INTRODUCTION:

Dental caries remains the most prevalent chronic ailment¹, with a relatively high prevalence among children², especially in developing countries³. According to the Indonesian Dentists Association (PDGI), 89% of patients withtooth caries are children⁴. Caries develops due to tooth demineralization⁵^,^6,7 initiated by metabolic processes within the bacterial biofilm on the tooth surface^8,9, which produces lactic acid¹⁰.

A biofilm is a community of microbial cells attached to a surface and encased within a polysaccharide matrix. The initiation of biofilm formation, or dental plaque, commences with the adherence of early colonizing bacteria such as Streptococcus gordonii to host tissue components. S. gordonii is an early colonizer due to its hightened affinity for the hard surfaces in the oral cavity compared to other bacteria species¹¹. Bacterial adhesion to biological surfaces is contingent upon a specific ligand-receptor interaction, with the targeted receptor for bacterial adhesion potentially manifesting as a protein or a glycoconjugate expressed by the host^12,13. Following the initial adhesion event, the production of extracellular polymeric substances (EPS) occurs, allowing other bacteria to colonize the tooth's surface until the microbial colony reaches maturity^8,14,15.

S. gordonii can induce ecological shifts in the oral cavity, resulting in enhanced acid production and acid resistance. This, in turn, fosters the proliferation of acidogenic bacteria, consequently rendering the oral environment more acidic. In such an acidic milieu, acid-resistant (aciduric) bacteria, such as Streptococcus mutans and non-mutants, tend to dominate, resulting in a rise in plaque cariogenicity¹¹. As a result of sugar metabolism, S. gordonii produces acid, thereby causing a reduction in the pH of the biofilm, also known as a decline in glycolytic pH. This decrease in glycolytic pH can contribute to tooth enamel demineralization and the development of dental caries¹⁶.

The growth of S. gordonii must be carefully regulated due to its commensal nature, which, under certain conditions, can manifest pathogenicity¹⁷. Researchers are actively pursuing the discovery of natural medicines sourced from herbal plants. This pursuit stems from the fact that 80% of the world's population continues to rely on traditional medicine to elleveite health issues, primarily due to its minimal side effects¹⁸. Traditional natural compounds have been consumed since ancient times as they exhibit less toxicity, affordability, minimal side effects, abundant therapeutic properties^19–21 Green tea is an herbal plant that widely consumed Indonesia²². It boasts high polyphenolic contents²³^,²⁴, primarily derived from catechins²⁵^,²⁶. One of the most common catechins in green tea is epigallocatechin gallate (EGCG), which constitutes approximately >50% of the total green tea compositioon²⁷. Other notable tea catechins are epigallocatechin 3-gallate, epigallocatechin, epicatechin, epicatechin 3-gallate, and catechin. These polyphenols were discovered to have antimicrobial properties and the ability to inhibit a wide variety of both gram-positive and gram-negative bacteria in vitro^28,29.

Previous research has indicated that EGCG exhibits an anti-biofilm effect against the gram-positive S. mutans³⁰. Additionally previous studies have demonstrated EGCG’s capacity to inhibit both acid production and the aggregation of S. gordonii²⁷. However, for the accumulation of additional empirical evidence in this regard, further investigation is warranted to elucidate the impact of EGCG on biofilm adherence and glycolytic pH in S. gordonii.

MATERIALS AND METHODS:

The Faculty of Dental Medicine issued a Certificate of Ethical Eligibility for this study, Universitas Airlangga 346/HRECC.FODM/VI/2022. We cultured experimental S. gordonii in Brain Heart Infusion Broth (BHIB) (Research Center, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia) for 24 at 37^°C using an incubator. We examined adherence and glycolytic pH as described previously, with a few modifications³¹. For the biofilm adherence assay, the bacteria were cultivated in polystyrene tubes for 24 h at 37^°C and then divide divided into five groups: the normal control group (BHIB + S. gordonii); the negative control group (BHIB + S. gordonii + 5% sucrose); and the treatment group (BHIB + S. gordonii + 5% sucrose), which treated with varying concentrations of ECGC (0.03, 0.06, and 0.12mg/mL). Afterward, the bacterial culture was carefully removed from the polystyrene tubes. Adhered cells were detached from the tube surfaces using 0.5 M NaOH, followed by centrifugation, washing, and immersion in saline solution. The bacterial solution was agitated using a vortex and then quantified at 570 nm wavelength using the SmartSpec Plus Spectrophotometer (Bio-Rad, Hercules, CA, USA).

