Inhibition of Streptococcus mutans biofilm accumulation and development of dental caries in vivo by 7-epiclusianone and fluoride

7-Epiclusianone (7-epi), a novel naturally occurring compound isolated from Rheedia brasiliensis, effectively inhibits the synthesis of exopolymers and biofilm formation by Streptococcus mutans. In the present study, the ability of 7-epi, alone or in combination with fluoride (F), to disrupt biofilm development and pathogenicity of S. mutans in vivo was examined using a rodent model of dental caries. Treatment (twice-daily, 60s exposure) with 7-epi, alone or in combination with 125 ppm F, resulted in biofilms with less biomass and fewer insoluble glucans than did those treated with vehicle-control, and they also displayed significant cariostatic effects in vivo (p < 0.05). The combination 7-epi + 125 ppm F was as effective as 250 ppm F (positive-control) in reducing the development of both smooth- and sulcal-caries. No histopathological alterations were observed in the animals after the experimental period. The data show that 7-epiclusianone is a novel and effective antibiofilm/anticaries agent, which may enhance the cariostatic properties of fluoride.     

Enhancement of fluoride activity against Streptococcus mutans biofilms by a substance separated from Polygonum cuspidatum

Polygonum cuspidatum is a plant with spreading rhizomes and numerous reddish-brown stems that has been used in Korean folk medicine to improve oral hygiene. Nevertheless, there are no reports related to its possible effect on the virulence of dental biofilms. In this study, the ability of a fraction (F1) separated from P. cuspidatum, alone or in combination with fluoride, to disrupt virulence factors and the composition of Streptococcus mutans biofilms was examined. F1 was mainly composed of resveratrol, emodin and physcion (approximately 16.2%, 18.9% and 2.07% of the weight of F1, respectively). F1 showed inhibitory effects on acid production and F-ATPase activity of S. mutans in biofilms, and could enhance fluoride activity against acid production and acid tolerance of S. mutans in biofilms. When S. mutans biofilms were briefly treated with F1 (10 min, a total of five times), the biomass accumulation, water-insoluble polysaccharides and intracellular iodophilic polysaccharides were reduced. Furthermore, the fluoride activity against biomass accumulation was enhanced by F1. These results suggest that F1 may be useful in the control of dental biofilms and in improving the cariostatic properties of fluoride without increasing its exposure.     

Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ?lytS and ?lytT; LTA – ?dltA and ?dltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.     

Effects of combined oleic acid and fluoride at sub-MIC levels on EPS formation and viability of Streptococcus mutans UA159 biofilms

Despite the widespread use of fluoride, dental caries, a biofilm-related disease, remains an important health problem. This study investigated whether oleic acid, a monounsaturated fatty acid, can enhance the effect of fluoride on extracellular polysaccharide (EPS) formation by Streptococcus mutans UA159 biofilms at sub-minimum inhibitory concentration levels, via microbiological and biochemical methods, confocal fluorescence microscopy, and real-time PCR. The combination of oleic acid with fluoride inhibited EPS formation more strongly than did fluoride or oleic acid alone. The superior inhibition of EPS formation was due to the combination of the inhibitory effects of oleic acid and fluoride against glucosyltransferases (GTFs) and GTF-related gene (gtfB, gtfC, and gtfD) expression, respectively. In addition, the combination of oleic acid with fluoride altered the bacterial biovolume of the biofilms without bactericidal activity. These results suggest that oleic acid may be useful for enhancing fluoride inhibition of EPS formation by S. mutans biofilms, without killing the bacterium.     

Diacetylcurcumin: a new photosensitizer for antimicrobial photodynamic therapy in Streptococcus mutans biofilms

This study evaluated the effect of antimicrobial photodynamic therapy (aPDT) on S. mutans using diacetylcurcumin (DAC) and verified DAC toxicity. In vitro, S. mutans biofilms were exposed to curcumin (CUR) and DAC and were light-irradiated. Biofilms were collected, plated and incubated for colony counts. DAC and CUR toxicity assays were conducted with Human Gingival Fibroblast cells (HGF). In vivo, G. mellonella larvae were injected with S. mutans and treated with DAC, CUR and aPDT. The hemolymph was plated and incubated for colony counts. Significant reductions were observed when DAC and CUR alone were used and when aPDT was applied. HGF assays demonstrated no differences in cell viability for most groups. DAC and CUR reduced the S. mutans load in G. mellonella larvae both alone and with aPDT. Systematic toxicity assays on G. mellonella demonstrated no effect of DAC and CUR or aPDT on the survival curve.     

