The effect of epigallocatechin gallate on Streptococcus Gordonii biofilm formation

Background: Dental caries, as a primary disease in dentistry, is strongly influenced by the presence of biofilms. One of the Gram-positive bacteria that acts as an initiator in the biofilm formation process is Streptococcus gordonii. As the primary catechin in green tea, epigallocatechin gallate (EGCG) is easily found in our daily lives, and it has a broad spectrum of antimicrobial effects. Several studies have revealed that EGCG inhibited the growth of Gram-positive bacteria, including inhibiting biofilm formation by damaging the bacterial cell wall and reducing glucosyltransferase activity. However, there is still limited information that explains the effect of EGCG on S. gordonii bacterial biofilms. Purpose: This study aims to analyze the effect of EGCG in inhibiting the formation of S. gordonii bacterial biofilms. Methods: This study was an in-vitro experimental laboratory study, with samples divided into five groups, namely, the group containing BHIB-bacteria, the BHIB-bacteria-5% sucrose groups, and the treatment groups containing BHIB-bacteria-5% sucrose-EGCG with concentrations of 12.5%, 6.25%, and 3.125%, respectively, incubated for 24 hours. The data was analyzed using the Kruskal–Wallis test. Results: There was a significant difference in the formation of biofilms in S. gordonii bacteria with the addition of 5% sucrose in BHIB compared with the group of S. gordonii bacteria in BHIB. The highest biofilm formation in the group containing bacteria-BHIB-5% sucrose, while the lowest biofilm formation occurred in the treatment group containing bacteria-BHIB-5% sucrose-12.5% EGCG with significant difference between the group. Conclusion: The addition of EGCG 12.5% inhibits the formation of S. gordonii biofilms.


INTRODUCTION
The oral cavity is an important part of the body as it serves as the main entrance for nutrients to enter the systemic body.Therefore, oral health requires increased attention. 1owever, many children face issues with their oral cavity, such as dental caries. 2 According to the World Health Organization, 3 60-90% of school-age children suffer from dental caries, especially in developing countries.
Dental disease is undoubtedly a public health concern and one of the most prevalent diseases worldwide, particularly dental caries, which is a biofilm-associated disease. 4Biofilm is an aggregate of bacteria with various species.Bacterial cells adhere to a surface and are enveloped in a matrix produced by the bacteria, known as extracellular polymeric substances (EPS).Streptococcus gordonii is a Gram-positive bacterium with a coccus-shaped alpha-hemolytic chain that plays a crucial role as a pioneer bacterium in the biofilm formation process. 5,6ecently, herbal medicine in dentistry has become the preference for many, with green tea being one of the choices. 7The compositions of green tea includes polyphenols, amino acids, proteins, fats, and various minerals and carbohydrates, with the main content being polyphenols. 8Almost all of the polyphenols in green tea are catechins. 9,10The primary catechin in green tea is epigallocatechin gallate (EGCG), which offers positive health effects, such as antitumor, antioxidant and antimicrobial effects. 11,12Furthermore, EGCG exhibits broad-spectrum antimicrobial activity.In several studies, EGCG has been shown to be more effective in inhibiting the growth of Gram-positive bacteria than that of Gramnegative bacteria. 12 previous study revealed that EGCG influences the expression of regulatory genes in the formation of biofilms and affects the morphology of microorganism cells. 13Another study demonstrated that EGCG can bind to Streptococcus mutans glucansucrase and inhibit enzyme activity. 14Therefore, this study aims to confirm the effect of EGCG against the formation of S. gordonii bacterial biofilms.

