Research ArticleCurcumin as a Promising Antibacterial Agent Effects onMetabolism and Biofilm Formation in S mutans

Bingchun Li12 Xinlong Li12 Huancai Lin 12 and Yan Zhou 12

1Department of Preventive Dentistry Guanghua School of Stomatology Sun Yat-sen University 56 Ling Yuan Road WestGuangzhou 510055 China2Guangdong Provincial Key Laboratory of Stomatology Sun Yat-sen University Guangzhou 510055 China

Correspondence should be addressed to Huancai Lin linhcgz126com and Yan Zhou zhouy103163com

Received 9 November 2017 Revised 16 January 2018 Accepted 29 January 2018 Published 28 February 2018

Academic Editor Ronald E Baynes

Copyright copy 2018 Bingchun Li et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Streptococcus mutans (S mutans) has been proved to be the main aetiological factor in dental caries Curcumin a natural producthas been shown to exhibit therapeutic antibacterial activity suggesting that curcuminmay be of clinical interestTheobjective of thisstudy is to evaluate the inhibitory effects of curcumin onmetabolism and biofilm formation in S mutans using a vitro biofilmmodelin an artificial oral environment S mutans biofilms were treated with varying concentrations of curcuminThe biofilmmetabolismand biofilmbiomasswere assessed by the 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazoliumbromide assay and the crystal violetassay Confocal laser scanning microscopy was used to analyse the composition and extracellular polysaccharide content of Smutans biofilm after curcumin treatment The biofilm structure was evaluated using a scanning electron microscope The geneexpression of virulence-related factors was assessed by real-time PCR The antibiofilm effect of curcumin was compared with thatof chlorhexidine The sessile minimum inhibitory concentration (SMIC

50) of curcumin against S mutans biofilm was 500 120583MCurcumin reduced the biofilmmetabolism from 5min to 24 h Curcumin inhibited the quantity of live bacteria and total bacteria inboth the short term (5min) and the long termMoreover curcumin decreased the production of extracellular polysaccharide in theshort termThe expression of genes related to extracellular polysaccharide synthesis carbohydrate metabolism adherence and thetwo-component transduction system decreased after curcumin treatmentThe chlorhexidine-treated group showed similar resultsWe speculate that curcumin has the capacity to be developed as an alternative agent with the potential to reduce the pathogenictraits of S mutans biofilm

1 Introduction

Dental caries one of the most prevalent infectious diseasesworldwide is a biofilm-mediated sugar-driven multifacto-rial dynamic disease [1] Although the pathobiology of dentalcaries is complicated it is widely recognized that the forma-tion of dental plaque biofilm is one of the important causes ofdental caries [2 3] Biofilm formation creates an anaerobicand acidic environment that results in the formation anddevelopment of caries [4]Therefore the eradication of dentalbiofilm is an effective method of controlling caries

Streptococcus mutans (S mutans) has been proved tobe the main aetiological factor in dental caries [5 6] Byadhering to solid surfaces S mutans can colonize the oralcavity and form a bacterial biofilm [7] Dental biofilms aremicrobial aggregates encased in a self-produced extracellular

polymer matrix The phenotype of the bacteria involved inbiofilm formation is quite different from the phenotype inthe planktonic state [8] The bacteria in a dental biofilmare far more resistant to unfavourable growth conditionssuch as biocides and hydrodynamic shear forces [9 10] Theextracellular polysaccharide (EPS) produced by S mutansthrough glycosyltransferases (Gtfs) has been shown to formthe matrix of the biofilm [11] The EPS mediates the irre-versible adherence between bacteria to form a high-cell-density biofilm [12] which increases the bacterial resistanceto antibiotics

Currently many natural products have been consideredas alternative or adjunctive anticaries therapies particularlybotanically derived molecules which offer advantages oversynthetic derivatives due to their natural evolution anddiminished likelihood of resistance [13] Curcumin themajor

HindawiBioMed Research InternationalVolume 2018 Article ID 4508709 11 pageshttpsdoiorg10115520184508709

2 BioMed Research International

constituent of Curcuma longa L or turmeric has beenconfirmed as a potential therapeutic antibacterial agent [14]Curcumin has been reported to inhibit various bacteriaStaphylococcus aureus Salmonella paratyphi Trichophytongypseum and Mycobacterium tuberculosis [14] Recent pub-lications have also reported that curcumin is active against aplethora of drug-resistant bacterial strains [14]

Studies have shown the effects of curcumin on oralbacteria associated with dental disease Song et al [15] foundthat curcumin could significantly inhibit the adhesiveness ofS mutans by its effects on collagen and fibronectin Hu etal [16 17] reported that curcumin is an S mutans sortaseAinhibitor and has promising anticaries characteristics basedon an antiadhesion-mediated mechanism Manoil et al [18]found that blue light-activated curcumin can photoinactivateplanktonic S mutans but the effect on S mutans biofilm waspoor These studies proposed the potential use of curcuminas an antibacterial agent and underlined the need to explorethe mechanism by which curcumin acts on oral bacteria

Our research aims to explore the effects of curcumin onthe biofilm formed by S mutans and compares the resultswith the effects of chlorhexidineThe study contributes to thepossibility of a natural medicine with fewer side effects andstronger antibacterial effects thereby promoting the widerclinical application of curcumin

2 Materials and Methods

21 Bacterial Strain and Growth Conditions S mutans strainUA159 (ATCC700610) was provided by the GuangdongMicrobial Culture Collection Center The S mutans growthin brain-heart infusion (BHI Difco Detroit MI USA) brothmedium was measured For biofilm formation an overnightculture of bacteria was adjusted toOD= 10 (1lowast108 CFUml)The adjusted bacteria were inoculated into 96-well flat-bottom plates The medium was 1 BHIS that is BHI withthe addition of 1 (wtvol) sucrose The bacteria were grownat 37∘C under anaerobic conditions (5 CO

2 10 H

2 85

N2)

22 Minimum Inhibitory Concentration Assay The mini-mum inhibitory concentration (MIC) of curcumin againstplanktonic Smutanswas determined bymicrodilutionmeth-ods following the Clinical Laboratory Standards Institute(CLSI) procedure [19] with some modifications as describedbelow Curcumin from a 250mM stock in dimethyl sul-foxide (DMSO) was diluted in BHI The resulting solutionwas then serially diluted in sterile BHI broth in a 96-wellround-bottom plate to obtain 2000 1000 500 250 125625 and 3125 120583M curcumin in BHI culture medium Thecorresponded concentrations of DMSOwere used as controlEach well contained 100 120583l of serially diluted curcumin towhich 100 120583l of an overnight culture of S mutans was addedbefore incubation at 37∘C under anaerobic conditions (5CO2 10 H

2 85 N

2) for 24 h Three parallel samples were

prepared at each concentration and BHI without curcuminwas used as a control The MIC was defined as the lowestconcentration at which no visible bacteria grew in the broth

The sessile minimum inhibitory concentration (SMIC) ofcurcumin against biofilm S mutanswas determined by the 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazoliumbromide(MTT) assay Overnight S mutans UA159 cultures wereadjusted with 1 BHIS to an initial optical density at 600 of01 (approximately 107 CFUml) and equal volumes (200 120583l)of diluted bacteria were added to 96-well plates and incubatedfor 24 h at 37∘C in a humidified atmosphere containing 5CO2 After 24 h the planktonic bacteria were removed care-

fully and the biofilms formed on the plates were washed withsterile phosphate buffered saline (PBS) The stock solutionof curcumin was diluted in BHIS broth to concentrationsranging from 15625120583M to 1000120583M and added to the testwells The corresponded concentrations of DMSO were usedas control After 24 h of incubation the BHIS broth withcurcumin was removed carefully and the biofilms formed onthe plates were washed again with sterile PBS To quantify thebiofilms 200 120583l of 05mgml MTT was added to each welland incubated at 37∘C in the dark in a humidified atmospherefor 3 h Then the supernatants were removed and 100120583lof 100 DMSO was added to each well For each well theabsorbance at 570 nmwas recorded using amicroplate readerThe percentage of biofilm viability was calculated as follows(absorbance of treated groupabsorbance of control group) times100 [22]

23 Assays of the Viability and Biomass of S mutans BiofilmThebiofilm viability and biomass were tested at 5min 10min15min 30min 60min and 24 h using the MTT assay andthe crystal violet (CV) assay [20]The SMIC

50 concentrationof curcumin was selected for the assay and 012 (wt)chlorhexidine was used in the experiment for comparisonand the 012 (wt) water was used as control For the CVassay the contents of the microplate were removed afterincubation and the wells were washed with PBS fixed with95 methanol washed again and stained with 01 (wtvol)crystal violet solution for 15min at room temperature Subse-quently the microplates were vigorously tapped on napkinsto remove any excess liquid and air-dried The remainingCV was dissolved in 100 120583l of 100 ethanol for 15min atroom temperature and finally 75 120583l from each sample wastransferred to a new 96-well plate and the extract was read at600 nm in a spectrophotometer [20 21]

24 Analysis of the Bacteria Composition and ExtracellularPolysaccharide (EPS) of S mutans Biofilm

241 Analysis of Live and Dead Bacteria by Confocal LaserScanning Microscopy (CLSM) As described previously a24 h biofilm of S mutans was formed on a 15mm confocaldish at 37∘C in a humidified atmosphere containing 5CO2 The planktonic bacteria were removed and the biofilm

was washed twice with sterile PBS The biofilm was treatedwith 500 120583M curcumin 012 chlorhexidine or 1 BHISas a control for 5min or 24 h After treatment the biofilmwas stained with L-7012 LIVEDEAD BacLight BacterialViability Kits (Molecular Probes Eugene OR USA) Thelivedead stain stored at minus20∘C was warmed to roomtemperature and centrifuged before useThe staining solution

BioMed Research International 3

Table 1 Nucleotide sequence of primers used for qRT-PCR

Gene Primer sequence (51015840-31015840)Forward Reverse

gtfB ACACTTTCGGGTGGCTTG GCTTAGATGTCACTTCGGTTGgtfC CCAAAATGGTATTATGGCTGTCG GAGTCTCTATCAAAGTAACGCAGTgtfD TTGACGGTGTTCGTGTTGAT AAAGCGATAGGCGCAGTTTAfruA TGTAGGTCTCGGTTTGTGGGAC TCTTGAGCCAATGCTTCTGGTGgbpB AGCAACAGAAGCACAACCATCAG CCACCATTACCCCAGTAGTTTCCFtf CTGACATAACTACGCCAAAG TGCTTAAATTAATACCAGCTTCluxS CCAGGGACATCTTTCCATGAGAT ACGGGATGATTGACTGTTCCCcomC GACTTTAAAGAAATTAAGACTG AAGCTTGTGTAAAACTTCTGTcomD CTCTGATTGACCATTCTTCTGG CATTCTGAGTTTATGCCCCTCcomE CCTGAAAAGGGCAATCACCAG GGGGCATAAACTCAGAATGTGTCGVicR TGACACGATTACAGCCTTTGATG CGTCTAGTTCTGGTAACATTAAGTCCAATAatpH ACCATACATTTCAGGCTG TTTTAGCACTTGGGATTGscrA GATTGCCCTCAGCAGTTGACAT GCTGGGAAACTTTGATGGAGACscrB ACAGCCTGTCCTGATTTATAGTC CTGGTAACCCAATCCATGAGACsrtA GAAGCTTCCTGTAATTGGCG TTCATCGTTCCAGCACCATAspaP GACTTTGGTAATGGTTATGCATCAA TTTGTATCAGCCGGATCAAGTG16sRNA CTTACCAGGTCTTGACATCCCG ACCCAACATCTCACGACACGAG

included two components SYTO9 and propidium iodidewhich were mixed in equal quantities and applied to thedish for 15min The excitation wavelengths for SYTO9 andpropidium iodide are 488 and 543 nm The biovolumes oflive and dead cells were quantified from the entire stackusing COMSTAT image-processing software The biovolumeis defined as the volume of the biomass (120583m3) divided by thearea of the substratum (confocal dish) (120583m2) [22]

242 Analysis of EPS by CLSM As described previouslya 24 h biofilm of S mutans was grown in 15-mm confocaldishes protected from light with 1120583M Alexa Fluor 647red fluorescent dye (Invitrogen Corp Carlsbad CA USA)to label the EPS [23] After incubation for 24 h at 37∘C ina humidified atmosphere containing 5 CO

2 treatments

consisting of 500120583M curcumin 012 chlorhexidine and1 BHIS as a control were added to separate dishes Thenafter incubation for 5min or 24 h the biofilms were gentlywashed with normal saline and incubated with 1 120583M SYTO9green fluorescent dye at room temperature for 15min tolabel the live bacteria The image collection gates were setto 655ndash690 nm for Alexa Fluor 647 and 495ndash515 nm forSYTO9 Images were obtained by CLSM and analysed usingthe image-processing software COMSTAT [24]

25 Scanning Electron Microscopy (SEM) S mutans 24 hbiofilm was incubated in 1 BHIS medium with or without500 120583M curcumin or 012 chlorhexidine at 37∘C for 5minor 24 h Next the S mutans biofilms were washed twice withPBS fixed with 25 glutaraldehyde overnight at 4∘C andwashed with PBS After dehydration by an alcohol gradient(30 50 70 80 85 90 95 and 100) the biofilmwas dried in a desiccator and sputter-coated with gold Then

the sample were examined at 2000x 5000x and 10000xmagnification by SEM [23 24]

26 Assay of Gene Expression by Real-Time PCR Biofilmsgrown for 24 h were treated with drugs for 5min or 24 hThe biofilms were harvested by centrifugation at 12000 rpmfor 5min and the total RNA was isolated by ultrasoniccrushing and using the RNeasy Mini Kit (QIAGEN Valen-cia CA USA) The total RNA concentration and puritywere determined using a NanoDrop 2000 spectrophotometer(Thermo Fisher Scientific Pittsburgh PA USA) The reversetranscription of the total RNA and quantitative real-timePCR were performed similarl to the procedures described inprevious studies [23] The primer sequences were based onthe previous literature and are listed in Table 1 [17 24ndash27]The 2minusΔΔCt method was used to calculate the gene expressionfold changes in S mutans

27 Statistical Analysis Each experiment was independentlyrepeated at least three times GraphPad Prism version 504(GraphPad Software San Diego CA) was used to assess thedata The differences between the experimental group andthe untreated control group were statistically analysed usingthe SPSS 170 software The data were assessed to determinewhether they were normally distributed One-way analysisof variance (ANOVA) and Tukeyrsquos test were performed formultiple groupsThe unpaired 119905-test was used for two groupsThe significance level of the 119875 value was lt005

3 Results

31 Determination of the MIC of Curcumin against Planktonicand Biofilm S mutans The results showed that curcumininhibited the growth of planktonic S mutans with an MIC

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Figure 2 Antibiofilm effect of different curcumin exposure times on S mutans biofilm A 24 h biofilm was incubated in curcumin at theSMIC

50for different times The Colony-Forming Units (CFU) of lived bacteria in biofilm were evaluated by the MTT assay (a) and the

percentage of biofilm biomass was evaluated by the crystal violet (CV) assay (b) The data represent the mean plusmn SD of three independenttests The asterisks (lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

of 125 120583M After treatment with concentrations higher than125 120583M no visible S mutans growth was observed TheMTTassay showed that after treatment with serial dilutions ofcurcumin the biofilm viability decreased significantly by50 at 500120583M The DMSO has no effect on viability ofbiofilm (Figure 1(a)) The growth curve at concentrationsof 500 120583M and for the control showed similar trends Thebacteria in both cases reached the stationary phase at 12 h(Figure 1(b)) The variation in biofilm biomass between500 120583M and 0 was independent of the growth kineticsTherefore a concentration of 500 120583M was defined as caus-ing 50 inhibition (SMIC

50) and selected for the nextstudy

32 Inhibition of the Metabolism of Biofilm Formation byCurcumin Both MTT and CV assays were conducted toevaluate the alteration in relevant biofilm characteristics ofS mutans under the influence of curcumin In addition012 chlorhexidine treatment was used to provide a com-parison group The results of the MTT assay showed thatcurcumin decreased the biofilm metabolism at both 5minand 24 h compared to that of untreated biofilm (119875 lt 0001Figure 2(a)) Chlorhexidine treatment produced the sametrend as curcumin and the biofilmmetabolismdecreased sig-nificantly more than for curcumin (119875 lt 0001 Figure 2(a))The CV assay showed that curcumin and chlorhexidine bothresulted in reduced S mutans biomass at 24 h Chlorhexidine

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Figure 3 CLSM imaging of S mutans biofilm grown in 1 BHIS After 24 h of growth the biofilm was treated with 1 BHIS (control)500 120583M curcumin or 012 chlorhexidine for 5min (a) and 24 h (b) Each micrograph represents 4 optical sections green representing livebacteria red representing dead bacteria combined green and red from two channel images and three-dimensional reconstructions of thecontrol biofilm without any treatment the 500 120583M curcumin-treated biofilm and the 012 chlorhexidine-treated biofilmThe total bacteriabiomass and the Colony-Forming Units (CFU) of lived bacteria are quantified in (c) and (d)The data represent the mean plusmn SDThe asterisks(lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

resulted in a significantly greater decrease than curcumin(119875 lt 0001 Figure 2(b)) As curcumin decreased the metab-olism of the S mutans biofilm in a short time (5min) we nextcompared the short-term (5min) and long-term (24 h) effectsof curcumin on S mutans biofilm

33 Evaluation of the Effect on the LiveDead Bacteria Ratio inSmutans Biofilm Theeffects of curcumin and chlorhexidineon the livedead bacteria ratio in S mutans biofilm wereassayed by CLSM (Figure 3) In the confocal micrographs

green florescence indicates lived cells while red fluorescenceindicates dead cellsThe images reflect different green and redflorescence intensities from those in the control group Lowergreen-stained density than in the control group was observedin both treated groups at 5min and 24 h (Figures 3(a) and3(b)) At 5min the live bacteria in the curcumin group andchlorhexidine group were reduced while the total bacteria inthe curcumin group were reduced (Figure 3(c)) At 24 h thelive bacteria in the curcumin group were reduced while thetotal bacteria were reduced in both groups (Figure 3(d))

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Figure 4 CLSM images of S mutans biofilm (a b) Three-dimensional reconstructions of the untreated (control) biofilm the 500 120583Mcurcumin-treated biofilm and the 012 chlorhexidine-treated biofilm at 5min (a) and 24 h (b) EPS was labelled in red (Alexa Fluor 647)bacterial cells were labelled in green (SYTO9) and red and green superimposed appear as yellow Images were obtained at 20x magnification(c) Image representing the volume of EPS calculated according to 5 random sites of each sample repeated three times (d) Change in biofilmthickness at 5min and 24 h respectively calculated from data obtained from fluorescence CLSM Data are presented as the mean plusmn SD Theasterisks (lowast) indicate significant differences (lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

34 Evaluation of the Effect on the EPS of S mutans BiofilmThe three-dimensional reconstruction images obtained fromCLSM of the S mutans biofilm (Figures 4(a) and 4(c))confirmed that the EPS decreased with both curcumin andchlorhexidine treatment after 5min (119875 lt 005) and this trendcontinued at 24 h The thickness of the biofilm showed nodifference among the three groups at 5min but was visiblyreduced at 24 h (Figures 4(b) and 4(d)) The SEM imageconfirmed the change in EPS (Figure 5) Treatment withcurcumin or chlorhexidine was able to reduce the overallquantity of S mutans EPS compared to the untreated controlas indicated by arrows Furthermore long-term treatment(24 h) resulted in a more obvious reduction than short-termtreatment (5min)

35 Evaluation of the Effect on the mRNA Levels of Smutans Biofilm Virulence Factors Compared with the levelsobserved in S mutans biofilms grown in 1 BHIS theexpression levels of gtfB gtfC and gtfD in S mutans biofilmsexposed to 500 120583M curcumin for 5min were significantlydecreased by 057- 059- and 059-fold respectively At 24 hthe expression levels of gtfB gtfC and gtfD in S mutanswere also decreased and there was no significant differencefrom the levels at 5min Meanwhile the expression of gtfBgtfC and gtfD was also downregulated under the influence

of 012 chlorhexidine for 5min and 24 h there was nosignificant difference between two time points (Figure 6(a))

In Figure 6(b) we observed no significant difference inthe expression of scrA at either 5min or 24 h However theexpression of atpH and scrB decreased in the presence of cur-cumin (119875 lt 005) The expression of atpH decreased furtherover time In contrast the expression of scrB increased overtime

The entire set of virulence genes involved in quorum-sensing signalling mechanisms was found to be down-regulated after treatment with curcumin Treatment withcurcumin for 5min repressed the expression levels of luxScomC comD and comE to 507 574 648 and 541respectively as compared to the control values Likewisechlorhexidine repressed the gene expression levels to 501523 607 and 523 respectively After treatment withthe drugs for 24 h only the expression of comDE showed asignificant difference

The expression levels of srtA and spaP in the biofilmstreated with curcumin for 5min were significantly downreg-ulated by approximately onefold compared with the levelsin untreated biofilm and the same tendency was shownby chlorhexidine (Figure 6(d)) After 24 h of treatment witheither drug the expression levels of srtA and spaP showed nosignificant difference

BioMed Research International 7

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Figure 5 Morphological characteristics of S mutans biofilm treated with or without drugs Representative SEM images of 24 h S mutansbiofilm grown in curcumin for 5min (b) and 24 h (e) and grown in chlorhexidine for 5min (c) and 24 h (f) The image of 24 h S mutansbiofilm grown in 1 BHIS for 5min (a) and 24 h (d) as a control Magnifications of 2000x 5000x and 10000x are shown for each conditionThe black arrows highlight the EPS of S mutans which was markedly reduced

4 Discussion

Dental caries a common infectious disease worldwide iscaused by biofilm known as dental plaque that result fromthe adhesion of bacteria to tooth surfaces The effective erad-ication of cariogenic biofilm and the pathogenic microorgan-isms within is the key way to prevent tooth decay [28] Smutans is the main aetiological microorganism involved inbiofilm [29] In this study we used curcumin a natural food-grade product to control the formation of S mutans biofilm

Previous studies have shown that curcumin could inhibitthe viability of S mutans with an MIC value of 125ndash175 120583M[17] Similar MIC results were obtained in this study but theSMIC values here were much higher than the MIC As is wellknown it is more difficult to inhibit the viability of S mutansbiofilm than planktonic bacteria because the planktonicbacteria are more susceptible to the effects of commonly usedantimicrobial drugs than the bacteria embedded in a biofilm[30 31]

Accordingly we evaluated the effect of curcumin on Smutans biofilm over time At the SMIC

50 curcumin was

shown to exert both a short-term effect and a long-termeffect on the viability of S mutant biofilm which was alsotrue for 012 chlorhexidine Previous studies showed theeffect of chlorhexidine on S mutant biofilm Yang et al [32]

reported that S mutans biofilm was susceptible to 012chlorhexidine with a biomass reduction of over 80observed after treatment with 012 chlorhexidine for 1 hTamura et al [33] found that 8 120583gml chlorhexidine waseffective in removing biofilm after 24 h All of these resultsillustrate that chlorhexidine has antibiofilm effects whichwasconsistent with our resultsThe short-term effect of curcuminon S mutans biofilm also suggested that curcumin has thepotential to be a new antibiofilm drug because it exhibitedsimilar effects to chlorhexidine

S mutans has a regulatory network to integrate its cellularresponse to environmental change [6]This study found thatafter treatment with curcumin the amounts of viable bacteriaand total bacteria were reduced as confirmed by the results ofCLSM Because curcumin exhibited no effect on the growthkinetics of S mutans the reduced quantity of live bacteriaindicated a direct effect of curcumin on S mutans

A biofilm is a highly dynamic and structured communityof microbial cells that are enmeshed in an extracellularmatrix of polymeric substances such as exopolysaccharidesproteins and nucleic acids [34] As reported previously theproduction of EPS by S mutans on a surface enhances thelocal accumulation and clustering of microbes In additionas the biofilm develops the spatial heterogeneities resultingfrom EPS synthesis form a complex 3D matrix architecture

8 BioMed Research International

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowast lowast

lowastlowast

24 h5min

ftf

gtfB

gtfC

gtfD

fruA

gbpB ftf

gtfC

gtfB

gtfD

fruA

gbpB

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

nChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(a)

lowastlowast

lowastlowastlowast

lowast lowast

24 h5min

scrB

scrA

atpH scrB

scrA

atpH

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(b)

lowast

lowastlowastlowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

24 h5min

vicRluxS

com

E

com

D

com

C

luxS

vicR

com

E

com

C

com

D

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(c)

lowastlowastlowast

lowastlowast

lowast

24 h5min

srtA

spaP

srtA

spaP

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(d)

Figure 6 Results of qRT-PCR to examine the gene expression of different virulence systems in S mutans UA159 (a) EPS synthesis system(b) carbohydrate metabolism system (c) quorum-sensing system (d) two-component transduction system All targets were amplified usingprimers Different gene expression levels were normalized to the level of 16sRNA gene transcripts Data are presented as the mean plusmn SDTheasterisks (lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

and create environmental and protective niches within thebiofilm that can directly modulate caries pathogenesis [3435] Curcumin was shown to destroy the structure of EPS inthe short term decreasing the biomass of EPS the quantityof total bacteria and the percentage of lived bacteria Withprolonged curcumin exposure the structure of the EPS wasbadly damaged and the thickness of the biofilm and thenumber of total bacteria were decreased demonstrably Khanet al reported that the extract of Trachyspermum ammi seedscould modulate the expression of specific virulence genes byS mutans which disrupted the accumulation and structuralorganization of EPS ultimately affecting the cariogenicityof S mutans [36] Xu et al found that the tea catechinepigallocatechin gallate could inhibit the formation of Smutans biofilm by suppressing the gtfs gene [37] Thesestudies serve as a reminder that the effects of natural productson the EPS of S mutans biofilm are usually exerted byregulating cariogenic genes

As previously reported the regulatory systems in Smutans such as the EPS synthesis system carbohydratemetabolism system quorum-sensing system and two-component transduction system are connected to the ecolog-ical balance and to bacterial carcinogenesis [6 38]The resultsshowed that curcumin decreased the expression of cariogenicgenes in the plaque of S mutans Although the majorityof the 24 h qPCR results were not statistically significant

the 119875 values between the control group and curcumin groupwere close to 005 and the decrease was pronounced Thuscurcumin has an inhibitory effect on certain genes in Smutans at 24 h

Gtfs encoded by the gtfBCD genes are indispensable forthe utilization of glucose and for EPS synthesis in S mutansThey are important factors in biofilm formation and thedevelopment of caries [24] GtfB synthesizes water-insolublepolysaccharide [39] which is the main component of theEPS Downregulation of the expression of gtfB in curcuminmight explain the decreased biomass of EPS in the shortterm Oral bacterial aggregation is mediated by interactionsbetween surface-associated glucan-binding proteins (GbpBs)that adhere to glucans thereby promoting plaque formation[40] The expression of gbpB was decreased in both treat-ment groups which may relate to the reduced thickness ofthe biofilm Fructosyltransferase (FTF) is an enzyme thatconverts sucrose to extracellular homopolymers of fructosethe fructans FTF is the product of the fruA gene and isan exo-120573-d-fructosidase that releases fructose from 120573(26)-and 120573(21)-linked fructans and cleaves fructose from sucroseand raffinose [41ndash43] Both ftf and fruA expressions weredysregulated after short-term exposure to curcumin

In S mutans the scrA gene in the phosphotransferasesystem (PTS) encodes a high-affinity permease which inter-nalizes sucrose Then intracellular sucrose-6-PO

4is first

BioMed Research International 9

hydrolyzed by ScrB a sucrose-6-PO4hydrolase to produce

fructose and glucose-6-PO4 In addition the downstream of

scrA and scrB could encode a regulator to change the expres-sion level of scrAB Both scrAB genes were downregulatedin our study which might contribute to the reduction inthe S mutans biofilm In the oral environment S mutansis subjected to rapid pH fluctuations arising from the avail-ability of carbohydrates The atpH gene in S mutans encodessubunit C of a multisubunit enzyme (F1F0-ATPase) involvedin intracellular pH regulation and acid tolerance [44 45]Gene expression analysis found that atpHwas downregulatedin S mutans biofilm after treatment with curcumin for 5minand 24 hThus curcumin inhibits the acid tolerance ability ofS mutans and probably decreases the cariogenic traits of Smutans

The quorum-sensing system is an essential component ofentire gene regulation networks responsible for the adapta-tion of bacteria in biofilms [46] Two well-studied quorum-sensing systems exist in S mutans the ComCDE systemwhich can enable intraspecies cell-cell communication andhas been proved to have a positive regulatory effect onthe expression of biofilm-related genes such as gtfB gtfCand gbpB in S mutans and the LuxS system which cancatalyse the formation of the signal peptide AI-2 to mediateinterspecies and intraspecies interaction in the multispeciesplaque community [6 47] The findings of the current worksuggest that curcumin can potentially contribute to reducingthe quantity of lived bacteria in the biofilm and decrease thebiomass of EPS by decreasing the cariogenic potential of Smutans biofilm via inhibiting the expression of ComCDEsystem and LuxS system The short-term inhibitory effectis more significant than the long-term effect There is adecreasing tendency in the VicR expression of S mutans aftercurcumin treatment suggesting that curcumin may act byinhibiting the expression of VicR The regulatory network ofcariogenic genes in S mutans is complex It was reported thatthe ComRComS is the second quorum-sensing system inS mutans [48] The cell-to-cell communication system in Smutans is not clear Further research is required in this field

S mutans possesses a series of cell-surface proteins suchas SpaP (also known as antigen III Pac P1 and antigenB) which are anchored to the cell envelope by sortase A(SrtA) Sortase A is encoded by the gene srtA and recognizesa motif consisting of leucine proline X threonine andglycine (LPXTG where X is any amino acid) [33 49] Naturalproducts attract substantial attention because they can inhibitsrtA in vitro and in vivo but do not significantly decreasethe viability of bacteria The current study demonstrates thatcurcumin can inhibit the expression of spaP and srtA withan SMIC

