Hydroxyapatite Discs Research Articles

Antibacterial effects of dentifrices against Streptococcus mutans in children: a comparative in vitro study.

Fluoridated dentifrices have antibacterial effects on children's teeth. On the other hand, the side effects encountered with the use of them have led researchers to look for safe alternatives. This study aimed to determine the antibacterial effect of different commercially available fluoride-free dentifrices on Streptococcus mutans (S. mutans) in comparison with different concentrations of fluoridated dentifrices. Study groups comprised of fluoride-free dentifrices, which contain Probiotic (Activated Charcoal Probiotic Dentifrice-Group P), Aloe Vera-Group AV and Salivary Proteins-Group SP. Fluoridated dentifrices containing 1450 ppm fluoride-Control Group 1 and 500 ppm fluoride-Control Group 2 served as control groups. Antibacterial activity was assessed by Minimum Inhibitory Concentrations and agar well diffusion assays on S. mutans. Biofilm inhibition assay was performed with dentifrices, which had antibacterial activities, and a negative control phosphate-buffered saline (Group PBS) on sterile hydroxyapatite discs against S. mutans. Statistical evaluation was performed. Only group AV showed an antibacterial effect on S. mutans, while control groups showed a similar antibacterial effect. The mean number of viable bacteria present in S. mutans biofilm in Control Group 1 and 2 and Group AV were statistically significantly lower than that in Group PBS, but there were no statistically significant differences between Control Groups and Group AV. Antibacterial activity of commercial dentifrices against S. mutans may be exerted by antibacterial components other than fluoride. Aloe vera-containing toothpaste showed an antibacterial effect on S. mutans, although not as much as the fluoride-containing toothpastes in the control groups. However, further in vivo and long-term studies are required.

Morphological changes and viability of Streptococcus mutans biofilm treated with erythrosine: A confocal laser scanning microscopy analysis.

Antimicrobial photodynamic therapy (a-PDT) is a modality that aims to induce microorganisms through visible light, a photosensitizer, and molecular oxygen. This therapy has shown promising results in controlling cariogenic biofilm in vitro and in vivo counterparts. This study investigated bacterial viability and morphological characterization of Streptococcus mutans mature biofilms after combination of erythrosine and a high potency dental curing light. Biofilms were formed on saliva-coated hydroxyapatite disks in batch culture. The samples were performed in triplicates. Fresh medium was replaced daily for five days and treated using 40 μM of E activated by HL 288 J/cm2 and total dose of 226 J at 1200 mW/cm2. Phosphate buffer saline and 0.12% of chlorhexidine were used as negative and positive control, respectively. After treatment, biofilms were assessed for microbial viability and morphological characterization by means of bio-volume and thickness. COMSTAT software was used for image analysis. Data were analyzed using two-way ANOVA followed by Tukey test with significance level 5%. The application of a-PDT and CHX treatments decreased S. mutans bacterial viability. The image analysis showed more red cells on biofilms when compared to other groups, demonstrating photobacterial killing. Erythrosine irradiated with a high potency curing light can potentially act as an antimicrobial tool in the treatment of cariogenic biofilms. The morphology and viability of microorganisms were impacted after treatment. Treatment with photodynamic therapy may be able to reduce the bio-volume and viability of bacteria present in biofilms. CLINICAL RELEVANCE AND RESEARCH HIGHLIGHTS: The use of the a-PDT technique has been applied in dentistry with satisfactory results. Some applications of this technique are in stomatology and endodontics. In the present study, we sought to understand the use of photodynamic therapy in the control of biofilm and the results found are compatible with the objective of microbiological control proposed by this technique, thus raising the alert for future studies in vivo using the combination of a-PDT with erythrosine, since they are easily accessible materials for the dental surgeon and can be applied in clinical practice.

Targeted Nanoparticles Based on Alendronate Polyethylene Glycol Conjugated Chitosan for the Delivery of siRNA and Curcumin for Bone Metastasized Breast Cancer Applications.

