ORIGINAL ARTICLEMetabolic activity of Streptococcus mutans biofilms andgene expression during exposure to xylitol and sucroseEva-Maria Decker1, Christian Klein1,2, Dimitri Schwindt1and Christiane von Ohle1TheobjectiveofthestudywastoanalyseStreptococcusmutansbiofilmsgrownunderdifferentdietaryconditionsbyusingmultifacetedmethodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generatedduring a period of 24 h in the following media: Schaedler broth as a control medium containing endogenous glucose, Schaedler brothwith an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy(CLSM)-based analyses of the microbial vitality, respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride, CTC) and production ofextracellular polysaccharides (EPS) were performed separately in the inner, middle and outer biofilm layers. In addition to themicrobiological sample testing, the glucose/sucrose consumption of the biofilm bacteria was quantified, and the expression ofglucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutansbiofilms, as monitored by the following experimental parameters: culture growth, vitality, CTC activity and EPS production. However,xylitol exposure caused a difference in gene expression compared to the control. GtfC was upregulated only in the presence of xylitol.Under xylitol exposure, gtfB was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three.Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose wasalmost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametricmeasurementsshowedthatxylitolinthepresenceofglucosecausedpredominantlygenotypicdifferencesbutdidnotinducemetabolicdifferencescomparedtothecontrol.Thus,theavailabilityofdietarycarbohydratesineitherapureorcombinedformseemstoaffectthecariogenic potential of S. mutans biofilms.International Journal of Oral Science (2014) 6, 195–204; doi:10.1038/ijos.2014.38; published 25 July 2014Keywords: biofilms;5-cyano-2,3-ditolyl tetrazolium chloride;extracellular polysaccharides;geneexpression;Streptococcus mutans;sucrose; viability; xylitolINTRODUCTIONSimilar to other bacteria, oral microorganisms have a strong tendencytoattachtoliquid/solidinterfacesandcolonizethem.Furthermore,asa survival mechanism, they grow as complex communities called bio-films. The genotypic and phenotypic expression profiles of biofilmbacteria differ strongly from those of their planktonic counterparts.For example, biofilm bacteria have increased antimicrobial resistance,are regulated by quorum sensing, and form a distinctive extracellularpolymericmatrixnetwork.1–3Dentalbiofilmformationcanleadtothedevelopment of oral infectious diseases, such as caries, gingivitis andperiodontal inflammation. Streptococcus mutans belongs toa group ofcolonizers of human teeth and has the ability to metabolize variouscarbohydrates into organic acids, which may lead to the cariogenicdestruction of tooth surfaces.4In particular, sucrose, the substrate forglucosyltransferase-mediated sucrose-dependent glucan production,promotes the adhesion of S. mutans to tooth surfaces.5–6Sucrose is afermentable disaccharide and may act as a substrate for extracellularpolysaccharide synthesis. Changes in the local environment at thetooth surface induced by sucrose exposure disturb the microbialbalance and may promote the growth of acidogenic populationsincluding S. mutans, actinomyces and lactobacilli.7Substitutingnutritive sweeteners for cariogenic sugars is an important measureforcariespreventioninoralhygienecare,similartochemotherapeutictreatment and fluoridation. Polyol xylitol cannot be metabolized intoacids by oral microorganisms for energy generation; instead, aftercellular uptake, it accumulates in the cell as a toxic sugar-phosphate.8ThearchitectureandgeneexpressionprofilesofS.mutansbiofilmsarealtered according to the availability of sugar.9–10The cariogenic properties of S. mutans biofilms are regulated byvariousgenes,whichareinvolvedinfiveessentialmetabolicpathways:(i) microbial adhesion,11–13(ii) biofilm formation,14–16(iii) extracel-lular polysaccharide synthesis,12–13(iv)carbohydrate uptake,17–18and(v) acid tolerance.19Several clinical studies have focused on the effects of xylitol on S.mutans,buttheresultsarecontradictory.Whilevariousinvestigationsshowed the plaque-reducing effects of xylitol,20–22other studies couldnot confirm these results.23–24Generally, clinical investigations ofbiofilmsarecomplicatedbytheheterogeneityoftheoralenvironment,1Department for Pediatric and Operative Dentistry, Periodontology and Endodontology, Center of Dentistry, Oral Medicine, and Maxillofacial Surgery, University of Tu ¨bingen,Tu ¨bingen, Germany and2Private Practice Zahngesundheit Waiblingen, Waiblingen, GermanyCorrespondence: Dr EM Decker, Department for Pediatric and Operative Dentistry, Periodontology and Endodontology, Center of Dentistry, Oral Medicine, and MaxillofacialSurgery, University of Tu ¨bingen, Osianderstrasse 2–8, Tu ¨bingen D-72076, GermanyE-mail: evi.decker@med.uni-tuebingen.deAccepted 3 June 2014OPENInternational Journal of Oral Science (2014) 6, 195–204? 2014 WCSS. 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