Tea Catechin Epigallocatechin gallate inhibits Streptococcus mutans Biofilm
Formation by Suppressing gtf Genes
Xin Xu1,2, Xue D. Zhou2, Christine D. Wu1*
1Department of Pediatric Dentistry, College of Dentistry, University of Illinois at
Chicago, Chicago, IL, USA; 2State Key Laboratory of Oral Diseases, Sichuan
University, Chengdu, China
Running title: EGCG Inhibits gtf genes of S. mutans
Key words: Streptococcus mutans, epigallocatechin gallate (EGCG), biofilm
formation, gtf, anticariogenic agents, tea polyphenols
*Corresponding author. Mailing address: Department of Pediatric Dentistry, University
of Illinois at Chicago, College of Dentistry, MC850, 801 S. Paulina Street, Room 469J,
Chicago, IL 60612-7212. Phone (312) 355-1990. Fax (312) 996-1981. E-mail:
Tea Catechin Epigallocatechin gallate inhibits Streptococcus mutans Biofilm
Formation by Suppressing gtf Genes
Objective: The anti-cariogenic properties of tea have been suggested for decades.
Tea polyphenols, especially Epigallocatechin gallate (EGCG), have been shown to
inhibit dental plaque accumulation, but the exact mechanisms are not clear at present.
We hypothesize that EGCG suppresses gtf genes in S. mutans at the transcriptional
level disrupting the initial attachment of S. mutans and thus the formation of mature
biofilms. Design: In this study, the effect of EGCG on the sucrose-dependent initial
attachment of S. mutans UA159 in a chemically defined medium was monitored over
4 h using a chamber slide model. The effects of EGCG on the aggregation and gtf B,
C, D gene expression of S. mutans UA159 were also examined. Results: It was
found that EGCG (7.8-31.25 μg/ml) exhibited dose-dependent inhibition of the initial
attachment of S. mutans UA159. EGCG did not induce cellular aggregation of S.
mutans UA159 at concentrations less than 78.125 μg/ml. Analysis of data obtained
from real-time PCR showed that EGCG at sub-MIC level (15.6 μg/ml) significantly
suppressed the gtf B, C, D genes of S. mutans UA159 compared with the non-treated
control (p < 0.05). Conclusions: These findings suggest that EGCG may represent a
novel, natural anti-plaque agent that inhibits the specific genes associated with
bacterial biofilm formation without necessarily affecting the growth of oral bacteria.
Tea Catechin Epigallocatechin gallate inhibits Streptococcus mutans Biofilm
Formation by Suppressing gtf Genes
Dental caries is one of the most prevalent and costly oral infectious diseases
throughout the world (1). The etiology of dental caries is associated with bacterial
metabolism of carbohydrates, leading to plaque acidification and demineralization of
the dental hard tissues. Classic bacterial virulence factors contributing to the initiation
and progression of dental caries consist of three components, i.e. stable biofilm
formation, efficient acid production, and sophisticated environmental stress
adaptation (2-5). Streptococcus mutans, one of the primary etiologic agents of dental
caries (6, 7), produces glucosyltransferases (GTFs) which synthesize intracellular
polysaccharides (IPS) and extracellular polysaccharides (EPS). The EPS, especially
water-insoluble glucans, mediate the initial adherence of S. mutans and other oral
bacteria on tooth surfaces and facilitate the formation of mature dental plaque biofilm
(8-10). A recent study has shown that deletion of gtfB and gtfC genes in S. mutans
resulted in diminished biofilm formation with minimal accumulation of bacteria and
polysaccharides in vitro (10). This suggested that suppression of gtf genes may
represent an alternative approach to disrupting biofilm formation.
Tea (infusion of dried leaves of Camellia sinensis) is the most popular and widely
consumed beverage in the world today (11). Its polyphenolic component has been
reported to possess antioxidant, antimicrobial, antimutagenic, antidiabetic,
hypocholesterolemic, anti-inflammatory, and cancer-preventive properties (12-14). Its
anti-cariogenic activity has also been demonstrated in humans and experimental
animals (15-21). Our previous study in adult humans found that rinsing with black tea
extract resulted in a significantly reduced plaque pH fall and a lower plaque index
compared with rinsing with water alone (11). Frequent short-term rinses with black tea
also inhibited subsequent regrowth and glycolysis of human supragingival plaque
bacteria (14). Our recent study has demonstrated that epigallocatechin gallate
(EGCG), the antimicrobial monomeric component of tea catechins (the major
polyphenolic component in tea) exhibited a wide range of physiological effects on S.
mutans, particularly on virulence factors associated with its acidogenicity and
Many researchers have reported that tea catechins, especially EGCG, reduced S.
mutans cell adherence by suppressing the activity of GTF enzymes (16, 23, 24). The
concentrations of EGCG reported for this inhibition were often in the mg/ml range
which were sufficient to inhibit growth or viability of oral streptococci (15, 22).
