变异链球菌LuxS基因缺陷突变株的构建及其生物膜结构的初步观察研究

目的:通过同源重组法构建变异链球菌LuxS基因缺陷突变菌株,比较其与变异链球菌UA159标准株在生物膜形成上的差异。方法:运用基因同源重组方法将氯霉素抗性基因(Cmr)与LuxS基因上下游区域的2个基因片段按一定顺序重组到PUC载体的多克隆位点中,构建出带氯霉素抗性标志的重组质粒,将载体质粒转化到含完整LuxS基因的变异链球菌UA159中,利用氯霉素抗性筛选出LuxS基因缺陷的突变株,并利用聚合酶链式反应(PCR)和哈氏弧菌发光实验进行检测。以釉质磨片为载体,在扫描电镜下观察上述两菌株含有20 g/L葡萄糖、20 g/L蔗糖的乳酪消化胨酵母(TPY)液体培养基中形成24 h的生物膜。结果:PCR基因扩增结果显示:突变株LuxS基因已被Cmr基因完全替换,成功的构建了变异链球菌LuxS基因突变株,并且突变株不能诱导哈氏弧菌BB170的生物发光。当用葡萄糖作为补充糖源时,变异链球菌UA159标准株和LuxS基因突变株所形成的生物膜表现型未见明显差异;而用蔗糖作为补充糖源时,两菌株所形成的生物膜有明显不同,UA159生物膜相对平滑均质,而LuxS基因突变株生物膜呈松散的蜂房状,基质间有较大的间隙,形成较大的团簇状菌落。结论:成功构建出LuxS基因缺陷的变异链球菌突变株,与变异链球菌UA159标准株其生物膜结构发生改变,提示LuxS会影响变异链球菌生物膜的形成。     

变异链球菌luxS基因对牙菌斑生物膜形成的影响研究

目的敲除变异链球菌中的luxS基因,构建luxS突变株,测试变异链球菌失去luxS基因后形成牙菌斑生物膜的能力。方法把luxS基因敲除重组质粒转入变异链球菌UA159中,得到转化菌,在含有卡拉霉素的培养基中进行筛选,得到变异链球菌luxS基因突变株,并利用聚合酶链式反应（PCR）和哈氏弧菌发光实验对变异链球菌luxS突变株进行检测,通过扫描电镜,对不同时间段变异链球菌luxS突变株和变异链球菌UA159在BHIS培养液（含1%蔗糖的脑心浸液）和BHIG培养液（含1%葡萄糖的脑心浸液）中形成的生物膜进行比较。结果成功的构建了变异链球菌luxS突变株,在BHIS培养液中,变异链球菌luxS突变株形成生物膜的能力比变异链球菌UA159弱,而在BHIG培养液中,二者无显著差异。结论变异链球菌luxS基因对牙菌斑生物膜的形成有重要的影响,并且luxS基因对变异链球菌生物膜的调控是蔗糖依赖型的。     

Role of sucrose in the fitness of Streptococcus mutans

Introduction:  Dental caries has been closely linked to fermentable carbohydrates as key environmental factors. Sucrose has been identified as the most cariogenic carbohydrate. Streptococcus mutans , considered to be the primary pathogen causing dental caries, is able to utilize sucrose as a nutrient source, partially for the production of intracellular storage components and for the production of extracellular glucans via the glucosyltransferases GtfB, GtfC, and GtfD. The following study explores the competitiveness and fitness of S. mutans when grown with different concentrations of sucrose.
Methods:  Growth competition with oral streptococci and antimicrobial susceptibility in static biofilm models grown without sucrose or with 0.1% or 0.5% sucrose were investigated using confocal laser scanning microscopy. The numbers of surviving S. mutans of both wild-type and an isogenic Gtf-negative mutant after antimicrobial treatment were determined as colony-forming units.
Results:  S. mutans was able to establish microcolonies with increasing sucrose concentration in the presence of other streptococcal competitors during biofilm development. The antimicrobial susceptibility decreased when sucrose was available as substrate and was dependent on the presence of the Gtfs.
Conclusion:  The increased resistance against antimicrobial treatment was associated with the availability of sucrose, but was not influenced much by the concentration used during this study. The resistance was strongly associated with the Gtf activity, excluding any intracellular metabolic effect of sucrose in the resistance mechanism.     

