Fig 1.
An MRSA overnight culture was treated with 10X MIC (20 μg/mL) gentamicin for 4 h and the titer of viable cells was determined. After the 4 h treatment with gentamicin, the culture was treated with additional antibiotics at the indicated concentrations (10X MIC) for an additional 4 h, followed by once again determining the titer of viable cells. Results are shown as means ± s.d.; n = 3. Gm: gentamicin, Cipro: ciprofloxacin, Van: vancomycin.
Fig 2.
Lysostaphin and nisin kill MRSA persisters by inducing membrane permeabilization.
MRSA persisters were treated with 0.1% DMSO (A), 10X MIC lysostaphin (B), or 10X MIC nisin (C). Membrane permeabilization (open circles) was measured spectrophotometrically by monitoring the uptake of SYTOX Green (excitation wavelength of 485 nm and an emission wavelength of 525 nm). Colony forming unit counts of persisters (solid circles) were measured by serial dilution and plating on TSA plates. The data points on the x-axis are below the level of detection (2x102 CFU/mL). Results are shown as means ± s.d.; n = 3.
Fig 3.
Validation of SYTOX Green assay robustness.
To test the robustness of the SYTOX Green assay, the Z’-factor was calculated from fluorescence intensity data from a 384-well plate where half of the wells contained 0.1% DMSO (negative control, open circles) and the remaining wells contained 10X MIC lysostaphin (A) or 10X MIC nisin (B) (positive controls, solid circles). Fluorescence was measured with an excitation wavelength of 485 nm and an emission wavelength of 525 nm after incubation in the dark for 1 h. The Z’-factors for each assay were 0.767 (A) and 0.712 (B).
Fig 4.
NH125 kills MRSA persisters by inducing membrane permeabilization.
(A) The chemical structure of NH125. (B) MRSA persisters were treated with 10 μg/mL NH125. Membrane permeabilization (open circles) was measured spectrophotometrically by monitoring the uptake of SYTOX Green (excitation wavelength of 485 nm and an emission wavelength of 525 nm). Colony forming unit counts of persisters (solid circles) was measured by serial dilution and plating on TSA plates. (C) MRSA persisters were treated with several concentrations of NH125: 10X MIC (20 μg/mL, circles), 5X MIC (10 μg/mL, squares), 2.5X MIC (5 μg/mL, triangles), and 1X MIC (2 μg/mL, inverted triangles). Colony forming unit counts of persisters was measured by serial dilution and plating on TSA plates. The data points on the x-axis are below the level of detection (2x102 CFU/mL). Results are shown as means ± s.d.; n = 3.
Table 1.
Minimal inhibitory concentration (MIC) against S. aureus MW2.
Fig 5.
NH125 eradicates MRSA biofilms.
(A) MRSA biofilms formed on 13 mm cellulose ester membranes for 24 hours were treated with 10X MIC of vancomycin (Van) or NH125 for 24 h. Survival was measured by comparing the number of viable cells in biofilms between non-treated and treated samples. (B) MRSA biofilms grown in a 96-well microtiter plate for 48 h were treated with the indicated concentration of vancomycin or NH125 for 24 h. The remaining biofilms were stained with 0.1% crystal violet dissolved with 95% ethanol and OD590 nm was measured. Results are shown as means ± s.d.; n = 3.