Bone metastasized breast cancer reduces the quality of life and median survival. Targeted delivery of small interfering RNA (siRNA) and chemotherapeutic drugs using nanoparticles (NPs) is a promising strategy to overcome current limitations in treating these metastatic breast cancers. This research develops alendronate conjugated polyethylene glycol functionalized chitosan (ALD-PEG-CHI) NP for the delivery of cell death siRNA (CD-siRNA) and curcumin (CUR) and explores its targeting ability and in vitro cell cytotoxicity. Polyethylene glycol functionalized CHI (mPEG-CHI) NPs serve as control. The size of CD-siRNA loaded NPs is below 100nm while CUR loaded NPs is below 200nm, with near neutral zeta potential for all NPs. The CUR encapsulation efficiency (EE) is 70% and 88% for targeted and control NPs, respectively, while complete encapsulation of CD-siRNA is achieved in both NP systems. The bone targeting ability of CY5-dsDNA loaded ALD-PEG-CHI NPs using hydroxyapatite discs is fivefold compared to control indicating ALD presentation at the targeting NP surface. Delivery of CD-siRNA loaded NPs and CUR loaded NPs show synergistic and additive growth inhibition effects against MCF-7 cells by mPEG-CHI and ALD-PEG-CHI NPs, respectively. Overall, these in vitro results illustrate the potential of the targeted NPs as an effective therapeutic system toward bone metastasized breast cancer.

Antimicrobial Activity of Five Calcium Silicate Based Root Canal Sealers against a Multispecies Engineered Biofilm: An In Vitro Study.

The present study's objective was to compare the impact of CerasealR, total fill BC SealerR, Bio-C SealerR, AH Plus BioceramicR, and K-BiocerR on the elimination of a multispecies' endodontic biofilm at 3, 7 and 14 days. A total of 20 freshly extracted, caries-free premolars were prepared for the study to create dentinal disks. For the multispecies biofilm formation, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans were cultured and used to inoculate hydroxyapatite discs. After incubation, the biofilms were placed on blotting papers in petri dishes with an orthodontic bend. Different root canal sealers, including CeraSeal, total Fill BC Sealer, Bio-C Sealer, AH Plus Bioceramic, K-Biocer, and Sealite, were injected into the bend, facilitating contact with the biofilms. The samples were divided into seven groups, including a negative control. At specific intervals, 3, 7, and 14 days, 3 biofilm samples from each group were collected, diluted, and plated on Agar media for colony counting and analysis. In all tested groups, the total bacterial count significantly decreased between day 3 and 14 (p < 0.05) with no statistically significant differences among the different sealers' groups at all-time points for the total bacterial count, E. faecalis count, and P. mirabilis count. However, Sealite demonstrated the most consistent effectiveness in reducing bacterial counts across multiple categories. The sealite group was capable of decreasing the C. albicans count significantly between day 3 and day 14 (p < 0.05) in comparison with the bioceramic groups. All sealers had antibacterial activity against the multispecies biofilm between day 3 and day 14. The ascending order of sealers in terms of their effectiveness in killing bacteria, based on the provided results, is as follows: Sealite, Bio-C Sealer, AH Plus, CeraSeal, TotalFill, and K-Biocer. However, there were no statistically significant differences in the bacterial counts among the different sealer groups at any time point. The role of sealers in combating biofilm-associated infections highlights their potential clinical utility in preserving root canal health. Understanding the antimicrobial properties of these sealers is vital for informed decision-making in selecting the most effective materials for improved treatment outcomes and long-term success in endodontic procedures.

Effects of DJK-5 and chlorhexidine on exopolysaccharide volume and pH in oral biofilms

BackgroundExopolysaccharides (EPS) are essential constituents of the extracellular matrix within oral biofilms and are significantly influenced by the local microenvironment. This study aimed to investigate the impact of two distinct antimicrobial agents, DJK-5 and chlorhexidine (CHX), on the EPS volume and pH levels in oral biofilms.MethodsOral biofilms obtained from two donors were cultured on hydroxyapatite discs for durations of 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks. Subsequently, these biofilms were subjected to treatment with 10 µg/mL DJK-5 or 2% CHX for 3 min. The impact of these antimicrobial treatments on factors such as the proportion of dead bacterial, in situ pH, and EPS volume within the biofilms was assessed using corresponding fluorescent probes. The examination was carried out utilizing confocal laser scanning microscopy, and the resulting images were analyzed with a focus on the upper and lower layers of the biofilm, respectively.ResultsDJK-5 exhibited a more potent bactericidal effect compared to CHX across the 3-day to 4-week duration of the biofilm (P < 0.05). The biofilms were acidic, with the upper layer being less acidic than the lower layer (P < 0.05). Both antimicrobial agents increased the pH, but DJK-5 had a greater effect than CHX (P < 0.05). The volume of EPS was significantly lower in DJK-5 treated biofilms compared to that of CHX, regardless of age or layer (P < 0.05).ConclusionDJK-5 exhibited superior effectiveness in reducing viable bacteria and EPS volume, as well as in raising extracellular pH, as compared to chlorhexidine.