50of 500 120583M The present study explored the effect

of curcumin on S mutans UA159 However S mutans hasheterogeneity in different strains Much more strains of Smutans are needed to be test in future

In conclusion curcumin has both a short-term and along-term antibacterial effect Additionally it is a food-gradenatural product with a similar effect to that of chlorhexidineand it takes effect faster than 012 chlorhexidine Curcumincould be an alternative strategy to treat oral disease Cur-cumin is a promising antibacterial agent with potential forwider clinical application in the future

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by Science and Technology Plan-ning Project ofGuangdongProvince China (2014A020212621and 2017A020215064)

References

[1] N B Pitts D T Zero P D Marsh et al ldquoDental cariesrdquo NatureReviews Disease Primers vol 3 Article ID 17030 2017

[2] D F A Pereira C J Seneviratne C Y Koga-Ito and L PSamaranayake ldquoIs the oral fungal pathogen Candida albicansa cariogenrdquo Oral Diseases 2017

[3] B NyvadW Crielaard AMira N Takahashi andD BeightonldquoDental caries from a molecular microbiological perspectiverdquoCaries Research vol 47 no 2 pp 89ndash102 2013

[4] R E Marquis ldquoOxygen metabolism oxidative stress and acid-base physiology of dental plaque biofilmsrdquo Journal of IndustrialMicrobiology and Biotechnology vol 15 no 3 pp 198ndash207 1995

[5] W H Bowen and H Koo ldquoBiology of streptococcus mutans-derived glucosyltransferases role in extracellular matrix forma-tion of cariogenic biofilmsrdquo Caries Research vol 45 no 1 pp69ndash86 2011

[6] E G Smith and G A Spatafora ldquoGene regulation in S mutanscomplex control in a complex environmentrdquo Journal of DentalResearch vol 91 no 2 pp 133ndash141 2012

[7] W Krzysciak A Jurczak D Koscielniak B Bystrowska and ASkalniak ldquoThe virulence of Streptococcus mutans and the abilityto form biofilmsrdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 33 no 4 pp 499ndash515 2014

[8] J Merritt J Kreth F Qi R Sullivan and W Shi ldquoNon-disruptive real-time analyses of themetabolic status and viabil-ity of Streptococcus mutans cells in response to antimicrobialtreatmentsrdquo Journal of Microbiological Methods vol 61 no 2pp 161ndash170 2005

[9] J Fricks-Lima C M Hendrickson M Allgaier et al ldquoDif-ferences in biofilm formation and antimicrobial resistance ofPseudomonas aeruginosa isolated from airways of mechanicallyventilated patients and cystic fibrosis patientsrdquo InternationalJournal of Antimicrobial Agents vol 37 no 4 pp 309ndash315 2011

[10] R Utsumi Bacterial Signal Transduction Networks and DrugTargets vol 631 Springer New York NY USA 2008

[11] H Koo andW H Bowen ldquoCandida albicans and Streptococcusmutans a potential synergistic alliance to cause virulent toothdecay in childrenrdquo Future Microbiology vol 9 no 12 pp 1295ndash1297 2014

[12] S Hamada T Koga and T Ooshima ldquoVirulence factors ofStreptococcus mutans and dental caries preventionrdquo Journal ofDental Research vol 63 no 3 pp 407ndash411 1984

[13] J-G Jeon P L Rosalen M L Falsetta and H Koo ldquoNaturalproducts in caries research current (limited) knowledge chal-lenges and future perspectiverdquoCaries Research vol 45 no 3 pp243ndash263 2011

[14] S-Y Teow K Liew S A Ali A S-B Khoo and S-CPeh ldquoAntibacterial Action of Curcumin against Staphylococcusaureus A Brief Reviewrdquo Journal of Tropical Medicine vol 2016Article ID 2853045 10 pages 2016

10 BioMed Research International

[15] J Song B Choi E-J Jin Y Yoon and K-H Choi ldquoCurcuminsuppresses Streptococcus mutans adherence to human toothsurfaces and extracellular matrix proteinsrdquo European Journalof Clinical Microbiology amp Infectious Diseases vol 31 no 7 pp1347ndash1352 2012

[16] P Hu P Huang and M W Chen ldquoCurcumin reduces Strep-tococcus mutans biofilm formation by inhibiting sortase Aactivityrdquo Archives of Oral Biolog vol 58 no 10 pp 1343ndash13482013

[17] P Hu P Huang and W M Chen ldquoCurcumin inhibits thesortase a activity of the streptococcus mutans UA159rdquo AppliedBiochemistry and Biotechnology vol 171 no 2 pp 396ndash4022013

[18] DManoil A Filieri C Gameiro et al ldquoFlow cytometric assess-ment of Streptococcus mutans viability after exposure to bluelight-activated curcuminrdquo Photodiagnosis and PhotodynamicTherapy vol 11 no 3 pp 372ndash379 2014

[19] M A Pfaller and D J Diekema ldquoProgress in antifungalsusceptibility testing of Candida spp by use of Clinical andLaboratory Standards Institute broth microdilution methods2010 to 2012rdquo Journal of Clinical Microbiology vol 50 no 9 pp2846ndash2856 2012

[20] M M Weerasekera G K Wijesinghe T A Jayarathna etal ldquoCulture media profoundly affect Candida Albicans andCandida tropicalis growth adhesion and biofilm developmentrdquoMemorias do InstitutoOswaldoCruz vol 111 no 11 pp 697ndash7022016

[21] J Chen T Li X Zhou et al ldquoCharacterization of the clusteredregularly interspaced short palindromic repeats sites in Strep-tococcus mutans isolated from early childhood caries patientsrdquoArchives of Oral Biolog vol 83 pp 174ndash180 2017

[22] M Sun Z Zhou J Dong J Zhang Y Xia and R ShuldquoAntibacterial and antibiofilm activities of docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA) against periodon-topathic bacteriardquo Microbial Pathogenesis vol 99 pp 196ndash2032016

[23] X Huang K Zhang M Deng et al ldquoEffect of arginine on thegrowth and biofilm formation of oral bacteriardquo Archives of OralBiolog vol 82 pp 256ndash262 2017

[24] S Liu W Qiu K Zhang et al ldquoNicotine enhances inter-species relationship between streptococcus mutans and candidaalbicansrdquo BioMed Research International vol 2017 Article ID7953920 9 pages 2017

[25] T B L Bedran L Grignon D P Spolidorio and D GrenierldquoSubinhibitory concentrations of triclosan promote Streptococ-cus mutans biofilm formation and adherence to oral epithelialcellsrdquo PLoS ONE vol 9 no 2 Article ID e89059 2014

[26] C M Levesque E Voronejskaia Y-C C Huang RWMair RP Ellen and D G Cvitkovitch ldquoInvolvement of sortase anchor-ing of cell wall proteins in biofilm formation by Streptococcusmutansrdquo Infection and Immunity vol 73 no 6 pp 3773ndash37772005

[27] M M Nascimento J A Lemos J Abranches V K Lin andR A Burne ldquoRole of RelA of Streptococcus mutans in globalcontrol of gene expressionrdquo Journal of Bacteriology vol 190 no1 pp 28ndash36 2008

[28] B Kunze M Reck A Dotsch et al ldquoDamage of Streptococcusmutans biofilms by carolacton a secondarymetabolite from themyxobacterium Sorangium cellulosumrdquo BMC Microbiologyvol 10 article no 199 2010

[29] V LeungDDufour andCM Levesque ldquoDeath and survival inStreptococcusmutansDiffering outcomes of a quorum-sensing

signaling peptiderdquo Frontiers in Microbiology vol 6 Article ID01176 2015

[30] A Tofino-Rivera M Ortega-Cuadros D Galvis-Pareja HJimenez-Rios L JMerini andMCMartınez-Pabon ldquoEffect ofLippia alba and Cymbopogon citratus essential oils on biofilmsof Streptococcusmutans and cytotoxicity in CHO cellsrdquo Journalof Ethnopharmacology vol 194 pp 749ndash754 2016

[31] K Jhajharia A Parolia K V Shetty and L K Mehta ldquoBiofilmin endodontics a reviewrdquo Journal of International Society ofPreventive amp Community Dentistry vol 5 no 1 pp 1ndash12 2015

[32] H Yang Y Bi X Shang et al ldquoAntibiofilm activities of anovel chimeolysin against Streptococcus mutans under physi-ological and cariogenic conditionsrdquo Antimicrobial Agents andChemotherapy vol 60 no 12 pp 7436ndash7443 2016

[33] H Tamura A Yamada and H Kato ldquoMolecular characteriza-tion of the dextran-binding lectin B gene dblB of Streptococcuscriceti in Streptococcus mutans strain GS-5 with mutations inboth gbpC and spaP genesrdquo Genes amp Genetic Systems vol 89no 2 pp 41ndash50 2014

[34] M I Klein G Hwang P H S Santos O H Campanella andH Koo ldquoStreptococcus mutans-derived extracellular matrixin cariogenic oral biofilmsrdquo Frontiers in Cellular and InfectionMicrobiology vol 5 article no 10 2015

[35] H Koo M L Falsetta and M I Klein ldquoThe exopolysaccharidematrix a virulence determinant of cariogenic biofilmrdquo Journalof Dental Research vol 92 no 12 pp 1065ndash1073 2013

[36] R KhanMAdilMDanishuddin P KVerma andAUKhanldquoIn vitro and in vivo inhibition of Streptococcusmutans biofilmby Trachyspermum ammi seeds An approach of alternativemedicinerdquo Phytomedicine vol 19 no 8-9 pp 747ndash755 2012

[37] X Xu X D Zhou and C DWu ldquoTea catechin epigallocatechingallate inhibits Streptococcus mutans biofilm formation bysuppressing gtf genesrdquo Archives of Oral Biolog vol 57 no 6 pp678ndash683 2012

[38] K Stephenson ldquoTwo-component and phosphorelay signal-transduction systems as therapeutic targetsrdquo Current Opinionin Pharmacology vol 2 no 5 pp 507ndash512 2002

[39] H Aoki T Shiroza H Hayakawa S Sato and H K KuramitsuldquoCloning of a Streptococcus mutans glycosyltransferase genecoding for insoluble glucan synthesisrdquo Infection and Immunityvol 53 no 3 pp 587ndash594 1986

[40] Z He Q Wang Y Hu et al ldquoUse of the quorum sensinginhibitor furanone C-30 to interfere with biofilm formation byStreptococcus mutans and its luxS mutant strainrdquo InternationalJournal of Antimicrobial Agents vol 40 no 1 pp 30ndash35 2012

[41] A Ogawa S Furukawa S Fujita et al ldquoInhibition of Strepto-coccus mutans biofilm formation by Streptococcus salivariusFruArdquo Applied and Environmental Microbiology vol 77 no 5pp 1572ndash1580 2011

[42] R A Burne and J E C Penders ldquoDifferential localization of theStreptococcus mutans GS-5 fructan hydrolase enzyme FruArdquoFEMS Microbiology Letters vol 121 no 2 pp 243ndash249 1994

[43] R A Burne K Schilling W H Bowen and R E YasbinldquoExpression purification and characterization of an Exo-120573-D-fructosidase of Streptococcus mutansrdquo Journal of Bacteriologyvol 169 no 10 pp 4507ndash4517 1987

[44] L Sadeghinejad D G Cvitkovitch W L Siqueira J Merritt JP Santerre and Y Finer ldquoMechanistic genomic and proteomicstudy on the effects of BisGMA-derived biodegradation producton cariogenic bacteriardquoDental Materials vol 33 no 2 pp 175ndash190 2017

BioMed Research International 11

[45] G R Bender S V W Sutton and R E Marquis ldquoAcidtolerance proton permeabilities and membrane ATPases oforal streptococcirdquo Infection and Immunity vol 53 no 2 pp 331ndash338 1986

[46] Y-H Li X-L Tian G Layton C Norgaard and G Sis-son ldquoAdditive attenuation of virulence and cariogenic poten-tial of Streptococcus mutans by simultaneous inactivationof the ComCDE quorum-sensing system and HKRR11 two-component regulatory systemrdquoMicrobiology vol 154 no 11 pp3256ndash3265 2008

[47] D G Cvitkovitch Y-H Li and R P Ellen ldquoQuorum sensingand biofilm formation in Streptococcal infectionsrdquoThe Journalof Clinical Investigation vol 112 no 11 pp 1626ndash1632 2003

[48] R Khan H V Rukke A P R Filho et al ldquoExtracellularidentification of a processed type II ComRComS pheromoneof Streptococcus mutansrdquo Journal of Bacteriology vol 194 no15 pp 3781ndash3788 2012

[49] P Huang P Hu S Y Zhou Q Li and W M Chen ldquoMorininhibits sortase A and subsequent biofilm formation in strepto-coccus mutansrdquo Current Microbiology vol 68 no 1 pp 47ndash522014

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International Journal of

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Hindawiwwwhindawicom Volume 2018

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Submit your manuscripts atwwwhindawicom

BioMed Research International 3

Table 1 Nucleotide sequence of primers used for qRT-PCR

Gene Primer sequence (51015840-31015840)Forward Reverse

gtfB ACACTTTCGGGTGGCTTG GCTTAGATGTCACTTCGGTTGgtfC CCAAAATGGTATTATGGCTGTCG GAGTCTCTATCAAAGTAACGCAGTgtfD TTGACGGTGTTCGTGTTGAT AAAGCGATAGGCGCAGTTTAfruA TGTAGGTCTCGGTTTGTGGGAC TCTTGAGCCAATGCTTCTGGTGgbpB AGCAACAGAAGCACAACCATCAG CCACCATTACCCCAGTAGTTTCCFtf CTGACATAACTACGCCAAAG TGCTTAAATTAATACCAGCTTCluxS CCAGGGACATCTTTCCATGAGAT ACGGGATGATTGACTGTTCCCcomC GACTTTAAAGAAATTAAGACTG AAGCTTGTGTAAAACTTCTGTcomD CTCTGATTGACCATTCTTCTGG CATTCTGAGTTTATGCCCCTCcomE CCTGAAAAGGGCAATCACCAG GGGGCATAAACTCAGAATGTGTCGVicR TGACACGATTACAGCCTTTGATG CGTCTAGTTCTGGTAACATTAAGTCCAATAatpH ACCATACATTTCAGGCTG TTTTAGCACTTGGGATTGscrA GATTGCCCTCAGCAGTTGACAT GCTGGGAAACTTTGATGGAGACscrB ACAGCCTGTCCTGATTTATAGTC CTGGTAACCCAATCCATGAGACsrtA GAAGCTTCCTGTAATTGGCG TTCATCGTTCCAGCACCATAspaP GACTTTGGTAATGGTTATGCATCAA TTTGTATCAGCCGGATCAAGTG16sRNA CTTACCAGGTCTTGACATCCCG ACCCAACATCTCACGACACGAG

included two components SYTO9 and propidium iodidewhich were mixed in equal quantities and applied to thedish for 15min The excitation wavelengths for SYTO9 andpropidium iodide are 488 and 543 nm The biovolumes oflive and dead cells were quantified from the entire stackusing COMSTAT image-processing software The biovolumeis defined as the volume of the biomass (120583m3) divided by thearea of the substratum (confocal dish) (120583m2) [22]

242 Analysis of EPS by CLSM As described previouslya 24 h biofilm of S mutans was grown in 15-mm confocaldishes protected from light with 1120583M Alexa Fluor 647red fluorescent dye (Invitrogen Corp Carlsbad CA USA)to label the EPS [23] After incubation for 24 h at 37∘C ina humidified atmosphere containing 5 CO

2 treatments

consisting of 500120583M curcumin 012 chlorhexidine and1 BHIS as a control were added to separate dishes Thenafter incubation for 5min or 24 h the biofilms were gentlywashed with normal saline and incubated with 1 120583M SYTO9green fluorescent dye at room temperature for 15min tolabel the live bacteria The image collection gates were setto 655ndash690 nm for Alexa Fluor 647 and 495ndash515 nm forSYTO9 Images were obtained by CLSM and analysed usingthe image-processing software COMSTAT [24]

25 Scanning Electron Microscopy (SEM) S mutans 24 hbiofilm was incubated in 1 BHIS medium with or without500 120583M curcumin or 012 chlorhexidine at 37∘C for 5minor 24 h Next the S mutans biofilms were washed twice withPBS fixed with 25 glutaraldehyde overnight at 4∘C andwashed with PBS After dehydration by an alcohol gradient(30 50 70 80 85 90 95 and 100) the biofilmwas dried in a desiccator and sputter-coated with gold Then

the sample were examined at 2000x 5000x and 10000xmagnification by SEM [23 24]

26 Assay of Gene Expression by Real-Time PCR Biofilmsgrown for 24 h were treated with drugs for 5min or 24 hThe biofilms were harvested by centrifugation at 12000 rpmfor 5min and the total RNA was isolated by ultrasoniccrushing and using the RNeasy Mini Kit (QIAGEN Valen-cia CA USA) The total RNA concentration and puritywere determined using a NanoDrop 2000 spectrophotometer(Thermo Fisher Scientific Pittsburgh PA USA) The reversetranscription of the total RNA and quantitative real-timePCR were performed similarl to the procedures described inprevious studies [23] The primer sequences were based onthe previous literature and are listed in Table 1 [17 24ndash27]The 2minusΔΔCt method was used to calculate the gene expressionfold changes in S mutans

27 Statistical Analysis Each experiment was independentlyrepeated at least three times GraphPad Prism version 504(GraphPad Software San Diego CA) was used to assess thedata The differences between the experimental group andthe untreated control group were statistically analysed usingthe SPSS 170 software The data were assessed to determinewhether they were normally distributed One-way analysisof variance (ANOVA) and Tukeyrsquos test were performed formultiple groupsThe unpaired 119905-test was used for two groupsThe significance level of the 119875 value was lt005

3 Results

31 Determination of the MIC of Curcumin against Planktonicand Biofilm S mutans The results showed that curcumininhibited the growth of planktonic S mutans with an MIC

4 BioMed Research International

CurcuminDMSO

lowast lowastlowastlowastlowast

lowastlowastlowastlowastlowast

107

1075

108

625 125 250 100015625 5000 3125

Concentration (M)

lgCF

U

(a)

500 －

0 －

00

02

＄

600

04

06

20 300 10

Time (h)

(b)

Figure 1 (a) Antibiofilm effects of different concentrations of curcumin and the correspondent concentrations of DMSO on S mutansbiofilmThe bacteria were inoculated in a 96-well microtiter plate containing 1 BHISmedium to form a 24 h biofilm then washed with PBSand incubated in 1 BHIS with different concentrations of curcumin (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001) After 24 h of incubation theColony-Forming Units (CFU) of lived bacteria in biofilm were evaluated by the MTT assay (b) Effect of SMIC

50curcumin on the growth of

S mutans

lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

ChlorhexidineCurcuminControl

MTT

107

108

lgCF

U

15 min 30min10 min 60min 24 h5min

(a)

lowastlowast

lowastlowast lowastlowast lowastlowastlowastlowast

lowastlowastlowast

CV Stain

0

50

100

150

b

iom

ass

15 min 30min10 min 60min 24 h5min

ChlorhexidineCurcuminControl

(b)

Figure 2 Antibiofilm effect of different curcumin exposure times on S mutans biofilm A 24 h biofilm was incubated in curcumin at theSMIC

50for different times The Colony-Forming Units (CFU) of lived bacteria in biofilm were evaluated by the MTT assay (a) and the

percentage of biofilm biomass was evaluated by the crystal violet (CV) assay (b) The data represent the mean plusmn SD of three independenttests The asterisks (lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

of 125 120583M After treatment with concentrations higher than125 120583M no visible S mutans growth was observed TheMTTassay showed that after treatment with serial dilutions ofcurcumin the biofilm viability decreased significantly by50 at 500120583M The DMSO has no effect on viability ofbiofilm (Figure 1(a)) The growth curve at concentrationsof 500 120583M and for the control showed similar trends Thebacteria in both cases reached the stationary phase at 12 h(Figure 1(b)) The variation in biofilm biomass between500 120583M and 0 was independent of the growth kineticsTherefore a concentration of 500 120583M was defined as caus-ing 50 inhibition (SMIC

50) and selected for the nextstudy

32 Inhibition of the Metabolism of Biofilm Formation byCurcumin Both MTT and CV assays were conducted toevaluate the alteration in relevant biofilm characteristics ofS mutans under the influence of curcumin In addition012 chlorhexidine treatment was used to provide a com-parison group The results of the MTT assay showed thatcurcumin decreased the biofilm metabolism at both 5minand 24 h compared to that of untreated biofilm (119875 lt 0001Figure 2(a)) Chlorhexidine treatment produced the sametrend as curcumin and the biofilmmetabolismdecreased sig-nificantly more than for curcumin (119875 lt 0001 Figure 2(a))The CV assay showed that curcumin and chlorhexidine bothresulted in reduced S mutans biomass at 24 h Chlorhexidine

BioMed Research International 5

5min

3D

Mer

ged

imag

eD

ead

bact

eria

ChlorhexidineCurcumin

Live

bac

teria

Control(20x)

050

100150

200

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350350

400 400

0

0

50100

1530

150

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400 400

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0

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1530

150200

050

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150

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350350

400 400

0

0

50100

1530

150

200

(a)24 h

3D

Mer

ged

imag

eD

ead

bact

eria

ChlorhexidineCurcumin

Live

bac

teria

Control(20x)

050

100150

200

250250

300300

350 350

400 400

0

0

50100

1530

150

200

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200

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400 400

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0

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22

150200

0

50

100

150

200

250

250300

300350

350

400400

0

0

50100

20

150200

(b)

lowastlowastlowastlowastlowastlowast

5min5min

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol 1078

1079

108

109

lived

bac

teria

(lgC

FU)

0

20

40

60

80

Tota

l Bac

teria

(Ｇ

3

Ｇ2)

(c)

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowast

24 h24 h

0

20

40

60

80

Tota

l Bac

teria

(Ｇ

3

Ｇ2)

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e 1078

1079

108

1081

1082

lived

bac

teria

(lgC

FU)

(d)

Figure 3 CLSM imaging of S mutans biofilm grown in 1 BHIS After 24 h of growth the biofilm was treated with 1 BHIS (control)500 120583M curcumin or 012 chlorhexidine for 5min (a) and 24 h (b) Each micrograph represents 4 optical sections green representing livebacteria red representing dead bacteria combined green and red from two channel images and three-dimensional reconstructions of thecontrol biofilm without any treatment the 500 120583M curcumin-treated biofilm and the 012 chlorhexidine-treated biofilmThe total bacteriabiomass and the Colony-Forming Units (CFU) of lived bacteria are quantified in (c) and (d)The data represent the mean plusmn SDThe asterisks(lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

resulted in a significantly greater decrease than curcumin(119875 lt 0001 Figure 2(b)) As curcumin decreased the metab-olism of the S mutans biofilm in a short time (5min) we nextcompared the short-term (5min) and long-term (24 h) effectsof curcumin on S mutans biofilm

33 Evaluation of the Effect on the LiveDead Bacteria Ratio inSmutans Biofilm Theeffects of curcumin and chlorhexidineon the livedead bacteria ratio in S mutans biofilm wereassayed by CLSM (Figure 3) In the confocal micrographs

green florescence indicates lived cells while red fluorescenceindicates dead cellsThe images reflect different green and redflorescence intensities from those in the control group Lowergreen-stained density than in the control group was observedin both treated groups at 5min and 24 h (Figures 3(a) and3(b)) At 5min the live bacteria in the curcumin group andchlorhexidine group were reduced while the total bacteria inthe curcumin group were reduced (Figure 3(c)) At 24 h thelive bacteria in the curcumin group were reduced while thetotal bacteria were reduced in both groups (Figure 3(d))

6 BioMed Research International

Mer

ged

EPS

Bact

eria

(20x)ChlorhexidineCurcuminControl5min

050

100150

200

250250

300300

350350

400 400

050

100150

200

050

100150

200

250 250300 300

350 350

400 400

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100150

200

0

50100

150

200

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300 300

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050

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200

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350 350

400 400

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400 400

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250250

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350 350

400 400

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250250

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350 350

400 400

050

100150

200

050

100150

200

250

250300300

350350

400 400

050

100150

200

(a)

Mer

ged

EPS

Bact

eria

(20x)ChlorhexidineCurcuminControl24 h

01530

01530

015 0

18

0

18

0

18

0

18

018

0

18

30

050

100150

200

250250

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350350

400 400

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100150

200

050

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150

200

250250

300 300

350 350

400 400

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100150

200

050

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150

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400 400

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0

50100

150

200

250250

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350350

400 400

050

100

150

200

0

50

100

150

200

250

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300

350350

400 400

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150

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050

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150

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350350

400 400

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100150

200

0

50100

150

200

250

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350

400 400

050

100150

200

0

50

100

150

200

250

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300

350350

400 400

050

100150

200

050

100

150

200

250

250300

300350

350

400400

050

100150

200

(b)

5min 5min

lowastlowastlowastlowastlowast

0

5

10

15

EPS

Biom

ass(

Ｇ

3 Ｇ

2 )

0

5

10

15

20

thic

knes

s of b

iofil

m (

m)

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e

(c)

24 h 24 h

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

0

10

20

30

40

EPS

Biom

ass(

Ｇ

3 Ｇ

2 )

01020304050

thic

knes

s of b

iofil

m (

m)

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e

(d)

Figure 4 CLSM images of S mutans biofilm (a b) Three-dimensional reconstructions of the untreated (control) biofilm the 500 120583Mcurcumin-treated biofilm and the 012 chlorhexidine-treated biofilm at 5min (a) and 24 h (b) EPS was labelled in red (Alexa Fluor 647)bacterial cells were labelled in green (SYTO9) and red and green superimposed appear as yellow Images were obtained at 20x magnification(c) Image representing the volume of EPS calculated according to 5 random sites of each sample repeated three times (d) Change in biofilmthickness at 5min and 24 h respectively calculated from data obtained from fluorescence CLSM Data are presented as the mean plusmn SD Theasterisks (lowast) indicate significant differences (lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

34 Evaluation of the Effect on the EPS of S mutans BiofilmThe three-dimensional reconstruction images obtained fromCLSM of the S mutans biofilm (Figures 4(a) and 4(c))confirmed that the EPS decreased with both curcumin andchlorhexidine treatment after 5min (119875 lt 005) and this trendcontinued at 24 h The thickness of the biofilm showed nodifference among the three groups at 5min but was visiblyreduced at 24 h (Figures 4(b) and 4(d)) The SEM imageconfirmed the change in EPS (Figure 5) Treatment withcurcumin or chlorhexidine was able to reduce the overallquantity of S mutans EPS compared to the untreated controlas indicated by arrows Furthermore long-term treatment(24 h) resulted in a more obvious reduction than short-termtreatment (5min)

35 Evaluation of the Effect on the mRNA Levels of Smutans Biofilm Virulence Factors Compared with the levelsobserved in S mutans biofilms grown in 1 BHIS theexpression levels of gtfB gtfC and gtfD in S mutans biofilmsexposed to 500 120583M curcumin for 5min were significantlydecreased by 057- 059- and 059-fold respectively At 24 hthe expression levels of gtfB gtfC and gtfD in S mutanswere also decreased and there was no significant differencefrom the levels at 5min Meanwhile the expression of gtfBgtfC and gtfD was also downregulated under the influence

of 012 chlorhexidine for 5min and 24 h there was nosignificant difference between two time points (Figure 6(a))

In Figure 6(b) we observed no significant difference inthe expression of scrA at either 5min or 24 h However theexpression of atpH and scrB decreased in the presence of cur-cumin (119875 lt 005) The expression of atpH decreased furtherover time In contrast the expression of scrB increased overtime

The entire set of virulence genes involved in quorum-sensing signalling mechanisms was found to be down-regulated after treatment with curcumin Treatment withcurcumin for 5min repressed the expression levels of luxScomC comD and comE to 507 574 648 and 541respectively as compared to the control values Likewisechlorhexidine repressed the gene expression levels to 501523 607 and 523 respectively After treatment withthe drugs for 24 h only the expression of comDE showed asignificant difference

The expression levels of srtA and spaP in the biofilmstreated with curcumin for 5min were significantly downreg-ulated by approximately onefold compared with the levelsin untreated biofilm and the same tendency was shownby chlorhexidine (Figure 6(d)) After 24 h of treatment witheither drug the expression levels of srtA and spaP showed nosignificant difference

BioMed Research International 7

10000x5000x2000x

(A3)(A2)(A1)

5minC

ontro

l

(a)

(B3)(B2)(B1)

10000x5000x2000x5min

Curc

umin

(b)

(C3)(C2)(C1)

10000x5000x2000x5min

Chlo

rhex

idin

e

(c)

10000x5000x2000x

(D3)(D2)(D1)

Con

trol

24 h

(d)

(E3)(E2)(E1)

10000x5000x2000x

Curc

umin

24 h

(e)

(F3)(F2)(F1)

10000x5000x2000x

Chlo

rhex

idin

e

24 h

(f)

Figure 5 Morphological characteristics of S mutans biofilm treated with or without drugs Representative SEM images of 24 h S mutansbiofilm grown in curcumin for 5min (b) and 24 h (e) and grown in chlorhexidine for 5min (c) and 24 h (f) The image of 24 h S mutansbiofilm grown in 1 BHIS for 5min (a) and 24 h (d) as a control Magnifications of 2000x 5000x and 10000x are shown for each conditionThe black arrows highlight the EPS of S mutans which was markedly reduced

