## 2 Materials and Methods
### Biological Validation Assays
The effect of different compounds on biofilm formation was tested by adding compounds at different concentrations to the bacterial culture (100\(\times\) diluted overnight culture; approximately 10\({}^{7}\) cells) in fresh LB medium. Two hundred microliter aliquots were pipetted into 96-well plates and placed for 24 or 48 h into a 37 \({}^{\circ}\)C incubator. The biofilm mass was measured by the CV staining assay described above. Biofilm persistence assays with ampicillin: Biofilms were grown for 24 or 48 h as described above. Planktonic cells were removed, and the biofilms were washed twice with 250 uL sterile PBS solution. Two hundred microliters of fresh LB medium with various concentrations of ampicillin were dispensed into the wells. After 18 h of incubation at 37 degC, the volume of biofilm was measured by CV staining, as described above. Synergistic effects of _in silico_ 'hit' compounds and antibiotics: Biofilms were grown for 24, 48, or 72 h as described above. Planktonic cells were then removed, and biofilms were washed twice with 250 uL sterile PBS solution. Two hundred microliter aliquots of fresh LB medium with multiple concentrations of the 'hit' compounds to be tested and ampicillin were dispensed into the wells. After 18 h of incubation at 37 degC, the biofilm mass was measured as described above. For the alamarBlue viability test, 4 uL of alamarBlue (Invitrogen, Carlsbad, CA, USA) was added and plates were incubated in a BioTek HT plate reader at 37 degC for 4 h. Cell viability was measured as fluorescence at 530/590 nm (excitation/emission) versus compound concentration or initial cell density. Cell growth curves: The effect of the hit compounds on bacterial growth was tested by adding compounds at multiple concentrations to the bacterial culture (100x diluted overnight culture; approximately 10\({}^{7}\) cells) in fresh LB medium. Two hundred microliters of aliquots were pipetted into 96-well plates and placed into a BioTek HT (BioTek, Winooski, VT, USA) or Tecan Infinite M200 Pro (Tecan, Mannedorf, Switzerland) plate reader for 18 h at 37 degC. Plates were shaken during incubation and the optical density (OD\({}_{630}\) or OD\({}_{600}\)) was measured every 15 min. Antibiotic susceptibility assays: For liquid cultures, the minimal inhibitory concentrations (MICs) of the antimicrobial drugs were determined using 96-well plates and the broth dilution method. Suspensions were then incubated at 37 degC for 18 h in a BioTek HT plate reader (see bacterial growth). Biofilm destruction experiments were performed with different antibiotic concentrations, and cell densities were measured after 18 h. Bacterial concentrations were calculated via optical density (OD\({}_{630}\)), and the lowest concentration causing 80% growth inhibition relative to the growth of the control was deemed to be the MIC. Scanning electron microscopy (SEM) of biofilm: _E. coli_ biofilms were grown in LB with multiple concentrations of the hit compounds on metal pins [51]. These metal pins were then washed twice in 1 x PBS. The biofilm-containing metal pins were then placed in a 5% glutaraldehyde solution for 1 h. Metal pins were then dehydrated using a gradient of ethanol from 50% to 100%--5 min in each solution. The pins were sputter coated with gold at a thickness of 60 A. SEM images were taken on a Zeiss Supra 50 VP Scanning Electron Microscope (Carl Zeiss AG, Oberkochen, Germany) with 5 kV beam acceleration. Statistical analysis: Statistical analyses were performed using OriginPro 8.5 (Originlab Corporation, Northampton, MA, USA). Relevant statistical data are included in the results and discussion for each experiment. Error bars indicate standard deviation from the mean. Asterisks represent statistical significance of at least \(p<0.05\).