{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T19:19:04Z","timestamp":1790882344135,"version":"4.1.0"},"reference-count":47,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,10,2]],"date-time":"2025-10-02T00:00:00Z","timestamp":1759363200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Objective<\/jats:title>\n                    <jats:p>\n                      This study aimed to investigate the effect of the natural extract of Stevioside on biofilm formation and cariogenic virulence factors of\n                      <jats:italic>Streptococcus mutans (S. mutans)<\/jats:italic>\n                      , and to explore its mechanism of action preliminarily, with the goal of identifying a safer and more effective non-cariogenic sweetener.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      The inhibitory effect of Stevioside on the growth of\n                      <jats:italic>S. mutans<\/jats:italic>\n                      biofilm was detected by crystal violet staining. The acid production capacity of\n                      <jats:italic>S. mutans<\/jats:italic>\n                      biofilms was evaluated by measuring the pH values and lactic acid contents. The bacterial viability within the biofilms was determined using the plate counting method. Scanning electron microscopy (SEM) and laser confocal microscopy (CLSM) were used to observe the biofilm structure, and the fluorescence intensity of live and dead bacteria and biofilm thickness were further analyzed. The anthrone sulfuric acid method quantified the production of Soluble Extracellular Polysaccharide (SEPS) and Insoluble Extracellular Polysaccharide (IEPS) in biofilm. Furthermore, real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of genes related to biofilm growth, acid production, acid tolerance, exopolysaccharide synthesis, two-component signal and quorum sensing.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Stevioside significantly inhibited the formation of\n                      <jats:italic>S. mutans<\/jats:italic>\n                      biofilm, and reduced acid production, bacterial activity and EPS production. SEM and CLSM confirmed the reduction of the dense three-dimensional structure of biofilm. In addition, compared with sucrose, the expression of related genes was down-regulated in Stevioside.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      Stevioside inhibits\n                      <jats:italic>S. mutans<\/jats:italic>\n                      virulence factors and biofilms and is a promising natural sucrose substitute for preventing dental caries.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fmicb.2025.1675322","type":"journal-article","created":{"date-parts":[[2025,10,2]],"date-time":"2025-10-02T05:30:58Z","timestamp":1759383058000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["Inhibition of Streptococcus mutans biofilm formation and virulence by natural extract Stevioside"],"prefix":"10.3389","volume":"16","author":[{"given":"Rong","family":"Ma","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peiling","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanyuan","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yimin","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinpu","family":"Chu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,10,2]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"116","DOI":"10.4103\/2231-0762.146215","article-title":"Effect of aqueous and alcoholic stevia (Stevia rebaudiana) extracts against Streptococcus mutans and Lactobacillus acidophilus in comparison to chlorhexidine: an in vitro study","volume":"4","author":"Ajagannanavar","year":"2014","journal-title":"J. Int. Soc. Prev. Commun. Dent."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"267","DOI":"10.3390\/dj11120267","article-title":"Sugar substitute stevia inhibits biofilm formation, exopolysaccharide production, and downregulates the expression of streptococcal genes involved in exopolysaccharide synthesis","volume":"11","author":"AlKanderi","year":"2023","journal-title":"Dent. J."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"94","DOI":"10.2174\/1871525718666200207105436","article-title":"Stevia as a natural sweetener: a review","volume":"18","author":"Arumugam","year":"2020","journal-title":"Cardiovasc. Hematol. Agents Med. 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Ecol."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"996","DOI":"10.1128\/jcm.22.6.996-1006.1985","article-title":"Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices","volume":"22","author":"Christensen","year":"1985","journal-title":"J. Clin. Microbiol."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"214","DOI":"10.1159\/000354410","article-title":"Sucrose substitutes affect the cariogenic potential of Streptococcus mutans biofilms","volume":"48","author":"Durso","year":"2014","journal-title":"Caries Res."