Baicalein Inhibits Streptococcus Mutans Biofilms and Dental Caries

antibiotics

Article Baicalein Inhibits Streptococcus mutans Bioﬁlms and Dental Caries-Related Virulence Phenotypes

Aparna Vijayakumar 1 , Hema Bhagavathi Sarveswari 1, Sahana Vasudevan 1, Karthi Shanmugam 1, Adline Princy Solomon 1,* and Prasanna Neelakantan 2,*

1 Quorum Sensing Laboratory, Centre for Research in Infectious Diseases (CRID), School of Chemical and Biotechnology, SASTRA Deemed to be University, Thanjavur 613401, India; [email protected] (A.V.); [email protected] (H.B.S.); [email protected] (S.V.); [email protected] (K.S.) 2 Faculty of Dentistry, The University of Hong Kong, Hong Kong * Correspondence: [email protected] or [email protected] (A.P.S.); [email protected] (P.N.); Tel.: +91-4362264101 (A.P.S.); +852-28590581 (P.N.); Fax: +91-4362264129 (A.P.S.); +852-25599013 (P.N.)

Abstract: Dental caries, the most common oral disease, is a major public healthcare burden and affects more than three billion people worldwide. The contemporary understanding of the need for a healthy microbiome and the emergence of antimicrobial resistance has resulted in an urgent need to identify compounds that curb the virulence of pathobionts without microbial killing. Through this study, we have demonstrated for the first time that 5,6,7-trihydroxyflavone (Baicalein) significantly downregulates crucial caries-related virulence phenotypes in Streptococcus mutans. Baicalein significantly inhibited biofilm formation by Streptococcus mutans UA159 (MBIC = 200 µM), without 50 significant growth inhibition. Notably, these concentrations of baicalein did not affect the commensal S. gordonii. Strikingly, baicalein significantly reduced cell surface hydrophobicity, autoaggregation Citation: Vijayakumar, A.; S. mutans Sarveswari, H.B.; Vasudevan, S.; and acid production by . Mechanistic studies (qRT-PCR) showed downregulation of various Shanmugam, K.; Solomon, A.P.; genes regulating biofilm formation, surface attachment, quorum sensing, acid production and compe- Neelakantan, P. Baicalein Inhibits tence. Finally, we demonstrate the potential translational value of baicalein by reporting synergistic Streptococcus mutans Biofilms and interaction with fluoride against S. mutans biofilms. Dental Caries-Related Virulence Phenotypes. Antibiotics 2021, 10, 215. Keywords: baicalein; biofilm; dental caries; hydrophobicity; hydroxyflavone; Streptococcus mutans https://doi.org/10.3390/antibiotics 10020215

Academic Editor: Eliana De Gregorio 1. Introduction Dental caries is a microbial-mediated oral disease initiated by the presence of stagnant Received: 19 January 2021 plaque biofilms, where pathogens such as S. mutans metabolise dietary sugars and produce Accepted: 19 February 2021 acids [1]. This results in dysbiosis, which sustains S. mutans biofilm growth, creating Published: 21 February 2021 acidic microenvironments that demineralise the dental hard tissues, leading to dental caries [2]. Acid production and the glucan-rich matrix in S. mutans biofilms enable the Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in spatial orientation of other cariogenic microbes constituting an elaborate microbial network published maps and institutional affil- leading to the development of caries [1,3]. In addition, multiple virulence factors, including iations. acid and stress tolerance, cell persistence, genetic competence and bacteriocin production collectively contribute to the progression of dental caries [4]. The currently available options in preventing or treating dental caries predominantly involve the use of fluoride. Fluoride compounds are somewhat effective in inhibiting dem- ineralisation and enhancing remineralisation of the tooth structure. Even though fluoride Copyright: © 2021 by the authors. exhibits bactericidal activity it cannot inhibit biofilm [5]. Furthermore, repeated usage of Licensee MDPI, Basel, Switzerland. This article is an open access article high concentrations of fluoride has been linked to the development of fluorosis, decrease distributed under the terms and in bone density and neural toxicity [6,7]. S. mutans strains exhibiting fluoride resistance conditions of the Creative Commons have already been reported [8]. Taken together, the high prevalence of dental caries and Attribution (CC BY) license (https:// the drawbacks of currently used strategies demonstrates the pressing need to identify creativecommons.org/licenses/by/ more effective, efficient, and non-toxic alternative treatment options, that can potentially 4.0/).

Antibiotics 2021, 10, 215. https://doi.org/10.3390/antibiotics10020215 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, x FOR PEER REVIEW 2 of 14

caries and the drawbacks of currently used strategies demonstrates the pressing need to identify more effective, efficient, and non-toxic alternative treatment options, that can potentially synergise with fluorides, so that they can be easily incorporated into commercially available oral care products. Although caries is a polymicrobial disease, S. mutans is considered as the primary Antibiotics 2021, 10, 215 driver for the initiation and progression of caries [3,9]. There has been considerable2 of 13 argu- ment on what the disease-drivers and commensal-keepers in caries are, with various groups demonstrating different microbiome profiles in caries in an age-dependent man- nersynergise [10–12]. with That fluorides, said, sothe that concept they can of be “keyston easily incorporatede pathogen” into is commercially probably only available relevant if the designedoral care products. strategies are microbicidal in nature. However, given the current understanding of theAlthough need for caries a health is a polymicrobialmicrobiome, disease, it is importantS. mutans tois consideredidentify compounds as the primary that can sup- pressdriver the for cariogenic the initiation virulence and progression determinants of caries instead [3,9]. There of affecting has been considerablethe growth ar-and survival gument on what the disease-drivers and commensal-keepers in caries are, with various of the microbes [13]. Natural products are indeed a safe approach for rapid clinical trans- groups demonstrating different microbiome profiles in caries in an age-dependent man- lationner [10 since–12]. they That said,are generally the concept recognised of “keystone as pathogen”safe by the is United probably States only relevantFood and if Drug Ad- ministrationthe designed [14]. strategies are microbicidal in nature. However, given the current under- standingBaicalein of the (5,6,7-trihydroxyflavone), need for a health microbiome, a it flavonoid is important isolated to identify from compounds the roots that of Scutellaria baicalensiscan suppress and the Scutellaria cariogenic lateriflora virulence determinantsis known to insteadexhibit of a affectingwide range the growthof biological and proper- tiessurvival [15–18]. of the Previous microbes [studies13]. Natural have products demonstrated are indeed that a safe baicalein, approach forboth rapid in naked, clinical as well as translation since they are generally recognised as safe by the United States Food and Drug nanoparticle-encapsulatedAdministration [14]. forms were biocompatible to human gingival epithelial cells [19]. Furthermore,Baicalein (5,6,7-trihydroxyflavone), from the systemic a flavonoidcontext, baicalein isolated from has the demonstrated roots of Scutellaria hepatoprotec- tivebaicalensis propertiesand Scutellaria [20]. There lateriflora is evidenceis known to to show exhibit the a wideanti-quorum range of biological sensing proper-activity and bio- filmties [inhibitory15–18]. Previous potential studies of Baicalein have demonstrated against Staphylococcus that baicalein, both aureus in naked, [21], Avian as well Pathogenic E.as coli nanoparticle-encapsulated (APEC) [22] and Candida forms albicans were biocompatible[23]. Based on to this human premise, gingival we epithelial hypothesised that baicaleincells [19]. inhibits Furthermore, S. mutans from the biofilms systemic and context, its virulence. baicalein has The demonstrated aim of this hepatopro- study was to char- tective properties [20]. There is evidence to show the anti-quorum sensing activity and acterisebiofilm inhibitorythe effects potential of baicalein of Baicalein on againstthe growthStaphylococcus and biofilm aureus [formation21], Avian Pathogenic of S. mutans along withE. coli its(APEC) associated [22] and virulenceCandida albicans factors.[23 Furthe]. Basedrmore, on this the premise, potential we hypothesised mechanisms that of these ef- fectsbaicalein were inhibits studiedS. mutansusing biofilmstranscriptomic and its virulence. analysis. The aim of this study was to charac- terise the effects of baicalein on the growth and biofilm formation of S. mutans along with 2.its Results associated and virulence Discussion factors. Furthermore, the potential mechanisms of these effects were studied using transcriptomic analysis. 2.1. Baicalein Inhibit S. mutans UA159 Biofilm and Its Virulence Potential 2. ResultsFigure and 1 shows Discussion the planktonic and biofilm inhibitory actions of baicalein. There was 2.1. Baicalein Inhibit S. mutans UA159 Biofilm and Its Virulence Potential no significant growth inhibition within the concentration range tested. However, biofilm quantificationFigure1 shows showed the planktonic a concentration-depend and biofilm inhibitoryent actions inhibition of baicalein. of biof Thereilm with was no ~50% inhibi- significant growth inhibition within the concentration range tested. However, biofilm quan- tiontification at 200 showed μM and a concentration-dependent ~80% inhibition at 400 inhibition μM with of biofilm negligible with ~50% impact inhibition on the at planktonic growth200 µM (<10% and ~80% inhibition). inhibition In at 400addition,µM with the negligible tested concentrations impact on the planktonic of baicalein growth had no significant(<10% impact inhibition). on the In growth addition, of the the tested commensal concentrations Streptococcus of baicalein gordonii had no (Figure significant 2). impact on the growth of the commensal Streptococcus gordonii (Figure2).

