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Subinhibitory Concentrations of Triclosan Promote Streptococcus mutans Biofilm Formation and Adherence to Oral Epithelial Cells

Triclosan is a general membrane-active agent with a broad-spectrum antimicrobial activity that is commonly used in oral care products. In this study, we investigated the effect of sub-minimum inhibitory concentrations (MICs) of triclosan on the capacity of the cariogenic bacterium Streptococcus mutans to form biofilm and adhere to oral epithelial cells. As quantified by crystal violet staining, biofilm formation by two reference strains of S. mutans was dose-dependently promoted, in the range of 2.2- to 6.2-fold, by 1/2 and 1/4 MIC of triclosan. Observations by scanning electron microscopy revealed the presence of a dense biofilm attached to the polystyrene surface. Growth of S. mutans in the presence of triclosan at sub-MICs also increased its capacity to adhere to a monolayer of gingival epithelial cells. The expression of several genes involved in adherence and biofilm formation in S. mutans was investigated by quantitative RT-PCR. It was found that sub-MICs of triclosan significantly increased the expression of comD, gtfC, and luxS, and to a lesser extent of gtfB and atlA genes. These findings stress the importance of maintaining effective bactericidal concentrations of therapeutic triclosan since sub-MICs may promote colonization of the oral cavity by S. mutans.


Introduction
Triclosan (2,4,49-trichloro-29-hydroxydiphenyl ether) is a nonionic molecule with broad-spectrum antimicrobial activities against both bacteria (Gram positive and negative) and fungi [1]. More specifically, it is a general membrane-active agent that causes structural perturbations resulting in a loss of permeabilitybarrier functions [2]. Triclosan has also been reported to possess an anti-inflammatory activity since it reduces cytokine secretion by host cells such as gingival fibroblasts [3,4]. Given this interesting dual action associated to triclosan and the fact that its high substantivity can be associated to a long-lasting effect, it has been used in oral care products (mouthwash, toothpaste) as an active agent for the reduction of dental plaque accumulation and the control of inflammatory gingivitis. The systematic review performed by Davies et al. [5] supports the benefits of triclosancontaining toothpaste for reduction of dental plaque and gingivitis. Being a general anti-plaque agent, triclosan may also be effective for the management of dental caries, a chronic infectious disease associated with a progressive destruction of the hard tooth structures (enamel, dentine, cementum) by the action of acidogenic/aciduric bacteria, mainly Streptococcus mutans, embedded in the dental biofilm [6,7]. To support that, it has been reported that triclosan-containing toothpastes enhance the anti-caries potential of fluoride in dentifrices [8]. Phan and Marquis [9] brought evidence that the ability of triclosan to inhibit glycolysis of S. mutans in biofilms may be, at least in part, responsible for its anti-caries effect.
The effective therapeutic results of antimicrobials are optimal when the concentration is above the minimum inhibitory concentration (MIC). Several studies have reported that antimicrobials at sub-MICs may modulate biological characteristics of bacteria, including their ability to colonize the host [10][11][12][13][14][15]. In this study, we investigated the effect of triclosan sub-MICs on S. mutans biofilm formation and adherence to oral epithelial cells. In addition, the effect of triclosan at sub-MICs on expression of several genes involved in adherence and biofilm formation was investigated.

Determination of MIC of Triclosan
Overnight cultures of S. mutans were diluted in THB-HK to obtain an optical density at 660 nm (OD 660 ) of 0.2 (corresponding to 1610 8 colony-forming units [CFU]/ml). Samples (100 ml) were added to the wells of a 96-well tissue culture plate containing serial dilutions (125 to 0.50 mg/ml) of triclosan (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) in culture medium (100 ml). Control bacteria were cultivated in the absence of triclosan. After incubation for 24 h at 37uC, bacterial growth was monitored by recording the OD 660 using a microplate reader. The MIC was defined as the lowest concentration of triclosan that completely inhibits S. mutans growth (OD 660 #0.05).

Biofilm Assay
S. mutans was grown (24 h) in a flat-bottomed 96-well microplate as above in the absence (control) and presence of triclosan at 1/2, 1/4, or 1/8 MIC. Similar assays were also performed using THB-HK supplemented with 0.25% sucrose. The medium, free-floating bacteria, and loosely-bound biofilm were then removed by aspiration, and the wells were washed three times with 50 mM phosphate-buffered saline (pH 7.2; PBS). The biofilms were stained with 0.04% crystal violet (100 ml) for 10 min. The wells were washed three times with PBS to remove unbound crystal violet dye and dried for 2 h at 37uC. After adding 100 ml of 95% (v/v) ethanol to each well, the plate was shaken for 10 min to release the dye from the biofilms and the absorbance at 550 nm (A 550 ) was recorded.

