Table 1.
The minimum inhibitory concentration (MIC, μg/mL) of ebselen and control antibiotics against enterococci clinical isolates.
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.
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.
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.
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.
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.