2.4. Antibiofilm and Antibacterial Activity Assays

A planktonic susceptibility testing of S. epidermidis, S. aureus and E. faecalis was performed by the reference broth microdilution assay according to Clinical and Laboratory Standards Institute (CLSI) guidelines [20]. The growth was evaluated by the difference between the OD600 absorbance measured at the beginning (t = 0 h) and at the end (t = 24 h) of the incubation time using a 96-well polystyrene flat-bottom microtiter plate and cation-adjusted Mueller-Hinton broth. As a negative control for bacterial growth the derivatives were replaced by 80 µL of water, with this being considered as representing 100% planktonic bacterial growth. Values higher than 100% represent a stimulation of bacterial growth when compared to the control. The minimum inhibitory concentration (MIC) to kill 100% of bacterial cells was determined, and 50 µL of a serial dilution was spread on to MH agar plates. Vancomycin 8 µg/mL (Sigma-Aldrich Co., St. Louis, MO, USA) was used as a positive control for the inhibition of bacterial growth.

The antibiofilm assays were performed with the incubation of the bacterial solution with the compounds in 96-well microtiter plates, as established by Trentin et al. [21]. Firstly, 80 µL of the bacterial suspension, 80 µL of the triterpenes derivatives solution (previously solubilized in DMSO 2% and water) in concentrations of 100, 25 or 5 µM in the wells, and 40 µL of tryptone soya broth (TSB, Oxoid Ltd., Basingstoke, England) were added. After that, the experiment followed the incubation period at 37 °C of 24 h, and after this the content of the wells was removed and washed three times with sterile saline. The remaining attached bacteria were heat-fixed at 60 °C for 1 h. The adherent biofilm layer that was formed was stained with 0.4% crystal violet for 15 min at room temperature. The stain bound to the cells was solubilized with 99.5% DMSO (Sigma-Aldrich Co.) for 30 min, and the absorbance was measured at 570 nm (Spectramax M2e Multimode Microplate Reader, Molecular Devices, San Jose, CA, USA).

This was used in the antibiofilm formation and antimicrobial assays, negative controls representing 100% of the biofilm formation or 100% of the planktonic cells viability, respectively. In both experiments the positive control used was a commercial antimicrobial vancomycin 8 µg/mL (Sigma–Aldrich Co.), while the vehicle control that was used was a DMSO 2% solution [21,22].