4 Discussion

Dental caries a common infectious disease worldwide iscaused by biofilm known as dental plaque that result fromthe adhesion of bacteria to tooth surfaces The effective erad-ication of cariogenic biofilm and the pathogenic microorgan-isms within is the key way to prevent tooth decay [28] Smutans is the main aetiological microorganism involved inbiofilm [29] In this study we used curcumin a natural food-grade product to control the formation of S mutans biofilm

Previous studies have shown that curcumin could inhibitthe viability of S mutans with an MIC value of 125ndash175 120583M[17] Similar MIC results were obtained in this study but theSMIC values here were much higher than the MIC As is wellknown it is more difficult to inhibit the viability of S mutansbiofilm than planktonic bacteria because the planktonicbacteria are more susceptible to the effects of commonly usedantimicrobial drugs than the bacteria embedded in a biofilm[30 31]

Accordingly we evaluated the effect of curcumin on Smutans biofilm over time At the SMIC

50 curcumin was

shown to exert both a short-term effect and a long-termeffect on the viability of S mutant biofilm which was alsotrue for 012 chlorhexidine Previous studies showed theeffect of chlorhexidine on S mutant biofilm Yang et al [32]

reported that S mutans biofilm was susceptible to 012chlorhexidine with a biomass reduction of over 80observed after treatment with 012 chlorhexidine for 1 hTamura et al [33] found that 8 120583gml chlorhexidine waseffective in removing biofilm after 24 h All of these resultsillustrate that chlorhexidine has antibiofilm effects whichwasconsistent with our resultsThe short-term effect of curcuminon S mutans biofilm also suggested that curcumin has thepotential to be a new antibiofilm drug because it exhibitedsimilar effects to chlorhexidine

S mutans has a regulatory network to integrate its cellularresponse to environmental change [6]This study found thatafter treatment with curcumin the amounts of viable bacteriaand total bacteria were reduced as confirmed by the results ofCLSM Because curcumin exhibited no effect on the growthkinetics of S mutans the reduced quantity of live bacteriaindicated a direct effect of curcumin on S mutans

A biofilm is a highly dynamic and structured communityof microbial cells that are enmeshed in an extracellularmatrix of polymeric substances such as exopolysaccharidesproteins and nucleic acids [34] As reported previously theproduction of EPS by S mutans on a surface enhances thelocal accumulation and clustering of microbes In additionas the biofilm develops the spatial heterogeneities resultingfrom EPS synthesis form a complex 3D matrix architecture

8 BioMed Research International

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowast lowast

lowastlowast

24 h5min

ftf

gtfB

gtfC

gtfD

fruA

gbpB ftf

gtfC

gtfB

gtfD

fruA

gbpB

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

nChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(a)

lowastlowast

lowastlowastlowast

lowast lowast

24 h5min

scrB

scrA

atpH scrB

scrA

atpH

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(b)

lowast

lowastlowastlowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

24 h5min

vicRluxS

com

E

com

D

com

C

luxS

vicR

com

E

com

C

com

D

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(c)

lowastlowastlowast

lowastlowast

lowast

24 h5min

srtA

spaP

srtA

spaP

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(d)

Figure 6 Results of qRT-PCR to examine the gene expression of different virulence systems in S mutans UA159 (a) EPS synthesis system(b) carbohydrate metabolism system (c) quorum-sensing system (d) two-component transduction system All targets were amplified usingprimers Different gene expression levels were normalized to the level of 16sRNA gene transcripts Data are presented as the mean plusmn SDTheasterisks (lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

and create environmental and protective niches within thebiofilm that can directly modulate caries pathogenesis [3435] Curcumin was shown to destroy the structure of EPS inthe short term decreasing the biomass of EPS the quantityof total bacteria and the percentage of lived bacteria Withprolonged curcumin exposure the structure of the EPS wasbadly damaged and the thickness of the biofilm and thenumber of total bacteria were decreased demonstrably Khanet al reported that the extract of Trachyspermum ammi seedscould modulate the expression of specific virulence genes byS mutans which disrupted the accumulation and structuralorganization of EPS ultimately affecting the cariogenicityof S mutans [36] Xu et al found that the tea catechinepigallocatechin gallate could inhibit the formation of Smutans biofilm by suppressing the gtfs gene [37] Thesestudies serve as a reminder that the effects of natural productson the EPS of S mutans biofilm are usually exerted byregulating cariogenic genes

As previously reported the regulatory systems in Smutans such as the EPS synthesis system carbohydratemetabolism system quorum-sensing system and two-component transduction system are connected to the ecolog-ical balance and to bacterial carcinogenesis [6 38]The resultsshowed that curcumin decreased the expression of cariogenicgenes in the plaque of S mutans Although the majorityof the 24 h qPCR results were not statistically significant

the 119875 values between the control group and curcumin groupwere close to 005 and the decrease was pronounced Thuscurcumin has an inhibitory effect on certain genes in Smutans at 24 h

Gtfs encoded by the gtfBCD genes are indispensable forthe utilization of glucose and for EPS synthesis in S mutansThey are important factors in biofilm formation and thedevelopment of caries [24] GtfB synthesizes water-insolublepolysaccharide [39] which is the main component of theEPS Downregulation of the expression of gtfB in curcuminmight explain the decreased biomass of EPS in the shortterm Oral bacterial aggregation is mediated by interactionsbetween surface-associated glucan-binding proteins (GbpBs)that adhere to glucans thereby promoting plaque formation[40] The expression of gbpB was decreased in both treat-ment groups which may relate to the reduced thickness ofthe biofilm Fructosyltransferase (FTF) is an enzyme thatconverts sucrose to extracellular homopolymers of fructosethe fructans FTF is the product of the fruA gene and isan exo-120573-d-fructosidase that releases fructose from 120573(26)-and 120573(21)-linked fructans and cleaves fructose from sucroseand raffinose [41ndash43] Both ftf and fruA expressions weredysregulated after short-term exposure to curcumin

In S mutans the scrA gene in the phosphotransferasesystem (PTS) encodes a high-affinity permease which inter-nalizes sucrose Then intracellular sucrose-6-PO

4is first

BioMed Research International 9

hydrolyzed by ScrB a sucrose-6-PO4hydrolase to produce

fructose and glucose-6-PO4 In addition the downstream of

scrA and scrB could encode a regulator to change the expres-sion level of scrAB Both scrAB genes were downregulatedin our study which might contribute to the reduction inthe S mutans biofilm In the oral environment S mutansis subjected to rapid pH fluctuations arising from the avail-ability of carbohydrates The atpH gene in S mutans encodessubunit C of a multisubunit enzyme (F1F0-ATPase) involvedin intracellular pH regulation and acid tolerance [44 45]Gene expression analysis found that atpHwas downregulatedin S mutans biofilm after treatment with curcumin for 5minand 24 hThus curcumin inhibits the acid tolerance ability ofS mutans and probably decreases the cariogenic traits of Smutans

The quorum-sensing system is an essential component ofentire gene regulation networks responsible for the adapta-tion of bacteria in biofilms [46] Two well-studied quorum-sensing systems exist in S mutans the ComCDE systemwhich can enable intraspecies cell-cell communication andhas been proved to have a positive regulatory effect onthe expression of biofilm-related genes such as gtfB gtfCand gbpB in S mutans and the LuxS system which cancatalyse the formation of the signal peptide AI-2 to mediateinterspecies and intraspecies interaction in the multispeciesplaque community [6 47] The findings of the current worksuggest that curcumin can potentially contribute to reducingthe quantity of lived bacteria in the biofilm and decrease thebiomass of EPS by decreasing the cariogenic potential of Smutans biofilm via inhibiting the expression of ComCDEsystem and LuxS system The short-term inhibitory effectis more significant than the long-term effect There is adecreasing tendency in the VicR expression of S mutans aftercurcumin treatment suggesting that curcumin may act byinhibiting the expression of VicR The regulatory network ofcariogenic genes in S mutans is complex It was reported thatthe ComRComS is the second quorum-sensing system inS mutans [48] The cell-to-cell communication system in Smutans is not clear Further research is required in this field

S mutans possesses a series of cell-surface proteins suchas SpaP (also known as antigen III Pac P1 and antigenB) which are anchored to the cell envelope by sortase A(SrtA) Sortase A is encoded by the gene srtA and recognizesa motif consisting of leucine proline X threonine andglycine (LPXTG where X is any amino acid) [33 49] Naturalproducts attract substantial attention because they can inhibitsrtA in vitro and in vivo but do not significantly decreasethe viability of bacteria The current study demonstrates thatcurcumin can inhibit the expression of spaP and srtA withan SMIC

50of 500 120583M The present study explored the effect

of curcumin on S mutans UA159 However S mutans hasheterogeneity in different strains Much more strains of Smutans are needed to be test in future

In conclusion curcumin has both a short-term and along-term antibacterial effect Additionally it is a food-gradenatural product with a similar effect to that of chlorhexidineand it takes effect faster than 012 chlorhexidine Curcumincould be an alternative strategy to treat oral disease Cur-cumin is a promising antibacterial agent with potential forwider clinical application in the future

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by Science and Technology Plan-ning Project ofGuangdongProvince China (2014A020212621and 2017A020215064)

References

[1] N B Pitts D T Zero P D Marsh et al ldquoDental cariesrdquo NatureReviews Disease Primers vol 3 Article ID 17030 2017

[2] D F A Pereira C J Seneviratne C Y Koga-Ito and L PSamaranayake ldquoIs the oral fungal pathogen Candida albicansa cariogenrdquo Oral Diseases 2017

[3] B NyvadW Crielaard AMira N Takahashi andD BeightonldquoDental caries from a molecular microbiological perspectiverdquoCaries Research vol 47 no 2 pp 89ndash102 2013

[4] R E Marquis ldquoOxygen metabolism oxidative stress and acid-base physiology of dental plaque biofilmsrdquo Journal of IndustrialMicrobiology and Biotechnology vol 15 no 3 pp 198ndash207 1995

[5] W H Bowen and H Koo ldquoBiology of streptococcus mutans-derived glucosyltransferases role in extracellular matrix forma-tion of cariogenic biofilmsrdquo Caries Research vol 45 no 1 pp69ndash86 2011

[6] E G Smith and G A Spatafora ldquoGene regulation in S mutanscomplex control in a complex environmentrdquo Journal of DentalResearch vol 91 no 2 pp 133ndash141 2012

[7] W Krzysciak A Jurczak D Koscielniak B Bystrowska and ASkalniak ldquoThe virulence of Streptococcus mutans and the abilityto form biofilmsrdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 33 no 4 pp 499ndash515 2014

[8] J Merritt J Kreth F Qi R Sullivan and W Shi ldquoNon-disruptive real-time analyses of themetabolic status and viabil-ity of Streptococcus mutans cells in response to antimicrobialtreatmentsrdquo Journal of Microbiological Methods vol 61 no 2pp 161ndash170 2005

[9] J Fricks-Lima C M Hendrickson M Allgaier et al ldquoDif-ferences in biofilm formation and antimicrobial resistance ofPseudomonas aeruginosa isolated from airways of mechanicallyventilated patients and cystic fibrosis patientsrdquo InternationalJournal of Antimicrobial Agents vol 37 no 4 pp 309ndash315 2011

[10] R Utsumi Bacterial Signal Transduction Networks and DrugTargets vol 631 Springer New York NY USA 2008

[11] H Koo andW H Bowen ldquoCandida albicans and Streptococcusmutans a potential synergistic alliance to cause virulent toothdecay in childrenrdquo Future Microbiology vol 9 no 12 pp 1295ndash1297 2014

[12] S Hamada T Koga and T Ooshima ldquoVirulence factors ofStreptococcus mutans and dental caries preventionrdquo Journal ofDental Research vol 63 no 3 pp 407ndash411 1984

[13] J-G Jeon P L Rosalen M L Falsetta and H Koo ldquoNaturalproducts in caries research current (limited) knowledge chal-lenges and future perspectiverdquoCaries Research vol 45 no 3 pp243ndash263 2011

[14] S-Y Teow K Liew S A Ali A S-B Khoo and S-CPeh ldquoAntibacterial Action of Curcumin against Staphylococcusaureus A Brief Reviewrdquo Journal of Tropical Medicine vol 2016Article ID 2853045 10 pages 2016

10 BioMed Research International

[15] J Song B Choi E-J Jin Y Yoon and K-H Choi ldquoCurcuminsuppresses Streptococcus mutans adherence to human toothsurfaces and extracellular matrix proteinsrdquo European Journalof Clinical Microbiology amp Infectious Diseases vol 31 no 7 pp1347ndash1352 2012

[16] P Hu P Huang and M W Chen ldquoCurcumin reduces Strep-tococcus mutans biofilm formation by inhibiting sortase Aactivityrdquo Archives of Oral Biolog vol 58 no 10 pp 1343ndash13482013

[17] P Hu P Huang and W M Chen ldquoCurcumin inhibits thesortase a activity of the streptococcus mutans UA159rdquo AppliedBiochemistry and Biotechnology vol 171 no 2 pp 396ndash4022013

[18] DManoil A Filieri C Gameiro et al ldquoFlow cytometric assess-ment of Streptococcus mutans viability after exposure to bluelight-activated curcuminrdquo Photodiagnosis and PhotodynamicTherapy vol 11 no 3 pp 372ndash379 2014

[19] M A Pfaller and D J Diekema ldquoProgress in antifungalsusceptibility testing of Candida spp by use of Clinical andLaboratory Standards Institute broth microdilution methods2010 to 2012rdquo Journal of Clinical Microbiology vol 50 no 9 pp2846ndash2856 2012

[20] M M Weerasekera G K Wijesinghe T A Jayarathna etal ldquoCulture media profoundly affect Candida Albicans andCandida tropicalis growth adhesion and biofilm developmentrdquoMemorias do InstitutoOswaldoCruz vol 111 no 11 pp 697ndash7022016

[21] J Chen T Li X Zhou et al ldquoCharacterization of the clusteredregularly interspaced short palindromic repeats sites in Strep-tococcus mutans isolated from early childhood caries patientsrdquoArchives of Oral Biolog vol 83 pp 174ndash180 2017

[22] M Sun Z Zhou J Dong J Zhang Y Xia and R ShuldquoAntibacterial and antibiofilm activities of docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA) against periodon-topathic bacteriardquo Microbial Pathogenesis vol 99 pp 196ndash2032016

[23] X Huang K Zhang M Deng et al ldquoEffect of arginine on thegrowth and biofilm formation of oral bacteriardquo Archives of OralBiolog vol 82 pp 256ndash262 2017

[24] S Liu W Qiu K Zhang et al ldquoNicotine enhances inter-species relationship between streptococcus mutans and candidaalbicansrdquo BioMed Research International vol 2017 Article ID7953920 9 pages 2017

[25] T B L Bedran L Grignon D P Spolidorio and D GrenierldquoSubinhibitory concentrations of triclosan promote Streptococ-cus mutans biofilm formation and adherence to oral epithelialcellsrdquo PLoS ONE vol 9 no 2 Article ID e89059 2014

[26] C M Levesque E Voronejskaia Y-C C Huang RWMair RP Ellen and D G Cvitkovitch ldquoInvolvement of sortase anchor-ing of cell wall proteins in biofilm formation by Streptococcusmutansrdquo Infection and Immunity vol 73 no 6 pp 3773ndash37772005

[27] M M Nascimento J A Lemos J Abranches V K Lin andR A Burne ldquoRole of RelA of Streptococcus mutans in globalcontrol of gene expressionrdquo Journal of Bacteriology vol 190 no1 pp 28ndash36 2008

[28] B Kunze M Reck A Dotsch et al ldquoDamage of Streptococcusmutans biofilms by carolacton a secondarymetabolite from themyxobacterium Sorangium cellulosumrdquo BMC Microbiologyvol 10 article no 199 2010

[29] V LeungDDufour andCM Levesque ldquoDeath and survival inStreptococcusmutansDiffering outcomes of a quorum-sensing

signaling peptiderdquo Frontiers in Microbiology vol 6 Article ID01176 2015

[30] A Tofino-Rivera M Ortega-Cuadros D Galvis-Pareja HJimenez-Rios L JMerini andMCMartınez-Pabon ldquoEffect ofLippia alba and Cymbopogon citratus essential oils on biofilmsof Streptococcusmutans and cytotoxicity in CHO cellsrdquo Journalof Ethnopharmacology vol 194 pp 749ndash754 2016

[31] K Jhajharia A Parolia K V Shetty and L K Mehta ldquoBiofilmin endodontics a reviewrdquo Journal of International Society ofPreventive amp Community Dentistry vol 5 no 1 pp 1ndash12 2015

[32] H Yang Y Bi X Shang et al ldquoAntibiofilm activities of anovel chimeolysin against Streptococcus mutans under physi-ological and cariogenic conditionsrdquo Antimicrobial Agents andChemotherapy vol 60 no 12 pp 7436ndash7443 2016

[33] H Tamura A Yamada and H Kato ldquoMolecular characteriza-tion of the dextran-binding lectin B gene dblB of Streptococcuscriceti in Streptococcus mutans strain GS-5 with mutations inboth gbpC and spaP genesrdquo Genes amp Genetic Systems vol 89no 2 pp 41ndash50 2014

[34] M I Klein G Hwang P H S Santos O H Campanella andH Koo ldquoStreptococcus mutans-derived extracellular matrixin cariogenic oral biofilmsrdquo Frontiers in Cellular and InfectionMicrobiology vol 5 article no 10 2015

[35] H Koo M L Falsetta and M I Klein ldquoThe exopolysaccharidematrix a virulence determinant of cariogenic biofilmrdquo Journalof Dental Research vol 92 no 12 pp 1065ndash1073 2013

[36] R KhanMAdilMDanishuddin P KVerma andAUKhanldquoIn vitro and in vivo inhibition of Streptococcusmutans biofilmby Trachyspermum ammi seeds An approach of alternativemedicinerdquo Phytomedicine vol 19 no 8-9 pp 747ndash755 2012

[37] X Xu X D Zhou and C DWu ldquoTea catechin epigallocatechingallate inhibits Streptococcus mutans biofilm formation bysuppressing gtf genesrdquo Archives of Oral Biolog vol 57 no 6 pp678ndash683 2012

[38] K Stephenson ldquoTwo-component and phosphorelay signal-transduction systems as therapeutic targetsrdquo Current Opinionin Pharmacology vol 2 no 5 pp 507ndash512 2002

[39] H Aoki T Shiroza H Hayakawa S Sato and H K KuramitsuldquoCloning of a Streptococcus mutans glycosyltransferase genecoding for insoluble glucan synthesisrdquo Infection and Immunityvol 53 no 3 pp 587ndash594 1986

[40] Z He Q Wang Y Hu et al ldquoUse of the quorum sensinginhibitor furanone C-30 to interfere with biofilm formation byStreptococcus mutans and its luxS mutant strainrdquo InternationalJournal of Antimicrobial Agents vol 40 no 1 pp 30ndash35 2012

[41] A Ogawa S Furukawa S Fujita et al ldquoInhibition of Strepto-coccus mutans biofilm formation by Streptococcus salivariusFruArdquo Applied and Environmental Microbiology vol 77 no 5pp 1572ndash1580 2011

[42] R A Burne and J E C Penders ldquoDifferential localization of theStreptococcus mutans GS-5 fructan hydrolase enzyme FruArdquoFEMS Microbiology Letters vol 121 no 2 pp 243ndash249 1994

[43] R A Burne K Schilling W H Bowen and R E YasbinldquoExpression purification and characterization of an Exo-120573-D-fructosidase of Streptococcus mutansrdquo Journal of Bacteriologyvol 169 no 10 pp 4507ndash4517 1987

[44] L Sadeghinejad D G Cvitkovitch W L Siqueira J Merritt JP Santerre and Y Finer ldquoMechanistic genomic and proteomicstudy on the effects of BisGMA-derived biodegradation producton cariogenic bacteriardquoDental Materials vol 33 no 2 pp 175ndash190 2017

BioMed Research International 11

[45] G R Bender S V W Sutton and R E Marquis ldquoAcidtolerance proton permeabilities and membrane ATPases oforal streptococcirdquo Infection and Immunity vol 53 no 2 pp 331ndash338 1986

[46] Y-H Li X-L Tian G Layton C Norgaard and G Sis-son ldquoAdditive attenuation of virulence and cariogenic poten-tial of Streptococcus mutans by simultaneous inactivationof the ComCDE quorum-sensing system and HKRR11 two-component regulatory systemrdquoMicrobiology vol 154 no 11 pp3256ndash3265 2008

[47] D G Cvitkovitch Y-H Li and R P Ellen ldquoQuorum sensingand biofilm formation in Streptococcal infectionsrdquoThe Journalof Clinical Investigation vol 112 no 11 pp 1626ndash1632 2003

[48] R Khan H V Rukke A P R Filho et al ldquoExtracellularidentification of a processed type II ComRComS pheromoneof Streptococcus mutansrdquo Journal of Bacteriology vol 194 no15 pp 3781ndash3788 2012

[49] P Huang P Hu S Y Zhou Q Li and W M Chen ldquoMorininhibits sortase A and subsequent biofilm formation in strepto-coccus mutansrdquo Current Microbiology vol 68 no 1 pp 47ndash522014

Hindawiwwwhindawicom

International Journal of

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Submit your manuscripts atwwwhindawicom

4 BioMed Research International

CurcuminDMSO

lowast lowastlowastlowastlowast

lowastlowastlowastlowastlowast

107

1075

108

625 125 250 100015625 5000 3125

Concentration (M)

lgCF

U

(a)

500 －

0 －

00

02

＄

600

04

06

20 300 10

Time (h)

(b)

Figure 1 (a) Antibiofilm effects of different concentrations of curcumin and the correspondent concentrations of DMSO on S mutansbiofilmThe bacteria were inoculated in a 96-well microtiter plate containing 1 BHISmedium to form a 24 h biofilm then washed with PBSand incubated in 1 BHIS with different concentrations of curcumin (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001) After 24 h of incubation theColony-Forming Units (CFU) of lived bacteria in biofilm were evaluated by the MTT assay (b) Effect of SMIC

50curcumin on the growth of

S mutans

lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

ChlorhexidineCurcuminControl

MTT

107

108

lgCF

U

15 min 30min10 min 60min 24 h5min

(a)

lowastlowast

lowastlowast lowastlowast lowastlowastlowastlowast

lowastlowastlowast

CV Stain

0

50

100

150

b

iom

ass

15 min 30min10 min 60min 24 h5min

ChlorhexidineCurcuminControl

(b)

Figure 2 Antibiofilm effect of different curcumin exposure times on S mutans biofilm A 24 h biofilm was incubated in curcumin at theSMIC

50for different times The Colony-Forming Units (CFU) of lived bacteria in biofilm were evaluated by the MTT assay (a) and the

percentage of biofilm biomass was evaluated by the crystal violet (CV) assay (b) The data represent the mean plusmn SD of three independenttests The asterisks (lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

of 125 120583M After treatment with concentrations higher than125 120583M no visible S mutans growth was observed TheMTTassay showed that after treatment with serial dilutions ofcurcumin the biofilm viability decreased significantly by50 at 500120583M The DMSO has no effect on viability ofbiofilm (Figure 1(a)) The growth curve at concentrationsof 500 120583M and for the control showed similar trends Thebacteria in both cases reached the stationary phase at 12 h(Figure 1(b)) The variation in biofilm biomass between500 120583M and 0 was independent of the growth kineticsTherefore a concentration of 500 120583M was defined as caus-ing 50 inhibition (SMIC

50) and selected for the nextstudy

32 Inhibition of the Metabolism of Biofilm Formation byCurcumin Both MTT and CV assays were conducted toevaluate the alteration in relevant biofilm characteristics ofS mutans under the influence of curcumin In addition012 chlorhexidine treatment was used to provide a com-parison group The results of the MTT assay showed thatcurcumin decreased the biofilm metabolism at both 5minand 24 h compared to that of untreated biofilm (119875 lt 0001Figure 2(a)) Chlorhexidine treatment produced the sametrend as curcumin and the biofilmmetabolismdecreased sig-nificantly more than for curcumin (119875 lt 0001 Figure 2(a))The CV assay showed that curcumin and chlorhexidine bothresulted in reduced S mutans biomass at 24 h Chlorhexidine

BioMed Research International 5

5min

3D

Mer

ged

imag

eD

ead

bact

eria

ChlorhexidineCurcumin

Live

bac

teria

Control(20x)

050

100150

200

250

250300300

350350

400 400

0

0

50100

1530

150

200

050

100

150

200

250

250300300

350350

400 400

0

0

50100

1530

150200

050

100

150

200

250250

300300

350350

400 400

0

0

50100

1530

150

200

(a)24 h

3D

Mer

ged

imag

eD

ead

bact

eria

ChlorhexidineCurcumin

Live

bac

teria

Control(20x)

050

100150

200

250250

300300

350 350

400 400

0

0

50100

1530

150

200

050

100150

200

250250

300300

350350

400 400

0

0

50100

22

150200

0

50

100

150

200

250

250300

300350

350

400400

0

0

50100

20

150200

(b)

lowastlowastlowastlowastlowastlowast

5min5min

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol 1078

1079

108

109

lived

bac

teria

(lgC

FU)

0

20

40

60

80

Tota

l Bac

teria

(Ｇ

3

Ｇ2)

(c)

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowast

24 h24 h

0

20

40

60

80

Tota

l Bac

teria

(Ｇ

3

Ｇ2)

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e 1078

1079

108

1081

1082

lived

bac

teria

(lgC

FU)

(d)

Figure 3 CLSM imaging of S mutans biofilm grown in 1 BHIS After 24 h of growth the biofilm was treated with 1 BHIS (control)500 120583M curcumin or 012 chlorhexidine for 5min (a) and 24 h (b) Each micrograph represents 4 optical sections green representing livebacteria red representing dead bacteria combined green and red from two channel images and three-dimensional reconstructions of thecontrol biofilm without any treatment the 500 120583M curcumin-treated biofilm and the 012 chlorhexidine-treated biofilmThe total bacteriabiomass and the Colony-Forming Units (CFU) of lived bacteria are quantified in (c) and (d)The data represent the mean plusmn SDThe asterisks(lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

resulted in a significantly greater decrease than curcumin(119875 lt 0001 Figure 2(b)) As curcumin decreased the metab-olism of the S mutans biofilm in a short time (5min) we nextcompared the short-term (5min) and long-term (24 h) effectsof curcumin on S mutans biofilm

33 Evaluation of the Effect on the LiveDead Bacteria Ratio inSmutans Biofilm Theeffects of curcumin and chlorhexidineon the livedead bacteria ratio in S mutans biofilm wereassayed by CLSM (Figure 3) In the confocal micrographs

green florescence indicates lived cells while red fluorescenceindicates dead cellsThe images reflect different green and redflorescence intensities from those in the control group Lowergreen-stained density than in the control group was observedin both treated groups at 5min and 24 h (Figures 3(a) and3(b)) At 5min the live bacteria in the curcumin group andchlorhexidine group were reduced while the total bacteria inthe curcumin group were reduced (Figure 3(c)) At 24 h thelive bacteria in the curcumin group were reduced while thetotal bacteria were reduced in both groups (Figure 3(d))

6 BioMed Research International

Mer

ged

EPS

Bact

eria

(20x)ChlorhexidineCurcuminControl5min

050

100150

200

250250

300300

350350

400 400

050

100150

200

050

100150

200

250 250300 300

350 350

400 400

050

100150

200

0

50100

150

200

250250

300 300

350 350

400 400

050

100

150

200

050

100150

200

250250

300300

350 350

400 400

050

100150

200

050

100150

200

250250

300 300

350 350

400 400

050

100150

200

050

100

150

200

250250

300300

350350

400 400

050

100150

200

050

100150

200

250250

300300

350 350

400 400

050

100150

200

050

100150

200

250250

300300

350 350

400 400

050

100150

200

050

100150

200

250

250300300

350350

400 400

050

100150

200

(a)

Mer

ged

EPS

Bact

eria

(20x)ChlorhexidineCurcuminControl24 h

01530

01530

015 0

18

0

18

0

18

0

18

018

0

18

30

050

100150

200

250250

300300

350350

400 400

050

100150

200

050

100

150

200

250250

300 300

350 350

400 400

050

100150

200

050

100

150

200

250

250300

300350

350

400 400

050

100150

200

0

50100

150

200

250250

300300

350350

400 400

050

100

150

200

0

50

100

150

200

250

250300

300

350350

400 400

050

100

150

200

050

100

150

200

250250

300300

350350

400 400

050

100150

200

0

50100

150

200

250

250300

300350

350

400 400

050

100150

200

0

50

100

150

200

250

250300

300

350350

400 400

050

100150

200

050

100

150

200

250

250300

300350

350

400400

050

100150

200

(b)

5min 5min

lowastlowastlowastlowastlowast

0

5

10

15

EPS

Biom

ass(

Ｇ

3 Ｇ

2 )

0

5

10

15

20

thic

knes

s of b

iofil

m (

m)

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e

(c)

24 h 24 h

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

0

10

20

30

40

EPS

Biom

ass(

Ｇ

3 Ｇ

2 )

01020304050

thic

knes

s of b

iofil

m (

m)

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e

(d)

Figure 4 CLSM images of S mutans biofilm (a b) Three-dimensional reconstructions of the untreated (control) biofilm the 500 120583Mcurcumin-treated biofilm and the 012 chlorhexidine-treated biofilm at 5min (a) and 24 h (b) EPS was labelled in red (Alexa Fluor 647)bacterial cells were labelled in green (SYTO9) and red and green superimposed appear as yellow Images were obtained at 20x magnification(c) Image representing the volume of EPS calculated according to 5 random sites of each sample repeated three times (d) Change in biofilmthickness at 5min and 24 h respectively calculated from data obtained from fluorescence CLSM Data are presented as the mean plusmn SD Theasterisks (lowast) indicate significant differences (lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

