Hrev_master Healthcare in Low-resource Settings 2025; volume 13:13016 Explication of the mixture of Piper betle and Eugenia polyantha on oral bacteria Neneng Nurjanah,1 Megananda Hiranya Putri,1 Yenni Hendriani Praptiwi,1 Dewi Sodja Laela,1 Susi Sukmasari2 1Department of Dental Nurse, Bandung Polytechnic of Health, Jawa Barat, Indonesia; 2Department of Paediatric Dentistry and Dental Public Health, Kulliyyah of Dentistry, International Islamic University Malaysia, Kuantan, Pahang, Malaysia. Abstract The aim of this study was to analyze the effectiveness of the mixtures of Eugenia polyantha and Piper betle leaves against oral bacteria. Four compositions of EP and PB were made and observed on tryptic soy agar, blood agar, MacConkey Agar (MCA), and Mannitol Salt Agar (MSA). Inhibition analysis was conducted at concentrations of 20%, 30%, 40%, 50%, and 60%. Our study results indicate that Staphylococcus aureus exhibited growth on BA, bacterial colonies were identified on MSA, and serological tests confirmed coagulation. A biochemical test con- ducted on a MCA sample revealed several species within the Enterobacteriaceae family, including Escherichia coli, Pseudomonas, Klebsiella, and Proteus. The highest level of inhi- bition was observed with the sixth toothbrush (K4). Significant differences in inhibition were noted among the various groups (K1, K2, K3, K4, and controls), with the diameter of inhibition for each combination yielding p<0.05. However, the differences in inhibition between the combinations themselves were not statisti- cally significant, with p>0.05. Streptococcus sp, Klebsiella sp, Enterobacteriaceae sp (Coliform sp), Pseudomonas, and Staphylococcus aureus are the contaminant bacteria on used tooth- brushes. The highest inhibition level and bactericidal properties were achieved by mixing 70% bay leaf (Eugenia polyantha) and 30% betel leaf infusion. Introduction The teeth, gums, tongue, mucous membranes, throat, and buccal mucosa provide various surfaces for microbial colonization. In total, there are more than 700 species of microorganisms present in the oral cavity.1,2 The organisms present as commensal and pathogenic microorganisms. Over time, pathogenic microorganisms can lead to tooth decay and periodontal diseases. Some of these microorganisms adhere to solid surfaces, forming dental biofilm, while others remain suspended in aqueous environments. Under adverse circumstances, bacteria can lead to bacteremia and spread to vital organs, such as the heart valves, forming a persistent plaque. Therefore, biofilm management or plaque control is needed to prevent oral diseases. Many studies have proven the effectiveness of mechanical plaque controls, such as toothbrushing, to manage dental biofilm.3 Nevertheless, toothbrushes may become infected with microorganisms from the buccal cavity, environment, hand aerosol, and storage locations.4,5 The microbiomes found on used toothbrushes are associated with the microbiome from various sites in the oral cavity.4,5 The microbiota can lead to cross-contamination with microorganisms in the oral cavity, such as S. mutans, Staphylococcus aureus, Streptococcus pyogenes and Candida albicans,6 Pseudomonas, Coliforme Corynebacterium., Bacillus sp, Gram (-) Neisseria sp.4,7 Moreover, antimicrobial-resistant bacteria has been found in used contaminated toothbrushes.8 Glass observed that the defect of oral tissue was exacerbated through a contaminated toothbrush instead of a sterile toothbrush. Some studies found that toothbrush handle material and the tightness of brush fibers have an impact on the retention of bacteria.9 Therefore, it is necessary to disinfect the toothbrush after use or before putting it in storage.10,11 Several methods are recommended for the decontamination of toothbrushes, including the immersion of toothbrushes into alcohol and spraying toothbrush fibers with antimicrobial solutions such as chlorhexidine, sodium hypochlorite, hydrogen Correspondence: Susi Sukmasari, Department of Paediatric Dentistry and Dental Public Health, Kulliyyah of Dentistry, International Islamic University Malaysia. Jl. Sultan Ahmad Shah, Bandar Indera Mahkota, Kuantan, 25200, Pahang, Malaysia. Tel.: +6095705458 - +60179040567 E-mail: sukmasari@iium.edu.my Key words: Piper betle; Eugenia polyantha; natural disinfection; used toothbrushes. Contributions: NN, conceptualization, data curation, formal analysis, methodology, validation, visualization, original draft writing; MHP, con- ceptualization, investigation, methodology, validation, and writing – original draft writing. YHP, DSL, supervision, and original draft writing; SS, writing, visualization, review & editing. Conflict of interest: the authors declare no potential conflict of interest. Ethics approval and consent to participate: the research has received eth- ical approval from the Health Research Ethics Commission, Bandung Polytechnique of Health. Availability of data and materials: all data generated or analyzed during this study are included in this published article. Funding: this research was supported by a research grant from the Director of Bandung Polytechnique of Health. Received: 5 September 2024. Accepted: 2 December 2024. Early access: 11 February 2025. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright: the Author(s), 2025 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2025; 13:13016 doi:10.4081/hls.2025.13016 Publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organi- zations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. [Healthcare in Low-resource Settings 2025;13:13016] [page 105] peroxide, essential oil vinegar, and warm salt water.5,12-19 However, the disadvantage of manufactured chemical products is that they are costly and can adversely affect long-term use. Considering the aforementioned factors, natural products that are standardized and safety-oriented are becoming more accepted globally. Some previous studies investigated some herbs as toothbrush disinfectants.10 Furthermore, mixing multiple herbs into one formula enhances antimicrobial potential.20,21 Piper Betle is a pop- ular antibacterial, mouthwash, and wound-healing component in Southeast Asian and Ayurvedic traditional medicine, and Eugenia polyantha is also used to treat gastrointestinal problems. Eugenia Polyantha is a tropical tree belonging to the kingdom Plantae, division Spermatophyta, subdivision Pinophyta, class Coniferopsida, order Myricales, family Myrtaceae, genus Eugenia, and species Polyanthum (Wight). Eugenia polyantha leaf has long been known as a spice with traditional healing use, antimicrobial, and antioxidant,22,23 which is easy to find in the mar- ket. The leaves are rich in phenolic compounds, flavonoids, phe- nol, tannins, saponins, steroids, terpenoids, and essential oils.24,25 As an antioxidant, Eugenia polyantha is stable in frying crude coconut oil.25 The solutions have been shown to inhibit the growth of oral bacteria and candida.7,26,27 Green betel (Piper betle (L)) is an herbaceous plant that grows or climbs on other tree trunks or wood. It is classified as follows: kingdom: Plantae; subkingdom: Tracheobionta; division: Spermatophyta; subdivision: Angiospermae; class: Dicotyledonae; order: Piperales, family: Piperaceae; genus: Piper; species: Piper betle (L). Research conducted by Nurjannah et al. showed that boiled Piper betle has an inhibitory effect on bacterial colonies carried by toothbrushes.7 Besides essential oils, other compounds are flavonoid acid, organic acids, amino acids, steroids, sugars, tannins, proteins, terpenoids, saponin, fats, starch, and carbohydrates.28 Piper betle has been recognized for its antibacterial and antifungal properties and is considered safe for oral use.29-31 Traditionally, it is used for skin diseases, hemorrhoids, smelly sweat, cleaning the eyes, reducing blood vessels, and as a cough medicine. In dentistry, Piper betel leaf infusion can be used to eliminate bad breath and to stop gum bleeding and toothache. The general purpose of this study was to analyze the effectiveness of the infusion containing the mixtures of Eugenia polyantha leaves and Piper betle leaves against bacterial growth on used toothbrushes. The analysis includes the determination of oral microorganisms on used toothbrushes, inhibition, and contact time. Materials and Methods Infusion preparation Fresh Eugenia polyantha leaves and Piper betle leaves were washed, drained, and sliced. We weighed 600 g of EP leaves and added 1 L of distilled water, and 350 g slices of PB leaves and added 1 L of distilled water. Both were heated in a water bath at a temperature of ±90ºC for 20 minutes, then cooled and filtered. The four compositions of the solutions between Eugenia polyantha and Piper betle were made as follows: K1 (40%:60%), K2 (50%:50%), K3 (60%:40%), K4 (70%:30%), with sterile water as a negative control, and ciprofloxacin as a positive control. The K1, K2, and K3 combination in that composition has been balanced between the two herbs for comparison. Meanwhile, K4 was made based on the observations conducted by Putri et al.32 Toothbrushing implementation Eight primary school students were chosen randomly as partic- ipants. Each participant was given a toothbrush with the same size and type of brush. Recommended tooth brushing techniques were demonstrated by the dentist prior to tooth brushing implementa- tion. Participants brushed their teeth systematically for 2 minutes, consisting of 20 seconds for each region, starting