Anti-Bacterial and Anti-Biofilm Activities of Anandamide against the Cariogenic Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium in the oral cavity involved in plaque formation and dental caries. The endocannabinoid anandamide (AEA), a naturally occurring bioactive lipid, has been shown to have anti-bacterial and anti-biofilm activities against Staphylococcus aureus. We aimed here to study its effects on S. mutans viability, biofilm formation and extracellular polysaccharide substance (EPS) production. S. mutans were cultivated in the absence or presence of various concentrations of AEA, and the planktonic growth was followed by changes in optical density (OD) and colony-forming units (CFU). The resulting biofilms were examined by MTT metabolic assay, Crystal Violet (CV) staining, spinning disk confocal microscopy (SDCM) and high-resolution scanning electron microscopy (HR-SEM). The EPS production was determined by Congo Red and fluorescent dextran staining. Membrane potential and membrane permeability were determined by diethyloxacarbocyanine iodide (DiOC2(3)) and SYTO 9/propidium iodide (PI) staining, respectively, using flow cytometry. We observed that AEA was bactericidal to S. mutans at 12.5 µg/mL and prevented biofilm formation at the same concentration. AEA reduced the biofilm thickness and biomass with concomitant reduction in total EPS production, although there was a net increase in EPS per bacterium. Preformed biofilms were significantly affected at 50 µg/mL AEA. We further show that AEA increased the membrane permeability and induced membrane hyperpolarization of these bacteria. AEA caused S. mutans to become elongated at the minimum inhibitory concentration (MIC). Gene expression studies showed a significant increase in the cell division gene ftsZ. The concentrations of AEA needed for the anti-bacterial effects were below the cytotoxic concentration for normal Vero epithelial cells. Altogether, our data show that AEA has anti-bacterial and anti-biofilm activities against S. mutans and may have a potential role in preventing biofilms as a therapeutic measure.

Sustained release varnish containing chlorhexidine for prevention of Streptococcus mutans biofilm formation on voice prosthesis surface: an in vitro study.

OBJECTIVES: In this study, we aimed to develop a novel, sustained release varnish (SRV) for voice prostheses (VP) releasing chlorhexidine (CHX), for the prevention of biofilm formation caused by the common oral bacteria Streptococcus mutans on VP surfaces. METHODS: This study was performed in an in vitro model as a step towards future in vivo trials. VPs were coated with a SRV containing CHX (SRV-CHX) or SRV alone (placebo-SRV) that were daily exposed to S. mutans. The polymeric materials of SRV were composed of ethylcellulose and PEG-400. Biofilm formation was assessed by DNA quantification (qPCR), crystal violet staining, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and kinetics experiments. RESULTS: The amount of DNA in the biofilms formed by S. mutans on VP surfaces coated once with SRV-CHX (1.024 ± 0.218 ng DNA/piece) was 58.5 ± 8.8% lower than that of placebo-SRV-coated VPs (2.465 ± 0.198 ng DNA/piece) after a 48-h exposure to S. mutans (p = 0.038). Reduced biofilm mass on SRV-CHX-coated VPs was visually confirmed by CLSM and SEM. CV staining of SRV-CHX single-coated VPs that have been exposed to S. mutans nine times showed a 98.1 ± 0.2% reduction in biofilm mass compared to placebo-SRV-coated VPs (p = 0.003). Kinetic experiments revealed that SRV-CHX triple-coated VPs could delay bacterial growth for 23 days. CONCLUSIONS: Coating VPs with SRV-CHX has an inhibitory effect on biofilm formation and prevents bacterial growth in their vicinities. This study is a proof-of-principle that paves the way for developing new clinical means for reducing both VPs' bacterial biofilm formation and device failure.

Targeting the Achilles' Heel of Multidrug-Resistant Staphylococcus aureus by the Endocannabinoid Anandamide.