The cariogenic characters of xylitol-resistant and xylitol-sensitive Streptococcus mutans in biofilm formation with salivary bacteria

Streptococcus mutans metabolize carbohydrates, such as glucose and sucrose, to produce acid and enhance biofilm formation with the early colonizing bacteria to induce dental caries. Xylitol has been used as a reliable substitute for carbohydrate to inhibit the acid production of S. mutans. However, long-term xylitol consumption leads to the emergence of xylitol-resistance in S. mutans. The aim of this study was to investigate the cariogenic trait of Xylitol-resistant (X(R)) S. mutans using biofilm formation and coaggregation of xylitol-sensitive (X(S)) and X(R) S. mutans with salivary bacteria and their glucosyltransferases expression. When X(S) or X(R) S. mutans were incubated in brain heart infusion broth with bacteria from human saliva, X(R)S. mutans exhibited reduction in biofilm formation in comparison to X(S) S. mutans. The coaggregation between X(R) S. mutans and S, gordonii, S. mitis, S. oralis or S. sanguinis was less pronounced than that of X(S) S. mutans in the presence of sucrose. However, there was no difference in the coaggregation between X(R) and X(S) S. mutans in the sucrose-free condition. The level of gtfB and gtfC mRNA expression of X(R) S. mutans was lower than that of X(S) S. mutans, whilst the level of gtfD mRNA expression did not differ between the two strains. The reduction of biofilm formation in X(S) S. mutans due to decrease in glucosyltransferases expression suggests that X(R) S. mutans may be less cariogenic than X(S) S. mutans.     

变异链球菌gtfs在不同pH条件下表达的差异性

目的检测具有不同产胞外多糖（EPS）能力的变异链球菌菌株在不同pH条件下产EPS的能力及其与gtfA、gtfB、gtfC、gtfD表达变化的关系。方法选取变异链球菌（血清型c）临床分离株502（高产糖株）和参考株UA159（低产糖株）,以实时定量逆转录聚合酶链反应（real-time RT-PCR）方法检测在不同pH条件下与变异链球菌产糖能力密切相关的毒力因子gtfA、gtfB、gtfC、gtfD的表达变化。结果在pH5.5条件下,高产糖株和低产糖株gtfA、gtfB、gtfD的表达均有不同程度的升高,而gtfC略降低,高产糖株gtfB、gtfC的表达水平高于低产糖株。结论gtfs的表达水平与变异链球菌的致龋力密切相关。     

The effects of orthodontic bonding steps on biofilm formation of Streptococcus mutans in the presence of saliva

Abstract Objective. To investigate the effects of various orthodontic bonding steps on biofilm formation of Streptococcus mutans in the presence of saliva. Materials and methods: Hydroxyapatite (HA) and orthodontic adhesive (AD) disks were prepared to a uniform size. HA disks were etched with 37% phosphoric acid gel in the etched group (HE). In the primed group (HP), Transbond XT primer was applied to the etched HA surface and light-cured. For biofilm formation, Streptococcus mutans was grown on each specimen in a biofilm medium with either glucose or sucrose in the presence of fluid-phase UWS (F-UWS) or surface adsorbed saliva (S-UWS). The adherent bacteria were quantified by enumeration of the total viable counts of bacteria. Biofilms formed on each surface were examined by scanning electron microscopy. Results. When glucose was used, both F-UWS and S-UWS suppressed biofilm formation of S. mutans. Compared to HA and HE, biofilm formation was significantly inhibited on HP and AD in the presence of glucose. Biofilm-forming patterns that were inhibited by saliva were restored in a sucrose-containing medium. F-UWS promoted biofilm formation on HA and HE, while S-UWS significantly promoted biofilm formation on HP. S. mutans developed biofilm better on HA and HE than on AD when sucrose was used as the sole carbohydrate source. Conclusions. This study suggests that the biofilm development by S. mutans is significantly influenced by the orthodontic bonding procedure. Biofilm formation of S. mutans was inhibited on AD more than other surfaces, irrespective of the presence of saliva or a carbohydrate source.     

