INTRODUCTION:

Biofilm is a problem in the world of health that has not yet received optimal attention in handling infections despite its significant role in causing chronic conditions in various countries.

Based on data from the National Institute of Health, an estimated 60-80% of infections in the human body are caused by biofilms. Biofilms are formed from a collection of microbial cells that are irreversibly attached to a surface and protected in the Extracellullar Polymeric Substances (EPS) matrix they produce^1–3_. In the oral cavity, biofilms pose a serious health threat, contributing to health problems such as dental caries, periodontitis, halitosis and gingivitis. These problems lead to an increased incidence of recurrent infections, treatment failure, and increased morbidity and mortality^4,5.

Streptococcus species are said to represent up to 60-80% of all bacteria present in the oral cavity, this occurs because these species have the ability to attach to the salivary layer and metabolize its components^6,7. Streptococcus mutans is referred to as the main bacterium in causing dental caries by both sucrose-dependent and sucrose-independent in forming biofilms on teeth. The sucrose-dependent mechanism involves extracellular polysaccharides that will help bacteria attach to the tooth surface and form biofilms. The sucrose-independent mechanism involves other means, such as with salivary proteins or other components to attach and form biofilms on the teeth⁸. Lactobacillus acidophilus contributes to the development of caries by producing lactic acid, which can damage tooth enamel⁹. Meanwhile, Pseudomonas aeruginosa and Staphylococcus aureus, although often found as nasocomial pathogens, can also cause oral infections, especially in individuals with weakened immune systems^10,11.

Treatment of infections caused by biofilms is a hidden challenge because microorganisms in biofilms show high resistance to antibiotics and antimicrobial agents compared to planktonic cells. In addition, the treatment of oral biofilms is still problematic because the complex biofilm structure with EPS makes it difficult to eradicate the microbes present^12,13_.Therefore, it is necessary to search for new drug candidates as an effective therapy against oral biofilms. This study focuses on filling the gap by exploring the potential of natural materials as antibiofilm agents.

Telang ternate (Clitoria ternatea) is a plant that has been used in traditional medicine for various diseases in Indonesia. Some preliminary studies have shown that extracts from telang ternate have significant antibacterial activity^14,15. However, research on the antibiofilm potential of active isolates of telang ternate is still limited. Given the ability of biofilms to increase bacterial resistance, it is important to explore the potential of these isolates in inhibiting biofilm formation by oral pathogenic bacteria.

MATERIALS AND METHODS:

Materials:

Isolat Murni Telang Ternate, Standard biofilm-forming S. mutans isolate (S. mutans ATCC 25175), L. acidophilus isolate (L. acidophilus ATCC 4356), P. Aeruginosa isolate (P. Aeruginosa ATCC 27853), S. aureus isolate (S. aureus ATCC 25923) from the Microbiology Laboratory, Faculty of Pharmacy, UGM, Fluconazole, DMSO 1%, NaCl, McFarland 0.5 standard, sterile aquadest, Sabaroud Dextrose Agar (Merck, Germany), RPMI 1640 (Merck, Germany), PBS (Phosphate Buffer Saline) solution, crystal violet 1% (Merck, Germany), Laminar Air Flow, incubator (IF-2B)(Sakura, Japan), micropipettepipetman (Gilson, France), multichannel micropipette (Socorex, Swiss), microplate flat-bottom polystyrene 96 well (Iwaki, Japan), microtiter plate reader (Optic Ivymen System 2100-C, Spain), autoclave (Sakura, Japan), Scanning Electron Microscopy, and analytic scales (AB204 -5, Switzerland).

Methods:

Bacterial Culture Preparation:

Test bacteria namely S. mutans, L. acidophilus, P. aeruginosa, and S. aureus were grown for 24 hours at 370C in BHI medium. The optical density (OD 600) of the microbial culture was adjusted to 0.1 (equivalent to McFarland 0.5 – 1.5 x 10⁸ CFU/ml)¹⁶_.

