Christine D. Wu

The Tea Catechin Epigallocatechin Gallate Suppresses Cariogenic Virulence Factors of Streptococcus mutans

ABSTRACT Streptococcus mutans, the primary etiologic agent of dental caries, possesses a series of virulence factors associated with its cariogenicity. Alternatives to traditional antimicrobial treatment, agents selectively inhibiting the virulence factors without necessarily suppressing the resident oral species, are promising. The anticariogenic properties of tea have been suggested in experimental animals and humans. Tea polyphenols, especially epigallocatechin gallate (EGCg), have been shown to inhibit the growth and glucosyltransferases activity of S. mutans. However, their effects on biofilm and cariogenic virulence factors of oral streptococci other than glucosyltransferases have not been well documented. In this study, we investigated the biological effect of EGCg on the virulence factors of S. mutans associated with its acidogenicity and acidurity. The antimicrobial effects of EGCg on S. mutans biofilm grown in chemically defined medium were also examined. EGCg inhibited growth of S. mutans planktonic cells at an MIC of 31.25 μg/ml and a minimal bactericidal concentration (MBC) of 62.5 μg/ml. EGCg also inhibited S. mutans biofilm formation at 15.6 μg/ml (minimum concentration that showed at least 90% inhibition of biofilm formation) and reduced viability of the preformed biofilm at 625 μg/ml (sessile MIC80). EGCg at sub-MIC levels inhibited acidogenicity and acidurity of S. mutans cells. Analysis of the data obtained from real-time PCR showed that EGCg significantly suppressed the ldh, eno, atpD, and aguD genes of S. mutans UA159. Inhibition of the enzymatic activity of F1Fo-ATPase and lactate dehydrogenase was also noted (50% inhibitory concentration between 15.6 and 31.25 μg/ml). These findings suggest that EGCg is a natural anticariogenic agent in that it exhibits antimicrobial activity against S. mutans and suppresses the specific virulence factors associated with its cariogenicity.

In Vitro Remineralization Effects of Grape Seed Extract on Artificial Root Caries

Grape seed extract (GSE) contains proanthocyanidins (PA), which has been reported to strengthen collagen-based tissues by increasing collagen cross-links. We used an in vitro pH-cycling model to evaluate the effect of GSE on the remineralization of artificial root caries. Sound human teeth fragments obtained from the cervical portion of the root were stored in a demineralization solution for 96 h at 37 degrees C to induce artificial root caries lesions. The fragments were then divided into three treatment groups including: 6.5% GSE, 1,000 ppm fluoride (NaF), and a control (no treatment). The demineralized samples were pH-cycled through treatment solutions, acidic buffer and neutral buffer for 8 days at 6 cycles per day. The samples were subsequently evaluated using a microhardness tester, polarized light microscopy (PLM) and confocal laser scanning microscopy (CLSM). Data were analyzed using ANOVA and Fishers tests (p<0.05). GSE and fluoride significantly increased the microhardness of the lesions (p<0.05) when compared to a control group. PLM data revealed a significantly thicker mineral precipitation band on the surface layer of the GSE-treated lesions when compared to the other groups (p>0.05), which was confirmed by CLSM. We concluded that grape seed extract positively affects the demineralization and/or remineralization processes of artificial root caries lesions, most likely through a different mechanism than that of fluoride. Grape seed extract may be a promising natural agent for non-invasive root caries therapy.

Tea catechin epigallocatechin gallate inhibits Streptococcus mutans biofilm formation by suppressing gtf genes

OBJECTIVE The anti-cariogenic properties of tea have been suggested for decades. Tea polyphenols, especially epigallocatechin gallate (EGCG), have been shown to inhibit dental plaque accumulation, but the exact mechanisms are not clear at present. We hypothesise that EGCG suppresses gtf genes in Streptococcus mutans at the transcriptional level disrupting the initial attachment of S. mutans and thus the formation of mature biofilms. DESIGN In this study, the effect of EGCG on the sucrose-dependent initial attachment of S. mutans UA159 in a chemically defined medium was monitored over 4 h using a chamber slide model. The effects of EGCG on the aggregation and gtf B, C, D gene expression of S. mutans UA159 were also examined. RESULTS It was found that EGCG (7.8-31.25 μg/ml) exhibited dose-dependent inhibition of the initial attachment of S. mutans UA159. EGCG did not induce cellular aggregation of S. mutans UA159 at concentrations less than 78.125 μg/ml. Analysis of data obtained from real-time PCR showed that EGCG at sub-MIC level (15.6 μg/ml) significantly suppressed the gtf B, C, D genes of S. mutans UA159 compared with the non-treated control (p < 0.05). CONCLUSIONS These findings suggest that EGCG may represent a novel, natural anti-plaque agent that inhibits the specific genes associated with bacterial biofilm formation without necessarily affecting the growth of oral bacteria.

