination on the growth of Escherichia coli and HeLa cells

Synergistic effect of ebselen with silver nitrate (AgNO3) in combination on the growth of E. coli. Escherichia coli DHB4 overnight cultures were diluted 1:1,000 into 100 μl of LB medium in 96 micro‐well plates, and treated with 100 μl serial dilutions of ebselen and AgNO3 in combination for 16 h, and cell viability was determined by measuring OD600 nm. Ag+ alone inhibited E. coli growth with a minimal inhibition concentration (MIC) of 42 μM after 16‐h treatment, while 2 μM ebselen dramatically decreased the MIC of Ag+ to 4.2 μM (P = 0.000028, Student's t‐test).

Effects of ebselen with AgNO3 in combination on the growth of HeLa cells. HeLa cells were treated with serial concentrations of ebselen and AgNO3 for 24 h, and cell toxicity was detected by MTT assay. 5 μM Ag+ and 2.5 μM ebselen in combination showed no synergistic toxicity on human HeLa cells (P = 0.98, Student's t‐test).

Data information: Data are presented as means ± SD of three independent experiments.
Figure EV1
Figure EV1. Effects of ebselen on the growth of E. coli
Escherichia coli DHB4 overnight cultures were diluted 1:1,000 into 100 μl of LB medium in 96 micro‐well plates and treated with different concentrations of ebselen for 16 h. The cell viability was determined by measuring the absorbance at 600 nm. Data are presented as means ± SD of three independent experiments.
Figure 2
Figure 2. Silver with ebselen in combination exhibited synergistic bactericidal effect
Escherichia coli DHB4 grown to OD600 nm of 0.4 were treated with serial dilutions of ebselen and AgNO3 in combination.

Cell viability was represented by measuring OD600 nm. The growth curves showed a synergistic bacteriostatic effect of Ag+ with ebselen in combination in LB medium. 5 μM Ag+ and 40 μM ebselen in combination inhibited E. coli growth 480 min post‐treatment (**P = 0.0075).

Changes of colony forming units of E. coli DHB4 on LB plates 0, 10, 60, 120, and 240 min post‐treatment. The synergistic bactericidal effect of 5 μM Ag+ and 80 μM ebselen in combination was confirmed by the colony formation assay on LB‐agar plates. 5 μM Ag+ and 80 μM ebselen in combination killed the majority of E. coli 60 min post‐treatment (***P = 0.00021).

FACS plots (C) and mean ± SD (D) of propidium iodide (PI)‐stained E. coli DHB4. 5 μM Ag+ and 20 μM ebselen in combination enhanced the frequency of propidium iodide (PI) staining (***P = 0.00083).

Data information: Data are presented as means ± SD of three independent experiments. **P < 0.01, ***P < 0.001 (Student's t‐test).
Figure EV2
Figure EV2. Antibacterial effect of ebselen on E. coli growth
Escherichia coli DHB4 cells were grown in 15‐ml tubes until an OD600 nm of 0.4 and treated with serial concentrations of ebselen for 24 h. The cell viability was determined by measuring the absorbance at 600 nm. Data are presented as means ± SD of three independent experiments. *< 0.05, **< 0.01, ***< 0.001 (Student's t‐test).
Figure 3
Figure 3. Silver with ebselen in combination directly disrupted bacterial Trx and GSH systems
Escherichia coli DHB4 grown to OD600 nm of 0.4 were treated with serial dilutions of ebselen and AgNO3 in combination.

TrxR activities were assayed using DTNB reduction in the presence of Trx in E. coli extracts, 50 mM Tris–HCl (pH 7.5), 200 μM NADPH, 1 mM EDTA, 1 mM DTNB, in the presence of 100 nM E. coli TrxR. 5 μM Ag+ and 20 μM ebselen in combination resulted in a dramatic loss of TrxR activities (***P = 0.00018).

Trx activities were assayed using DTNB reduction in the presence of Trx in E. coli extracts, 50 mM Tris–HCl (pH 7.5), 200 μM NADPH, 1 mM EDTA, 1 mM DTNB, 5 μM E. coli Trx. 5 μM Ag+, and 20 μM ebselen in combination resulted in a dramatic loss of Trx activities (**P = 0.0036).

Changes of Trx1 redox state in E. coli upon ebselen and AgNO3 treatment. Escherichia coli were precipitated in 5% TCA and alkylated with 15 mM AMS, and the percent of reduced Trx1 were analyzed by Western blot.

Changes of Trx2 redox state in E. coli upon ebselen and AgNO3 treatment. Escherichia coli were precipitated in 5% TCA and alkylated with 15 mM AMS, diamide‐oxidized Trx2 was used as a Trx2 positive control, and the percent of reduced Trx2 were analyzed by Western blot.

GSH amounts were measured by GR‐coupled DTNB reduction assay in E. coli extracts, 50 mM Tris–HCl (pH 7.5), 200 μM NADPH, 1 mM EDTA, 1 mM DTNB, 50 nM GR. 5 μM Ag+, and 20 μM ebselen in combination depleted the functional GSH in 10 min compared with control (***P = 0.000021).