Inhibition of mid-phase (24 hours) and maturation (48 hours) biofilm formation by microbroth dillution method:

Antibiofilm testing was carried out by microdilution method using bacteria in Brain Heart Infusion media suspension, isolates were tested at concentrations of 1, 0.5, 0.25, and 0.125% with the positive control being Listerine®. The test solution is placed in a 96 wells U-bottom microplate with a total volume of 100μL per well. The assay is performed on one test plate (with bacteria) and one blank plate (without bacteria). After incubation for 18-24hours at 36.6°C, the microplate was washed with water and dried. 1% crystal violet dye (125 μL) was added, allowed to stand for 15 minutes, then washed again. Then 96% ethanol (200μL) was added and allowed to stand for 15 minutes. Optical Density (OD) test results were read using a microplate reader after transferring 150μL of solution to a 96 wells flat-bottom microplate^2,12. The OD value is then used to calculate the percent inhibition with the following equation:

Average OD of negative control – Average OD of test sample

% Inhibition = ----------------------------------------------------------- x 100

Average OD of negative control

The sample level that inhibits at least 50% of biofilm formation is considered the Minimum Biofilm Inhibition Conceration (MBIC50)¹⁷.

Eradication Test Of Biofilm Formation:

The ternate telang active isolate will also be examined on biofilms of several Oral Pathogenic Bacteria strains using the same method of microbroth dilution. Biofilms were inoculated in microtiter plates and incubated at 37⁰C for 48hours. The plate was then washed three times with 150μL of sterile distilled water to remove non-adherent cells. Media containing purified isolates at various concentrations (1%, 0.5%, 0.25%, 0.125% b/v) was added to each well and re-incubated at 370C for 48 hours. Listerine 1% b/v was used as a positive control. After incubation, the plate was washed three times with 200μL of 1% Crystal violet solution to each well, incubated for 15 minutes, washed with PBS, and then added 200μL of 96% ethanol to dissolve the biofilm. OD readings were taken with a microplate reader at a wavelength of 595nm².

Statistical Methods:

Statistical analysis was performed using ANOVA and normality test was performed using Shapiro-Wilk, with a p value of 0.05 or less. The data were analyzed using the Statistical Package for the Social Sciences (SPSS).

RESULT:

Inhibitory Activity of Telang Ternate Active Isolate on Mid-Phase (24 hours) Biofilms of S. mutans, L. acidophilus, P. aeruginosa, and S. aureus

Table 1. Data of Percent Biofilm Inhibition of Telang Ternate Active Isolate 24 hours

*Oral Pathogenic Bacteria*	Biofilm Growth Inhibition (%)
*Oral Pathogenic Bacteria*	1%	0.5%	0.25%	0.125%
Streptococcus Mutans	85.17 ± 0.01	81.06 ± 0.01	76.15 ± 0.01	69.67 ± 0.01
Lactobacillus acidophilus	83.04 ± 0.01	80.12 ± 0.01	74.54 ± 0.01	67.17 ± 0.01
Pseudomanas aeruginosa	80.67 ± 0.01	78.73 ± 0.01	72.66 ± 0.01	64.39 ± 0.01
Staphylococcus aureus	78.62 ± 0.01	76.98 ± 0.01	66.38 ± 0.01	61.94 ± 0.01
Listerine	82.71 ± 0.01

The active isolate of telang ternate was tested for its ability to eradicate biofilms in the mid-phase of several bacteria, and showed the following results: at a concentration of 1% b/v, the active isolate was able to inhibit biofilms of S. mutans by 85.17%±0.01, L. acidophilus by 83.04%±0.01, P. aeruginosa by 80.67% ±0.01, and S. aureus by 78.62%±0.01. (Figure 1) shows that the positive control reduced biofilms by 86.71%± 0.01, making it more effective than Telang Ternate extract at the concentrations tested.

