Ursolic acid disrupts cariogenic biofilms with selective antibacterial activity against Streptococcus mutans and Streptococcus sobrinus
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Abstract
Prevention of dental caries requires effective control of biofilm-forming Streptococcus spp. The present study evaluated the antimicrobial and antibiofilm properties of ursolic acid (UA) and beta-sitosterol against cariogenic bacteria. UA demonstrated selective antimicrobial activity, with MIC/MBC values of 8 and 16 μM against Streptococcus mutans and Streptococcus sobrinus, respectively. At 16 μM, UA reduced biofilm adherence by 95% and 90% in S. mutans and S. sobrinus, respectively. Confocal microscopy revealed a dose-dependent reduction in biofilm thickness, decreasing from 35.2 ± 2.3 μm to 17.3 ± 1.5 μm for S. mutans and from 64.3 ± 1.8 μm to 42.1 ± 1.2 μm for S. sobrinus following treatment with 64 μM UA. At this concentration, bacterial viability decreased to 40% and 62% for S. mutans and S. sobrinus, respectively. In comparison, 0.12% chlorhexidine (positive control) reduced biofilm thickness to 15.1 ± 2.5 μm and 32.5 ± 1.9 μm, with viability decreasing to 36% and 52% for S. mutans and S. sobrinus, respectively. Unlike UA, beta-sitosterol showed negligible antibacterial activity. These findings demonstrate the potential of UA as a promising natural therapeutic agent against cariogenic biofilms, although its efficacy remains lower than that of chlorhexidine.
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References
Amanpour, S., Akbari Javar, M., Sarhadinejad, Z., Doustmohammadi, M., Moghadari, M., & Sarhadynejad, Z. (2023). A systematic review of medicinal plants and herbal products’ effectiveness in oral health and dental cure with health promotion approach. Journal of Education and Health Promotion, 12(1), Article 306. https://doi.org/10.4103/jehp.jehp_1297_22
Balakrishnan, M., Simmonds, R. S., & Tagg, J. R. (2000). Dental caries is a preventable infectious disease. Australian Dental Journal, 45(4), 235–245. https://doi.org/10.1111/j.1834-7819.2000.tb00257.x
Bowen, W. H., & Koo, H. (2011). Biology of Streptococcus mutans- derived glucosyltransferases: Role in extracellular matrix formation of cariogenic biofilms. Caries Research, 45(1), 69–86. https://doi.org/10.1159/000324598
Brookes, Z. L. S., Bescos, R., Belfield, L. A., Ali, K., & Roberts, A. (2020). Current uses of chlorhexidine for management of oral disease: A narrative review. Journal of Dentistry, 103, Article 103497. https://doi.org/10.1016/j.jdent.2020.103497
Burne, R. A., Ahn, S.-J., Wen, Z. T., Zeng, L., Lemos, J. A., Abranches, J., & Nascimento, M. (2009). Opportunities for disrupting cariogenic biofilms. Advances in Dental Research, 21(1), 17–20. https://doi.org/10.1177/0895937409335593
Chatzigiannidou, I., Teughels, W., Van de Wiele, T., & Boon, N. (2020). Oral biofilms exposure to chlorhexidine results in altered microbial composition and metabolic profile. npj Biofilms and Microbiomes, 6, Article 13. https://doi.org/10.1038/s41522-020-0124-3
Chen, R., Du, M., & Liu, C. (2022). Strategies for dispersion of cariogenic biofilms: Applications and mechanisms. Frontiers in Microbiology, 13, Article 981203. https://doi.org/10.3389/fmicb.2022.981203
Choi, A., Dong, K., Williams, E., Pia, L., Batagower, J., Bending, P., Shin, I., Peters, D. I., & Kaspar, J. R. (2024). Human saliva modifies growth, biofilm architecture, and competitive behaviors of oral streptococci. mSphere, 9(2), Article e00771-23. https://doi.org/10.1128/msphere.00771-23
Dutta, P. P., Marbaniang, K., Sen, S., Dey, B. K., & Talukdar, N. C. (2023). A review on phytochemistry of Paederia foetida Linn. Phytomedicine Plus, 3(1), Article 100411. https://doi.org/10.1016/j.phyplu.2023.100411
Forssten, S. D., Björklund, M., & Ouwehand, A. C. (2010). Streptococcus mutans, caries and simulation models. Nutrients, 2(3), 290–298. https://doi.org/10.3390/nu2030290
Gouda, N. A., Alshammari, S. O., Abourehab, M. A. S., Alshammari, Q. A., & Elkamhawy, A. (2023). Therapeutic potential of natural products in inflammation: Underlying molecular mechanisms, clinical outcomes, technological advances, and future perspectives. Inflammopharmacology, 31(6), 2857–2883. https://doi.org/10.1007/s10787-023-01366-y
Ham, Y., & Kim, T.-J. (2023). Synergistic inhibitory activity of Glycyrrhizae Radix and Rubi Fructus extracts on biofilm formation of Streptococcus mutans. BMC Complementary Medicine and Therapies, 23, Article 22. https://doi.org/10.1186/s12906-023-03861-9
Hasan, S., Danishuddin, M., Adil, M., Singh, K., Verma, P. K., & Khan, A. U. (2012). Efficacy of E. officinalis on the cariogenic properties of Streptococcus mutans: A novel and alternative approach to suppress quorum-sensing mechanism. PLoS ONE, 7(7), Article e40319. https://doi.org/10.1371/journal.pone.0040319
Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K., & Molin, S. (2000). Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology, 146(10), 2395–2407. https://doi.org/10.1099/00221287-146-10-2395
Hope, C. K., & Wilson, M. (2004). Analysis of the effects of chlorhexidine on oral biofilm vitality and structure based on viability profiling and an indicator of membrane integrity. Antimicrobial Agents and Chemotherapy, 48(5), 1461–1468. https://doi.org/10.1128/AAC.48.5.1461-1468.2004
Kim, M. J., Kim, C. S., Park, J.-Y., Lim, Y. K., Park, S.-N., Ahn, S.-J., Jin, D.-C., Kim, T. H. & Kook, J.-K. (2011). Antimicrobial effects of ursolic acid against mutans streptococci isolated from Koreans. International Journal of Oral Biology, 36(1), 7–11.