34 Evaluation of the Effect on the EPS of S mutans BiofilmThe three-dimensional reconstruction images obtained fromCLSM of the S mutans biofilm (Figures 4(a) and 4(c))confirmed that the EPS decreased with both curcumin andchlorhexidine treatment after 5min (119875 lt 005) and this trendcontinued at 24 h The thickness of the biofilm showed nodifference among the three groups at 5min but was visiblyreduced at 24 h (Figures 4(b) and 4(d)) The SEM imageconfirmed the change in EPS (Figure 5) Treatment withcurcumin or chlorhexidine was able to reduce the overallquantity of S mutans EPS compared to the untreated controlas indicated by arrows Furthermore long-term treatment(24 h) resulted in a more obvious reduction than short-termtreatment (5min)

35 Evaluation of the Effect on the mRNA Levels of Smutans Biofilm Virulence Factors Compared with the levelsobserved in S mutans biofilms grown in 1 BHIS theexpression levels of gtfB gtfC and gtfD in S mutans biofilmsexposed to 500 120583M curcumin for 5min were significantlydecreased by 057- 059- and 059-fold respectively At 24 hthe expression levels of gtfB gtfC and gtfD in S mutanswere also decreased and there was no significant differencefrom the levels at 5min Meanwhile the expression of gtfBgtfC and gtfD was also downregulated under the influence

of 012 chlorhexidine for 5min and 24 h there was nosignificant difference between two time points (Figure 6(a))

In Figure 6(b) we observed no significant difference inthe expression of scrA at either 5min or 24 h However theexpression of atpH and scrB decreased in the presence of cur-cumin (119875 lt 005) The expression of atpH decreased furtherover time In contrast the expression of scrB increased overtime

The entire set of virulence genes involved in quorum-sensing signalling mechanisms was found to be down-regulated after treatment with curcumin Treatment withcurcumin for 5min repressed the expression levels of luxScomC comD and comE to 507 574 648 and 541respectively as compared to the control values Likewisechlorhexidine repressed the gene expression levels to 501523 607 and 523 respectively After treatment withthe drugs for 24 h only the expression of comDE showed asignificant difference

The expression levels of srtA and spaP in the biofilmstreated with curcumin for 5min were significantly downreg-ulated by approximately onefold compared with the levelsin untreated biofilm and the same tendency was shownby chlorhexidine (Figure 6(d)) After 24 h of treatment witheither drug the expression levels of srtA and spaP showed nosignificant difference

BioMed Research International 7

10000x5000x2000x

(A3)(A2)(A1)

5minC

ontro

l

(a)

(B3)(B2)(B1)

10000x5000x2000x5min

Curc

umin

(b)

(C3)(C2)(C1)

10000x5000x2000x5min

Chlo

rhex

idin

e

(c)

10000x5000x2000x

(D3)(D2)(D1)

Con

trol

24 h

(d)

(E3)(E2)(E1)

10000x5000x2000x

Curc

umin

24 h

(e)

(F3)(F2)(F1)

10000x5000x2000x

Chlo

rhex

idin

e

24 h

(f)

Figure 5 Morphological characteristics of S mutans biofilm treated with or without drugs Representative SEM images of 24 h S mutansbiofilm grown in curcumin for 5min (b) and 24 h (e) and grown in chlorhexidine for 5min (c) and 24 h (f) The image of 24 h S mutansbiofilm grown in 1 BHIS for 5min (a) and 24 h (d) as a control Magnifications of 2000x 5000x and 10000x are shown for each conditionThe black arrows highlight the EPS of S mutans which was markedly reduced

4 Discussion

Dental caries a common infectious disease worldwide iscaused by biofilm known as dental plaque that result fromthe adhesion of bacteria to tooth surfaces The effective erad-ication of cariogenic biofilm and the pathogenic microorgan-isms within is the key way to prevent tooth decay [28] Smutans is the main aetiological microorganism involved inbiofilm [29] In this study we used curcumin a natural food-grade product to control the formation of S mutans biofilm

Previous studies have shown that curcumin could inhibitthe viability of S mutans with an MIC value of 125ndash175 120583M[17] Similar MIC results were obtained in this study but theSMIC values here were much higher than the MIC As is wellknown it is more difficult to inhibit the viability of S mutansbiofilm than planktonic bacteria because the planktonicbacteria are more susceptible to the effects of commonly usedantimicrobial drugs than the bacteria embedded in a biofilm[30 31]

Accordingly we evaluated the effect of curcumin on Smutans biofilm over time At the SMIC

50 curcumin was

shown to exert both a short-term effect and a long-termeffect on the viability of S mutant biofilm which was alsotrue for 012 chlorhexidine Previous studies showed theeffect of chlorhexidine on S mutant biofilm Yang et al [32]

reported that S mutans biofilm was susceptible to 012chlorhexidine with a biomass reduction of over 80observed after treatment with 012 chlorhexidine for 1 hTamura et al [33] found that 8 120583gml chlorhexidine waseffective in removing biofilm after 24 h All of these resultsillustrate that chlorhexidine has antibiofilm effects whichwasconsistent with our resultsThe short-term effect of curcuminon S mutans biofilm also suggested that curcumin has thepotential to be a new antibiofilm drug because it exhibitedsimilar effects to chlorhexidine

S mutans has a regulatory network to integrate its cellularresponse to environmental change [6]This study found thatafter treatment with curcumin the amounts of viable bacteriaand total bacteria were reduced as confirmed by the results ofCLSM Because curcumin exhibited no effect on the growthkinetics of S mutans the reduced quantity of live bacteriaindicated a direct effect of curcumin on S mutans

A biofilm is a highly dynamic and structured communityof microbial cells that are enmeshed in an extracellularmatrix of polymeric substances such as exopolysaccharidesproteins and nucleic acids [34] As reported previously theproduction of EPS by S mutans on a surface enhances thelocal accumulation and clustering of microbes In additionas the biofilm develops the spatial heterogeneities resultingfrom EPS synthesis form a complex 3D matrix architecture

8 BioMed Research International

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowast lowast

lowastlowast

24 h5min

ftf

gtfB

gtfC

gtfD

fruA

gbpB ftf

gtfC

gtfB

gtfD

fruA

gbpB

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

nChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(a)

lowastlowast

lowastlowastlowast

lowast lowast

24 h5min

scrB

scrA

atpH scrB

scrA

atpH

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(b)

lowast

lowastlowastlowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

24 h5min

vicRluxS

com

E

com

D

com

C

luxS

vicR

com

E

com

C

com

D

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(c)

lowastlowastlowast

lowastlowast

lowast

24 h5min

srtA

spaP

srtA

spaP

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(d)

Figure 6 Results of qRT-PCR to examine the gene expression of different virulence systems in S mutans UA159 (a) EPS synthesis system(b) carbohydrate metabolism system (c) quorum-sensing system (d) two-component transduction system All targets were amplified usingprimers Different gene expression levels were normalized to the level of 16sRNA gene transcripts Data are presented as the mean plusmn SDTheasterisks (lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

and create environmental and protective niches within thebiofilm that can directly modulate caries pathogenesis [3435] Curcumin was shown to destroy the structure of EPS inthe short term decreasing the biomass of EPS the quantityof total bacteria and the percentage of lived bacteria Withprolonged curcumin exposure the structure of the EPS wasbadly damaged and the thickness of the biofilm and thenumber of total bacteria were decreased demonstrably Khanet al reported that the extract of Trachyspermum ammi seedscould modulate the expression of specific virulence genes byS mutans which disrupted the accumulation and structuralorganization of EPS ultimately affecting the cariogenicityof S mutans [36] Xu et al found that the tea catechinepigallocatechin gallate could inhibit the formation of Smutans biofilm by suppressing the gtfs gene [37] Thesestudies serve as a reminder that the effects of natural productson the EPS of S mutans biofilm are usually exerted byregulating cariogenic genes

As previously reported the regulatory systems in Smutans such as the EPS synthesis system carbohydratemetabolism system quorum-sensing system and two-component transduction system are connected to the ecolog-ical balance and to bacterial carcinogenesis [6 38]The resultsshowed that curcumin decreased the expression of cariogenicgenes in the plaque of S mutans Although the majorityof the 24 h qPCR results were not statistically significant

the 119875 values between the control group and curcumin groupwere close to 005 and the decrease was pronounced Thuscurcumin has an inhibitory effect on certain genes in Smutans at 24 h

Gtfs encoded by the gtfBCD genes are indispensable forthe utilization of glucose and for EPS synthesis in S mutansThey are important factors in biofilm formation and thedevelopment of caries [24] GtfB synthesizes water-insolublepolysaccharide [39] which is the main component of theEPS Downregulation of the expression of gtfB in curcuminmight explain the decreased biomass of EPS in the shortterm Oral bacterial aggregation is mediated by interactionsbetween surface-associated glucan-binding proteins (GbpBs)that adhere to glucans thereby promoting plaque formation[40] The expression of gbpB was decreased in both treat-ment groups which may relate to the reduced thickness ofthe biofilm Fructosyltransferase (FTF) is an enzyme thatconverts sucrose to extracellular homopolymers of fructosethe fructans FTF is the product of the fruA gene and isan exo-120573-d-fructosidase that releases fructose from 120573(26)-and 120573(21)-linked fructans and cleaves fructose from sucroseand raffinose [41ndash43] Both ftf and fruA expressions weredysregulated after short-term exposure to curcumin

In S mutans the scrA gene in the phosphotransferasesystem (PTS) encodes a high-affinity permease which inter-nalizes sucrose Then intracellular sucrose-6-PO

4is first

BioMed Research International 9

hydrolyzed by ScrB a sucrose-6-PO4hydrolase to produce

fructose and glucose-6-PO4 In addition the downstream of

scrA and scrB could encode a regulator to change the expres-sion level of scrAB Both scrAB genes were downregulatedin our study which might contribute to the reduction inthe S mutans biofilm In the oral environment S mutansis subjected to rapid pH fluctuations arising from the avail-ability of carbohydrates The atpH gene in S mutans encodessubunit C of a multisubunit enzyme (F1F0-ATPase) involvedin intracellular pH regulation and acid tolerance [44 45]Gene expression analysis found that atpHwas downregulatedin S mutans biofilm after treatment with curcumin for 5minand 24 hThus curcumin inhibits the acid tolerance ability ofS mutans and probably decreases the cariogenic traits of Smutans

The quorum-sensing system is an essential component ofentire gene regulation networks responsible for the adapta-tion of bacteria in biofilms [46] Two well-studied quorum-sensing systems exist in S mutans the ComCDE systemwhich can enable intraspecies cell-cell communication andhas been proved to have a positive regulatory effect onthe expression of biofilm-related genes such as gtfB gtfCand gbpB in S mutans and the LuxS system which cancatalyse the formation of the signal peptide AI-2 to mediateinterspecies and intraspecies interaction in the multispeciesplaque community [6 47] The findings of the current worksuggest that curcumin can potentially contribute to reducingthe quantity of lived bacteria in the biofilm and decrease thebiomass of EPS by decreasing the cariogenic potential of Smutans biofilm via inhibiting the expression of ComCDEsystem and LuxS system The short-term inhibitory effectis more significant than the long-term effect There is adecreasing tendency in the VicR expression of S mutans aftercurcumin treatment suggesting that curcumin may act byinhibiting the expression of VicR The regulatory network ofcariogenic genes in S mutans is complex It was reported thatthe ComRComS is the second quorum-sensing system inS mutans [48] The cell-to-cell communication system in Smutans is not clear Further research is required in this field

S mutans possesses a series of cell-surface proteins suchas SpaP (also known as antigen III Pac P1 and antigenB) which are anchored to the cell envelope by sortase A(SrtA) Sortase A is encoded by the gene srtA and recognizesa motif consisting of leucine proline X threonine andglycine (LPXTG where X is any amino acid) [33 49] Naturalproducts attract substantial attention because they can inhibitsrtA in vitro and in vivo but do not significantly decreasethe viability of bacteria The current study demonstrates thatcurcumin can inhibit the expression of spaP and srtA withan SMIC

50of 500 120583M The present study explored the effect

of curcumin on S mutans UA159 However S mutans hasheterogeneity in different strains Much more strains of Smutans are needed to be test in future

In conclusion curcumin has both a short-term and along-term antibacterial effect Additionally it is a food-gradenatural product with a similar effect to that of chlorhexidineand it takes effect faster than 012 chlorhexidine Curcumincould be an alternative strategy to treat oral disease Cur-cumin is a promising antibacterial agent with potential forwider clinical application in the future

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by Science and Technology Plan-ning Project ofGuangdongProvince China (2014A020212621and 2017A020215064)

References

[1] N B Pitts D T Zero P D Marsh et al ldquoDental cariesrdquo NatureReviews Disease Primers vol 3 Article ID 17030 2017

[2] D F A Pereira C J Seneviratne C Y Koga-Ito and L PSamaranayake ldquoIs the oral fungal pathogen Candida albicansa cariogenrdquo Oral Diseases 2017

[3] B NyvadW Crielaard AMira N Takahashi andD BeightonldquoDental caries from a molecular microbiological perspectiverdquoCaries Research vol 47 no 2 pp 89ndash102 2013

[4] R E Marquis ldquoOxygen metabolism oxidative stress and acid-base physiology of dental plaque biofilmsrdquo Journal of IndustrialMicrobiology and Biotechnology vol 15 no 3 pp 198ndash207 1995

[5] W H Bowen and H Koo ldquoBiology of streptococcus mutans-derived glucosyltransferases role in extracellular matrix forma-tion of cariogenic biofilmsrdquo Caries Research vol 45 no 1 pp69ndash86 2011

[6] E G Smith and G A Spatafora ldquoGene regulation in S mutanscomplex control in a complex environmentrdquo Journal of DentalResearch vol 91 no 2 pp 133ndash141 2012

[7] W Krzysciak A Jurczak D Koscielniak B Bystrowska and ASkalniak ldquoThe virulence of Streptococcus mutans and the abilityto form biofilmsrdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 33 no 4 pp 499ndash515 2014

[8] J Merritt J Kreth F Qi R Sullivan and W Shi ldquoNon-disruptive real-time analyses of themetabolic status and viabil-ity of Streptococcus mutans cells in response to antimicrobialtreatmentsrdquo Journal of Microbiological Methods vol 61 no 2pp 161ndash170 2005

[9] J Fricks-Lima C M Hendrickson M Allgaier et al ldquoDif-ferences in biofilm formation and antimicrobial resistance ofPseudomonas aeruginosa isolated from airways of mechanicallyventilated patients and cystic fibrosis patientsrdquo InternationalJournal of Antimicrobial Agents vol 37 no 4 pp 309ndash315 2011

[10] R Utsumi Bacterial Signal Transduction Networks and DrugTargets vol 631 Springer New York NY USA 2008

[11] H Koo andW H Bowen ldquoCandida albicans and Streptococcusmutans a potential synergistic alliance to cause virulent toothdecay in childrenrdquo Future Microbiology vol 9 no 12 pp 1295ndash1297 2014

[12] S Hamada T Koga and T Ooshima ldquoVirulence factors ofStreptococcus mutans and dental caries preventionrdquo Journal ofDental Research vol 63 no 3 pp 407ndash411 1984

[13] J-G Jeon P L Rosalen M L Falsetta and H Koo ldquoNaturalproducts in caries research current (limited) knowledge chal-lenges and future perspectiverdquoCaries Research vol 45 no 3 pp243ndash263 2011

[14] S-Y Teow K Liew S A Ali A S-B Khoo and S-CPeh ldquoAntibacterial Action of Curcumin against Staphylococcusaureus A Brief Reviewrdquo Journal of Tropical Medicine vol 2016Article ID 2853045 10 pages 2016

10 BioMed Research International

[15] J Song B Choi E-J Jin Y Yoon and K-H Choi ldquoCurcuminsuppresses Streptococcus mutans adherence to human toothsurfaces and extracellular matrix proteinsrdquo European Journalof Clinical Microbiology amp Infectious Diseases vol 31 no 7 pp1347ndash1352 2012

[16] P Hu P Huang and M W Chen ldquoCurcumin reduces Strep-tococcus mutans biofilm formation by inhibiting sortase Aactivityrdquo Archives of Oral Biolog vol 58 no 10 pp 1343ndash13482013

[17] P Hu P Huang and W M Chen ldquoCurcumin inhibits thesortase a activity of the streptococcus mutans UA159rdquo AppliedBiochemistry and Biotechnology vol 171 no 2 pp 396ndash4022013

[18] DManoil A Filieri C Gameiro et al ldquoFlow cytometric assess-ment of Streptococcus mutans viability after exposure to bluelight-activated curcuminrdquo Photodiagnosis and PhotodynamicTherapy vol 11 no 3 pp 372ndash379 2014

[19] M A Pfaller and D J Diekema ldquoProgress in antifungalsusceptibility testing of Candida spp by use of Clinical andLaboratory Standards Institute broth microdilution methods2010 to 2012rdquo Journal of Clinical Microbiology vol 50 no 9 pp2846ndash2856 2012

[20] M M Weerasekera G K Wijesinghe T A Jayarathna etal ldquoCulture media profoundly affect Candida Albicans andCandida tropicalis growth adhesion and biofilm developmentrdquoMemorias do InstitutoOswaldoCruz vol 111 no 11 pp 697ndash7022016

[21] J Chen T Li X Zhou et al ldquoCharacterization of the clusteredregularly interspaced short palindromic repeats sites in Strep-tococcus mutans isolated from early childhood caries patientsrdquoArchives of Oral Biolog vol 83 pp 174ndash180 2017

[22] M Sun Z Zhou J Dong J Zhang Y Xia and R ShuldquoAntibacterial and antibiofilm activities of docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA) against periodon-topathic bacteriardquo Microbial Pathogenesis vol 99 pp 196ndash2032016

[23] X Huang K Zhang M Deng et al ldquoEffect of arginine on thegrowth and biofilm formation of oral bacteriardquo Archives of OralBiolog vol 82 pp 256ndash262 2017

[24] S Liu W Qiu K Zhang et al ldquoNicotine enhances inter-species relationship between streptococcus mutans and candidaalbicansrdquo BioMed Research International vol 2017 Article ID7953920 9 pages 2017

[25] T B L Bedran L Grignon D P Spolidorio and D GrenierldquoSubinhibitory concentrations of triclosan promote Streptococ-cus mutans biofilm formation and adherence to oral epithelialcellsrdquo PLoS ONE vol 9 no 2 Article ID e89059 2014

[26] C M Levesque E Voronejskaia Y-C C Huang RWMair RP Ellen and D G Cvitkovitch ldquoInvolvement of sortase anchor-ing of cell wall proteins in biofilm formation by Streptococcusmutansrdquo Infection and Immunity vol 73 no 6 pp 3773ndash37772005

[27] M M Nascimento J A Lemos J Abranches V K Lin andR A Burne ldquoRole of RelA of Streptococcus mutans in globalcontrol of gene expressionrdquo Journal of Bacteriology vol 190 no1 pp 28ndash36 2008

[28] B Kunze M Reck A Dotsch et al ldquoDamage of Streptococcusmutans biofilms by carolacton a secondarymetabolite from themyxobacterium Sorangium cellulosumrdquo BMC Microbiologyvol 10 article no 199 2010

[29] V LeungDDufour andCM Levesque ldquoDeath and survival inStreptococcusmutansDiffering outcomes of a quorum-sensing

signaling peptiderdquo Frontiers in Microbiology vol 6 Article ID01176 2015

[30] A Tofino-Rivera M Ortega-Cuadros D Galvis-Pareja HJimenez-Rios L JMerini andMCMartınez-Pabon ldquoEffect ofLippia alba and Cymbopogon citratus essential oils on biofilmsof Streptococcusmutans and cytotoxicity in CHO cellsrdquo Journalof Ethnopharmacology vol 194 pp 749ndash754 2016

[31] K Jhajharia A Parolia K V Shetty and L K Mehta ldquoBiofilmin endodontics a reviewrdquo Journal of International Society ofPreventive amp Community Dentistry vol 5 no 1 pp 1ndash12 2015

[32] H Yang Y Bi X Shang et al ldquoAntibiofilm activities of anovel chimeolysin against Streptococcus mutans under physi-ological and cariogenic conditionsrdquo Antimicrobial Agents andChemotherapy vol 60 no 12 pp 7436ndash7443 2016

[33] H Tamura A Yamada and H Kato ldquoMolecular characteriza-tion of the dextran-binding lectin B gene dblB of Streptococcuscriceti in Streptococcus mutans strain GS-5 with mutations inboth gbpC and spaP genesrdquo Genes amp Genetic Systems vol 89no 2 pp 41ndash50 2014

[34] M I Klein G Hwang P H S Santos O H Campanella andH Koo ldquoStreptococcus mutans-derived extracellular matrixin cariogenic oral biofilmsrdquo Frontiers in Cellular and InfectionMicrobiology vol 5 article no 10 2015

[35] H Koo M L Falsetta and M I Klein ldquoThe exopolysaccharidematrix a virulence determinant of cariogenic biofilmrdquo Journalof Dental Research vol 92 no 12 pp 1065ndash1073 2013

[36] R KhanMAdilMDanishuddin P KVerma andAUKhanldquoIn vitro and in vivo inhibition of Streptococcusmutans biofilmby Trachyspermum ammi seeds An approach of alternativemedicinerdquo Phytomedicine vol 19 no 8-9 pp 747ndash755 2012

[37] X Xu X D Zhou and C DWu ldquoTea catechin epigallocatechingallate inhibits Streptococcus mutans biofilm formation bysuppressing gtf genesrdquo Archives of Oral Biolog vol 57 no 6 pp678ndash683 2012

[38] K Stephenson ldquoTwo-component and phosphorelay signal-transduction systems as therapeutic targetsrdquo Current Opinionin Pharmacology vol 2 no 5 pp 507ndash512 2002

[39] H Aoki T Shiroza H Hayakawa S Sato and H K KuramitsuldquoCloning of a Streptococcus mutans glycosyltransferase genecoding for insoluble glucan synthesisrdquo Infection and Immunityvol 53 no 3 pp 587ndash594 1986

[40] Z He Q Wang Y Hu et al ldquoUse of the quorum sensinginhibitor furanone C-30 to interfere with biofilm formation byStreptococcus mutans and its luxS mutant strainrdquo InternationalJournal of Antimicrobial Agents vol 40 no 1 pp 30ndash35 2012

[41] A Ogawa S Furukawa S Fujita et al ldquoInhibition of Strepto-coccus mutans biofilm formation by Streptococcus salivariusFruArdquo Applied and Environmental Microbiology vol 77 no 5pp 1572ndash1580 2011

[42] R A Burne and J E C Penders ldquoDifferential localization of theStreptococcus mutans GS-5 fructan hydrolase enzyme FruArdquoFEMS Microbiology Letters vol 121 no 2 pp 243ndash249 1994

[43] R A Burne K Schilling W H Bowen and R E YasbinldquoExpression purification and characterization of an Exo-120573-D-fructosidase of Streptococcus mutansrdquo Journal of Bacteriologyvol 169 no 10 pp 4507ndash4517 1987

[44] L Sadeghinejad D G Cvitkovitch W L Siqueira J Merritt JP Santerre and Y Finer ldquoMechanistic genomic and proteomicstudy on the effects of BisGMA-derived biodegradation producton cariogenic bacteriardquoDental Materials vol 33 no 2 pp 175ndash190 2017

BioMed Research International 11

[45] G R Bender S V W Sutton and R E Marquis ldquoAcidtolerance proton permeabilities and membrane ATPases oforal streptococcirdquo Infection and Immunity vol 53 no 2 pp 331ndash338 1986

[46] Y-H Li X-L Tian G Layton C Norgaard and G Sis-son ldquoAdditive attenuation of virulence and cariogenic poten-tial of Streptococcus mutans by simultaneous inactivationof the ComCDE quorum-sensing system and HKRR11 two-component regulatory systemrdquoMicrobiology vol 154 no 11 pp3256ndash3265 2008

[47] D G Cvitkovitch Y-H Li and R P Ellen ldquoQuorum sensingand biofilm formation in Streptococcal infectionsrdquoThe Journalof Clinical Investigation vol 112 no 11 pp 1626ndash1632 2003

[48] R Khan H V Rukke A P R Filho et al ldquoExtracellularidentification of a processed type II ComRComS pheromoneof Streptococcus mutansrdquo Journal of Bacteriology vol 194 no15 pp 3781ndash3788 2012

[49] P Huang P Hu S Y Zhou Q Li and W M Chen ldquoMorininhibits sortase A and subsequent biofilm formation in strepto-coccus mutansrdquo Current Microbiology vol 68 no 1 pp 47ndash522014

Hindawiwwwhindawicom

International Journal of

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Submit your manuscripts atwwwhindawicom

BioMed Research International 5

5min

3D

Mer

ged

imag

eD

ead

bact

eria

ChlorhexidineCurcumin

Live

bac

teria

Control(20x)

050

100150

200

250

250300300

350350

400 400

0

0

50100

1530

150

200

050

100

150

200

250

250300300

350350

400 400

0

0

50100

1530

150200

050

100

150

200

250250

300300

350350

400 400

0

0

50100

1530

150

200

(a)24 h

3D

Mer

ged

imag

eD

ead

bact

eria

ChlorhexidineCurcumin

Live

bac

teria

Control(20x)

050

100150

200

250250

300300

350 350

400 400

0

0

50100

1530

150

200

050

100150

200

250250

300300

350350

400 400

0

0

50100

22

150200

0

50

100

150

200

250

250300

300350

350

400400

0

0

50100

20

150200

(b)

lowastlowastlowastlowastlowastlowast

5min5min

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol 1078

1079

108

109

lived

bac

teria

(lgC

FU)

0

20

40

60

80

Tota

l Bac

teria

(Ｇ

3

Ｇ2)

(c)

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowast

24 h24 h

0

20

40

60

80

Tota

l Bac

teria

(Ｇ

3

Ｇ2)

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e 1078

1079

108

1081

1082

lived

bac

teria

(lgC

FU)

(d)

Figure 3 CLSM imaging of S mutans biofilm grown in 1 BHIS After 24 h of growth the biofilm was treated with 1 BHIS (control)500 120583M curcumin or 012 chlorhexidine for 5min (a) and 24 h (b) Each micrograph represents 4 optical sections green representing livebacteria red representing dead bacteria combined green and red from two channel images and three-dimensional reconstructions of thecontrol biofilm without any treatment the 500 120583M curcumin-treated biofilm and the 012 chlorhexidine-treated biofilmThe total bacteriabiomass and the Colony-Forming Units (CFU) of lived bacteria are quantified in (c) and (d)The data represent the mean plusmn SDThe asterisks(lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

resulted in a significantly greater decrease than curcumin(119875 lt 0001 Figure 2(b)) As curcumin decreased the metab-olism of the S mutans biofilm in a short time (5min) we nextcompared the short-term (5min) and long-term (24 h) effectsof curcumin on S mutans biofilm

33 Evaluation of the Effect on the LiveDead Bacteria Ratio inSmutans Biofilm Theeffects of curcumin and chlorhexidineon the livedead bacteria ratio in S mutans biofilm wereassayed by CLSM (Figure 3) In the confocal micrographs

green florescence indicates lived cells while red fluorescenceindicates dead cellsThe images reflect different green and redflorescence intensities from those in the control group Lowergreen-stained density than in the control group was observedin both treated groups at 5min and 24 h (Figures 3(a) and3(b)) At 5min the live bacteria in the curcumin group andchlorhexidine group were reduced while the total bacteria inthe curcumin group were reduced (Figure 3(c)) At 24 h thelive bacteria in the curcumin group were reduced while thetotal bacteria were reduced in both groups (Figure 3(d))

6 BioMed Research International

Mer

ged

EPS

Bact

eria

(20x)ChlorhexidineCurcuminControl5min

050

100150

200

250250

300300

350350

400 400

050

100150

200

050

100150

200

250 250300 300

350 350

400 400

050

100150

200

0

50100

150

200

250250

300 300

350 350

400 400

050

100

150

200

050

100150

200

250250

300300

350 350

400 400

050

100150

200

050

100150

200

250250

300 300

350 350

400 400

050

100150

200

050

100

150

200

250250

300300

350350

400 400

050

100150

200

050

100150

200

250250

300300

350 350

400 400

050

100150

200

050

100150

200

250250

300300

350 350

400 400

050

100150

200

050

100150

200

250

250300300

350350

400 400

050

100150

200

(a)

Mer

ged

EPS

Bact

eria

(20x)ChlorhexidineCurcuminControl24 h

01530

01530

015 0

18

0

18

0

18

0

18

018

0

18

30

050

100150

200

250250

300300

350350

400 400

050

100150

200

050

100

150

200

250250

300 300

350 350

400 400

050

100150

200

050

100

150

200

250

250300

300350

350

400 400

050

100150

200

0

50100

150

200

250250

300300

350350

400 400

050

100

150

200

0

50

100

150

200

250

250300

300

350350

400 400

050

100

150

200

050

100

150

200

250250

300300

350350

400 400

050

100150

200

0

50100

150

200

250

250300

300350

350

400 400

050

100150

200

0

50

100

150

200

250

250300

300

350350

400 400

050

100150

200

050

100

150

200

250

250300

300350

350

400400

050

100150

200

(b)

5min 5min

lowastlowastlowastlowastlowast

0

5

10

15

EPS

Biom

ass(

Ｇ

3 Ｇ

2 )

0

5

10

15

20

thic

knes

s of b

iofil

m (

m)

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e

(c)

24 h 24 h

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

0

10

20

30

40

EPS

Biom

ass(

Ｇ

3 Ｇ

2 )

01020304050

thic

knes

s of b

iofil

m (

m)

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e

(d)

Figure 4 CLSM images of S mutans biofilm (a b) Three-dimensional reconstructions of the untreated (control) biofilm the 500 120583Mcurcumin-treated biofilm and the 012 chlorhexidine-treated biofilm at 5min (a) and 24 h (b) EPS was labelled in red (Alexa Fluor 647)bacterial cells were labelled in green (SYTO9) and red and green superimposed appear as yellow Images were obtained at 20x magnification(c) Image representing the volume of EPS calculated according to 5 random sites of each sample repeated three times (d) Change in biofilmthickness at 5min and 24 h respectively calculated from data obtained from fluorescence CLSM Data are presented as the mean plusmn SD Theasterisks (lowast) indicate significant differences (lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

