Structures of the aryl rhodanines used in this study.

Aryl rhodanines specifically inhibit the early phase of biofilm growth in S. aureus ATCC 35556. (A) MBX-1240 specifically inhibits the early phases of staphylococcal biofilm formation. MBX-1240 (4× the MBIC) was added to biofilm cultures at various times during growth, and the effect on biofilm formation after a total of 22 h of growth was quantified by staining with CV and measuring OD₆₀₀. The percent inhibition of biofilm growth compared to that of an untreated control was calculated and plotted on the primary y axis as a function of the time of addition (% Inhibition Biofilm; closed triangles). A biofilm growth curve was carried out in parallel in which biofilm growth was quantified at various times after inoculation as described above and was plotted on the secondary y axis (Biofilm Growth [OD₆₀₀ CV bound]; open squares). (B) MBX-1240 does not inhibit planktonic growth at concentrations of up to 8× the MBIC.

Aryl rhodanines do not affect autolysis or PNAG synthesis. (A) MBX-1240 does not inhibit Triton X-100-induced autolysis of S. aureus ATCC 35556 at concentrations of up to 8× the MBIC. Triton X-100-induced autolysis of cultures grown to mid-log phase and resuspended in buffer containing 0.05% Triton X-100 was monitored as the change in OD₆₀₀ over time. Key: 0× the MBIC, closed triangles; 2× the MBIC, open squares; 4× the MBIC, open circles; 8× the MBIC, closed circles; autolysin-defective ΔatlA::erm mutant, open triangles. (B) MBX-1240 does not inhibit autolysis of stationary-phase cultures. Autolysis of ATCC 35556(pAJ22) was monitored as the levels of β-galactosidase activity (Miller units) in the culture supernatant that were release during stationary phase with the Miller assay. Key: 0× the MBIC, squares; 1× the MBIC, triangles; 4× the MBIC, diamonds; 8× the MBIC, circles; β-galactosidase activity in Miller units, open symbols and dashed lines; OD₆₀₀, closed symbols and solid lines. (C) MBX-1240 does not inhibit PNAG synthesis by S. aureus ATCC 35556 at concentrations of up to 8× the MBIC. PNAG extracts were prepared from S. aureus ATCC 35556 cultures grown in the presence of various concentrations of MBX-1240 (0× to 8× the MBIC), serially diluted 1:4, spotted onto nitrocellulose, and probed with WGA-HRP. The amount of WGA-HRP bound was detected by adding a chemiluminescent substrate and exposing the blot to film. The specificity of the assay is demonstrated by the lack of reaction in the extract prepared from a PNAG-defective (Δica::Tc) strain. Dln, dilution.

Aryl rhodanines inhibit biofilm formation on silicone disks. Biofilms of S. aureus ATCC 35556 were grown on silicone disks in the presence of various concentrations of MBX-1240 and MBX-1384 (0× to 8× the MBIC) for 18 h, at which time nonadherent cells were removed by repeated washing. Adherent cells were heat fixed and stained with CV. The OD₆₀₀ of the eluted CV from each sample was measured, and the average values obtained from a total of three silicone disks is plotted as a function of the compound concentration. The Student t test was used to determine whether differences in biofilm formation between treated and untreated controls are significant. Significant differences are indicated by the asterisks (P < 0.001) above the bars. The error bars represent the standard deviation.

Aryl rhodanines inhibit primary adhesion. (A) Aryl rhodanines inhibit attachment of S. aureus ATCC 35556 to tissue culture-treated polystyrene in a static assay. A mid-log-phase culture was harvested, resuspended in PBS to an OD₆₀₀ of 0.5, and aliquoted. Aryl rhodanines were added to aliquots to achieve various concentrations (0× to 8× the MBIC), which were then transferred to 96-well tissue culture-treated polystyrene assay plates and incubated at 37°C for 30 min. After repeated washing, adherent cells were stained with BacLight Live/Dead stain (Molecular Probes) and fluorescence emission (excitation wavelength, 485 nm; emission wavelength, 530 nm) was measured to quantify adherent cells. Each bar represents the average of at least eight assay wells, and the error bars represent the standard deviation. The asterisks indicate a statistically significant difference between the untreated control and a treated sample determined with a two-tailed Student t test (*, P ≤ 0.05; **, P ≤ 0.001). (B) Pretreatment of S. aureus ATCC 35556 with aryl rhodanines inhibits primary attachment. A cell suspension was prepared as described above and pretreated with aryl rhodanines for 30 min. Cells were harvested to remove free compound, resuspended in PBS, and used in an adhesion assay as described above. (C) Aryl rhodanines inhibit attachment to PVC in a static assay. The assay described in panel A was used to measure adhesion to PVC. (D) Aryl rhodanines inhibit attachment to silicone disks in a static assay. The assay described in panel A was used to measure adhesion to medical grade silicone disks. Each bar represents the average of three silicone disks, and the error bars represent the standard deviation. (E) MBX-1240 inhibits attachment to silicone disks in the presence of shear force. Cell suspensions containing various concentrations of MBX-1240 were prepared as described above, transferred to a culture tube containing a silicone disk, and incubated at 37°C with moderate shaking. After repeated washes, the disk was transferred to a fresh tube and adherent bacteria were removed by sonication. The numbers of adherent bacteria were determined by plating appropriate dilutions and counting colonies. Each bar represents the average value obtained from two silicone disks. Light and dark bars represent log₁₀ CFU/disk and fraction of control, respectively.