However, in the oral cavity, due to saliva dilution, sustaining such high inhibitory
concentrations of polyphenols over a long period of time after tea consumption would
We have recently found that EGCG inhibited in vitro biofilm formation of S.
mutans at an MBIC (minimum biofilm inhibition concentration) of 15.6 μg/ml, a
concentration lower than the minimum growth inhibitory concentration (MIC) against
S. mutans planktonic cells (22). This suggested the involvement of additional
mechanism(s) by which EGCG may exhibit anti-plaque biofilm activity in the oral
cavity without necessarily inhibiting growth of oral bacteria. To our knowledge, the
effect of tea polyphenols on S. mutans gtf gene expression at the transcriptional level
has yet to be examined. Moreover, the mode of action of EGCG on the
sucrose-dependent initial attachment of S. mutans towards biofilm formation has not
been well documented. We hypothesize that EGCG suppresses gtf gene expression
in S. mutans, thus inhibiting biofilm formation. In this study, we investigated: 1) the
effect of sub-bacteriostatic levels of EGCG on the sucrose-dependent initial
attachment of S. mutans to surfaces, and 2) the effect of EGCG on the transcriptional
expression of S. mutans gtf B, C, D genes.
2. Materials and methods
2.1. Chemicals, test bacterium and growth conditions
Epigallocatechin gallate from green tea (EGCG, 95% HPLC) and all chemicals,
unless otherwise stated, were purchased from Sigma-Aldrich Corp (Saint Louis, MO,
USA). S. mutans UA159 was grown in a chemically defined medium (CDM) (25) at
37°C in an anaerobic chamber (37 ºC, 10% H2, 5% CO2, and 85% N2; Forma
Scientific, Inc., Marietta, OH, USA). Artificial saliva used in attachment assay was
prepared according to the formula as described previously (26).
2.2. Sucrose-dependent Initial Attachment Assay.
EGCG at sub-MIC concentration was used for the sucrose-dependent initial
attachment of S. mutans cells. The minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC) of EGCG against S. mutans UA159 were
pre-determined in CDM using a micro-dilution method as described previously (22).
The initial attachment of S. mutans UA159 on the glass surface was determined
using a four-well chamber slide (culture area of 1.8 cm² per well; Nunc Lab-Tek,
Rochester, NY, USA). S. mutans were collected in mid-log phase from the broth
culture, washed two times with PBS, and re-suspended in CDM (1 × 106 CFU/ml)
supplemented with 1% (w/v) sucrose and EGCG (7.8 - 31.25 μg/ml, 1/4 MIC - MIC).
The cells suspension was placed onto artificial saliva pre-coated (37°C for 1 h)
chamber slides and incubated under anaerobic condition. After 1h, 2h and 4 h
incubation, cell free cultures supernatant were removed from respective chamber
slide and the chambers were gently washed three times with deionized water to
remove un-attached cells. The attached cells were stained with the fluorescent
Live/Dead BackLightTM stain (Molecular Probes Inc., Eugene, Oregon, USA) and
examined under a Leica DMRE microscope (Leica, Wetzlar, Germany). Images were
captured at 20 × magnification using a digital camera (Meyer Instruments, Inc.,
Houston, TX, USA) and analyzed by Image-Pro Plus 5.1 (Media Cybernetics Inc.,
Bethesda, MD, USA). Total average coverage area of bacterial cells on the surface
was obtained from at least 4 different images of the same sample. Control contained