from the upper right to the upper front, upper left, lower left, lower front, and lower right regions. After tooth brushing, without rinsing, the toothbrush was put into a sterile test tube containing NaCl. The sterile test tube was firmly sealed and transported to the lab for testing. After the successful breeding of microorganisms, the used toothbrushes were placed in the medical waste bin and discarded. Specimen breeding and microorganism identifica- tion 50 µL of bacterial suspension from the participant’s used toothbrush was inoculated on Tryptic Soy Broth (TSB), incubated at 37ºC for 24 hours, then observed for turbidity. TSB serves as a base medium for a variety of specialized growing conditions. It is high in nutrients and is widely used to cultivate microorganisms for diagnostic and sensitivity testing. To identify the type of con- taminant bacteria on the toothbrush, the gram stain was performed in TSB agar. It was planted for a four-way streak on the following selective medium to determine microorganisms in mixed cultures. Colony characteristics were observed on TSA. TSA is com- monly used in clinical microbiology to culture and isolate non-fas- tidious microorganisms for further testing. It is an enriched, non- selective medium that supports the growth of both gram-positive and gram-negative bacteria. Coccus gram-positive and gram-negative were isolated on Blood Agar (BA). Blood agar is an enhanced medium containing general nutrients and animal blood. It is used to cultivate bacteria and detect pathogens by hemolytic activity. Gram-negative bacteria were identified using MacConkey Agar (MCA), which inhibits the growth of gram-positive bacteria due to the presence of bile salts and crystal violet. Agar is a selective and differential medium that isolates and distinguishes gram-negative enteric bacteria based on their capac- ity to ferment lactose. Lactose fermenters (such as Escherichia coli) produce acid, which causes them to appear pink/red on the medium. Non-lactose fermenters (such as Salmonella and Shigella) stay colorless or pale. Mannitol salt agar (MSA) is a selective and differential medi- um used to isolate Staphylococcus species, particularly Staphylococcus aureus, using mannitol fermentation differentia- tion. Inhibition analysis Using a sterile cotton swab, 100 µL of bacterial suspension from each TSB was evenly spread over the surface of Mueller- Hinton Agar (MHA). Six mm wells were made using a sterile tube. Poured 50µL of each mixture (K1, K2, K3, and K4) infusion at concentrations of 20%, 30%, 40%, 50%, 60%, sterile distilled water as a negative control, and 5 µg ciprofloxacin as a positive control into the well. The media was wrapped in brown paper, labeled, and incubated at 37˚C for 24 hours. The clear area was analyzed as an inhibition zone on each well. The inhibition zone, or clear area, was coded as 1 and 0 for no clear area. Contact time analysis The micropipetting solution from each tube containing microorganisms from the used toothbrush was added to the first Article [page 106] [Healthcare in Low-resource Settings 2025;13:13016] tube containing 9 mL of 0.85% NaCl and mixed until homoge- neous. The solution was pipetted from the first tube to the second tube, and this process was repeated until the fifth tube. Then, 1 mL of solution from each NaCl tube was transferred into a labeled petri dish. Next, approximately 15 mL of Plate Count Agar (PCA) media was added at a temperature of 40-45ºC. The treatment was conducted for eight used toothbrushes. The number of bacterial colonies in each PCA medium using a colony counter was counted. Points 1 and 2 were carried out for contact times of 10, 20, 30, 40, 50, and 60 minutes, and each treatment was repeated 4 times. Statistical analysis All data were statistically analyzed by using SPSS software package 25 for Windows. The normality test was used prior to the differential test. The Levene test based on the mean (α=0.05) was used to analyze the homogeneity. The Kruskal-Wallis test was used to see the effectiveness differences of the infusions (K1, K2, K3, K4, control +, and control –) on inhibition of bacterial growth. The Mann-Whitney test was carried out to see the inhibition efficacy differences between each combination on bacterial growth. Results After incubating, the solution containing the used toothbrush in TSB became cloudy, as shown in Figure 1. To study the presence of bacteria contained in toothbrushes and to determine gram-posi- tive and gram-negative colonies, the bacterial suspension of the TSB media was inoculated in the blood agar. The result shows that the shape, elevation, color, and characteristics of bacterial colonies were round, convex, and cloudy for β-hemolysis, while