Comparison of biofilm models for producing artificial active white spot lesions.

OBJECTIVE: This study compared three protocols for developing artificial white spot lesions (WSL) using biofilm models. METHODOLOGY: In total, 45 human enamel specimens were sterilized and allocated into three groups based on the biofilm model: Streptococcus sobrinus and Lactobacillus casei (Ss+Lc), Streptococcus sobrinus (Ss), or Streptococcus mutans (Sm). Specimens were incubated in filter-sterilized human saliva to form the acquired pellicle and then subjected to the biofilm challenge consisting of three days of incubation with bacteria (for demineralization) and one day of remineralization, which was performed once for Ss+Lc (four days total), four times for Ss (16 days total), and three times for Sm (12 days total). After WSL creation, the lesion fluorescence, depth, and chemical composition were assessed using Quantitative Light-induced Fluorescence (QLF), Polarized Light Microscopy (PLM), and Raman Spectroscopy, respectively. Statistical analysis consisted of two-way ANOVA followed by Tukey's post hoc test (α=0.05). WSL created using the Ss+Lc protocol presented statistically significant higher fluorescence loss (ΔF) and integrated fluorescence (ΔQ) in comparison to the other two protocols (p<0.001). RESULTS: In addition, Ss+Lc resulted in significantly deeper WSL (137.5 µm), followed by Ss (84.1 µm) and Sm (54.9 µm) (p<0.001). While high mineral content was observed in sound enamel surrounding the WSL, lesions created with the Ss+Lc protocol showed the highest demineralization level and changes in the mineral content among the three protocols. CONCLUSION: The biofilm model using S. sobrinus and L. casei for four days was the most appropriate and simplified protocol for developing artificial active WSL with lower fluorescence, higher demineralization, and greater depth.

Spent culture supernatant of Streptococcus gordonii mitigates inflammation of human periodontal cells and inhibits proliferation of pathogenic oral microbes.

BACKGROUND: The oral commensal bacterial species Streptococcus gordonii has been reported to regulate the inflammation of oral epithelial cells stimulated by the periodontal pathogen Porphyromonas gingivalis. This study investigated the activities of S. gordonii metabolites in S. gordonii spent culture supernatants (Sg-SCS) on periodontal-related bacterial growth and periodontitis-associated inflammatory cytokines. METHODS: Sg-SCS was collected from S. gordonii cultures grown in Dulbecco Modified Eagle Medium and added to the growth media of representative health- and disease-related oral species: S. gordonii, Streptococcus sanguinis, Streptococcus mitis, Streptococcus oralis, P. gingivalis, Tannerella forsythia, and Treponema denticola. The Sg-SCS was also tested for its ability to regulate the expression of proinflammatory cytokines by human macrophages, epithelial cells, and gingival fibroblasts upon stimulation with P. gingivalis-derived lipopolysaccharide (Pg-LPS). RESULTS: Sg-SCS significantly reduced transcript and protein levels of interleukin (IL)-1ß, 6, and 8 induced by Pg-LPS stimulation in multiple types of periodontal cells. mRNA sequencing and bioinformatics analyses indicated that Sg-SCS significantly affects 10 inflammatory pathways. Additionally, Sg-SCS exhibited suppression of the growth of periodontal disease-related bacteria, including T. denticola and P. gingivalis, along with the primary plaque-colonizing species S. oralis. At a low concentration, Sg-SCS also inhibits P. gingivalis adhesion. CONCLUSIONS: These results strongly suggest that S. gordonii-derived SCS contains metabolites that have anti-inflammatory properties and an ability to inhibit periodontitis-associated pathogenic bacteria. Further investigation will be needed to identify the individual metabolites within the Sg-SCS to develop a novel metabolite-based approach to treating and preventing periodontitis.

Choline and geranic acid (CAGE) ionic liquids inhibit both elastase activity and growth of oral bacteria.

Choline and geranic acid (CAGE) ionic liquids have recently been shown to have applications in the delivery of macromolecules and poorly soluble drugs across epithelial barriers and in bacterial growth inhibition. Ionic liquids are known to denature proteins by the disruption of forces that guide natural protein folding, and the inflammatory enzyme elastase was recently shown to be inhibited by a variety of ionic liquids other than CAGE. Inhibition of collagenolytic enzymes, including elastase, has been shown to improve outcomes in cases of periodontitis via amelioration of periodontal inflammation and alveolar bone resorption. In this study, we investigated whether CAGE prepared with varying stoichiometries was able to inhibit elastase at varying concentrations and whether these CAGE formulations could inhibit the growth of key pathogenic bacterial species associated with oral health conditions. We found that CAGE was capable of inhibiting both porcine elastase and human neutrophil elastase at concentrations as low as 5 mM, and that CAGE formulations were effective at inhibiting the growth of all tested pathogenic oral bacteria. The inhibition of elastase by CAGE may be a mechanism by which CAGE can improve outcomes in periodontitis independent from CAGE's known antibacterial properties.

Role of mutans streptococci, acid tolerant bacteria and oral Candida species in predicting the onset of early childhood caries.

Aim: Early childhood caries is the most common chronic infectious disease in children in the United States. This study, which is part of a larger, longitudinal study exploring oral microbiological components of caries development in children, reports on the impact of total mutans streptococci (MS), total acid tolerant bacteria and Candida species on the development of dental caries in a subset of these children. Of particular interest was the relationship between caries development and co-colonization of mutans streptococci and Candida species. Methods: Children between the ages of 12 and 47 months displaying no evidence of dental caries were recruited for a longitudinal study (n = 130). Twelve age- and gender-matched pairs were selected. In each pair, one child developed caries during the study, and one did not. Whole mouth plaque samples were collected by swab at baseline and every 6 months thereafter for a duration of 18 months and spiral plated for microbial counts (CFU/ml). Cut-offs based on percent of total cultivable flora were designated for all microbial measures. A scoring system designated the Plaque Microbial Index (PMI) was developed for use in statistical analyses to assess potential predictive factors for caries risk assessment. Results: Children who developed caries were significantly more likely to harbor higher percentages of acid tolerant bacteria (p = 0.003), MS (p < 0.001) and have Candida species present (p < 0.001) at ≥1 visit leading up to caries onset. Mean PMI scores derived from the aforementioned microbial measures, were higher for caries active children than caries free children (p = 0.000147). Co-colonization of MS and Candida species was significantly associated with caries development (p < 0.001) and detection of both at the same visit had a 100% positive predictive value and 60% negative predictive value for caries development. Conclusion: In children who developed caries, there was a statistically significant association with the percent of total flora that was acid tolerant, the percent of MS, the presence of Candida and co-colonization of MS and Candida species. Combining these microbial measures into PMI scores further delineated children who developed caries from those who remained caries-free. These microbiological measures show potential as predictive factors and risk assessment tools for caries development.

A Social Media Intervention for Promoting Oral Health Behaviors in Adolescents: A Non-Randomized Pilot Clinical Trial.

Poor oral hygiene and excessive consumption of soda are among the main drivers of systemic health issues in adolescents in the United States. This non-randomized pilot clinical trial focused on the effects of a health text message system and smartphone-based intervention on adolescent tooth-brushing behavior and dietary choices, with a convenience sample of 94 participants aged 12 to 14 years old. A group of 75 participants agreed to use a tooth-brushing app and received a health text message; the other group of 15 agreed to use the tooth-brushing app, but did not receive a health text message. Saliva specimens were collected directly before and at the end of each experiment; changes in the salivary presence of cariogenic bacteria over the duration of the study were evaluated and compared with the demographics and behavioral variables. Within the text message group, 5% of participants increased the frequency of daily tooth brushing. Within the non-intervention group, 29% of participants increased the frequency of their daily tooth brushing. There were reductions in the total salivary bacteria and total streptococci in both groups (p < 0.001), but no change in the presence of cariogenic Mutans streptococci. Raising adolescents' consciousness of oral health behavior resulted in marginal to moderate improvements to oral hygiene and dietary choices, as well as reductions in total salivary bacteria.

Systematic review and meta-analysis of oral squamous cell carcinoma associated oral microbiome.

The intersection between the human oral microbiome and oral health is an emerging area of study which has gained momentum over the last decade. This momentum has motivated a search for associations between the oral microbiome and oral cancer, in hopes of identifying possible biomarkers that facilitate earlier diagnosis and improved prognosis for patients with that disease. The present study examined the relationship between the microbiome in the human oral cavity and oral squamous cell carcinoma (OSCC). We searched the literature for case-control studies which focused on the relationship between the human oral microbiome and OSCC. We aggregated three types of data from these studies: bacteriome data at the genus level, predicted functional pathway data, and gene abundance data. From these data, we noted several microbial genera which may be associated with oral cancer status, including Fusobacterium. We also identified functional pathways which merit further investigation, including RNA degradation (ko03018) and primary immunodeficiency (ko05340). In addition, our analysis of gene abundance data identified the gene K06147 (ATP-binding cassette, subfamily B, bacterial) as being over abundant in OSCC samples. Our results are generalizations which identified some currents that we believe could guide further research. Our work faced several limitations related to the heterogeneity of the available data. Wide variation in methods for sample collection, methods for controlling for known behavioral risk factors, computing platform choice, and methods for case-control design all posed confounding factors in this work. We examined the current methods of data collection, data processing, and data reporting in order to offer suggestions toward the establishment of best practices within this field. We propose that these limitations should be addressed through the implementation of standardized data analytic practices that will conform to the rigor and reproducibility standards required of publicly funded research.

Vanillin-bioglass cross-linked 3D porous chitosan scaffolds with strong osteopromotive and antibacterial abilities for bone tissue engineering.

Chitosan scaffolds crosslinked by current methods insufficiently meet the demands of bone tissue engineering applications. We developed a novel effective crosslinking technique by using the natural and safe vanillin together with bioglass microparticles to generate an antibacterial, osteoconductive, and mechanically robust 3D porous chitosan-vanillin-bioglass (CVB) scaffold. In addition to the significantly improved mechanical properties, the CVB scaffolds had high porosity (>90%) and interconnected macroporous structures. Our data suggested that the crosslinking mainly resulted from the Schiff base reactions between the aldehydes of vanillin and amines of chitosan, together with the hydrogen and ionic bonds formed within them. Importantly, the CVB scaffolds not only showed good biocompatibility, bioactivity, and strong antibacterial ability but also significantly promoted osteoblastic differentiation, mineralization in vitro, and ectopic bone formation in vivo. Thus, the CVB scaffolds hold great promise for bone tissue engineering applications based on their robust mechanical properties, osteoconductivity, and antibacterial abilities.

Solubilized ubiquinol for preserving corneal function.

Defective cellular metabolism, impaired mitochondrial function, and increased cell death are major problems that adversely affect donor tissues during hypothermic preservation prior to transplantation. These problems are thought to arise from accumulated reactive oxygen species (ROS) inside cells. Oxidative stress acting on the cells of organs and tissues preserved in hypothermic conditions before surgery, as is the case for cornea transplantation, is thought to be a major reason behind cell death prior to surgery and decreased graft survival after transplantation. We have recently discovered that ubiquinol - the reduced and active form of coenzyme Q10 and a powerful antioxidant - significantly enhances mitochondrial function and reduces apoptosis in human donor corneal endothelial cells. However, ubiquinol is highly lipophilic, underscoring the need for an aqueous-based formulation of this molecule. Herein, we report a highly dispersible and stable formulation comprising a complex of ubiquinol and gamma cyclodextrin (γ-CD) for use in aqueous-phase ophthalmic products. Docking studies showed that γ-CD has the strongest binding affinity with ubiquinol compared to α- or ß-CD. Complexed ubiquinol showed significantly higher stability compared to free ubiquinol in different aqueous ophthalmic products including Optisol-GS® corneal storage medium, balanced salt solution for intraocular irrigation, and topical Refresh® artificial tear eye drops. Greater ROS scavenging activity was noted in a cell model with high basal metabolism and ROS generation (A549) and in HCEC-B4G12 human corneal endothelial cells after treatment with ubiquinol/γ-CD compared to free ubiquinol. Furthermore, complexed ubiquinol was more effective at lowering ROS, and at far lower concentrations, compared to free ubiquinol. Complexed ubiquinol inhibited lipid peroxidation and protected HCEC-B4G12 cells against erastin-induced ferroptosis. No evidence of cellular toxicity was detected in HCEC-B4G12 cells after treatment with complexed ubiquinol. Using a vertical diffusion system, a topically applied inclusion complex of γ-CD and a lipophilic dye (coumarin-6) demonstrated transcorneal penetrance in porcine corneas and the capacity for the γ-CD vehicle to deliver drug to the corneal endothelium. Using the same model, topically applied ubiquinol/γ-CD complex penetrated the entire thickness of human donor corneas with markedly greater ubiquinol retention in the endothelium compared to free ubiquinol. Lastly, the penetrance of ubiquinol/γ-CD complex was assayed using human donor corneas preserved for 7 days in Optisol-GS® per standard industry practices, and demonstrated higher amounts of ubiquinol retained in the corneal endothelium compared to free ubiquinol. In summary, ubiquinol complexed with γ-CD is a highly stable composition that can be incorporated into a variety of aqueous-phase products for ophthalmic use including donor corneal storage media and topical eye drops to scavenge ROS and protect corneal endothelial cells against oxidative damage.

Dental adhesive microtensile bond strength following a biofilm-based in vitro aging model.

OBJECTIVE: Laboratory tests are routinely used to test bonding properties of dental adhesives. Various aging methods that simulate the oral environment are used to complement these tests for assessment of adhesive bond durability. However, most of these methods challenge hydrolytic and mechanical stability of the adhesive- enamel/dentin interface, and not the biostability of dental adhesives. To compare resin-dentin microtensile bond strength (µTBS) after a 15-day Streptococcus mutans (SM) or Streptococcus sobrinus (SS) bacterial exposure to the 6-month water storage (WS) ISO 11405 type 3 test. METHODOLOGY: A total of 31 molars were flattened and their exposed dentin was restored with Optibond-FL adhesive system and Z-100 dental composite. Each restored molar was sectioned and trimmed into four dumbbell-shaped specimens, and randomly distributed based on the following aging conditions: A) 6 months of WS (n=31), B) 5.5 months of WS + 15 days of a SM-biofilm challenge (n=31), C) 15 days of a SM-biofilm challenge (n=31) and D) 15 days of a SS-biofilm challenge (n=31). µTBS were determined and the failure modes were classified using light microscopy. RESULTS: Statistical analyses showed that each type of aging condition affected µTBS (p<0.0001). For Group A (49.7±15.5MPa), the mean µTBS was significantly greater than in Groups B (19.3±6.3MPa), C (19.9±5.9MPa) and D (23.6±7.9MPa). For Group D, the mean µTBS was also significantly greater than for Groups B and C, but no difference was observed between Groups B and C. CONCLUSION: A Streptococcus mutans- or Streptococcus sobrinus-based biofilm challenge for 15 days resulted in a significantly lower µTBS than did the ISO 11405 recommended 6 months of water storage. This type of biofilm-based aging model seems to be a practical method for testing biostability of resin-dentin bonding.

Antimicrobial efficacy of platinum-doped silver nanoparticles.

Silver nanoparticles (AgNPs) have been proposed to combat oral infection due to their efficient ionic silver (Ag+ ) release. However, concentrations required for antimicrobial efficacy may not be therapeutically viable. In this work, platinum-doped silver nanoparticles (Pt-AgNPs) were explored to evaluate their potential for enhanced Ag+ release, which could lead to enhanced antimicrobial efficacy against S. aureus, P. aeruginosa, and E. coli. AgNPs doped with 0.5, 1, and 2 mol% platinum (Pt0.5 -AgNPs, Pt1 -AgNPs, and Pt2 -AgNPs) were synthesized by a chemical reduction method. Transmission electron microscopy revealed mixed morphologies of spherical, oval, and ribbon-like nanostructures. Surface-enhanced Raman scattering revealed that the surface of Pt-AgNPs was covered with up to 93% Pt. The amount of Ag+ released increased 16.3-fold for Pt2 -AgNPs, compared to AgNPs. The initial lag phase in bacterial growth curve was prolonged for Pt-AgNPs. This is consistent with a Ag+ release profile that exhibited an initial burst followed by sustained release. Doping AgNPs with platinum significantly increased the antimicrobial efficacy against all species. Pt2 -AgNPs exhibited the lowest minimum inhibitory concentrations, followed by Pt1 -AgNPs, Pt0.5 -AgNPs, and AgNPs, respectively. Doping AgNPs with a small amount of platinum promoted the release of Ag+ , based on the sacrificial anodic effect, and subsequently enhanced their antimicrobial efficacy.

Evaluating the relationship between acidogenicity and acid tolerance for oral streptococci from children with or without a history of caries.

Background: Dental caries etiology is attributed to a dysbiotic imbalance within the plaque microbiome leading to a dominance of strong acidogens. Some studies that investigate the link between acidogens and caries quantify the recovery of acid tolerant strains on acid agar as a measure of acidogenic potential. This methodology assumes that acidogenic potential and acid tolerance are directly related. Aim: The validity of that assumption was investigated by statistically evaluating that relationship using streptococci recovered from children with or without a history of dental caries. Methods: Thirty streptococcal isolates were isolated from each of 13 subjects. Acidogenicity was quantified by measuring the terminal pH after overnight growth in Brain Heart Infusion (BHI) and Chemically Defined Medium (CDM). Acid tolerance was quantified by measuring the lowest pH acid agar displaying growth. Results: A significant difference in acidogenicity in CDM between levels of acid tolerance was found, but no significant difference in acidogenicity in BHI was noted. Moreover, there were no significant interactions between acid tolerance and caries history on acidogenicity measures in either medium. Conclusion: An ability to grow on acid agar below pH 5.0 is best aligned with strong acidogenicity and best able to distinguish between subjects with differing caries histories.

The effect of aging methods on the fracture toughness and physical stability of an oxirane/acrylate, ormocer, and Bis-GMA-based resin composites.

PURPOSE: To determine the effect of aging methods on the fracture toughness of a conventional Bis-GMA-based resin composite (Filtek Supreme), an ormocer-based resin composite (Admira), and an experimental hydrophobic oxirane/acrylate interpenetrating network resin system (OASys)-based composite. METHODS: A 25 × 5 × 2.8-mm stainless-steel mold with 2.5 mm single-edge center notch, following ASTM standards [E399-90], was used to fabricate 135 specimens (n = 15) of the composite materials and randomly distributed into groups. For the baseline group, specimens were fabricated and then tested after 24-h storage in water. For the biofilm challenge, specimens were randomly placed in a six-well tissue culture plate and kept at 37 °C with bacterial growth media (Brain Heart Infusion (BHI); Streptococcus mutans) changed daily for 15 days. For the water storage challenge, specimens were kept in 5 ml of deionized distilled autoclaved water for 30 days at 37 °C. µCT evaluation by scanning the specimens was performed before and after the proposed challenge. Fracture toughness (KIc) testing was carried out following the challenges. RESULTS: µCT surface area and volume analyses showed no significant changes regardless of the materials tested or the challenge. Filtek and Admira fracture toughness was significantly lower after the biofilm and water storage challenges. OASys mean fracture toughness values after water aging were significantly higher than that of baseline. Toughness values for OASys composites after biofilm aging were not statistically different when compared to either water or baseline values. CONCLUSION: The fracture toughness of Bis-GMA and ormocer-based dental resin composites significantly decreased under water and bacterial biofilm assault. However, such degradation in fracture toughness was not visible in OASys-based composites. CLINICAL SIGNIFICANCE: Current commercial dental composites are affected by the oral environment, which might contribute to the long-term performance of these materials.

Dental adhesive microtensile bond strength following a biofilm-based in vitro aging model

Abstract Laboratory tests are routinely used to test bonding properties of dental adhesives. Various aging methods that simulate the oral environment are used to complement these tests for assessment of adhesive bond durability. However, most of these methods challenge hydrolytic and mechanical stability of the adhesive- enamel/dentin interface, and not the biostability of dental adhesives. Objective To compare resin-dentin microtensile bond strength (μTBS) after a 15-day Streptococcus mutans (SM) or Streptococcus sobrinus (SS) bacterial exposure to the 6-month water storage (WS) ISO 11405 type 3 test. Methodology A total of 31 molars were flattened and their exposed dentin was restored with Optibond-FL adhesive system and Z-100 dental composite. Each restored molar was sectioned and trimmed into four dumbbell-shaped specimens, and randomly distributed based on the following aging conditions: A) 6 months of WS (n=31), B) 5.5 months of WS + 15 days of a SM-biofilm challenge (n=31), C) 15 days of a SM-biofilm challenge (n=31) and D) 15 days of a SS-biofilm challenge (n=31). μTBS were determined and the failure modes were classified using light microscopy. Results Statistical analyses showed that each type of aging condition affected μTBS (p<0.0001). For Group A (49.7±15.5MPa), the mean μTBS was significantly greater than in Groups B (19.3±6.3MPa), C (19.9±5.9MPa) and D (23.6±7.9MPa). For Group D, the mean μTBS was also significantly greater than for Groups B and C, but no difference was observed between Groups B and C. Conclusion A Streptococcus mutans- or Streptococcus sobrinus-based biofilm challenge for 15 days resulted in a significantly lower μTBS than did the ISO 11405 recommended 6 months of water storage. This type of biofilm-based aging model seems to be a practical method for testing biostability of resin-dentin bonding.

Are the mutans streptococci still considered relevant to understanding the microbial etiology of dental caries?

The mutans streptococci were once the primary focus of research dedicated to understanding the etiology of dental caries. That focus has now shifted to an emphasis on the ecological balances and complexities within the entirety of the plaque microbiome. Within that framework there are considerable differences of opinion regarding the importance and relative contributions of the mutans streptococci. This article explores the basis for the various viewpoints, the limitations of current knowledge, and the confounders that make it difficult to arrive at a consensus.

PCR-Based Identification of Oral Streptococcal Species.

The microbial etiology of dental caries is still debated. Among the hypothesized contributors are the "low pH streptococci," a designation given to unusually acid proficient strains among the primary plaque colonizers S. oralis, S. mitis, S. gordonii, and S. anginosus. However, accurate assignment of species is difficult among the oral streptococci. Our objective was to develop a streamlined method for identifying strains of S. oralis and S. mitis from plaque samples so that they could be analyzed in a separate study devoted to low pH streptococci and caries. Two independent PCR amplifications of a locus highly conserved among streptococci were used for presumptive species identification. Multilocus sequence analysis (MLSA) was used to measure accuracy. Sensitivity was 100% for selecting S. oralis and S. mitis among the clones sampled. Specificity was good except for the most closely related species that could not be reliably distinguished even by MLSA. The results with S. oralis and S. mitis were used to identify new primer sets that expanded the utility of the approach to other oral streptococcal species. These novel primer sets offer a convenient means of presumptive identification that will have utility in many studies where large scale, in-depth genomic analyses are not practical.

Acidogenicity and acid tolerance of Streptococcus oralis and Streptococcus mitis isolated from plaque of healthy and incipient caries teeth.

BACKGROUND: Non-mutans low pH oral streptococci are postulated to contribute to caries etiology. OBJECTIVE: This study was undertaken to investigate whether the acidogenicity and acid tolerance of clinical strains of Streptococcus oralis and Streptococcus mitis correlate with health or early-stage enamel caries. DESIGN: S. oralis and S. mitis were isolated from plaque samples taken from the occlusal surfaces of second molars sampled at two different visits 4 years apart. All sites were sound at Visit 1; subjects were segregated into one of three groups based on the status of the site at Visit 2 and caries elsewhere in the dentition. Strains of S. oralis and S. mitis were evaluated for acidogenicity and acid tolerance, and the results correlated with the clinical status of the sites from which they were isolated. Mutans streptococci (MS) isolated from the plaque samples were also quantified, and the presence or absence of growth on pH 5.5 media or on media selective for bifidobacteria was recorded. RESULTS: No significant positive correlations were found between the acidogenicity properties of the S. oralis and S. mitis clones and caries at either visit. Similar results were obtained for acid tolerance of S. oralis clones but were inconclusive for S. mitis clones. A statistically significant positive correlation between MS levels and caries (or future caries) was evident at both visits, but there were no statistical correlations with the growth on pH 5.5 media or media selective for bifidobacteria. CONCLUSIONS: The low pH potential likely varies considerably among oral streptococcal species and is least likely to be found among strains of S. mitis. Accordingly, the concept and constitution of 'low pH streptococci' may need to be re-evaluated.

Discovery of antifungal constituents from the Miao medicinal plant Isodon flavidus.

ETHNOPHARMACOLOGICAL RELEVANCE: Leigong Mountain is an area in the Southwest of China where there is a high incidence rate of athlete's foot, but the Miao people, a Chinese minority who reside in this mountainous area have suffered less from this disease due to their use of the herbal medicine Isodon flavidus (Hand.-Mazz.) H. Hara. AIM OF THE STUDY: The present study is to identify the active chemical constituents responsible for antifungal effects of the folk medicine plant. MATERIALS AND METHODS: The natural compounds were separated from the methanol extract of the twigs and leaves of I. flavidus by phytochemical study using chromatographic methods, and their chemical structures were determined by analysis of the spectroscopic data including 1D and 2D NMR spectra. The absolute configuration of fladin A (1) was further confirmed by X-ray crystallographic analysis. The compounds were evaluated for their antifungal activity against the athlete's foot fungus Trichophyton rubrum. They were further evaluated for their antimicrobial and anti-biofilm activity against the dental pathogens Streptococcus mutans, Porphyromonas gingivalis and Candida albicans. RESULTS: Phytochemical and biological studies of I. flavidus led to the discovery of two antifungal compounds, fladin A (1) and lophanic acid (2). Fladin A (1) is a novel diterpene with an unprecedented cyclic ether group formed between C-4 and C-9. Lophanic acid (2) displayed inhibition activity against the athlete's foot fungus Trichophyton rubrum with an MIC value of 7.8µg/mL, and fladin A (1) also showed inhibition activity against the fungus with a MIC value of 62.5µg/mL. CONCLUSIONS: Our identification of two antifungal compounds provided strong evidence for the Miao people to use I. flavidus as a medicinal plant for treatment of athlete's foot disease. The very different chemical structures of the active compounds from those in the market presents them as potential antifungal lead compounds for follow-up study.

Codon-optimized fluorescent mTFP and mCherry for microscopic visualization and genetic counterselection of streptococci and enterococci.

Despite the powerful potential of fluorescent proteins for labeling bacteria, their use has been limited in multi-species oral biofilm models. Fermentative metabolism by streptococcal species that initiate biofilm colonization results in an acidic, reduced microenvironment that may limit the activities of some fluorescent proteins which are influenced by pH and oxygen availability. The need to reliably distinguish morphologically similar strains within biofilms was the impetus for this work. Teal fluorescent protein (mTFP1) and red fluorescent protein (mCherry) were chosen because their fluorescent properties made them promising candidates. Since tRNA availability has been implicated in efficient translation of sufficient quantities of protein for maximum fluorescence, a streptococcal codon optimization approach was used. DNA was synthesized to encode either protein using codons most frequently used in streptococci; each coding region was preceded by an engineered ribosomal binding site and restriction sites for cloning a promoter. Plasmids carrying this synthesized DNA under control of the Streptococcus mutans lactate dehydrogenase promoter conferred fluorescence to nine representative streptococcal and two Enterococcus faecalis strains. Further characterization in Streptococcus gordonii showed that mTFP1 and mCherry expressions could be detected in cells grown planktonically, in biofilms, or in colonies on agar when expressed on an extrachromosomal plasmid or in single copy integrated into the chromosome. This latter property facilitated counterselection of chromosomal mutations demonstrating value for bacterial strain construction. Fluorescent and non-fluorescent bacteria were distinguishable at acidic pH. These codon-optimized versions of mTFP1 and mCherry have promising potential for use in multiple experimental applications.

Recovery of Viable Bacteria from Probiotic Products that Target Oral Health.

Probiotic therapy has predominantly been directed toward promoting and maintaining intestinal health. In recent years, however, probiotic regimens that target oral health have appeared on the market. These regimens are often delivered in the form of lozenges. Despite the oral health claims made by the manufacturers of these products, there is little independent evidence in the literature to support such claims. In theory, probiotic organisms can be beneficial by several different means including direct inhibition of pathogens and boosting of the host immune response, with the underlying assumption that these mechanisms require a critical number of viable organisms. In this study, five brands of probiotics marketed for oral health were tested for the recovery of viable bacteria. For only one brand could viable bacteria be recovered within one log of the manufacturer's stated starting amount of bacteria. Nearly a billion viable bacteria could be recovered from a lozenge of this brand. The other brands claimed similar starting amounts of bacteria at the time of manufacture but at least a three-log drop off was observed in the amount of viable bacteria recovered from those products. Refrigeration of the probiotics significantly improved the recovery for one brand, but recoveries for all but one brand remained below the recommended daily dosage for probiotic regimens. It is concluded that probiotic brands differ significantly in the quantities of bacteria that remain viable with most failing to meet recommended dosage targets.