Effect of a genetically engineered bacteriophage on Enterococcus faecalis biofilms

ObjectiveEnterococcus faecalis is a Gram-positive, facultative anaerobic bacterium that is associated with failed endodontic cases and nosocomial infections. E. faecalis can form biofilms, penetrate dentinal tubules and survive in root canals with scarce nutritional supplies. These properties can make E. faecalis resistant to conventional endodontic disinfection therapy. Furthermore, treatment may be complicated by the fact that many E. faecalis strains are resistant to antibiotics. A potential alternative to antibiotic therapy is phage therapy. ϕEf11 is a temperate phage that infects strains of E. faecalis. It was previously sequenced and genetically engineered to modify its properties in order to render it useful as a therapeutic agent in phage therapy. In the current study, we have further genetically modified the phage to create phage ϕEf11/ϕFL1C(Δ36)^PnisA. The aim of this study was to evaluate the efficacy of bacteriophage ϕEf11/ϕFL1C(Δ36)^PnisA, to disrupt biofilms of two Enterococcus faecalis strains: JH2-2 (vancomycin-sensitive) and V583 (vancomycin-resistant).Methods24 h static biofilms of E. faecalis strains JH2-2(pMSP3535 nisR/K) and V583 (pMSP3535nisR/K), formed on cover slips, were inoculated with bacteriophage ϕEf11/ϕFL1C(Δ36)^PnisA. After 24 and 48 h incubation, the bacterial biomass was imaged by confocal microscopy and viable cells were quantified by colony forming unit measurement.ResultsThe results showed a 10-100-fold decrease in viable cells (CFU/biofilm) after phage treatment, which was consistent with comparisons of treated and untreated biofilm images visualized as max projections of the Z-series.ConclusionThe biomass of both vancomycin-sensitive and vancomycin-resistant E. faecalis biofilms is markedly reduced following infection by bacteriophage ϕEf11/ϕFL1C(Δ36)^PnisA.     

Effect of arginine on the growth and biofilm formation of oral bacteria

BackgroundAlkali production via arginine deiminase system (ADS) of oral bacteria plays a significant role in oral ecology, pH homeostasis and inhibition of dental caries. ADS activity in dental plaque varies greatly between individuals, which may profoundly affect their susceptibility to caries.ObjectiveTo investigate the effect of arginine on the growth and biofilm formation of oral bacteria.Methods and resultsPolymicrobial dental biofilms derived from saliva were formed in a high-throughput active attachment biofilm model and l-arginine (Arg) was shown to reduce the colony forming units (CFU) counts of such biofilms grown for various periods or biofilms derived from saliva of subjects with different caries status. Arg hardly disturbed bacterial growth of Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus gordonii in BHI medium, but only inhibited biofilm formation of S. mutans. Scanning electron microscope (SEM) showed S. mutans biofilms harboured fewer cells grown with Arg than that without Arg, even in the initial 2 h and 8 h phase. Confocal laser scanning microscope (CLSM) images of poly-microbial dental and S. mutans biofilms revealed the biofilms grown with Arg had lower exopolysaccharide (EPS)/bacteria ratios than those without Arg (P = 0.004, 0.002, respectively). Arg could significantly reduce the production of water-insoluble EPS in S. mutans biofilms (P < 0.001); however, quantitative real-time PCR (qRT-PCR) did not show significantly influence in gene expression of gtfB, gtfC or gtfD (P = 0.32, 0.06, 0.44 respectively).ConclusionsArg could reduce the biomass of poly-microbial dental biofilms and S. mutans biofilms, which may be due to the impact of Arg on water-insoluble EPS. Considering the contribution to pH homeostasis in dental biofilms, Arg may serve as an important agent keeping oral biofilms healthy thus prevent dental caries.     

Thymus vulgaris L. extract has antimicrobial and anti-inflammatory effects in the absence of cytotoxicity and genotoxicity

ObjectivesThis study evaluated the biological effects of the T. vulgaris L. extract., such as antimicrobial activity on planktonic cultures and mono- and polymicrobial biofilms, cytotoxicity, anti-inflammatory activity and genotoxicity.MethodsMonomicrobial biofilms of Candida albicans, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutans and Pseudomonas aeruginosa and polymicrobial biofilms composed by C. albicans with each bacterium were formed for 48 h and exposed for 5 min to the plant extract. Murine macrophages (RAW 264.7), human gingival fibroblasts (FMM-1), human breast carcinoma cells (MCF-7) and cervical carcinoma cells (HeLa) were also exposed to the plant extract for 5 min and the cell viability were analyzed by MTT, neutral red (NR) and crystal violet (CV) assays. Interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) produced by RAW 264.7 was quantified by ELISA, after 24 h exposure to the plant extract, both in the absence and presence of lipopolysaccharide (LPS) from Escherichia coli. Genotoxicity of the plant extract was evaluated by micronucleus formation (MN) in 1000 cells. The results were analyzed by T-Test or ANOVA and Tukey’s Test (P ≤ 0.05).ResultsAll biofilms showed significant reductions in CFU/mL (colony-forming units per milliliter). Cell viability was above 50% for all cell lines. Anti-inflammatory effect on the synthesis of IL-1β and TNF-α was observed. The MN was similar or lower than the control group in all cells.ConclusionsT. vulgaris L. extract was effective against all biofilms, promoted high cell viability, anti-inflammatory effect and presented no genotoxicity.     