round, convex, and murky or slimy color for non-hemolysis. The suspected Staphylococcus aureus on Mannitol Salt Agar (MSA) is based on a round shape, 1-2 mm in size, convex eleva- tion, gold color, and hemolysis. The serological test detected the presence of bacteria coagulating from all used toothbrushes. Inoculation on MCA Table 1 shows the observed results for the shape and size, ele- vation, color, and characteristics of bacterial colonies on MC agar. The bacterial characteristics were lactose fermenter and non-lac- Article [Healthcare in Low-resource Settings 2025;13:13016] [page 107] Table 1. Results of observations of bacterial colonies on MacConkey agar. Sample code Colony Shape and Size Elevation Color Characteristic TB 1 1 Round, 2-3 mm Convex Pink Lactose fermenter 2 Round, 1-1.5 mm Convex Yellow Non lactose fermenter TB 2 1 Round, 3-4 mm Convex Pink, slippery Lactose fermenter TB 3 1 Round, 2mm Convex Pink, slippery Lactose fermenter 2 Round, 3mm Convex Pink, rough Lactose fermenter TB 4 1 Round, 3-4 mm Convex Pink, slippery,slimy Lactose fermenter TB 5 1 Round, 2mm Convex Pink, slippery Lactose fermenter 2 Round, 3mm Convex Pink, rough Lactose fermenter TB 6 1 Round, 3mm Convex Pink, mucoid Lactose fermenter 2 Round, 2-3 mm Convex Yellow Non lactose fermenter TB 7 1 Round, 2-3 mm Convex Pink Lactose fermenter 2 Round, 4mm Convex Yellow Non lactose fermenter 3 Round, 3mm Convex Yellow, mucoid Non lactose fermenter TB 8 1 Round, 3-4 mm Convex Pink, slippery Lactose fermenter TB, toothbrush. Figure 1. The bacterial turbidity of solution containing used toothbrushes in tryptic soy broth (TSB). tose fermenter. To determine the growth characteristics of the bac- teria from the Enterobacteriaceae group, a biochemical test was carried out on each colony on MC agar. The bacteria were Escherichia coli, Enterobacter cloacae, Pseudomonas sp, Klebsiellaozaena, and Proteus sp. The results of biochemical tests are shown in Table 2. Inhibition test of bay leaf and betel leaf infusion on bac- terial growth on toothbrushes The mean inhibitory levels of the four combinations K1, K2, K3, and K4 compared to the positive control (ciprofloxacin 5 mg) are presented. The diameter of the inhibitory zone on the negative control on all toothbrushes was 0, while the positive control on all toothbrushes was 38, 30, 28, 25, 19, 27, 24, and 28, respectively. The four combinations of the mixtures were below positive con- trol. The inhibition zone of the combinations was K1: 4, 13, 16, 17, 14, 17, 16, and 0, K2: 15, 16, 13, 14, 15, 17, 14, and 0, K3: 10, 14, 13, 13, 14, 17, 12, and 12, K4: 17, 17, 13, 13, 0, 5, 19, 12, and 0. Table 3 displays the average number of bacteria at different time intervals (initial, 10, 20, and 30 minutes) after immersing the toothbrushes in a mixture of Eugenia polyantha and Piper betle infusion (K4). Statistical analysis Based on the Kruskal-Wallis test, there was a significant differ- ence between the diameter of the inhibition of betel leaf infusion in the six data groups (K1 K2 K3 K4 cipro 50 μg/ 50 μL, and sterile aqua dest as a negative control). The Whitney test showed the diameter of inhibition for each combination of K1, K2, K3, K4, ciprofloxacin, and aqua dest was p<0.05. However, the diameter of the inhibition of betel leaves among combinations was not signifi- cantly different (p>0.05). Discussion In this study, all incubated solutions containing samples from used toothbrushes were cloudy. This indicates that oral bacteria from the used toothbrushes successfully grew in TSB. In healthy individuals, toothbrush contamination occurs immediately after use and increases with repeated use.3,6 Moreover, the used tooth- brush will likely be contaminated by pathogenic opportunists in a sick condition.4 A recent study found that used toothbrushes har- Article Table 2. Biochemical test results from MacConkey agar media. Sample code Colony Observation result Suspect Indole MR VP SC TB 1 LF + + – – Escherichia coli LF – – + + Enterobacter cloacae NLF – – – – Pseudomonas sp TB 2 LF + + – – Escherichia coli LF – – + – Klebsiellaozaena TB 3 LF + + – – Escherichia coli LF – – + – Klebsiellaozaena NLF – – – – Pseudomonas sp NLF + + –/+ +/– Proteus sp TB 4 LF + + – – Escherichia coli LF – – + – Klebsiellaozaena TB 5 LF + + – – Escherichia coli LF – – + – Klebsiellaozaena TB 6 LF + + – – Escherichia coli NLF – – – – Pseudomonas sp TB 7 LF + + – – Escherichia coli LF – – + – Klebsiellaozaena NLF – – – – Pseudomonas sp NLF + + –/+ +/– Proteus sp TB 8 LF + + – – Escherichia coli LF – – + – Klebsiellaozaena TB, toothbrush; LF, lactose fermenter; NLF, non-lactose fermenter; MR, methyl red; VPs, Voges-Proskauer; SC, Simmons Citrate. TB, toothbrush. Table 3. Average number of bacteria on a