Anti-biofilm activity of a novel nanoemulsion containing Curcuma xanthorrhiza oil

ObjectivesWe aimed to solubilize Curcuma xanthorrhiza oil (CXO) using nanoemulsification and evaluate its inhibitory effects against biofilm formation. MethodsThe components of CXO were evaluated through high-performance liquid chromatography (HPLC) analysis. Healthy human saliva was inoculated onto hydroxyapatite discs to form microcosm biofilms for four days and treated six times with each antimicrobial agent: distilled water (DW), CXO emulsion (EM), CXO nanoemulsion (NE), and positive controls (Listerine and chlorhexidine). Biofilm fluorescence imaging was performed using quantitative light-induced fluorescence, and cell viability and dry-weight measurements were obtained. We compared the bacterial cell and extracellular polysaccharide (EPS) biovolume and thickness using confocal laser scanning microscopy (CLSM). ResultsHPLC analysis revealed that CXO was composed of approximately 47% xanthorrhizol. Compared with DW, NE exhibited significantly lower red fluorescence intensity and area (42% and 37%, p < 0.001 and p < 0.001, respectively), and reduced total and aciduric bacterial cell viability (7.3% and 3.9%, p < 0.001, p = 0.01, respectively). Furthermore, the bacterial cell and EPS biovolume and thickness in NE decreased by 40–80% compared to DW, similar to chlorhexidine. Conversely, EM showed a significant difference only in cell viability against total bacteria when compared with DW (p = 0.003), with EPS biovolume and thickness exhibiting higher values than DW. ConclusionsNanoemulsification successfully solubilized CXO and demonstrated superior anti-biofilm effects compared to the emulsion form. Clinical significanceThese findings suggest the potential use of NE as a novel antimicrobial agent for preventing oral diseases.

Probiotics for oral health: do they deliver what they promise?

Probiotics have demonstrated oral health benefits by influencing the microbiome and the host. Although promising, their current use is potentially constrained by several restrictions. One such limiting factor lies in the prevailing preparation of a probiotic product. To commercialize the probiotic, a shelf stable product is achieved by temporarily inactivating the live probiotic through drying or freeze drying. Even though a lyophilized probiotic can be kept dormant for an extended period of time, their viability can be severely compromised, making their designation as probiotics questionable. Additionally, does the application of an inactive probiotic directly into the oral cavity make sense? While the dormancy may allow for survival on its way towards the gut, does it affect their capacity for oral colonisation? To evaluate this, 21 probiotic product for oral health were analysed for the number of viable (probiotic), culturable (CFU) and dead (postbiotic) cells, to verify whether the commercial products indeed contain what they proclaim. After isolating and uniformly lyophilizing three common probiotic species in a simple yet effective lyoprotective medium, the adhesion to saliva covered hydroxyapatite discs of lyophilized probiotics was compared to fresh or reactivated lyophilized probiotics. Unfortunately, many of the examined products failed to contain the claimed amounts of viable cells, but also the strains used were inadequately characterized and lacked clinical evidence for that unknown strain, questioning their label of a 'probiotic'. Additionally, lyophilized probiotics demonstrated low adhesive capacity compared to their counterparts, prompting the question of why fresh or reactivated probiotics are not currently used.