For the glycolytic pH assay, bacteria were cultivated in polystyrene tubes for 24 h at 37^°C and then divided into five groups: the normal control group (BHIB + S. gordonii); the negative control group (BHIB + S gordonii), to which 5% sucrose was subsequently introduced; and three treatment groups (BHIB + S. gordonii) supplemented with EGCG at concentrations of 0.03, 0.06, and 0.12mg/mL, in addition to 5% sucrose. The bacterial cultures in the polystyrene tubes were centrifuged and then carefully removed. Adhered cells were washed using salt solution containing 50mM KCl and 1 mM MgCl₂, followed by agitation using a vortex. After another round centrifugation, the solution was decanted from the polystyrene tubes. The bacterial culture was then resuspended using a salt solution containing 50mM KCl and 1mM MgCl₂. In the case of the control polystyrene tubes, aquades was added; in the case of polystyrene tubes in the treatment group, EGCG was added at concentrations of 0.03, 0.06, and 0.12 mg/mL in each tube. pH in both the control and treatment groups was adjusted to7.2-7.4 using 0.2M KOH solution before 5% sucrose was added. All the polystyrene tubes were incubated for 60 min, and pH was quantified using a pH meter (Mettler Toledo Seven Compact pH/Ion Meter).

RESULT:

The results of diluting of EGCG from 100% to concentrations of 3.125%, 6.25%, 12.5% revealed minimum inhibitory concentrations (MIC) against S. gordonii bacteria at 0.03, 0.06, and 0.12mg/mL, respectively. The primary objective of this study was to assess the impact of EGCG on biofilm adherence (Figure 1) and glycolytic pH (Figure 2) in S.gordonii. Data were collected by measuring the mean and standard deviation of biofilm adherence.

The adherence variable tests showed that the normal control group (S. gordonii cultured in BHIB) exhibited an average value of 23.56mg/mL and a standard deviation value of 0.41; the negative control group (S. gordonii cultured in BHIB and 5% sucrose) had an average value of 44.48mg/mL and a standard deviation value of 0.57; treatment group I (S. gordonii cultured in BHIB, 5% sucrose, and EGCG at a concentration of 0.03 mg/mL) had an average value of 37.55mg/mL and a standard deviation value of 4.38; treatment group II (S.gordonii cultured in BHIB, 5% sucrose, and EGCG at a concentration of 0.06mg/mL) had an average value of 34.17mg/mL and a standard deviation value of 3.57; and treatment group III (S. gordonii cultured in BHIB, 5% sucrose, and EGCG at a concentration of 0.12mg/mL) had an average value of 30.49mg/mL and a standard deviation value of 6.37.

Figure 1. Biofilm adherence in Streptococcus gordonii after treatment with epigallocatechin gallate. Values followed with similar letters are not considerably different based on Mann Whitney test. *indicates significant difference (*p<0.05)

To assess the normality of the data distribution, we conducted the Shapiro Wilk test on the adherence variable data of S. gordonii exposed to EGCG. Following the assessment of the normality of the adherence data for each group, we observed that all the data exhibited a normal distribution, as indicated by a significance value >0.05. Furthermore, to evaluate the homogeneity of variance, we conducted Levene's test, which a yielded a results 0.021. This result indicates that the data is did not conform to a homogeneous distribution, given the significance value of <0.05. Consequently, in light of the non-homogenous distribution, we opted to employ the Kruskal Wallis and Mann Whitney data analysis tests to compare differences across the treatment groups.

