Repurposing ebselen for treatment of multidrug-resistant staphylococcal infections

Novel antimicrobials and new approaches to developing them are urgently needed. Repurposing already-approved drugs with well-characterized toxicology and pharmacology is a novel way to reduce the time, cost, and risk associated with antibiotic innovation. Ebselen, an organoselenium compound, is known to be clinically safe and has a well-known pharmacology profile. It has shown potent bactericidal activity against multidrug-resistant clinical isolates of staphylococcus aureus, including methicillin- and vancomycin-resistant S. aureus (MRSA and VRSA). We demonstrated that ebselen acts through inhibition of protein synthesis and subsequently inhibited toxin production in MRSA. Additionally, ebselen was remarkably active and significantly reduced established staphylococcal biofilms. The therapeutic efficacy of ebselen was evaluated in a mouse model of staphylococcal skin infections. Ebselen 1% and 2% significantly reduced the bacterial load and the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and monocyte chemo attractant protein-1 (MCP-1) in MRSA USA300 skin lesions. Furthermore, it acts synergistically with traditional antimicrobials. This study provides evidence that ebselen has great potential for topical treatment of MRSA skin infections and lays the foundation for further analysis and development of ebselen as a potential treatment for multidrug-resistant staphylococcal infections.

Mechanism of action. Given the potent anti-staphylococcal activity of ebselen in vitro, we investigated its anti-staphylococcal mechanism of action by macromolecular synthesis assay. As shown in Fig. 1, ebselen primarily inhibited protein synthesis at 1X the MIC. However, additional secondary effects were observed at a higher concentration (8X MIC). At higher concentration, ebselen inhibited DNA, RNA and lipid synthesis similar to control antibiotics such as ciprofloxacin, rifampicin and cerulenin respectively.
Ebselen inhibits MRSA toxin production. The effect of ebselen on production of important toxins such as Panton-Valentine leucocidin (PVL) and α -hemolysin (Hla) was tested by ELISA. The concentrations of each toxin were compared as unadjusted concentrations (ng/ml) and corrected for organism inoculum for each treatment (ng/ml to log 10 colony-forming units, CFU/ml). Ebselen significantly suppressed toxin production in MRSA USA300 (Fig. 2).
Cytotoxicity study. Safety of ebselen in mammalian cells was evaluated against human keratinocyte cells (HaCat) by MTS assay. Ebselen did not show toxicity up to 32 μ g/ml. The results demonstrated that half maximal inhibitory concentration (IC 50 ) required by ebselen to inhibit 50% of HaCat cells was found to be 58.78 + 0.64 μ g/ml (Fig. 4).

The therapeutic efficacy of ebselen in a mouse model of MRSA skin infection.
(I) Bacterial load: Five groups of mice were treated topically either with vehicle alone (petroleum jelly) or control antibiotic (2% mupirocin) or ebselen (0.5%, 1%, or 2%) twice a day for five days. One group of mice was treated with linezolid orally. As shown in Fig. 5a, ebselen (1% and 2%) significantly reduced the mean bacterial counts compared with the control group (P ≤ 0.01). The group treated with 2% mupirocin had the highest reduction in CFU (2.28 ± 0.25 log 10 ), followed by 2% ebselen (1.71 ± 0.11 log 10 ), linezolid (25 mg/kg) (1.55 ± 0.01 log 10 ), and 1% ebselen (1.02 ± 0.17 log 10 ). (II) Effect of vehicle: In order to investigate the effect of the vehicle on the efficacy of ebselen in the treatment of MRSA skin infections, two groups of mice were treated topically either with vehicle alone (lipoderm base) 17 or ebselen 1% formulated in lipoderm base twice a day for five days. Ebselen 1% significantly reduced the mean bacterial counts by 1.37 ± 0.20 log 10 compared with the control group (P ≤ 0.01) (Fig. 5b). Although ebselen formulated in lipoderm base had higher reduction in bacterial count (1.37 ± 0.20 log 10 ) than ebselen formulated in petroleum jelly (1.02 ± 0.17 log 10 ), the difference was not statistically significant ( Fig. 5a,b).