Oral Biofilm Cryotherapy as a Novel Ecological Modulation Approach

Oral cryotherapy is used in dentistry as a safe, simple, and low-cost treatment for a variety of oral lesions. It is well known for its ability to aid in the healing process. However, its effect on oral biofilms is unknown. As a result, the purpose of this study was to assess the effects of cryotherapy on in vitro oral biofilms. In vitro multispecies oral biofilms were grown on the surface of hydroxyapatite discs in symbiotic or dysbiotic states. CryoPen X+ was used to treat the biofilms, whereas untreated biofilms served as control. One set of biofilms was collected for study immediately after cryotherapy, whereas another group was reincubated for 24 h to permit biofilm recovery. Changes in biofilm structure were analyzed with a confocal laser scanning microscope (CLSM) and a scanning electron microscope (SEM), while biofilm ecology and community compositional changes were analyzed with viability DNA extraction and quantitative polymerase chain reaction (v-qPCR) analysis. One cryo-cycle immediately reduced biofilm load by 0.2 to 0.4 log10 Geq/mL, which increased with additional treatment cycles. Although the bacterial load of the treated biofilms recovered to the same level as the control biofilms within 24 h, the CLSM detected structural alterations. Compositional alterations were also detected by SEM, corroborating the v-qPCR findings that showed ≈≤10% incidence of pathogenic species compared to nontreated biofilms that encompassed ≈45% and 13% pathogenic species in dysbiotic and symbiotic biofilms, respectively. Spray cryotherapy showed promising results in a novel conceptual approach to the control of oral biofilms. Acting selectively by targeting oral pathobionts and retaining commensals, spray cryotherapy could modify the ecology of in vitro oral biofilms to become more symbiotic and prevent the evolution of dysbiosis without the use of antiseptics/antimicrobials.

Effects of fluoride on in vitro hydroxyapatite demineralisation analysed by 19F MAS-NMR

IntroductionFluoride plays a major role in inhibiting enamel dissolution and promoting fluorapatite formation. Porous hydroxyapatite (HAP) discs can be used as an enamel analogue in artificial demineralisation/remineralisation studies.MethodThe aim of the study was to monitor the fluoride-mineral phases formed on HAP surfaces as a function of fluoride concentration ([F−]) under demineralising conditions, using 19F magic angle spinning nuclear magnetic resonance (MAS-NMR) spectroscopy, and compare the results with a previous study using an enamel substrate. Porous HAP blocks were immersed in demineralisation solutions (0.1 M acetic acid, pH 4.0) with increasing [F−] (0–1450 ppm).ResultsAt below 50 ppm [F−], 19F MAS-NMR showed fluoride-substituted apatite formation; above 50 ppm [F−], calcium fluoride (CaF2) was formed in increasing proportions. These results mirrored those of previous similar studies with an enamel substrate. Further increases in fluoride caused no further measurable reduction in demineralisation but increased the proportion of CaF2 formed. The total calcium concentration [Ca] and total phosphorus [P] concentrations in the solution were measured by inductively coupled plasma atomic emission spectroscopy. At high fluoride concentrations, the solution total [P] increased, and the molar Ca:P ratios decreased to values consistent with the formation of CaF2. However, Ca:P ratios found at low [F−] were higher than those in the previous enamel study and consistent with the formation of a partially fluoridated apatite.ConclusionsUnder demineralising conditions, CaF2 formed on HAP at an [F−] of 50 ppm and above, whereas fluoridated apatite formed at an [F−] below 50 ppm. The results were consistent with those obtained when an enamel substrate was used.

Effect of two graphene derivatives on Enterococcus faecalis biofilms and cytotoxicity.

This study investigated the antibacterial efficacy and cytotoxicity of two graphene derivatives -graphene oxide (GO) and reduced graphene oxide (RGO)- against Enterococcus faecalis (E. faecalis) in biofilms formed on hydroxyapatite discs. The viability and biomass of biofilms treated with GO or RGO were evaluated by colony-forming unit counting and crystal violet staining. The morphological features of the biofilms were observed by scanning electron microscopy (SEM), and the bio-volume was quantitatively analyzed by confocal laser scanning microscopy. Furthermore, the cytotoxicity of GO and RGO was evaluated. GO and RGO had similar antibacterial effects on E. faecalis in biofilms (p>0.05). The SEM images showed the ability of GO and RGO to disrupt E. faecalis. Furthermore, GO had significantly higher cytotoxicity than RGO (p<0.05). The results suggest that RGO has the potential to be used as a more effective disinfecting agent than GO since it showed less cytotoxicity while exerting similar antibacterial efficacy.