Figure 1. Dose response curve representing the action of Baicalein on S. mutans. MBIC50 (Minimum Figure 1. Dose response curve representing the action of Baicalein on S. mutans. MBIC50 (Minimum Bioﬁlm Inhibitory Concentration) = 200 µM; MIC50 (Minimum Inhibitory Concentration) > 400 µM. Biofilm Inhibitory Concentration) = 200 μM; MIC50 (Minimum Inhibitory Concentration) > 400 μM. Antibiotics 2021, 10, x FOR PEER REVIEW 3 of 14

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Figure 2. Effect of Baicalein on S. gordonii growth at varying concentrations. Student t test was used for the significance analysis. p < 0.05 was considered significant. ns denotes non-significance.

Figure 2. Effect of Baicalein on S. gordonii growth at varying concentrations. Student t test was used FigureStreptococcus 2. Effect of Baicalein mutans on is S.known gordonii to growth be one at ofvarying the pioneers concentrations. of tooth Student surface t test colonization was for the significance analysis. p < 0.05 was considered significant. ns denotes non-significance. andused is for often the significance considered analysis. the model p < 0.05 organism was considered for studying significant. novel ns therapies.denotes non-significance. The biofilm matrix productionStreptococcus in mutans S. mutansis known is enhanced to be one of in the the pioneers presence of tooth of a surface sucrose colonization rich environment. However,and isStreptococcus often bacterial considered mutans cell-to-substrate the modelis known organism to beinteractions for one studying of the novelpioneerscan therapies.also of take tooth The place surface biofilm in matrix acolonization sucrose-inde- pendentandproduction is often manner, in consideredS. mutans mediatedis the enhanced model by hydrophobic in organism the presence for inte of studying aractions sucrose novel rich [24]. environment. therapies. Cell surface The However hydrophobicity biofilm, ma- istrixbacterial an production important cell-to-substrate determinantin S. mutans interactions isof enhancedbacterial can also surfin take theace place presence attachment. in a sucrose-independent of a Non-hydrophobic sucrose rich manner,environment. cells (those withHowever,mediated a low by bacterialhydrophobicity hydrophobic cell-to-substrate interactions score) display [interactions24]. Cell very surface weakcan hydrophobicity also surface take adhesion, place is anin important a and sucrose-inde- consequently determinant of bacterial surface attachment. Non-hydrophobic cells (those with a low poorpendent biofilm manner, formation. mediated Baicalein by hydrophobic decreased inte theractions cell surface [24]. Cellhydrophobicity surface hydrophobicity at the 5th and ishydrophobicity an important score)determinant display veryof bacterial weak surface surface adhesion, attachment. and consequently Non-hydrophobic poor biofilm cells (those 7thformation. hour of Baicaleintreatment decreased (Figure the 3). cell At surface the 9th hydrophobicity hour, a significant at the 5th decrease and 7th in hour the of hydropho- with a low hydrophobicity score) display very weak surface adhesion, and consequently bicitytreatment was (Figureobserved3). At wherein the 9th hour,the hydrophobicity a significant decrease index in thewas hydrophobicity 25%, compared was to the un- poor biofilm formation. Baicalein decreased the cell surface hydrophobicity at the 5th and treatedobserved control wherein (hydrophobicity the hydrophobicity index index >70%). was 25%, compared to the untreated control 7th(hydrophobicity hour of treatment index >70%).(Figure 3). At the 9th hour, a significant decrease in the hydrophobicity was observed wherein the hydrophobicity index was 25%, compared to the untreated control (hydrophobicity index >70%).

Figure 3. Effect of baicalein on S. mutans cell surface hydrophobicity. Student t-test was used for Figure 3. Effect of baicalein on S. mutans cell surface hydrophobicity. Student t-test was used for analysing significance and p < 0.05 was considered significant. ** denotes p ≤ 0.05. analysing significance and p < 0.05 was considered significant. * denotes p ≤ 0.05, ** denotes p ≤ 0.01Figure andAutoaggregation, 3. *** Effect denotes of baicalein p ≤ 0.001. a phenomenon on S. mutans cell where surface bacterial hydrophobicity. cells of the Student same species t-test was secrete used for analysingspecific polymerssignificance to and interact p < 0.05 and was aggregate, considered isenhanced significant. by * denotes hydrophobic p ≤ 0.05, interactions ** denotes p ≤ 0.01amongst andAutoaggregation, *** cells denotes [25], and p ≤ is0.001. considereda phenomenon an initial where step in biofilmbacterial formation. cells of Baicalein the same treatment species secrete specificshowed apolymers marked reduction to interact in autoaggregation and aggregate, at all is the enhanced three time points:by hydrophobic Early log phase interactions amongst(5th hour),Autoaggregation, cells mid-log [25], phase and a (7th isphenomenon considered hour) and late anwhere log initia phase bactl step (9therial hour) in cells biofilm (Figure of the 4formation.). Thesame differences species Baicalein secrete treat- were more distinct in the late log phase where baicalein treatment reduced the aggregation mentspecific showed polymers a marked to interact reduction and aggregate, in autoaggr is egationenhanced at byall hydrophobicthe three time interactions points: Early amongstby at least cells 20% [25], relative and to is the considered control. Light an microscopicinitial step imagesin biofilm clearly formation. demonstrated Baicalein the treat- loginhibition phase of(5th autoaggregation hour), mid-log by baicaleinphase (7th at different hour) timeand pointslate log considered phase (9th (Figure hour)4A). (Figure 4). ment showed a marked reduction in autoaggregation at all the three time points: Early The differences were more distinct in the late log phase where baicalein treatment reduced log phase (5th hour), mid-log phase (7th hour) and late log phase (9th hour) (Figure 4). the aggregation by at least 20% relative to the control. Light microscopic images clearly The differences were more distinct in the late log phase where baicalein treatment reduced demonstrated the inhibition of autoaggregation by baicalein at different time points con- the aggregation by at least 20% relative to the control. Light microscopic images clearly sidered (Figure 4A). demonstrated the inhibition of autoaggregation by baicalein at different time points considered (Figure 4A). Antibiotics 2021, 10, x FOR PEER REVIEW 4 of 14 Antibiotics 2021, 10, 215 4 of 13

