Deletion of gtfC of Streptococcus mutans has no influence on the composition of a mixed-species in vitro biofilm model of supragingival plaque

Glucosyltransferases from Streptococcus mutans are thought to play an important role in bacterial adherence to the tooth surface. The goal of the present study was to determine the effect of the deletion of the gtfC gene, which encodes a glucosyltransferase that catalyses primarily the formation of insoluble glucan (mutan), on colonization of S. mutans in a mixed-species biofilm model of supragingival plaque. A gtfC deletion mutant of S. mutans UA159 grew poorly in biofilms on a polystyrene surface in Todd–Hewitt medium containing sucrose, but biofilm formation in the semi-defined fluid universal medium (FUM) was not affected. The S. mutans gtfC mutant colonized with the same efficiency as the wild-type strain when grown together with five other species in a mixed-species biofilm on hydroxyapatite in a mixture of FUM and saliva with pulses of sucrose and showed the same ability to demineralize enamel in vitro . Colonization of mutant and wild-type strains was also equal in an association experiment in specific-pathogen-free rats. However, the gtfC mutant gave rise to more dentinal fissure lesions and smooth surface caries than the wild-type strain; this could be caused by a change in diffusion properties as a result of to the lack of mutan.     

Effects of xylitol on survival of mutans streptococci in mixed-six-species in vitro biofilms modelling supragingival plaque

Xylitol has been claimed to reduce mutans streptococci (MS) in dental plaque by energy-consuming futile metabolic cycles. This study aimed to investigate the effects of xylitol on MS in an in vitro 6-species oral biofilm model. Each multispecies biofilm contained either a laboratory reference strain, a fresh isolate, a xylitol-sensitive or a xylitol-resistant strain of Streptococcus mutans or Streptococcus sobrinus. Biofilms, grown on pellicle-coated hydroxyapatite discs, were fed with a glucose/sucrose-supplemented medium 3 times daily for 45 min and incubated in saliva between feedings. Before or after feeding, biofilms were exposed to either 7.5% xylitol, 7.5% sorbitol or to saliva (control) for 20 min. After 64.5 h, biofilms were harvested and the microbial composition was analysed by non-selective and selective culturing. Strain variability in the ability to colonize biofilms was observed. However, the response patterns in the biofilms to the 4 polyol treatments were similar. None of the MS were inhibited by xylitol provided either before or after feeding. Sorbitol given before feeding did not affect microbial growth whereas sorbitol provided after feeding showed a slight, albeit statistically significant increase in MS counts for some of the tested strains. It did so at the expense of Streptococcus oralis, which decreased in numbers. The present findings do not support the contention that xylitol reduces MS in plaque by futile metabolic cycles.     

Effect of arginine on the growth and biofilm formation of oral bacteria

BackgroundAlkali production via arginine deiminase system (ADS) of oral bacteria plays a significant role in oral ecology, pH homeostasis and inhibition of dental caries. ADS activity in dental plaque varies greatly between individuals, which may profoundly affect their susceptibility to caries.ObjectiveTo investigate the effect of arginine on the growth and biofilm formation of oral bacteria.Methods and resultsPolymicrobial dental biofilms derived from saliva were formed in a high-throughput active attachment biofilm model and l-arginine (Arg) was shown to reduce the colony forming units (CFU) counts of such biofilms grown for various periods or biofilms derived from saliva of subjects with different caries status. Arg hardly disturbed bacterial growth of Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus gordonii in BHI medium, but only inhibited biofilm formation of S. mutans. Scanning electron microscope (SEM) showed S. mutans biofilms harboured fewer cells grown with Arg than that without Arg, even in the initial 2 h and 8 h phase. Confocal laser scanning microscope (CLSM) images of poly-microbial dental and S. mutans biofilms revealed the biofilms grown with Arg had lower exopolysaccharide (EPS)/bacteria ratios than those without Arg (P = 0.004, 0.002, respectively). Arg could significantly reduce the production of water-insoluble EPS in S. mutans biofilms (P < 0.001); however, quantitative real-time PCR (qRT-PCR) did not show significantly influence in gene expression of gtfB, gtfC or gtfD (P = 0.32, 0.06, 0.44 respectively).ConclusionsArg could reduce the biomass of poly-microbial dental biofilms and S. mutans biofilms, which may be due to the impact of Arg on water-insoluble EPS. Considering the contribution to pH homeostasis in dental biofilms, Arg may serve as an important agent keeping oral biofilms healthy thus prevent dental caries.     