toothbrush after soaking with a mixture of Eugenia polyantha and Piper betle Infusion (K4). Dilution of the infusion mixture Average number of bacteria (CFU/mL) at contact time (minutes) 0 10 20 30 1: 4 81,667 62,967 23,689 32,900 1: 9 >111,000 >111,000 >111,000 >111,000 [page 108] [Healthcare in Low-resource Settings 2025;13:13016] bored antimicrobial-resistant bacteria such as Staphylococcus aureus, E. coli, and Pseudomonas.8 Therefore, based on the find- ings, to reduce the risk of contamination, the used toothbrush should be reconsidered to disinfect immediately after use. The commensal oral flora was identified by cultivating sam- ples on various types of media The findings of this study were con- sistent with previous research that identified Streptococcus growth on blood agar, Klebsiella sp, Enterobacteriaceae sp (Coliform sp), Pseudomonas growth on McConkey agar, and Staphylococcus aureus on mannitol salt agar.33-35 In addition, as a normal flora in dental plaque, Streptococcus mutans is also higher in teeth with cavities. The selected participants had more than three cavities due to caries. As a result, the findings indicated that the growth of microorganism colonies on blood agar, Mannitol Salt Agar (MSA), and MacConkey Agar (MC) consisted of oral microorganisms that were transferred by toothbrushes. All compositions of the mixtures can inhibit the growth of bac- terial colonies, which was evidenced by the formation of an inhi- bition zone around the bacterial colonies grown on Mueller-Hinton Agar (MHA). The inhibition zone of the mixture was varied for each toothbrush, ranging from 12 mm to 19 mm; the lowest inhi- bition zone was found on toothbrush number 8 with an inhibition zone of 12 mm (K3), and the highest inhibition zone was on tooth- brush number 6, which is 19 mm (K4). Therefore, the infusion composition of the combination of 70% Eugenia polyantha + 30% Piper betle has the greatest inhibitory power against the growth of decontaminant bacteria compared to the other compositions. However, the average diameter of the K4 inhibition zone (70% Eugenia polyantha + 30% Piper betle) is 13.47 mm, which is rel- atively slightly smaller when compared to the average diameter of the inhibition zone for bay leaf infusion in the previous study.36 This is probably due to a reduction in the concentration of each part at the combined concentration. This study concluded that both formulations of Eugenia polyantha and Piper betle have potential effects on gingivitis pre- vention. They had an effectiveness level almost similar to chlorhexidine gluconate 2%. The F-II formula demonstrated superior physical indicator val- ues, as these were closer to the standard values. For its antibacterial properties and improved physical indicators, which align better with herbal standard values, the F-II formula is recommended as a mouthwash for gingivitis. The absence of bacterial growth on the PCA medium indicated that a 70% Eugenia polyantha and 30% Piper betle (K4) combina- tion exhibited bactericidal properties. In addition, the K4 mixture was diluted at 1:4 and 1:9. The results revealed that diluting K4 in a 1:4 ratio was better than 1:9. However, bacterial colonies in K4 dilutions at ratio 1:4 with contact times of 0, 10, 20, and 30 min- utes were >300. O’Toole stated that if the number of colonies reaches 300, the bacterial count will not meet the required stan- dards.37 Therefore, further examination with a different dilution ratio is necessary. Furthermore, the extract mixture was tested against four isolated pathogenic bacteria: Streptococcus sp, Klebsiella sp, Enterobacteriaceae sp (Coliform sp), Pseudomonas and Staphylococcus. The aqueous extract exhibited antibacterial activity against gram-positive and gram-negative bacteria. Several studies have found that these plants effectively inhibit bacterial growth. Piper betle is widely used in Ayurvedic and Southeast Asian traditional medicine for antimicrobial, mouthwash, and wound healing. Eugenia polyantha is also used in traditional medicine to treat gastrointestinal problems and as an antibiotic. Although Piper betle is often used as a mouthwash, this study dis- covered that a higher concentration of Eugenia polyantha pro- duced better results. The widespread use of Eugenia polyantha for gastrointestinal disorders, along with the similarity of microbiome strains in oral and intestinal populations, highlights its potential effectiveness and merits further investigation. Conclusions Streptococcus sp., Klebsiella sp., Enterobacteriaceae sp. (Coliform sp.), Pseudomonas and Staphylococcus aureus are the contaminant bacterial colonies that remain on used toothbrushes after washing with water. 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