The compound S-8 was synthesized in our laboratory and was characterized by Nuclear Magnetic Resonance (NMR) analysis, melting point, and elemental analysis using the same procedure as described previously (Feldman et al., 2014). The purity of the compound was above 95%.

The assay was performed as described previously (Sztajer et al., 2014) with minor modifications. Briefly, pre-cultures of S. mutans UA159 and C. albicans SC5314 were inoculated from single colony grown for 18 h and harvested by centrifugation (5000 r.p.m., 10 min, 4°C). Next, both microbial cultures were diluted to OD₆₀₀ = 0.1 in RPMI + 1% sucrose. The growth medium was supplemented with S-8 at concentrations of 8 and 16 μg/ml. In some experiments Tryptic Soy Broth (TSB) + 1% sucrose medium was used. Equal volumes of each strain (0.1 ml) were inoculated into 96-well microtitre plates. Wells containing mixed cultures without S-8 served as a control. Biofilms were allowed to develop for 48 h, at 5% CO₂ at 37°C. Biofilm formation was monitored by quantitative PCR.

Extraction and quantification of DNA was performed as described previously (Periasamy and Kolenbrander, 2009). Briefly, formed biofilms were washed with PBS. Next, 160 mL 0.05 M NaOH (BioLab Ltd., Jerusalem, Israel) and 40 mL water (DEPC treated; Bio Basic Canada Inc, Markham, ON, Canada) were added to each well. The plates were immersed in a water bath at 60°C for 1 h, and 18.5 μL Tris buffer, pH 7.0 (Eastman Kodak Company, Rochester, NY, USA), was added to each well after the heating period. The extracted DNA samples were stored at -20°C until use. The DNA samples from the mixed biofilms were quantified by means of a quantitative PCR reaction with specific primers for S. mutans 16S rRNA and C. albicans 18S rRNA using a ABI Prism 7300 (Applied Biosystems, Foster City, CA, USA). The amount of DNA was quantified according to a specific standard curve. The bacterial and fungal genomic DNA for the standard curve analysis was extracted from an overnight culture of S. mutans UA159 and C. albicans SC5314, respectively, using GenElute Bacterial genomic DNA kit and Plant/Fungi DNA Isolation Kit, respectively (Sigma–Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The genomic DNA was stored at -20°C. The data are presented as a percentage of total biomass in mixed biofilms treated with S-8 and compared to untreated controls. The biomass of each microbe was presented as percentage of the total biomass in each sample. Three separate experiments were performed.

Dual species biofilms treated with S-8 at 8 and 16 μg/ml or untreated controls were incubated in 96-well microtitre plates under the conditions described above. For labeling S. mutans EPS, 1 mM AlexaFluor 555-labeled dextran conjugate (10,000 MW, Molecular Probes Inc., Eugene, OR, USA) was added to each well before biofilm formation, as described previously (Assaf et al., 2015). Forty-eight hour-biofilms were washed with PBS and incubated for 45 min in PBS containing the fluorescent stain concanavalin A-Alexa Fluor 647 conjugate (ConA; 25 mg/ml) (Invitrogen, Carsbad, CA, USA). ConA (excitation wavelength 650 nm and emission at 668 nm) binds to the glucose and mannose residues of fungal cell wall exopolysaccharides (EPS) (Chandra et al., 2001), and fluoresces red. Stained EPS (blue color for bacteria and red color for fungi) were observed with a Zeiss LSM510 CLS microscope (Carl Zeiss, Oberkochen, Germany). Three-dimensional images of the formed biofilms and EPS distribution were constructed using Zen 2009 software (Carl Zeiss). At least three random fields were observed and analyzed. Three independent experiments were performed. The amount of individual EPS production by S. mutans and C. albicans in each sample was calculated as blue and red fluorescence intensity, respectively, using Image J v3.91 software (http://rsb.info.nih.gov/ij). The data were presented as individual EPS production by C. albicans and S. mutans cells in each layer of biofilm (10 μm). The percentage of total EPS production by mixed biofilms treated with S-8 at 8 μg/ml and 16 μg/ml was calculated as area under the curve (AUC) and compared to untreated control. The impact of each individual microbial EPS was calculated as AUC and compared to EPS production within mixed biofilm in each sample.

After biofilm formation, the wells were washed with PBS, followed by fixation in 4% formaldehyde for 1 h at room temperature. The morphology of bacteria and fungi in mixed biofilms untreated, and treated with 8 and 16 μg/ml of S-8 was then visualized using an analytical Quanta 200 Environmental High Resolution Scanning Electron Microscope (EHRSEM) (FEI, Eindhoven, The Netherlands) at 5,000X magnification. At least four random fields were observed and analyzed. Three independent experiments were performed.

The assay was performed similarly to that described previously (Feldman et al., 2014). Briefly, dual species biofilms were grown in the absence or presence of S-8 at 8 and 16 μg/ml in 6-well plates under the conditions mentioned above. After washing with PBS, biofilm cells were removed from the bottom of the plates with a sterile scraper following disruption in a Fast Prep Cell Disrupter (Bio 101, Savant Instruments, Inc., NY, USA). Total RNA was extracted from mixed biofilms using Tri-Reagent (Sigma–Aldrich). RNA concentration was determined spectrophotometrically using a Nanodrop ND-1000 Instrument (Wilmington, DE, USA). Two micro gram of template was reverse-transcribed with Super Script First Strand (Invitrogen, Life Technologies, Carlsbad, CA, USA). The integrity and purity of the RNA was assessed using an Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA). Expression of C. albicans hyphal/yeast-specific, adhesion and hydrophobicity-associated genes, as well as oxidative stress relative genes (hwp1, ywp1, als3, csh1, sod1, sod2, cat1) and S. mutans biofilm, stress, and QS-associated genes (gtfB, gtfC, gtfD, gbpB, brpA, spaA, groEL, nox, sodA, comDE, luxS) were analyzed. The relative expression levels of the target genes were analyzed using an ABI-Prism 7300 Instrument (Applied Biosystems, Foster City, CA, USA). Platinum SYBR Green PCR Master Mix (Invitrogen) was used to monitor the amplified product in real time, following the manufacturer’s protocol. Primers for the tested genes are listed in Supplementary Table S1. For each set of primers, a standard amplification curve (critical threshold cycle vs. exponential of concentration) was plotted, and only those with slope ≈-3 were considered reliable. The PCR involved denaturation at 95°C for 10 min, followed by 40 cycles of amplification (95°C for 10 s, 55°C for 10 s, and 72°C for 10 s) and quantification. The expression of 18S rRNA and 16S rRNA was used for normalization and to calculate the relative changes in target gene expression of C. albicans and S. mutans, respectively. Control reactions were also performed with RNA that had not been reverse-transcribed to ensure that no genomic DNA was amplified during the PCRs. Gene expression is expressed in relative values, setting the expression level of the untreated with S-8 control to 1 for each gene. The assays were performed in triplicate and repeated three times.

The assay measuring the cell proliferation rate was performed as described previously (Jahn et al., 1995; Hahnel et al., 2012) with some modifications. Briefly, dual species biofilms of C. albicans and S. mutans formed in 96-well plate with or without added S-8 were washed three times with sterile PBS and overlaid with 100 mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). To determine planktonic cell viability, 50 μl of the MTT solution (100 mM) was added to the supernatants aspirated from the biofilms and placed in a new 96-well plate. Plates containing biofilms as well as supernatants were then incubated for 1 h at 37°C. Under these conditions, the lightly yellowish MTT will be reduced to an insoluble blue tetrazolium salt accumulated within the biofilms. The amount of intracellular tetrazolium salts was quantified spectrophotometrically by measuring the absorbance of the solution at 570 nm. The accumulation of tetrazolium salt by the reduction of MTT by cellular dehydrogenases is proportional to the number of viable cells growing in biofilm and planktonic condition. Four independent experiments were performed.

qPCR analysis demonstratedan inhibitory effect of S-8 on total mixed biofilm formation of S. mutans and C. albicans. No change in biofilm development was observed using up to 4 μg/ml of S-8. However, increasing S-8 concentration to 8 μg/ml and 16 μg/ml resulted in biofilm inhibition by 42 and 59%, respectively, as compared to the untreated control (Figure 1). Data obtained by the MTT assay demonstrated results similar to qPCR dose-dependent inhibition of dual species biofilm by S-8 (data not shown). The influence of S-8 on each individual specie within the mixed biofilm was differed. The relative effect of S-8 on S. mutans within the mixed biofilm was negligible at all tested doses as compared to the untreated co-species biofilm (Figure 1). In contrast, the portion Candida in the mixed biofilm was dramatically reduced by 55 and 81% under S-8 treatment with 8 and 16 μg/ml, respectively, as compared to C. albicans fraction from untreated co-species biofilm (Figure 1).

Three-dimensional images constructed using CLSM, demonstrated alteration in C. albicans/S. mutans mixed biofilm structure under S-8 treatment. The thickness of the co-species biofilm was reduced dose-dependently, by 60 μm (30%) at 8 μg/ml of S-8 (Figure 2B), and by 90 μm (45%) at 16 μg/ml (Figure 2C), as compared to controls (Figure 2A). Within the biofilm structure, total EPS formation was reduced by 29% in the presence of S-8 at both tested doses of 8 and 16 μg/ml as compared to control (Figure 2F). However, the pattern of EPS production by each individual microbe calculated as AUC for C. albicans (Figure 2D) and S. mutans (Figure 2E) due to S-8 treatment varied. Untreated mixed biofilm consisted of slightly higher amounts of fungal EPS (58%), compared to bacterial EPS (42%) (Figures 2A,F). However, exposure to 8 μg/ml of S-8 caused a substantial change in EPS composition in co-species biofilm: a majority of S. mutans EPS (71%) and minority of EPS of C. albicans (29%) (Figures 2B,F). S-8 at a concentration of 16 μg/ml further modified EPS impact in favor of S. mutans (78%), while fungal polysaccharides consisted only 22% of total EPS (Figures 2C,F).

Microscopic observation showed that S-8 dramatically alters biofilm morphologic composition. As shown in Figure 3, untreated control biofilm (Figure 3A) consisted predominantly of candidal branched hyphae (#, blue arrow) and characterized by highly dense mycelium, while streptococcal cells (^∗, red arrow) are spread across fungal filaments. However, S-8 already at 8 μg/ml influenced bacterial and fungal morphology. S. mutans are seen aggregated in clusters ($, red arrow), while density of fungal mycelium decreased (Figure 3B). Furthermore, S-8 at dose of 16 μg/ml leads to the alteration of yeast–to-hyphae transition resulting in the appearance of mainly yeast form of C. albicans (&, blue arrow) (Figure 3C). Bacteria appeared mostly attached to each other forming aggregates ($, red arrow) (Figure 3C).

To evaluate the mechanisms underlying S-8’s effect on biofilm formation and composition, we analyzed changes in C. albicans and S. mutans gene expression levels in mixed biofilms exposed to S-8 (Figure 4). We investigated fungal genes which are essential in biofilm development including those associated with adhesion and hyphal formation (als3 and hwp1), cell surface hydrophobicity (csh1), and yeast wall protein (ywp1). The transcriptional levels of genes involved in the adhesion process, such as the hyphal-specific genes hwp1 and als3, were significantly downregulated by S-8 at 16 μg/ml by 3.5- and 5.4-fold, respectively, as compared to untreated control (Figure 4A). CSH plays an important role during biofilm formation in Candida albicans. The csh1 gene, which codes for the CSH-associated protein, is the first candidate gene that has been demonstrated to play a role in affecting the CSH phenotype in C. albicans. S-8 at 8 and 16 μg/ml dose-dependently reduced csh1 expression in mixed biofilm by 1.2- and 12.5-fold, respectively, as compared to control (Figure 4A). The transcription of ywp1 gene, regarded as a specific marker for the yeast form of C. albicans and also as an inhibitor of adhesion activities, was upregulated twofold by S-8 at 16 μg/ml, as compared to control (Figure 4A).

In parallel, we tested biofilm-associated genes of S. mutans in mixed biofilm including EPS glucan formation genes (gtfB, gtfC, gtfD), glucan-binding protein (gbpB), cell surface antigen (spaA), surface-associated protein (brpA), QS-associated genes (comDE, luxS) and stress-responsive proteins (groEL, sodA, nox). All three tested gtfs genes were dramatically upregulated in a dose-dependent manner by S-8 in the co-spcies biofilm. Briefly, the presence of 16 μg/ml of S-8 in mixed biofilm, increased mRNA transcription of gtfB by 26-fold, gtfC by sevenfold, and gtfD by 13-fold, as compared to untreated control (Figure 4B). Moreover, gbpB gene, encoding to surface-associated glucan-binding protein, and thereby promoting plaque formation, was also upregulated by 3.5-fold under exposure of S-8 at 16 μg/ml as compared to control (Figure 4B). In addition, brpA transcript translated to another surface-associated protein playing important role in regulation of biofilm formation, was strongly elevated by 18-fold with in presence of 16 μg/ml of S-8, as compared to control (Figure 4B). In contrast, surface antigen spaA expression remained unchanged in the presence of S-8 (Figure 4B). Furthermore, the agent caused a marked induction of both QS-associated pathways gene expression: luxS which synthetizes autoinducer 2 (AI-2) and comDE involved in sensing and response regulation of competence stimulating peptide (CSP). A dose-dependent increase by 3- and 10.5-fold of luxS transcript was detected following biofilm treatment with S-8 at doses of 8 and 16 μg/ml, respectively, while comDE mRNA was increased by 7.7-fold by S-8 at 16 μg/ml, as compared to untreated control (Figure 4B).

Our data show a notable induction by S-8 of oxidative stress related genes of S. mutans. Streptococcal nox and sodA genes were upregulated by 2.5-fold, already by 8 μg/ml of S-8, while increasing the dose of the agent up to 16 μg/ml, caused a further significant induction of these genes by 13- and 25-fold, respectively, as compared to control (Figure 4B). However, another stress-associated S. mutans groEL gene was not affected by S-8 (Figure 4B). In contrast to the induction by S-8 of bacterial genes related to oxidative stress response, fungal genes associated with defense against oxidative stress were downregulated in mixed biofilms exposed to the agent. C. albicans sod1 and cat1 expressions were decreased by more than twofold at S-8 = 8 μg/ml, while increasing the dose of the agent to 16 μg/ml, caused a drastic downregulation of these genes by 13.5- and 28.5-fold, respectively, as compared to controls (Figure 4A). Finally, candidal sod2 mRNA was reduced by10-fold at 16 μg/ml of S-8, as compared to control (Figure 4A).

Washed suspensions of C. albicans/S. mutans biofilms were tested for their ability to scavenge ROS generated by the peroxide cocktail. S-8 untreated biofilms strongly scavenged ROS in a time-dependent mode, while biofilms treated by tested concentrations of S-8 had only minimal scavenging properties (Figure 5A). Moreover, initial amount of ROS (time 0) was almost twofold lower in untreated biofilms as compared to biofilms formed in the presence of S-8 (Figure 5A). In other assays, biofilms treated with S-8 were mixed with 100 μM of H₂O₂ and incubated at room temperature for 5 min and the amounts of unscavenged peroxide were measured by the Thurman reagent (Thurman et al., 1972). Increasing concentration of S-8 prevented the uptake of peroxide by the biofilm (Figure 5B). In addition, using Thurman reagent, we observed a marked induction of hydrogen peroxide release by co-species biofilms treated with 16 μg/ml of S-8, as compared to control (Figure 5C).

In other experiments, production of endogenous ROS by co-species biofilm was monitored using a fluorometric assay. A dose-dependent increase in ROS accumulation was recorded in biofilms exposed to 8 and 16 μg/ml of S-8 as compared to untreated control (black columns, Figure 6). Moreover, co incubation of dual species biofilms with S-8 and a SOD inhibitor, DDC, resulted in increased endogenous ROS levels in mixed biofilms as compared to results with S-8 treatment alone (white columns, Figure 6). The most pronounced generation of ROS was detected when mixed biofilms were treated with DDC and the highest tested dose of S-8 = 16 μg/ml. Addition of the antioxidant, AA, significantly reduced ROS accumulation in all untreated and S-8 treated samples (gray columns, Figure 6). The MTT assay showed that the viability of biofilm and planktonic cells exposed either to DDC or AA alone or in combination with S-8 was not significantly altered (data not shown).