Potential mechanisms for the effects of tea extracts on the attachment,biofilm formation and cell size of Streptococcus mutans

Tea can inhibit the attachment of Streptococcus mutans to surfaces and subsequent biofilm formation. Five commercial tea extracts were screened for their ability to inhibit attachment and biofilm formation by two strains of S. mutans on glass and hydroxyapatite surfaces. The mechanisms of these effects were investigated using scanning electron microscopy (SEM) and phytochemical screening. The results indicated that extracts of oolong tea most effectively inhibited attachment and extracts of pu-erh tea most effectively inhibited biofilm formation. SEM images showed that the S. mutans cells treated with extracts of oolong tea, or grown in medium containing extracts of pu-erh tea, were coated with tea components and were larger with more rounded shapes. The coatings on the cells consisted of flavonoids, tannins and indolic compounds. The ratio of tannins to simple phenolics in each of the coating samples was ～3:1. This study suggests potential mechanisms by which tea components may inhibit the attachment and subsequent biofilm formation of S. mutans on tooth surfaces, such as modification of cell surface properties and blocking of the activity of proteins and the structures used by the bacteria to interact with surfaces.     

Co-operative inhibition by fluoride and zinc of glucosyl transferase production and polysaccharide synthesis by mutans streptococci in suspension cultures and biofilms

Fluoride and zinc, alone or in combination at concentrations of 0.2 mM, inhibited production-secretion of glucosyltranferases by Streptococcus mutans UA159 growing in suspension cultures. Inhibition did not involve growth inhibition or starvation. Fluoride and zinc also inhibited glucan production, especially insoluble glucan, in fed-batch biofilms. Inhibition of biofilms appeared to be associated with starvation as indicated by markedly decreased ATP pools and iodophilic polysaccharide levels in biofilm cells. As insoluble glucans are important for virulence of mutans streptococci, the inhibitory actions of fluoride and zinc could significantly affect cariogenicity.     

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis

Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.     

In vitro activity of xanthorrhizol against Streptococcus mutans biofilms

AIMS: We determined the effect of xanthorrhizol (XTZ) purified from the rhizome of Curcuma xanthorrhiza Roxb. on the Streptococcus mutans biofilms in vitro. METHODS AND RESULTS: The biofilms of S. mutans at different phases of growth were exposed to XTZ at different concentrations (5, 10 and 50 micromol l(-1)) and for different time exposures (1, 10, 30 and 60 min). The results demonstrated that the activity of XTZ in removing S. mutans biofilm was dependent on the concentration, exposure time and the phase growth of biofilm. A concentration of 5 micromol l(-1) of XTZ completely inhibited biofilm formation by S. mutans at adherent phases of growth, whereas 50 micromol l(-1) of XTZ removed 76% of biofilm at plateau accumulated phase when exposed to S. mutans biofilm for 60 min. CONCLUSIONS: Xanthorrhizol isolated from an edible plant (C. xanthorrhiza Roxb.) shows promise as an antibacterial agent for inhibiting and removing S. mutans biofilms in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: XTZ could be used as a potential antibacterial agent against biofilm formation by S. mutans.     

Activity of Nidus Vespae extract and chemical fractions against Streptococcus mutans biofilms

AIMS: To evaluate the effect of Nidus Vespae extract and chemical fractions on the viability and architecture of Streptococcus mutans biofilms. METHODS AND RESULTS: The raw material was first extracted using 95% ethanol/water. Subsequent fractions were prepared from this extract using cyclohexane/ethyl acetate, petroleum ether/ethyl acetate and chloroform/methanol. The biomass dry weight and total protein of samples treated with Nidus Vespae extract and chemical fractions were significantly less than those treated with the vehicle control (P < 0.05). Biofilms treated with Nidus Vespae also resulted in lower percentage of polysaccharide composition. The pH decrease in the biofilm matrix was retarded by Nidus Vespae compared with the vehicle control. Architecture of biofilms treated with Nidus Vespae was different than in the vehicle control and 0.05% chlorhexidine. CONCLUSIONS: Chloroform/methanol fraction was the most effective treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: The significant antibiofilm activity demonstrated by Nidus Vespae shows it to be a promising source of novel anticariogenic agents.     

Quasi-irreversible inhibition of enolase of Streptococcus mutans by flouride

Abstract Fluoride at concentrations greater than 0.01 mM was found to be a quasi-irreversible inhibitor of enolase of permeabilized cells of Streptococcus mutans GS-5 and also of isolated yeast enolase. The inhibition appeared to be of the type that has been described for P-ATPases, but was not dependent on added Al³⁺ or Be²⁺ ions. Fluoride inhibition of enolase was not reversed by repeatedly washing the permeabilized cells in chilled fluoride-free medium but could be reversed by the product, phosphoenolpyruvate, or by very high levels of the substrate, 2-phosphoglycerate. Irreversible inhibition of glycolysis was not evident after fluoride treatment of intact cells, washing to remove unbound or loosely bound fluoride and addition of glucose, presumably because intracellular levels of phosphoenolpyruvate were sufficiently high to preclude irreversible fluoride inhibition of enolase.     

Incorporation of fatty acids by Streptococcus mutans

In a series of investigations into the cariogenicity of Streptococcus mutans, we studied the incorporation of exogenous fatty acids with reference to glucosyltransferase secretion and membrane fatty acid changes. When cells were grown with different fatty acids, both saturated and unsaturated fatty acids were readily incorporated into the membrane lipids and were biotransformed and elongated preferentially to the longer 16- and 18-carbon-chain fatty acids. This incorporation and chain-elongation led to significant changes in fatty acids composition. By adding fatty acids to the medium, it was possible to appropriately modify the degree of unsaturation and the relative ratio between specific fatty acids in the membrane lipids of S. mutans.     

