Compounds
Glabridin (Wako Chemicals, Richmond, VA, USA) and licochalcone A (Enzo Life Sciences, Inc., Farmingdale, NY, USA) were prepared in 95% (v/v) ethanol, while licoricidin (EMMX Biotechnology, Lake Forest, CA, USA) was prepared in dimethyl sulfoxide. Stock solutions (20 mg/mL) were kept in the dark at 4 °C for up to 1 month. Given that these stock solutions were highly diluted in the assays described below, neither ethanol nor dimethyl sulfoxide caused any biological effects (data not shown). Nisin A (Sigma-Aldrich Canada Co., Oakville, ON, Canada) was prepared in 0.02 N HCl at a concentration of 750 μg/mL and was sterilized by filtration through a 0.22-μm pore size membrane filter. Chlorhexidine digluconate (Sigma-Aldrich Canada Co.,), which was used as a positive antimicrobial control, was prepared in distilled water at a concentration of 5 mg/mL and was sterilized by filtration through a 0.22-μm pore size membrane filter.
Bacteria and growth conditions
The reference strain E. faecalis ATCC 19433 as well as two clinical isolates of E. faecalis (0DOT, and 1DOT) were used. They were grown in Brain Heart Infusion broth (BHI; BBL Microbiology Systems, Mississauga, ON, Canada) supplemented with 0.5% glucose at 37 °C in an anaerobic chamber (80% N2/10% H2/10% CO2).
Determination of minimum inhibitory and minimum bactericidal concentrations
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of nisin and the licorice polyphenols were assessed using the microdilution method as described by the Clinical and Laboratory Standards Institute (Wayne 2012). The bacterial suspension and two-fold serial dilutions of the compounds (from 200 µg/mL) diluted in culture medium were added to the wells of a 96-well microplate. Wells with no compounds (only carrier solvent) were used as controls (100% growth). After a 24-h incubation, microbial growth was assessed by recording the optical density at 660 nm (OD660) using a Synergy 2 microplate reader (BioTek Instruments, Winooski, VT, USA). The MIC values are the lowest concentrations of the compounds that completely inhibited bacterial growth. To determine the MBC values, 5-μL aliquots from the wells with no visible growth were spread on BHI agar plates, which were incubated for 3 days at 37 °C. The MBC values are the lowest concentrations at which no colony formation occurred. Assays were performed in triplicate in two independent experiments to ensure reproducibility. A representative set of data is presented.
Biofilm formation
E. faecalis ATCC 19433 was grown (24 h) in the wells of a flat-bottomed 96-well microplate as described above in the absence (control) and presence of either nisin or licorice polyphenols at sub-inhibitory concentrations corresponding to 1/4 MIC. The medium, free-floating bacteria, and loosely-bound biofilm were then removed by aspiration, and the wells were gently washed three times with 50 mM phosphate-buffered saline (pH 7.2; PBS). The biofilms were stained with 0.04% crystal violet (100 µL) for 10 min. The wells were washed three times with PBS to remove unbound crystal violet dye and were dried for 2 h at 37 °C. After adding 100 µL of 95% (v/v) ethanol to each well, the microplate was shaken for 10 min to release the dye from the biofilms, and the absorbance at 550 nm (A550) was recorded using a Synergy 2 microplate reader. Assays were performed in triplicate in two independent experiments, and the means ± standard deviations were calculated.
Synergistic antibacterial interactions
The synergistic antibacterial effects against E. faecalis ATCC 19433 of nisin in combination with the licorice polyphenols were evaluated using the checkerboard method (Eliopoulos and Moellering 1996). Compound A was serially diluted in culture medium (100 µL) along the ordinate of a 96-well microplate, while compound B was serially diluted in culture medium (100 µL) along the abscissa. A bacterial suspension prepared in fresh culture medium and adjusted to a McFarland standard of 0.5 was used as an inoculum. The microplate wells were inoculated with 100 µL of the suspension, and the microplate was incubated at 37 °C for 24 h in an anaerobic chamber. Wells with no bacteria or no compounds were included in the assay. After the incubation period, bacterial growth was assessed by recording the OD660 using a Synergy 2 microplate reader. The lowest concentration at which no growth occurred was considered the MIC. The fractional inhibitory concentration index (FICI) was calculated using the following equation: FICI = (MICA+B/MICA) + (MICB+A/MICB). An FICI ≤ 0.5 was considered as indicating a synergistic effect, an FICI > 0.5 and ≤ 4.0 as indicating no effect, and an FICI > 4.0 as indicating an antagonistic effect. Assays were performed in triplicate to ensure reproducibility. A representative set of data is presented.
