{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T11:07:56Z","timestamp":1783508876962,"version":"3.55.0"},"reference-count":51,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,9,28]],"date-time":"2021-09-28T00:00:00Z","timestamp":1632787200000},"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:p>The self-produced matrix of biofilms, consisting of extracellular polymeric substances, plays an important role in biofilm adhesion to surfaces and the structural integrity of biofilms. In dentistry, biofilms cause multiple diseases such as caries, periodontitis, and pulpitis. Disruption of these biofilms adhering to dental hard tissues may pose a major challenge since biofilms show higher tolerance to antimicrobials and antibiotics than planktonic cells. In this study, the effect of low concentrations of chlorhexidine (CHX) on enzyme-treated multispecies oral biofilm was investigated in an <jats:italic>in vitro<\/jats:italic> model. Six-species biofilms were enzymatically treated by anaerobic growth in a medium containing DNase I and proteinase K. Biofilms were exposed to a low concentration of CHX at defined time points. After 64h, biofilms were either harvested and quantified by cultural analyses or stained for confocal laser scanning microscopy (CLSM) analyses using either Live\/Dead kit or different fluorescent dyes. A mixture of YoPro1 and SYTOX<jats:sup>\u2122<\/jats:sup> Green, Fluorescent Brightener 28 (Calcofluor), and SYPRO<jats:sup>\u2122<\/jats:sup> Ruby Protein Gel Stain was used to stain total DNA, exopolysaccharides, and extracellular proteins, respectively. Extracellular DNA (eDNA) was visualized <jats:italic>via<\/jats:italic> an indirect immunofluorescence assay (Mouse anti-DNA IgG, Goat anti-Mouse IgG, Streptavidin-Cy3). Overall, the total colony-forming units significantly decreased after combined treatment with a low concentration of CHX and enzymes compared to the group treated with CHX alone (<jats:italic>p<\/jats:italic>&amp;lt;0.001). These findings also apply to five species individually (<jats:italic>Streptococcus mutans, Streptococcus oralis, Actinomyces oris, Veillonella dispar,<\/jats:italic> and <jats:italic>Candida albicans<\/jats:italic>) occurring in the biofilms, with <jats:italic>Fusobacterium nucleatum<\/jats:italic> being the only exception. Furthermore, CLSM images showed less dense biofilms and a reduction in cell numbers after combined treatment compared to the group without enzymes. The combination of enzymes capable of disturbing the matrix integrity with antimicrobial agents thus appears to be a promising approach for biofilm disruption and killing.<\/jats:p>","DOI":"10.3389\/fmicb.2021.741863","type":"journal-article","created":{"date-parts":[[2021,9,28]],"date-time":"2021-09-28T07:20:39Z","timestamp":1632813639000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":21,"title":["Low Concentrations of Chlorhexidine Inhibit the Formation and Structural Integrity of Enzyme-Treated Multispecies Oral Biofilms"],"prefix":"10.3389","volume":"12","author":[{"given":"Kay Andrin","family":"Gr\u00e4nicher","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lamprini","family":"Karygianni","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Attin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Thurnheer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,9,28]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","DOI":"10.3402\/jom.v5i0.20015","article-title":"Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K","volume":"5","author":"Ali Mohammed","year":"2013","journal-title":"J. Oral Microbiol."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"587","DOI":"10.1128\/iai.53.3.587-594.1986","article-title":"Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis","volume":"53","author":"Aoki","year":"1986","journal-title":"Infect. Immun."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"353","DOI":"10.1016\/j.jpor.2018.01.005","article-title":"Effectiveness of a combination denture-cleaning method versus a mechanical method: comparison of denture cleanliness, patient satisfaction, and oral health-related quality of life","volume":"62","author":"Baba","year":"2018","journal-title":"J. Prosthodont. Res."