Antibiotic-resistant Staphylococcus aureus is a major health issue that requires new therapeutic approaches. Accumulating data suggest that it is possible to sensitize these bacteria to antibiotics by combining them with inhibitors targeting efflux pumps, the low-affinity penicillin-binding protein PBP2a, cell wall teichoic acid, or the cell division protein FtsZ. We have previously shown that the endocannabinoid Anandamide (N-arachidonoylethanolamine; AEA) could sensitize drug-resistant S. aureus to a variety of antibiotics, among others, through growth arrest and inhibition of drug efflux. Here, we looked at biochemical alterations caused by AEA. We observed that AEA increased the intracellular drug concentration of a fluorescent penicillin and augmented its binding to membrane proteins with concomitant altered membrane distribution of these proteins. AEA also prevented the secretion of exopolysaccharides (EPS) and reduced the cell wall teichoic acid content, both processes known to require transporter proteins. Notably, AEA was found to inhibit membrane ATPase activity that is necessary for transmembrane transport. AEA did not affect the membrane GTPase activity, and the GTPase cell division protein FtsZ formed the Z-ring of the divisome normally in the presence of AEA. Rather, AEA caused a reduction in murein hydrolase activities involved in daughter cell separation. Altogether, this study shows that AEA affects several biochemical processes that culminate in the sensitization of the drug-resistant bacteria to antibiotics.

Anti-Bacterial Effect of Cannabidiol against the Cariogenic Streptococcus mutans Bacterium: An In Vitro Study.

Dental caries is caused by biofilm-forming acidogenic bacteria, especially Streptococcus mutans, and is still one of the most prevalent human bacterial diseases. The potential use of cannabidiol (CBD) in anti-bacterial therapies has recently emerged. Here we have studied the anti-bacterial and anti-biofilm activity of CBD against S. mutans. We measured minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC). The bacterial growth and changes in pH values were measured in a kinetic study. The biofilm biomass was assessed by Crystal Violet staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) metabolic assay. Spinning Disk Confocal Microscopy (SDCM) was used to assess biofilm structure, bacterial viability and extracellular polysaccharide (EPS) production. CBD inhibited S. mutans planktonic growth and biofilm formation in a dose-dependent manner, with similar MIC and MBIC values (5 µg/mL). CBD prevented the bacteria-mediated reduction in pH values that correlated with bacterial growth inhibition. SDCM showed a decrease of 50-fold in live bacteria and EPS production. CBD significantly reduced the viability of preformed biofilms at 7.5 µg/mL with an 80 ± 3.1% reduction of metabolic activity. At concentrations above 20 µg/mL, there was almost no bacterial recovery in the CBD-treated preformed biofilms even 48 h after drug withdrawal. Notably, precoating of the culture plate surfaces with CBD prior to incubation with bacteria inhibited biofilm development. Additionally, CBD was found to induce membrane hyperpolarization in S. mutans. Thus, CBD affects multiple processes in S. mutans including its cariogenic properties. In conclusion, we show that CBD has a strong inhibitory effect against cariogenic bacteria, suggesting that it is a potential drug adjuvant for reducing oral pathogenic bacterial load as well as protecting against dental caries.

Saturated and aromatic aldehydes originating from skin and cutaneous bacteria activate the Nrf2-keap1 pathway in human keratinocytes.

Skin homeostasis is constantly challenged by environmental factors, affecting its delicate redox balance. The skin is also home to a wide variety of bacterial species, including Staphylococci. The cutaneous redox state is governed by the Nrf2-keap1 pathway, which is responsible for the induction of phase II cytoprotective enzymes, thus sustaining a healthy oxidative state. As part of normal metabolism, both bacteria and cutaneous tissue emit copious amounts of volatile organic compounds (VOCs), one subgroup of which are aldehydes. α,ß-unsaturated aldehydes are known activators of Nrf2-keap1 pathway by direct oxidation of the keap1 protein. However, we did not encounter reports of Nrf2 activation by saturated or aromatic aldehydes, neither bacteria nor skin-derived. We hypothesized that non-α,ß-unsaturated aldehydes derived from skin or cutaneous bacteria may act as Nrf2-keap1 pathway activators and therefore afford protection against environmental insults. The saturated aldehydes nonanal and decanal (known skin metabolites) and the aromatic aldehyde benzaldehyde (known skin and Staphylococcus epidermidis metabolite) were shown to induce the Nrf2-keap1 pathway in human keratinocytes. We also identified a newly described aromatic aldehyde, 3-furaldehyde (3-FA), emitted from S. aureus and S. epidermidis cultures, which also activated the pathway. Moreover, Nrf2-keap1 induction led to a significant protection against UVB-induced apoptosis. The mechanism involved in this activation has been partially elucidated. This work emphasizes the importance of cutaneous bacteria, as well as healthy skin lipid peroxidation processes in the maintenance and regulation of the cellular antioxidant response, namely with regard to coping with environmental stressors.