Photodynamic nano hydroxyapatite with biofilm penetration capability for dental plaque eradication and prevention of demineralization

Dental caries represents one of the most prevalent diseases worldwide, characteristic of the growth of dental plaque and demineralization of tooth enamel. Current medications for eradication of dental plaques and prevention of demineralization suffer from several limitations to overcome, calling for novel strategies with great potency in eliminating cariogenic bacteria and dental plaque that forms, as well as in inhibiting the demineralization of enamel, into an integrated system. Considering the potency of photodynamic therapy in bacteria inactivation and the composition of enamel, we herein report that the novel photodynamic nano hydroxyapatite (nHAP), named Ce6 @QCS/nHAP, was useful for this purpose. Ce6 @QCS/nHAP, comprised of quaternary chitosan (QCS)-coated nHAP loaded with chlorin e6 (Ce6), exhibited good biocompatibility and non-compromised photodynamic activity. In vitro studies revealed that Ce6 @QCS/nHAP could effectively associate with cariogenic Streptococcus mutans (S. mutans), leading to a significant antibacterial effect through photodynamic killing and physical inactivation against the planktonic microbe. Three-dimensional fluorescence imaging suggested that Ce6 @QCS/nHAP exhibited a superior S. mutans biofilm penetration capacity to free Ce6, resulting in effective dental plaque eradiation when light irradiation was applied. The number of surviving bacteria in biofilm was at least 2.8 log units lower in the Ce6 @QCS/nHAP group compared to that in the free Ce6 group. Further, in the S. mutans biofilm-infected artificial tooth model, treatment with Ce6 @QCS/nHAP also resulted in the significant prevention of hydroxyapatite disks from demineralization, with lower percentage of fragmentation and weight loss These data suggest that our photodynamic nanosystem can effectively eradicate dental plaque while also significantly protecting artificial tooth from demineralization, opening up new possibilities in treating bacterium-associated dental caries.

In vitro versus in situ biofilms for evaluating the antimicrobial effectiveness of herbal mouthrinses.

For centuries, diverse mouthrinses have been applied for medicinal purposes in the oral cavity. In view of the growing resistance of oral microorganisms against conventional antimicrobial agents e.g. chlorhexidine, the implementation of alternative treatments inspired by nature has lately gained increasing interest. The aim of the present study was to compare in vitro biofilm models with in situ biofilms in order to evaluate the antimicrobial potential of different natural mouthrinses. For the in vitro study a six-species supragingival biofilm model containing A. oris, V. dispar, C. albicans, F. nucleatum, S. mutans and S. oralis was used. Biofilms were grown anaerobically on hydroxyapatite discs and treated with natural mouthrinses Ratanhia, Trybol and Tebodont. 0.9% NaCl and 10% ethanol served as negative controls, while 0.2% CHX served as positive control. After 64h hours, biofilms were harvested and quantified by cultural analysis CFU. For the in situ study, individual test splints were manufactured for the participants. After 2h and 72h the biofilm-covered samples were removed and treated with the mouthrinses and controls mentioned above. The biofilms were quantified by CFU and stained for vitality under the confocal laser scanning microscope. In the in vitro study, 0.2% CHX yielded the highest antimicrobial effect. Among all mouthrinses, Tebodont (4.708 ± 1.294 log10 CFU, median 5.279, p<0.0001) compared with 0.9% NaCl showed the highest antimicrobial potential. After 72h there was no significant reduction in CFU after 0.2% CHX treatment. Only Trybol showed a statistically significant reduction of aerobic growth of microorganisms in situ (5.331 ± 0.7350 log10 CFU, median 5.579, p<0.0209). After treatment with the positive control 0.2% CHX, a significant percentage of non-vital bacteria (42.006 ± 12.173 log10 CFU, median 42.150) was detected. To sum up, a less pronounced effect of all mouthrinses was shown for the in situ biofilms compared to the in vitro biofilms.

Influence of Lactobacillus reuteri, Bifidobacterium animalis subsp. lactis, and prebiotic inulin on dysbiotic dental biofilm composition ex vivo.