The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces

Although Candida containing biofilms contribute to the development of oral candidosis, the characteristics of multi-species Candida biofilms and how oral bacteria modulate these biofilms is poorly understood. The aim of this study was to investigate interactions between Candida albicans and either Candida glabrata or Streptococcus mutans in biofilms grown on various surfaces, with or without saliva. Hydroxyapatite (HA), polymethylmetacrylate (PMMA) and soft denture liner (SL) discs were used as substratum. Counts of viable micro-organisms in the accumulating biofilm layer were determined and converted to colony forming units per unit surface area. Confocal laser scanning microscopy was used to characterize biofilms and to quantitate the number of hyphae in each condition tested. Viable counts of C. albicans and C. glabrata per mm(2) decreased in the order HA>PMMA>SL (p<0.05). Biofilms grown on saliva-coated specimens harboured fewer C. glabrata than uncoated specimens (p<0.05). Glucose and the presence of S. mutans suppressed C. albicans hyphal formation. Dual Candida species biofilms did not show competitive interaction between the two species. We conclude that Candida biofilms are significantly affected by saliva, substratum type and by the presence of other micro-organisms.     

Differences between single- and dual-species biofilms of Streptococcus mutans and Veillonella parvula in growth, acidogenicity and susceptibility to chlorhexidine

Streptococcus mutans, considered a primary pathogen in dental caries, thrives in dental plaque, which is a multispecies biofilm. Metabolic interactions between S. mutans and Veillonella parvula have been suggested. In this study we developed a biofilm model to quantify single-species (S. mutans or V. parvula) and dual-species (S. mutans and V. parvula) biofilm formation, and we identified the differences between the respective biofilms in terms of growth, acid formation, and response to chlorhexidine. Polystyrene 96-well microtiter plates were used for biofilm formation. These biofilms were exposed to various chlorhexidine concentrations (0.025-0.4 mg ml(-1)) and treatment conditions. Growth of the biofilms and the effects of chlorhexidine were evaluated by viable counts. Viability of the two species in all biofilm types was similar ( approximately 10(8) colony-forming units per well) after 72 h. Lactic acid accumulation of dual-species biofilms was significantly lower at 48 and 72 h than single-species biofilms of S. mutans. Dual-species biofilms were less susceptible to chlorhexidine than single-species biofilms when a neutralization step was included. These results indicate that bacteria in dual-species biofilms have different properties from bacteria in single-species biofilms.     

Recalcitrance of Streptococcus mutans biofilms towards detergent-stimulated detachment

The biofilm mode of growth protects the plaque microorganisms against environmental attacks, such as from antimicrobials or detergents. Detergents have a demonstrated ability to detach initially adhering bacteria from enamel surfaces, but the ability of detergent components to detach plaque bacteria is not always obvious from in vivo experiments and reports on their clinical efficacy are inconsistent. It is likely that antimicrobials or detergents are unable to penetrate the plaque and reach the bacteria that actually link the plaque mass to the substratum surface. Attenuated total reflectance/Fourier transform infrared spectroscopy was used to measure the transport of sodium lauryl sulphate (SLS) through Streptococcus mutans HG 985 biofilms. The transport of SLS to the base of the S. mutans biofilms was not hindered, while moreover an accumulation of SLS near the base of the biofilms was found, suggesting that SLS was adsorbed to biofilm components. X-ray photoelectron spectroscopy confirmed the ability of S. mutans, grown on sucrose supplemented medium, to adsorb SLS, and simultaneously indicated that exposure of cells to SLS might lead to a loss of surface proteins. Furthermore, experiments in a parallel-plate flow chamber demonstrated that initially adhering S. mutans HG 985 could be stimulated to detach by SLS, but that, depending on the growth stage of the biofilm, only maximally 27% of biofilm bacteria could be stimulated to detach by a 4% (w/v) SLS solution.     