Scanning Electron Microscopy
The S. mutans (ATCC 25175) biofilm was examined by scanning electron microscopy. One ml of S. mutans resuspended to an OD 660 of 0.1 in culture THB-HK 6 triclosan at 1/2 or 1/4 MIC was added into wells of a 6-well plate containing a 13 mm-diameter plastic coverslip. After 24 h incubation, medium and free-floating bacteria were removed. The biofilms were incubated overnight in fixation buffer (4% (w/v) paraformaldehyde, 2.5% (w/v) glutaraldehyde, 2 mM CaCl 2 in 0.2 M cacodylate buffer, pH 7.2), washed with 0.1 M cacodylate buffer pH 7.0 (3620 min) and postfixed for 90 min at room temperature in 1% (w/v) osmic acid containing 2 mM potassium ferrocyanide and 6% (w/v) sucrose in cacodylate buffer. Samples were dehydrated through a graded series of ethanol (50, 70, 95 and 100%), critical point dried, gold sputtered and examined using a JEOL JSM6360LV scanning electron microscope operating at 30 kV.
Assay for Adherence to Oral Epithelial Cells S. mutans ATCC 25175 cells cultivated in the absence (control) or presence of triclosan at 1/2, 1/4 or 1/8 MIC were labeled with fluorescein isothyocyanate (FITC) as previously reported [16]. The immortalized human gingival epithelial cell line OBA-9 used in this study, kindly provided by Dr. Marcia Mayer (Departamento de Microbiologia, Institute of Biomedical Sciences, Universidade de São Paulo, São Paulo, Brazil), was initially described by Kusumoto et al. [17]. The epithelial cells were cultured (96-well microplate) in Keratinocyte-Serum Free Medium (K-SFM, Life Technologies Inc., Burlington, ON, Canada) containing insulin, epidermal growth factor, and fibroblast growth factor, and supplemented with 100 mg/ml of penicillin G/streptomycin at 37uC in a 5% CO 2 atmosphere until they reached confluence. The adherence assay of S. mutans to epithelial cells was carried out as described in a previous study [16]. After removing unbound bacteria and washing wells, the relative fluorescence units (RUF; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the degree of bacterial adherence were determined using a microplate reader.

Determination of Cell Surface Hydrophobicity
The relative cell surface hydrophobicity of S. mutans ATCC 29175 grown in THB-HK 6 triclosan at 1/2, 1/4, or 1/8 MIC was determined by measuring their absorption to n-hexadecane according to the procedure described by Rosenberg et al. [18].

RNA Isolation and Quantitative RT-PCR
To investigate the effect of sub-MICs of triclosan on expression of several genes involved in adherence and biofilm formation, S. mutans ATCC 25175 was grown to mid-log phase (OD 660 = 0.45) and then triclosan was added at 1/2, 1/4 or 1/8 MIC prior to further incubate at 37uC for 2 h. Control cells were incubated in the absence of triclosan. Bacteria were collected by centrifugation (7,0006g for 5 min) and treated with an RNAprotect bacterial reagent (Qiagen Canada Inc., Montreal, QC, Canada). Bacterial cells were then lysed and RNA was isolated and purified using the RNeasy minikit (Qiagen Canada Inc.). The amounts of mRNA were quantified with the Experion TM system (Bio-Rad Laboratories, Mississauga, ON, Canada). The reverse transcriptionpolymerase chain reaction (RT-PCR) analysis was performed as follows. RNA from each sample (100 ng/ml) was reversetranscribed using Maloney murine leukemia virus reverse transcriptase and random hexamers in a Bio-Rad MyCycler TM thermal cycler (Bio-Rad Laboratories). Reverse transcription conditions were 5 min at 70uC, 10 min at 25uC, 50 min at 37uC, and 15 min at 70uC. Real-time PCR was used for quantification of atlA, comD, gtfB, gtfC, and luxS mRNA expression. 16S rRNA gene was used as an internal control for data normalization. The primers used for the quantitative RT-PCR were purchased from Life Technologies Inc. (Burlington, ON, Canada) and are listed in Table 1. The sequences of primers were obtained from a previous study [19] while the primers for atlA were designed in this study. Triplicate reactions were prepared with 25 ml of PCR mixture containing 12.5 ml of IQ SYBR Green Supermix, 5 ml of cDNA, 1 ml of gene-specific primer, and 6.5 ml of RNase-and DNase-free water. The samples were amplified using a Bio-Rad MyCycler TM thermal cycler (Bio-Rad Laboratories). The amplification conditions for atlA, comD, gtfC, luxS and 16S rRNA were 95uC for 3 min followed by 30 cycles at 95uC for 45 s, 60uC for 45 s and 72uC for 30 s, while that of gtfB was 95uC for 3 min followed by 40 cycles at 95uC for 45 s, 55uC for 45 s and 72uC for 30 s. To validate the specificity of each primer pair, temperature curve analyses were performed.