Figure 4. Effect of baicalein on autoaggregation. (A) Microscopic analysis of the reduction in autoaggregation for the bai- Figure 4. Effect of baicalein on autoaggregation. (A) Microscopic analysis of the reduction in autoaggregation for the calein treated cells in time dependent manner when compared to control. (B) Reduction in % autoaggregation. Student t- baicaleintest was treated used for cells analysing in time dependent significance manner and p when< 0.05 comparedwas considered to control. significant. (B) Reduction * denotes in p % ≤ autoaggregation.0.05, ** denotes p Student≤ 0.01 and t-test*** wasdenotes used p for≤ 0.001. analysing significance and p < 0.05 was considered significant. * denotes p ≤ 0.05, ** denotes p ≤ 0.01 and *** denotes p ≤ 0.001. The SEM analysis was concurrent with the findings of the cell surface hydrophobicity andThe autoaggregation SEM analysis was assays, concurrent and the with changes the findings observed of in the topography, cell surface hydrophobicitydisruption in bio- andfilm autoaggregation architecture, decreased assays, and adhered the changes cell count observed and inreduction topography, in cell disruption clustering in can biofilm be at- architecture, decreased adhered cell count and reduction in cell clustering can be attributed tributed to the ability of baicalein to affect these factors negatively. SEM analysis indicated to the ability of baicalein to affect these factors negatively. SEM analysis indicated a a decrease in S. mutans biofilm biomass when treated with 200 μM of baicalein. A signifi- decrease in S. mutans biofilm biomass when treated with 200 µM of baicalein. A significant cant reduction in bacterial cell chain length and a 5-fold decrease in adhered cell number reduction in bacterial cell chain length and a 5-fold decrease in adhered cell number was was observed in the baicalein treated biofilms (Figure 5). Therefore, baicalein impedes the observed in the baicalein treated biofilms (Figure5). Therefore, baicalein impedes the ability of S. mutans to successfully colonize the surface, thereby reducing initial biofilm ability of S. mutans to successfully colonize the surface, thereby reducing initial biofilm formation. This initial biofilm serves as a potential adhesion site for other pathogenic oral formation. This initial biofilm serves as a potential adhesion site for other pathogenic oral bacteria in a polymicrobial environment [1]. These findings highlight the possibility of bacteria in a polymicrobial environment [1]. These findings highlight the possibility of using baicalein as an antifouling coating on substrates. using baicalein as an antifouling coating on substrates. The CLSM analysis further strengthened the experimental evidence on biofilm inhibitory effect of baicalein (Figure6). The architectural analysis of the untreated biofilm and baicalein treated biofilm showed ~70% reduction in the biofilm biomass and ~ 75% reduction in the average thickness of the biofilm matrix. The ~80% reduction of the diffusion distance in the baicalein treated biofilm when compared to the untreated control, confirms the reduced solute movement in the biofilm [26]. These results corroborate the biofilm inhibitory effects of baicalein. Furthermore, the higher proportion of green-to- red cells indicate that baicalein can achieve the anti-virulent effects without bactericidal effects. The reduced total number of cells in the test group is attributed to the biofilm- inhibitory effect of baicalein and the consequent loss of free-floating cells, rather than bactericidal effects. Antibiotics 2021, 10, x FOR PEER REVIEW 5 of 14 Antibiotics 2021, 10, 215 5 of 13

FigureFigure 5. (A) 5. SEM (A) SEM (Scanning (Scanning Electron Electron Microscopy) Microscopy) micrograph micrograph depictin depictingg the theeffect effect of baicalein of baicalein on biofilm on bioﬁlmformation. formation. Ar- rowsAntibiotics indicate 2021 the, 10 long, x FOR and PEER short REVIEW chains of S. mutans in control and treatment, respectively. (B) Reduction in number6 of of14

Arrowscells indicate in the thebaicalein long andtreated short when chains comp ofaredS. mutans to untreatedin control control and biofilms treatment, where respectively. the treated cells (B )displayed Reduction an inadhered number of cells incell the count baicalein of <100 treated cells when when compared compared to tothe untreated untreated controlcontrol bioﬁlmswhich had where around the >500 treated adhered cells cells. displayed an adhered cell count of <100 cells when compared to the untreated control which had around >500 adhered cells. The CLSM analysis further strengthened the experimental evidence on biofilm inhibitory effect of baicalein (Figure 6). The architectural analysis of the untreated biofilm and baicalein treated biofilm showed ~70% reduction in the biofilm biomass and ~ 75% reduction in the average thickness of the biofilm matrix. The ~80% reduction of the diffusion distance in the baicalein treated biofilm when compared to the untreated control, confirms the reduced solute movement in the biofilm [26]. These results corroborate the biofilm inhibitory effects of baicalein. Furthermore, the higher proportion of green-to-red cells indicate that baicalein can achieve the anti-virulent effects without bactericidal effects. The reduced total number of cells in the test group is attributed to the biofilm-inhibitory effect of baicalein and the consequent loss of free-floating cells, rather than bactericidal effects.