Biogeographical Landscape of the Human Face Skin Microbiome Viewed in High Definition.

The bacterial community that colonizes the human face imparts physiochemical and physiological effects on the facial skin. These skin-microbe interactions impact dermatological, cosmetic and skincare applications due to the centrality of the human face in daily interactions. However, fine-scale characterization of the human face skin microbiome is lacking. Using 16S rRNA sequencing and 3D cartography, this study plotted and characterized the facial skin microbiome in high- definition, based on 1,649 samples from 12 individuals. Analysis yielded a number of novel insights, including that of the relative uniformity of skin microbiome composition within skin sites, site localization of certain microbes, and the interpersonal variability of the skin microbiome. The results show that high-resolution topographical mapping of the skin microbiome is a powerful tool for studying the human skin microbiome. Despite a decade of skin microbiome research, there is still much to be discovered.

Voice Prosthesis Coated with Sustained Release Varnish Containing Clotrimazole Shows Long-Term Protection against Candida albicans: An In Vitro Study.

Fungal biofilm formation on voice prosthesis (VP) is a major health problem that requires repeated replacement of the prosthesis. Candida albicans is one of the pathogens that frequently inhabits the VP. We proposed that coating VPs with sustained-release varnish (SRV) containing clotrimazole (CTZ) might prevent fungal biofilm formation. The long-term antifungal activities of SRV-CTZ- versus SRV-placebo-coated VPs was tested daily by measuring the inhibition zone of C. albicans seeded on agar plates or by measuring the fungal viability of C. albicans in suspension. The extent of biofilm formation on coated VPs was analyzed by confocal microscopy and scanning electron microscopy. We observed that SRV-CTZ-coated VPs formed a significant bacterial inhibition zone around the VPs and prevented the growth of C. albicans in suspension during the entire testing period of 60 days. Fungal biofilms were formed on placebo-coated VPs, while no significant biofilms were observed on SRV-CTZ-coated VPs. HPLC analysis shows that CTZ is continuously released during the whole test period of 60 days at a concentration above the minimal fungistatic concentration. In conclusion, coating VPs with an SRV-CTZ film is a potential effective method for prevention of fungal infections and biofilm formation on VPs.

Effect of epigallocatechin gallate on dental biofilm of Streptococcus mutans: An in vitro study.

BACKGROUND: Streptococcus mutans (S. mutans) plays a major role in the formation of dental caries. The aim of this study was to examine the effect of the green tea polyphenol, epigallocatechin gallate (EGCG), on biofilm formation of S. mutans. METHODS: Following exposure to increasing concentrations of EGCG, the planktonic growth was measured by optical density and the biofilm biomass was quantified by crystal violet staining. Exopolysaccharides (EPS) production was visualized by confocal scanning laser microscopy, and the bacterial DNA content was determined by quantitative polymerase chain reaction (qPCR). Gene expression of selected genes was analyzed by real time (RT)-qPCR and membrane potential was examined by flow cytometry. RESULTS: We observed that EGCG inhibited in a dose-dependent manner both the planktonic growth and the biofilm formation of S. mutans. Significant reduction of S. mutans biofilm formation, DNA content, and EPS production was observed at 2.2-4.4 mg/ml EGCG. EGCG reduced the expression of gtfB, gtfC and ftf genes involved in EPS production, and the nox and sodA genes involved in the protection against oxidative stress. Moreover, EGCG caused an immediate change in membrane potential. CONCLUSIONS: EGCG, a natural polyphenol, has a significant inhibitory effect on S. mutans dental biofilm formation and EPS production, and thus might be a potential drug in preventing dental caries.

Tooth mousse containing casein phosphopeptide-amorphous calcium phosphate prevents biofilm formation of Streptococcus mutans.