Statistical Analysis
Unless specified otherwise, assays were run in triplicate and the means 6 standard deviations were calculated. Data were analyzed using the Student t-test.

Results
Using a microdilution broth method, the MIC of triclosan for S. mutans ATCC 25175 and ATCC 35668 was 7.8 mg/ml. Thereafter, biofilm formation by S. mutans was investigated following growth in culture medium 6 triclosan at 1/2, 1/4, or 1/8 MIC. As reported in Table 2, biofilm formation by both strains of S. mutans was dose-dependently induced by sub-MICs of triclosan, as determined by crystal violet staining. At 1/2 and 1/4 MIC of triclosan, the biofilm of S. mutans ATCC 25175 was increased by 6.2-and 5-fold, while that of strain ATCC 35668 was increased by 3-and 2.2-fold, respectively. The biofilm was not significantly affected following growth in the presence of triclosan at 1/8 MIC. The effect of adding 0.25% sucrose to THB-HK on biofilm formation induced by sub-MICs of triclosan was also tested. As shown in Table 2, in the absence of triclosan, an important biofilm was formed by both strains of S. mutans. The triclosan sub-MICsinducing effect on biofilm formation was much less significant in the presence of sucrose. Planktonic cells, estimated by recording the OD 660 of the bacterial suspensions surrounding the biofilm, were significantly decreased following growth in the presence of 1/ 2 MIC of triclosan in the presence or not of sucrose ( Table 2). Given that the triclosan-induced biofilm formation was optimal for S. mutans ATCC 25175 grown in the absence of sucrose, this strain and condition were selected for further analyses.
Scanning electron microscopy analysis was performed to observe the triclosan sub-MICs-induced biofilm formation by S. mutans ATCC 25175. As shown in Figure 1A, individual short chains of S. mutans were observed attached to the polystyrene surface when growth was carried out in THB. However, when the culture medium was supplemented with 1/2 and 1/4 MIC of triclosan ( Figures 1B and 1C), a thick biofilm made of aggregates and microcolonies of S. mutans almost completely covered the surface of the polystyrene support. Sucrose, a well-known biofilmpromoting agent used as positive control, also induced the formation of biofilm ( Figure 1D).
Thereafter, we further investigated the impact of triclosan at sub-MICs on the host colonization properties of S. mutans ATCC 25175 by evaluating the effect on adherence to gingival epithelial cells. As reported in Figure 2, triclosan at 1/2 and 1/4 MIC promoted the adherence of FITC-labeled S. mutans to a monolayer of gingival epithelial cells. More specifically, at 1/2 MIC of triclosan, the adherence of S. mutans to epithelial cells was increased by 42.5%.
We then attempted to identify the mechanism by which triclosan at sub-MICs may increase the capacity of S. mutans to form biofilm and adhere to epithelial cells. Since the hydrophobic properties of the bacterial cell surface may be involved in adherence and biofilm formation, we tested the effect of growing S. mutans in the presence of triclosan at sub-MICs on cell surface hydrophobicity. No significant modifications in cell surface hydrophobicity were observed (data not shown).
The expression profile of five genes related to adherence and biofilm formation in S. mutans was determined following incubation (2 h) of S. mutans in the absence and presence of triclosan at 1/2 and 1/4 MIC. As reported in Figure 3, the genes gtfC (glucosyltransferase C), comD (histidine kinase sensor protein), and luxS (autoinducer 2 synthase) were those for which the expression was the most upregulated. More specifically, triclosan at 1/2 MIC, increased gtfC, comD, and luxS expression by 3.6-, 3.1-, and 4-fold, respectively. Although the upregulation of atlA (autolysin) and gtfB (glucosyltransferase B) expression was less pronounced, it was significantly increased following incubation of S. mutans with triclosan at sub-MICs.