Klaophimai, S., Pouyfung, P., Klaophimai, A., & Chairatvit, K. (2023). Bactericidal and anti-biofilm properties against Streptococcus mutans and Streptococcus sobrinus of rhinacanthin-C isolated from Rhinacanthus nasutus. Songklanakarin Journal of Science and Technology, 45(5), 591–598.
Kolenbrander, P. E., Andersen, R. N., Blehert, D. S., Egland, P. G., Foster, J. S., & Palmer, R. J. (2002). Communication among oral bacteria. Microbiology and Molecular Biology Reviews, 66(3), 486–505. https://doi.org/10.1128/MMBR.66.3.486-505.2002
Krzyściak, W., Jurczak, A., Kościelniak, D., Bystrowska, B., & Skalniak, A. (2014). The virulence of Streptococcus mutans and the ability to form biofilms. European Journal of Clinical Microbiology & Infectious Diseases, 33(4), 499–515. https://doi.org/10.1007/s10096-013-1993-7
Lamarque, G. C. C., Méndez, D. A. C., Gutierrez, E., Dionisio, E. J., Machado, M. A. A. M., Oliveira, T. M., Rios, D., & Cruvinel, T. (2019). Could chlorhexidine be an adequate positive control for antimicrobial photodynamic therapy in- in vitro studies? Photodiagnosis and Photodynamic Therapy, 25, 58–62. https://doi.org/10.1016/j.pdpdt.2018.11.004
Li, B., Li, X., Lin, H., & Zhou, Y. (2018). Curcumin as a promising antibacterial agent: Effects on metabolism and biofilm formation in S. mutans. BioMed Research International, 2018(1), Article 4508709. https://doi.org/10.1155/2018/4508709
Li, X., Yin, L., Ramage, G., Li, B., Tao, Y., Zhi, Q., Lin, H., & Zhou, Y. (2019). Assessing the impact of curcumin on dual-species biofilms formed by Streptococcus mutans and Candida albicans. MicrobiologyOpen, 8(12), Article e937. https://doi.org/10.1002/mbo3.937
Lin, Y., Chen, J., Zhou, X., & Li, Y. (2021). Inhibition of Streptococcus mutans biofilm formation by strategies targeting the metabolism of exopolysaccharides. Critical Reviews in Microbiology, 47(5), 667–677. https://doi.org/10.1080/1040841X.2021.1915959
Liu, G., Qin, P., Cheng, X., Wu, L., Zhao, W., & Gao, W. (2024). Evaluation of the mechanistic basis for the antibacterial activity of ursolic acid against Staphylococcus aureus. Frontiers in Microbiology, 15, Article 1389242. https://doi.org/10.3389/fmicb.2024.1389242
Liu, Y., Huang, Y., Fan, C., Chi, Z., Bai, M., Sun, L., Yang, L., Yu, C., Song, Z., Yang, X., Yi, J., Wang, S., Liu, L., Wang, G., & Zheng, L. (2021). Ursolic acid targets glucosyltransferase and inhibits its activity to prevent Streptococcus mutans biofilm formation. Frontiers in Microbiology, 12, Article 743305. https://doi.org/10.3389/fmicb.2021.743305
Loesche, W. J. (1986). Role of Streptococcus mutans in human dental decay. Microbiological Reviews, 50(4), 353–380. https://doi.org/10.1128/mr.50.4.353-380.1986
Lu, L., Hu, W., Tian, Z., Yuan, D., Yi, G., Zhou, Y., Cheng, Q., Zhu, J., & Li, M. (2019). Developing natural products as potential anti-biofilm agents. Chinese Medicine, 14, Article 11. https://doi.org/10.1186/s13020-019-0232-2
Lyu, X., Wang, L., Shui, Y., Jiang, Q., Chen, L., Yang, W., He, X., Zeng, J., & Li, Y. (2021). Ursolic acid inhibits multi-species biofilms developed by Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii. Archives of Oral Biology, 125, Article 105107. https://doi.org/10.1016/j.archoralbio.2021.105107
Manosroi, J., Manosroi, A., & Rungruangsri, U. (2006). Translation of Lanna medicinal-plant recipes for research and development of modern pharmaceuticals and the understanding of the Lanna Thai cultures/histories. Chiang Mai University Journal of Natural Sciences, 5(3), 437–442. http://cmuir.cmu.ac.th/jspui/handle/6653943832/61980
