Periodontal diseases are a prevalent group of oral health disorders characterized by the inflammation and destruction of supporting tissues around teeth. They are increasingly recognized as risk factors for certain systemic diseases [1]. The etiology of these diseases is primarily attributed to microbial colonization and subsequent biofilm formation on the tooth surfaces [2]. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are the predominant bacterial species associated with periodontal pathogenesis. These microorganisms play a pivotal role in the initiation and progression of periodontal infections because of their virulent attributes and their ability to manipulate the host immune response [3,4].

Efforts to combat periodontal diseases have led researchers to explore novel therapeutic agents that target these pathogenic bacteria. Genistein, a soy isoflavone, has garnered attention for its multifaceted biological properties, including its anti-inflammatory effects both in vitro and in vivo [5]. Furthermore, genistein exhibits antibacterial effects against a range of bacterial species. However, not all bacteria are susceptible to genistein. For example, the growth of Staphylococcus aureus and Helicobacter pylori is inhibited by genistein, whereas that of Escherichia coli is not [6-8].

Although the antibacterial potential of genistein has been recognized, its specific activity against periodontal bacteria remains unexplored. Given the pressing requirement for new treatment approaches for periodontal diseases, it is crucial to assess the potential of genistein as an antibacterial agent against periodontal pathogens. This study aimed to fill the knowledge gap regarding the antibacterial effect of genistein against periodontal bacteria, particularly P. gingivalis and A. actinomycetemcomitans.

Bacterial culture

P. gingivalis ATCC 33277 was grown in tryptic soy broth supplemented with yeast extract (1 mg/mL), hemin (5 μg/mL), and menadione (1 μg/mL). Tryptic soy agar for P. gingivalis was additionally supplemented with 5% sheep blood. A. actinomycetemcomitans ATCC 33384 was grown in tryptic soy broth supplemented with yeast extract (1 mg/mL). Tryptic soy agar for A. actinomycetemcomitans was also supplemented with 5% sheep blood. The strains were grown anaerobically (85% N₂, 10% H₂, and 5% CO₂) at 37°C for 18–24 hr.

Antimicrobial assays

Genistein was purchased from Merck (Burlington, MA, USA). The antibacterial activity of genistein was evaluated using the broth microdilution method [9]. Freshly prepared bacterial cultures were added to the culture medium containing serially diluted genistein in wells of microtiter plates. The inoculum size was controlled by measuring the optical density (OD) at 600 nm and extrapolating the CFU/mL using preset standard curves. Successive twofold dilutions of genistein were prepared at a volume of 100 μL, and 100 μL samples of each bacterium were added to the prepared plates. The final inoculum concentration was 1×10⁷ CFU/mL. The wells of the plates included one growth control and one sterility control. After incubation for 48 hr, microbial growth was measured using a microplate reader at 600 nm. The minimum inhibitory concentration (MIC) was defined as the lowest dilution at which no growth was detected.

Assessment of bacterial viability

In vitro bactericidal activities were evaluated by measuring viable bacterial counts. In brief, 10 mL of P. gingivalis or A. actinomycetemcomitans was cultured in 50-mL tubes to obtain colony counts. The approximate bacterial inoculum size was 1×10⁶ CFU/mL. Bacterial cultures were treated with genistein. After 1 hr of incubation, 100 μL aliquots were obtained from the tubes. Subsequently, 10-fold dilutions were prepared and cultured on tryptic soy agar plates for 48–72 hr. The number of colonies formed was counted.

Crystal violet assay for assessing cytotoxicity

YD15, an oral epithelial cell line derived from mucoepidermoid carcinoma, was purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). YD15 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. The cells were seeded at 1×10⁵ cells per well in 48-well plates and allowed to adhere overnight. The next morning, the cells were treated with genistein and incubated for 24 hr. Cell viability was assessed using the crystal violet assay, which measures the number of viable adherent cells [10]. After removing nonadherent cells by repeatedly washing the cultures with phosphate-buffered saline (PBS), the cells were fixed with 0.5 mL of 100% methanol for 10 minutes and stained with 1% crystal violet solution at room temperature for 10 minutes. The plates were thoroughly washed with PBS, and 33% acetic acid was then added to each well. The OD of dissolved dye, corresponding to the number of viable cells, was measured using a microplate reader at 570 nm.

Statistical analysis

Our experiments were independently conducted three times to ensure the reproducibility of the results. The data are presented as means with standard deviations. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey–Kramer multiple comparisons test in GraphPad InStat (GraphPad Software, La Jolla, CA, USA). Differences were considered significant at the level p<0.05.