34 Evaluation of the Effect on the EPS of S mutans BiofilmThe three-dimensional reconstruction images obtained fromCLSM of the S mutans biofilm (Figures 4(a) and 4(c))confirmed that the EPS decreased with both curcumin andchlorhexidine treatment after 5min (119875 lt 005) and this trendcontinued at 24 h The thickness of the biofilm showed nodifference among the three groups at 5min but was visiblyreduced at 24 h (Figures 4(b) and 4(d)) The SEM imageconfirmed the change in EPS (Figure 5) Treatment withcurcumin or chlorhexidine was able to reduce the overallquantity of S mutans EPS compared to the untreated controlas indicated by arrows Furthermore long-term treatment(24 h) resulted in a more obvious reduction than short-termtreatment (5min)

35 Evaluation of the Effect on the mRNA Levels of Smutans Biofilm Virulence Factors Compared with the levelsobserved in S mutans biofilms grown in 1 BHIS theexpression levels of gtfB gtfC and gtfD in S mutans biofilmsexposed to 500 120583M curcumin for 5min were significantlydecreased by 057- 059- and 059-fold respectively At 24 hthe expression levels of gtfB gtfC and gtfD in S mutanswere also decreased and there was no significant differencefrom the levels at 5min Meanwhile the expression of gtfBgtfC and gtfD was also downregulated under the influence

of 012 chlorhexidine for 5min and 24 h there was nosignificant difference between two time points (Figure 6(a))

In Figure 6(b) we observed no significant difference inthe expression of scrA at either 5min or 24 h However theexpression of atpH and scrB decreased in the presence of cur-cumin (119875 lt 005) The expression of atpH decreased furtherover time In contrast the expression of scrB increased overtime

The entire set of virulence genes involved in quorum-sensing signalling mechanisms was found to be down-regulated after treatment with curcumin Treatment withcurcumin for 5min repressed the expression levels of luxScomC comD and comE to 507 574 648 and 541respectively as compared to the control values Likewisechlorhexidine repressed the gene expression levels to 501523 607 and 523 respectively After treatment withthe drugs for 24 h only the expression of comDE showed asignificant difference

The expression levels of srtA and spaP in the biofilmstreated with curcumin for 5min were significantly downreg-ulated by approximately onefold compared with the levelsin untreated biofilm and the same tendency was shownby chlorhexidine (Figure 6(d)) After 24 h of treatment witheither drug the expression levels of srtA and spaP showed nosignificant difference

BioMed Research International 7

10000x5000x2000x

(A3)(A2)(A1)

5minC

ontro

l

(a)

(B3)(B2)(B1)

10000x5000x2000x5min

Curc

umin

(b)

(C3)(C2)(C1)

10000x5000x2000x5min

Chlo

rhex

idin

e

(c)

10000x5000x2000x

(D3)(D2)(D1)

Con

trol

24 h

(d)

(E3)(E2)(E1)

10000x5000x2000x

Curc

umin

24 h

(e)

(F3)(F2)(F1)

10000x5000x2000x

Chlo

rhex

idin

e

24 h

(f)

Figure 5 Morphological characteristics of S mutans biofilm treated with or without drugs Representative SEM images of 24 h S mutansbiofilm grown in curcumin for 5min (b) and 24 h (e) and grown in chlorhexidine for 5min (c) and 24 h (f) The image of 24 h S mutansbiofilm grown in 1 BHIS for 5min (a) and 24 h (d) as a control Magnifications of 2000x 5000x and 10000x are shown for each conditionThe black arrows highlight the EPS of S mutans which was markedly reduced

4 Discussion

Dental caries a common infectious disease worldwide iscaused by biofilm known as dental plaque that result fromthe adhesion of bacteria to tooth surfaces The effective erad-ication of cariogenic biofilm and the pathogenic microorgan-isms within is the key way to prevent tooth decay [28] Smutans is the main aetiological microorganism involved inbiofilm [29] In this study we used curcumin a natural food-grade product to control the formation of S mutans biofilm

Previous studies have shown that curcumin could inhibitthe viability of S mutans with an MIC value of 125ndash175 120583M[17] Similar MIC results were obtained in this study but theSMIC values here were much higher than the MIC As is wellknown it is more difficult to inhibit the viability of S mutansbiofilm than planktonic bacteria because the planktonicbacteria are more susceptible to the effects of commonly usedantimicrobial drugs than the bacteria embedded in a biofilm[30 31]

Accordingly we evaluated the effect of curcumin on Smutans biofilm over time At the SMIC

50 curcumin was

shown to exert both a short-term effect and a long-termeffect on the viability of S mutant biofilm which was alsotrue for 012 chlorhexidine Previous studies showed theeffect of chlorhexidine on S mutant biofilm Yang et al [32]

reported that S mutans biofilm was susceptible to 012chlorhexidine with a biomass reduction of over 80observed after treatment with 012 chlorhexidine for 1 hTamura et al [33] found that 8 120583gml chlorhexidine waseffective in removing biofilm after 24 h All of these resultsillustrate that chlorhexidine has antibiofilm effects whichwasconsistent with our resultsThe short-term effect of curcuminon S mutans biofilm also suggested that curcumin has thepotential to be a new antibiofilm drug because it exhibitedsimilar effects to chlorhexidine

S mutans has a regulatory network to integrate its cellularresponse to environmental change [6]This study found thatafter treatment with curcumin the amounts of viable bacteriaand total bacteria were reduced as confirmed by the results ofCLSM Because curcumin exhibited no effect on the growthkinetics of S mutans the reduced quantity of live bacteriaindicated a direct effect of curcumin on S mutans

A biofilm is a highly dynamic and structured communityof microbial cells that are enmeshed in an extracellularmatrix of polymeric substances such as exopolysaccharidesproteins and nucleic acids [34] As reported previously theproduction of EPS by S mutans on a surface enhances thelocal accumulation and clustering of microbes In additionas the biofilm develops the spatial heterogeneities resultingfrom EPS synthesis form a complex 3D matrix architecture

8 BioMed Research International

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowast lowast

lowastlowast

24 h5min

ftf

gtfB

gtfC

gtfD

fruA

gbpB ftf

gtfC

gtfB

gtfD

fruA

gbpB

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

nChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(a)

lowastlowast

lowastlowastlowast

lowast lowast

24 h5min

scrB

scrA

atpH scrB

scrA

atpH

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(b)

lowast

lowastlowastlowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

24 h5min

vicRluxS

com

E

com

D

com

C

luxS

vicR

com

E

com

C

com

D

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(c)

lowastlowastlowast

lowastlowast

lowast

24 h5min

srtA

spaP

srtA

spaP

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(d)

Figure 6 Results of qRT-PCR to examine the gene expression of different virulence systems in S mutans UA159 (a) EPS synthesis system(b) carbohydrate metabolism system (c) quorum-sensing system (d) two-component transduction system All targets were amplified usingprimers Different gene expression levels were normalized to the level of 16sRNA gene transcripts Data are presented as the mean plusmn SDTheasterisks (lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

and create environmental and protective niches within thebiofilm that can directly modulate caries pathogenesis [3435] Curcumin was shown to destroy the structure of EPS inthe short term decreasing the biomass of EPS the quantityof total bacteria and the percentage of lived bacteria Withprolonged curcumin exposure the structure of the EPS wasbadly damaged and the thickness of the biofilm and thenumber of total bacteria were decreased demonstrably Khanet al reported that the extract of Trachyspermum ammi seedscould modulate the expression of specific virulence genes byS mutans which disrupted the accumulation and structuralorganization of EPS ultimately affecting the cariogenicityof S mutans [36] Xu et al found that the tea catechinepigallocatechin gallate could inhibit the formation of Smutans biofilm by suppressing the gtfs gene [37] Thesestudies serve as a reminder that the effects of natural productson the EPS of S mutans biofilm are usually exerted byregulating cariogenic genes

As previously reported the regulatory systems in Smutans such as the EPS synthesis system carbohydratemetabolism system quorum-sensing system and two-component transduction system are connected to the ecolog-ical balance and to bacterial carcinogenesis [6 38]The resultsshowed that curcumin decreased the expression of cariogenicgenes in the plaque of S mutans Although the majorityof the 24 h qPCR results were not statistically significant

the 119875 values between the control group and curcumin groupwere close to 005 and the decrease was pronounced Thuscurcumin has an inhibitory effect on certain genes in Smutans at 24 h

Gtfs encoded by the gtfBCD genes are indispensable forthe utilization of glucose and for EPS synthesis in S mutansThey are important factors in biofilm formation and thedevelopment of caries [24] GtfB synthesizes water-insolublepolysaccharide [39] which is the main component of theEPS Downregulation of the expression of gtfB in curcuminmight explain the decreased biomass of EPS in the shortterm Oral bacterial aggregation is mediated by interactionsbetween surface-associated glucan-binding proteins (GbpBs)that adhere to glucans thereby promoting plaque formation[40] The expression of gbpB was decreased in both treat-ment groups which may relate to the reduced thickness ofthe biofilm Fructosyltransferase (FTF) is an enzyme thatconverts sucrose to extracellular homopolymers of fructosethe fructans FTF is the product of the fruA gene and isan exo-120573-d-fructosidase that releases fructose from 120573(26)-and 120573(21)-linked fructans and cleaves fructose from sucroseand raffinose [41ndash43] Both ftf and fruA expressions weredysregulated after short-term exposure to curcumin

In S mutans the scrA gene in the phosphotransferasesystem (PTS) encodes a high-affinity permease which inter-nalizes sucrose Then intracellular sucrose-6-PO

4is first

BioMed Research International 9

hydrolyzed by ScrB a sucrose-6-PO4hydrolase to produce

fructose and glucose-6-PO4 In addition the downstream of

scrA and scrB could encode a regulator to change the expres-sion level of scrAB Both scrAB genes were downregulatedin our study which might contribute to the reduction inthe S mutans biofilm In the oral environment S mutansis subjected to rapid pH fluctuations arising from the avail-ability of carbohydrates The atpH gene in S mutans encodessubunit C of a multisubunit enzyme (F1F0-ATPase) involvedin intracellular pH regulation and acid tolerance [44 45]Gene expression analysis found that atpHwas downregulatedin S mutans biofilm after treatment with curcumin for 5minand 24 hThus curcumin inhibits the acid tolerance ability ofS mutans and probably decreases the cariogenic traits of Smutans

The quorum-sensing system is an essential component ofentire gene regulation networks responsible for the adapta-tion of bacteria in biofilms [46] Two well-studied quorum-sensing systems exist in S mutans the ComCDE systemwhich can enable intraspecies cell-cell communication andhas been proved to have a positive regulatory effect onthe expression of biofilm-related genes such as gtfB gtfCand gbpB in S mutans and the LuxS system which cancatalyse the formation of the signal peptide AI-2 to mediateinterspecies and intraspecies interaction in the multispeciesplaque community [6 47] The findings of the current worksuggest that curcumin can potentially contribute to reducingthe quantity of lived bacteria in the biofilm and decrease thebiomass of EPS by decreasing the cariogenic potential of Smutans biofilm via inhibiting the expression of ComCDEsystem and LuxS system The short-term inhibitory effectis more significant than the long-term effect There is adecreasing tendency in the VicR expression of S mutans aftercurcumin treatment suggesting that curcumin may act byinhibiting the expression of VicR The regulatory network ofcariogenic genes in S mutans is complex It was reported thatthe ComRComS is the second quorum-sensing system inS mutans [48] The cell-to-cell communication system in Smutans is not clear Further research is required in this field

S mutans possesses a series of cell-surface proteins suchas SpaP (also known as antigen III Pac P1 and antigenB) which are anchored to the cell envelope by sortase A(SrtA) Sortase A is encoded by the gene srtA and recognizesa motif consisting of leucine proline X threonine andglycine (LPXTG where X is any amino acid) [33 49] Naturalproducts attract substantial attention because they can inhibitsrtA in vitro and in vivo but do not significantly decreasethe viability of bacteria The current study demonstrates thatcurcumin can inhibit the expression of spaP and srtA withan SMIC

50of 500 120583M The present study explored the effect

of curcumin on S mutans UA159 However S mutans hasheterogeneity in different strains Much more strains of Smutans are needed to be test in future

In conclusion curcumin has both a short-term and along-term antibacterial effect Additionally it is a food-gradenatural product with a similar effect to that of chlorhexidineand it takes effect faster than 012 chlorhexidine Curcumincould be an alternative strategy to treat oral disease Cur-cumin is a promising antibacterial agent with potential forwider clinical application in the future

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by Science and Technology Plan-ning Project ofGuangdongProvince China (2014A020212621and 2017A020215064)

References

[1] N B Pitts D T Zero P D Marsh et al ldquoDental cariesrdquo NatureReviews Disease Primers vol 3 Article ID 17030 2017

[2] D F A Pereira C J Seneviratne C Y Koga-Ito and L PSamaranayake ldquoIs the oral fungal pathogen Candida albicansa cariogenrdquo Oral Diseases 2017

[3] B NyvadW Crielaard AMira N Takahashi andD BeightonldquoDental caries from a molecular microbiological perspectiverdquoCaries Research vol 47 no 2 pp 89ndash102 2013

[4] R E Marquis ldquoOxygen metabolism oxidative stress and acid-base physiology of dental plaque biofilmsrdquo Journal of IndustrialMicrobiology and Biotechnology vol 15 no 3 pp 198ndash207 1995

[5] W H Bowen and H Koo ldquoBiology of streptococcus mutans-derived glucosyltransferases role in extracellular matrix forma-tion of cariogenic biofilmsrdquo Caries Research vol 45 no 1 pp69ndash86 2011

[6] E G Smith and G A Spatafora ldquoGene regulation in S mutanscomplex control in a complex environmentrdquo Journal of DentalResearch vol 91 no 2 pp 133ndash141 2012

[7] W Krzysciak A Jurczak D Koscielniak B Bystrowska and ASkalniak ldquoThe virulence of Streptococcus mutans and the abilityto form biofilmsrdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 33 no 4 pp 499ndash515 2014

[8] J Merritt J Kreth F Qi R Sullivan and W Shi ldquoNon-disruptive real-time analyses of themetabolic status and viabil-ity of Streptococcus mutans cells in response to antimicrobialtreatmentsrdquo Journal of Microbiological Methods vol 61 no 2pp 161ndash170 2005

[9] J Fricks-Lima C M Hendrickson M Allgaier et al ldquoDif-ferences in biofilm formation and antimicrobial resistance ofPseudomonas aeruginosa isolated from airways of mechanicallyventilated patients and cystic fibrosis patientsrdquo InternationalJournal of Antimicrobial Agents vol 37 no 4 pp 309ndash315 2011

[10] R Utsumi Bacterial Signal Transduction Networks and DrugTargets vol 631 Springer New York NY USA 2008

[11] H Koo andW H Bowen ldquoCandida albicans and Streptococcusmutans a potential synergistic alliance to cause virulent toothdecay in childrenrdquo Future Microbiology vol 9 no 12 pp 1295ndash1297 2014

[12] S Hamada T Koga and T Ooshima ldquoVirulence factors ofStreptococcus mutans and dental caries preventionrdquo Journal ofDental Research vol 63 no 3 pp 407ndash411 1984

[13] J-G Jeon P L Rosalen M L Falsetta and H Koo ldquoNaturalproducts in caries research current (limited) knowledge chal-lenges and future perspectiverdquoCaries Research vol 45 no 3 pp243ndash263 2011

[14] S-Y Teow K Liew S A Ali A S-B Khoo and S-CPeh ldquoAntibacterial Action of Curcumin against Staphylococcusaureus A Brief Reviewrdquo Journal of Tropical Medicine vol 2016Article ID 2853045 10 pages 2016

10 BioMed Research International

[15] J Song B Choi E-J Jin Y Yoon and K-H Choi ldquoCurcuminsuppresses Streptococcus mutans adherence to human toothsurfaces and extracellular matrix proteinsrdquo European Journalof Clinical Microbiology amp Infectious Diseases vol 31 no 7 pp1347ndash1352 2012

[16] P Hu P Huang and M W Chen ldquoCurcumin reduces Strep-tococcus mutans biofilm formation by inhibiting sortase Aactivityrdquo Archives of Oral Biolog vol 58 no 10 pp 1343ndash13482013

[17] P Hu P Huang and W M Chen ldquoCurcumin inhibits thesortase a activity of the streptococcus mutans UA159rdquo AppliedBiochemistry and Biotechnology vol 171 no 2 pp 396ndash4022013

[18] DManoil A Filieri C Gameiro et al ldquoFlow cytometric assess-ment of Streptococcus mutans viability after exposure to bluelight-activated curcuminrdquo Photodiagnosis and PhotodynamicTherapy vol 11 no 3 pp 372ndash379 2014

[19] M A Pfaller and D J Diekema ldquoProgress in antifungalsusceptibility testing of Candida spp by use of Clinical andLaboratory Standards Institute broth microdilution methods2010 to 2012rdquo Journal of Clinical Microbiology vol 50 no 9 pp2846ndash2856 2012

[20] M M Weerasekera G K Wijesinghe T A Jayarathna etal ldquoCulture media profoundly affect Candida Albicans andCandida tropicalis growth adhesion and biofilm developmentrdquoMemorias do InstitutoOswaldoCruz vol 111 no 11 pp 697ndash7022016

[21] J Chen T Li X Zhou et al ldquoCharacterization of the clusteredregularly interspaced short palindromic repeats sites in Strep-tococcus mutans isolated from early childhood caries patientsrdquoArchives of Oral Biolog vol 83 pp 174ndash180 2017

[22] M Sun Z Zhou J Dong J Zhang Y Xia and R ShuldquoAntibacterial and antibiofilm activities of docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA) against periodon-topathic bacteriardquo Microbial Pathogenesis vol 99 pp 196ndash2032016

[23] X Huang K Zhang M Deng et al ldquoEffect of arginine on thegrowth and biofilm formation of oral bacteriardquo Archives of OralBiolog vol 82 pp 256ndash262 2017

[24] S Liu W Qiu K Zhang et al ldquoNicotine enhances inter-species relationship between streptococcus mutans and candidaalbicansrdquo BioMed Research International vol 2017 Article ID7953920 9 pages 2017

[25] T B L Bedran L Grignon D P Spolidorio and D GrenierldquoSubinhibitory concentrations of triclosan promote Streptococ-cus mutans biofilm formation and adherence to oral epithelialcellsrdquo PLoS ONE vol 9 no 2 Article ID e89059 2014

[26] C M Levesque E Voronejskaia Y-C C Huang RWMair RP Ellen and D G Cvitkovitch ldquoInvolvement of sortase anchor-ing of cell wall proteins in biofilm formation by Streptococcusmutansrdquo Infection and Immunity vol 73 no 6 pp 3773ndash37772005

[27] M M Nascimento J A Lemos J Abranches V K Lin andR A Burne ldquoRole of RelA of Streptococcus mutans in globalcontrol of gene expressionrdquo Journal of Bacteriology vol 190 no1 pp 28ndash36 2008

[28] B Kunze M Reck A Dotsch et al ldquoDamage of Streptococcusmutans biofilms by carolacton a secondarymetabolite from themyxobacterium Sorangium cellulosumrdquo BMC Microbiologyvol 10 article no 199 2010

[29] V LeungDDufour andCM Levesque ldquoDeath and survival inStreptococcusmutansDiffering outcomes of a quorum-sensing

signaling peptiderdquo Frontiers in Microbiology vol 6 Article ID01176 2015

[30] A Tofino-Rivera M Ortega-Cuadros D Galvis-Pareja HJimenez-Rios L JMerini andMCMartınez-Pabon ldquoEffect ofLippia alba and Cymbopogon citratus essential oils on biofilmsof Streptococcusmutans and cytotoxicity in CHO cellsrdquo Journalof Ethnopharmacology vol 194 pp 749ndash754 2016

[31] K Jhajharia A Parolia K V Shetty and L K Mehta ldquoBiofilmin endodontics a reviewrdquo Journal of International Society ofPreventive amp Community Dentistry vol 5 no 1 pp 1ndash12 2015

[32] H Yang Y Bi X Shang et al ldquoAntibiofilm activities of anovel chimeolysin against Streptococcus mutans under physi-ological and cariogenic conditionsrdquo Antimicrobial Agents andChemotherapy vol 60 no 12 pp 7436ndash7443 2016

[33] H Tamura A Yamada and H Kato ldquoMolecular characteriza-tion of the dextran-binding lectin B gene dblB of Streptococcuscriceti in Streptococcus mutans strain GS-5 with mutations inboth gbpC and spaP genesrdquo Genes amp Genetic Systems vol 89no 2 pp 41ndash50 2014

[34] M I Klein G Hwang P H S Santos O H Campanella andH Koo ldquoStreptococcus mutans-derived extracellular matrixin cariogenic oral biofilmsrdquo Frontiers in Cellular and InfectionMicrobiology vol 5 article no 10 2015

[35] H Koo M L Falsetta and M I Klein ldquoThe exopolysaccharidematrix a virulence determinant of cariogenic biofilmrdquo Journalof Dental Research vol 92 no 12 pp 1065ndash1073 2013

[36] R KhanMAdilMDanishuddin P KVerma andAUKhanldquoIn vitro and in vivo inhibition of Streptococcusmutans biofilmby Trachyspermum ammi seeds An approach of alternativemedicinerdquo Phytomedicine vol 19 no 8-9 pp 747ndash755 2012

[37] X Xu X D Zhou and C DWu ldquoTea catechin epigallocatechingallate inhibits Streptococcus mutans biofilm formation bysuppressing gtf genesrdquo Archives of Oral Biolog vol 57 no 6 pp678ndash683 2012

[38] K Stephenson ldquoTwo-component and phosphorelay signal-transduction systems as therapeutic targetsrdquo Current Opinionin Pharmacology vol 2 no 5 pp 507ndash512 2002

[39] H Aoki T Shiroza H Hayakawa S Sato and H K KuramitsuldquoCloning of a Streptococcus mutans glycosyltransferase genecoding for insoluble glucan synthesisrdquo Infection and Immunityvol 53 no 3 pp 587ndash594 1986

[40] Z He Q Wang Y Hu et al ldquoUse of the quorum sensinginhibitor furanone C-30 to interfere with biofilm formation byStreptococcus mutans and its luxS mutant strainrdquo InternationalJournal of Antimicrobial Agents vol 40 no 1 pp 30ndash35 2012

[41] A Ogawa S Furukawa S Fujita et al ldquoInhibition of Strepto-coccus mutans biofilm formation by Streptococcus salivariusFruArdquo Applied and Environmental Microbiology vol 77 no 5pp 1572ndash1580 2011

[42] R A Burne and J E C Penders ldquoDifferential localization of theStreptococcus mutans GS-5 fructan hydrolase enzyme FruArdquoFEMS Microbiology Letters vol 121 no 2 pp 243ndash249 1994

[43] R A Burne K Schilling W H Bowen and R E YasbinldquoExpression purification and characterization of an Exo-120573-D-fructosidase of Streptococcus mutansrdquo Journal of Bacteriologyvol 169 no 10 pp 4507ndash4517 1987

[44] L Sadeghinejad D G Cvitkovitch W L Siqueira J Merritt JP Santerre and Y Finer ldquoMechanistic genomic and proteomicstudy on the effects of BisGMA-derived biodegradation producton cariogenic bacteriardquoDental Materials vol 33 no 2 pp 175ndash190 2017

BioMed Research International 11

[45] G R Bender S V W Sutton and R E Marquis ldquoAcidtolerance proton permeabilities and membrane ATPases oforal streptococcirdquo Infection and Immunity vol 53 no 2 pp 331ndash338 1986

[46] Y-H Li X-L Tian G Layton C Norgaard and G Sis-son ldquoAdditive attenuation of virulence and cariogenic poten-tial of Streptococcus mutans by simultaneous inactivationof the ComCDE quorum-sensing system and HKRR11 two-component regulatory systemrdquoMicrobiology vol 154 no 11 pp3256ndash3265 2008

[47] D G Cvitkovitch Y-H Li and R P Ellen ldquoQuorum sensingand biofilm formation in Streptococcal infectionsrdquoThe Journalof Clinical Investigation vol 112 no 11 pp 1626ndash1632 2003

[48] R Khan H V Rukke A P R Filho et al ldquoExtracellularidentification of a processed type II ComRComS pheromoneof Streptococcus mutansrdquo Journal of Bacteriology vol 194 no15 pp 3781ndash3788 2012

[49] P Huang P Hu S Y Zhou Q Li and W M Chen ldquoMorininhibits sortase A and subsequent biofilm formation in strepto-coccus mutansrdquo Current Microbiology vol 68 no 1 pp 47ndash522014

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Submit your manuscripts atwwwhindawicom

6 BioMed Research International

Mer

ged

EPS

Bact

eria

(20x)ChlorhexidineCurcuminControl5min

050

100150

200

250250

300300

350350

400 400

050

100150

200

050

100150

200

250 250300 300

350 350

400 400

050

100150

200

0

50100

150

200

250250

300 300

350 350

400 400

050

100

150

200

050

100150

200

250250

300300

350 350

400 400

050

100150

200

050

100150

200

250250

300 300

350 350

400 400

050

100150

200

050

100

150

200

250250

300300

350350

400 400

050

100150

200

050

100150

200

250250

300300

350 350

400 400

050

100150

200

050

100150

200

250250

300300

350 350

400 400

050

100150

200

050

100150

200

250

250300300

350350

400 400

050

100150

200

(a)

Mer

ged

EPS

Bact

eria

(20x)ChlorhexidineCurcuminControl24 h

01530

01530

015 0

18

0

18

0

18

0

18

018

0

18

30

050

100150

200

250250

300300

350350

400 400

050

100150

200

050

100

150

200

250250

300 300

350 350

400 400

050

100150

200

050

100

150

200

250

250300

300350

350

400 400

050

100150

200

0

50100

150

200

250250

300300

350350

400 400

050

100

150

200

0

50

100

150

200

250

250300

300

350350

400 400

050

100

150

200

050

100

150

200

250250

300300

350350

400 400

050

100150

200

0

50100

150

200

250

250300

300350

350

400 400

050

100150

200

0

50

100

150

200

250

250300

300

350350

400 400

050

100150

200

050

100

150

200

250

250300

300350

350

400400

050

100150

200

(b)

5min 5min

lowastlowastlowastlowastlowast

0

5

10

15

EPS

Biom

ass(

Ｇ

3 Ｇ

2 )

0

5

10

15

20

thic

knes

s of b

iofil

m (

m)

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e

(c)

24 h 24 h

lowastlowastlowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

0

10

20

30

40

EPS

Biom

ass(

Ｇ

3 Ｇ

2 )

01020304050

thic

knes

s of b

iofil

m (

m)

Con

trol

Curc

umin

Chlo

rhex

idin

e

Con

trol

Curc

umin

Chlo

rhex

idin

e

(d)

Figure 4 CLSM images of S mutans biofilm (a b) Three-dimensional reconstructions of the untreated (control) biofilm the 500 120583Mcurcumin-treated biofilm and the 012 chlorhexidine-treated biofilm at 5min (a) and 24 h (b) EPS was labelled in red (Alexa Fluor 647)bacterial cells were labelled in green (SYTO9) and red and green superimposed appear as yellow Images were obtained at 20x magnification(c) Image representing the volume of EPS calculated according to 5 random sites of each sample repeated three times (d) Change in biofilmthickness at 5min and 24 h respectively calculated from data obtained from fluorescence CLSM Data are presented as the mean plusmn SD Theasterisks (lowast) indicate significant differences (lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

34 Evaluation of the Effect on the EPS of S mutans BiofilmThe three-dimensional reconstruction images obtained fromCLSM of the S mutans biofilm (Figures 4(a) and 4(c))confirmed that the EPS decreased with both curcumin andchlorhexidine treatment after 5min (119875 lt 005) and this trendcontinued at 24 h The thickness of the biofilm showed nodifference among the three groups at 5min but was visiblyreduced at 24 h (Figures 4(b) and 4(d)) The SEM imageconfirmed the change in EPS (Figure 5) Treatment withcurcumin or chlorhexidine was able to reduce the overallquantity of S mutans EPS compared to the untreated controlas indicated by arrows Furthermore long-term treatment(24 h) resulted in a more obvious reduction than short-termtreatment (5min)

35 Evaluation of the Effect on the mRNA Levels of Smutans Biofilm Virulence Factors Compared with the levelsobserved in S mutans biofilms grown in 1 BHIS theexpression levels of gtfB gtfC and gtfD in S mutans biofilmsexposed to 500 120583M curcumin for 5min were significantlydecreased by 057- 059- and 059-fold respectively At 24 hthe expression levels of gtfB gtfC and gtfD in S mutanswere also decreased and there was no significant differencefrom the levels at 5min Meanwhile the expression of gtfBgtfC and gtfD was also downregulated under the influence

of 012 chlorhexidine for 5min and 24 h there was nosignificant difference between two time points (Figure 6(a))

In Figure 6(b) we observed no significant difference inthe expression of scrA at either 5min or 24 h However theexpression of atpH and scrB decreased in the presence of cur-cumin (119875 lt 005) The expression of atpH decreased furtherover time In contrast the expression of scrB increased overtime

The entire set of virulence genes involved in quorum-sensing signalling mechanisms was found to be down-regulated after treatment with curcumin Treatment withcurcumin for 5min repressed the expression levels of luxScomC comD and comE to 507 574 648 and 541respectively as compared to the control values Likewisechlorhexidine repressed the gene expression levels to 501523 607 and 523 respectively After treatment withthe drugs for 24 h only the expression of comDE showed asignificant difference