Maruo, I. T., Rosa, E. A. R., Maruo, H., Tanaka, O., Guariza Filho, O., Ignácio, S. A., & Camargo, E. S. (2008). Effect of chlorhexidine mouth rinse on streptococci counts of tooth-tissue-borne palatal expander biofilm. Orthodontics & Craniofacial Research, 11(3), 136–142. https://doi.org/10.1111/j.1601-6343.2007.00418.x
Pitts, N. B., Twetman, S., Fisher, J., & Marsh, P. D. (2021). Understanding dental caries as a non-communicable disease. British Dental Journal, 231(12), 749–753. https://doi.org/10.1038/s41415-021-3775-4
Poppolo Deus, F., & Ouanounou, A. (2022). Chlorhexidine in dentistry: Pharmacology, uses, and adverse effects. International Dental Journal, 72(3), 269–277. https://doi.org/10.1016/j.identj.2022.01.005
Pouyfung, P., Kuraeiad, S., Yimthiang, S., & Khamphaya, T. (2023). Long-term oral administration of Paederia foetida decreases cytochrome P450 mRNA expression: The predictive approaches in a rat model. Journal of Applied Pharmaceutical Science, 13(2), 021–028. https://doi.org/10.7324/JAPS.2023.130203
Shen, Y., Stojicic, S., & Haapasalo, M. (2011). Antimicrobial efficacy of chlorhexidine against bacteria in biofilms at different stages of development. Journal of Endodontics, 37(5), 657–661. https://doi.org/10.1016/j.joen.2011.02.007
Smith, K. P., & Kirby, J. E. (2018). The inoculum effect in the era of multidrug resistance: Minor differences in inoculum have dramatic effect on MIC determination. Antimicrobial Agents and Chemotherapy, 62(8), Article e00433-18. https://doi.org/10.1128/AAC.00433-18
Subramenium, G. A., Vijayakumar, K., & Pandian, S. K. (2015). Limonene inhibits streptococcal biofilm formation by targeting surface-associated virulence factors. Journal of Medical Microbiology, 64(8), 879–890. https://doi.org/10.1099/jmm.0.000105
Takahashi, N., & Nyvad, B. (2010). The role of bacteria in the caries process: Ecological perspectives. Sage Journal, 90(3), 294–303. https://doi.org/10.1177/0022034510379602
Trombetta, D., Castelli, F., Sarpietro, M. G., Venuti, V., Cristani, M., Daniele, C., Saija, A., Mazzanti, G., & Bisignano, G. (2005). Mechanisms of antibacterial action of three monoterpenes. Antimicrobial Agents and Chemotherapy, 49(6), 2474–2478. https://doi.org/10.1128/AAC.49.6.2474-2478.2005
Veenman, F., van Dijk, A., Arredondo, A., Medina-Gomez, C., Wolvius, E., Rivadeneira, F., Àlvarez, G., Blanc, V., & Kragt, L. (2024). Oral microbiota of adolescents with dental caries: A systematic review. Archives of Oral Biology, 161, Article 105933. https://doi.org/10.1016/j.archoralbio.2024.105933
Vorregaard, M. (2008). Comstat2-a modern 3D image analysis environment for biofilms [Master’s thesis, Technical University of Denmark]. DTU Research Output. https://www2.imm.dtu.dk/pubdb/edoc/imm5628.pdf
Waldman, L. J., Butera, T., Boyd, J. D., & Grady, M. E. (2023). Sucrose-mediated formation and adhesion strength of Streptococcus mutans biofilms on titanium. Biofilm, 6, Article 100143. https://doi.org/10.1016/j.bioflm.2023.100143
Wang, L., Jiang, Y., Han, T., Zheng, C., & Qin, L. (2014). A phytochemical, pharmacological and clinical profile of Paederia foetida and P. scandens. Natural Product Communications, 9(6), 879–886. https://doi.org/10.1177/1934578X1400900640
Zhang, Q., Ma, Q., Wang, Y., Wu, H., & Zou, J. (2021). Molecular mechanisms of inhibiting glucosyltransferases for biofilm formation in Streptococcus mutans. International Journal of Oral Science, 13, Article 30. https://doi.org/10.1038/s41368-021-00137-1