Biofilm killing
The ability of nisin/glabridin, nisin/licoricidin, and nisin/licochalcone A to kill E. faecalis ATCC 19433 biofilms was investigated. Briefly, 24-h biofilms were pre-formed in the wells of a 96-well microplate, washed once with PBS, and treated for 30 min with the above combinations. Each compound of a combination was used at a concentration corresponding to its MIC value (in PBS). Following these treatments, the biofilms were washed once with PBS. A series of biofilms was stained with 0.04% crystal violet as described above to determine biofilm desorption. A second series of biofilms was used to determine bacterial viability using a commercial luminescence assay (BacTiter-Glo™; Madison, WI, USA) that measures adenosine triphosphate (ATP), an indicator of metabolically active viable bacteria. Luminescence was quantified using a Synergy 2 microplate reader. All the assays were performed in triplicate in two independent experiments, and the means ± standard deviations were calculated.
In vitro biocompatibility assay
The human oral epithelial cell line B11, which has already been characterized (Groeger et al. 2008), was kindly provided by S. Groeger (Justus Liebig University Giessen, Germany). Cells were cultured in keratinocyte serum-free medium (K-SFM; Life Technologies Inc., Burlington, ON, Canada) supplemented with growth factors (50 µg/mL of bovine pituitary extract and 5 ng/mL of human epidermal growth factor) and 100 µg/mL of penicillin G-streptomycin. The primary human gingival fibroblast cell line HGF-1 (CRL-2014) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies Inc.) supplemented with 4 mM l-glutamine, 15% heat-inactivated fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA), and 100 µg/mL of penicillin G-streptomycin. The previously characterized human SCAP cell line (RP-89) was cultured in -minimum essential medium (MEM; Life Technologies Inc.) supplemented with 10% FBS, 2 mmol/L l-glutamine, and 100 µg/mL of penicillin G/streptomycin (Ruparel et al. 2013). All cell cultures were incubated at 37 °C in a 5% CO2 atmosphere. To evaluate the effect of nisin/glabridin, nisin/licoricidin, and nisin/licochalcone A on cell viability, cells were seeded (1 × 105 cells in 100 µL) in the wells of a 96-well tissue culture plate and were incubated at 37 °C in a 5% CO2 atmosphere until they reached confluence. The cells were treated with the combinations for 2 h. Each compound of a combination was used at concentrations corresponding to the MIC and twofold MIC values obtained against E. faecalis ATCC 19433. Thereafter, an MTT (3-[4, 5-diethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was performed according to the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany) to assess cell viability. The assays were performed in triplicate in two independent experiments, and the means ± standard deviations were calculated.
NF-κB activation assay
The human monoblastic leukemia cell line U937-3xκB-LUC was kindly provided by R. Blomhoff (University of Oslo, Norway). It consists of the U937 cell line stably transfected with a luciferase gene coupled to a promoter of three NF-κB binding sites (Carlsen et al. 2002). Monocytes were grown at 37 °C in a 5% CO2 atmosphere in RPMI-1640 medium (Life Technologies Inc.) supplemented with 10% heat-inactivated FBS, 100 µg/mL of penicillin–streptomycin, and 75 µg/mL of hygromycin B. In a first analysis, the ability of E. faecalis ATCC 19433 to induce NF-κB activation was assessed. Briefly, 100 µL of the monocyte suspension (107 cells/mL) was seeded in the wells of a black bottom, black wall 96-well microplate (Greiner Bio-One North America Inc., Monroe, NC, USA), and an overnight culture of E. faecalis suspended in culture medium was added at a multiplicity of infection (MOI) of 100, 50, 25, 12, 6, or 3. The plate was incubated at 37 °C (5% CO2) for a further 6 h. NF-κB activation was then assessed using the Bright-Glo™ Luciferase Assay System (Promega, Madison, WI, USA) by adding 100 µL of luciferase substrate to the wells at room temperature. Luminescence was recorded using the luminometer option of the Synergy 2 microplate reader (BioTek Instruments) within 3 min of adding the substrate. To investigate the ability of nisin/glabridin, nisin/licoricidin, and nisin/licochalcone A (each compound at their MIC, ½ MIC, and ¼ MIC obtained against E. faecalis ATCC 19433) to inhibit NF-κB activation, the combinations were added 30 min prior to challenge U937-3xκB-LUC cells with E. faecalis at an MOI of 100. All the assays were carried out in triplicate in two independent experiments, and the means ± standard deviations were calculated.
Statistical analysis
Statistical analyses were performed using a one-way analysis of variance with a post hoc Bonferroni multiple comparison test (GraphPad Software Inc., La Jolla, CA, USA). All results were considered statistically significant at p < 0.01.