Candida tropicalis biofilm inhibition by ZnO nanoparticles and EDTA

ObjectiveBiofilm of Candida tropicalis denote as a complex cellular congregation with major implication in pathogenesis. This lifestyle of fungus as a biofilm can inhibit immune system and antifungal therapy in treatment of infectious disease especially medical device associated chronic disease. In this study effects of Zinc Oxide (ZnO) nanoparticles and EDTA were evaluated on C. tropicalis biofilm by using different techniques. ZnO nanoparticles were synthesized from Egg albumin.DesignTo assay the formation of biofilm of yeast cells like Fluconazole-susceptible C. tropicalis (ATCC 13,803) and fluconazole-resistant standard strains of C. tropicalis (ATCC 750) were grown in 24 well plates and antifungal effect of ZnO and EDTA were evaluated on C. tropicalis biofilm using ATP bioluminescence and tetrasodium salt (XTT) reduction assays.ResultsSynthesized ZnO NPs and EDTA had effective antifungal properties at the concentration of 5.2, 8.6 μg/ml for Fluconazole susceptible strain and 5.42, 10.8 μg/ml Fluconazole resistant strains of C. tropicalis biofilms compared to fluconazole drug.ConclusionIn present study we conclude, ZnO considered as a new agent in field of prevention C. tropicalis biofilms especially biofilms formed surface of medical device.     

Salivary pellicles equalise surfaces’ charges and modulate the virulence of Candida albicans biofilm

IntroductionNumerous environmental factors influence the pathogenesis of Candida biofilms and an understanding of these is necessary for appropriate clinical management.AimsTo investigate the role of material type, pellicle and stage of biofilm development on the viability, bioactivity, virulence and structure of C. albicans biofilms.MethodsThe surface roughness (SR) and surface free energy (SFE) of acrylic and titanium discs was measured. Pellicles of saliva, or saliva supplemented with plasma, were formed on acrylic and titanium discs. Candida albicans biofilms were then generated for 1.5 h, 24 h, 48 h and 72 h. The cell viability in biofilms was analysed by culture, whilst DNA concentration and the expression of Candida virulence genes (ALS1, ALS3 and HWP1) were evaluated using qPCR. Biofilm metabolic activity was determined using XTT reduction assay, and biofilm structure analysed by Scanning Electron Microscopy (SEM).ResultsWhilst the SR of acrylic and titanium did not significantly differ, the saliva with plasma pellicle increased significantly the total SFE of both surface. The number of viable microorganisms and DNA concentration increased with biofilm development, not differing within materials and pellicles. Biofilms developed on saliva with plasma pellicle surfaces had significantly higher activity after 24 h and this was accompanied with higher expression of virulence genes at all periods.ConclusionInduction of C. albicans virulence occurs with the presence of plasma proteins in pellicles, throughout biofilm growth. To mitigate such effects, reduction of increased plasmatic exudate, related to chronic inflammatory response, could aid the management of candidal biofilm-related infections.     

Novel root canal sealer with dimethylaminohexadecyl methacrylate,nano-silver and nano-calcium phosphate to kill bacteria inside root dentin and increase dentin hardness

ObjectivesRoot canal re-infection and weakening of roots are two main challenges in endodontics. The objectives of the study were: (1) to develop a novel root canal sealer containing dimethylaminohexadecyl methacrylate (DMAHDM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP), and (2) to investigate the effects on the physical, anti-biofilm, remineralizing ions, and hardness of human dentin for the first time.MethodsMethacrylate-resin dual-cured root canal sealer contained 5% DMAHDM, 0.15% NAg, and NACP at 10%, 20% and 30% mass fractions. The flow, film thickness, and Ca and P ions release were investigated. The effects of NACP on radicular dentin hardness after treatment with sodium hypochlorite (NaOCL) and ethylenediaminetetraacetic acid (EDTA) were assessed. Antibacterial properties were measured against Enterococcus faecalis (E. faecalis)-impregnated dentin blocks; colony-forming units (CFU) and live/dead assays were measured.ResultsIncorporating DMAHDM, NAg and NACP did not adversely influence the flow and film thickness properties. Sealer with 30% NACP neutralized the acid and increased the solution pH (p < 0.05). Sealer containing 30% NACP regenerated dentin minerals lost due to NaOCL and EDTA treatment, and increased the dentin hardness to match that of sound dentin (p > 0.1). Incorporating 5% DMAHDM and 0.15% NAg reduced biofilm CFU of E. faecalis-impregnated dentin blocks by nearly 3 logs when compared control group (p < 0.05).SignificanceThe novel therapeutic root canal sealer with triple bioactive agents of DMAHDM, NAg and NACP neutralized acid, raised the pH, regenerated dentin minerals, increased root dentin hardness, and reduced dentin-block-impregnated biofilm CFU by 3 logs. This new sealer with highly desirable antibacterial and remineralization properties are promising to increase the success rate of endodontic therapy and strengthen the tooth root structures.     