MATERIALS AND METHODS
This research constitutes a laboratory experiment with a post-test only control group design.The sample in this study was S. gordonii (ATCC 51656, VPI E1A-1A [PK488], Manassas, United States) available at the Research Center for the Faculty of Dental Medicine, Airlangga University.The laboratory instruments and materials included a micropipette, incubator, polystyrene test tube, inoculation loop, fridge (−80 o ) (Thermo Fisher Scientific, Waltham, MA, USA), anaerobic jar (Thermo Fisher Scientific, Oxoid, Waltham, MA, USA), clean bench (Sanyo, Osaka, Japan), vortex (Thermo Fisher Scientific, Waltham, MA, USA), centrifugation instrument (Thermo Fisher Scientific, Waltham, MA, USA) and an enzyme-linked immunosorbent assay (ELISA) reader (Epoch, Biotek, Germany).
One inoculation loop of pure S. gordonii bacteria was inoculated into BHIB medium and then incubated in an incubator for 24 hours at 37°C.The EGCG used in this study was in the form of a solution.Epigallocatechin gallate preparation in experiments or studies typically involves solubilizing EGCG powder in a suitable solvent.Here, powder-form EGCG was added to sterile water to obtain concentrations of 3.125%, 6.25%, and 12.5%.
S. gordonii bacteria, which had been cultured in BHIB, BHIB-5% sucrose and BHIB-5% sucrose-EGCG (3.125%, 6.25%, 12.5%) for 24 hours at 37°C were taken in 100-µL samples and then placed into a microtiter dish.The bacteria were then incubated at 37°C for 48 hours, followed by the removal of bacterial cells that did not adhere to the microtiter plate.Next, the planktonic cells were rinsed using 200 µL of PBS.Following this, all wells were stained with 200 µl of 0.1% crystal violet for 15 minutes at room temperature; subsequently, the dye was removed by rinsing twice with sterile water, PBS and 100 µl of 95% alcohol.The microtiter plate was shaken for 10 minutes using a shaker.The formed biofilm was measured using the ELISA reader at 540 nm (GloMax® Discover Microplate Reader, Promega, Madison, Wisconsin, USA). 15The Kruskal-Wallis test was used to determine the significance of intergroup differences.Statistical significance was defined as a p-value of <0.05 (Statistical Analysis for Social Science (SPSS), IBM corporation, Illinois, Chicago, USA).

RESULTS
The results of this study indicated a significant difference in the formation of biofilms in S. gordonii bacteria with the addition of 5% sucrose in BHIB, measuring (25.95 mg/mL±0.694)compared with the group of S. gordonii bacteria in BHIB (11.92 mg/mL±3.288)(Figure 1).There was a notable difference between these groups (p<0.01), as described in Table 1.As Figure 2      illustrate, there was a decrease in biofilm formation with an increase in EGCG concentration.The graph highlights the highest biofilm formation in the group containing bacteria-BHIB-5% sucrose (25.95 mg/mL±0.694),while the lowest biofilm formation occurred in the treatment group containing bacteria-BHIB-5% sucrose-12.5% EGCG (16.48 mg/mL±1.262),as described in Figure 4 and Table 2.A significant difference was observed between the group containing bacteria-BHIB-5% sucrose and the treatment group containing bacteria-BHIB-5% sucrose-12.5% EGCG (p<0.01;Table 1).

