Oral Biol Res 2023; 47(4): 159-163  https://doi.org/10.21851/obr.47.04.202312.159
Antibacterial effect of genistein against periodontal pathogens
So-Hee Kim1 and In-Chol Kang2*
1Research Fellow, Department of Oral Microbiology, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
2Professor, Department of Oral Microbiology, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
Correspondence to: In-Chol Kang, Department of Oral Microbiology, School of Dentistry, Chonnam National University, 33, Yongbong-ro, Buk-gu, Gwangju 61186, Republic of Korea.
Tel: +82-62-530-4851, E-mail: ickang@jnu.ac.kr
Received: September 22, 2023; Revised: November 6, 2023; Accepted: November 15, 2023; Published online: December 31, 2023.
© Oral Biology Research. All rights reserved.

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Genistein, a soy isoflavone, has been widely used as an anti-inflammatory agent in vitro and in vivo. Although it exhibits antibacterial activity against several bacterial species, its effect on periodontal bacteria remains unknown. This study aimed to investigate the antibacterial activity of genistein against two typical periodontal pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. The broth microdilution method revealed that genistein inhibited bacterial growth at minimum inhibitory concentrations of 80–160 μM. Viable bacterial counts further confirmed its bactericidal activity against P. gingivalis and A. actinomycetemcomitans, as evidenced by decreased bacterial counts in the presence of genistein. Remarkably, the viability of YD15, a human oral epithelial cell line, was only slightly reduced at a high concentration of genistein. These findings indicate that genistein demonstrates antibacterial activity against periodontal pathogens, which could bear significant clinical implications for periodontal treatment.
Keywords: Aggregatibacter actinomycetemcomitans; Antibacterial agents; Genistein; Periodontal diseases; Porphyromonas gingivalis
Introduction

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.

Materials and Methods

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.

Results

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.
Discussion

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.

Funding

None.

Conflicts of Interest

The authors declare that they have no competing interests.

References
  1. Genco RJ, Sanz M. Clinical and public health implications of periodontal and systemic diseases: an overview. Periodontol 2000 2020;83:7-13. doi: 10.1111/prd.12344.
    Pubmed CrossRef
  2. Zhang M, Whiteley M, Lewin GR. Polymicrobial interactions of oral microbiota: a historical review and current perspective. mBio 2022;13:e0023522. doi: 10.1128/mbio.00235-22.
    Pubmed KoreaMed CrossRef
  3. Herbert BA, Novince CM, Kirkwood KL. Aggregatibacter actinomycetemcomitans, a potent immunoregulator of the periodontal host defense system and alveolar bone homeostasis. Mol Oral Microbiol 2016;31:207-227. doi: 10.1111/omi.12119.
    Pubmed KoreaMed CrossRef
  4. Xu W, Zhou W, Wang H, Liang S. Roles of Porphyromonas gingivalis and its virulence factors in periodontitis. Adv Protein Chem Struct Biol 2020;120:45-84. doi: 10.1016/bs.apcsb.2019.12.001.
    Pubmed KoreaMed CrossRef
  5. Goh YX, Jalil J, Lam KW, Husain K, Premakumar CM. Genistein: a review on its anti-inflammatory properties. Front Pharmacol 2022;13:820969. doi: 10.3389/fphar.2022.820969.
    Pubmed KoreaMed CrossRef
  6. Bae EA, Han MJ, Kim DH. In vitro anti-Helicobacter pylori activity of irisolidone isolated from the flowers and rhizomes of Pueraria thunbergiana. Planta Med 2001;67:161-163. doi: 10.1055/s-2001-11499.
    Pubmed CrossRef
  7. Hong H, Landauer MR, Foriska MA, Ledney GD. Antibacterial activity of the soy isoflavone genistein. J Basic Microbiol 2006;46:329-335. doi: 10.1002/jobm.200510073.
    Pubmed CrossRef
  8. Ulanowska K, Tkaczyk A, Konopa G, Wegrzyn G. Differential antibacterial activity of genistein arising from global inhibition of DNA, RNA and protein synthesis in some bacterial strains. Arch Microbiol 2006;184:271-278. doi: 10.1007/s00203-005-0063-7.
    Pubmed CrossRef
  9. Brook I, Wexler HM, Goldstein EJ. Antianaerobic antimicrobials: spectrum and susceptibility testing. Clin Microbiol Rev 2013;26:526-546. doi: 10.1128/CMR.00086-12.
    Pubmed KoreaMed CrossRef
  10. Elsehly WM, Mourad GM, Mehanna RA, Kholief MA, El-Nikhely NA, Awaad AK, Attia MH. The potential implications of estrogenic and antioxidant-dependent activities of high doses of methyl paraben on MCF7 breast cancer cells. J Biochem Mol Toxicol 2022;36:e23012. doi: 10.1002/jbt.23012.
    Pubmed CrossRef
  11. Vila J, Moreno-Morales J, Ballesté-Delpierre C. Current landscape in the discovery of novel antibacterial agents. Clin Microbiol Infect 2020;26:596-603. doi: 10.1016/j.cmi.2019.09.015.
    Pubmed CrossRef
  12. Hajishengallis G, Darveau RP, Curtis MA. The keystone-pathogen hypothesis. Nat Rev Microbiol 2012;10:717-725. doi: 10.1038/nrmicro2873.
    Pubmed KoreaMed CrossRef
  13. Mehta J, Eaton C, AlAmri M, Lin GH, Nibali L. The association between Aggregatibacter actinomycetemcomitans JP2 clone and periodontitis: a systematic review and meta-analysis. J Periodontal Res 2023;58:465-482. doi: 10.1111/jre.13102.
    Pubmed CrossRef
  14. Bhattarai G, Poudel SB, Kook SH, Lee JC. Anti-inflammatory, anti-osteoclastic, and antioxidant activities of genistein protect against alveolar bone loss and periodontal tissue degradation in a mouse model of periodontitis. J Biomed Mater Res A 2017;105:2510-2521. doi: 10.1002/jbm.a.36109.
    Pubmed CrossRef
  15. Gutiérrez-Venegas G, Bando-Campos CG. The flavonoids luteolin and quercetagetin inhibit lipoteichoic acid actions on H9c2 cardiomyocytes. Int Immunopharmacol 2010;10:1003-1009. doi: 10.1016/j.intimp.2010.05.012.
    Pubmed CrossRef
  16. Luo LJ, Liu F, Lin ZK, Xie YF, Xu JL, Tong QC, Shu R. Genistein regulates the IL-1 beta induced activation of MAPKs in human periodontal ligament cells through G protein-coupled receptor 30. Arch Biochem Biophys 2012;522:9-16. doi: 10.1016/j.abb.2012.04.007.
    Pubmed CrossRef


This Article

e-submission

Archives