The expression levels of srtA and spaP in the biofilmstreated with curcumin for 5min were significantly downreg-ulated by approximately onefold compared with the levelsin untreated biofilm and the same tendency was shownby chlorhexidine (Figure 6(d)) After 24 h of treatment witheither drug the expression levels of srtA and spaP showed nosignificant difference

BioMed Research International 7

10000x5000x2000x

(A3)(A2)(A1)

5minC

ontro

l

(a)

(B3)(B2)(B1)

10000x5000x2000x5min

Curc

umin

(b)

(C3)(C2)(C1)

10000x5000x2000x5min

Chlo

rhex

idin

e

(c)

10000x5000x2000x

(D3)(D2)(D1)

Con

trol

24 h

(d)

(E3)(E2)(E1)

10000x5000x2000x

Curc

umin

24 h

(e)

(F3)(F2)(F1)

10000x5000x2000x

Chlo

rhex

idin

e

24 h

(f)

Figure 5 Morphological characteristics of S mutans biofilm treated with or without drugs Representative SEM images of 24 h S mutansbiofilm grown in curcumin for 5min (b) and 24 h (e) and grown in chlorhexidine for 5min (c) and 24 h (f) The image of 24 h S mutansbiofilm grown in 1 BHIS for 5min (a) and 24 h (d) as a control Magnifications of 2000x 5000x and 10000x are shown for each conditionThe black arrows highlight the EPS of S mutans which was markedly reduced

4 Discussion

Dental caries a common infectious disease worldwide iscaused by biofilm known as dental plaque that result fromthe adhesion of bacteria to tooth surfaces The effective erad-ication of cariogenic biofilm and the pathogenic microorgan-isms within is the key way to prevent tooth decay [28] Smutans is the main aetiological microorganism involved inbiofilm [29] In this study we used curcumin a natural food-grade product to control the formation of S mutans biofilm

Previous studies have shown that curcumin could inhibitthe viability of S mutans with an MIC value of 125ndash175 120583M[17] Similar MIC results were obtained in this study but theSMIC values here were much higher than the MIC As is wellknown it is more difficult to inhibit the viability of S mutansbiofilm than planktonic bacteria because the planktonicbacteria are more susceptible to the effects of commonly usedantimicrobial drugs than the bacteria embedded in a biofilm[30 31]

Accordingly we evaluated the effect of curcumin on Smutans biofilm over time At the SMIC

50 curcumin was

shown to exert both a short-term effect and a long-termeffect on the viability of S mutant biofilm which was alsotrue for 012 chlorhexidine Previous studies showed theeffect of chlorhexidine on S mutant biofilm Yang et al [32]

reported that S mutans biofilm was susceptible to 012chlorhexidine with a biomass reduction of over 80observed after treatment with 012 chlorhexidine for 1 hTamura et al [33] found that 8 120583gml chlorhexidine waseffective in removing biofilm after 24 h All of these resultsillustrate that chlorhexidine has antibiofilm effects whichwasconsistent with our resultsThe short-term effect of curcuminon S mutans biofilm also suggested that curcumin has thepotential to be a new antibiofilm drug because it exhibitedsimilar effects to chlorhexidine

S mutans has a regulatory network to integrate its cellularresponse to environmental change [6]This study found thatafter treatment with curcumin the amounts of viable bacteriaand total bacteria were reduced as confirmed by the results ofCLSM Because curcumin exhibited no effect on the growthkinetics of S mutans the reduced quantity of live bacteriaindicated a direct effect of curcumin on S mutans

A biofilm is a highly dynamic and structured communityof microbial cells that are enmeshed in an extracellularmatrix of polymeric substances such as exopolysaccharidesproteins and nucleic acids [34] As reported previously theproduction of EPS by S mutans on a surface enhances thelocal accumulation and clustering of microbes In additionas the biofilm develops the spatial heterogeneities resultingfrom EPS synthesis form a complex 3D matrix architecture

8 BioMed Research International

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowast lowast

lowastlowast

24 h5min

ftf

gtfB

gtfC

gtfD

fruA

gbpB ftf

gtfC

gtfB

gtfD

fruA

gbpB

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

nChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(a)

lowastlowast

lowastlowastlowast

lowast lowast

24 h5min

scrB

scrA

atpH scrB

scrA

atpH

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(b)

lowast

lowastlowastlowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

24 h5min

vicRluxS

com

E

com

D

com

C

luxS

vicR

com

E

com

C

com

D

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(c)

lowastlowastlowast

lowastlowast

lowast

24 h5min

srtA

spaP

srtA

spaP

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(d)

Figure 6 Results of qRT-PCR to examine the gene expression of different virulence systems in S mutans UA159 (a) EPS synthesis system(b) carbohydrate metabolism system (c) quorum-sensing system (d) two-component transduction system All targets were amplified usingprimers Different gene expression levels were normalized to the level of 16sRNA gene transcripts Data are presented as the mean plusmn SDTheasterisks (lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

and create environmental and protective niches within thebiofilm that can directly modulate caries pathogenesis [3435] Curcumin was shown to destroy the structure of EPS inthe short term decreasing the biomass of EPS the quantityof total bacteria and the percentage of lived bacteria Withprolonged curcumin exposure the structure of the EPS wasbadly damaged and the thickness of the biofilm and thenumber of total bacteria were decreased demonstrably Khanet al reported that the extract of Trachyspermum ammi seedscould modulate the expression of specific virulence genes byS mutans which disrupted the accumulation and structuralorganization of EPS ultimately affecting the cariogenicityof S mutans [36] Xu et al found that the tea catechinepigallocatechin gallate could inhibit the formation of Smutans biofilm by suppressing the gtfs gene [37] Thesestudies serve as a reminder that the effects of natural productson the EPS of S mutans biofilm are usually exerted byregulating cariogenic genes

As previously reported the regulatory systems in Smutans such as the EPS synthesis system carbohydratemetabolism system quorum-sensing system and two-component transduction system are connected to the ecolog-ical balance and to bacterial carcinogenesis [6 38]The resultsshowed that curcumin decreased the expression of cariogenicgenes in the plaque of S mutans Although the majorityof the 24 h qPCR results were not statistically significant

the 119875 values between the control group and curcumin groupwere close to 005 and the decrease was pronounced Thuscurcumin has an inhibitory effect on certain genes in Smutans at 24 h

Gtfs encoded by the gtfBCD genes are indispensable forthe utilization of glucose and for EPS synthesis in S mutansThey are important factors in biofilm formation and thedevelopment of caries [24] GtfB synthesizes water-insolublepolysaccharide [39] which is the main component of theEPS Downregulation of the expression of gtfB in curcuminmight explain the decreased biomass of EPS in the shortterm Oral bacterial aggregation is mediated by interactionsbetween surface-associated glucan-binding proteins (GbpBs)that adhere to glucans thereby promoting plaque formation[40] The expression of gbpB was decreased in both treat-ment groups which may relate to the reduced thickness ofthe biofilm Fructosyltransferase (FTF) is an enzyme thatconverts sucrose to extracellular homopolymers of fructosethe fructans FTF is the product of the fruA gene and isan exo-120573-d-fructosidase that releases fructose from 120573(26)-and 120573(21)-linked fructans and cleaves fructose from sucroseand raffinose [41ndash43] Both ftf and fruA expressions weredysregulated after short-term exposure to curcumin

In S mutans the scrA gene in the phosphotransferasesystem (PTS) encodes a high-affinity permease which inter-nalizes sucrose Then intracellular sucrose-6-PO

4is first

BioMed Research International 9

hydrolyzed by ScrB a sucrose-6-PO4hydrolase to produce

fructose and glucose-6-PO4 In addition the downstream of

scrA and scrB could encode a regulator to change the expres-sion level of scrAB Both scrAB genes were downregulatedin our study which might contribute to the reduction inthe S mutans biofilm In the oral environment S mutansis subjected to rapid pH fluctuations arising from the avail-ability of carbohydrates The atpH gene in S mutans encodessubunit C of a multisubunit enzyme (F1F0-ATPase) involvedin intracellular pH regulation and acid tolerance [44 45]Gene expression analysis found that atpHwas downregulatedin S mutans biofilm after treatment with curcumin for 5minand 24 hThus curcumin inhibits the acid tolerance ability ofS mutans and probably decreases the cariogenic traits of Smutans

The quorum-sensing system is an essential component ofentire gene regulation networks responsible for the adapta-tion of bacteria in biofilms [46] Two well-studied quorum-sensing systems exist in S mutans the ComCDE systemwhich can enable intraspecies cell-cell communication andhas been proved to have a positive regulatory effect onthe expression of biofilm-related genes such as gtfB gtfCand gbpB in S mutans and the LuxS system which cancatalyse the formation of the signal peptide AI-2 to mediateinterspecies and intraspecies interaction in the multispeciesplaque community [6 47] The findings of the current worksuggest that curcumin can potentially contribute to reducingthe quantity of lived bacteria in the biofilm and decrease thebiomass of EPS by decreasing the cariogenic potential of Smutans biofilm via inhibiting the expression of ComCDEsystem and LuxS system The short-term inhibitory effectis more significant than the long-term effect There is adecreasing tendency in the VicR expression of S mutans aftercurcumin treatment suggesting that curcumin may act byinhibiting the expression of VicR The regulatory network ofcariogenic genes in S mutans is complex It was reported thatthe ComRComS is the second quorum-sensing system inS mutans [48] The cell-to-cell communication system in Smutans is not clear Further research is required in this field

S mutans possesses a series of cell-surface proteins suchas SpaP (also known as antigen III Pac P1 and antigenB) which are anchored to the cell envelope by sortase A(SrtA) Sortase A is encoded by the gene srtA and recognizesa motif consisting of leucine proline X threonine andglycine (LPXTG where X is any amino acid) [33 49] Naturalproducts attract substantial attention because they can inhibitsrtA in vitro and in vivo but do not significantly decreasethe viability of bacteria The current study demonstrates thatcurcumin can inhibit the expression of spaP and srtA withan SMIC

50of 500 120583M The present study explored the effect

of curcumin on S mutans UA159 However S mutans hasheterogeneity in different strains Much more strains of Smutans are needed to be test in future

In conclusion curcumin has both a short-term and along-term antibacterial effect Additionally it is a food-gradenatural product with a similar effect to that of chlorhexidineand it takes effect faster than 012 chlorhexidine Curcumincould be an alternative strategy to treat oral disease Cur-cumin is a promising antibacterial agent with potential forwider clinical application in the future

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by Science and Technology Plan-ning Project ofGuangdongProvince China (2014A020212621and 2017A020215064)

References

[1] N B Pitts D T Zero P D Marsh et al ldquoDental cariesrdquo NatureReviews Disease Primers vol 3 Article ID 17030 2017

[2] D F A Pereira C J Seneviratne C Y Koga-Ito and L PSamaranayake ldquoIs the oral fungal pathogen Candida albicansa cariogenrdquo Oral Diseases 2017

[3] B NyvadW Crielaard AMira N Takahashi andD BeightonldquoDental caries from a molecular microbiological perspectiverdquoCaries Research vol 47 no 2 pp 89ndash102 2013

[4] R E Marquis ldquoOxygen metabolism oxidative stress and acid-base physiology of dental plaque biofilmsrdquo Journal of IndustrialMicrobiology and Biotechnology vol 15 no 3 pp 198ndash207 1995

[5] W H Bowen and H Koo ldquoBiology of streptococcus mutans-derived glucosyltransferases role in extracellular matrix forma-tion of cariogenic biofilmsrdquo Caries Research vol 45 no 1 pp69ndash86 2011

[6] E G Smith and G A Spatafora ldquoGene regulation in S mutanscomplex control in a complex environmentrdquo Journal of DentalResearch vol 91 no 2 pp 133ndash141 2012

[7] W Krzysciak A Jurczak D Koscielniak B Bystrowska and ASkalniak ldquoThe virulence of Streptococcus mutans and the abilityto form biofilmsrdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 33 no 4 pp 499ndash515 2014

[8] J Merritt J Kreth F Qi R Sullivan and W Shi ldquoNon-disruptive real-time analyses of themetabolic status and viabil-ity of Streptococcus mutans cells in response to antimicrobialtreatmentsrdquo Journal of Microbiological Methods vol 61 no 2pp 161ndash170 2005

[9] J Fricks-Lima C M Hendrickson M Allgaier et al ldquoDif-ferences in biofilm formation and antimicrobial resistance ofPseudomonas aeruginosa isolated from airways of mechanicallyventilated patients and cystic fibrosis patientsrdquo InternationalJournal of Antimicrobial Agents vol 37 no 4 pp 309ndash315 2011

[10] R Utsumi Bacterial Signal Transduction Networks and DrugTargets vol 631 Springer New York NY USA 2008

[11] H Koo andW H Bowen ldquoCandida albicans and Streptococcusmutans a potential synergistic alliance to cause virulent toothdecay in childrenrdquo Future Microbiology vol 9 no 12 pp 1295ndash1297 2014

[12] S Hamada T Koga and T Ooshima ldquoVirulence factors ofStreptococcus mutans and dental caries preventionrdquo Journal ofDental Research vol 63 no 3 pp 407ndash411 1984

[13] J-G Jeon P L Rosalen M L Falsetta and H Koo ldquoNaturalproducts in caries research current (limited) knowledge chal-lenges and future perspectiverdquoCaries Research vol 45 no 3 pp243ndash263 2011

[14] S-Y Teow K Liew S A Ali A S-B Khoo and S-CPeh ldquoAntibacterial Action of Curcumin against Staphylococcusaureus A Brief Reviewrdquo Journal of Tropical Medicine vol 2016Article ID 2853045 10 pages 2016

10 BioMed Research International

[15] J Song B Choi E-J Jin Y Yoon and K-H Choi ldquoCurcuminsuppresses Streptococcus mutans adherence to human toothsurfaces and extracellular matrix proteinsrdquo European Journalof Clinical Microbiology amp Infectious Diseases vol 31 no 7 pp1347ndash1352 2012

[16] P Hu P Huang and M W Chen ldquoCurcumin reduces Strep-tococcus mutans biofilm formation by inhibiting sortase Aactivityrdquo Archives of Oral Biolog vol 58 no 10 pp 1343ndash13482013

[17] P Hu P Huang and W M Chen ldquoCurcumin inhibits thesortase a activity of the streptococcus mutans UA159rdquo AppliedBiochemistry and Biotechnology vol 171 no 2 pp 396ndash4022013

[18] DManoil A Filieri C Gameiro et al ldquoFlow cytometric assess-ment of Streptococcus mutans viability after exposure to bluelight-activated curcuminrdquo Photodiagnosis and PhotodynamicTherapy vol 11 no 3 pp 372ndash379 2014

[19] M A Pfaller and D J Diekema ldquoProgress in antifungalsusceptibility testing of Candida spp by use of Clinical andLaboratory Standards Institute broth microdilution methods2010 to 2012rdquo Journal of Clinical Microbiology vol 50 no 9 pp2846ndash2856 2012

[20] M M Weerasekera G K Wijesinghe T A Jayarathna etal ldquoCulture media profoundly affect Candida Albicans andCandida tropicalis growth adhesion and biofilm developmentrdquoMemorias do InstitutoOswaldoCruz vol 111 no 11 pp 697ndash7022016

[21] J Chen T Li X Zhou et al ldquoCharacterization of the clusteredregularly interspaced short palindromic repeats sites in Strep-tococcus mutans isolated from early childhood caries patientsrdquoArchives of Oral Biolog vol 83 pp 174ndash180 2017

[22] M Sun Z Zhou J Dong J Zhang Y Xia and R ShuldquoAntibacterial and antibiofilm activities of docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA) against periodon-topathic bacteriardquo Microbial Pathogenesis vol 99 pp 196ndash2032016

[23] X Huang K Zhang M Deng et al ldquoEffect of arginine on thegrowth and biofilm formation of oral bacteriardquo Archives of OralBiolog vol 82 pp 256ndash262 2017

[24] S Liu W Qiu K Zhang et al ldquoNicotine enhances inter-species relationship between streptococcus mutans and candidaalbicansrdquo BioMed Research International vol 2017 Article ID7953920 9 pages 2017

[25] T B L Bedran L Grignon D P Spolidorio and D GrenierldquoSubinhibitory concentrations of triclosan promote Streptococ-cus mutans biofilm formation and adherence to oral epithelialcellsrdquo PLoS ONE vol 9 no 2 Article ID e89059 2014

[26] C M Levesque E Voronejskaia Y-C C Huang RWMair RP Ellen and D G Cvitkovitch ldquoInvolvement of sortase anchor-ing of cell wall proteins in biofilm formation by Streptococcusmutansrdquo Infection and Immunity vol 73 no 6 pp 3773ndash37772005

[27] M M Nascimento J A Lemos J Abranches V K Lin andR A Burne ldquoRole of RelA of Streptococcus mutans in globalcontrol of gene expressionrdquo Journal of Bacteriology vol 190 no1 pp 28ndash36 2008

[28] B Kunze M Reck A Dotsch et al ldquoDamage of Streptococcusmutans biofilms by carolacton a secondarymetabolite from themyxobacterium Sorangium cellulosumrdquo BMC Microbiologyvol 10 article no 199 2010

[29] V LeungDDufour andCM Levesque ldquoDeath and survival inStreptococcusmutansDiffering outcomes of a quorum-sensing

signaling peptiderdquo Frontiers in Microbiology vol 6 Article ID01176 2015

[30] A Tofino-Rivera M Ortega-Cuadros D Galvis-Pareja HJimenez-Rios L JMerini andMCMartınez-Pabon ldquoEffect ofLippia alba and Cymbopogon citratus essential oils on biofilmsof Streptococcusmutans and cytotoxicity in CHO cellsrdquo Journalof Ethnopharmacology vol 194 pp 749ndash754 2016

[31] K Jhajharia A Parolia K V Shetty and L K Mehta ldquoBiofilmin endodontics a reviewrdquo Journal of International Society ofPreventive amp Community Dentistry vol 5 no 1 pp 1ndash12 2015

[32] H Yang Y Bi X Shang et al ldquoAntibiofilm activities of anovel chimeolysin against Streptococcus mutans under physi-ological and cariogenic conditionsrdquo Antimicrobial Agents andChemotherapy vol 60 no 12 pp 7436ndash7443 2016

[33] H Tamura A Yamada and H Kato ldquoMolecular characteriza-tion of the dextran-binding lectin B gene dblB of Streptococcuscriceti in Streptococcus mutans strain GS-5 with mutations inboth gbpC and spaP genesrdquo Genes amp Genetic Systems vol 89no 2 pp 41ndash50 2014

[34] M I Klein G Hwang P H S Santos O H Campanella andH Koo ldquoStreptococcus mutans-derived extracellular matrixin cariogenic oral biofilmsrdquo Frontiers in Cellular and InfectionMicrobiology vol 5 article no 10 2015

[35] H Koo M L Falsetta and M I Klein ldquoThe exopolysaccharidematrix a virulence determinant of cariogenic biofilmrdquo Journalof Dental Research vol 92 no 12 pp 1065ndash1073 2013

[36] R KhanMAdilMDanishuddin P KVerma andAUKhanldquoIn vitro and in vivo inhibition of Streptococcusmutans biofilmby Trachyspermum ammi seeds An approach of alternativemedicinerdquo Phytomedicine vol 19 no 8-9 pp 747ndash755 2012

[37] X Xu X D Zhou and C DWu ldquoTea catechin epigallocatechingallate inhibits Streptococcus mutans biofilm formation bysuppressing gtf genesrdquo Archives of Oral Biolog vol 57 no 6 pp678ndash683 2012

[38] K Stephenson ldquoTwo-component and phosphorelay signal-transduction systems as therapeutic targetsrdquo Current Opinionin Pharmacology vol 2 no 5 pp 507ndash512 2002

[39] H Aoki T Shiroza H Hayakawa S Sato and H K KuramitsuldquoCloning of a Streptococcus mutans glycosyltransferase genecoding for insoluble glucan synthesisrdquo Infection and Immunityvol 53 no 3 pp 587ndash594 1986

[40] Z He Q Wang Y Hu et al ldquoUse of the quorum sensinginhibitor furanone C-30 to interfere with biofilm formation byStreptococcus mutans and its luxS mutant strainrdquo InternationalJournal of Antimicrobial Agents vol 40 no 1 pp 30ndash35 2012

[41] A Ogawa S Furukawa S Fujita et al ldquoInhibition of Strepto-coccus mutans biofilm formation by Streptococcus salivariusFruArdquo Applied and Environmental Microbiology vol 77 no 5pp 1572ndash1580 2011

[42] R A Burne and J E C Penders ldquoDifferential localization of theStreptococcus mutans GS-5 fructan hydrolase enzyme FruArdquoFEMS Microbiology Letters vol 121 no 2 pp 243ndash249 1994

[43] R A Burne K Schilling W H Bowen and R E YasbinldquoExpression purification and characterization of an Exo-120573-D-fructosidase of Streptococcus mutansrdquo Journal of Bacteriologyvol 169 no 10 pp 4507ndash4517 1987

[44] L Sadeghinejad D G Cvitkovitch W L Siqueira J Merritt JP Santerre and Y Finer ldquoMechanistic genomic and proteomicstudy on the effects of BisGMA-derived biodegradation producton cariogenic bacteriardquoDental Materials vol 33 no 2 pp 175ndash190 2017

BioMed Research International 11

[45] G R Bender S V W Sutton and R E Marquis ldquoAcidtolerance proton permeabilities and membrane ATPases oforal streptococcirdquo Infection and Immunity vol 53 no 2 pp 331ndash338 1986

[46] Y-H Li X-L Tian G Layton C Norgaard and G Sis-son ldquoAdditive attenuation of virulence and cariogenic poten-tial of Streptococcus mutans by simultaneous inactivationof the ComCDE quorum-sensing system and HKRR11 two-component regulatory systemrdquoMicrobiology vol 154 no 11 pp3256ndash3265 2008

[47] D G Cvitkovitch Y-H Li and R P Ellen ldquoQuorum sensingand biofilm formation in Streptococcal infectionsrdquoThe Journalof Clinical Investigation vol 112 no 11 pp 1626ndash1632 2003

[48] R Khan H V Rukke A P R Filho et al ldquoExtracellularidentification of a processed type II ComRComS pheromoneof Streptococcus mutansrdquo Journal of Bacteriology vol 194 no15 pp 3781ndash3788 2012

[49] P Huang P Hu S Y Zhou Q Li and W M Chen ldquoMorininhibits sortase A and subsequent biofilm formation in strepto-coccus mutansrdquo Current Microbiology vol 68 no 1 pp 47ndash522014

Hindawiwwwhindawicom

International Journal of

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

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Cell BiologyInternational Journal of

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Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 7

10000x5000x2000x

(A3)(A2)(A1)

5minC

ontro

l

(a)

(B3)(B2)(B1)

10000x5000x2000x5min

Curc

umin

(b)

(C3)(C2)(C1)

10000x5000x2000x5min

Chlo

rhex

idin

e

(c)

10000x5000x2000x

(D3)(D2)(D1)

Con

trol

24 h

(d)

(E3)(E2)(E1)

10000x5000x2000x

Curc

umin

24 h

(e)

(F3)(F2)(F1)

10000x5000x2000x

Chlo

rhex

idin

e

24 h

(f)

Figure 5 Morphological characteristics of S mutans biofilm treated with or without drugs Representative SEM images of 24 h S mutansbiofilm grown in curcumin for 5min (b) and 24 h (e) and grown in chlorhexidine for 5min (c) and 24 h (f) The image of 24 h S mutansbiofilm grown in 1 BHIS for 5min (a) and 24 h (d) as a control Magnifications of 2000x 5000x and 10000x are shown for each conditionThe black arrows highlight the EPS of S mutans which was markedly reduced

4 Discussion

Dental caries a common infectious disease worldwide iscaused by biofilm known as dental plaque that result fromthe adhesion of bacteria to tooth surfaces The effective erad-ication of cariogenic biofilm and the pathogenic microorgan-isms within is the key way to prevent tooth decay [28] Smutans is the main aetiological microorganism involved inbiofilm [29] In this study we used curcumin a natural food-grade product to control the formation of S mutans biofilm

Previous studies have shown that curcumin could inhibitthe viability of S mutans with an MIC value of 125ndash175 120583M[17] Similar MIC results were obtained in this study but theSMIC values here were much higher than the MIC As is wellknown it is more difficult to inhibit the viability of S mutansbiofilm than planktonic bacteria because the planktonicbacteria are more susceptible to the effects of commonly usedantimicrobial drugs than the bacteria embedded in a biofilm[30 31]

Accordingly we evaluated the effect of curcumin on Smutans biofilm over time At the SMIC

50 curcumin was

shown to exert both a short-term effect and a long-termeffect on the viability of S mutant biofilm which was alsotrue for 012 chlorhexidine Previous studies showed theeffect of chlorhexidine on S mutant biofilm Yang et al [32]

reported that S mutans biofilm was susceptible to 012chlorhexidine with a biomass reduction of over 80observed after treatment with 012 chlorhexidine for 1 hTamura et al [33] found that 8 120583gml chlorhexidine waseffective in removing biofilm after 24 h All of these resultsillustrate that chlorhexidine has antibiofilm effects whichwasconsistent with our resultsThe short-term effect of curcuminon S mutans biofilm also suggested that curcumin has thepotential to be a new antibiofilm drug because it exhibitedsimilar effects to chlorhexidine

S mutans has a regulatory network to integrate its cellularresponse to environmental change [6]This study found thatafter treatment with curcumin the amounts of viable bacteriaand total bacteria were reduced as confirmed by the results ofCLSM Because curcumin exhibited no effect on the growthkinetics of S mutans the reduced quantity of live bacteriaindicated a direct effect of curcumin on S mutans

A biofilm is a highly dynamic and structured communityof microbial cells that are enmeshed in an extracellularmatrix of polymeric substances such as exopolysaccharidesproteins and nucleic acids [34] As reported previously theproduction of EPS by S mutans on a surface enhances thelocal accumulation and clustering of microbes In additionas the biofilm develops the spatial heterogeneities resultingfrom EPS synthesis form a complex 3D matrix architecture

8 BioMed Research International

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowast lowast

lowastlowast

24 h5min

ftf

gtfB

gtfC

gtfD

fruA

gbpB ftf

gtfC

gtfB

gtfD

fruA

gbpB

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

nChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(a)

lowastlowast

lowastlowastlowast

lowast lowast

24 h5min

scrB

scrA

atpH scrB

scrA

atpH

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(b)

lowast

lowastlowastlowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

24 h5min

vicRluxS

com

E

com

D

com

C

luxS

vicR

com

E

com

C

com

D

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(c)

lowastlowastlowast

lowastlowast

lowast

24 h5min

srtA

spaP

srtA

spaP

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(d)

Figure 6 Results of qRT-PCR to examine the gene expression of different virulence systems in S mutans UA159 (a) EPS synthesis system(b) carbohydrate metabolism system (c) quorum-sensing system (d) two-component transduction system All targets were amplified usingprimers Different gene expression levels were normalized to the level of 16sRNA gene transcripts Data are presented as the mean plusmn SDTheasterisks (lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

and create environmental and protective niches within thebiofilm that can directly modulate caries pathogenesis [3435] Curcumin was shown to destroy the structure of EPS inthe short term decreasing the biomass of EPS the quantityof total bacteria and the percentage of lived bacteria Withprolonged curcumin exposure the structure of the EPS wasbadly damaged and the thickness of the biofilm and thenumber of total bacteria were decreased demonstrably Khanet al reported that the extract of Trachyspermum ammi seedscould modulate the expression of specific virulence genes byS mutans which disrupted the accumulation and structuralorganization of EPS ultimately affecting the cariogenicityof S mutans [36] Xu et al found that the tea catechinepigallocatechin gallate could inhibit the formation of Smutans biofilm by suppressing the gtfs gene [37] Thesestudies serve as a reminder that the effects of natural productson the EPS of S mutans biofilm are usually exerted byregulating cariogenic genes

As previously reported the regulatory systems in Smutans such as the EPS synthesis system carbohydratemetabolism system quorum-sensing system and two-component transduction system are connected to the ecolog-ical balance and to bacterial carcinogenesis [6 38]The resultsshowed that curcumin decreased the expression of cariogenicgenes in the plaque of S mutans Although the majorityof the 24 h qPCR results were not statistically significant

the 119875 values between the control group and curcumin groupwere close to 005 and the decrease was pronounced Thuscurcumin has an inhibitory effect on certain genes in Smutans at 24 h

Gtfs encoded by the gtfBCD genes are indispensable forthe utilization of glucose and for EPS synthesis in S mutansThey are important factors in biofilm formation and thedevelopment of caries [24] GtfB synthesizes water-insolublepolysaccharide [39] which is the main component of theEPS Downregulation of the expression of gtfB in curcuminmight explain the decreased biomass of EPS in the shortterm Oral bacterial aggregation is mediated by interactionsbetween surface-associated glucan-binding proteins (GbpBs)that adhere to glucans thereby promoting plaque formation[40] The expression of gbpB was decreased in both treat-ment groups which may relate to the reduced thickness ofthe biofilm Fructosyltransferase (FTF) is an enzyme thatconverts sucrose to extracellular homopolymers of fructosethe fructans FTF is the product of the fruA gene and isan exo-120573-d-fructosidase that releases fructose from 120573(26)-and 120573(21)-linked fructans and cleaves fructose from sucroseand raffinose [41ndash43] Both ftf and fruA expressions weredysregulated after short-term exposure to curcumin

In S mutans the scrA gene in the phosphotransferasesystem (PTS) encodes a high-affinity permease which inter-nalizes sucrose Then intracellular sucrose-6-PO