In vitro synergism between berberine and miconazole against planktonic and biofilm Candida cultures

OBJECTIVES To investigate the antimycotic activity of the plant alkaloid berberine (BBR), alone and in combination with antifungal azoles, against planktonic and biofilm Candida cultures. DESIGN The minimum inhibitory concentrations (MICs) of BBR, miconazole (MCZ), and fluconazole (FLC) towards Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida parapsilosis, and Candida tropicalis were determined by a microdilution method. For C. albicans, the synergistic effects of BBR combined with MCZ or FLC were examined in a paper disc agar diffusion assay and checkerboard microdilution assay. The effect of the BBR/MCZ combination was further investigated in a C. albicans biofilm formation model with a dual-chamber flow cell. The effect on metabolic activity of biofilm cells was established using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)/menadione. RESULTS Berberine inhibited the growth of various Candida species (MICs 0.98-31.25mg/L) in the following order of susceptibility: C. krusei > C. kefyr > C. glabrata > C. tropicalis > C. parapsilosis and C. albicans. Synergism between BBR and MCZ or FLC was observed in the disc diffusion assay as well as in suspension showing an FIC index <0.5 (∑FIC=0.19). Whilst neither BBR (16 mg/L) nor MCZ (0.8 mg/L) alone significantly inhibited biofilm formation of C. albicans, their combination reduced biofilm formation by >91% after 24 h, as established from the reduction in surface area coverage (P<0.01). The BBR/MCZ combination also exhibited synergy against the metabolic activity of pre-formed C. albicans biofilms in polystyrene microtiter plates (∑FIC=0.25). CONCLUSION Berberine exhibits synergistic effects with commonly used antimycotic drugs against C. albicans, either in planktonic or in biofilm growth phases.

Quaternary ammonium silane-functionalized, methacrylate resin composition with antimicrobial activities and self-repair potential.

The design of antimicrobial polymers to address healthcare issues and minimize environmental problems is an important endeavor with both fundamental and practical implications. Quaternary ammonium silane-functionalized methacrylate (QAMS) represents an example of antimicrobial macromonomers synthesized by a sol-gel chemical route; these compounds possess flexible Si-O-Si bonds. In present work, a partially hydrolyzed QAMS co-polymerized with 2,2-[4(2-hydroxy 3-methacryloxypropoxy)-phenyl]propane is introduced. This methacrylate resin was shown to possess desirable mechanical properties with both a high degree of conversion and minimal polymerization shrinkage. The kill-on-contact microbiocidal activities of this resin were demonstrated using single-species biofilms of Streptococcus mutans (ATCC 36558), Actinomyces naeslundii (ATCC 12104) and Candida albicans (ATCC 90028). Improved mechanical properties after hydration provided the proof-of-concept that QAMS-incorporated resin exhibits self-repair potential via water-induced condensation of organic modified silicate (ormosil) phases within the polymerized resin matrix.

An ORMOSIL-containing orthodontic acrylic resin with concomitant improvements in antimicrobial and fracture toughness properties

Global increase in patients seeking orthodontic treatment creates a demand for the use of acrylic resins in removable appliances and retainers. Orthodontic removable appliance wearers have a higher risk of oral infections that are caused by the formation of bacterial and fungal biofilms on the appliance surface. Here, we present the synthetic route for an antibacterial and antifungal organically-modified silicate (ORMOSIL) that has multiple methacryloloxy functionalities attached to a siloxane backbone (quaternary ammonium methacryloxy silicate, or QAMS). By dissolving the water-insoluble, rubbery ORMOSIL in methyl methacrylate, QAMS may be copolymerized with polymethyl methacrylate, and covalently incorporated in the pressure-processed acrylic resin. The latter demonstrated a predominantly contact-killing effect on Streptococcus mutans ATCC 36558 and Actinomyces naselundii ATCC 12104 biofilms, while inhibiting adhesion of Candida albicans ATCC 90028 on the acrylic surface. Apart from its favorable antimicrobial activities, QAMS-containing acrylic resins exhibited decreased water wettability and improved toughness, without adversely affecting the flexural strength and modulus, water sorption and solubility, when compared with QAMS-free acrylic resin. The covalently bound, antimicrobial orthodontic acrylic resin with improved toughness represents advancement over other experimental antimicrobial acrylic resin formulations, in its potential to simultaneously prevent oral infections during appliance wear, and improve the fracture resistance of those appliances.

Evaluating biofilm growth of two oral pathogens

Aims: To determine the expediency of a microtitre assay system for establishing, quantifying and antimicrobial testing of two representative oral pathogens.

ISOLATION OF ALKALOIDS FROM GOLDENSEAL (HYDRASTIS CANADENSIS RHIZOMES) USING pH-ZONE REFINING COUNTERCURRENT CHROMATOGRAPHY

Goldenseal (the rhizomes of Hydrastis canadensis) has a long history of use in North American folk medicine and today is one of the top-selling herbal dietary supplements in the United States. The alkaloids present in the plant have been shown to be responsible for a broad range of biological activities, and the purpose of this work was to isolate preparative quantities of alkaloids present in Goldenseal for subsequent biological evaluation. Berberine chloride, canadaline, canadine, β-hydrastine, and isocorypalmine were separated from a methanolic extract of Goldenseal by a combination of solvent/solvent partition, pH-zone refining countercurrent chromatographic, and recrystallization techniques.

The antibiofilm activity of lingonberry flavonoids against oral pathogens is a case connected to residual complexity

The antimicrobial activity of lingonberry (Vaccinium vitis-idaea L.) was evaluated against two oral pathogens, Streptococcus mutans and Fusobacterium nucleatum. Long-bed gel permeation chromatography (GPC; Sephadex LH-20) yielded purified flavonoids, with the most efficient minimum inhibitory concentrations (MICs) against planktonic cells in the anthocyanin and procyanidin primary fractions against F. nucleatum (63-125 μg/ml) and in the procyanidin rich fraction against S. mutans (16-31 μg/ml). The purified flavonol glycosides and procyanidins inhibited biofilm formation of S. mutans (MICs 16-31 μg/ml), while the corresponding reference compounds showed no activity. Secondary GPC purification yielded flavonol glycosides devoid of antibiofilm activity in the 50% MeOH fraction, while elution with 70% acetone recovered a brownish material with activity against S. mutans biofilm (MIC 8 μg/ml). Even after HPLC-PDA, NMR, and MALDI-TOF analyses, the structural identity of this material remained unknown, while its color and analytical characteristics appear to be consistent with flavonoid oxidation products.

The preventive effect of grape seed extract on artificial enamel caries progression in a microbial biofilm-induced caries model

OBJECTIVES The aim of this study was to evaluate the effect of grape seed extract (GSE) on enamel caries lesion formation in an in vitro Streptococcus mutans biofilm model. METHODS Enamel fragments were prepared from bovine incisors and divided into six treatment groups (n=12): inoculated Brain Heart Infusion with 1% sucrose (BHIS), 1mg/mL GSE, 2mg/mL GSE, 3mg/mL GSE, 10ppm fluoride as NaF, and uninoculated BHIS. For biofilm formation, tooth fragments were incubated anaerobically in polystyrene 6-well tissue culture plates containing BHIS, the respective agents, and S. mutans (1×10(5)CFU/mL) for 24h at 37°C. Culture medium was replaced with fresh BHIS and respective agents daily over a 7-day period. Following caries lesion formation, lesion depth (LD) and relative optical density (ROD) were determined by polarized light microscopy (PLM) and confocal laser scanning microscopy (CLSM), respectively, to evaluate lesion progression. RESULTS LDs of the 2mg/mL GSE group (122.86±13.41μm) and the 3mg/mL GSE group (111.92±11.39μm) were significantly smaller than those of the 1mg/mL GSE (198.33±17.70μm) and control groups (210.86±15.50μm) (p<0.05). Compared with the 2mg/mL and 3mg/mL groups, the control and 1mg/mL GSE groups showed significantly lower ROD values when depth was less than 200μm, indicating greater mineral loss. CONCLUSIONS Dose-dependent GSE inhibits in vitro enamel caries formation due to its ability to suppress growth of S. mutans and the formation of biofilm. CLINICAL SIGNIfiCANCE: Grape seed extract may be a novel virulence-targeted natural antimicrobial agent for caries prevention.