Figure 6. (A)Figure CLSM 6. (Confocal(A) CLSM (Confocal Laser ScanningLaser Scanning Microscopy) Microscopy) micrograph micrographss illustrating illustrating the effect the on effectthe sub-inhibitory on the sub-inhibitory con- concentration ofcentration baicalein of baicalein (200 µ M)(200on μM) biofilm on biofilm architecture architecture and viability viability of th ofe biofilm the biofilm cells through cells throughLive/Dead Live/Deadstaining. Bai- staining. Baicalein treatedcalein cell treated cultures cell cultures show show sparing sparin greeng green color color cells with with no noconfluent confluent mass, mass,indicating indicating inhibition of inhibition biofilm for- of biofilm mation. (B) COMSTAT analysis of the CLSM images revealed a reduction in the biofilm biomass, average thickness, and formation. (B)diffusion COMSTAT distances analysis in the ofbaicalein the CLSM treated images biofilms revealedcompared to a reductionthe untreated in control. the biofilm (C) ImageJ biomass, analysis averageshows the thickness, and diffusion distancesproportion inof viable the baicalein cells in the treated biofilm formed biofilms with compared baicalein treatment to the untreatedat the concentration control. of (200C) μ ImageJM (MBIC analysis50). shows the proportion of viable cells in the biofilm formed with baicalein treatment at the concentration of 200 µM (MBIC ). Acid production serves two roles in the caries process. Its primary role is to maintain50 an acidic environment where the non-aciduric microbes cannot survive, contributing to dysbiosis. Secondly, acid production is also responsible for the acidic microenvironments within the biofilm, resulting in demineralization of the contacting tooth surface, leading to cavity formation [27,28]. Our results showed that baicalein did not allow the pH to drop below 5.2–5.5 which is considered the critical pH for enamel demineralization [29]. The pH of Baicalein solution in BHI media and supplemented with 20 mM sucrose was maintained at pH 7.4 ± 0.1 and in the sterile distilled water was maintained at pH 6.4 ± 0.1. Baicalein treated groups demonstrated a relative increase in pH from the 5th hour of incubation. The pH of ~5.2 was maintained until 9 h of treatment, while the pH dropped to ~4 for the control group (Figure 7). Antibiotics 2021, 10, 215 6 of 13

Acid production serves two roles in the caries process. Its primary role is to maintain an acidic environment where the non-aciduric microbes cannot survive, contributing to dysbiosis. Secondly, acid production is also responsible for the acidic microenvironments within the bioﬁlm, resulting in demineralization of the contacting tooth surface, leading to cavity formation [27,28]. Our results showed that baicalein did not allow the pH to drop below 5.2–5.5 which is considered the critical pH for enamel demineralization [29]. The pH of Baicalein solution in BHI media and supplemented with 20 mM sucrose was maintained Antibiotics 2021, 10, x FOR PEER REVIEWatpH 7.4 ± 0.1 and in the sterile distilled water was maintained at pH 6.4 ± 0.1. Baicalein7 of 14

treated groups demonstrated a relative increase in pH from the 5th hour of incubation. The pH of ~5.2 was maintained until 9 h of treatment, while the pH dropped to ~4 for the control group (Figure7).

Figure 7. S. mutans Figure 7. EffectEffect of of baicalein baicalein on on S. mutans acidacid production. production. The The time time dependent dependent effect effect on onglycolytic glycolytic pHpH drop. drop. Student Student t-testt-test was was used used for for analysing analysing significance significance and and p p< <0.05 0.05 was was considered considered signifi- significant. cant.* denotes * denotesp ≤ p0.05, ≤ 0.05, ** denotes ** denotesp ≤ p0.01 ≤ 0.01 and and *** *** denotes denotesp ≤ p 0.001.≤ 0.001. 2.2. Baicalein Downregulates Several Genes That Regulate Metabolism, Biofilm, and Virulence 2.2. Baicalein Downregulates Several Genes That Regulate Metabolism, Biofilm, and Virulence Gene expression studies showed that baicalein can negatively affect the expression of variousGene interconnected expression studies virulent showed genes withthat baicalein multiple overlappingcan negatively functions affect whichthe expression are a part ofof various the inherent interconnectedS. mutans genevirulent network. genes Itwith is to multiple be noted overlapping that there is functions a significant which difference are a partin the of downregulationthe inherent S. mutans of certain gene genes network. between It theis to mid-log be noted phase that biofilm there cellsis a andsignificant late-log differencephase biofilm in the cells. downregulation The spatial–temporal of certain genes variations between are the naturally mid-log observed phase biofilm in the cells case andof biofilmlate-log genephase expression biofilm cells. [30 ].The This spatial–temporal was proven in variations the case of areE. naturally coli [31,32 observed]. While thein thetemporal case of variationbiofilm gene in S. expression mutans biofilms [30]. This is yet was to beproven explored, in the it cancase be of understoodE. coli [31,32]. that Whilethe variations the temporal in the variation gene expression in S. mutans pattern biofilms in the is biofilmyet to be cells explored, are apparent. it can be A under- similar stoodtrend that is observed the variations in our in study the gene where expression the downregulation pattern in the is biofilm more in cells the mid-logare apparent. phase Aas similar compared trend to is the observed late-log in phase. our study Due towhere the complexity the downregulation of the biofilm is more cells in at the different mid- logdevelopmental phase as compared stages, to the the difference late-log phase. in the geneDue expressionto the complexity pattern of is the expected. biofilm cells at differentThe developmental time points for stages, the qRT-PCR the difference analysis in were thechosen gene expression based on the pattern time duringis expected. which theThe planktonic time points cell culture for the reaches qRT-PCR it logarithmic analysis were phase chosen (7th hour: based Mid on logthe phasetime during and 9th whichhour: the Late planktonic log phase). cell It shouldculture bereaches noted it that logarithmic at 7th and phase 9th hour, (7th therehour: was Mid a log significant phase andreduction 9th hour: in theLate cell-cell log phase). hydrophobicity It should be and noted auto that aggregation. at 7th and Hence,9th hour, we there chose was these a significanttwo points reduction to understand in the the cell-cell temporal hydrophobicity regulation in and gene auto expression aggregation. in treatment Hence, withwe chosebaicalein. these qRT-PCRtwo points analysis to understand demonstrated the tempor thatal the regulation effect of baicalein in gene expression on downregulation in treat- mentof several with baicalein. genes on S.qRT-PCR mutans biofilmanalysis cells demonstrated was temporal, that i.e., the theeffect downregulation of baicalein on at down- the 7th regulationhour was of relatively several highergenes on than S. mutans that observed biofilm at cells the was 9th hourtemporal, (Figure i.e.,8). the An downregula- approximate tion4 log at 10thefold 7th reductionhour was relatively of comDE higherexpression than that was observed observed at at the the 9th 7th hour hour, (Figure indicating 8). An a approximatehindrance in 4 thelog10 bacterial fold reduction two-component of comDE system expression as well was as observed competence at the development. 7th hour, indicatingSimilarly, a ~3 hindrance log10 fold inreduction the bacterial was two-co observedmponent in the system expression as well of asluxS competencewhich implies development.inhibition ofSimilarly, quorum ~3 sensing log10 fold and reduction a reduction was in observed cell competence. in the expression However, of at luxS the 9th which hour impliesafter treatment, inhibition the of quorum fold reductions sensingin and expression a reduction reduced in cell tocompetence. approximately However, 1.4 log at10 thefold 9thand hour 1.9 logafter10 treatment,fold for comDE, the foldand reductionsluxS, respectively. in expression Other reduced quorum to sensing approximately genes such 1.4 as log10 fold and 1.9 log10 fold for comDE, and luxS, respectively. Other quorum sensing genes such as comA and comB which displayed a 3.9 log10 fold and 2 log10 fold reduction respectively in the 7th displayed almost no change in expression in the 9th hour. Similarly, a relative downregulation of biofilm related genes atlA, ftsz, vicR, gbpB and brpA in the 7th hour, was decreased at the 9th hour. A similar trend was also observed in other virulence genes such as idh, bsml, bsmH, comX and atpD. By contrast, brpA and spaP, which are responsible for surface interaction and adhesion, and other virulence genes such as dnaK, immA and relA sustained their downregulation at both time points. Interestingly the fold reduction for immB increased from 1.4 log10 fold in the 7th hour of treatment to 2.8 log10 fold in the 9th hour of treatment. Antibiotics 2021, 10, x FOR PEER REVIEW 8 of 14