BACKGROUND: Streptococcus mutans is a common cariogenic bacterium in the oral cavity involved in plaque formation. Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) has been introduced into tooth mousse to encourage remineralization of dental enamel. The aim of this research was to study the effect of tooth mousse containing CPP-ACP (GC Tooth Mousse®) or CPP-ACP with 0.2% fluoride (CPP-ACPF; GC Tooth Mousse Plus®; GCP) on S. mutans planktonic growth and biofilm formation. METHODS: S. mutans was cultivated in the presence of different dilutions of the tooth mousse containing CPP-ACP or CPP-ACPF, and the planktonic growth was determined by ATP viability assay and counting colony-forming units (CFUs). The resulting biofilms were examined by crystal violet staining, MTT metabolic assay, confocal laser scanning microscopy (CLSM), and scanning electron microscope (SEM). RESULTS: The CPP-ACP tooth mousse (GC) at a dilution of 5-50 mg/ml (0.5-5%) did not inhibit planktonic growth, and even increased the ATP content and the number of viable bacteria after a 24 h incubation. The same was observed for the CPP-ACPF tooth mousse (GCP), except for the higher concentrations (25 and 50 mg/ml) that led to a drop in the bacterial count. Importantly, both compounds significantly decreased S. mutans biofilm formation at dilutions as low as 1.5-3 mg/ml. 12.5 mg/ml GC and 6.25 mg/ml GCP inhibited biofilm formation by 90% after 4 h. After 24 h, the MBIC90 was 6.25 mg/ml for both. CLSM images confirmed the strong inhibitory effect GC and GCP had on biofilm formation when using 5 mg/ml tooth mousse. SEM images of those bacteria that managed to form biofilm in the presence of 5 mg/ml tooth mousse, showed alterations in the bacterial morphology, where the streptococci appear 25-30% shorter on the average than the control bacteria. CONCLUSION: Our data show that the tooth mousse containing CPP-ACP reduces biofilm formation of the cariogenic bacterium S. mutans without killing the bacteria. The use of natural substances which inhibit biofilm development without killing the bacteria, has therapeutic benefits, especially in orthodontic pediatric patients.

Sustained-release delivery of antimicrobial drugs for the treatment of periodontal diseases: Fantasy or already reality?

Periodontal diseases are prevalent in humans. Conventional means of combating these diseases involve basic oral hygiene, mostly toothbrushing, use of mouthwashes, and flossing. Supplementary means of treatment, either clinical or pharmaceutical, are often necessary. The use of sustained-release delivery systems, applied locally to the periodontal pocket, seems to be one feasible approach: local sustained-release delivery of antibacterial agents to treat periodontal diseases is conceivable. The use of local (intrapocket) sustained-release delivery systems has numerous clinical, pharmacologic, and toxicologic advantages over conventional treatments for periodontal diseases. Sustained-release technology has been proven to be effective over the last few decades. Films, gels, and fibers are the three main classical intrapocket pharmaceutical delivery systems. Research today is more focused on improving drug delivery, and less on introducing new drugs. New approaches, eg, those making use of nanotechnology, are emerging for local drug-delivery systems. The local sustained-release delivery system concept is innovative and a few products are already commercially available.

Exposure of Streptococcus mutans and Streptococcus sanguinis to blue light in an oral biofilm model.

The potential anti-cariogenic effect of blue light was evaluated using an oral biofilm model. Two species, Streptococcus mutans and Streptococcus sanguinis, were cultivated ex vivo on bovine enamel blocks for 24 h, either separately or mixed together, then exposed to blue light (wavelengths 400-500 nm) using 112 J/cm2. Twenty four or 48 h after exposure to light the biofilm structure and biomass were characterized and quantified using SEM and qPCR, respectively. Bacterial viability was analyzed by CLSM using live/dead bacterial staining. Gene expression was examined by RT-qPCR. After exposure to light, S. mutans biomass in mono-species biofilm was increased mainly by dead bacteria, relative to control. However, the bacterial biomass of S. mutans when grown in mixed biofilm and of S. sanguinis in mono-species biofilm was reduced after light exposure, with no significant change in viability when compared to control. Furthermore, when grown separately, an upregulation of gene expression related to biofilm formation of S. mutans, and downregulation of similar genes of S. sanguinis, were measured 24 h after exposure to blue light. However, in mixed biofilm, a downregulation of those genes in both species was observed, although not significant in S. mutans. In conclusion, blue light seems to effectively alter the bacterial biomass by reducing the viability and virulence characteristics in both bacterial species and may promote the anti-cariogenic balance between them, when grown in a mixed biofilm. Therefore, exposure of oral biofilm to blue light has the potential to serve as a complementary approach in preventive dentistry.