According to the results of the glycolytic pH variable tests, the normal control group (S. gordonii cultured in BHIB) exhibited an average pH value of 7.02 with a standard deviation of 0.15. In contrast, the negative control group (S. gordonii cultured in BHIB and 5% sucrose) displayed an average pH value of 4.80 with a standard deviation of 0.05. The treatment groups yielded the following results: treatment group I (S. gordonii cultured in BHIB and 5% sucrose with the addition of EGCG at a concentration of 0.03 mg/mL) showed an average pH value of 5.07 with a standard deviation of 0.10. In treatment group II (S. gordonii cultured in BHIB and 5% sucrose with EGCG at a concentration of 0.06 mg/mL), the average pH value was 5.23 with a standard deviation of 0.07. Lastly, treatment group III (S. gordonii cultured in BHIB and 5% sucrose with EGCG at a concentration of 0.12 mg/mL) had an average pH value of 5.13 with a standard deviation of 0.11 (Figure 2).

Figure 2. Glycolytic pH in Streptococcus gordonii following treatment with epigallocatechin gallate. Values followed with similar letters are not considerably different based on Duncan test *indicates significant difference (*p<0.05).

The glycolytic pH data of S. gordonii exposed to EGCG was analysed for normality using the Kolmogorov Smirnov test. The resulting p-value was 0.381, indicating that the glycolytic pH data followed a normal distribution as the p-value exceeded the significance threshold of 0.05. To assess the homogeneity of variation, we performed Levene’s test which yielded a p-value of 0.837. Since this p-value was also greater than 0.05, it suggested that the data exhibited a homogenous distribution. To further investigate variations in the glycolytic pH data, we conducted a comparison test employing the one-way ANOVA test followed the Duncan test.

DISCUSSION:

In this study, S. gordonii were treated with a 5% sucrose solution. The addition of sucrose at this concentration of has proven to be effective promoting the formation of streptococcal bacterial biofilms, characterized by their high cariogenic potential⁶. The results showed that the average concentration of bacterial biofilms in samples treated with 5% sucrose was higher than that observed in untreated samples. The findings our study are consistent with the theory of glucosyltransferase activity; glucosyltransferase catalyzes the synthesis of sucrose into EPS glucan. This glucan production plays a role in bacterial adherence to the tooth pellicle. An increase in glucan levels enhances adherence and subsequent biofilm accumulation on the tooth surface ³².^.S. gordonii, a gram-positive bacterium, is an early colonizer. Early colonizers contribute to the initial attachment of bacteria colonies or biofilms to the tooth surface^11,17,33

The adherence of early colonizing bacteria is an essential stage in microbe pathogenesis. S. gordonii expresses a diverse array of adhesins, that mediate its adherence to host tissues³⁴. S. gordonii possesses serine-rich repeat adhesins Hsa, which facilitates the attachment of S. gordonii to the host's saliva, and GspB adhesin, responsible for the attachment of the bacteria to the dentin of the teeth³⁵. The capacity of S. gordonii to form a biofilms can be attributed to the presence of glucan polymers produced through the enzymatic activity of glucosyltransferase³⁶. These of bacterial colonies or biofilms assemblages, can initiate the process of cariogenesis, ultimately leading to dental caries. Cariogenesis arises from the metabolic formation of carbohydrates by bacterial colonies, resulting in the production of lactic acid. This acidification of the environment leads to a decrease in pH and demineralization of the tooth surface³⁷.

Numerous strategies exist to mitigate the growth of bacteria and dental plaque formation, such as the use antibiotics and mouthwash. However, employing these methods over an extended period is not advisable due to their potential side effects, that disrupt the normal oral flora³⁸. Therefore, many studies have been conducted to explore alternative herbal components to prevent S. gordonii biofilm adherence, with the anticipation of fewer side effects¹⁸.

One of the herbal ingredients that can be used is EGCG. EGCG is a catechin component of green tea polyphenols, recognized for its multifarious advantages, including anti-bacterial, anti-cariogenic, anti-inflammatory, anti-oxidant, anti-cancer, and anti-viral properties²⁸. Previous studies have elucidated, EGCG to be effective in inhibiting the formation of bacterial biofilms of S. mutans, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Phorpyromonas gingivalis, and Pseudomonas aeruginosa^39,40.

