A young red wine was used in this investigation (var. Pinot Noir, vintage 2010), provided by Bodegas Miguel Torres S.A. (Vilafranca del Penedès, Barcelona, Spain). The phenolic content present in the wine include: total anthocyanins = 0.447 mg of malvidin-3-glucoside mL− 1, total catechins = 1.612 mg of (+)-catechin mL− 1 and total polyphenols = 1.758 mg of gallic acid equiv. mL− 1. The principal individual phenolic compounds found in this wine were flavan-3-ols, flavonols, alcohols, anthocyanins, stilbenes and hydroxycinnamic acids, determined by Ultra-High-Performance Liquid Chromatography-ElectroSpray Ionization-tandem Mass spectrometry (UHPLC-ESI-MS/MS) for other studies .
A rotary evaporator was used for the preparation of dealcoholized red wine, removing the EtOH and adding distilled water to reconstitute it until the original volume.
Two commercially available oenological phenolic extracts were used: Provinols™, a red wine extract, kindly supplied by Safic-Alcan Especialidades S.A.U. (Barcelona, Spain) and a grape seed extract, Vitaflavan®, kindly provided by Piriou (Les Derives Resiniques & Terpeniques S.A., France). The total phenolic content of the extracts was 474 mg of gallic acid equiv. g− 1 for Provinols™ and 629 mg of gallic acid equiv. g− 1 for Vitaflavan®. The phenolic compositions of both oenological extracts has been determined by UHPLC-ESI-MS/MS in previous studies [34, 35]. Both the wine extract and grape seed extracts were dissolved in distilled water containing 4% dimethyl sulfoxide (DMSO) (v/v), until reaching a final concentration of 20 mg mL− 1.
Bacterial strains and culture conditions
Six bacterial strains, including Streptococcus oralis CECT 907 T, Veillonella parvula NCTC 11810, Actinomyces naeslundii ATCC 19039, F. nucleatum DMSZ 20482, A. actinomycetemcomitans DSMZ 8324 and P. gingivalis ATCC 33277 were used. Bacteria were cultured in blood agar plates (Blood Agar Oxoid No 2; Oxoid, Basingstoke, UK), supplemented with 5% (v/v) sterile horse blood (Oxoid), 5.0 mg L− 1 hemin (Sigma, St. Louis, MO, USA) and 1.0 mg L− 1 menadione (Merck, Darmstadt, Germany) at 37 °C for 24–72 h in anaerobic conditions (10% H2, 10% CO2, and balance N2).
A multi-species in vitro biofilm model was developed as previously described by Sánchez and colleagues . For the inoculum preparation, the microorganisms were individually cultivated in anaerobic conditions on a protein rich medium containing brain-heart infusion (BHI) (Becton, Dickinson and Company, USA) supplemented with 2.5 g L− 1 mucin (Oxoid, Thermo Scientific, Hampshire, UK), 1.0 g L− 1 yeast extract (Oxoid, Thermo Scientific, Hampshire, UK), 0.1 g L− 1 cysteine (Sigma-Aldrich, Barcelona, Spain), 2.0 g L− 1 sodium bicarbonate (Merck, NJ, USA), 5.0 mg L− 1 hemin (Sigma-Aldrich, Barcelona, Spain), 1.0 mg L− 1 menadione (Merck, NJ, USA) and 0.25% (v/v) glutamic acid (Sigma-Aldrich, Barcelona, Spain). The bacterial cultures were harvested at mid-exponential phase (measured by spectrophotometry), and a mixed bacteria suspension in modified BHI medium containing 103 colony-forming units (CFU) mL− 1 for S. oralis, 105 CFU mL− 1 for V. parvula and A. naeslundii, and 106 CFU mL− 1 for F. nucleatum, A. actinomycetemcomitans and P. gingivalis was prepared. The biofilms were grown on sterile calcium hydroxyapatite (HA) discs of 7 mm of diameter and 1.8 mm (standard deviation, SD = 0.2) of thickness (Clarkson Chromatography Products, Williamsport, PA, USA) discs deposited in 24-wells cell culture plates (Greiner Bio-one, Frickenhausen, Germany), inoculating each well with 1.5 mL of mixed bacteria, for 72 h at 37 °C in anaerobic condition. All assays were performed independently at least three times and in triplicate (n = 9).
The antimicrobial activity of wines and oenological extracts was examined on 72 h biofilms by determining the reduction in the number of viable CFU mL− 1 using the quantitative polymerase chain reaction (qPCR). For the oenological extracts, 30 and 60 s were selected as exposure times since they are bioactive products, commercially available, and for them, the standard exposure times established for other antimicrobial commercially available products (e.g. products with chlorhexidine), was selected [37,38,39]. On the other hand, in the case of wine solutions, the product was considered as a new possible bioactive agent, evaluated for the first time, therefore, not only the standard 60 s interval was selected as exposure time, but also an “extreme” exposure time of 5 min, with the aim of detecting any possible effect of red wine solutions (dealcoholized or not). Two different protocols were performed:
For red wine (dealcoholized or not), biofilms were dipped during 1 and 5 min in the wine solutions at room temperature. Phosphate buffer saline (PBS) was used as negative control and, in order to discard a bactericidal effect of the EtOH contained in the wine, also 12% ethanol was applied.