DISCUSSION
S. gordonii plays an essential role in creating dental biofilm, which can lead to the development of dental caries.This bacterium, as an early coloniser, can aggregate with other microorganisms such as C. albicans, P.gingivalis, and other Streptococcus species to form biofilms on enamel surfaces. 16S. gordonii can metabolise glucose by binding with the α-amylase enzyme. 17Simultaneously, glucose metabolism leads to the production of organic acids that can demineralize the enamel structure. 18n addition, S. gordonii has a faster multiplication rate compared with other Streptococcus species.This bacterium also aids S. mutans, the primary pathogenic bacterium in dental caries, 17 by facilitating glucose metabolism, exhibiting greater resistance to acidic environments (low pH), 19 and activating the glucosyltransferase (GTF) enzyme.The ability of S. gordonii to facilitate and aggregate with other microorganisms in forming dental biofilm on the enamel surfaces plays a crucial role in the dental caries process. 20he results indicate that the formation of biofilms in the BHIB-bacteria-5% sucrose group was significantly higher than that in the BHIB-bacteria group (p<0.01).Thus, 5% sucrose induces the formation of S. gordonii biofilms.Sucrose is widely recognised as one of the most cariogenic carbohydrates among dietary sugars.It can be fermented and serves as a substrate for the synthesis of polysaccharides, particularly EPSs in dental plaque, which play a crucial role in the development of caries. 21Previous research has demonstrated that increasing sucrose to 5% can lead to the formation of the most cariogenic biofilms. 22iofilm formation on tooth surfaces is associated with a high sucrose diet. 23Sucrose is highly soluble in water and rapidly diffuses into biofilms. 24In the presence of sucrose, bacteria synthesise soluble and insoluble glucans through the enzyme, Gtf.Synthesised glucan plays a role in mediating attachment to the tooth surface and biofilm growth by attracting planktonic cells.The accumulation of planktonic cells and EPS leads to the formation of colonies that develop into mature biofilms. 18his study aimed to determine the effect of EGCG on S. gordonii biofilm formation.Based on the results, the biofilm formation of S. gordonii decreases with an increase in EGCG concentration.A 12.5% concentration of EGCG significantly inhibited the biofilm formation of S. gordonii (p<0.05).Previous studies have revealed that EGCG possesses antibacterial and anti-biofilm effects against Gram-positive bacteria, such as S. mutans. 13,25As a constituent of the catechins in green tea, EGCG plays a vital role in the antimicrobial effect of this type of tea.Green tea exerts antibacterial activity by acting on the protective components of saliva (secretory immunoglobulin, histatin, mucin, lysozyme, lactoferrin and oral peroxidase). Previous research demonstrated that EGCG can hinder biofilm formation through three distinct mechanisms. 27n fact, EGCG disrupts biofilm formation via several avenues, including the inhibition of bacterial growth by impeding bacterial metabolism, the promotion of bacterial aggregation, and the inhibition of Gtf activity. 27In brief, EGCG enters the cell membrane, damaging the lipid bilayer and consequently weakening the bacterial wall, a pivotal factor in bacterial defense and growth.Furthermore, EGCG can impede the activity of the phosphoenolpyruvatephosphotransferase system (PEP-PTS), an enzyme crucial for transporting glucose into bacteria, with the system encompassing enzymes located in both the cell membrane and cytoplasm.The inhibition of PEP-PTS activity can curtail glucose absorption into bacterial cells, thereby affecting bacterial metabolism and interfering with acid production. 27Furthermore, EGCG hinders acidogenic and aciduric properties by suppressing the enzymatic activity of ATPase F1F0, alongside downregulating proteins in the ATP synthesized field. 28Since ATP plays an important role as an energy source for bacteria if ATP synthesis decreases, bacterial growth will be inhibited, in severe cases, leading to bacterial cell lysis, resulting in bacterial demise. 29n line with previous research, it was demonstrated that EGCG disrupts biofilm formation through at least three different mechanisms, including the inhibition of bacterial growth through the inhibition of bacterial metabolism, the promotion of bacterial aggregation, and the inhibition of Gtf activity. 27A study conducted by Zayed et al. 21affirmed that EGCG reduces the expression of three genes that code for the Gtf enzyme.The Gtf enzyme is instrumental in converting sucrose into glucan, a foundational component of EPS crucial to biofilm formation, thereby inhibiting biofilm formation.This study limitation is only conducted in in vitro study with effect of EGCG in the S. gordonii bacterias with simple method.
The conclusion of the study is the addition of EGCG 12.5% inhibits the formation of S. gordonii biofilms.
The results of this study offer preliminary insights into how EGCG can impede biofilms in S. gordonii bacteria, indicating its potential as a material for preventing dental caries.Further research is needed to determine the effect of EGCG on the formation of Streptococcus gordonii bacterial biofilms in the oral cavity using other methods such as scanning electron microscopy or confocal scanning laser microscopy.Further in vivo and clinical research are expected.In addition, more detailed research is expected on the effects of EGCG on Gtf enzyme activity and also on the bacterial metabolic process in Streptococcus gordonii.

and Figure 3 40*Figure 1 .
Figure 1.Biofilm formation of Streptococcus gordonii after incubating in brain heart infusion broth with/without sucrose 5%.

Figure 3 .
Figure 3. Colony forming of Streptococcus gordonii to perform the Minimum Bactericidal Concentration (MBC).

Figure 2 .
Figure 2. Dilution method to perform the Minimum Inhibitory Concentration (MIC).

Table 1 .
The significance differences between the groups

Table 2 .
Mean and standard deviation of biofilm formation EGCG can inhibit the growth of both Gram-positive and Gram-negative bacteria.Several studies indicate that EGCG is more effective in inhibiting the growth of Gram-positive bacteria than Gram-negative bacteria.Other research asserts that EGCG can inhibit the growth of Mycobacterium tuberculosis, Stenotrophomonas maltophilia, Staphylococcus aureus, Helicobacter pylori, Staphylococcus aureus and Streptococci spp.