Role of phosphoglucosamine mutase on virulence properties of Streptococcus mutans

Introduction:  Streptococcus mutans has been strongly implicated as the principal etiological agent in dental caries. As a gram-positive bacterium, S. mutans has a thick and compact cell wall to maintain the cell shape and protect the cells against mechanical or osmotic damage. Previous studies have proved that peptidoglycan is the main component of the cell wall involved in the autolysis or biofilm formation processes.
Methods:  In this study, we investigated the gene SMU.1426c in the amino-sugar metabolism pathway of S. mutans UA159, which encodes phosphoglucosamine mutase (GlmM). The glmM gene that functions in the biosynthesis of peptidoglycan has been well investigated in Escherichia coli . Here a glmM mutant strain of S. mutans UA159 was constructed and several virulence properties were investigated.
Results:  The mutant devoid of the glmM gene displayed long chains, reduced growth rate and increased autolysis. Biofilm formation by the mutant was found to be attenuated.
Conclusion:  These results proved that peptidoglycan biosynthesis plays an important part in a series of bacterial morphologies. The glmM gene may have a constructive role in the virulence properties of S. mutans .     

变形链球菌luxS基因缺失对生物膜早期形成的影响

目的 利用变形链球菌luxS基因敲除突变株,研究这一种属间密度感应系统对生物膜早期形成的影响.方法通过在生物膜培养悬液中加入与菌细胞直径相近的磁性小珠,利用这些小珠在磁场中受到生物膜的位移约束力的原理,采用生物膜定量分析仪,定量比较、分析变形链球菌luxS基因knockout突变株与野生株在生物膜形成上的差异.结果变形链球菌luxS基因突变株与野生株在生物膜形成模式上有显著差别,突变株生物膜自第6小时起开始形成,生物膜形成指数(biofilm index,BFI)差值ABFI=2.015,约第10小时突变株形成的生物膜可完全限制磁珠在磁场中的位移(ABFI=7.025);而野生株生物膜约第10小时开始形成(ABFI=1.875),明显晚于突变株.12h后两菌株生物膜形成未见明显差异(P>0.05).结论变形链球菌luxS基因的缺失可影响生物膜的早期形成.     

Real-time monitoring of Streptococcus mutans biofilm formation using a quartz crystal microbalance

The ability of Streptococcus mutans, a well-known etiological agent in dental caries, to attach and form a biofilm is an important key to its virulence. The effects of various environmental factors (i.e. sucrose concentration, flow rate and temperature as well as genetic manipulations) on the capability of S. mutans (UA 140) to attach, form and detach were monitored in situ using quartz crystal microbalance. The biofilm growth rate was much slower than that of planktonic growth. Greater availability of sucrose contributed to biofilms with less lag time, lower doubling times and earlier detachment. Flow rate experiments showed that as the shear stress was reduced, the maximum mass accumulated also decreased. However, the detachment process was independent of shear force, perhaps indicative of quorum sensing. Increasing the incubation temperature from 37 to 40 degrees C extended the lag period and inhibited the ability of the biofilm to attach readily. Absence of either the ciaH, luxS, gtfB or gtfC genes also greatly affected the ability of the S. mutans to adhere to a surface in comparison to the wild type. Quartz crystal microbalance results indicate that the gtfC gene possibly has a greater contribution to biofilm attachment than the gtfB gene, that the presence of the luxS gene is critical for attachment and that the ciaH gene primarily affects the initial reversible attachment of the biofilm.     

变异链球菌luxS基因对多糖基质代谢的影响

目的:利用变异链球菌luxS基因敲除的突变株,研究该基因缺陷对于生物膜多糖基质的影响。方法:通过向生物膜培养悬液中加入与细菌直径相近的磁性小珠,利用这些小珠由于生物膜约束而在磁场中位移受限的原理,采用生物膜定量分析仪,定量比较luxS突变株与野生株在利用外源性碳水化合物合成生物膜多糖基质能力方面的差异。采用SPSS 10.0软件包对实验数据,进行Dunnet双侧t检验。结果:变异链球菌luxS基因突变株与野生株均可利用碳水化合物合成胞外多糖基质,且外源性糖的加入可显著促进生物膜的形成。在加入1%蔗糖时,2菌株生物膜均在1h内迅速形成,而两者之间无显著差异。在加入1%葡萄糖时,两菌株的生物膜形成速度均有所加快,突变株的改变则更为明显。结论:luxS基因参与调节细菌对多糖基质代谢的过程,而其对于生物膜形成的影响也更多是通过调节多糖基质代谢实现的。     