S. mutans UA159 cells grown in the absence of EGCG.
2.3. Bacterial Aggregation Assay.
Aggregation of S. mutans UA159 cells in the presence of EGCG was determined
according to the method modified from Matsumoto, et al (27). S. mutans UA159 cells
were collected in mid-log phase by centrifugation, washed three times with PBS,
re-suspended in PBS or CDM with 0.1% sucrose to a concentration, upon ten times
dilution, yielding an OD660nm of 0.3 in 96-well microtiter plate (200 μl). This highly
concentrated cell suspension was then used to test the aggregation-inducing
capability of EGCG. 100 μl of the cell suspension and an equal volume of twofold
serial dilution of EGCG in PBS or CDM were mixed in the 96-well microtiter plate and
incubated at 37°C for 2 h. 100 μl of the reaction mixture was then carefully transferred
to a new 96-well microtiter plate without disturbing the precipitated cells at the bottom,
and OD660nm was recorded. The aggregation percentage of S. mutans cells were
calculated according to the formula: Aggregation (%) = 100 × [1 - (ODExp - ODBlk) /
(ODNTC - OD????)], where ODExp was OD660nm of each experimental well (with serial
concentrations of EGCG), ODNTC was OD660nm of the non-treated control (without
EGCG), and ODBlk and OD???? were OD660nm readings of the blank for experimental
wells and the non-treated control respectively. The minimum concentration that
induced cellular aggregation was defined as the lowest concentration of EGCG
promoting no less than 10% of cellular aggregation compared with the non-treated
2.4 RNA Isolation, Purification, Reverse Transcription and Quantitative
S. mutans UA159 was grown in CDM supplemented with sub-MIC concentration of
EGCG (15.6 μg/ml). Cells were collected at late exponential phase by centrifugation
and RNA was immediately stabilized using an RNAprotect Bacteria Reagent
(QIAGEN, Valencia, CA, USA). Cells were then pelleted and re-suspended in 100 μl
of lysis buffer (20 mM Tris-HCl, 3 mM EDTA, 20mg/mL lysozyme, 60 mAU/ml
proteinase K, 1000 U/ml mutanolysin, [pH 8.0]) and incubated at 37ºC with gentle
agitation for 45 min. The lysate was further sonicated by means of a cuphorn (Thermo
Fisher Scientific Inc., Pittsburgh, PA, USA) on ice for 2 cycles of ultrasonication for 60
s and then purified using an RNeasy Mini Kit (QIAGEN). Reverse transcription was
performed by use of a 1st Strand cDNA Synthesis Kit with random hexamer primers
(Invitrogen, Madison, WI, USA).
Real-time PCR was used to quantify gtf B, C, D mRNA expression with 16S rRNA
as an internal control. All primers for real-time PCR were designed with Primer3 (28)
and obtained commercially from Sigma-Aldrich Corp. (Table 1). Real-time PCR
amplification was performed on the iCycler iQ detection system (Applied Biosystems,
Foster City, CA). The reaction mixture (25 μl) contained 1 X SYBR green PCR Master
Mix (Applied Biosystems), template cDNA, and forward and reverse primers (10 μM
each). Thermal cycling conditions were the same as described previously (22).
Threshold cycle values (CT) were determined, and data were analyzed by StepOneTM
Software v2.0 (Applied Biosystems) according to the 2-?? CT method.
2.5 Statistical Analysis.
All experiments were performed in triplicate and reproduced at least three separate
times. Differences between the experimental group and the untreated control group
were analyzed by SPSS (version 15.0 for Windows). One-way analysis of variance
(ANOVA) was performed, and a post hoc Tukey test was used for the comparison of
multiple means. Significance was set at a p value of < 0.05.
3.1. EGCG inhibits the sucrose-dependent initial attachment of S. mutans
without inducing significant cellular aggregation in vitro.
EGCG inhibited the in vitro growth of S. mutans UA159 in CDM (MIC = 31.25 μg/ml)
and was bactericidal at an MBC of 62.5 μg/ml. Given the importance of biofilm
formation in the cariogenic virulence of S. mutans cells, inhibition of the formation of
biofilm by increasing concentrations of EGCG was investigated in a chamber slide
model. Fluorescent Live/Dead BackLightTM stain revealed sucrose-dependent initial
attachment of S. mutans UA159 cells to glass surfaces toward biofilm formation
(Figure 1A). The area of cell coverage of non-treated control was 500.4 ± 186.23 μm2
at 1 h incubation, increasing to 7401.7 ± 1879.99 μm2 at 2 h, and 34347.2 ± 5418.04
μm2 at 4 h. In the presence of EGCG (7.8-31.25 μg/ml), a dosage dependent
inhibition on the sucrose-dependent initial attachment of S. mutans UA159 cells to
surfaces was observed. EGCG at 31.25 μg/ml inhibited the area of cell coverage of S.
mutans UA159 cells by 79.57% at 1 h, 98.33% at 2 h, and 91.78% at 4h compared
with the non-treated control (Figure 1B).
To determine whether the reduced bacterial attachment observed above was due
to possible cellular aggregation induced by EGCG, we investigated the effect of
EGCG on aggregation of S. mutans UA159 cells in both PBS and CDM. Although a
dosage-dependent aggregation inducing effect of EGCG on S. mutans UA159 cells
was observed ranging from 78.125-1250 μg/ml, EGCG at the test concentrations
employed in attachment assays (7.8-31.25 μg/ml) did not induce significant cellular
aggregation (Figure 2). The minimum EGCG concentration that induced cellular
aggregation in CDM was 78.125 μg/ml, which was more than two-folds higher than
the concentrations used in the attachment assay (7.8-31.25 μg/ml).