Figure 1. Biofilm Inhibition Activity of Telang Ternate Active Isolate 24 Hours

Inhibitory Activity of Telang Ternate Active Isolates on Mid-Phase (48hours) Biofilms of S. mutans, L. acidophilus, P. aeruginosa, and S. aureus

Table 2. Data of Percent Biofilm Inhibition of Telang Ternate Active Isolate 48 hours

*Oral Pathogenic Bacteria*	Biofilm Growth Inhibition (%)
*Oral Pathogenic Bacteria*	1%	0.5%	0.25%	0.125%
Streptococcus Mutans	78.36 ± 0.01	74.70 ± 0.01	70.71 ± 0.01	63.30 ± 0.01
Lactobacillus acidophilus	77.38 ± 0.01	71.51 ± 0.01	64.85 ± 0.01	58.89 ± 0.01
Pseudomanas aeruginosa	77.52 ± 0.01	70.15 ± 0.01	62.88 ± 0.01	57.76 ± 0.01
Staphylococcus aureus	71.93 ± 0.01	66.35 ± 0.01	61.84 ± 0.01	57.15 ± 0.01
Listerine 1%	80.71 ± 0.01

The active isolate of Telang Ternate has been shown to be effective in preventing biofilm formation during the 48-hour maturation phase, according to the results of this study. The extract showed the highest level of activity at a concentration of 1% b/v against various bacteria, resulting in inhibition of 78.36%±0.01 for S. mutans, 77.38%±0.01 for L. acidophilus, 77.52%±0.01 for P. aeruginosa, and 71.93%±0.01 for S. aureus. In comparison, the control drug Listerine showed an inhibitory activity of 80.71%±0.01 at the same concentration. However, as the biofilm fully formed during the maturation phase, the potency of the extract decreased, making it less effective against stronger and more resistant biofilms as shown in (Figure 2).

Figure 2. Biofilm Inhibition Activity of Telang Ternate Active Isolate 48 Hours

Inhibitory Activity of Telang Ternate Active Isolate in the Biofilm Eradication Phase of S. mutans, L. acidophilus, P. aeruginosa, and S. aureus

Table 3. Data on Percent Biofilm Inhibition of Telang Ternate Active Isolate Eradication Phase

*Oral Pathogenic Bacteria*	Biofilm Growth Inhibition (%)
*Oral Pathogenic Bacteria*	1%	0.5%	0.25%	0.125%
Streptococcus Mutans	75.28 ± 0.01	72.05 ± 0.01	62.14 ± 0.01	58.74 ± 0.01
Lactobacillus acidophilus	74.34 ± 0.01	69.22 ± 0.01	60.86 ± 0.01	52.60 ± 0.01
Pseudomanas aeruginosa	74.52 ± 0.01	67.75 ± 0.01	58.68 ± 0.01	53.98 ± 0.01
Staphylococcus aureus	69.90 ± 0.01	62.08 ± 0.01	59.01 ± 0.01	54.09 ± 0.01
Listerine 1%	77.33 ± 0.01

The active isolate of Telang Ternate was tested for its ability to eradicate biofilms of several bacteria, showing the following results: at a concentration of 1% b/v, the extract inhibited biofilms of S. mutans by 75.28%±0.01, L. acidophilus by 74.34%±0.01, P. aeruginosa by 74.52%±0.01, and S. aureus by 69.90%±0.01. (Figure 3) shows that Listerine as a positive control reduced biofilms by 77.33%±0.01, making it more effective than Telang Ternate isolate at the concentration tested.

Figure 3. Biofilm Inhibition Activity of Eradicated Telang Ternate Active Isolate

DISCUSSION:

This study aims to evaluate the inhibitory activity of active isolates from the telang ternate plant against biofilm formation of various bacteria such as S. mutans, L. acidophilus, P. aeruginosa and S. aureus that cause infections in the oral cavity. The results showed that the active isolate of telang ternate has a significant ability to inhibit biofilm formation by these oral pathogenic bacteria with a positive control comparison using Listerine, which is widely recognized as a commercial antibacterial agent for oral care¹⁸.

Biofilm inhibition by telang ternate active isolate was significant against all bacteria tested in the middle phase in Figure 1. On S. mutans, which is known as one of the main causes of dental caries, telang ternate active isolate showed biofilm inhibition that was almost equivalent to listerine with a percentage value reaching 85.17%±0.01 in comparison to 86.71%±0.01 inhibition shown by Listerine. This shows the potential of telang ternate isolate to be a natural alternative in caries prevention. Biofilm growth is prevented by active compounds contained in telang ternate, based on table 1, it is known that telang ternate contains flavonoids that can disrupt communication between bacterial cells through the Quarum Sensing mechanism which will inhibit the ability of S. mutans to colonize and form biofilms. In addition, tannins contained in the active isolate of telang ternate can precipitate adhesion proteins to attach to the tooth surface, reducing the ability of bacteria to form biofilms that are resistant to mechanical cleaning^19,20_.