LuxS基因缺失对变异链球菌生物学性状的影响

目的:观察LuxS基因突变对变异链球菌生物性状的影响。方法:将变链菌标准株和LuxS突变株分别培养于TSA、TSA-Cmr、BHI培养基培养48 h,通过菌落形态观察、常规生化检测、革兰染色涂片观察和生长曲线,比较两种菌株的生长特性;体外建立LuxS基因突变株和标准株的生物被膜模型,结晶紫染色,观察比较两菌株形成生物被膜的能力。结果:在含氯霉素的TSA-Cmr固体培养基中增塑,标准株基本不能生长,而突变株的生长状况基本正常。在不含氯霉素的TSA固体培养基中培养,两种菌株的菌落形态没有明显差别,革兰染色镜下观察,可见标准株菌体呈长链状排列且相互缠绕,而突变株菌体多呈短链状排列,形成长链的较少。生长曲线观察发现:两菌株在生长模式上基本一致,均呈典型的"S"型曲线,只是在进入生长的稳定期后,两者在细菌饱和度上有一定差异,即11.5～22.5 h之间各时间点标准株的A值均明显高于LuxS突变株(P<0.05)。在BHI培养基中两菌株均能形成生物被膜,但结构存在差异:标准株形成光滑且均匀分布的生物被膜,而突变株的生物被膜形态较粗糙。结论:LuxS基因突变对变异链球菌的生长以及生物被膜形成能力等生物学性状有一定影响。     

Functional profiling in Streptococcus mutans: construction and examination of a genomic collection of gene deletion mutants

A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene‐specific barcoded deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non‐essential genes. As S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out‐compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared with the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose‐ or sucrose‐containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co‐cultures of deletion strains were grown either in vitro in a chemostat to steady‐state values of pH 7 and pH 5 or in vivo in an animal model for oral infection. Taken together, these data represent a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.     

The adaptive response of Streptococcus mutans towards oral care products: involvement of the ClpP serine protease

In the oral cavity a balanced physiological response is essential for Streptococcus mutans to survive various types of external challenges. In this study we examined the role of the ClpP serine protease in the response of S. mutans towards sodium fluoride, sodium chloride, hydrogen peroxide, and chlorhexidine. By constructing a clpP promoter-green fluorescent protein reporter strain, we showed increased fluorescence intensities under all types of stress, indicating a need for ClpP under all these challenges. We constructed a clpP knockout mutant, which proved to be more sensitive to all the challenges than the wild-type strain. This knockout strain also displayed a reduced growth rate, hyperaggregation, and increased biofilm formation. Furthermore, an increased resistance to toxic levels of hydrogen peroxide and chlorhexidine after pre-incubation with sublethal levels of the corresponding compounds was found in the wild-type strain but not in the knockout mutant. In conclusion, ClpP is involved in the general stress response of S. mutans and assists the bacteria to resist killing through adaptation.     

Identification of a functional capsule locus in Streptococcus mitis

The polysaccharide capsule of Streptococcus pneumoniae is a hallmark for virulence in humans. In its close relative Streptococcus mitis, a common human commensal, analysis of the sequenced genomes of six strains revealed the presence of a putative capsule locus in four of them. We constructed an isogenic S. mitis mutant from the type strain that lacked the 19 open reading frames in the capsule locus (Δcps mutant), using a deletion strategy similar to previous capsule functional studies in S. pneumoniae. Transmission electron microscopy and atomic force microscopy revealed a capsule‐like structure in the S. mitis type strain that was absent or reduced in the Δcps mutant. Since S. mitis are predominant oral colonizers of tooth surfaces, we addressed the relevance of the capsule locus for the S. mitis overall surface properties, autoaggregation and biofilm formation. The capsule deletion resulted in a mutant with approximately two‐fold increase in hydrophobicity. Binding to the Stains‐all cationic dye was reduced by 40%, suggesting a reduction in the overall negative surface charge of the mutant. The mutant exhibited also increased autoaggregation in coaggregation buffer, and up to six‐fold increase in biofilm levels. The results suggested that the capsule locus is associated with production of a capsule‐like structure in S. mitis and indicated that the S. mitis capsule‐like structure may confer surface attributes similar to those associated with the capsule in S. pneumoniae.     