3.2. EGCG inhibits gtf B, C, D genes expression of S. mutans.
In order to determine the effect of EGCG on the virulence factors associated with
cells attachment and biofilm formation of S. mutans, real-time PCR was used to
quantify gtf B, C, D mRNA expression with 16S rRNA as an internal control. Melt
curves revealed the absence of non-specific products in all amplification reactions.
EGCG at sub-MIC level (15.6 μg/ml) significantly inhibited the gtf B, C, D genes
expression by 60.88%, 60.49% and 66.37%, respectively compared to the
non-treated control (Figure 3, p < 0.05).
Dental plaque is a complex bacterial biofilm community whose composition is
governed by factors such as bacterial adherence, co-aggregation, and growth and
survival in the environment (29). Biofilm organisms frequently express phenotypes
quite distinct from those of their free planktonic counterparts, e.g., enhanced
resistance to antibiotics or antimicrobial chemicals (30). Stable biofilm formation is
considered one of the key factors of caries pathogenesis (5). Streptococcus mutans,
a prominent member of the dental plaque community, synthesizes extracellular
adherent glucans from dietary sucrose via GTFs, thus promoting the accumulation of
oral bacteria on tooth surfaces (9). The early stage of S. mutans biofilm,
characterized by the sucrose-dependent bacterial attachment to tooth surfaces,
represents an important initial step towards the subsequent formation of the mature
biofilm (31). Therefore, compounds capable of inhibiting this initial attachment of S.
mutans would effectively prevent dental plaque formation and maturation.
In our previous study, we reported that EGCG disrupted in vitro S. mutans biolfilm
formation at a minimum biofilm inhibition concentration (MBIC90) of 15.6 μg/ml (22). In
the present study, we have demonstrated that EGCG at sub-MIC levels was able to
inhibit the sucrose-dependent initial attachment of S. mutans thus leading to inhibition
of subsequent mature biofilm formatiom (Figure 1). It is known that bacterial
aggregation may result in cellular clearance and reduced cell attachment onto
surfaces (32, 33). Since tea polyphenols have been reported to induce cell
aggregation (27), one may argue that the inhibition of initial attachment of S. mutans
to surfaces by EGCG observed in this study could have been attributed to cellular
aggregation. However, this is not the case because the minimum EGCG
concentration (78.125 μg/ml) needed to induce cellular aggregation of S. mutans was
more than twice the concentrations (7.8-31.25 μg/ml) that inhibited their attachment
Tea polyphenols, especially EGCG, have been reported to inhibit activity of S.
mutans GTFs through their interaction with enzyme proteins (23, 24, 34). The
effective EGCG concentrations reported in these previous studies have mostly been
above the milligram per milliliter level (16, 23, 24). The average concentration of tea
catechins in a typical cup of tea (230 ml) is approximately 1 mg/ml (15). Immediately
after tea consumption, EGCG may be concentrated enough in the oral cavity to inhibit
growth and GTFs activity, thus reducing biofilm formation of S. mutans. However, a
gradual decrease in EGCG concentration to the sub-MIC level may occur due to the
dilution by saliva. At this point, EGCG, although at a lower concentration, may still be
capable of suppressing gtf gene expression leading to disruption of S. mutans biofilm
formation as demonstrated in this study.
Based on our current findings, we conclude that EGCG at sub-lethal levels is able to
reduces S. mutans biofilm formation by suppressing gtf expression associated with
cell adherence and biofilm formation. Given the difficulties of maintaining effective
levels of various therapeutic agents to achieve antimicrobial efficacy in the oral cavity,
EGCG represents a promising natural anticariogenic agent that prevents plaque
biofilm formation without necessarily suppressing the growth of oral bacteria.
We thank Dr. Daniel J. Smith at the Forsyth Institute (Boston, MA, USA) for
providing the formulation of the chemically defined medium for growth of oral
streptococci. We thank Dr. Luisa A. DiPietro at the College of Dentistry, the University
of Illinois at Chicago (UIC), Chicago, IL, for the use of the real-time PCR equipment.
This study was supported by the Department of Pediatric Dentistry, UIC College of
Dentistry, Chicago, IL. Dr. Xin Xu is the recipient of a scholarship granted by the State
Scholarship Fund, the China Scholarship Council.
Conflict of interest statement
The authors declare that there is no potential conflict of interest that would prejudice
the impartiality of this scientific work.
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