Influence of Streptococcus mutans on Enterococcus faecalis Biofilm Formation

IntroductionAn important virulence factor of Enterococcus faecalis is its ability to form biofilms. Most studies on biofilm formation have been carried out by using E. faecalis monocultures. Given the polymicrobial nature of root canal infections, it is important to understand biofilm formation of E. faecalis in the presence of other microorganisms.MethodsEight clinical strains of E. faecalis were tested for biofilm formation on hydroxyapatite disks in the presence and absence of a Streptococcus mutans biofilm.ResultsSignificantly more E. faecalis viable cells were found in biofilms in the presence of S. mutans. This phenomenon was, however, strain-dependent. Of the 8 strains tested, biofilm formation of strains AA-OR34, ER5/1, and V583 was not influenced by S. mutans biofilms.ConclusionsThe results from this study, especially the strain difference, underline the importance of studying biofilm formation in a more realistic multispecies setting.     

Alkaline Tolerance and Biofilm Formation of Root Canal Isolates of Enterococcus faecalis: An In Vitro Study

IntroductionThe aim of this study was to compare the effects of glucose and glycerol (provided as principal fermentable supplements) on alkaline tolerance and biofilm-forming capabilities of root canal–derived strains of Enterococcus faecalis and those from other environments.MethodsThe planktonic growth kinetics and the biofilm-forming capabilities of E. faecalis isolates (identified by 16S ribosomal DNA sequencing) were compared when supplied with glucose and glycerol at pH levels of 8 and 11 in a microtiter plate. The metabolic activity of the biofilms plate that formed at a neutral pH level (supplied with either glucose or glycerol) was measured after subsequent adjustment to a pH level of 11.ResultsTen isolates (7 from root canals and 3 from other sources) were examined. The lag phase and the doubling time increased under elevated alkalinity irrespective of either the fermentable supplement (glucose or glycerol) or the origin of the isolate. Biofilm formation and metabolic activity varied among strains, but neither was related to the source of isolation. In general, biofilm formation was enhanced when grown in glucose compared with glycerol and at a pH of 8 compared with a pH of 11 (irrespective of the fermentable supplement). The provision of glycerol did not increase either the planktonic growth rate or biofilm development compared with glucose but significantly increased the metabolic activity of biofilms, especially at a pH of 11 compared with a pH of 8.ConclusionsIn the nutrient-deprived environment of a necrotic or root-filled root canal, glycerol may be an alternative energy source that can promote increased metabolic activity of E. faecalis under alkaline treatment conditions.     

Residual antibiofilm effects of various concentrations of double antibiotic paste used during regenerative endodontics after different application times

ObjectiveWe investigated the residual antibiofilm effects of different concentrations of double antibiotic paste (DAP) applied on radicular dentin for 1 or 4 weeks.DesignDentin samples were prepared (n = 120), sterilized and pretreated for 1 or 4 weeks with the clinically used concentration of DAP (500 mg/mL), low concentrations of DAP (1, 5 or 50 mg/mL) loaded into a methylcellulose system, calcium hydroxide (Ca(OH)₂), or placebo paste. After the assigned treatment time, treatment pastes were rinsed off and the samples were kept independently in phosphate buffered saline for 3 weeks. Pretreated dentin samples were then inoculated with Enterococcus faecalis and bacterial biofilms were allowed to grow for an additional 3 weeks. Biofilms were then retrieved from dentin using biofilm disruption assays, diluted, spiral plated, and quantified. Fisher’s Exact and Wilcoxon rank sum tests were used for statistical comparisons (α=0.05).ResultsDentin pretreatment for 4 weeks with 5, 50 or 500 mg/mL of DAP demonstrated significantly higher residual antibiofilm effects and complete eradication of E. faecalis biofilms in comparison to a 1 week pretreatment with similar concentrations. However, dentin pretreated with 1 mg/mL of DAP or Ca(OH)₂ did not provide a substantial residual antibiofilm effect regardless of the application time.ConclusionsDentin pretreatment with 5 mg/mL of DAP or higher for 4 weeks induced significantly higher residual antibiofilm effects in comparison to a 1 week pretreatment with the same concentrations.     