The Effects of Surface Roughness of Composite Resin on Biofilm Formation of Streptococcus mutans in the Presence of Saliva

SUMMARY The purpose of this study was to investigate the effects of surface roughness of resin composite on biofilm formation of Streptococcus mutans in the presence of saliva. To provide uniform surface roughness on composites, disks were prepared by curing composite against 400-grit silicon carbide paper (SR400), 800-grit silicon carbide paper (SR800), or a glass slide (SRGlass). The surface roughness was examined using confocal laser microscopy. For biofilm formation, S. mutans was grown for 24 hours with each disk in a biofilm medium with either glucose or sucrose in the presence of fluid-phase or surface-adsorbed saliva. The adherent bacteria were quantified via enumeration of the total viable counts of bacteria. Biofilms were examined using scanning electron microscopy. This study showed that SR400 had deeper and larger, but fewer depressions than SR800. Compared to SRGlass and SR800, biofilm formation was significantly increased on SR400. In addition, the differences in the effect of surface roughness on the amount of biofilm formation were not significantly influenced by either the presence of saliva or the carbohydrate source. Considering that similar differences in surface roughness were observed between SR400 and SR800 and between SR800 and SRGlass, this study suggests that surface topography (size and depth of depressions) may play a more important role than surface roughness in biofilm formation of S. mutans .     

Effects of oral streptococci on biofilm formation by cariogenic bacteria in dual species cultures

The effects of oral commensal streptococci (Streptococcus sanguinis, Streptococcus gordonii, Streptococcus mitis, and Streptococcus salivarius) on biofilm formation by cariogenic mutans streptococci (Streptococcus mutans and Streptococcus sobrinus) were investigated. Cell suspensions were cultured on 96-well microtiter plates coated with or without salivary components (SC), and in flow cell systems coated with SC in tryptic soy broth including 0.25% sucrose without dextrose (TSB). The resultant biofilm formations were stained using safranin or a LIVE/DEAD BacLight Viability Kit, and examined with absorbance at 492 nm or using confocal laser scanning microscopy. Mutans streptococci and S. sanguinis biofilms were formed significantly on the polystyrene surfaces in TSB. Further, in combination cultures, S. sanguinis formed a sufficient biofilm when cultured with S. mutans. However, when S. sanguinis was cultured with S. sobrinus, biofilm formation was slightly inhibited. S. gordonii also inhibited biofilm formation in the culture with S. sobrinus, but not when cultured with S. mutans. S. mitis and S. salivarius collapsed the biofilm morphology and inhibited volume development in some conditions when cultured with S. mutans or S. sobrinus. Biofilm formation by mutans streptococci was challenged and collapsed on the whole by culturing with each of the other oral streptococci. These results indicate that co-culturing of multiple species of mutans streptococci and other oral streptococci has physical effects related to previous attachment and colonization on the surface, as well as biological effects to regulate biofilm formation.     

变形链球菌luxS基因突变对口腔混合细菌生物膜的影响

目的 研究致龋菌变形链球菌luxS基因在口腔细菌混合培养形成牙菌斑生物膜中的作用。方法 将变形链球菌野生株(UA159)及其2种luxS基因突变株（luxS基因高表达株和luxS基因缺陷株）分别与口腔细菌嗜酸乳杆菌（ATCC4356）按照1∶1比例接种于牛心脑浸液培养基,体外混合培养不同时间,包括生物膜形成过程中的初期（4 h）、中期（14 h）、晚期（24 h）,通过MTT法检测混合菌在生物膜形成的量。通过激光共聚焦显微镜观察混合细菌24 h形成的生物膜结构,实时定量PCR检测变形链球菌相关基因（ftf, smu630, brpA, gbpB, gtfB, vicR, comDE和relA）的表达。采用SPSS17.0软件包对数据进行统计学分析。结果 变形链球菌野生株及其2种luxS基因突变株与嗜酸乳杆菌混合培养14 h后,生物膜的量分别为0.481±0.024、0.591±0.023和0.279±0.019;24 h后,混合细菌形成生物膜的量趋势与该时间点一致,变形链球菌高表达株高于野生株,而缺陷株明显降低;但4 h后形成的生物膜组间无显著差异。激光共聚焦显微镜结果表明,高表达株和野生株的集聚程度更高,形成生物膜的结构更加紧密;而缺陷株生物膜菌间结构比较稀疏。以变形链球菌野生株和嗜酸乳杆菌混合形成的生物膜中相关基因的表达为标准,高表达株相关基因的表达均增加,缺陷株表达均降低,且各组间存在显著差异（P<0.05）。结论 变形链球菌luxS基因影响与具核梭杆菌混合培养形成的牙菌斑生物膜,为进一步研究该基因在生物膜中的作用及其调控机制提供了依据。     