For the oenological extracts, biofilms were dipped during 30 s and 1 min at room temperature, due to their high phenolic content. PBS was used as negative control, and in order to discard a bactericidal effect of the DMSO used for dissolve the extracts, 4% DMSO solution was also tested.
After the antimicrobial treatment, biofilms were sequentially rinsed in 2 mL of sterile PBS three times (immersion time per rinse, 10 s), in order to remove possible remains of the oenological solutions or extracts and unbound bacteria. Then, biofilms were disrupted by vortex for 2 min in 1 mL of PBS. To discriminate between DNA from live and dead bacteria, propidium monoazide (PMA) (Biotium Inc., Hayword, CA, USA) was used. The use of this PMA dye combined with qPCR has shown the ability to detect the DNA from viable bacteria . For this, 100 μM of PMA was added to 250 μL of disaggregated biofilm. Following an incubation period of 10 min at 4 °C in the dark, the samples were subjected to light-exposure for 30 min, using PMA-Lite LED Photolysis Device (Biotium Inc.), and then centrifuged at 12,000 rpm for 3 min prior to DNA extraction.
Bacterial DNA was isolated from all biofilms using a commercial kit ATP Genomic DNA Mini Kit® (ATP biotech. Taipei, Taiwan), following manufacturer’s instructions and the hydrolysis 5’nuclease probe assay qPCR method was used for detecting and quantifying the bacterial DNA. The qPCR amplification was performed following a protocol previously optimized by our research group, using primers and probes targeted against 16S rRNA gene [obtained through Life Technologies Invitrogen (Carlsbad, CA, USA)] .
Each DNA sample was analysed in duplicate. Quantification cycle (Cq) values, describing the PCR cycle number at which fluorescence rises above the baseline, were determined using the provided software package (LC 480 Software 1.5; Roche Diagnostic GmbH; Mannheim, Germany). Quantification of viable cells by qPCR was based on standard curves. The correlation between Cq values and CFU mL− 1 was automatically generated through informatics analysis (LC 480 Software 1.5; Roche).
All assays were developed with a linear quantitative detection range established by the slope range of 3.3–3.5 cycles/log decade, r2 > 0.998 and an efficiency range of 1.9–2.0.
Confocal laser scanning microscopy (CLSM)
Non-invasive confocal imaging of fully hydrated biofilms was carried out using a fixed-stage Ix83 Olympus inverted microscope coupled to an Olympus FV1200 confocal system (Olympus; Shinjuku, Tokyo, Japan). LIVE/DEAD® BacLight™ Bacterial Viability Kit solution (Molecular Probes B. V., Leiden, The Netherlands) was used to stained the biofilms at room temperature. The fluorochromes were incubated (ratio 1:1) during 9 ± 1 min to obtain the optimum fluorescence signal at the corresponding wave lengths (Syto9: 515–530 nm; Propidium Iodide (PI): > 600 nm. The CLSM software was set to take a z-series of scans (xyz) of 1 μm thickness (8 bits, 1024 × 1024 pixels). Image stacks were analyzed by using the Olympus® software (Olympus). Image analysis and live/dead cell ratio (i.e. the area occupied by living cells divided by the area occupied by dead cells) was performed with Fiji software (ImageJ Version 2.0.0-rc-65 / 1.52b, Open source image processing software).
The selected outcome variables to study the antibacterial effect of wine solutions and oenological extracts were the counts of viable bacteria present on the biofilms, expressed as viable CFU mL− 1 of A. actinomycetemcomitans, P. gingivalis, F. nucleatum and total bacteria by qPCR, and the live/dead cell ratio of the whole biofilm by CLSM. An experiment-level analysis was performed for each parameter of the study (n = 9 for qPCR and n = 3 for CLSM results). Shapiro–Wilk goodness-of-fit tests and distribution of data were used to assess normality. Data were expressed as means ± SD.
In the case of the experiments with red wine, the effect of each solution [red wine (dealcoholized or not), PBS and 12% EtOH], the time of exposure (1 or 5 min) and their interaction with the main outcome variable (counts expressed as CFU mL− 1 or live/dead cell ratio), was compared by means of a parametric ANOVA test for independent samples, and a general linear model was constructed for each bacterium (A. actinomycetemcomitans, P. gingivalis and F. nucleatum) and for total bacteria for qPCR results and for total bacteria for live/dead cell ratio of whole biofilm obtained by CLSM, using the method of maximum likelihood and Bonferroni corrections for multiple comparisons. A similar model was constructed in the case of the experiments with oenological extracts, in order to compare the effect of each solution (wine extract, grape seed extract, PBS and DMSO), the time of exposure (30 s or 1 min) and their interaction with the main outcome variable (CFU mL− 1 and live/dead cell ratio of whole biofilms).
Results were considered statistically significant at p < 0.05. A software package (IBM SPSS Statistics 24.0; IBM Corporation, Armonk, NY, USA) was used for all data analysis.