In the test bacteria L. acidophilus, which is often known as a beneficial probiotic bacteria under certain conditions, is also able to form biofilms by contributing to oral disorders such as candidiasis, biofilms that appear due to the presence of L. acidophilus bacteria can also protect other opurtunistic pathogens in the oral cavity²¹. The active isolate from telang ternate again showed the ability to inhibit biofilm with a percentage reaching 83.04%±0.01 in the middle phase, this was not much significant with the positive control, Listerine with a percent inhibition reaching 86.71%±0.01. Compounds present in the telang ternate plant such as flavonoids and tannins can disrupt the adhesion of these bacteria on the surface of the mouth and inhibit the initial formation of biofilm²².

P.aeruginosa is a gram negative bacterium that is often involved in nasocomial infections and has a high ability to form biofilms that are resistant to antibiotics²³. This bacterium is also undeniably capable of causing infections in the oral cavity. The active isolate of telang ternate showed high effectiveness in inhibiting biofilm formation by these bacteria with a percentage reaching 80.67%±0.01. Alkaloids in telang ternate can damage the integrity of the bacterial cell membrane, causing leakage of cell contents and bacterial death, flavonoids contained in this active isolate will also interfere with quaorum sensing and inhibit the ability of bacteria to communicate in forming biofilms²⁴. This is very important, considering that P.aeruginosa often shows resistance to many conventional antibiotics.

Staphylococcus aureus is a gram-positive bacterium that is often found in oral and respiratory tract infections with the ability to form biofilms that protect them from the body's immune response and antibiotics²⁵. The active isolate of telang ternate was proven to be able to inhibit biofilm formation with this bacterium reaching an inhibition percentage of 78.62%±0.01. The active compounds contained in telang ternate can disrupt the bacterial cell wall, reducing the ability of bacteria to colonize and form biofilms. Not only that, these compounds can damage bacterial cell membranes and reduce surface tension, which affects the ability of bacteria to adhere and form biofilms²⁶.

In the maturation phase, the bacteria began to produce extracellular matrix that formed a more complex biofilm structure as well, the inhibitory activity of the telang ternate isolate began to show a slight decrease. Nevertheless, the active isolate of the telang ternate is still effective in disrupting the synthesis and structure of the biofilm matrix. In the degradation phase, the biofilm reached full maturity, and the inhibitory activity of the telang ternate active isolate decreased more significantly²⁷^-34. Although the active compounds present in telang ternate are still able to partially damage the biofilm structure and reduce its integrity, their effectiveness is still reduced because the EPS matrix in this phase provides stronger protection for bacteria^34-42

The findings of this study indicate that the active isolate of telang ternate has great potential as a biofilm inhibiting agent on oral pathogenic bacteria. This antibiofilm activity is most likely due to the combination of bioactive compounds in telang ternate that work through its mechanism. Although the inhibitory effectiveness decreased in the biofilm maturation and degradation phases, the active isolates of telang ternate still showed significant activity in all phases. Further research is needed to identify the specific active compounds and molecular mechanisms underlying this activity, as well as to test its safety and effectiveness in clinical trials.

CONCLUSION:

This study shows that active isolates from telang ternate have significant potential in inhibiting biofilm formation due to oral pathogenic bacteria. The effectiveness of this inhibition varied depending on the phase of biofilm development, with the highest activity in the mid-phase and a gradual decrease in the maturation and eradication phases. The bioactive compounds in the telang ternate contribute through various mechanisms in inhibiting biofilm formation, as well as damaging the extracellular matrix that protects the bacteria. These findings open up opportunities for the development of oral care products based on telang ternate active isolates that can provide a more holistic and safe approach to oral health management.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

Our grateful thanks to the Direktorat Riset, Teknologi, dan Pengabdian Kepada Masyarakat (DRTPM) for research funding assistance with contract number: 113/E5/PG.02.00.PL/2024

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Received on 13.07.2024 Revised on 22.01.2025

Accepted on 15.04.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3761-3766.

DOI: 10.52711/0974-360X.2025.00541

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.