A significant downregulation in the quorum sensing genes, including luxS, comDE, comA and comB, indicates that baicalein could significantly reduce the synchronized expression of virulence [33,34]. The comDE, comA and comB also play a role in conferring cell competence and eDNA in S.mutans is known for its role in biofilm formations and matu- Antibiotics 2021, 10, 215 7 of 13 ration [8]. Furthermore, the significant fold reduction observed in the genes crucial for biofilm formation further validated our findings from the biofilm inhibition assay. The downregulation of brpA might also responsible for causing a ripple effect in the expression ofcomA otherand virulencecomB which genes displayedincluding aatlA 3.9 logwhich10 fold is responsible and 2 log10 forfold biofilm reduction formation respectively and spaPin the which 7th displayedplays a role almost in sucrose no change independent in expression cell adhesion in the 9th [4]. hour. The Similarly, reduction a relativein the expressiondownregulation of dnaK of sheds bioﬁlm light related on baicalein’s genes atlA, ability ftsz, to vicR hinder, gbpB stressand tolerancebrpA in the and 7th recov- hour, erywas [35]. decreased Genes, such at the as 9th immA hour. and A similarimmB, which trend wasare responsible also observed for in the other secretion virulence of bacte- genes riocinssuch asalsoidh play, bsml, a bsmHrole in, comX conferringand atpD immuni. By contrast,ty againstbrpA antimicrobialsand spaP, which including are responsible fluoride andfor chlorohexidine. surface interaction The and sustained adhesion, downregulation and other virulence of immA genes and such a significant as dnaK, immAtime de-and pendentrelA sustained increase their in downregulation downregulation of atimmB both on time treatment points. with Interestingly baicalein theshowed fold reductionthat this compoundfor immB increasedcould possibly from synergise 1.4 log10 fold with in conventional the 7th hour therapeutics of treatment [36] to 2.8 log10 fold in the 9th hour of treatment.

Figure 8. The transcriptional changes (fold reduction) in the expression of various genes in S. mutans Figurebiofilm 8. cellsThe transcriptional on treatment with changes baicalein. (fold reduction) in the expression of various genes in S. mutans biofilm cells on treatment with baicalein. A significant downregulation in the quorum sensing genes, including luxS, comDE, 2.3.comA Baicaleinand comB and Fluoride, indicates Synergistica that baicaleinlly Inhibit could S. mutans significantly Biofilm reduce Formation the synchronized ex- pressionPhytochemicals, of virulence including [33,34]. TheflavonoidscomDE, like comA baicaleinand comB are alsoknown play inhibitors a role in of conferring energy metabolism,cell competence and studies and eDNA have inalsoS.mutans proven isthat known a synergistic for its role effect in biofilmcan be obtained formations when and administeredmaturation [in8]. combination Furthermore, with the conventional significant fold antimicrobials reduction observed [37]. Fluoride in the treatment genes crucial is for biofilm formation further validated our findings from the biofilm inhibition assay. The downregulation of brpA might also responsible for causing a ripple effect in the expression of other virulence genes including atlA which is responsible for biofilm formation and spaP which plays a role in sucrose independent cell adhesion [4]. The reduction in the expression of dnaK sheds light on baicalein’s ability to hinder stress tolerance and recovery [35]. Genes, such as immA and immB, which are responsible for the secretion of bacteriocins also play a role in conferring immunity against antimicrobials including fluoride and chlorohexidine. The sustained downregulation of immA and a significant time Antibiotics 2021, 10, 215 8 of 13

dependent increase in downregulation of immB on treatment with baicalein showed that this compound could possibly synergise with conventional therapeutics [36]

Antibiotics 2021, 10, x FOR PEER REVIEW2.3. Baicalein and Fluoride Synergistically Inhibit S. mutans Bioﬁlm Formation 9 of 14 AntibioticsAntibioticsAntibiotics 2021 20212021,, , 10 1010,, , x xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 999 of ofof 14 1414

Phytochemicals, including flavonoids like baicalein are known inhibitors of energy metabolism, and studies have also proven that a synergistic effect can be obtained when administered in combination with conventional antimicrobials [37]. Fluoride treatment is currently used widely for as a means to prevent dental caries by inhibiting the demineraliza- currentlycurrentlycurrentlycurrently used widelyusedusedused widelywidelywidely for as forforfora means asasas aaa meansmeansmeans to prev tototoent prevprevprev dentalententent dentaldentaldental caries caries cariescariesby inhibiting bybyby inhibitinginhibitinginhibiting the deminer- thethethe deminer-deminer-deminer- tion of the tooth surface, and by enhancing remineralization [38]. However, the presence of alizationalizationalizationalization of the of ofoftooth the thethe tooth toothsurface,tooth surface, surface,surface, and by and andand enhancing by byby enhancing enhancingenhancing remineralization remineralization remineralizationremineralization [38]. However, [38]. [38].[38]. However, However,However, the pres- the thethe pres- pres-pres- biofilms prevents the adsorption of fluoride on to the tooth surface [39]. Having confirmed ence ofenceenceence biofilms ofofof biofilmsbiofilmsbiofilms prevents preventspreventsprevents the adsorption thethethe adsorptionadsorptionadsorption of fluoride ofofof fluoridefluoridefluoride on to onontheon toto totooth thethethe toothtoothsurfacetooth surfacesurfacesurface [39]. Having [39].[39].[39]. HavingHavingHaving that baicalein can inhibit biofilm formation, we further asked if it could synergize with confirmedconfirmedconfirmedconfirmed that baicalein that thatthat baicalein baicaleinbaicalein can inhibit can cancan inhibit inhibitinhibit biofilm biofilm biofilmbiofilm formation, formation, formation,formation, we further we wewe further furtherfurther asked asked asked askedif it could if ifif it itit could couldcouldsyner- syner- syner-syner- fluoride to inhibit biofilms. The Combination Index (CI) values were less than 1.0 for all gize withgizegizegize fluoride withwithwith fluoridefluoridefluoride to inhibit tototo inhibitinhibitinhibit biofilms. biofilms.biofilms.biofilms. The Combination TheTheThe CombinationCombinationCombination Index Index IndexIndex(CI) values (CI)(CI)(CI) valuesvaluesvalues were werelesswerewere than lesslessless thanthanthan combinations of sodium fluoride and baicalein showing synergy (Table S2). The dose-effect 1.0 for1.01.0 all for forcombinations allall combinationscombinations of sodium ofof sodiumsodium fluoride fluoridefluoride and baicalein andand baicaleinbaicalein showing showingshowing synergy synergysynergy (Table (Table(Table S2). The S2).S2). TheThe curve1.0 (Figure for all9A) combinations revealed a significant of sodium increase fluoride in and the baicalein Fa (Fraction showing affected) synergy values (Table for var- S2). The dose-effectdose-effectdose-effect curve curvecurve(Figure (Figure(Figure 9A) revealed 9A)9A) revealedrevealed a significant aa significantsignificant increase increaseincrease in the in inFa thethe (Fraction FaFa (Fraction(Fraction affected) affected)affected) ious combinationsdose-effect curve of baicalein (Figure and9A) fluoriderevealed when a significant compared increase to individual in the Fa drug (Fraction treatment. affected) valuesvaluesvalues for various forfor variousvarious combinations combinationscombinations of baicalein ofof baicaleinbaicalein and fluoride andand fluoridefluoride when whenwhencompared comparedcompared to individual toto individualindividual Further,values the medianfor various effect combinations plot (Figure9 ofB) baicalein showed a and significant fluoride increase when compared in the combinato- to individual drug treatment.drugdrug treatment.treatment. Further, Further,Further, the median thethe medianmedian effect effectpleffectot (Figure plplotot (Figure(Figure 9B) showed 9B)9B) showedshowed a significant aa significantsignificant increase increaseincrease rial dose-responsedrug treatment. when Further, compared the median to the dose-response effect plot (Figure of only 9B) fluoride. showed Thea significant left-skewed increase in thein inincombinatorial the thethe combinatorial combinatorialcombinatorial dose-response dose-response dose-responsedose-response when when whenwhencompared compared comparedcompared to the to totodose-response the thethe dose-response dose-responsedose-response of only of ofof fluoride. only onlyonly fluoride. fluoride.fluoride. combinatorialThe left-skewed curve combinatorial points towards curve points a more to efficientwards a drugmore responseefficient drug when response compared when to the treatmentTheTheThe left-skewed left-skewedleft-skewed by only combinatorial combinatorialcombinatorial baicalein. The curve curvecurve CI plot points pointspoints (Figure to totowardswardswards9C) a aa for more moremore the efficient efficientefficient set for concentrationdrug drugdrug response responseresponse when whenwhen compared to the treatment by only baicalein. The CI plot (Figure 9C) for the set for con- combinationscomparedcomparedcompared tested tototo thethethe indicates treatmenttreatmenttreatment synergy. bybyby onlyonlyonly baicalein.baicalein.baicalein. TheTheThe CICICI plotplotplot (Figure(Figure(Figure 9C)9C)9C) forforfor thethethe setsetset forforfor con-con-con- centrationcentrationcentrationcentration combinations combinations combinationscombinations tested tested tested testedindicates indicates indicatesindicates synergy. synergy. synergy.synergy.