变异链球菌和血链球菌全代谢途径比对分析

为了比较变异链球菌和血链球菌全代谢途径,依据KEGG数据库(http://www.genome.ad.jp/kegg)对变异链球菌和血链球菌的全部代谢途径作逐项比对。结果显示,二者参与了85个代谢途径,包括多数以相同的酶参与的中央代谢途径,即糖酵解、三羧酸循环、磷酸戊糖途径等,和多数以不同的酶参与的双组分感应系统等。通过变异链球菌和血链球菌整体代谢网络对比,了解了变异链球菌和血链球菌理论上的全部代谢途径,为全面揭示二者代谢交流研究奠定了基础。     

Treatment of Streptococcus mutans biofilms with a nonthermal atmospheric plasma

AIMS: A nonthermal atmospheric plasma, designed for biomedical applications, was tested for its antimicrobial activity against biofilm cultures of a key cariogenic bacterium Streptococcus mutans. METHODS AND RESULTS: The Strep. mutans biofilms were grown with and without 0.15% sucrose. A chlorhexidine digluconate rinse (0.2%) was used as a positive antimicrobial reference. The presence of sucrose and the frequency of plasma application during growth were shown to have a significant effect on the response to treatment and antibacterial activity. CONCLUSIONS: A single plasma treatment for 1 min on biofilms cultured without sucrose caused no re-growth within the observation period. However, with either single or repeated plasma treatments of 1 min, on biofilms cultured with 0.15% sucrose, growth was only reduced. SIGNIFICANCE AND IMPACT OF THE STUDY: In summary, there may be a role for nonthermal plasma therapies in dental procedures. Sucrose and associated growth conditions may be a factor in the survival of oral biofilms after treatment.     

Importance of biofilm formation for corrosion inhibition of SAE 1018 steel by axenic aerobic biofilms

To investigate if corrosion inhibition by aerobic biofilms is a general phenomenon, carbon steel (SAE 1018) coupons were exposed to a complex liquid medium (Luria–Bertani) and seawater-mimicking medium (VNSS) containing fifteen different pure-culture bacterial suspensions representing seven genera. Compared to sterile controls, the mass loss in the presence of these bacteria (which are capable of developing a biofilm to various degrees) decreased by 2- to 15-fold. The extent of corrosion inhibition in LB medium depended on the nature of the biofilm: an increased proportion of live cells, observed with confocal scanning laser microscopy (CSLM) and image analysis, decreased corrosion. Corrosion inhibition in LB medium was greatest with Pseudomonas putida (good biofilm formation), while metal coupons exposed to Streptomyces lividans in LB medium (poor biofilm formation) corroded in a manner similar to the sterile controls. Pseudomonas mendocina KR1 reduced corrosion the most in VNSS. It appears that only a small layer of active, respiring cells is required to inhibit corrosion, and the corrosion inhibition observed is due to the attached biofilm. Received 09 December 1996/ Accepted in revised form 19 March 1997     

Biofilm formation by Candida albicans and Streptococcus mutans in the presence of farnesol: a quantitative evaluation

The aim of this study was to evaluate the effect of the QS molecule farnesol on single and mixed species biofilms formed by Candida albicans and Streptococcus mutans. The anti-biofilm effect of farnesol was assessed through total biomass quantification, counting of colony forming units (CFUs) and evaluation of metabolic activity. Biofilms were also analyzed by scanning electron microscopy (SEM). It was observed that farnesol reduced the formation of single and mixed biofilms, with significant reductions of 37% to 90% and 64% to 96%, respectively, for total biomass and metabolic activity. Regarding cell viability, farnesol treatment promoted significant log reductions in the number of CFUs, ie 1.3–4.2 log₁₀ and 0.67–5.32 log₁₀, respectively, for single and mixed species biofilms. SEM images confirmed these results, showing decreases in the number of cells in all biofilms. In conclusion, these findings highlight the role of farnesol as an alternative agent with the potential to reduce the formation of pathogenic biofilms.     

Cell death in Streptococcus mutans biofilms: a link between CSP and extracellular DNA

Streptococcal competence-stimulating peptides (CSPs) were once thought to passively communicate population density in a process known classically as quorum sensing. However, recent evidence has shown that these peptides may also be inducible 'alarmones,' capable of conveying sophisticated messages in a population including the induction of altruistic cellular suicide under stressful conditions. We have previously characterized the alarmone response in Streptococcus mutans , a cariogenic resident of the oral flora, in which a novel bacteriocin-like peptide causes cell death in a subset of the population. Our objective in this work was to characterize the mechanism of immunity to cell death in S. mutans . Toward this goal, we have identified the conditions under which immunity is induced, and identified the regulatory system responsible for differential (and protective) expression of immunity. We also showed that CSP-induced death contributes to S. mutans biofilm formation through the release of chromosomal DNA into the extracellular matrix, providing a long sought-after mechanistic explanation for the role of CSP in S. mutans biofilm formation.