Probiotic bacterial supplementation has shown promising results in the treatment of periodontitis and the maintenance of periodontal health. The purpose of this investigation was to evaluate the influence of Lactobacillus reuteri or Bifidobacterium animalis subsp. lactis supplementation with and without prebiotic inulin on biofilm composition using an ex vivo biofilm model. Subgingival plaque specimens from three periodontitis-affected human donors were used to grow biofilms on hydroxyapatite disks in media supplemented with varying combinations of prebiotic inulin, Lactobacillus reuteri, and Bifidobacterium animalis subsp. lactis. Relative abundances of bacterial genera present in mature biofilms were evaluated using 16S rRNA next-generation sequencing. Diversity metrics of microbial communities were evaluated using a next-generation microbiome bioinformatics platform. Inulin supplementation produced statistically significant dose-dependent increases in relative abundances of Lactobacillus and Bifidobacterium species (p<0.001) with concomitant decreases in relative abundances of Streptococcus, Veillonella, Fusobacterium, Parvimonas, and Prevotella species (p<0.001). Inoculation with L. reuteri or B. animalis subsp. lactis increased the relative abundance of only the supplemented probiotic genera (p<0.05). Supplemental inulin led to a statistically significant decrease in biofilm alpha diversity (p<0.001). The described ex vivo model appears suitable for investigating the effects of probiotic bacteria, prebiotic oligosaccharides, and combinations thereof on biofilm composition and complexity. Within the limitations imposed by this model, results from the present study underscore the potential for prebiotic inulin to modify biofilm composition favorably. Additional research further elucidating biologic rationale and controlled clinical research defining therapeutic benefits is warranted.

Antimicrobial Effects of Non-Thermal Atmospheric Pressure Plasma on Oral Microcosm Biofilms.

We comparatively evaluated the antibacterial effects of non-thermal atmospheric pressure plasma (NTAPP) on oral microcosm biofilms. Oral microcosm biofilms, which are derived from inoculation with human saliva, were cultured on 48 hydroxyapatite disks for 6 days. The prepared biofilms were divided into three different daily treatment groups: distilled water for 1 min, 0.12% chlorhexidine (CHX) for 1 min, and NTAPP for 5 min. Using a quantitative light-induced fluorescence-digital camera, the red fluorescence intensity of the biofilms was measured as red/green ratios (RatioR/G) before and after treatment. Total and aciduric bacteria were counted as colony-forming units. Using live/dead bacterial staining, bacterial viability was calculated as the RatioG/G+R. RatioR/G was approximately 0.91-fold lower in the NTAPP group than in the CHX group on day 1 of treatment (p = 0.001), and approximately 0.94-fold lower on both days 2 and 3 (p < 0.001). The number of total bacteria was higher in the NTAPP group than in the CHX group, but not significantly different. The number of aciduric bacteria was lowest in the CHX group (p < 0.001). However, bacterial viability was lowest in the NTAPP group. Restricted bacterial aggregation was observed in the NTAPP group. These findings suggest that NTAPP may more effectively reduce the pathogenicity of oral microcosm biofilms than 0.12% CHX.

Identification and Characterization of MUC5B Binding Peptides by Phage Display

ObjectivesMUC5B plays a multifactorial role in oral health. As a consequence, decreased MUC5B output leads to impaired salivary functions and xerostomia. Synthetic combinatorial technologies have been used to develop functional peptide libraries by phage display e.g. for therapeutic purposes. In this light, our primary aim was to identify peptide sequences with specific selectivity for salivary MUC5B in vitro using phage display. Our secondary aims were to analyze their effect on salivary spinnbarkeit in situ and their effect on acid-induced demineralization in vitro. MethodsMUC5B binding phages were selected by phage display. Peptide affinity to MUC5B was evaluated using MUC5B coated hydroxyapatite (HA) granules. The MUC5B binding peptides (MBPs) were then examined for their effects on salivary spinnbarkeit and protective effect on acid-induced demineralization in vitro. A competitive ELISA was performed to identify the binding epitope on MUC5B using F2, a MUC5B specific antibody. ResultsMBP-12 and MBP-14 displayed the highest affinity to MUC5B. MBP-12 mildly stabilized the spinnbarkeit of serous saliva after overnight incubation and of mucous saliva at all timepoints tested. The addition of MBP-12 to a pellicle of unstimulated saliva on HA discs showed no additive protective effect against acid-induced demineralization. Epitope characterization suggested sulfo-Lewisa SO3–3Gal_1–3GlcNAc (galactose residue) as MBP-12 binding site on MUC5B. ConclusionsThe use of phage display in generating MBPs was successful. Characterization of the MBPs revealed a mild effect on spinnbarkeit in case of mucous saliva. Possibly, combinatorial peptide libraries might contribute to the development of novel formulations to treat xerostomia.