Adaptation of Bacillus species to dairy associated environment facilitates their biofilm forming ability.

Biofilm-forming Bacillus species are often involved in contamination of dairy products and therefore present a major microbiological challenge in the field of food quality and safety. In this study, we sequenced and analyzed the genomes of milk- and non-milk-derived Bacillus strains, and evaluated their biofilm-formation potential in milk. Unlike non-dairy Bacillus isolates, the dairy-associated Bacillus strains were characterized by formation of robust submerged and air-liquid interface biofilm (pellicle) during growth in milk. Moreover, genome comparison analysis revealed notable differences in putative biofilm-associated determinants between the dairy and non-dairy Bacillus isolates, which correlated with biofilm phenotype. These results suggest that biofilm formation by Bacillus species might represent a presumable adaptation strategy to the dairy environment.

Efficacy and potential use of novel sustained release fillers as intracanal medicaments against Enterococcus faecalis biofilm in vitro.

BACKGROUND: Enterococcus faecalis is a bacterium frequently isolated after failed root canal therapy. This study analyzed the antibacterial and antibiofilm effects in vitro of sustained-release fillers (SRF) containing cetylpyridinium chloride (CPC) against vancomycin resistant E. faecalis. METHODS: First, the solidification capability was tested by introducing liquid SRF into phosphate buffered saline, followed by 30 s of vortexing. The antimicrobial effects of SRF-CPC against static monospecies biofilms were analyzed with a metabolic assay. Inhibition of biofilm formation was tested by exposing daily refreshed E. faecalis suspensions to SRF-CPC for 9 weeks. To evaluate the effects of SRF-CPC against preformed biofilms, biofilms were grown for 1, 3 and 7 days, and then treated with SRF-CPC for 24 h. Biofilm kill time was tested by applying SRF-CPC to a 3-day-old biofilm and measuring its viability at different time points. All experiments were compared to Placebo SRFs and to untreated control biofilms. Data were analyzed with two-way ANOVA followed by Tukey's test. Results were considered significant at P < 0.05. RESULTS: The liquid SRF solidified within seconds and no structural changes were observed after 30 s of vortexing at maximum speed. SRF-CPC inhibited E. faecalis biofilm formation for 7 weeks and significantly reduced its viability in weeks 8 and 9. Mature biofilms grown for 1, 3 and 7 days were destructed by SRF-CPC in less than 24 h. Fifty percent of a 3-day-old biofilm was destructed in 2 h and complete destruction occurred in less than 12 h. (P < 0.05 in all cases, compared to SRII-Placebo). CONCLUSIONS: SRF-CPC's physical properties and long-lasting anti-biofilm effects make it a promising coadjuvant medication for endodontic therapy.

Temporal Stability of the Healthy Human Skin Microbiome Following Dead Sea Climatotherapy.

Dead Sea climatotherapy (DSC) is a therapeutic modality for a variety of chronic skin conditions, yet there has been scarce research on the relationship between the cutaneous microbiota and disease states in response to DSC. We characterized the skin bacterial and fungal microbiome of healthy volunteers who underwent DSC. Bacterial community diversity remained similar before and after treatment, while fungal diversity was significantly reduced as a result of the treatment. Individuals showed greater inter-individual than temporal bacterial community variance, yet the opposite was true for fungal community composition. We further identified Malassezia as the genus driving temporal mycobiome variations. The results indicate that the microbiome remains stable throughout DSC, while the mycobiome undergoes dramatic community changes. The results of this study will serve as an important baseline for future investigations of microbiome and mycobiome temporal phenomena in diseased states.

Sustained effects of blue light on Streptococcus mutans in regrown biofilm.

In prior studies, exposure of Streptococcus mutans in biofilm to blue light using high fluences of up to 680 J/cm(2) did not interfere with bacterial capability to reform an initial biofilm; however, a delayed antibacterial effect was observed. Our aim was to determine the sustained effecttts of blue light-emitting diode (LED) curing light on the pathogenicity of the newly formed biofilm. S. mutans were grown to form biofilm that was exposed to blue light (wavelengths, 460-480 nm) for 1, 3, and 7 min (equivalent to 37, 112, and 262 J/cm(2), respectively). Then, bacteria were suspended and allowed to regrow into new biofilms. The regrown biofilms were assessed for bacterial quantification by optical density (OD) measurement and quantitative polymerase chain reaction (qPCR), bacterial viability and extracellular polysaccharide production by fluorescent staining using confocal scanning laser microscopy, acid production by bacteria (acidogenicity), and bacterial survival at low pH (aciduricity) using qPCR. Bacterial growth in the regrown biofilms was increased when samples were previously exposed to light; however, under the confocal microscopy, a higher proportion of dead bacteria and a reduction in polysaccharide production were observed. The acidogenicity from the regrown biofilm was lowered as fluences of light increased. The aciduricity of the regrown biofilm was decreased, meaning less growth of bacteria into biofilm in low pH with increasing fluences. Blue light has sustained effects on S. mutans bacteria grown into new biofilm. Although bacterial growth in biofilm increased, bacterial viability and virulence characteristics were impaired. The cariogenic potential over time of S. mutans previously exposed to blue light when grown on tooth surfaces is yet to be determined.

Sustained release of a novel anti-quorum-sensing agent against oral fungal biofilms.

Thiazolidinedione-8 (S-8) has recently been identified as a potential anti-quorum-sensing/antibiofilm agent against bacteria and fungi. Based on these results, we investigated the possibility of incorporating S-8 in a sustained-release membrane (SRM) to increase its pharmaceutical potential against Candida albicans biofilm. We demonstrated that SRM containing S-8 inhibits fungal biofilm formation in a time-dependent manner for 72 h, due to prolonged release of S-8. Moreover, the SRM effectively delivered the agent in its active form to locations outside the membrane reservoir. In addition, eradication of mature biofilm by the SRM containing S-8 was also significant. Of note, S-8-containing SRM affected the characteristics of mature C. albicans biofilm, such as thickness, exopolysaccharide (EPS) production, and morphogenesis of fungal cells. The concept of using an antibiofilm agent with no antifungal activity incorporated into a sustained-release delivery system is new in medicine and dentistry. This concept of an SRM containing a quorum-sensing quencher with an antibiofilm effect could pave the way for combating oral fungal infectious diseases.

Effect of the synthetic cannabinoid HU-210 on quorum sensing and on the production of quorum sensing-mediated virulence factors by Vibrio harveyi.

BACKGROUND: Bacterial populations communicate through the cell density-dependent mechanism of quorum sensing (QS). Vibrio harveyi, one of the best studied model organisms for QS, was used to explore effects of the synthetic cannabinoid HU-210 on QS and different QS-regulated physiological processes in bacteria. RESULTS: Analysis of QS-regulated bioluminescence in wild-type and mutant strains of V. harveyi revealed that HU-210 affects the autoinducer-2 (AI-2) pathway, one of three known QS cascades of V. harveyi. Furthermore, QS-mediated biofilm formation and swimming motility in the mutant strain BB152 (AI-1(-), AI-2(+)) were significantly reduced in the presence of HU-210. HU-210 inhibited QS-mediated virulence factor production without any inhibitory effect on bacterial growth. It also alters the expression of several genes, which are regulated by QS, specifically downregulating the genes of the AI-2 QS cascade. CONCLUSION: First evidence is being provided for interference of bacterial signal-transduction systems by a synthetic cannabinoid. The effect of HU-210 was specific to the AI-2 cascade in V. harveyi. AI-2 is known as a "universal autoinducer" and interference with its activity opens a broad spectrum of applications for synthetic cannabinoids in future research as a potential anti-QS agent.

Comparison of the efficacy of a novel sustained release clotrimazole varnish and clotrimazole troches for the treatment of oral candidiasis.

OBJECTIVES: Candida albicans is a common fungal infection and is commensal in 40-65 % of healthy adults. The development and pharmacokinetics of a novel sustained release clotrimazole varnish (Clot-SRV) for topical oral use have been reported. The aim of this study was to compare the efficacy of this varnish with clotrimazole troche treatment of oral candidiasis. MATERIALS AND METHODS: Of the 12 patients with denture stomatitis treated for 14 days, six used Clot-SRV (study group) and six clotrimazole troches (control). The patients were instructed to use Clot-SRV (50 mg of clotrimazole) once a day, and the control group was instructed to use five troches of 10 mg clotrimazole/day. Microbiological samples were obtained from saliva, buccal mucosa, palate, and denture. The degree of erythema was recorded at three time points, and subjective opinions noted using a questionnaire. RESULTS: At the end of the study, the control group had relatively more cases of erythema on all examined surfaces; patients who applied the Clot-SRV had significantly lower levels of candida on the denture surfaces and in saliva, and had better compliance to the medication. CONCLUSIONS: The novel clotrimazole sustained release varnish may be an important part of a new protocol for oral candidiasis, with improved clinical outcomes.

Anti-Candida albicans biofilm effect of novel heterocyclic compounds.

OBJECTIVES: The aims of this study were to develop new anti-biofilm drugs, examine their activity against Candida albicans biofilm and investigate their structure-activity relationship and mechanism of action. METHODS: A series of thiazolidinedione and succinimide derivatives were synthesized and their ability to inhibit C. albicans biofilm formation and destroy pre-formed biofilm was tested. The biofilms' structure, metabolic activity and viability were determined by XTT assay and propidium iodide and SYTO 9 live/dead stains combined with confocal microscopic analysis. The effect of the most active compounds on cell morphology, sterol distribution and cell wall morphology and composition was then determined by specific fluorescent stains and transmission electron microscopy. RESULTS: Most of the compounds were active at sub-MICs. Elongation of the aliphatic side chain resulted in reduced anti-biofilm activity and the sulphur atom contributed to biofilm killing, indicating a structure-activity relationship. The compounds differed in their effects on biofilm viability, yeast-to-hyphal form transition, hyphal morphology, cell wall morphology and composition, and sterol distribution. The most effective anti-biofilm compounds were the thiazolidinedione S8H and the succinimide NA8. CONCLUSIONS: We developed novel anti-biofilm agents that both inhibited and destroyed C. albicans biofilm. With some further development, these agents might be suitable for therapeutic purposes.

Effects of CO2 laser irradiation on tooth enamel coated with biofilm.

BACKGROUND AND OBJECTIVES: CO2 laser irradiation of tooth enamel can inhibit demineralization of tooth enamel, by changing enamel composition and resistance to acid attack. The aim of this work was to examine these effects of CO2 laser irradiation on enamel covered by biofilm. MATERIALS AND METHODS: Streptococcus mutans was grown on bovine enamel surfaces for 48 hours to form a mature biofilm. Samples were irradiated by CO2 laser (wavelength of 10.6 µm) at a power of 0.08 W in a super-pulse mode for 1 second and 24 pulses/second, with an energy density of 0.77 J/cm(2) per pulse. Untreated controls and laser treated samples with and without biofilm were examined for the morphology of the biofilm and the enamel surface by scanning electron microscopy (SEM). Structural biofilm viability was assessed using confocal laser scanning microscopy with live/dead staining. The biofilm was removed in a sonication water bath and the non-treated and irradiated enamel samples were chemically analyzed using energy dispersive X-ray spectrometry (EDS) and Fourier transform infrared spectroscopy (FTIR). RESULTS: Irradiated samples showed a melt zone with micro-cracks in the center of the irradiating beam position, which was smaller when irradiated enamel was covered by biofilm. Confocal microscopy images demonstrated higher proportion of dead bacteria at the margins of the irradiated spot area, while at the spot center the bacteria were evaporated exposing the enamel surface to direct laser irradiation. EDS analysis showed an increase in Ca/P ratio after irradiation of enamel covered with biofilm. FTIR analysis showed an approximately 40% carbonate loss in the irradiated enamel samples, including those with biofilms. CONCLUSION: Biofilms protect enamel surfaces from possible morphological irradiation damage without interfering with the resultant chemical changes that may increase the enamel resistance to acid attack. Therefore, under certain exposure regimens that are thermally and mechanically safe for enamel, CO2 laser irradiation of biofilms on dental hard tissues is suggested as a potential novel preventive treatment for controlling dental caries.