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"e20180593","DOI":"10.1590\/1678-7757-2018-0593","article-title":"Role of Candida albicans on enamel demineralization and on acidogenic potential of Streptococcus mutans in vitro biofilms","volume":"27","author":"Eidt","year":"2019","journal-title":"J. Appl. Oral Sci."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"3237","DOI":"10.1007\/s00784-020-03197-5","article-title":"Growth and viability of Streptococcus mutans in sucrose with different concentrations of Stevia rebaudiana Bertoni","volume":"24","author":"Escobar","year":"2020","journal-title":"Clin. Oral Investig."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"258","DOI":"10.1016\/j.archoralbio.2010.09.018","article-title":"Correlation of biological properties with glucan-binding protein B expression profile in Streptococcus mutans clinical isolates","volume":"56","author":"Fujita","year":"2011","journal-title":"Arch. Oral Biol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"1116","DOI":"10.1016\/j.archoralbio.2013.03.005","article-title":"Cariogenic potential of commercial sweeteners in an experimental biofilm caries model on enamel","volume":"58","author":"Giacaman","year":"2013","journal-title":"Arch. Oral Biol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"1","DOI":"10.3109\/09637480903193049","article-title":"Stevia (Stevia rebaudiana) a bio-sweetener: a review","volume":"61","author":"Goyal","year":"2009","journal-title":"Int. J. Food Sci. Nutr."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"e01012-20","DOI":"10.1128\/aem.01012-20","article-title":"Effect of rubusoside, a natural sucrose substitute, on Streptococcus mutans biofilm cariogenic potential and virulence gene expression in vitro","volume":"86","author":"Guan","year":"2020","journal-title":"Appl. Environ. Microbiol."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"1128668","DOI":"10.3389\/fmicb.2023.1128668","article-title":"Inhibitory effects of stevioside on Streptococcus mutans and Candida albicans dual-species biofilm","volume":"14","author":"Guo","year":"2023","journal-title":"Front. Microbiol."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"330","DOI":"10.1128\/am.16.2.330-334.1968","article-title":"Plate-dilution frequency technique for assay of microbial ecology","volume":"16","author":"Harris","year":"1968","journal-title":"Appl. Microbiol."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"2807","DOI":"10.1093\/nar\/gkaa112","article-title":"Cyclic di-AMP, a second messenger of primary importance: tertiary structures and binding mechanisms","volume":"48","author":"He","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"349","DOI":"10.1007\/s12088-015-0534-8","article-title":"Plausible drug targets in the Streptococcus mutans quorum sensing pathways to combat dental biofilms and associated risks","volume":"55","author":"Kaur","year":"2015","journal-title":"Indian J. Microbiol."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"86","DOI":"10.1016\/j.jdsr.2016.12.002","article-title":"Role of Streptococcus mutans two-component systems in antimicrobial peptide resistance in the oral cavity","volume":"53","author":"Kawada-Matsuo","year":"2017","journal-title":"Jpn. Dent. Sci. Rev."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"623980","DOI":"10.3389\/fcimb.2020.623980","article-title":"Synergism of Streptococcus mutans and Candida albicans reinforces biofilm maturation and acidogenicity in saliva: an in vitro study","volume":"10","author":"Kim","year":"2021","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"837","DOI":"10.1128\/aem.01299-08","article-title":"Structural and molecular basis of the role of starch and sucrose in Streptococcus mutans biofilm development","volume":"75","author":"Klein","year":"2009","journal-title":"Appl. Environ. Microbiol."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"116","DOI":"10.1159\/000296306","article-title":"Influence of cranberry proanthocyanidins on formation of biofilms by Streptococcus mutans on saliva-coated apatitic surface and on dental caries development in vivo","volume":"44","author":"Koo","year":"","journal-title":"Caries Res."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"3024","DOI":"10.1128\/jb.01649-09","article-title":"Exopolysaccharides produced by Streptococcus mutans glucosyltransferases modulate the establishment of microcolonies within multispecies biofilms","volume":"192","author":"Koo","year":"","journal-title":"J. Bacteriol."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"167","DOI":"10.21775\/cimb.032.167","article-title":"The VicRK two-component system regulates Streptococcus mutans virulence","volume":"32","author":"Lei","year":"2019","journal-title":"Curr. Issues Mol. Biol."},{"key":"ref26","doi-asserted-by":"publisher","DOI":"10.1128\/microbiolspec.gpp3-0051-2018","article-title":"The biology of Streptococcus mutans","volume":"7","author":"Lemos","year":"2019","journal-title":"Microbiol. Spectr."