Evaluation of extracellular polymeric substances matrix volume, surface roughness and bacterial adhesion property of oral biofilm

Background/purposeOral biofilms are highly structured bacterial colonies embedded in a highly hydrated extracellular polymeric substances (EPS) matrix. This study aimed to investigate the characteristics of oral biofilm at different stages of maturation. Materials and methodsOral multispecies biofilms were grown anaerobically from plaque bacteria on collagen coated hydroxyapatite discs in brain heart infusion broth for one and three weeks. The volume of live bacteria and EPS matrix of the biofilms were determined by using corresponding fluorescent probes and confocal laser scanning microscopy. Atomic force microscopy (AFM) was used to quantitatively probe and correlate cell surface adhesion force of biofilms. The surface roughness was quantified in terms of the root mean square average of the height deviations. Adhesion was measured from force–distance data for the retraction of the cell from the surface. ResultsThe volume of live bacteria and EPS of 3-week-old biofilms was higher than 1-week-old biofilms. The surface roughness value in 1-week-old biofilms was significantly higher than that in 3-week-old biofilms. AFM force–distance curve results showed that the adhesion force at the cell–cell interface was significantly more at-tractive than those at bacterial cells surface of both stages biofilms. Adhesion forces between the AFM tip and the surface of bacterial cell were fairly constant, whereas the cell–cell interface experienced greater adhesion forces in the biofilm's development. ConclusionAs oral biofilms become mature, EPS volume and cell–cell adhesion forces increase while the surface roughness decreases.

Weak direct current exerts synergistic effect with antibiotics and reduces the antibiotic resistance: An in vitro subgingival plaque biofilm model.

Weak direct current (DC) exerts killing effect and synergistic killing effect with antibiotics in some specific bacteria biofilms. However, the potential of weak DC alone or combined with periodontal antibiotics in controlling periodontal pathogens and plaque biofilms remains unclear. The objective of this study was to investigate whether weak DC could exert the anti-biofilm effect or enhance the killing effect of metronidazole (MTZ) and/or amoxicillin-clavulanate potassium (AMC) on subgingival plaque biofilms, by constructing an in vitro subgingival plaque biofilm model. The pooled subgingival plaque and saliva of patients with periodontitis (n=10) were collected and cultured anaerobically on hydroxyapatite disks in vitro for 48 h to construct the subgingival plaque biofilm model. Then such models were stimulated with 0μA DC alone (20 min/12 h), 1000 μA DC alone (20 min/12 h), 16 μg/ml MTZ, 16 μg/ml AMC or their combination, respectively. Through viable bacteria counting, metabolic activity assay, quantitative real-time PCR absolute quantification and 16S rDNA sequencing analysis, the anti-biofilm effect of 1000 μA DC and enhanced killing effects of 1000 μA DC combined with antibiotics (MTZ, AMC or MTZ+AMC) were explored. The old subgingival plaque model (48 h) had no significant difference in total bacterial loads from subgingival plaque in situ, which achieved a similarity of 80%. The 1000 μA DC plus MTZ or AMC for 12 h showed a stronger synergistic killing effect than the same combination for 20 min. The metabolic activity was reduced to the lowest by DC plus MTZ+AMC, as 37.4% of that in the control group, while average synergistic killing effect reached 1.06 log units and average total bacterial loads decreased to 0.87 log units. Furthermore, the relative abundance of the genera Porphyromonas, Prevotella, Treponema_2, and Tannerella were decreased significantly. The presence of weak DC (1000 μA) improved the killing effect of antibiotics on subgingival plaque biofilms, which might provide a novel strategy to reduce their antibiotic resistance.

Effect of Nystatin on Candida albicans - Streptococcus mutans duo-species biofilms

