Methods Bacterial selleck chemical growth conditions and MIC assays Bacterial strains used in this work are listed in Additional file 1: Table S2. Overnight cultures of bacteria were inoculated at an OD600 of 0.025 in LB broth supplemented with antibiotic in the absence and presence of DSF or its structural analogue (Table 1). One

hundred microliters of inoculated culture were grown in each well at 28°C or 37°C as AZD6244 clinical trial indicated with shaking at 200 rpm for 24 hours (Additional file 1: Table S2). MIC was defined as the lowest concentration of antibiotic in which bacterial growth in the well was not measureable by determination of the turbidity at 600 nm, and determined following the method from the Clinical and Laboratory Standards Institute (CLSI) [38]. Bacterial growth analysis Overnight bacterial cultures grown in LB broth were inoculated in the same medium to an OD600 of 0.025 in the absence and presence of DSF or its analogue at a final concentration of 50 μM. Three hundred microliters of inoculated culture were grown in each well at 28°C or 37°C as indicated in Additional file 1: Table S2 in a low intensity shaking model using the Bioscreen-C Automated Growth Curves Analysis System (OY Growth Curves AB Ltd., Finland). Biofilm formation assays Biofilm formation was assayed

using 96-well polypropylene microtitre dishes. Overnight bacterial cultures grown in LB broth were inoculated in the same medium to an OD600 of 0.01 in the absence and presence of DSF signal at different concentrations as indicated. One hundred microliters of inoculated culture were grown in each well at 37°C JNJ-64619178 in vitro with shaking at 150 rpm for 18 h. The cultures were removed and 200 μl of 1% crystal violet (w/v) was added. Following staining at room temperature for 15 min, the dye was removed and the wells

were rinsed three times with water. For quantification of the attached bacterial cells, the stained wells were decolorized with 200 μl of 95% ethanol. The quantity of crystal violet was determined by measuring the absorbance at 595 nm. Persistence Bumetanide assays Persistence was measured by determining the number of cfu/mL after exposure to 10 μg/mL gentamicin. Overnight cultures were diluted 100-fold in 10 mL of fresh medium and incubated at 37°C at 250 rpm to an OD600 of 1.0. Cultures were incubated with shaking at 150 rpm at 37°C supplemented with gentamicin in the absence and presence of DSF signal at a final concentration of 50 μM. For determination of cfu, 1-mL aliquots were removed at the indicated time points and cells were serially diluted in fresh medium and plated on solid medium. Persisters were calculated after incubation at 37°C overnight. Cytotoxicity assays in HeLa cell model The synergistic effect of DSF signal with antibiotic on the virulence of B. cereus was assayed by using HeLa cells.