FigureFigureFigureFigure Figure9.9. (A) Dose-response9. 9.9. ( ((AAA))) Dose-response Dose-responseDose-response curve curve curveplotted curve plottedcurve plotted plottedplotted between between between betweenbetween the the fraction fraction the thethe fraction fractionfraction affected affected affected affectedaffected (fa) (fa) of of (fa) (fa)biofilm(fa) biofilm of ofof biofilm biofilmbiofilm formed formed formed formedformed and and drug and andand drug dose drug drugdrug dose ( μdose dosedoseg/mL). (µg/mL). ( ((μμμg/mL).g/mL). g/mL).(B) ( ((BBB))) (MedianB) MedianMedianMedianMedian effect effect ploteffect effecteffect plot depicting plot plotplot depicting depicting depictingdepicting the thelogarithmic the thethe logarithmic logarithmic logarithmiclogarithmic transform transform transform transformtransform of the of theofraofof tio the thethe ratio of ra rara thetiotio oftio theof fractionofof the thethe fraction fraction fractionfraction affected affected affected affectedaffected (fa) (fa)and (fa) (fa)(fa) and fraction and andand fraction fraction fractionfraction unaffected unaffected unaffected unaffectedunaffected (fu) (fu) (fu)(fu) (fu)as a asfunctionasas aas function aaa functionfunctionfunction of the of ofoflogarithmicof the thethethe logarithmic logarithmiclogarithmiclogarithmic transform transform transformtransformtransform of the of ofofdosageof the thethethe dosage dosagedosagedosage of individual of ofofof individual individual individualindividual treatment- treatment—( treatment-treatment-treatment- ( ) baicalein, ((( ) ) ) baicalein,baicalein, baicalein, ( ) Fluoride (( ( ) ) ) FluorideFluorideFluoride and combi- andandand and combi-combi-combi- combinationnationnationnation nation( ) of (((Baicalein ) ) ) ofof BaicaleinBaicalein Baicalein and Fluoride. andand and Fluoride.Fluoride.Fluoride. (C) Combination (((CCC (C))) Combination)CombinationCombination Combination Index IndexIndex(CI)Index Index versus (CI)(CI)(CI) (CI) versusversus versusFraction versus FractionFractionFraction Fractionaffected affectedaffectedaffected (fa) affected plot (fa)(fa)(fa) for (fa) plotplotplot various plot forforfor for variousvariousvarious combina- various combina-combina-combinations of baicalein and fluoride when tested for S. mutans biofilm inhibition ( ) various combnations of baicalein and flu- combinationstionstionstions of ofof of baicalein baicaleinbaicalein baicalein and andand and fluoride fluoridefluoride fluoride when whenwhen when tested testedtested tested for forfor for S. S.S. S. mutans mutansmutans mutans biofilm biofilmbiofilmbiofilm inhibition inhibitioninhibition inhibition ( (( ( ) ) ) various variousvarious) various combnations combnationscombnations combnations of ofof of baicalein baicaleinbaicalein baicalein and andand flu- flu-fluoride. and fluoride.oride.oride.oride.