Changes of proteins S‐glutathionylation in E. coli. Cells were cultured, washed, and re‐suspended in lysis buffer containing 30 mM IAM. After lysed by sonication, Western blotting assay was performed with IgG2a mouse monoclonal antibody (VIROGEN, 101‐A/D8) for glutathione–protein complexes.

Data information: Data are presented as means ± SD of three independent experiments. **< 0.01, ***< 0.001 (Student's t‐test).
Figure 4
Figure 4. Inhibitory effects of silver on E. coli Trx system in vitro

Inhibition of E. coli TrxR by AgNO3. Pure recombinant 100 nM TrxR and 5 μM Trx mixture were incubated with AgNO3 solution in the presence of 200 μM NADPH, and then, their activities were detected by DTNB reduction assay.

Fluorescence spectra of a complex between reduced E. coli 10 μM Trx1 with AgNO3. Reduced 10 μM E. coli Trx1 protein was incubated with a serial concentration of AgNO3 solution, and the fluorescent spectra was detected with an excitation wavelength at 280 nm. Oxidized Trx1 (Trx‐S2) was used as a control.

Inhibition of Trx by AgNO3. After the treatment described in (B), Trx activity was assayed by a DTNB method in the presence of E. coli Trx1.

Inhibition reversibility of E. coli Trx1 by AgNO3. Silver‐inhibited E. coli Trx1 was passed through a desalting column to remove small molecules, and then, Trx activity was measured. Escherichia coli Trx1 without the inhibition was used as a control. The inhibition of Trx1 by Ag+ was irreversible since the Trx1 activity was not recovered after desalting (***P = 0.00021).

Data information: In (D), data are presented as means ± SD of three independent experiments. ***< 0.001 (Student's t‐test).
Figure 5
Figure 5. ROS was a determining factor for synergistic bactericidal effect of silver and ebselen

E. coli DHB4 grown to OD600 nm of 0.4 were treated with 20 μM ebselen and 5 μM AgNO3, and FACS histograms (A) and mean MFI ± SD (B) of H2DCF‐DA‐stained E. coli were detected. ROS level was detected by flow cytometry (CyAn adp, Beckman Coulter). Treatment with either 5 μM Ag+ or 20 μM ebselen alone did not change ROS concentrations, while the combination of 5 μM Ag+ and 20 μM ebselen resulted in increased levels of ROS (***P = 0.00012; Student's t‐test).

Detection of H2O2 using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen). Reactions containing 50 μM Amplex® Red reagent, 0.1 U/ml HRP, and the samples in 50 mM sodium phosphate buffer, pH 7.4, were incubated for 30 min at room temperature and detected with absorbance at 560 nm. Background determined for a non‐H2O2 control reaction has been subtracted from each value. The enhanced H2O2 generated by 5 μM Ag+ and 20 μM ebselen‐treated E. coli DHB4 cells were verified (***P = 0.00057; Student's t‐test).

Data information: In (B and C), data are presented as means ± SD of three independent experiments.
Figure EV3
Figure EV3. Bactericidal effects of silver and ebselen in the LB medium containing heparinized mice blood
Blood was extracted from three healthy mice and collected in heparinized tubes. One hundred E. coli DHB4 cells were harvested during the logarithmic phase, and drug combination was added to 100 μl heparinized mice blood. After incubation at 37°C for 6 h, duplicate 100‐μl aliquots from each blood sample were spread onto LB agar, and CFU/ml was enumerated using the following formula: [(colonies) × (dilution factor)]/(amount plated) after overnight incubation. 0.1% (v/v) DMSO‐treated cells were used as the positive control. Data are presented as means ± SD of three independent experiments. *< 0.05, **< 0.01 (Student's t‐test).
Figure 6
Figure 6. Mode of action of silver and ebselen in in vivo mild and acute mice peritonitis model

Mild mice peritonitis model. Mice were infected by intraperitoneal administration of 100 μl of 1.7 × 106 E. coli ZY‐1 cells. After 24 h, 12 mice per group received antibacterial treatments (25 mg ebselen/kg and 6 mg AgNO3/kg body weight). 12, 24, and 36 h after treatment, the peritoneal fluid was collected for analysis of E. coli CFU (n = 12 mice for each group), and data are presented as means ± SD of three independent experiments. **< 0.01 (Student's t‐test).

Acute mice peritonitis model. Inoculation was performed by intraperitoneal injection of 100 μl of 6.0 × 106 CFU/ml E. coli ZY‐1 inoculums. After inoculation for 1 h, 10 mice per group received antibacterial treatments, and the mice were observed for 7 days to evaluate overall survival (n = 10 mice for each group), and the experiment was performed in duplicate.

Figure EV4
Figure EV4. Inhibitory effects of silver on mammalian TrxR in vitro
Pure recombinant 10 nM human TrxR or 10 nM rat TrxR were incubated with serial concentrations of AgNO3 solution in the presence of 250 μM NADPH, and then, their activities were detected by DTNB reduction assay.

Inhibition of human TrxR by AgNO3. 5 nM silver can inhibit human TrxR (***P = 0.000054).

Inhibition of rat TrxR by AgNO3. 5 nM silver can inhibit human TrxR (***P = 0.000019).

Data information: Data are presented as means ± SD of three independent experiments. ***< 0.001 (Student's t‐test).

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