蔗糖环境对寡发酵链球菌与变异链球菌双菌种生物膜形成的影响

目的探讨蔗糖环境对寡发酵链球菌与变异链球菌双菌种生物膜形成的影响,并与血链球菌和变异链球菌的双菌种生物膜形成进行比较。方法运用菌落计数法观察蔗糖环境下唾液包被的玻璃生物模型中单/双菌株寡发酵链球菌,变异链球菌和血链球菌24 h生物膜形成情况;运用激光共聚焦显微镜观察蔗糖环境下寡发酵链球菌,变异链球菌和血链球菌单/双菌株生物模型中24h生物膜形成厚度。结果无糖环境下,单菌株模型中,菌落数:血链球菌(55.67±5.36)>变异链球菌(53.48±2.63)(P>0.05)>寡发酵链球菌(46.24±2.34)(P<0.05);生物膜厚度:血链球菌(17.23±3.82)>变异链球菌(15.16±4.21)(P>0.05)>寡发酵链球菌(10.54±4.37)(P<0.05)。双菌株模型中,变异链球菌菌落数降低幅度:血链球菌组>寡发酵链球菌组(P<0.05)。生物膜厚度:血链球菌组(8.12±2.82)<寡发酵链球菌组(11.27±3.55)(P<0.05)。蔗糖环境下,单菌株模型中,菌落数:变异链球菌(58.54±2.74)>血链球菌(51.87±5.35)>寡发酵链球菌(48.57±3.05)(P<0.05)生物膜厚度:变异链球菌(20.63±5.71)>血链球菌(13.37±4.93)>寡发酵链球菌(12.45±4.62)(P<0.05)双菌株模型中;变异链球菌菌落数降低幅度,寡发酵链球菌组>血链球菌组(P<0.05),生物膜厚度:寡发酵链球菌组(6.67±2.19)<血链球菌组(10.45±2.72)(P<0.05)。结论外界糖环境影响寡发酵链球菌和血链球菌对变异链球菌的抑制作用,蔗糖环境下,寡发酵链球菌的抑制作用强于血链球菌。     

Effects of Streptococcus mutans gtfC deficiency on mixed oral biofilms in vitro

The aim of this study was to examine the influence of glucosyltransferase-gene-negative (gtf-) Streptococcus mutans strains unable to synthesize water-insoluble or soluble glucan on the structure and macromolecular diffusion properties of in vitro grown mixed oral biofilms. Biofilms modeling supragingival plaque consisted of Actinomyces naeslundii OMZ 745, Candida albicans OMZ 110, Fusobacterium nucleatum KP-F2, Streptococcus oralis SK 248, Veillonella dispar ATCC 17748T and one of the S. mutans strains UA159, OMZ 966, OMZ 937 or OMZ 977. Biofilms were grown anaerobically on sintered hydroxyapatite disks for 64.5 h at 37 degrees C. To perform confocal laser scanning microscopy analyses, microorganisms were stained with Syto 13 and extracellular polysaccharides (EPS) with Calcofluor. Macromolecular diffusion properties were measured following timed biofilm exposure to Texas-Red-labeled 70-kDa dextran. Results showed that replacing wild-type S. mutans by a gtfC- mutant led to an increase in the volume fraction occupied by cells from 29 to 48% and a decrease of the EPS volume fraction from 51 to 33%. No such changes were observed when the S. mutans wild-type strain was replaced by a gtfB- or gtfD- mutant. The diffusion coefficient of 70-kDa dextran in biofilms containing the gtfC- S. mutans was 16-fold higher than in biofilms with the wild-type strain indicating a strong macromolecular sieving effect of GTF C-generated glucans. Our data demonstrate the influence of EPS on the structure and macromolecular diffusion properties of an oral biofilm model and uncover our still limited knowledge of the function of EPS in biofilms and plaque.