4is first

BioMed Research International 9

hydrolyzed by ScrB a sucrose-6-PO4hydrolase to produce

fructose and glucose-6-PO4 In addition the downstream of

scrA and scrB could encode a regulator to change the expres-sion level of scrAB Both scrAB genes were downregulatedin our study which might contribute to the reduction inthe S mutans biofilm In the oral environment S mutansis subjected to rapid pH fluctuations arising from the avail-ability of carbohydrates The atpH gene in S mutans encodessubunit C of a multisubunit enzyme (F1F0-ATPase) involvedin intracellular pH regulation and acid tolerance [44 45]Gene expression analysis found that atpHwas downregulatedin S mutans biofilm after treatment with curcumin for 5minand 24 hThus curcumin inhibits the acid tolerance ability ofS mutans and probably decreases the cariogenic traits of Smutans

The quorum-sensing system is an essential component ofentire gene regulation networks responsible for the adapta-tion of bacteria in biofilms [46] Two well-studied quorum-sensing systems exist in S mutans the ComCDE systemwhich can enable intraspecies cell-cell communication andhas been proved to have a positive regulatory effect onthe expression of biofilm-related genes such as gtfB gtfCand gbpB in S mutans and the LuxS system which cancatalyse the formation of the signal peptide AI-2 to mediateinterspecies and intraspecies interaction in the multispeciesplaque community [6 47] The findings of the current worksuggest that curcumin can potentially contribute to reducingthe quantity of lived bacteria in the biofilm and decrease thebiomass of EPS by decreasing the cariogenic potential of Smutans biofilm via inhibiting the expression of ComCDEsystem and LuxS system The short-term inhibitory effectis more significant than the long-term effect There is adecreasing tendency in the VicR expression of S mutans aftercurcumin treatment suggesting that curcumin may act byinhibiting the expression of VicR The regulatory network ofcariogenic genes in S mutans is complex It was reported thatthe ComRComS is the second quorum-sensing system inS mutans [48] The cell-to-cell communication system in Smutans is not clear Further research is required in this field

S mutans possesses a series of cell-surface proteins suchas SpaP (also known as antigen III Pac P1 and antigenB) which are anchored to the cell envelope by sortase A(SrtA) Sortase A is encoded by the gene srtA and recognizesa motif consisting of leucine proline X threonine andglycine (LPXTG where X is any amino acid) [33 49] Naturalproducts attract substantial attention because they can inhibitsrtA in vitro and in vivo but do not significantly decreasethe viability of bacteria The current study demonstrates thatcurcumin can inhibit the expression of spaP and srtA withan SMIC

50of 500 120583M The present study explored the effect

of curcumin on S mutans UA159 However S mutans hasheterogeneity in different strains Much more strains of Smutans are needed to be test in future

In conclusion curcumin has both a short-term and along-term antibacterial effect Additionally it is a food-gradenatural product with a similar effect to that of chlorhexidineand it takes effect faster than 012 chlorhexidine Curcumincould be an alternative strategy to treat oral disease Cur-cumin is a promising antibacterial agent with potential forwider clinical application in the future

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by Science and Technology Plan-ning Project ofGuangdongProvince China (2014A020212621and 2017A020215064)

References

[1] N B Pitts D T Zero P D Marsh et al ldquoDental cariesrdquo NatureReviews Disease Primers vol 3 Article ID 17030 2017

[2] D F A Pereira C J Seneviratne C Y Koga-Ito and L PSamaranayake ldquoIs the oral fungal pathogen Candida albicansa cariogenrdquo Oral Diseases 2017

[3] B NyvadW Crielaard AMira N Takahashi andD BeightonldquoDental caries from a molecular microbiological perspectiverdquoCaries Research vol 47 no 2 pp 89ndash102 2013

[4] R E Marquis ldquoOxygen metabolism oxidative stress and acid-base physiology of dental plaque biofilmsrdquo Journal of IndustrialMicrobiology and Biotechnology vol 15 no 3 pp 198ndash207 1995

[5] W H Bowen and H Koo ldquoBiology of streptococcus mutans-derived glucosyltransferases role in extracellular matrix forma-tion of cariogenic biofilmsrdquo Caries Research vol 45 no 1 pp69ndash86 2011

[6] E G Smith and G A Spatafora ldquoGene regulation in S mutanscomplex control in a complex environmentrdquo Journal of DentalResearch vol 91 no 2 pp 133ndash141 2012

[7] W Krzysciak A Jurczak D Koscielniak B Bystrowska and ASkalniak ldquoThe virulence of Streptococcus mutans and the abilityto form biofilmsrdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 33 no 4 pp 499ndash515 2014

[8] J Merritt J Kreth F Qi R Sullivan and W Shi ldquoNon-disruptive real-time analyses of themetabolic status and viabil-ity of Streptococcus mutans cells in response to antimicrobialtreatmentsrdquo Journal of Microbiological Methods vol 61 no 2pp 161ndash170 2005

[9] J Fricks-Lima C M Hendrickson M Allgaier et al ldquoDif-ferences in biofilm formation and antimicrobial resistance ofPseudomonas aeruginosa isolated from airways of mechanicallyventilated patients and cystic fibrosis patientsrdquo InternationalJournal of Antimicrobial Agents vol 37 no 4 pp 309ndash315 2011

[10] R Utsumi Bacterial Signal Transduction Networks and DrugTargets vol 631 Springer New York NY USA 2008

[11] H Koo andW H Bowen ldquoCandida albicans and Streptococcusmutans a potential synergistic alliance to cause virulent toothdecay in childrenrdquo Future Microbiology vol 9 no 12 pp 1295ndash1297 2014

[12] S Hamada T Koga and T Ooshima ldquoVirulence factors ofStreptococcus mutans and dental caries preventionrdquo Journal ofDental Research vol 63 no 3 pp 407ndash411 1984

[13] J-G Jeon P L Rosalen M L Falsetta and H Koo ldquoNaturalproducts in caries research current (limited) knowledge chal-lenges and future perspectiverdquoCaries Research vol 45 no 3 pp243ndash263 2011

[14] S-Y Teow K Liew S A Ali A S-B Khoo and S-CPeh ldquoAntibacterial Action of Curcumin against Staphylococcusaureus A Brief Reviewrdquo Journal of Tropical Medicine vol 2016Article ID 2853045 10 pages 2016

10 BioMed Research International

[15] J Song B Choi E-J Jin Y Yoon and K-H Choi ldquoCurcuminsuppresses Streptococcus mutans adherence to human toothsurfaces and extracellular matrix proteinsrdquo European Journalof Clinical Microbiology amp Infectious Diseases vol 31 no 7 pp1347ndash1352 2012

[16] P Hu P Huang and M W Chen ldquoCurcumin reduces Strep-tococcus mutans biofilm formation by inhibiting sortase Aactivityrdquo Archives of Oral Biolog vol 58 no 10 pp 1343ndash13482013

[17] P Hu P Huang and W M Chen ldquoCurcumin inhibits thesortase a activity of the streptococcus mutans UA159rdquo AppliedBiochemistry and Biotechnology vol 171 no 2 pp 396ndash4022013

[18] DManoil A Filieri C Gameiro et al ldquoFlow cytometric assess-ment of Streptococcus mutans viability after exposure to bluelight-activated curcuminrdquo Photodiagnosis and PhotodynamicTherapy vol 11 no 3 pp 372ndash379 2014

[19] M A Pfaller and D J Diekema ldquoProgress in antifungalsusceptibility testing of Candida spp by use of Clinical andLaboratory Standards Institute broth microdilution methods2010 to 2012rdquo Journal of Clinical Microbiology vol 50 no 9 pp2846ndash2856 2012

[20] M M Weerasekera G K Wijesinghe T A Jayarathna etal ldquoCulture media profoundly affect Candida Albicans andCandida tropicalis growth adhesion and biofilm developmentrdquoMemorias do InstitutoOswaldoCruz vol 111 no 11 pp 697ndash7022016

[21] J Chen T Li X Zhou et al ldquoCharacterization of the clusteredregularly interspaced short palindromic repeats sites in Strep-tococcus mutans isolated from early childhood caries patientsrdquoArchives of Oral Biolog vol 83 pp 174ndash180 2017

[22] M Sun Z Zhou J Dong J Zhang Y Xia and R ShuldquoAntibacterial and antibiofilm activities of docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA) against periodon-topathic bacteriardquo Microbial Pathogenesis vol 99 pp 196ndash2032016

[23] X Huang K Zhang M Deng et al ldquoEffect of arginine on thegrowth and biofilm formation of oral bacteriardquo Archives of OralBiolog vol 82 pp 256ndash262 2017

[24] S Liu W Qiu K Zhang et al ldquoNicotine enhances inter-species relationship between streptococcus mutans and candidaalbicansrdquo BioMed Research International vol 2017 Article ID7953920 9 pages 2017

[25] T B L Bedran L Grignon D P Spolidorio and D GrenierldquoSubinhibitory concentrations of triclosan promote Streptococ-cus mutans biofilm formation and adherence to oral epithelialcellsrdquo PLoS ONE vol 9 no 2 Article ID e89059 2014

[26] C M Levesque E Voronejskaia Y-C C Huang RWMair RP Ellen and D G Cvitkovitch ldquoInvolvement of sortase anchor-ing of cell wall proteins in biofilm formation by Streptococcusmutansrdquo Infection and Immunity vol 73 no 6 pp 3773ndash37772005

[27] M M Nascimento J A Lemos J Abranches V K Lin andR A Burne ldquoRole of RelA of Streptococcus mutans in globalcontrol of gene expressionrdquo Journal of Bacteriology vol 190 no1 pp 28ndash36 2008

[28] B Kunze M Reck A Dotsch et al ldquoDamage of Streptococcusmutans biofilms by carolacton a secondarymetabolite from themyxobacterium Sorangium cellulosumrdquo BMC Microbiologyvol 10 article no 199 2010

[29] V LeungDDufour andCM Levesque ldquoDeath and survival inStreptococcusmutansDiffering outcomes of a quorum-sensing

signaling peptiderdquo Frontiers in Microbiology vol 6 Article ID01176 2015

[30] A Tofino-Rivera M Ortega-Cuadros D Galvis-Pareja HJimenez-Rios L JMerini andMCMartınez-Pabon ldquoEffect ofLippia alba and Cymbopogon citratus essential oils on biofilmsof Streptococcusmutans and cytotoxicity in CHO cellsrdquo Journalof Ethnopharmacology vol 194 pp 749ndash754 2016

[31] K Jhajharia A Parolia K V Shetty and L K Mehta ldquoBiofilmin endodontics a reviewrdquo Journal of International Society ofPreventive amp Community Dentistry vol 5 no 1 pp 1ndash12 2015

[32] H Yang Y Bi X Shang et al ldquoAntibiofilm activities of anovel chimeolysin against Streptococcus mutans under physi-ological and cariogenic conditionsrdquo Antimicrobial Agents andChemotherapy vol 60 no 12 pp 7436ndash7443 2016

[33] H Tamura A Yamada and H Kato ldquoMolecular characteriza-tion of the dextran-binding lectin B gene dblB of Streptococcuscriceti in Streptococcus mutans strain GS-5 with mutations inboth gbpC and spaP genesrdquo Genes amp Genetic Systems vol 89no 2 pp 41ndash50 2014

[34] M I Klein G Hwang P H S Santos O H Campanella andH Koo ldquoStreptococcus mutans-derived extracellular matrixin cariogenic oral biofilmsrdquo Frontiers in Cellular and InfectionMicrobiology vol 5 article no 10 2015

[35] H Koo M L Falsetta and M I Klein ldquoThe exopolysaccharidematrix a virulence determinant of cariogenic biofilmrdquo Journalof Dental Research vol 92 no 12 pp 1065ndash1073 2013

[36] R KhanMAdilMDanishuddin P KVerma andAUKhanldquoIn vitro and in vivo inhibition of Streptococcusmutans biofilmby Trachyspermum ammi seeds An approach of alternativemedicinerdquo Phytomedicine vol 19 no 8-9 pp 747ndash755 2012

[37] X Xu X D Zhou and C DWu ldquoTea catechin epigallocatechingallate inhibits Streptococcus mutans biofilm formation bysuppressing gtf genesrdquo Archives of Oral Biolog vol 57 no 6 pp678ndash683 2012

[38] K Stephenson ldquoTwo-component and phosphorelay signal-transduction systems as therapeutic targetsrdquo Current Opinionin Pharmacology vol 2 no 5 pp 507ndash512 2002

[39] H Aoki T Shiroza H Hayakawa S Sato and H K KuramitsuldquoCloning of a Streptococcus mutans glycosyltransferase genecoding for insoluble glucan synthesisrdquo Infection and Immunityvol 53 no 3 pp 587ndash594 1986

[40] Z He Q Wang Y Hu et al ldquoUse of the quorum sensinginhibitor furanone C-30 to interfere with biofilm formation byStreptococcus mutans and its luxS mutant strainrdquo InternationalJournal of Antimicrobial Agents vol 40 no 1 pp 30ndash35 2012

[41] A Ogawa S Furukawa S Fujita et al ldquoInhibition of Strepto-coccus mutans biofilm formation by Streptococcus salivariusFruArdquo Applied and Environmental Microbiology vol 77 no 5pp 1572ndash1580 2011

[42] R A Burne and J E C Penders ldquoDifferential localization of theStreptococcus mutans GS-5 fructan hydrolase enzyme FruArdquoFEMS Microbiology Letters vol 121 no 2 pp 243ndash249 1994

[43] R A Burne K Schilling W H Bowen and R E YasbinldquoExpression purification and characterization of an Exo-120573-D-fructosidase of Streptococcus mutansrdquo Journal of Bacteriologyvol 169 no 10 pp 4507ndash4517 1987

[44] L Sadeghinejad D G Cvitkovitch W L Siqueira J Merritt JP Santerre and Y Finer ldquoMechanistic genomic and proteomicstudy on the effects of BisGMA-derived biodegradation producton cariogenic bacteriardquoDental Materials vol 33 no 2 pp 175ndash190 2017

BioMed Research International 11

[45] G R Bender S V W Sutton and R E Marquis ldquoAcidtolerance proton permeabilities and membrane ATPases oforal streptococcirdquo Infection and Immunity vol 53 no 2 pp 331ndash338 1986

[46] Y-H Li X-L Tian G Layton C Norgaard and G Sis-son ldquoAdditive attenuation of virulence and cariogenic poten-tial of Streptococcus mutans by simultaneous inactivationof the ComCDE quorum-sensing system and HKRR11 two-component regulatory systemrdquoMicrobiology vol 154 no 11 pp3256ndash3265 2008

[47] D G Cvitkovitch Y-H Li and R P Ellen ldquoQuorum sensingand biofilm formation in Streptococcal infectionsrdquoThe Journalof Clinical Investigation vol 112 no 11 pp 1626ndash1632 2003

[48] R Khan H V Rukke A P R Filho et al ldquoExtracellularidentification of a processed type II ComRComS pheromoneof Streptococcus mutansrdquo Journal of Bacteriology vol 194 no15 pp 3781ndash3788 2012

[49] P Huang P Hu S Y Zhou Q Li and W M Chen ldquoMorininhibits sortase A and subsequent biofilm formation in strepto-coccus mutansrdquo Current Microbiology vol 68 no 1 pp 47ndash522014

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

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Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

8 BioMed Research International

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowast lowast

lowastlowast

24 h5min

ftf

gtfB

gtfC

gtfD

fruA

gbpB ftf

gtfC

gtfB

gtfD

fruA

gbpB

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

nChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(a)

lowastlowast

lowastlowastlowast

lowast lowast

24 h5min

scrB

scrA

atpH scrB

scrA

atpH

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(b)

lowast

lowastlowastlowast

lowastlowastlowastlowastlowast lowastlowast

lowastlowastlowastlowast lowastlowast

lowastlowast

24 h5min

vicRluxS

com

E

com

D

com

C

luxS

vicR

com

E

com

C

com

D

000510152025

Relat

ive R

NA

expr

essio

n

00

05

10

15

20

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(c)

lowastlowastlowast

lowastlowast

lowast

24 h5min

srtA

spaP

srtA

spaP

00

05

10

15

Relat

ive R

NA

expr

essio

n

00

05

10

15

Relat

ive R

NA

expr

essio

n

ChlorhexidineCurcuminControl

ChlorhexidineCurcuminControl

(d)

Figure 6 Results of qRT-PCR to examine the gene expression of different virulence systems in S mutans UA159 (a) EPS synthesis system(b) carbohydrate metabolism system (c) quorum-sensing system (d) two-component transduction system All targets were amplified usingprimers Different gene expression levels were normalized to the level of 16sRNA gene transcripts Data are presented as the mean plusmn SDTheasterisks (lowast) indicate significant differences (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)

and create environmental and protective niches within thebiofilm that can directly modulate caries pathogenesis [3435] Curcumin was shown to destroy the structure of EPS inthe short term decreasing the biomass of EPS the quantityof total bacteria and the percentage of lived bacteria Withprolonged curcumin exposure the structure of the EPS wasbadly damaged and the thickness of the biofilm and thenumber of total bacteria were decreased demonstrably Khanet al reported that the extract of Trachyspermum ammi seedscould modulate the expression of specific virulence genes byS mutans which disrupted the accumulation and structuralorganization of EPS ultimately affecting the cariogenicityof S mutans [36] Xu et al found that the tea catechinepigallocatechin gallate could inhibit the formation of Smutans biofilm by suppressing the gtfs gene [37] Thesestudies serve as a reminder that the effects of natural productson the EPS of S mutans biofilm are usually exerted byregulating cariogenic genes

As previously reported the regulatory systems in Smutans such as the EPS synthesis system carbohydratemetabolism system quorum-sensing system and two-component transduction system are connected to the ecolog-ical balance and to bacterial carcinogenesis [6 38]The resultsshowed that curcumin decreased the expression of cariogenicgenes in the plaque of S mutans Although the majorityof the 24 h qPCR results were not statistically significant

the 119875 values between the control group and curcumin groupwere close to 005 and the decrease was pronounced Thuscurcumin has an inhibitory effect on certain genes in Smutans at 24 h

Gtfs encoded by the gtfBCD genes are indispensable forthe utilization of glucose and for EPS synthesis in S mutansThey are important factors in biofilm formation and thedevelopment of caries [24] GtfB synthesizes water-insolublepolysaccharide [39] which is the main component of theEPS Downregulation of the expression of gtfB in curcuminmight explain the decreased biomass of EPS in the shortterm Oral bacterial aggregation is mediated by interactionsbetween surface-associated glucan-binding proteins (GbpBs)that adhere to glucans thereby promoting plaque formation[40] The expression of gbpB was decreased in both treat-ment groups which may relate to the reduced thickness ofthe biofilm Fructosyltransferase (FTF) is an enzyme thatconverts sucrose to extracellular homopolymers of fructosethe fructans FTF is the product of the fruA gene and isan exo-120573-d-fructosidase that releases fructose from 120573(26)-and 120573(21)-linked fructans and cleaves fructose from sucroseand raffinose [41ndash43] Both ftf and fruA expressions weredysregulated after short-term exposure to curcumin

In S mutans the scrA gene in the phosphotransferasesystem (PTS) encodes a high-affinity permease which inter-nalizes sucrose Then intracellular sucrose-6-PO

4is first

BioMed Research International 9

hydrolyzed by ScrB a sucrose-6-PO4hydrolase to produce

fructose and glucose-6-PO4 In addition the downstream of

scrA and scrB could encode a regulator to change the expres-sion level of scrAB Both scrAB genes were downregulatedin our study which might contribute to the reduction inthe S mutans biofilm In the oral environment S mutansis subjected to rapid pH fluctuations arising from the avail-ability of carbohydrates The atpH gene in S mutans encodessubunit C of a multisubunit enzyme (F1F0-ATPase) involvedin intracellular pH regulation and acid tolerance [44 45]Gene expression analysis found that atpHwas downregulatedin S mutans biofilm after treatment with curcumin for 5minand 24 hThus curcumin inhibits the acid tolerance ability ofS mutans and probably decreases the cariogenic traits of Smutans

The quorum-sensing system is an essential component ofentire gene regulation networks responsible for the adapta-tion of bacteria in biofilms [46] Two well-studied quorum-sensing systems exist in S mutans the ComCDE systemwhich can enable intraspecies cell-cell communication andhas been proved to have a positive regulatory effect onthe expression of biofilm-related genes such as gtfB gtfCand gbpB in S mutans and the LuxS system which cancatalyse the formation of the signal peptide AI-2 to mediateinterspecies and intraspecies interaction in the multispeciesplaque community [6 47] The findings of the current worksuggest that curcumin can potentially contribute to reducingthe quantity of lived bacteria in the biofilm and decrease thebiomass of EPS by decreasing the cariogenic potential of Smutans biofilm via inhibiting the expression of ComCDEsystem and LuxS system The short-term inhibitory effectis more significant than the long-term effect There is adecreasing tendency in the VicR expression of S mutans aftercurcumin treatment suggesting that curcumin may act byinhibiting the expression of VicR The regulatory network ofcariogenic genes in S mutans is complex It was reported thatthe ComRComS is the second quorum-sensing system inS mutans [48] The cell-to-cell communication system in Smutans is not clear Further research is required in this field

S mutans possesses a series of cell-surface proteins suchas SpaP (also known as antigen III Pac P1 and antigenB) which are anchored to the cell envelope by sortase A(SrtA) Sortase A is encoded by the gene srtA and recognizesa motif consisting of leucine proline X threonine andglycine (LPXTG where X is any amino acid) [33 49] Naturalproducts attract substantial attention because they can inhibitsrtA in vitro and in vivo but do not significantly decreasethe viability of bacteria The current study demonstrates thatcurcumin can inhibit the expression of spaP and srtA withan SMIC

50of 500 120583M The present study explored the effect

of curcumin on S mutans UA159 However S mutans hasheterogeneity in different strains Much more strains of Smutans are needed to be test in future

In conclusion curcumin has both a short-term and along-term antibacterial effect Additionally it is a food-gradenatural product with a similar effect to that of chlorhexidineand it takes effect faster than 012 chlorhexidine Curcumincould be an alternative strategy to treat oral disease Cur-cumin is a promising antibacterial agent with potential forwider clinical application in the future

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by Science and Technology Plan-ning Project ofGuangdongProvince China (2014A020212621and 2017A020215064)

References

[1] N B Pitts D T Zero P D Marsh et al ldquoDental cariesrdquo NatureReviews Disease Primers vol 3 Article ID 17030 2017

[2] D F A Pereira C J Seneviratne C Y Koga-Ito and L PSamaranayake ldquoIs the oral fungal pathogen Candida albicansa cariogenrdquo Oral Diseases 2017

[3] B NyvadW Crielaard AMira N Takahashi andD BeightonldquoDental caries from a molecular microbiological perspectiverdquoCaries Research vol 47 no 2 pp 89ndash102 2013

[4] R E Marquis ldquoOxygen metabolism oxidative stress and acid-base physiology of dental plaque biofilmsrdquo Journal of IndustrialMicrobiology and Biotechnology vol 15 no 3 pp 198ndash207 1995

[5] W H Bowen and H Koo ldquoBiology of streptococcus mutans-derived glucosyltransferases role in extracellular matrix forma-tion of cariogenic biofilmsrdquo Caries Research vol 45 no 1 pp69ndash86 2011

[6] E G Smith and G A Spatafora ldquoGene regulation in S mutanscomplex control in a complex environmentrdquo Journal of DentalResearch vol 91 no 2 pp 133ndash141 2012

[7] W Krzysciak A Jurczak D Koscielniak B Bystrowska and ASkalniak ldquoThe virulence of Streptococcus mutans and the abilityto form biofilmsrdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 33 no 4 pp 499ndash515 2014

[8] J Merritt J Kreth F Qi R Sullivan and W Shi ldquoNon-disruptive real-time analyses of themetabolic status and viabil-ity of Streptococcus mutans cells in response to antimicrobialtreatmentsrdquo Journal of Microbiological Methods vol 61 no 2pp 161ndash170 2005

[9] J Fricks-Lima C M Hendrickson M Allgaier et al ldquoDif-ferences in biofilm formation and antimicrobial resistance ofPseudomonas aeruginosa isolated from airways of mechanicallyventilated patients and cystic fibrosis patientsrdquo InternationalJournal of Antimicrobial Agents vol 37 no 4 pp 309ndash315 2011

[10] R Utsumi Bacterial Signal Transduction Networks and DrugTargets vol 631 Springer New York NY USA 2008

[11] H Koo andW H Bowen ldquoCandida albicans and Streptococcusmutans a potential synergistic alliance to cause virulent toothdecay in childrenrdquo Future Microbiology vol 9 no 12 pp 1295ndash1297 2014

[12] S Hamada T Koga and T Ooshima ldquoVirulence factors ofStreptococcus mutans and dental caries preventionrdquo Journal ofDental Research vol 63 no 3 pp 407ndash411 1984

[13] J-G Jeon P L Rosalen M L Falsetta and H Koo ldquoNaturalproducts in caries research current (limited) knowledge chal-lenges and future perspectiverdquoCaries Research vol 45 no 3 pp243ndash263 2011

[14] S-Y Teow K Liew S A Ali A S-B Khoo and S-CPeh ldquoAntibacterial Action of Curcumin against Staphylococcusaureus A Brief Reviewrdquo Journal of Tropical Medicine vol 2016Article ID 2853045 10 pages 2016

10 BioMed Research International

[15] J Song B Choi E-J Jin Y Yoon and K-H Choi ldquoCurcuminsuppresses Streptococcus mutans adherence to human toothsurfaces and extracellular matrix proteinsrdquo European Journalof Clinical Microbiology amp Infectious Diseases vol 31 no 7 pp1347ndash1352 2012

[16] P Hu P Huang and M W Chen ldquoCurcumin reduces Strep-tococcus mutans biofilm formation by inhibiting sortase Aactivityrdquo Archives of Oral Biolog vol 58 no 10 pp 1343ndash13482013

[17] P Hu P Huang and W M Chen ldquoCurcumin inhibits thesortase a activity of the streptococcus mutans UA159rdquo AppliedBiochemistry and Biotechnology vol 171 no 2 pp 396ndash4022013

[18] DManoil A Filieri C Gameiro et al ldquoFlow cytometric assess-ment of Streptococcus mutans viability after exposure to bluelight-activated curcuminrdquo Photodiagnosis and PhotodynamicTherapy vol 11 no 3 pp 372ndash379 2014

[19] M A Pfaller and D J Diekema ldquoProgress in antifungalsusceptibility testing of Candida spp by use of Clinical andLaboratory Standards Institute broth microdilution methods2010 to 2012rdquo Journal of Clinical Microbiology vol 50 no 9 pp2846ndash2856 2012

[20] M M Weerasekera G K Wijesinghe T A Jayarathna etal ldquoCulture media profoundly affect Candida Albicans andCandida tropicalis growth adhesion and biofilm developmentrdquoMemorias do InstitutoOswaldoCruz vol 111 no 11 pp 697ndash7022016

[21] J Chen T Li X Zhou et al ldquoCharacterization of the clusteredregularly interspaced short palindromic repeats sites in Strep-tococcus mutans isolated from early childhood caries patientsrdquoArchives of Oral Biolog vol 83 pp 174ndash180 2017

[22] M Sun Z Zhou J Dong J Zhang Y Xia and R ShuldquoAntibacterial and antibiofilm activities of docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA) against periodon-topathic bacteriardquo Microbial Pathogenesis vol 99 pp 196ndash2032016

[23] X Huang K Zhang M Deng et al ldquoEffect of arginine on thegrowth and biofilm formation of oral bacteriardquo Archives of OralBiolog vol 82 pp 256ndash262 2017

[24] S Liu W Qiu K Zhang et al ldquoNicotine enhances inter-species relationship between streptococcus mutans and candidaalbicansrdquo BioMed Research International vol 2017 Article ID7953920 9 pages 2017

[25] T B L Bedran L Grignon D P Spolidorio and D GrenierldquoSubinhibitory concentrations of triclosan promote Streptococ-cus mutans biofilm formation and adherence to oral epithelialcellsrdquo PLoS ONE vol 9 no 2 Article ID e89059 2014

[26] C M Levesque E Voronejskaia Y-C C Huang RWMair RP Ellen and D G Cvitkovitch ldquoInvolvement of sortase anchor-ing of cell wall proteins in biofilm formation by Streptococcusmutansrdquo Infection and Immunity vol 73 no 6 pp 3773ndash37772005

[27] M M Nascimento J A Lemos J Abranches V K Lin andR A Burne ldquoRole of RelA of Streptococcus mutans in globalcontrol of gene expressionrdquo Journal of Bacteriology vol 190 no1 pp 28ndash36 2008

[28] B Kunze M Reck A Dotsch et al ldquoDamage of Streptococcusmutans biofilms by carolacton a secondarymetabolite from themyxobacterium Sorangium cellulosumrdquo BMC Microbiologyvol 10 article no 199 2010

[29] V LeungDDufour andCM Levesque ldquoDeath and survival inStreptococcusmutansDiffering outcomes of a quorum-sensing

signaling peptiderdquo Frontiers in Microbiology vol 6 Article ID01176 2015

[30] A Tofino-Rivera M Ortega-Cuadros D Galvis-Pareja HJimenez-Rios L JMerini andMCMartınez-Pabon ldquoEffect ofLippia alba and Cymbopogon citratus essential oils on biofilmsof Streptococcusmutans and cytotoxicity in CHO cellsrdquo Journalof Ethnopharmacology vol 194 pp 749ndash754 2016

[31] K Jhajharia A Parolia K V Shetty and L K Mehta ldquoBiofilmin endodontics a reviewrdquo Journal of International Society ofPreventive amp Community Dentistry vol 5 no 1 pp 1ndash12 2015

[32] H Yang Y Bi X Shang et al ldquoAntibiofilm activities of anovel chimeolysin against Streptococcus mutans under physi-ological and cariogenic conditionsrdquo Antimicrobial Agents andChemotherapy vol 60 no 12 pp 7436ndash7443 2016