Proteases of an early colonizer can hinder Streptococcus mutans colonization in vitro

Streptococcus mutans is the primary cariogen that produces several virulence factors that are modulated by a competence-stimulating peptide (CSP) signaling system. In this study, we sought to determine if proteases produced by early dental plaque colonizers such as Streptococcus gordonii interfere with the subsequent colonization of S. mutans BM71 on the existing streptococcal biofilms. We demonstrated that S. mutans BM71 colonized much less efficiently in vitro on streptococcal biofilms than on Actinomyces naeslundii biofilms. Several oral streptococci, relative to A. naeslundii, produced proteases that inactivated the S. mutans CSP. We further demonstrated that cell protein extracts from S. gordonii, but not from A. naeslundii, interfered with S. mutans BM71 colonization. In addition, S. mutans BM71 colonized more efficiently on the sgc protease knockout mutant of S. gordonii than on the parent biofilms. In conclusion, proteases of early colonizers can interfere with subsequent colonization by S. mutans in vitro.     

Removal of Streptococcus mutans biofilm by bubbles

BACKGROUND: Air bubbles had been shown to remove particles and bacteria from surfaces, but they had not yet been studied regarding the removal of mature biofilm from a surface. METHODS: Streptococcus mutans were grown as a biofilm on glass coverslips and were exposed to a fluid stream with or without bubbles. Three parameters (stream velocity, gas fraction, and bubble size) were varied in the bubble stream to determine which conditions best remove the biofilm. RESULTS: At low velocities bubbles enhance biofilm removal compared with the liquid alone. Stream conditions that were shown to be the most effective in removing biofilm were large bubbles at low gas fractions. CONCLUSIONS: These results suggest that flowing bubble streams may be a desirable feature to incorporate into oral hygiene products to remove accumulated biofilms such as dental plaque.     

Selection of dairy bacterial strains as probiotics for oral health

The aim of the present study was to select bacterial strains with potential properties as oral probiotics, namely for the prevention of dental caries. We examined 23 dairy microorganisms, out of which we identified two Streptococcus thermophilus and two Lactcoccus lactis strains that were able to adhere to saliva-coated hydroxyapatite beads to the same extent as Streptococcus sobrinus OMZ176. Two of them, Strep. thermophilus NCC1561 and Lactoc. lactis ssp. lactis NCC2211, were further successfully incorporated into a biofilm mimicking the dental plaque. Furthermore, they could grow in such a biofilm together with five strains of oral bacterial species, representative of supragingival plaque. In this system, Lactoc. lactis NCC2211 was able to modulate the growth of the oral bacteria, and in particular to diminish the colonization of Streptococcus oralis OMZ607, Veillonella dispar OMZ493, Actinomyces naeslundii OMZ745 and of the cariogenic Strep. sobrinus OMZ176. These findings encourage further research with selected non-pathogenic dairy bacterial strains with the aim to decrease the cariogenic potential of dental plaque.     

Light therapy: complementary antibacterial treatment of oral biofilm

Conventional antibacterial treatment fails to eradicate biofilms associated with common infections of the oral cavity. Unlike chemical agents, which are less effective than anticipated, owing to diffusion limitations in biofilms, light is more effective on bacteria in biofilm than in suspension. Effectiveness depends also on the type and parameters of the light. We tested the phototoxic effects of non-coherent blue light (wavelengths, 400-500 nm) and CO(2) laser (wavelength, 10.6 μm), which have different mechanisms of action on the oral bacterium Streptoccocus mutans, in biofilm and on tooth enamel. Exposure of S. mutans in biofilm to blue light had a delayed effect on bacterial viability throughout the biofilm and a sustained antibacterial effect on biofilm newly formed by previously irradiated bacteria. A synergistic antibacterial effect between blue light and H(2)O(2) may enhance the phototoxic effect, which involves a photochemical mechanism mediated by reactive oxygen species (ROS) formation. The effect of CO(2) laser irradiation on the viability of S. mutans in biofilm on enamel samples appeared to be higher in the deep layers, due to heating of the enamel surface by the absorbed energy. Biofilms do not interfere with the chemical changes resulting from irradiation, which may increase the enamel's resistance to acid attack.     

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.     