Synergistic activity of ebselen with topical antimicrobials in vitro.
The antimicrobial activity of ebselen in combination with topical antimicrobials (mupirocin, fusidic acid, retapamulin and daptomycin) was investigated in vitro by the Bliss model of synergism against four clinical isolates. With the exception of the VRSA5 strain and the antibiotic daptomycin, ebselen acted synergistically with all tested antibiotics against S. aureus clinical isolates (Fig. 7).

Discussion
For the past few decades the rise of multi-drug resistant S. aureus has been an emerging issue in hospital and community settings 5,18 . More importantly, the management of S. aureus strains associated with skin infections is becoming a serious issue in community settings 19,20 . Although there are several drugs recently approved by the FDA to combat Gram-positive pathogens such as tedizolid and dalbavancin 21,22 , there is still a pressing need for new antimicrobials to circumvent this burgeoning problem. Moreover, pharmaceutical companies are not interested in investing in antibiotic research and development because of low return compared to other drugs being developed for chronic ailments 5,23,24 . As an alternative to the traditional de novo antibiotic development, repurposing non-antimicrobial drugs is a novel and less expensive way to speed up the drug-development process 7 .
In an intensive search for antimicrobial activity among non-antibiotic drugs, we and others 10,15 identified ebselen as a potent antimicrobial agent against Gram-positive pathogens including MRSA. Ebselen, an organoselenium compound, is known to be clinically safe with a well-known pharmacology profile Figure 2. Effect of ebselen on toxin production. Toxin production (ng/ml) in S. aureus MRSA USA300 after antibiotic/drug exposure for 1 hour corrected for organism burden. The results are given as means ± SD (n = 3). **indicate statistical significant different from control (DMSO or water). P values of (*P ≤ 0.05) (**P ≤ 0.01) are considered as significant. Detailed P values are listed: α Hemolysin: Control vs linezolid, 0.0144; Control vs ebselen, 0.0147. PVs-LK: Control vs linezolid, 0.0024; Control vs ebselen, 0.0288. and it is currently undergoing clinical trials for the prevention and treatment of various disorders such as cardiovascular disease, arthritis, stroke, atherosclerosis, and cancer 12,[25][26][27][28] . Ebselen showed potent bactericidal activity against multiple clinical isolates of MRSA, including MRSA USA100, USA200, USA500, USA1000, and USA1100, which are resistant to various antimicrobials, including penicillin, fluoroquinolone, macrolides, and aminoglycosides. It also showed potent activity against multidrug-resistant clinical isolates of S. aureus strains, including a linezolid-resistant strain (NRS119), vancomycin-resistant strains (VRSA1-VRSA10), and a mupirocin-resistant strain (NRS107). Moreover, ebselen demonstrated excellent activity against MRSA USA300, a community-associated strain responsible for outbreaks of staphylococcal skin and soft-tissue infections (SSTI) in the United States 29 . Although the antimicrobial activity of ebselen has been reported before 10,15 , its mechanism of action in S. aureus and its in vivo efficacy have never been explored. Ebselen, in our study, inhibited protein synthesis in S. aureus. Inhibition of protein synthesis at a concentration equivalent to the MIC demonstrates that, protein synthesis is likely primary antibacterial mechanism of action of ebselen. In addition, secondary effects on DNA, RNA, lipid synthesis and to a lesser extent on cell wall synthesis were also noticed at higher concentrations (8X MIC). It is possible that disruption of protein synthesis could lead to downstream inhibition of other pathways. This provides valuable insight into ebselen's potential target in S. aureus. However, further work is needed to identify the cellular target of ebselen in S. aureus. For treatment of infections caused by toxin-producing pathogens such as S. aureus, inhibition of protein synthesis is an important consideration in the selection of antimicrobial agents 30 . Because antimicrobials that suppress translation in S. aureus markedly suppress the formation of toxins such as PVL and Hla, which will lead to better treatment outcomes [30][31][32][33] . In the light of our results, showing potent inhibition of bacterial protein synthesis, we tested the effect of ebselen on production of two important toxins in MRSA USA300 (Hla and PVL) by ELISA. Ebselen significantly suppressed toxin production after 1 hour incubation with MRSA. Inhibition of protein synthesis and the subsequent inhibition of toxin production are great advantages of ebselen as an antimicrobial agent.