Discussion
Triclosan is a broad spectrum antimicrobial agent used in oral care products to control dental plaque [20]. Although numerous studies investigated the antibacterial properties of triclosan towards oral bacteria [20,21], there are no data in the literature on the effects of this compound at sub-MICs. Since there are a number of in vivo circumstances where concentrations of triclosan may be at subinhibitory levels, we investigated the effects of sub-MICs of this antimicrobial agent on the cariogenic bacterium S. mutans in regard to its capacity to colonize the host.
Previous studies have shown that antimicrobial agents at subMICs can either increase or decrease biofilm formation by bacterial pathogens [10,11,13,14]. Our study brought clear evidence that triclosan at sub-MICs significantly increases the biofilm formation capability of S. mutans. To the best of our knowledge, this is the first report on the effect of sub-MICs of triclosan on bacterial adherence properties. Prior to our study, only one research group reported on the effect of an antimicrobial agent on biofilm formation by S. mutans. More specifically, Dong et al. [19] recently showed that sub-MICs of chlorhexidine appear to solidify and strengthen S. mutans biofilm. The ability of nicotine to enhance S. mutans biofilm formation has also been reported [22].
Although the primary natural location of S. mutans is the dental biofilm, we showed that growing S. mutans in the presence of triclosan at sub-MICs increased its capacity of adherence to epithelial cells. If the epithelial barriers are breached, adhered S. mutans may invade tissue, enter the bloodstream, and ultimately induce infective endocarditis. Since S. mutans is an important causative agent of subacute infective endocarditis in particular in subjects with predisposing cardiac conditions [23], further studies should investigate the effects of sub-MICs of triclosan on adherence to endothelial cells.
We then attempted to identify the mechanism by which triclosan at sub-MICs may increase S. mutans biofilm formation and adherence to epithelial cells. The cell surface hydrophobicity of bacteria is known to contribute to their adherence properties [24]. Wu et al. [25] reported that sub-MICs of specific antibiotics can increase the surface hydrophobicity of another important cariogenic bacterium, Streptococcus sobrinus, a phenomenon that may increase their adherence property. In the present study, triclosan at sub-MICs had no effect on the surface hydrophobicity of S. mutans, a result that ruled out the involvement of this mechanism in the increased adherence properties of S. mutans.
S. mutans can use sucrose to synthesize extracellular polysaccharides via glucosyltransferases, more specifically GtfB and GtfC [26]. In this study, although the expression of gtfB and gtfC was increased in S. mutans exposed to triclosan at sub-MICs, it is likely not responsible for the increased biofilm formation observed since sucrose was not used in the culture medium.
Biofilm formation is largely influenced by bacterial communication via quorum-sensing signaling system [27]. More specifically, in S. mutans, the comD gene product, an histidine kinase sensor protein for the competence-stimulating peptide (CSP), is known to play a critical role in biofilm formation [27]. Moreover, LuxS is produced by many Gram positive bacteria, including S. mutans, and is involved in the production of autoinducer 2, another signaling molecule playing a role in biofilm formation [28]. Our study showed that both comC and luxS genes were significantly upregulated when S. mutans was cultivated in the presence of sub-MICs of triclosan. This is likely contributing to the increased biofilm formed under this condition. Dong et al. [19] also reported on the capacity of sub-MICs of antimicrobial agents, more specifically sodium fluoride and tea polyphenols, to increase the mRNA expression of comD and luxS.
The autolysin AtlA (also known as Smu0630) of S. mutans has been reported to play a critical role in biofilm formation regardless of the carbohydrate source. [29]. Interestingly, AtlA has been identified as a fibronectin-binding protein that contributes to bacterial survival in the bloodstream and consequently as a virulence factor for infective endocarditis [30]. This cell surface adhesion whose expression was found to be upregulated in S. mutans exposed to sub-MICs of triclosan may contribute to the increased adherence to epithelial cells.

Conclusions
Our study showed that sub-MICs of triclosan can enhance biofilm formation and epithelial cell adherence of S. mutans. We also brought evidence that this may be modulated by an increased expression of specific genes coding for cell surface adhesins or involved in quorum-sensing. Collectively, our data stress the importance of maintaining MIC of therapeutic triclosan to efficiently prevent colonization of the oral cavity by S. mutans.