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"587","DOI":"10.3389\/fmicb.2019.00587","article-title":"Resistance toward chlorhexidine in oral bacteria \u2013 is there cause for concern?","volume":"10","author":"Cieplik","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"711","DOI":"10.1146\/annurev.mi.49.100195.003431","article-title":"Microbial biofilms","volume":"49","author":"Costerton","year":"1995","journal-title":"Annu. Rev. Microbiol."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"3642","DOI":"10.1016\/j.jmb.2018.07.026","article-title":"Functional amyloid and other protein fibers in the biofilm matrix","volume":"430","author":"Erskine","year":"2018","journal-title":"J. Mol. Biol."},{"key":"ref7","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms4040041","article-title":"EPS-then and now","volume":"4","author":"Flemming","year":"2016","journal-title":"Microorganisms"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"623","DOI":"10.1038\/nrmicro2415","article-title":"The biofilm matrix","volume":"8","author":"Flemming","year":"2010","journal-title":"Nat. Rev. Microbiol."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"563","DOI":"10.1038\/nrmicro.2016.94","article-title":"Biofilms: an emergent form of bacterial life","volume":"14","author":"Flemming","year":"2016","journal-title":"Nat. Rev. Microbiol."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"459","DOI":"10.1128\/iai.42.2.459-470.1983","article-title":"Antigenic heterogeneity of Bacteroides intermedius as recognized by monoclonal antibodies","volume":"42","author":"Gm\u00fcr","year":"1983","journal-title":"Infect. Immun."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"3085","DOI":"10.1093\/jac\/dkx265","article-title":"Synergistic antibiotic activity against planktonic and biofilm-embedded Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis","volume":"72","author":"Gonzalez Moreno","year":"2017","journal-title":"J. Antimicrob. Chemother."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"363","DOI":"10.1177\/00220345010800011201","article-title":"Validation of an in vitro biofilm model of supragingival plaque","volume":"80","author":"Guggenheim","year":"2001","journal-title":"J. Dent. Res."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"570","DOI":"10.1562\/0031-8655(2002)075<0570:HODIMB>2.0.CO;2","article-title":"Heterogeneity of diffusion inside microbial biofilms determined by fluorescence correlation spectroscopy under two-photon excitation","volume":"75","author":"Guiot","year":"2002","journal-title":"Photochem. Photobiol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"1999","DOI":"10.1128\/iai.56.8.1999-2005.1988","article-title":"Isolation and characterization of the Streptococcus mutans gtfC gene, coding for synthesis of both soluble and insoluble glucans","volume":"56","author":"Hanada","year":"1988","journal-title":"Infect. Immun."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"1461","DOI":"10.1128\/AAC.48.5.1461-1468.2004","article-title":"Analysis of the effects of chlorhexidine on oral biofilm vitality and structure based on viability profiling and an indicator of membrane integrity","volume":"48","author":"Hope","year":"2004","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"470","DOI":"10.1128\/AEM.02073-07","article-title":"Differential roles of poly-N-acetylglucosamine surface polysaccharide and extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis biofilms","volume":"74","author":"Izano","year":"2008","journal-title":"Appl. Environ. Microbiol."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"467","DOI":"10.1111\/lam.12134","article-title":"Life after death: the critical role of extracellular DNA in microbial biofilms","volume":"57","author":"Jakubovics","year":"2013","journal-title":"Lett. Appl. Microbiol."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1038\/ja.2011.113","article-title":"Recombinant human DNase I decreases biofilm and increases antimicrobial susceptibility in staphylococci","volume":"65","author":"Kaplan","year":"2012","journal-title":"J. Antibiot."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"983","DOI":"10.3390\/jcm9040983","article-title":"Combined DNase and proteinase treatment interferes with composition and structural integrity of multispecies oral biofilms","volume":"9","author":"Karygianni","year":"","journal-title":"J. Clin. Med."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"668","DOI":"10.1016\/j.tim.2020.03.016","article-title":"Biofilm matrixome: extracellular components in structured microbial communities","volume":"28","author":"Karygianni","year":"","journal-title":"Trends Microbiol."