Bacterial biofilms, which serve to protect the bacteria and hinder penetration of antibacterial drugs, contribute significantly to the treatment failure of Staphylococcus infections 34 . Given the potent antibacterial activity of ebselen against planktonic multidrug-resistant strains, we also considered the possibility that ebselen would be active against established bacterial biofilms of S. aureus and S. epidermidis (a leading cause of hospital-acquired implant-based infections) 35 . Ebselen was superior in reducing adherent biofilms of both S. aureus and S. epidermidis when compared to conventional antibiotics (linezolid, mupirocin, vancomycin and rifampicin).
In view of our results demonstrating the potent antimicrobial and antibiofilm activities of ebselen in vitro against MRSA, we moved forward with an in vivo experiment in a mouse model of MRSA skin infection. Ebselen 1% and 2% in petroleum jelly significantly reduced the mean bacterial counts compared with the control group (P ≤ 0.01). The lipoderm base enhanced the antimicrobial activity of ebselen but the reduction in bacterial load was not significant from petroleum jelly vehicle.
Since the clinical severity of S. aureus skin infections is driven by the excess host pro-inflammatory cytokines rather than by bacterial burden 36,37 , ebselen with its recognized immune-modulatory, anti-inflammatory, and antioxidant activities 11,38 has great potential for treatment of for treatment of skin infections 37,39 . In this study, topical treatment with ebselen 1 and 2% significantly reduced IL-1β , IL-6, TNF-α and MCP-1 which might benefit the healing of infected wounds [40][41][42][43][44] . Linezolid also inhibits IL-1β which is in line with previous findings 37,45 . Prolonged inflammation especially due to inflammatory cytokines such as IL-6, TNF-α , and MCP-1, greatly delays healing in chronic wounds 39 . Ebselen significantly (P ≤ 0.01) inhibits all three cytokines (IL-6, TNF-α , and MCP-1), which should provide a favorable outcome in wound healing 39 .
With the increasing incidence of MRSA strains resistant to topical drugs of choice, such as mupirocin and fusidic acid, combination therapies are being explored [46][47][48][49] . To investigate whether ebselen has the potential to act synergistically with topical antimicrobials against multidrug-resistant strains, the Bliss independence model was utilized 50 . Ebselen acted synergistically with topical antimicrobials against resistant strains of S. aureus, thus providing a strong platform to combine ebselen with topical antimicrobials in treating staphylococcal skin infections and reducing the likelihood of strains developing resistance to monotherapy. As a novel drug candidate for treating topical MRSA infections, ebselen has many advantageous qualities, including (i) potent antibacterial activity against clinical isolates of MRSA irrespective of the resistant patterns, (ii) suppression of bacterial protein synthesis and suppression of toxin production, (iii) potent anti-biofilm activity, (iv) suppression of excess pro-inflammatory cytokines, and (v) synergistic action with topical antimicrobials. These results lay the foundation for further analysis and development of ebselen as a potential treatment/prophylaxis for infected wounds and skin infections of public-health importance.