ObjectiveTo assess the effect of Nystatin on Candida albicans and Streptococcus mutans duo-species biofilms using an in vitro cariogenic biofilm model. DesignBiofilms were formed on saliva-coated hydroxyapatite discs under high sugar challenge (1 % sucrose and 1 % glucose), with inoculation of 105CFU/ml S. mutans and 103CFU/ml C. albicans. Between 20 and 68 h, biofilms were treated with 28,000 IU Nystatin solution, 5 min/application, 4 times/day, to mimic the clinical application. Biofilm's three-dimensional structure was assessed using multi-photon confocal microscopy. The expression of C. albicans and S. mutans virulence genes was assessed via real-time PCR. Duplicate discs were used in 3 independent repeats. t-test and Mann-Whitney U test were used to compare outcomes between treatment and control group. ResultsNystatin treatment eliminated C. albicans in biofilms at 44 h. Nystatin-treated group had a significant reduction of biofilm dry-weight and reduced S. mutans abundance by 0.5 log CFU/ml at 44 and 68 h (p < 0.05). Worth noting that biomass distribution across the vertical layout was altered by Nystatin treatment, resulting in less volume on the substrate layers in Nystatin-treated biofilms compared to the control. Reduction of microcolonies size and volume was also observed in Nystatin-treated biofilms (p < 0.05). Nystatin-treated biofilms formed unique halo-shaped microcolonies with reduced core EPS coverage. Furthermore, Nystatin-treated biofilms had significant down-regulations of S. mutans gtfD and atpD genes (p < 0.05). ConclusionsNystatin application altered the formation and characteristics of C. albicans and S. mutans duo-species biofilms. Therefore, developing clinical regimens for preventing or treating dental caries from an antifungal perspective is warranted.

Dietary sugars modulate bacterial-fungal interactions in saliva and inter-kingdom biofilm formation on apatitic surface.

Bacteria and fungi can interact to form inter-kingdom biofilms in the oral cavity. Streptococcus mutans and Candida albicans are frequently detected in saliva and in dental biofilms associated with early childhood caries (tooth-decay), a prevalent oral disease induced by dietary sugars. However, how different sugars influence this bacterial-fungal interaction remains unclear. Here, we investigate whether specific sugars affect the inter-kingdom interaction in saliva and subsequent biofilm formation on tooth-mimetic surfaces. The microbes were incubated in saliva containing common dietary sugars (glucose and fructose, sucrose, starch, and combinations) and analyzed via fluorescence imaging and quantitative computational analyses. The bacterial and fungal cells in saliva were then transferred to hydroxyapatite discs (tooth mimic) to allow microbial binding and biofilm development. We found diverse bacterial-fungal aggregates which varied in size, structure, and spatial organization depending on the type of sugars. Sucrose and starch+sucrose induced the formation of large mixed-species aggregates characterized by bacterial clusters co-bound with fungal cells, whereas mostly single-cells were found in the absence of sugar or in the presence of glucose and fructose. Notably, both colonization and further growth on the apatitic surface were dependent on sugar-mediated aggregation, leading to biofilms with distinctive spatial organizations and 3D architectures. Starch+sucrose and sucrose-mediated aggregates developed into large and highly acidogenic biofilms with complex network of bacterial and fungal cells (yeast and hyphae) surrounded by an intricate matrix of extracellular glucans. In contrast, biofilms originated from glucose and fructose-mediated consortia (or without sugar) were sparsely distributed on the surface without structural integration, growing predominantly as individual species with reduced acidogenicity. These findings reveal the impact of dietary sugars on inter-kingdom interactions in saliva and how they mediate biofilm formation with distinctive structural organization and varying acidogenicity implicated with human tooth-decay.

Effect of Bacteriocin-Like Inhibitory Substance (BLIS) from Enterococcus faecium DB1 on Cariogenic Streptococcus mutans Biofilm Formation.

The aim of the study was to investigate the effect of bacteriocin-like inhibitory substance (BLIS) from Enterococcus faecium DB1 on cariogenic Streptococcus mutans biofilm. Crystal violet staining, fluorescence, and scanning electron microscopy analyses demonstrated that the BLIS from Enterococcus faecium DB1 (DB1 BLIS) inhibited S. mutans biofilm. When DB1 BLIS was co-incubated with S. mutans, biofilm formation by S. mutans was significantly reduced (p<0.05). DB1 BLIS also destroyed the preformed biofilm of S. mutans. In addition, DB1 BLIS decreased the viability of S. mutans biofilm cells during the development of biofilm formation and in the preformed biofilm. DB1 BLIS significantly decreased the growth of S. mutans planktonic cells. Furthermore, S. mutans biofilm on the surface of saliva-coated hydroxyapatite discs was reduced by DB1 BLIS. Taken together, DB1 BLIS might be useful as a preventive and therapeutic agent against dental caries caused by S. mutans.