3. Material3.3.3. Material MaterialMaterial and Methods and andand Methods MethodsMethods 3.1. Bacterial3.1.3.1.3.1. Bacterial BacterialBacterial Strains Strains StrainsStrains andand Culture an ananddd Culture CultureCulture Conditions Conditions ConditionsConditions S. mutansS.S.S. mutansmutansmutans UA159UA159 UA159UA159UA159 (a(a well-characterizedwell-characterized (a (a(a well-characterizedwell-characterizedwell-characterized cariogeniccariogenic cariogeniccariogeniccariogenic pathogenpathogen pathogenpathogenpathogen andand bioﬁlmbiofilm and andand biofilmbiofilmbiofilm former)former) former) former)former) andand and andand StreptococcusStreptococcusStreptococcusStreptococcusStreptococcus gordonii gordoniigordoniigordonii ATCC35105 ATCC35105ATCC35105ATCC35105 (an(an oraloral (((ananan commensal)commensal) oraloraloral commensal)commensal)commensal) were weregrownwerewere growngrowngrown in Brain ininin BrainBrain HeartBrain Heart HeartHeart Infu- Infu-Infu-Infu- sion brothsionsionsion brothbroth(BHIB;broth (BHIB;(BHIB;(BHIB; Himedia HimediaHimediaHimedia Laboratories Laboratories LaboratoriesLaboratoriesLaboratories Pvt. Pvt. Ltd, Pvt.Ltd.,Pvt.Pvt. Mumbai, Ltd,Ltd,Ltd, Mumbai, Mumbai,Mumbai,Mumbai, India) India) India)India) India)supplemented supplemented supplementedsupplementedsupplemented with with 20 withwithwith 202020 − ◦ 20mM mM sucrose.mMmMmM sucrose. sucrose.sucrose.sucrose. The The cultures TheTheThe cultures culturesculturescultures were were wereweremaintawere maintained maintamaintamaintained asinedinedined as glycerol glycerol asasas glycerolglycerolglycerol stocks stocks stocksstocksstocks stored storedstoredstored at −8080 atatat °C. C.−−−808080 For °C.°C.°C. allall ForForFor allallall assays,assays,assays,assays, the inoculum the thethe inoculum inoculuminoculum was prepared was waswas prepared preparedprepared by harvestingharvesti by byby harvesti harvestiharvesting thengngng cells the thethe until cells cellscells theyuntil untiluntil reach they theythey reachreach thereach log the thethe phase, log loglog phase, phase,phase, centrifugedcentrifugedcentrifuged andand resuspendedresuspended andand resuspendedresuspended inin freshfresh inin BHIBfreshBHIBfresh BHIB containingBHIBcontaining cocontainingntaining 2020 mMmM 2020 sucrosesucrose mMmM sucrosesucrose andand adjustedadjusted andand adjustedadjusted toto toto centrifuged and8 resuspended−1 in fresh BHIB containing 20 mM sucrose and adjusted to OD595 0.10.1 (~1.5 (~1.5 × 10×8 10CFUCFU8 8mL − mL1). The − −11 ).bacterial The bacterial cells were cells incubated were incubated anaerobically anaerobically (5% CO2) ODODOD595595595 0.1 0.10.1 (~1.5 (~1.5(~1.5 × ×× 10 10108 CFU CFUCFU mL mLmL −1).).). The TheThe bacterial bacterialbacterial cells cellscells were werewere incubated incubatedincubated anaerobically anaerobicallyanaerobically (5% (5%(5% CO COCO222))) (5% CO ) at 370C under static conditions [40]. All experiments were performed in tripli- at 37⁰Catatat under2 37 3737⁰⁰⁰CCC under under understatic static conditionsstaticstatic conditions conditionsconditions [40]. All [40]. [40].[40]. experiments All AllAll experiments experimentsexperiments were performedwere werewere performed performedperformed in triplicates in inin triplicates triplicatestriplicates on three on onon three threethree cates on three independent occasions. independentindependentindependentindependent occasions. occasions. occasions.occasions.

3.2. Test3.2.3.2.3.2. Compound Test TestTest Compound CompoundCompound Preparation Preparation PreparationPreparation BaicaleinBaicaleinBaicaleinBaicalein (>98% (>98% (>98% (>98%purity) purity) purity)purity) and Dimethyl and andand Dimethyl DimethylDimethyl sulfoxide sulfoxide sulfoxidesulfoxide (DMSO) (DMSO) (DMSO)(DMSO) was purchased was waswas purchased purchasedpurchased from Tokyofrom fromfrom Tokyo TokyoTokyo ChemicalChemicalChemicalChemical Industry IndustryIndustryIndustry Co., Ltd Co.,Co.,Co., (Japan). LtdLtdLtd (Japan).(Japan).(Japan). The stoc TheTheThek stocstocsolutionstockkk solutionsolutionsolution was prepared waswaswas preparedpreparedprepared by dissolving bybyby dissolvingdissolvingdissolving bai- bai-bai-bai- caleincaleincalein caleinin DMSO. in inin DMSO. DMSO.DMSO. The working The TheThe working workingworking solution solution solutionsolution of baicalein of ofof baicalein baicaleinbaicalein was prepared was waswas prepared preparedprepared by diluting by byby diluting dilutingdiluting it in BHIB it itit in inin BHIB BHIBtoBHIB to toto maintainmaintainmaintainmaintain the final the thethe DMSO final finalfinal DMSO DMSODMSO concentration concentration concentrationconcentration of 0.9% of ofof [23].0.9% 0.9%0.9% [23]. [23].[23].

3.3. Effect3.3.3.3.3.3. Effect EffectonEffect Planktonic on onon Planktonic PlanktonicPlanktonic Growth Growth GrowthGrowth Antibiotics 2021, 10, 215 9 of 13

3.2. Test Compound Preparation Baicalein (>98% purity) and Dimethyl sulfoxide (DMSO) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The stock solution was prepared by dissolving baicalein in DMSO. The working solution of baicalein was prepared by diluting it in BHIB to maintain the ﬁnal DMSO concentration of 0.9% [23].

3.3. Effect on Planktonic Growth The test for effect of baicalein on growth of S. mutans and S. gordonii was performed using 96 well microtiter plates (MTP) with two-fold serial dilution in BHIB supplemented with 20 mM sucrose resulting in concentrations ranging from 0.39 µM to 400 µM. The start- ing inoculum of the test strains (~1.5 × 108 CFU/mL) were prepared as mentioned earlier and added to the respective wells. The culture plates were incubated at 37 ◦C for 24 h in 5% CO2. After 24 h, bacterial growth was calculated based on the OD595 values [40].

3.4. Effects on Biofilm Biomass The biofilm inhibitory effect of baicalein was tested using 96 well microtiter plate method [41]. The 96 well MTP method and incubation conditions were concordant with the growth analysis assay. After incubation, the planktonic cells were removed by gently washing the wells three times with phosphate buffer saline (PBS). The biofilms were then air-dried and stained with 0.2% crystal violet solution. The excess stain was washed with PBS, dried, and the biofilm was de-stained with 33% acetic acid. The OD595 was measured and the concentration at which baicalein inhibited 50% biofilm (MBIC50) was calculated.

3.5. Bacterial Surface Hydrophobicity Assay and Bacterial Aggregation Assay The cell surface hydrophobicity and bacterial autoaggregation assays were done as previously [42] with slight modiﬁcations. Brieﬂy, S. mutans culture (OD595 = 0.5) was aliquoted in BHIB with or without 200 µM baicalein and incubated at 37 ◦C under anaerobic conditions. Then, 2 mL of bacterial suspension in triplicates were collected at three different log phases (early, mid, and late) and centrifuged at 5000× g at 4 ◦C for 5 min. Finally, the cells were washed twice and resuspended in PBS. For the cell surface hydrophobicity assay, Xylene (20% v/v) was added to the PBS culture suspension obtained above, and vortexed vigorously to separate the aqueous phase, after which OD595 values was measured for the aqueous phase. The time-dependent decrease in absorbance was considered as a quantitative measure of the cell surface hydrophobicity index (HPBI). The HPBI was calculated with the following formula: HPBI = (A0 − A)/A0 × 100, where A0 and A are the absorbance reading before and after extraction with xylene. A HPBI >70% was scored as hydrophobic [24,42]. To determine the bacterial aggregation, absorbance at A595 (A0) values for the cell suspension mentioned above were initially recorded, then the absorbance (At) was sub- sequently measures following incubation for 2 hours. The extent of autoaggregation was calculated using the following formula: Aggregation % = (1 − (At/A0)) × 100, where At represents the absorbance at time t = 5, 7 and 9 h. The aggregated cells of the baicalein- treated and untreated control groups were also visualized microscopically after crystal violet staining for qualitative analysis.