Materials and Methods
Bacterial strains and reagents. Staphylococcus strains used in this study are presented in Table 1.  Similarly, protein synthesis was stopped after 40 min using 12 μ l of 5% TCA. Reaction wells containing cell wall and lipid synthesis were stopped after 40 min using 100 μ l of 8% SDS and 375 μ l of chloroform/ methanol (1:2) respectively. Reactions (DNA, RNA and protein) were incubated on ice for 30 min and the TCA precipitated materials were collected on a 25 mm GF/1.2 μ M PES 96 well filter plate. After washing five times with cold 5% TCA, the filters were dried and counted using a Packard Top Count microplate scintillation counter. For cell wall synthesis, reaction tubes were then heated at 95 °C for 30 min, cooled, centrifuged, and spotted onto nitrocellulose membrane filters (0.8 μ M). After washing three times with 0.1% SDS, the filters were rinsed two times with deionized water, allowed to dry, and then counted using a Beckman LS3801liquid scintillation counter. For lipid synthesis, reactions tubes were centrifuged at 13,000 rpm in a microfuge for 10 min, and then 150 μ l of the organic phase was transferred to a scintillation vial and allowed to dry for at least 1 hour. Samples were then counted using liquid scintillation counting. Based on the incorporation of radiolabeled precursors of DNA, RNA, protein, cell wall and lipid synthesis, results were expressed as percent inhibition of macromolecular synthesis pathways.
Measuring toxin production by ELISA. We tested the effect of ebselen on production of two important toxins Hla and PVL by ELISA as described before 30,51 . Briefly, Overnight grown MRSA USA300 bacterial culture was diluted approximately to 5 × 10 8 CFU/ml in TSB. 10X MICs of drugs and antibiotics were added and incubated in the shaking incubator at 37 °C. After 1 hr the bacterial culture was centrifuged and the supernatants were used for toxin detection.
ELISA plates (Nunc) were coated with 2 μ g/ml of sheep anti-Hla IgG (Toxin technology) in 100 μ l of coating buffer and left overnight at 4 °C. Plates were then washed 3 times with Tris-buffered saline (TBS) containing 0.05% tween 20 (wash buffer) and then blocking solution containing TBS with 2% bovine serum albumin was added. After 1 hour incubation at 37 °C, plates were washed 3 times with wash buffer. A total of 100 μ L of bacterial supernatants were added and incubated the plates at 37 °C for 2 hours. Purified Hla (Toxin technology) was used to generate a standard curve. Plates were again washed 3 times with wash buffer and 100 μ L of sheep anti-Hla HRP conjugate at a dilution of 1:300 was added. After 1 hour of incubation at 37 °C and final washing, 100 μ L of 3, 3′ , 5, 5′ -tetramethylbenzidine substrate (Sigma-Aldrich) was added, and the reaction was stopped after 10 minutes with 100 μ L of 0.2N H2S04. Plates were read on a spectrophotometer at optical density (OD) 450, and data were analyzed with SoftMax Pro (Molecular Devices). The nominal range of this assay was 0.1-6 μ g /mL.
For PVL Luk-S toxin, ELISA plates (Nunc) were coated as before with 2 μ g /ml of mouse anti-PVL Luk-S monoclonal antibody (IBT Bioservices). Purified S. aureus LukS-PV (His-tag) (IBT Bioservices) was used to generate a standard curve. The experiment was carried as before except detection antibodies rabbit anti-PVL Luk-S (2 μ g/ml) and rabbit IgG HRP conjugate (R&D Systems) at a dilution of 1:6000 was used. The concentrations of each toxin was compared as unadjusted concentrations (ng/ml) and corrected for organism inoculum for each treatment (ng/ml to log 10 CFU/ml).