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"e01137-19","DOI":"10.1128\/mBio.01137-19","article-title":"Identification of extracellular DNA-binding proteins in the biofilm matrix","volume":"10","author":"Kavanaugh","year":"2019","journal-title":"MBio"},{"key":"ref22","doi-asserted-by":"publisher","first-page":"100","DOI":"10.1159\/000324809","article-title":"Dental caries in rats associated with Candida albicans","volume":"45","author":"Klinke","year":"2011","journal-title":"Caries Res."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"471","DOI":"10.1038\/nrmicro2381","article-title":"Oral multispecies biofilm development and the key role of cell-cell distance","volume":"8","author":"Kolenbrander","year":"2010","journal-title":"Nat. Rev. Microbiol."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"740","DOI":"10.1038\/nrmicro.2017.99","article-title":"Targeting microbial biofilms: current and prospective therapeutic strategies","volume":"15","author":"Koo","year":"2017","journal-title":"Nat. Rev. Microbiol."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"a010306","DOI":"10.1101\/cshperspect.a010306","article-title":"Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era","volume":"3","author":"Kostakioti","year":"2013","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1038\/ja.2012.98","article-title":"Dispersal of Bap-mediated Staphylococcus aureus biofilm by proteinase K","volume":"66","author":"Kumar Shukla","year":"2013","journal-title":"J. Antibiot."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"2355","DOI":"10.1128\/JB.01493-14","article-title":"Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery","volume":"196","author":"Liao","year":"2014","journal-title":"J. Bacteriol."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"84","DOI":"10.1016\/j.jcf.2011.09.008","article-title":"Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy","volume":"11","author":"Manzenreiter","year":"2012","journal-title":"J. Cyst. Fibros."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1111\/j.1600-051X.2005.00790.x","article-title":"Dental plaque: biological significance of a biofilm and community life-style","volume":"32","author":"Marsh","year":"2005","journal-title":"J. Clin. Periodontol."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"26","DOI":"10.1016\/j.ijfoodmicro.2014.06.025","article-title":"DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms","volume":"187","author":"Nguyen","year":"2014","journal-title":"Int. J. Food Microbiol."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"e111312","DOI":"10.1371\/journal.pone.0111312","article-title":"\u03b1-Mangostin disrupts the development of Streptococcus mutans biofilms and facilitates its mechanical removal","volume":"9","author":"Nguyen","year":"2014","journal-title":"PLoS One"},{"key":"ref32","doi-asserted-by":"publisher","first-page":"1157","DOI":"10.1111\/iej.12420","article-title":"Synergistic effect of 2% chlorhexidine combined with proteolytic enzymes on biofilm disruption and killing","volume":"48","author":"Niazi","year":"2015","journal-title":"Int. Endod. J."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"756","DOI":"10.1111\/iej.12214","article-title":"The effectiveness of enzymic irrigation in removing a nutrient-stressed endodontic multispecies biofilm","volume":"47","author":"Niazi","year":"2014","journal-title":"Int. Endod. J."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"341","DOI":"10.3109\/1040841X.2013.841639","article-title":"The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms","volume":"41","author":"Okshevsky","year":"2015","journal-title":"Crit. Rev. Microbiol."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"179","DOI":"10.1111\/idh.12323","article-title":"Hygiene practices in removable prosthodontics: A systematic review","volume":"16","author":"Papadiochou","year":"2018","journal-title":"Int. J. Dent. Hyg."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"65","DOI":"10.2436\/20.1501.01.208","article-title":"Functional amyloids in bacteria","volume":"17","author":"Romero","year":"2014","journal-title":"Int. Microbiol."