Extracellular polysaccharides do not inhibit the reaction between Streptococcus mutans and its specific immunoglobulin G (IgG) or penetration of the IgG through S. mutans biofilm

The present study investigated whether extracellular polysaccharides inhibit reaction between Streptococcus mutans and its specific immunoglobulin G (IgG) and penetration of the IgG through S. mutans biofilm. The planktonic organisms with or without extracellular polysaccharides were prepared, incubated with rabbit IgG against whole cell of S. mutans and fluorescein isothiocyanate (FITC)-conjugated goat affinity purified antibody to rabbit IgG. Biofilms with or without extracellular polysaccharides were formed on cover glasses and incubated with rabbit IgG against S. mutans and FITC-conjugated goat antibody to rabbit IgG. Then, biofilms were stained with propidium iodide. The amount of specific IgG binding on S. mutans was determined by FITC intensity with a fluorescence microplate reader. The penetration of IgG through biofilms was determined by confocal laser scanning microscopy. The results showed that the fluorescence intensity of FITC in planktonic organisms with extracellular polysaccharides was similar to that in planktonic organisms without extracellular polysaccharides, indicating that extracellular polysaccharides did not inhibit the reaction between S. mutans and its specific IgG. Although biofilms of S. mutans with extracellular polysaccharides were much thicker and denser than those without extracellular polysaccharides, the speed with which IgG penetrated through both of the biofilms did not differ significantly, suggesting that penetration of IgG through S. mutans biofilm was not affected by extracellular polysaccharides.     

MPC-polymer reduces adherence and biofilm formation by oral bacteria

Oral biofilms such as dental plaque cause dental caries and periodontitis, as well as aspiration pneumonia and infectious endocarditis by translocation. Hence, the suppression of oral biofilm formation is an issue of considerable importance. Mechanical removal, disinfectants, inhibition of polysaccharide formation, and artificial sugar have been used for the reduction of oral biofilm. From the viewpoint of the inhibition of bacterial adherence, we investigated whether aqueous biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer can reduce streptococcal colonization and biofilm formation. We examined the effects of MPC-polymer on streptococcal adherence to saliva-coated hydroxyapatite and oral epithelial cells, and the adherence of Fusobacterium nucleatum to streptococcal biofilm. MPC-polymer application markedly inhibited both the adherence and biofilm formation of Streptococcus mutans on saliva-coated hydroxyapatite and streptococcal adherence to oral epithelial cells, and reduced the adherence of F. nucleatum to streptococcal biofilms. A small-scale clinical trial revealed that mouthrinsing with MPC-polymer inhibited the increase of oral bacterial numbers, especially of S. mutans. These findings suggest that MPC-polymer is a potent inhibitor of bacterial adherence and biofilm development, and may be useful to prevent dental-plaque-related diseases. (UMIN Clinical Trial Registry UMIN000003471).     

Photosensitization of in vitro biofilms by toluidine blue O combined with a light-emitting diode

In natural ecosystems, micro-organisms grow preferentially attached to surfaces, forming matrix-enclosed biofilms. The aim of this study was to determine photodestruction levels in biofilms after subjecting them to photodynamic therapy. Biofilms of Streptococcus mutans, S. sobrinus, and S. sanguinis were grown on enamel slabs for 3, 5 or 7 d. Both the number of viable micro-organisms and the concentration of water-insoluble polysaccharides were analysed, and mineral loss (DeltaZ) analyses were performed on the enamel slabs. The antimicrobial potential of toluidine blue O (0.1 mg ml(-1)), associated with 85.7 J cm(-2) of a light-emission diode, was evaluated on the viability of 5-d biofilms. Both the number of micro-organisms and the concentration of water-insoluble polysaccharide increased with the age of the biofilms. A significant reduction ( approximately 95%) in viability was observed for S. mutans and S. sobrinus biofilms following photosensitization, with a > 99.9% reduction in the viability of S. sanguinis biofilms. In conclusion, a biofilm model was shown to be suitable for studying changes in bacterial numbers and enamel mineralization and for demonstrating the potential value of photosensitization in the control of in vitro biofilms.     

改良恒化器中牙周致病菌和致龋菌的激光共聚焦显微镜动态观察

目的　模拟口腔条件下研究牙周致病菌和致龋菌的动态关系。方法　选用牙龈卟啉单胞菌、伴放线放线杆菌、具核梭杆菌、中间普氏菌、变形链球菌、血链球菌、黏性放线菌和嗜酸乳杆菌8种牙周致病菌和致龋菌。按分组接种于模拟口腔的改良恒化器中 ,连续培养 1、2 4、4 8和 96h ,活菌革兰荧光染色与激光共聚焦显微镜结合测量羟基磷灰石圆片表面生物膜厚度 ,连续断层扫描及三维重建。结果　随时间变化各组生物膜厚度均显著增加 (P <0 0 0 1) ;同一时间点血链球菌生物膜明显厚于伴放线放线杆菌 ,8种菌明显比血链球菌和伴放线放线杆菌形成的生物膜厚。三维重建显示 ,G-牙周致病菌主要分布于G 致龋菌菌团中或膜表层。结论　人工菌斑生物膜中G 致龋菌首先定植 ,G -牙周致病菌的量和比例随时间增加 ;牙周致病菌和致龋菌的生态平衡与疾病之间的关系值得深入研究     