Biofilm assay. Biofilm assay was performed as described before 34 . Briefly, biofilm-forming clinical isolates of S. aureus (ATCC 6538) and S. epidermidis (ATCC 35984) were inoculated in 96-well flat-bottom cell culture plates (polystyrene) in TSB supplemented with 1% glucose at 37 °C for 24 h. Then culture medium was removed, and wells were carefully washed with PBS four times to remove planktonic bacteria. Ebselen and antibiotics (linezolid, mupirocin, vancomycin and rifampicin) were added at different concentrations in TSB, and plates were incubated at 37 °C for 24 h. The wells were rinsed by submerging the entire plate in a tub containing tap water. Biofilms were stained with 0.1% (wt/vol) crystal violet for 30 min. After staining, the dye was removed and the wells were washed four times with water. The plates were dried for 1 h and ethanol (95%) was added to solubilize the dye bound to the biofilm. The OD of biofilm mass was measured at 595-nm absorbance by using a micro plate reader (Bio-Tek Instruments Inc.) Cytotoxicity assay. Human keratinocyte (HaCat) cells were seeded at a density of 10,000 cells per well in a 96-well tissue culture plate (CytoOne, CC7682-7596) in DMEM media containing 10% fetal bovine serum (FBS) and incubated overnight at 37 °C. Then cells were treated with ebselen at different concentrations from 0 to 128 μ g/ml for 24 hours. Treated cells were washed four times with PBS and the DMEM media containing MTS assay reagent, 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA) was added. After 4 hrs of incubation at 37 °C, absorbance was measured using ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). Percent cell viability of ebselen treated cells were calculated in relative to the untreated cells.

Mice infection.
All experiments were performed in accordance with relevant guidelines and regulations. Eight weeks old female BALB/c mice were used for this study (Harlan Laboratories, Indianapolis, IN). All animal procedures were approved by Purdue University Animal Care and Use Committee (PACUC). The murine model of MRSA skin infection has been described before 52 . Mice were injected intradermally with 40 μ l of MRSA USA300 (1.65 × 10 8 ) CFU per mouse. Forty-eight hours after infection and formation of open wound, the mice were divided into eight groups (n = 5). Four groups were treated topically with either 0.5%, 1%, or 2% ebselen in petroleum jelly (ointment-skin protectant) or 1% ebselen in lipoderm (dermal and transdermal delivery cream base). Two groups received the vehicles alone (petroleum jelly or lipoderm). One group was treated topically with 2% mupirocin in petroleum jelly and the last group was treated orally with linezolid (25 mg/kg). All groups were treated twice a day for 5 days. Twenty-four hours after the last treatment, the area around the wound was lightly swabbed with 70% ethanol and the wound was excised for bacterial counting on MSA after homogenization.
Cytokines detection. Skin homogenates were centrifuged and the supernatants were used to detect the cytokine level by ELISA. Tumor necrosis factor-α (TNF-α ), interleukin-6 (IL-6), interleukin-1 beta (IL-1β ), and monocyte chemo attractant protein-1(MCP-1) Duo-set ELISA Kits (R&D Systems, Inc.) were used for the quantification of cytokines The experiment was carried out as per the manufacture instructions 53 .
Scientific RepoRts | 5:11596 | DOi: 10.1038/srep11596 Bliss model of synergism. Synergism was calculated using the Bliss Independence Model, which calculates a degree of synergy using the formula: S = (f A0 /f 00 )(f 0B /f 00 ) − (f AB /f 00 ), where f AB refers to bacterial growth rate in the presence of the combined drugs at a concentration A, for one of the antibiotics, and B for the ebselen; f A0 and f 0B refer to the bacterial growth rates in the presence of antibiotics (or) ebselen at a concentration of A and B, respectively; f 00 refers to the bacterial growth rate in the absence of drugs; and S corresponds to the degree of synergy, a parameter that determines a synergistic interaction for positive values and an antagonistic interaction for negative ones. Growth rates at 12 hr are determined and the degree of synergism was calculated as described before 50 . Statistical analyses. Statistical analyses were assessed by Graph Pad Prism 6.0 (Graph Pad Software, La Jolla, CA). P values were calculated by the two-tailed Student t test. P values of <0.05 were considered as significant.