The addition of EGCG at concentrations of 0.03, 0.06, and 0.12mg/mL resulted on a significant reduction in S. gordonii biofilm adherence compared to the control group, which was not treated with EGCG. EGCG exerts its inhibitory effect on S. gordonii adherence by inhibiting glucosyltransferase activity, a virulence factor of S. gordonii, involved in the synthesis of EPS. EPS which plays a vital role in bacterial adherence, are glucan adhesins. A reduction in the synthesis of glucan adhesin, leads to decrese adherence of S. gordonii to the tooth pellicle⁴¹. Bacterial adherence is the initial step in the formation of a stable biofilm, ultimately leading to caries⁴².

The findings of this study showed a significant increase in the glycolytic pH of S. gordonii when EGCG was introduced at concentrations of 0.03, 0.06, and 0.12 mg/mL compared to the control group, which was not treated with EGCG. Previous studies on EGCG have demonstrated its capacityt to diminish the production of non-mutant streptococcal acids, such as Streptococcus sanguinis, S. gordonii, and Streptococcus. salivarius, while also promoting the aggregation of non-mutant streptococci²⁷.

Excessive carbohydrate consumption induces a matrix-rich milieu, fostering the proliferation of acid-producing microorganisms. This, in turn, causes ecological shift in the cariogenic microbiota, ultimately leading to caries⁴³. The pH of bacteria is regulated by the enzyme F0F1-ATPase, which facilities proton efflux from the cell. EGCG possesses the ability to inhibit this enzyme, thereby increasing the the acidity of the cytoplasm and diminishing proton effluxfrom the cell. As the cytoplasmabecomes more acidic, S. gordonii bacteria adapt to this altered environment, consequently leading to reduce activity of glycolysis-related enzymes. Bacteria produce less acid if they are less sensitive to acid and have fewer glycolytic enzymes^16,27

EGCG can inhibit the activity of the enzyme lactate dehydrogenase (LDH), which is present in both S mutan ⁴³ and S gordonii bacteria⁴⁴. This Inhibition of LDH leads to an elevation in NADH levels, a potential reducyion in redox balance, an imbalance between NAD+ and NADH, and an accumulation of glycolytic mediators in the cells. Consequently, the bacterial cytoplasm becomes more acidic, impeding the glycolytic process and resulting in a decline in ATP levels due to the disruption and accumulation of these glycolytic intermediaries. By halting glycolysis, S. gordonii bacteria can generate less acid , thereby preventing the pH drop attributed glycolysis⁴⁵.

The mechanism through which EGCG inhibits acid production involves the inhibition of the enzyme enolase. Enolase plays a pivotal role in the biosynthesis of phosphoenolpyruvate (PEP), which is indispensable for the functioning of the phosphotransferase system (PTS). The PTS governs the transportation of sugar to cells experiencing sugar deficiency. The inhibition of enolase leads to a diminished production of PEP, consequently reducing the influx of sugar into cells via the PTS. This sugar scarcity, in turn, suppresses glycolysis, ultimately diminishing the quantity of acid generated by bacteria^43,45.

In conclusion, EGCG possesses the capacity to impede both the adherence of biofilms and the glycolytic pH reduction in S. gordonii. Nonotheless, additional research is imperative to ascertain the potential of EGCG as a substance for deterring biofilm adherence and glycolytic pH other bacteria implicated in caries formation, such as Lactobacillus and Bifidobacterium. Furthermore, it is anticipated that forthcoming will investigations will delve into the impact of EGCG in preventing biofilm adherence and glycolytic pH reduction by S. gordonii in vivo or clinical studies.

ABBREVIATIONS:

Brain Heart Infusion Broth (BHIB); epigallocatechin gallate (EGCG); extracellular polymeric substances (EPS); lactate dehydrogenase (LDH); minimum inhibitory concentrations (MIC); phosphotransferase system (PTS)

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This study was supported by the Faculty of Dental Medicine, Universitas Airlangga provided financial support for Internal Research Program of 2022 [251/UN3/2022].

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Received on 30.09.2023 Modified on 25.01.2024

Research J. Pharm. and Tech 2024; 17(10):4711-4716.

DOI: 10.52711/0974-360X.2024.00726