3.6. Scanning Electron Microscopic Imaging Baicalein-treated and untreated control biofilms were grown on glass slides ◦ (5 mm × 5 mm) in BHIB containing 20 mM sucrose for 24 h at 37 C with 5% CO2. The samples were then fixed by immersing in 25% glutaraldehyde for 1 h and dehydrated with ascending concentrations of ethanol (40%, 60%, 80% and 100%). The slides were then gold sputter-coated, and the biofilm topography was analyzed under a Scanning Electron Microscope (VEGA3, TESCAN Analytics, Brno, Czech Republic) at 10 kV. The obtained images were analyzed using the ImageJ software v1.53a to determine the number of adhered bacterial cells. Antibiotics 2021, 10, 215 10 of 13

3.7. Confocal Laser Microscopic Imaging To further elucidate the effect of sub-inhibitory concentration of baicalein on the cell viability and architecture of the biofilm, confocal laser microscopy was performed. Biofilms were grown on sterile coverslips in BHIB containing 200 µM baicalein supplemented with 20 mM sucrose. The untreated biofilm was also grown to serve as the control. The biofilms ◦ were incubated for 24 h with 5% CO2 at 37 C. The slides were then washed with sterile PBS to remove the planktonic cells and the biofilm was stained using BacLight Bacterial Viability Kit (ThermoFisher Scientific, Waltham, USA). The biofilms were then observed using a confocal laser scanning microscope (Olympus FLUOVIEW, FV1000, Tokyo, Japan). Z-stacks were obtained from at least 5 regions and the obtained images were further analyzed using COMSTAT package in MATLAB R2017b [43]. Additionally, the cell count was enumerated using the ImageJ software.

3.8. Glycolytic pH Drop Assay The effect of baicalein on acid accumulation in bioﬁlms was determined using the glycolytic pH drop assay. S. mutans bacterial cells were harvested at the mid-exponential phase and washed with a salt solution comprising of 50 mM KCl and 1 mM MgCl2 and then resuspended in the same with or without 200 µM baicalein. The samples were adjusted to a pH of 7.2–7.4 using 0.2 M KOH and incubated in anaerobic conditions at 37 ◦C. The pH was recorded (Digital pH meter with pH electrode, Elico Ltd., Hyderabad, India) at three different time points (5, 7 and 9 h) [44]. The pH of Baicalein solution in BHI media and supplemented with 20 mM sucrose was maintained at pH 7.4 ± 0.1 and in the sterile distilled water was maintained at pH 6.4 ± 0.1.

3.9. Gene Expression Analysis The influence of baicalein on the gene profiles of S. mutans biofilm cells was evaluated using Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Biofilms were grown in 6 well plates in BHIB supplemented with 20 mM sucrose with or without 200 µM ◦ baicalein (MBIC50) and incubated at 37 C in anaerobic conditions. At different time points (7th and 9th hour), the biofilm cells were harvested by gently scraping with a sterile scalpel in the presence of PBS and washed thrice with the same. Total RNA was extracted following the manufacturer’s guidelines of HiMedia RNA Extraction Kit (MB613). The integrity and purity of the extracted RNA were verified using standard agarose gel electrophoresis and NanoDrop (Thermo Scientific, Waltham, MA, USA) respectively. The total RNA was converted to cDNA using the iScript™ cDNA Synthesis Kit using the manufacturer-recommended protocol. Relative quantification of gene expression was performed with 16sRNA as the house-keeping gene, using the Fast-Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) (Table S1). Table S1 lists the primer sequences used for this study. Fold changes in gene expression levels were calculated using the 2−∆∆Ct method. The experiment was performed in quadruplicates at each time point and was repeated on three independent occasions [40].

3.10. Combinatorial Effects of Baicalein and Fluoride The checkerboard assay was performed to assess the combinatorial effect of baicalein and fluoride on S. mutans biofilm formation. Bacterial cultures were inoculated in 96 well plates as described in previous experiments, with combinations of varying concentrations of baicalein and fluoride. The concentrations ranged from 12.5 to 200 µM for baicalein and from 0 to 31.25 ppm for fluoride. Untreated wells with only BHIB and sucrose served as controls. Biofilm quantification was done as described earlier and the resultant combined effect on the biofilm formation was evaluated using the CompuSyn software for the calculation of CI (Combinatorial Index) to determine the extent of synergy [45]. CI value of <1 indicates synergy, >1 indicates antagonism and 1 refers to additive interaction [46]. Antibiotics 2021, 10, 215 11 of 13

3.11. Statistical Analysis The results were expressed as mean ± SD. GraphPad Prism software version 6.05 (GraphPad Software Inc., San Diego, CA, USA) was used for performing the statistical analysis. Signiﬁcance was checked with Dunnett t-test for multiple comparisons and paired Student’s t-test (p ≤ 0.05).

4. Conclusions Our results provide the first experimental evidence to support the hypothesis that the biocompatible natural molecule baicalein can inhibit two high value targets of S. mutans for the development of caries, i.e., biofilm formation and acid production, without growth inhibition or inhibition of commensal bacteria such as S. gordonii. This indicates that baicalein has the potential to reverse dysbiosis in multi-species biofilms. This study highlighted the antibiofilm, anti-quorum sensing and anti-virulence potential of baicalein on Streptococ- cus mutans. Baicalein also synergized with fluoride to inhibit S. mutans biofilms, indicating its potential to be developed into anti-caries modalities. Novel delivery approaches are now being developed for further investigations using multi-species biofilm models on different substrates.

Supplementary Materials: The following are available online at https://www.mdpi.com/2079-638 2/10/2/215/s1, Table S1. List of Genes and Primer Sequences used for transcriptome analysis by qRT-PCR; Table S2. The CI values for individual combinations of Baicalein and Fluoride. For all the tested combinations CI < 1 indicating synergy. (CI = 1 indicates addictiveness while CI > 1 indicates antagonism). Author Contributions: A.V.: Investigation, formal analysis, writing. H.B.S.: Investigation, formal analysis, writing. S.V.: Investigation, formal analysis, writing. K.S.: Conceptualization, methodology, supervision. A.P.S.: Conceptualization, methodology, writing, project administration, funding acquisition. P.N.: Conceptualization, methodology, writing, project administration, funding acquisition. All authors have read and agreed to published version of the manuscript. Funding: This work was supported by funds from the MHRD, India Scheme for Promotion of Aca- demic and Research Collaboration—SPARC Cell, SPARC/2018-2019/P27 to SAP and PN. The authors gratefully acknowledge SASTRA Deemed to be University, Thanjavur for extending infrastructure support and the DST-FIST programme (SR/FST/LSI-327/2007 and SR/FST/ETI-331/2013) for the imaging facility. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors gratefully acknowledge SASTRA Deemed to be University, Thanjavur for extending infrastructure support and the DST-FIST program (SR/FST/ETI-331/2013) for the imaging facility. Conflicts of Interest: The authors declare no conflict of interest. Transparency Declaration: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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