[33] H Tamura A Yamada and H Kato ldquoMolecular characteriza-tion of the dextran-binding lectin B gene dblB of Streptococcuscriceti in Streptococcus mutans strain GS-5 with mutations inboth gbpC and spaP genesrdquo Genes amp Genetic Systems vol 89no 2 pp 41ndash50 2014

[34] M I Klein G Hwang P H S Santos O H Campanella andH Koo ldquoStreptococcus mutans-derived extracellular matrixin cariogenic oral biofilmsrdquo Frontiers in Cellular and InfectionMicrobiology vol 5 article no 10 2015

[35] H Koo M L Falsetta and M I Klein ldquoThe exopolysaccharidematrix a virulence determinant of cariogenic biofilmrdquo Journalof Dental Research vol 92 no 12 pp 1065ndash1073 2013

[36] R KhanMAdilMDanishuddin P KVerma andAUKhanldquoIn vitro and in vivo inhibition of Streptococcusmutans biofilmby Trachyspermum ammi seeds An approach of alternativemedicinerdquo Phytomedicine vol 19 no 8-9 pp 747ndash755 2012

[37] X Xu X D Zhou and C DWu ldquoTea catechin epigallocatechingallate inhibits Streptococcus mutans biofilm formation bysuppressing gtf genesrdquo Archives of Oral Biolog vol 57 no 6 pp678ndash683 2012

[38] K Stephenson ldquoTwo-component and phosphorelay signal-transduction systems as therapeutic targetsrdquo Current Opinionin Pharmacology vol 2 no 5 pp 507ndash512 2002

[39] H Aoki T Shiroza H Hayakawa S Sato and H K KuramitsuldquoCloning of a Streptococcus mutans glycosyltransferase genecoding for insoluble glucan synthesisrdquo Infection and Immunityvol 53 no 3 pp 587ndash594 1986

[40] Z He Q Wang Y Hu et al ldquoUse of the quorum sensinginhibitor furanone C-30 to interfere with biofilm formation byStreptococcus mutans and its luxS mutant strainrdquo InternationalJournal of Antimicrobial Agents vol 40 no 1 pp 30ndash35 2012

[41] A Ogawa S Furukawa S Fujita et al ldquoInhibition of Strepto-coccus mutans biofilm formation by Streptococcus salivariusFruArdquo Applied and Environmental Microbiology vol 77 no 5pp 1572ndash1580 2011

[42] R A Burne and J E C Penders ldquoDifferential localization of theStreptococcus mutans GS-5 fructan hydrolase enzyme FruArdquoFEMS Microbiology Letters vol 121 no 2 pp 243ndash249 1994

[43] R A Burne K Schilling W H Bowen and R E YasbinldquoExpression purification and characterization of an Exo-120573-D-fructosidase of Streptococcus mutansrdquo Journal of Bacteriologyvol 169 no 10 pp 4507ndash4517 1987

[44] L Sadeghinejad D G Cvitkovitch W L Siqueira J Merritt JP Santerre and Y Finer ldquoMechanistic genomic and proteomicstudy on the effects of BisGMA-derived biodegradation producton cariogenic bacteriardquoDental Materials vol 33 no 2 pp 175ndash190 2017

BioMed Research International 11

[45] G R Bender S V W Sutton and R E Marquis ldquoAcidtolerance proton permeabilities and membrane ATPases oforal streptococcirdquo Infection and Immunity vol 53 no 2 pp 331ndash338 1986

[46] Y-H Li X-L Tian G Layton C Norgaard and G Sis-son ldquoAdditive attenuation of virulence and cariogenic poten-tial of Streptococcus mutans by simultaneous inactivationof the ComCDE quorum-sensing system and HKRR11 two-component regulatory systemrdquoMicrobiology vol 154 no 11 pp3256ndash3265 2008

[47] D G Cvitkovitch Y-H Li and R P Ellen ldquoQuorum sensingand biofilm formation in Streptococcal infectionsrdquoThe Journalof Clinical Investigation vol 112 no 11 pp 1626ndash1632 2003

[48] R Khan H V Rukke A P R Filho et al ldquoExtracellularidentification of a processed type II ComRComS pheromoneof Streptococcus mutansrdquo Journal of Bacteriology vol 194 no15 pp 3781ndash3788 2012

[49] P Huang P Hu S Y Zhou Q Li and W M Chen ldquoMorininhibits sortase A and subsequent biofilm formation in strepto-coccus mutansrdquo Current Microbiology vol 68 no 1 pp 47ndash522014

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 9

hydrolyzed by ScrB a sucrose-6-PO4hydrolase to produce

fructose and glucose-6-PO4 In addition the downstream of

scrA and scrB could encode a regulator to change the expres-sion level of scrAB Both scrAB genes were downregulatedin our study which might contribute to the reduction inthe S mutans biofilm In the oral environment S mutansis subjected to rapid pH fluctuations arising from the avail-ability of carbohydrates The atpH gene in S mutans encodessubunit C of a multisubunit enzyme (F1F0-ATPase) involvedin intracellular pH regulation and acid tolerance [44 45]Gene expression analysis found that atpHwas downregulatedin S mutans biofilm after treatment with curcumin for 5minand 24 hThus curcumin inhibits the acid tolerance ability ofS mutans and probably decreases the cariogenic traits of Smutans

The quorum-sensing system is an essential component ofentire gene regulation networks responsible for the adapta-tion of bacteria in biofilms [46] Two well-studied quorum-sensing systems exist in S mutans the ComCDE systemwhich can enable intraspecies cell-cell communication andhas been proved to have a positive regulatory effect onthe expression of biofilm-related genes such as gtfB gtfCand gbpB in S mutans and the LuxS system which cancatalyse the formation of the signal peptide AI-2 to mediateinterspecies and intraspecies interaction in the multispeciesplaque community [6 47] The findings of the current worksuggest that curcumin can potentially contribute to reducingthe quantity of lived bacteria in the biofilm and decrease thebiomass of EPS by decreasing the cariogenic potential of Smutans biofilm via inhibiting the expression of ComCDEsystem and LuxS system The short-term inhibitory effectis more significant than the long-term effect There is adecreasing tendency in the VicR expression of S mutans aftercurcumin treatment suggesting that curcumin may act byinhibiting the expression of VicR The regulatory network ofcariogenic genes in S mutans is complex It was reported thatthe ComRComS is the second quorum-sensing system inS mutans [48] The cell-to-cell communication system in Smutans is not clear Further research is required in this field

S mutans possesses a series of cell-surface proteins suchas SpaP (also known as antigen III Pac P1 and antigenB) which are anchored to the cell envelope by sortase A(SrtA) Sortase A is encoded by the gene srtA and recognizesa motif consisting of leucine proline X threonine andglycine (LPXTG where X is any amino acid) [33 49] Naturalproducts attract substantial attention because they can inhibitsrtA in vitro and in vivo but do not significantly decreasethe viability of bacteria The current study demonstrates thatcurcumin can inhibit the expression of spaP and srtA withan SMIC

50of 500 120583M The present study explored the effect

of curcumin on S mutans UA159 However S mutans hasheterogeneity in different strains Much more strains of Smutans are needed to be test in future

In conclusion curcumin has both a short-term and along-term antibacterial effect Additionally it is a food-gradenatural product with a similar effect to that of chlorhexidineand it takes effect faster than 012 chlorhexidine Curcumincould be an alternative strategy to treat oral disease Cur-cumin is a promising antibacterial agent with potential forwider clinical application in the future

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by Science and Technology Plan-ning Project ofGuangdongProvince China (2014A020212621and 2017A020215064)

References

[1] N B Pitts D T Zero P D Marsh et al ldquoDental cariesrdquo NatureReviews Disease Primers vol 3 Article ID 17030 2017

[2] D F A Pereira C J Seneviratne C Y Koga-Ito and L PSamaranayake ldquoIs the oral fungal pathogen Candida albicansa cariogenrdquo Oral Diseases 2017

[3] B NyvadW Crielaard AMira N Takahashi andD BeightonldquoDental caries from a molecular microbiological perspectiverdquoCaries Research vol 47 no 2 pp 89ndash102 2013

[4] R E Marquis ldquoOxygen metabolism oxidative stress and acid-base physiology of dental plaque biofilmsrdquo Journal of IndustrialMicrobiology and Biotechnology vol 15 no 3 pp 198ndash207 1995

[5] W H Bowen and H Koo ldquoBiology of streptococcus mutans-derived glucosyltransferases role in extracellular matrix forma-tion of cariogenic biofilmsrdquo Caries Research vol 45 no 1 pp69ndash86 2011

[6] E G Smith and G A Spatafora ldquoGene regulation in S mutanscomplex control in a complex environmentrdquo Journal of DentalResearch vol 91 no 2 pp 133ndash141 2012

[7] W Krzysciak A Jurczak D Koscielniak B Bystrowska and ASkalniak ldquoThe virulence of Streptococcus mutans and the abilityto form biofilmsrdquo European Journal of Clinical Microbiology ampInfectious Diseases vol 33 no 4 pp 499ndash515 2014

[8] J Merritt J Kreth F Qi R Sullivan and W Shi ldquoNon-disruptive real-time analyses of themetabolic status and viabil-ity of Streptococcus mutans cells in response to antimicrobialtreatmentsrdquo Journal of Microbiological Methods vol 61 no 2pp 161ndash170 2005

[9] J Fricks-Lima C M Hendrickson M Allgaier et al ldquoDif-ferences in biofilm formation and antimicrobial resistance ofPseudomonas aeruginosa isolated from airways of mechanicallyventilated patients and cystic fibrosis patientsrdquo InternationalJournal of Antimicrobial Agents vol 37 no 4 pp 309ndash315 2011

[10] R Utsumi Bacterial Signal Transduction Networks and DrugTargets vol 631 Springer New York NY USA 2008

[11] H Koo andW H Bowen ldquoCandida albicans and Streptococcusmutans a potential synergistic alliance to cause virulent toothdecay in childrenrdquo Future Microbiology vol 9 no 12 pp 1295ndash1297 2014

[12] S Hamada T Koga and T Ooshima ldquoVirulence factors ofStreptococcus mutans and dental caries preventionrdquo Journal ofDental Research vol 63 no 3 pp 407ndash411 1984

[13] J-G Jeon P L Rosalen M L Falsetta and H Koo ldquoNaturalproducts in caries research current (limited) knowledge chal-lenges and future perspectiverdquoCaries Research vol 45 no 3 pp243ndash263 2011

[14] S-Y Teow K Liew S A Ali A S-B Khoo and S-CPeh ldquoAntibacterial Action of Curcumin against Staphylococcusaureus A Brief Reviewrdquo Journal of Tropical Medicine vol 2016Article ID 2853045 10 pages 2016

10 BioMed Research International

[15] J Song B Choi E-J Jin Y Yoon and K-H Choi ldquoCurcuminsuppresses Streptococcus mutans adherence to human toothsurfaces and extracellular matrix proteinsrdquo European Journalof Clinical Microbiology amp Infectious Diseases vol 31 no 7 pp1347ndash1352 2012

[16] P Hu P Huang and M W Chen ldquoCurcumin reduces Strep-tococcus mutans biofilm formation by inhibiting sortase Aactivityrdquo Archives of Oral Biolog vol 58 no 10 pp 1343ndash13482013

[17] P Hu P Huang and W M Chen ldquoCurcumin inhibits thesortase a activity of the streptococcus mutans UA159rdquo AppliedBiochemistry and Biotechnology vol 171 no 2 pp 396ndash4022013

[18] DManoil A Filieri C Gameiro et al ldquoFlow cytometric assess-ment of Streptococcus mutans viability after exposure to bluelight-activated curcuminrdquo Photodiagnosis and PhotodynamicTherapy vol 11 no 3 pp 372ndash379 2014

[19] M A Pfaller and D J Diekema ldquoProgress in antifungalsusceptibility testing of Candida spp by use of Clinical andLaboratory Standards Institute broth microdilution methods2010 to 2012rdquo Journal of Clinical Microbiology vol 50 no 9 pp2846ndash2856 2012

[20] M M Weerasekera G K Wijesinghe T A Jayarathna etal ldquoCulture media profoundly affect Candida Albicans andCandida tropicalis growth adhesion and biofilm developmentrdquoMemorias do InstitutoOswaldoCruz vol 111 no 11 pp 697ndash7022016

[21] J Chen T Li X Zhou et al ldquoCharacterization of the clusteredregularly interspaced short palindromic repeats sites in Strep-tococcus mutans isolated from early childhood caries patientsrdquoArchives of Oral Biolog vol 83 pp 174ndash180 2017

[22] M Sun Z Zhou J Dong J Zhang Y Xia and R ShuldquoAntibacterial and antibiofilm activities of docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA) against periodon-topathic bacteriardquo Microbial Pathogenesis vol 99 pp 196ndash2032016

[23] X Huang K Zhang M Deng et al ldquoEffect of arginine on thegrowth and biofilm formation of oral bacteriardquo Archives of OralBiolog vol 82 pp 256ndash262 2017

[24] S Liu W Qiu K Zhang et al ldquoNicotine enhances inter-species relationship between streptococcus mutans and candidaalbicansrdquo BioMed Research International vol 2017 Article ID7953920 9 pages 2017

[25] T B L Bedran L Grignon D P Spolidorio and D GrenierldquoSubinhibitory concentrations of triclosan promote Streptococ-cus mutans biofilm formation and adherence to oral epithelialcellsrdquo PLoS ONE vol 9 no 2 Article ID e89059 2014

[26] C M Levesque E Voronejskaia Y-C C Huang RWMair RP Ellen and D G Cvitkovitch ldquoInvolvement of sortase anchor-ing of cell wall proteins in biofilm formation by Streptococcusmutansrdquo Infection and Immunity vol 73 no 6 pp 3773ndash37772005

[27] M M Nascimento J A Lemos J Abranches V K Lin andR A Burne ldquoRole of RelA of Streptococcus mutans in globalcontrol of gene expressionrdquo Journal of Bacteriology vol 190 no1 pp 28ndash36 2008

[28] B Kunze M Reck A Dotsch et al ldquoDamage of Streptococcusmutans biofilms by carolacton a secondarymetabolite from themyxobacterium Sorangium cellulosumrdquo BMC Microbiologyvol 10 article no 199 2010

[29] V LeungDDufour andCM Levesque ldquoDeath and survival inStreptococcusmutansDiffering outcomes of a quorum-sensing

signaling peptiderdquo Frontiers in Microbiology vol 6 Article ID01176 2015

[30] A Tofino-Rivera M Ortega-Cuadros D Galvis-Pareja HJimenez-Rios L JMerini andMCMartınez-Pabon ldquoEffect ofLippia alba and Cymbopogon citratus essential oils on biofilmsof Streptococcusmutans and cytotoxicity in CHO cellsrdquo Journalof Ethnopharmacology vol 194 pp 749ndash754 2016

[31] K Jhajharia A Parolia K V Shetty and L K Mehta ldquoBiofilmin endodontics a reviewrdquo Journal of International Society ofPreventive amp Community Dentistry vol 5 no 1 pp 1ndash12 2015

[32] H Yang Y Bi X Shang et al ldquoAntibiofilm activities of anovel chimeolysin against Streptococcus mutans under physi-ological and cariogenic conditionsrdquo Antimicrobial Agents andChemotherapy vol 60 no 12 pp 7436ndash7443 2016

[33] H Tamura A Yamada and H Kato ldquoMolecular characteriza-tion of the dextran-binding lectin B gene dblB of Streptococcuscriceti in Streptococcus mutans strain GS-5 with mutations inboth gbpC and spaP genesrdquo Genes amp Genetic Systems vol 89no 2 pp 41ndash50 2014

[34] M I Klein G Hwang P H S Santos O H Campanella andH Koo ldquoStreptococcus mutans-derived extracellular matrixin cariogenic oral biofilmsrdquo Frontiers in Cellular and InfectionMicrobiology vol 5 article no 10 2015

[35] H Koo M L Falsetta and M I Klein ldquoThe exopolysaccharidematrix a virulence determinant of cariogenic biofilmrdquo Journalof Dental Research vol 92 no 12 pp 1065ndash1073 2013

[36] R KhanMAdilMDanishuddin P KVerma andAUKhanldquoIn vitro and in vivo inhibition of Streptococcusmutans biofilmby Trachyspermum ammi seeds An approach of alternativemedicinerdquo Phytomedicine vol 19 no 8-9 pp 747ndash755 2012

[37] X Xu X D Zhou and C DWu ldquoTea catechin epigallocatechingallate inhibits Streptococcus mutans biofilm formation bysuppressing gtf genesrdquo Archives of Oral Biolog vol 57 no 6 pp678ndash683 2012

[38] K Stephenson ldquoTwo-component and phosphorelay signal-transduction systems as therapeutic targetsrdquo Current Opinionin Pharmacology vol 2 no 5 pp 507ndash512 2002

[39] H Aoki T Shiroza H Hayakawa S Sato and H K KuramitsuldquoCloning of a Streptococcus mutans glycosyltransferase genecoding for insoluble glucan synthesisrdquo Infection and Immunityvol 53 no 3 pp 587ndash594 1986

[40] Z He Q Wang Y Hu et al ldquoUse of the quorum sensinginhibitor furanone C-30 to interfere with biofilm formation byStreptococcus mutans and its luxS mutant strainrdquo InternationalJournal of Antimicrobial Agents vol 40 no 1 pp 30ndash35 2012

[41] A Ogawa S Furukawa S Fujita et al ldquoInhibition of Strepto-coccus mutans biofilm formation by Streptococcus salivariusFruArdquo Applied and Environmental Microbiology vol 77 no 5pp 1572ndash1580 2011

[42] R A Burne and J E C Penders ldquoDifferential localization of theStreptococcus mutans GS-5 fructan hydrolase enzyme FruArdquoFEMS Microbiology Letters vol 121 no 2 pp 243ndash249 1994

[43] R A Burne K Schilling W H Bowen and R E YasbinldquoExpression purification and characterization of an Exo-120573-D-fructosidase of Streptococcus mutansrdquo Journal of Bacteriologyvol 169 no 10 pp 4507ndash4517 1987

[44] L Sadeghinejad D G Cvitkovitch W L Siqueira J Merritt JP Santerre and Y Finer ldquoMechanistic genomic and proteomicstudy on the effects of BisGMA-derived biodegradation producton cariogenic bacteriardquoDental Materials vol 33 no 2 pp 175ndash190 2017

BioMed Research International 11

[45] G R Bender S V W Sutton and R E Marquis ldquoAcidtolerance proton permeabilities and membrane ATPases oforal streptococcirdquo Infection and Immunity vol 53 no 2 pp 331ndash338 1986

[46] Y-H Li X-L Tian G Layton C Norgaard and G Sis-son ldquoAdditive attenuation of virulence and cariogenic poten-tial of Streptococcus mutans by simultaneous inactivationof the ComCDE quorum-sensing system and HKRR11 two-component regulatory systemrdquoMicrobiology vol 154 no 11 pp3256ndash3265 2008

[47] D G Cvitkovitch Y-H Li and R P Ellen ldquoQuorum sensingand biofilm formation in Streptococcal infectionsrdquoThe Journalof Clinical Investigation vol 112 no 11 pp 1626ndash1632 2003

[48] R Khan H V Rukke A P R Filho et al ldquoExtracellularidentification of a processed type II ComRComS pheromoneof Streptococcus mutansrdquo Journal of Bacteriology vol 194 no15 pp 3781ndash3788 2012

[49] P Huang P Hu S Y Zhou Q Li and W M Chen ldquoMorininhibits sortase A and subsequent biofilm formation in strepto-coccus mutansrdquo Current Microbiology vol 68 no 1 pp 47ndash522014

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

10 BioMed Research International

[15] J Song B Choi E-J Jin Y Yoon and K-H Choi ldquoCurcuminsuppresses Streptococcus mutans adherence to human toothsurfaces and extracellular matrix proteinsrdquo European Journalof Clinical Microbiology amp Infectious Diseases vol 31 no 7 pp1347ndash1352 2012

[16] P Hu P Huang and M W Chen ldquoCurcumin reduces Strep-tococcus mutans biofilm formation by inhibiting sortase Aactivityrdquo Archives of Oral Biolog vol 58 no 10 pp 1343ndash13482013

[17] P Hu P Huang and W M Chen ldquoCurcumin inhibits thesortase a activity of the streptococcus mutans UA159rdquo AppliedBiochemistry and Biotechnology vol 171 no 2 pp 396ndash4022013

[18] DManoil A Filieri C Gameiro et al ldquoFlow cytometric assess-ment of Streptococcus mutans viability after exposure to bluelight-activated curcuminrdquo Photodiagnosis and PhotodynamicTherapy vol 11 no 3 pp 372ndash379 2014

[19] M A Pfaller and D J Diekema ldquoProgress in antifungalsusceptibility testing of Candida spp by use of Clinical andLaboratory Standards Institute broth microdilution methods2010 to 2012rdquo Journal of Clinical Microbiology vol 50 no 9 pp2846ndash2856 2012

[20] M M Weerasekera G K Wijesinghe T A Jayarathna etal ldquoCulture media profoundly affect Candida Albicans andCandida tropicalis growth adhesion and biofilm developmentrdquoMemorias do InstitutoOswaldoCruz vol 111 no 11 pp 697ndash7022016

[21] J Chen T Li X Zhou et al ldquoCharacterization of the clusteredregularly interspaced short palindromic repeats sites in Strep-tococcus mutans isolated from early childhood caries patientsrdquoArchives of Oral Biolog vol 83 pp 174ndash180 2017

[22] M Sun Z Zhou J Dong J Zhang Y Xia and R ShuldquoAntibacterial and antibiofilm activities of docosahexaenoicacid (DHA) and eicosapentaenoic acid (EPA) against periodon-topathic bacteriardquo Microbial Pathogenesis vol 99 pp 196ndash2032016

[23] X Huang K Zhang M Deng et al ldquoEffect of arginine on thegrowth and biofilm formation of oral bacteriardquo Archives of OralBiolog vol 82 pp 256ndash262 2017

[24] S Liu W Qiu K Zhang et al ldquoNicotine enhances inter-species relationship between streptococcus mutans and candidaalbicansrdquo BioMed Research International vol 2017 Article ID7953920 9 pages 2017

[25] T B L Bedran L Grignon D P Spolidorio and D GrenierldquoSubinhibitory concentrations of triclosan promote Streptococ-cus mutans biofilm formation and adherence to oral epithelialcellsrdquo PLoS ONE vol 9 no 2 Article ID e89059 2014

[26] C M Levesque E Voronejskaia Y-C C Huang RWMair RP Ellen and D G Cvitkovitch ldquoInvolvement of sortase anchor-ing of cell wall proteins in biofilm formation by Streptococcusmutansrdquo Infection and Immunity vol 73 no 6 pp 3773ndash37772005

[27] M M Nascimento J A Lemos J Abranches V K Lin andR A Burne ldquoRole of RelA of Streptococcus mutans in globalcontrol of gene expressionrdquo Journal of Bacteriology vol 190 no1 pp 28ndash36 2008

[28] B Kunze M Reck A Dotsch et al ldquoDamage of Streptococcusmutans biofilms by carolacton a secondarymetabolite from themyxobacterium Sorangium cellulosumrdquo BMC Microbiologyvol 10 article no 199 2010

[29] V LeungDDufour andCM Levesque ldquoDeath and survival inStreptococcusmutansDiffering outcomes of a quorum-sensing

signaling peptiderdquo Frontiers in Microbiology vol 6 Article ID01176 2015

[30] A Tofino-Rivera M Ortega-Cuadros D Galvis-Pareja HJimenez-Rios L JMerini andMCMartınez-Pabon ldquoEffect ofLippia alba and Cymbopogon citratus essential oils on biofilmsof Streptococcusmutans and cytotoxicity in CHO cellsrdquo Journalof Ethnopharmacology vol 194 pp 749ndash754 2016

[31] K Jhajharia A Parolia K V Shetty and L K Mehta ldquoBiofilmin endodontics a reviewrdquo Journal of International Society ofPreventive amp Community Dentistry vol 5 no 1 pp 1ndash12 2015

[32] H Yang Y Bi X Shang et al ldquoAntibiofilm activities of anovel chimeolysin against Streptococcus mutans under physi-ological and cariogenic conditionsrdquo Antimicrobial Agents andChemotherapy vol 60 no 12 pp 7436ndash7443 2016

[33] H Tamura A Yamada and H Kato ldquoMolecular characteriza-tion of the dextran-binding lectin B gene dblB of Streptococcuscriceti in Streptococcus mutans strain GS-5 with mutations inboth gbpC and spaP genesrdquo Genes amp Genetic Systems vol 89no 2 pp 41ndash50 2014

[34] M I Klein G Hwang P H S Santos O H Campanella andH Koo ldquoStreptococcus mutans-derived extracellular matrixin cariogenic oral biofilmsrdquo Frontiers in Cellular and InfectionMicrobiology vol 5 article no 10 2015

[35] H Koo M L Falsetta and M I Klein ldquoThe exopolysaccharidematrix a virulence determinant of cariogenic biofilmrdquo Journalof Dental Research vol 92 no 12 pp 1065ndash1073 2013

[36] R KhanMAdilMDanishuddin P KVerma andAUKhanldquoIn vitro and in vivo inhibition of Streptococcusmutans biofilmby Trachyspermum ammi seeds An approach of alternativemedicinerdquo Phytomedicine vol 19 no 8-9 pp 747ndash755 2012

[37] X Xu X D Zhou and C DWu ldquoTea catechin epigallocatechingallate inhibits Streptococcus mutans biofilm formation bysuppressing gtf genesrdquo Archives of Oral Biolog vol 57 no 6 pp678ndash683 2012

[38] K Stephenson ldquoTwo-component and phosphorelay signal-transduction systems as therapeutic targetsrdquo Current Opinionin Pharmacology vol 2 no 5 pp 507ndash512 2002

[39] H Aoki T Shiroza H Hayakawa S Sato and H K KuramitsuldquoCloning of a Streptococcus mutans glycosyltransferase genecoding for insoluble glucan synthesisrdquo Infection and Immunityvol 53 no 3 pp 587ndash594 1986

[40] Z He Q Wang Y Hu et al ldquoUse of the quorum sensinginhibitor furanone C-30 to interfere with biofilm formation byStreptococcus mutans and its luxS mutant strainrdquo InternationalJournal of Antimicrobial Agents vol 40 no 1 pp 30ndash35 2012

[41] A Ogawa S Furukawa S Fujita et al ldquoInhibition of Strepto-coccus mutans biofilm formation by Streptococcus salivariusFruArdquo Applied and Environmental Microbiology vol 77 no 5pp 1572ndash1580 2011

[42] R A Burne and J E C Penders ldquoDifferential localization of theStreptococcus mutans GS-5 fructan hydrolase enzyme FruArdquoFEMS Microbiology Letters vol 121 no 2 pp 243ndash249 1994

[43] R A Burne K Schilling W H Bowen and R E YasbinldquoExpression purification and characterization of an Exo-120573-D-fructosidase of Streptococcus mutansrdquo Journal of Bacteriologyvol 169 no 10 pp 4507ndash4517 1987

[44] L Sadeghinejad D G Cvitkovitch W L Siqueira J Merritt JP Santerre and Y Finer ldquoMechanistic genomic and proteomicstudy on the effects of BisGMA-derived biodegradation producton cariogenic bacteriardquoDental Materials vol 33 no 2 pp 175ndash190 2017

BioMed Research International 11

[45] G R Bender S V W Sutton and R E Marquis ldquoAcidtolerance proton permeabilities and membrane ATPases oforal streptococcirdquo Infection and Immunity vol 53 no 2 pp 331ndash338 1986

[46] Y-H Li X-L Tian G Layton C Norgaard and G Sis-son ldquoAdditive attenuation of virulence and cariogenic poten-tial of Streptococcus mutans by simultaneous inactivationof the ComCDE quorum-sensing system and HKRR11 two-component regulatory systemrdquoMicrobiology vol 154 no 11 pp3256ndash3265 2008

[47] D G Cvitkovitch Y-H Li and R P Ellen ldquoQuorum sensingand biofilm formation in Streptococcal infectionsrdquoThe Journalof Clinical Investigation vol 112 no 11 pp 1626ndash1632 2003

[48] R Khan H V Rukke A P R Filho et al ldquoExtracellularidentification of a processed type II ComRComS pheromoneof Streptococcus mutansrdquo Journal of Bacteriology vol 194 no15 pp 3781ndash3788 2012

[49] P Huang P Hu S Y Zhou Q Li and W M Chen ldquoMorininhibits sortase A and subsequent biofilm formation in strepto-coccus mutansrdquo Current Microbiology vol 68 no 1 pp 47ndash522014

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

BioMed Research International 11

[45] G R Bender S V W Sutton and R E Marquis ldquoAcidtolerance proton permeabilities and membrane ATPases oforal streptococcirdquo Infection and Immunity vol 53 no 2 pp 331ndash338 1986

[46] Y-H Li X-L Tian G Layton C Norgaard and G Sis-son ldquoAdditive attenuation of virulence and cariogenic poten-tial of Streptococcus mutans by simultaneous inactivationof the ComCDE quorum-sensing system and HKRR11 two-component regulatory systemrdquoMicrobiology vol 154 no 11 pp3256ndash3265 2008

[47] D G Cvitkovitch Y-H Li and R P Ellen ldquoQuorum sensingand biofilm formation in Streptococcal infectionsrdquoThe Journalof Clinical Investigation vol 112 no 11 pp 1626ndash1632 2003

[48] R Khan H V Rukke A P R Filho et al ldquoExtracellularidentification of a processed type II ComRComS pheromoneof Streptococcus mutansrdquo Journal of Bacteriology vol 194 no15 pp 3781ndash3788 2012

[49] P Huang P Hu S Y Zhou Q Li and W M Chen ldquoMorininhibits sortase A and subsequent biofilm formation in strepto-coccus mutansrdquo Current Microbiology vol 68 no 1 pp 47ndash522014

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom