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, 13 (6), e0199710
eCollection

Repurposing Ebselen for Decolonization of Vancomycin-Resistant Enterococci (VRE)

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Repurposing Ebselen for Decolonization of Vancomycin-Resistant Enterococci (VRE)

Ahmed AbdelKhalek et al. PLoS One.

Abstract

Enterococci represent one of the microbial world's most challenging enigmas. Colonization of the gastrointestinal tract (GIT) of high-risk/immunocompromised patients by enterococci exhibiting resistance to vancomycin (VRE) can lead to life-threating infections, including bloodstream infections and endocarditis. Decolonization of VRE from the GIT of high-risk patients represents an alternative method to suppress the risk of the infection. It could be considered as a preventative measure to protect against VRE infections in high-risk individuals. Though multiple agents (ramoplanin and bacitracin) have been evaluated clinically, no drugs are currently approved for use in VRE decolonization of the GIT. The present study evaluates ebselen, a clinical molecule, for use as a decolonizing agent against VRE. When evaluated against a broad array of enterococcal isolates in vitro, ebselen was found to be as potent as linezolid (minimum inhibitory concentration against 90% of clinical isolates tested was 2 μg/ml). Though VRE has a remarkable ability to develop resistance to antibacterial agents, no resistance to ebselen emerged after a clinical isolate of vancomycin-resistant E. faecium was serially-passaged with ebselen for 14 days. Against VRE biofilm, a virulence factor that enables the bacteria to colonize the gut, ebselen demonstrated the ability to both inhibit biofilm formation and disrupt mature biofilm. Furthermore, in a murine VRE colonization reduction model, ebselen proved as effective as ramoplanin in reducing the bacterial shedding and burden of VRE present in the fecal content (by > 99.99%), cecum, and ileum of mice. Based on the promising results obtained, ebselen warrants further investigation as a novel decolonizing agent to quell VRE infection.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Time-kill assay for ebselen and the control antibiotics, linezolid and ramoplanin.
Tested at (A) 3 × MIC and (B) 6 × MIC. E. faecium HM-952 was aerobically incubated with the indicated concentrations of the drugs, in triplicate, for 24 hours at 37 °C and samples were collected at the indicated time points to enumerate bacteria.
Fig 2
Fig 2. Multi-step resistance selection of E. faecium HM-952 in presence of ebselen, gentamicin, linezolid, or ramoplanin.
The MIC of all the test agents was determined daily (for 14 passages) to test for the development of resistance (increase in MIC) to the tested isolate. A 4-fold increase in the MIC is indicative of resistance formation.
Fig 3
Fig 3. The anti-biofilm activity of ebselen against E. faecalis NR-31972.
(A) Biofilm inhibition activity of ebselen. Sub-inhibitory concentrations of each drug were added to bacteria in tryptic soya broth (TSB) + 1% glucose and incubated for 24 hours at 37 °C. The biofilm mass (OD595) was measured after staining with crystal violet and destaining with ethanol. Data is presented as biofilm mass relative to DMSO-treated wells, (*) indicates significant difference from DMSO treated wells using 2-way ANOVA with Dunnett’s pairwise comparison (P< 0.001). (B) Biofilm eradication activity of ebselen. Bacteria were incubated for 24 hours in TSB + 1% glucose to allow for the formation of mature biofilm. Supra-inhibitory concentrations of the drugs were then added and incubated with the bacterial biofilm for additional 24 hours before the biofilm density was measured (OD595) by crystal violet staining. Data is presented as biofilm mass relative to DMSO-treated wells, (*) indicates significant difference from linezolid-treated wells, while (#) indicates significant difference from ramoplanin-treated wells using 2-way ANOVA with Dunnett’s post hoc comparison test at P<0.05.
Fig 4
Fig 4. Bacterial counts of E. faecium HM-952 in the fecal samples of infected mice.
Infected mice were orally treated with ebselen (0.5 mg/kg) and ramoplanin (10 mg/kg) daily for 8 days. One group was left untreated. Fecal samples were freshly collected from mice in each group on days 0, 5, 10, 15, and 20 post treatment. (*) denotes significant difference from the untreated group (P < 0.001).
Fig 5
Fig 5. Bacterial counts of E. faecium HM-952 in (A) cecum and (B) ileum contents of mice.
Infected mice (n = 5 per group) were orally treated with ebselen (0.5 mg/kg) or ramoplanin (10 mg/kg) daily for eight days. One group was left untreated. Cecum and ileum contents were collected one day after the last treatment was administered (day 21 of experiment). Asterisk (*) denotes significant difference from the untreated group (P < 0.05). No significant difference was found between ebselen-treated and ramoplanin-treated groups.

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