Genistein-mediated inhibition of periodontal bacterial growth

The antibacterial activity of genistein against P. gingivalis and A. actinomycetemcomitans was determined using the broth microdilution method. As shown in Fig. 1, the growth of both bacteria was inhibited by genistein. P. gingivalis was more sensitive to genistein. The growth of P. gingivalis and A. actinomycetemcomitans, was completely inhibited by genistein at concentrations of 80 and 160 μM, respectively.

Fig. 1. Determination of antibacterial activity of genistein against Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa). Serial doses of genistein were added to bacterial cultures (1×10⁷ CFU/mL) in 96-well plates and incubated for 48 hr. The growth of bacteria was determined by measuring the optical density (OD) of the cultures at 600 nm. Data are means±standard deviation of triplicate wells. Similar results were obtained in two other independent experiments. The asterisk indicates significant difference (p<0.05) compared to without genistein.

Bactericidal effect of genistein against periodontal bacteria

We next investigated whether genistein could effectively kill P. gingivalis and A. actinomycetemcomitans within a short time frame. Bacterial cultures were exposed to genistein for 1 hr, after which viable counts were determined. The viability of P. gingivalis decreased by more than 2-fold upon treatment with 80 μM genistein, while the viable counts of A. actinomycetemcomitans similarly decreased by over 2-fold following treatment with 160 μM genistein (Fig. 2).

Fig. 2. Killing of Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) by genistein. Bacterial cultures (1×10⁶ CFU/mL) were treated with genistein. After 1 hr, colony counts were determined. Data are means±standard deviation of a representative experiment performed in triplicate. Similar results were obtained in two other independent experiments. (A) P. gingivalis treated with 80 μM genistein and (B) A. actinomycetemcomitans treated with 160 μM genistein. The asterisk indicates significant difference (p<0.05) compared to without genistein.

Cytotoxicity of genistein against oral epithelial cells

We investigated the effect of genistein on the viability of YD15, an oral epithelial cell line. YD15 cells were exposed to varying concentrations of genistein (50–200 μM), and cell viability was assessed using crystal violet assays. The results revealed only a slight reduction in cell viability at a high concentration of genistein (200 μM) (Fig. 3).

Fig. 3. Effect of genistein on viability of YD15 cells. 1×10⁵ YD15 cells were seeded in 48-well plates. The next day, cells were treated with varying concentrations of genistein (50–200 μM) and incubated for 24 hr. Cell viability was assessed using crystal violet assays. Data are means±standard deviation of a representative experiment performed in triplicate. Similar results were obtained in two other independent experiments. The asterisk indicates significant difference (p<0.05) compared to without genistein.

Antibiotics are often used to treat bacterial infections; however, the rise of antibiotic-resistant bacteria has prompted the search for new antibacterial agents [11]. This is the first study to demonstrate the antibacterial activity of genistein, a soybean phytochemical, against periodontal bacteria. Furthermore, viable bacterial counts indicated that genistein exhibits moderate bactericidal activity against P. gingivalis and A. actinomycetemcomitans. Thus, the growth inhibition observed in these bacteria can be attributed, at least in part, to the bactericidal effect of genistein.

In this study, we selected P. gingivalis and A. actinomycetemcomitans as representative periodontal bacteria because of their well-established status as key periodontopathogens. P. gingivalis, a keystone pathogen, plays a pivotal role in chronic periodontitis [12], while A. actinomycetemcomitans, a producer of leukotoxin, is the primary causative agent of aggressive periodontitis [13]. Notably, we found that P. gingivalis was more sensitive to genistein. The MICs of genistein for P. gingivalis and A. actinomycetemcomitans were 80 μM and 160 μM, respectively. Although our study focused on only two periodontal bacteria, the inhibitory effect of genistein may extend to numerous other periodontal pathogens, each with varying sensitivity.

Genistein, known for its ability to inhibit protein tyrosine kinases, can strongly inhibit the production of proinflammatory mediators in diverse cell types stimulated by periodontal bacteria, typically at concentrations around 50 μM [14-16]. This dual action of genistein as an anti-inflammatory and antibacterial agent makes it a promising candidate for treating periodontal diseases.

We also assessed the effect of genistein on oral epithelial cells, and found that the effective concentrations at which genistein was effective against periodontal bacteria did not compromise the viability of these cells. This result suggests that genistein holds therapeutic potential, at specific concentrations against periodontal bacteria, without causing substantial harm to oral epithelial cells.

In conclusion, our study highlights the ability of genistein to effectively inhibit the growth of P. gingivalis and A. actinomycetemcomitans, while maintaining the viability of oral epithelial cells. Considering its established anti-inflammatory properties and its novel antibacterial capabilities against periodontal bacteria, genistein can serve as a valuable alternative to conventional periodontal treatments.

None.

The authors declare that they have no competing interests.