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"S5","DOI":"10.1111\/jcpe.12682","article-title":"Role of microbial biofilms in the maintenance of oral health and in the development of dental caries and periodontal diseases. Consensus report of group 1 of the joint EFP\/ORCA workshop on the boundaries between caries and periodontal disease","volume":"44","author":"Sanz","year":"2017","journal-title":"J. Clin. Periodontol."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"50","DOI":"10.1016\/j.mimet.2016.03.002","article-title":"Confocal microscopy imaging of the biofilm matrix","volume":"138","author":"Schlafer","year":"2017","journal-title":"J. Microbiol. Methods"},{"key":"ref39","doi-asserted-by":"publisher","first-page":"436","DOI":"10.1159\/000477447","article-title":"Extracellular DNA contributes to dental biofilm stability","volume":"51","author":"Schlafer","year":"2017","journal-title":"Caries Res."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"S106","DOI":"10.1111\/jcpe.12331","article-title":"Efficacy of adjunctive anti-plaque chemical agents in managing gingivitis: a systematic review and meta-analysis","volume":"42","author":"Serrano","year":"2015","journal-title":"J. Clin. Periodontol."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"9188","DOI":"10.1073\/pnas.87.23.9188","article-title":"Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum","volume":"87","author":"Shak","year":"1990","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"93","DOI":"10.1159\/000057866","article-title":"An in vitro oral biofilm model for comparing the efficacy of antimicrobial mouthrinses","volume":"36","author":"Shapiro","year":"2002","journal-title":"CRE"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"S1","DOI":"10.4103\/ijmr.IJMR_410_15","article-title":"Staphylococcus aureus biofilm removal by targeting biofilm-associated extracellular proteins","volume":"146","author":"Shukla","year":"2017","journal-title":"Indian J. Med. Res."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"135","DOI":"10.2165\/00063030-200519030-00001","article-title":"The use of human deoxyribonuclease (rhDNase) in the management of cystic fibrosis","volume":"19","author":"Suri","year":"2005","journal-title":"BioDrugs"},{"key":"ref45","doi-asserted-by":"publisher","first-page":"1702","DOI":"10.1128\/AEM.69.3.1702-1709.2003","article-title":"Mass transport of macromolecules within an in vitro model of supragingival plaque","volume":"69","author":"Thurnheer","year":"2003","journal-title":"Appl. Environ. Microbiol."},{"key":"ref46","doi-asserted-by":"publisher","first-page":"1779","DOI":"10.1007\/s00784-013-1157-2","article-title":"Static biofilm removal around ultrasonic tips in vitro","volume":"18","author":"Thurnheer","year":"2014","journal-title":"Clin. Oral Investig."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"163","DOI":"10.1159\/000091065","article-title":"Effects of Streptococcus mutans gtfC deficiency on mixed oral biofilms in vitro","volume":"40","author":"Thurnheer","year":"2006","journal-title":"Caries Res."},{"key":"ref48","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1186\/s10020-020-00215-w","article-title":"Nebulized in-line endotracheal dornase alfa and albuterol administered to mechanically ventilated COVID-19 patients: a case series","volume":"26","author":"Weber","year":"2020","journal-title":"Mol. Med."},{"key":"ref49","doi-asserted-by":"publisher","first-page":"1487","DOI":"10.1126\/science.295.5559.1487","article-title":"Extracellular DNA required for bacterial biofilm formation","volume":"295","author":"Whitchurch","year":"2002","journal-title":"Science"},{"key":"ref50","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":"ref51","doi-asserted-by":"publisher","first-page":"226","DOI":"10.1111\/omi.12166","article-title":"FISHing for gutta-percha-adhered biofilms in purulent post-treatment apical periodontitis","volume":"32","author":"Zehnder","year":"2017","journal-title":"Mol. Oral Microbiol."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2021.741863\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,9,28]],"date-time":"2021-09-28T07:20:41Z","timestamp":1632813641000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2021.741863\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,9,28]]},"references-count":51,"alternative-id":["10.3389\/fmicb.2021.741863"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2021.741863","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,9,28]]},"article-number":"741863"}}