人工口腔牙菌斑生物膜药敏实验模型的建立

目的 :设计一种新的口腔保健品体外药敏实验模型。方法 :以瑞典LKB2 0 2 3液相层析系统的恒温操作箱为基础 ,建立一种新的口腔保健品体外药敏实验模型。以茶多酚、鞣酸、氟化钠为实验药物 ,比较了纸片法、琼脂稀释法和液体稀释法的药敏 ,再以人工口腔牙菌斑生物法比较这些药品对变形链球菌生物膜的敏感性。结果 :纸片法抑菌实验结果全部阴性 ,琼脂稀释法和液体稀释法比较 ,液体稀释法比较敏感。而生物膜法则需要较高浓度的药品才能起到抑菌作用 ,与液体稀释法相比 ,对于NaF两者相差 3 2倍 ,对于茶多酚和鞣酸相差 16倍。结论 :人工口腔牙菌斑生物膜药敏实验模型是一种较接近人口腔状况保健品基础实验模型     

哈茨木霉菌非水溶性葡聚糖酶的理化及生物学性质研究

目的研究源于哈茨木霉菌的非水溶性葡聚糖酶的理化性质，及其对口腔链球菌黏附和人工生物膜结构的影响。方法比较6种木霉菌产生非水溶性葡聚糖酶的能力，选出酶活性最高的菌株；确定该酶最适pH值和反应温度；RT—PCR扩增酶基因，连T载体，测序。通过黏附试验检测非水溶性葡聚糖酶抑制链球菌黏附的效果，并应用激光共聚焦显微镜观察其对人工生物膜结构的影响。结果哈茨木霉菌Th1产生的非水溶性葡聚糖酶活性最高，其反应最适pH值为5．5，温度40℃；基因序列与GenBank中的已知序列同源性达到92％。非水溶性葡聚糖酶能显著抑制链球菌的黏附；与未加酶组相比，加酶组生物膜高度降低，结构松散。结论哈茨木霉菌Th1产生的非水溶性葡聚糖酶能明显抑制细菌的黏附，影响生物膜的形成。     

Effect of Weissella cibaria isolates on the formation of Streptococcus mutans biofilm

The objective of this study was to isolate and identify lactic acid bacteria able to inhibit the in vitro formation of Streptococcus mutans biofilm as well as the in vivo formation of oral biofilm. Two strains, CMS1 and CMS3, exhibiting profound inhibitory effects on the formation of S. mutans biofilm and the proliferation of S. mutans, were isolated from children's saliva and identified as Weissella cibaria by 16S rDNA sequencing. The water-soluble polymers produced from sucrose by the W. cibaria isolates also inhibited the formation of S. mutans biofilm. According to the results of thin-layer chromatographic analysis, the hydrolysates of water-soluble polymers produced by the isolates were identical to those of dextran, forming mostly alpha-(1-6) glucose linkages. In the clinical study, the subjects mouthrinsed with a solution containing W. cibaria CMS1 evidenced plaque index reduction of approximately 20.7% (p < 0.001). These results indicate that the W. cibaria isolates possess the ability to inhibit biofilm formation, both in vitro and in vivo.     

Virulence properties of cariogenic bacteria

The importance of Streptococcus mutans in the etiology of dental caries has been well documented. However, there is growing recognition that the cariogenic potential of dental plaque may be determined by the composite interactions of the total plaque bacteria rather than solely the virulence properties of a single organism. This study will examine how the interactions of S. mutans with other biofilm constituents may influence the cariogenicity of plaque samples. In order to begin to investigate the effects of nonmutans streptococci on the cariogenic potential of S. mutans, we have examined the effects of Streptococcus gordonii on the virulence properties of the former organisms. These studies have indicated that S.gordonii can attenuate several potential virulence properties of S. mutans including bacteriocin production, genetic transformation, and biofilm formation. Therefore, modulation of the interactions between plaque bacteria might be a novel approach for attenuating dental caries initiation.