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"602305","DOI":"10.3389\/fmicb.2020.602305","article-title":"The multifaceted nature of streptococcal antigen I\/II proteins in colonization and disease pathogenesis","volume":"11","author":"Manzer","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"441","DOI":"10.1016\/j.cden.2010.03.002","article-title":"Microbiology of dental plaque biofilms and their role in oral health and caries","volume":"54","author":"Marsh","year":"2010","journal-title":"Dent. Clin. N. 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Scand."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"89","DOI":"10.2147\/ccide.s55761","article-title":"The effect of xylitol on dental caries and oral flora","volume":"6","author":"Nayak","year":"2014","journal-title":"Clin. Cosmet. Investig. Dent."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"749","DOI":"10.1038\/s41415-021-3775-4","article-title":"Understanding dental caries as a non-communicable disease","volume":"231","author":"Pitts","year":"2021","journal-title":"Br. Dent. J."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"17030","DOI":"10.1038\/nrdp.2017.30","article-title":"Dental caries","volume":"3","author":"Pitts","year":"2017","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref35","doi-asserted-by":"publisher","first-page":"3823426","DOI":"10.1155\/2018\/3823426","article-title":"Stevia prevents acute and chronic liver injury induced by carbon tetrachloride by blocking oxidative stress through Nrf2 upregulation","volume":"2018","author":"Ramos-Tovar","year":"2018","journal-title":"Oxidative Med. Cell. Longev."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"1186S","DOI":"10.1093\/jn\/nxy102","article-title":"Stevia leaf to stevia sweetener: exploring its science, benefits, and future potential","volume":"148","author":"Samuel","year":"2018","journal-title":"J. Nutr."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"475","DOI":"10.1080\/1040841x.2020.1796579","article-title":"Guardian genes ensuring subsistence of oral Streptococcus mutans","volume":"46","author":"Shanmugam","year":"2020","journal-title":"Crit. Rev. Microbiol."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"1341","DOI":"10.1177\/0022034515590377","article-title":"Diet and dental caries","volume":"94","author":"Sheiham","year":"2015","journal-title":"J. Dent. Res."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"96","DOI":"10.1016\/j.mib.2014.02.008","article-title":"Biofilm dispersion and quorum sensing","volume":"18","author":"Solano","year":"2014","journal-title":"Curr. Opin. Microbiol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"e58271","DOI":"10.1371\/journal.pone.0058271","article-title":"CovR and VicRK regulate cell surface biogenesis genes required for biofilm formation in Streptococcus mutans","volume":"8","author":"Stipp","year":"2013","journal-title":"PLoS One"},{"key":"ref41","doi-asserted-by":"publisher","first-page":"1628","DOI":"10.1177\/0022034515606045","article-title":"Oral microbiome metabolism","volume":"94","author":"Takahashi","year":"2015","journal-title":"J. Dent. Res."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"505","DOI":"10.1177\/154405910208100715","article-title":"Novel sucrose-dependent adhesion co-factors in Streptococcus mutans","volume":"81","author":"Tao","year":"2002","journal-title":"J. Dent. Res."},{"key":"ref43","doi-asserted-by":"publisher","first-page":"5633","DOI":"10.1128\/aem.01049-07","article-title":"Acid tolerance of biofilm cells of Streptococcus mutans","volume":"73","author":"Welin-Neilands","year":"2007","journal-title":"Appl. Environ. Microbiol."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"e1002623","DOI":"10.1371\/journal.ppat.1002623","article-title":"The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm","volume":"8","author":"Xiao","year":"2012","journal-title":"PLoS Pathog."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"19943","DOI":"10.1038\/s41598-019-56486-9","article-title":"Diversity of SpaP in genetic and salivary agglutinin mediated adherence among Streptococcus mutans strains","volume":"9","author":"Yang","year":"2019","journal-title":"Sci. Rep."},{"key":"ref46","doi-asserted-by":"publisher","first-page":"833","DOI":"10.1128\/jb.02042-12","article-title":"Comprehensive mutational analysis of sucrose-metabolizing pathways in Streptococcus mutans reveals novel roles for the sucrose phosphotransferase system permease","volume":"195","author":"Zeng","year":"2012","journal-title":"J. Bacteriol."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"201","DOI":"10.21775\/cimb.032.201","article-title":"ComDE two-component signal transduction systems in oral streptococci: structure and function","volume":"32","author":"Zu","year":"2019","journal-title":"Curr. Issues Mol. 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