Antibacterial and antibiofilm efficacy of k21-E in root canal disinfection

ObjectivesThe aim of the current project was to study the antimicrobial efficacy of a newly developed irrigant, k21/E against E. faecalis biofilm.MethodsRoot canals were instrumented and randomly divided into the following groups: irrigation with saline, 6% NaOCl (sodium hypochlorite), 6% NaOCl + 2% CHX (Chlorhexidine), 2% CHX, 0.5% k21/E (k21 - quaternary ammonium silane) and 1% k21/E. E. faecalis were grown (3-days) (1 × 10⁷ CFU mL⁻¹), treated, and further cultured for 11-days. Specimens were subjected to SEM, confocal and Raman analysis and macrophage vesicles characterized along with effect of lipopolysaccharide treatment. 3T3 mouse-fibroblasts were cultured for alizarin-red with Sortase-A active sites and Schrödinger docking was performed. TEM analysis of root dentin substrate with matrix metalloproteinases profilometry was also included. A cytotoxic test analysis for cell viability was measured by absorbance of human dental pulp cells after exposure to different irrigant solutions for 24 h. The test percentages have been highlighted in Table 1.ResultsAmong experimental groups, irrigation with 0.5% k21/E showed phase separation revealing significant bacterial reduction and lower phenylalanine 1003 cm⁻¹ and Amide III 1245 cm⁻¹ intensities. Damage was observed on bacterial cell membrane after use of k21/E. No difference in exosomes distribution between control and 0.5%k21/E was observed with less TNFα (*p < 0.05) and preferential binding of SrtA. TEM images demonstrated integrated collagen fibers in control and 0.5%k21/E specimens and inner bacterial membrane damage after k21/E treatment. The k21 groups appeared to be biocompatible to the dental pulpal cells grown for 24 h.SignificanceCurrent investigations highlight potential advantages of 0.5% k21/E as irrigation solution for root canal disinfection.     

Effect of different pre-irradiation times on curcumin-mediated photodynamic therapy against planktonic cultures and biofilms of Candida spp

ObjectivesThe aim of this study was to evaluate the effects of pre-irradiation time (PIT) on curcumin (Cur)-mediated photodynamic therapy (PDT) against planktonic and biofilm cultures of reference strains of Candida albicans, Candida glabrata and Candida dubliniensis.Materials and methodsSuspensions and biofilms of Candida species were maintained in contact with different concentrations of Cur for time intervals of 1, 5, 10 and 20 min before irradiation and LED (light emitting diode) activation. Additional samples were treated only with Cur, without illumination, or only with light, without Cur. Control samples received neither light nor Cur. After PDT, suspensions were plated on Sabouraud Dextrose Agar, while biofilm results were obtained using the XTT-salt reduction method. Confocal Laser Scanning Microscopy (CLSM) observations were performed to supply a better understanding of Cur penetration through the biofilms after 5 and 20 min of contact with the cultures.ResultsDifferent PITs showed no statistical differences in Cur-mediated PDT of Candida spp. cell suspensions. There was complete inactivation of the three Candida species with the association of 20.0 μM Cur after 5, 10 and 20 min of PIT. Biofilm cultures showed significant reduction in cell viability after PDT. In general, the three Candida species evaluated in this study suffered higher reductions in cell viability with the association of 40.0 μM Cur and 20 min of PIT. Additionally, CLSM observations showed different intensities of fluorescence emissions after 5 and 20 min of incubation.ConclusionPhotoinactivation of planktonic cultures was not PIT-dependent. PIT-dependence of the biofilm cultures differed among the species evaluated. Also, CLSM observations confirmed the need of higher time intervals for the Cur to penetrate biofilm structures.     

Effect of the quorum‐sensing luxS gene on biofilm formation by Enterococcus faecalis

Enterococcus faecalis is the species of bacterium most frequently isolated from the root canals of teeth that exhibit chronic apical periodontitis refractory to endodontic treatment. In this study, we evaluated the effect of the S‐ribosylhomocysteine lyase (luxS) quorum‐sensing gene on E. faecalis biofilm formation by constructing a knockout mutant. The biofilms formed by both E. faecalis and its luxS mutant strain were evaluated using the MTT method. Important parameters that influence biofilm formation, including cell‐surface hydrophobicity and the nutrient content of the growth medium, were also studied. Biofilm structures were observed using confocal laser scanning microscopy (CLSM), and expression of biofilm‐related genes was investigated using RT‐PCR. The results showed that the luxS gene can affect biofilm formation, whereas it does not affect the bacterial growth rate. Deletion of the luxS gene also increased cell‐surface hydrophobicity. Biofilm formation was accelerated by the addition of increasing concentrations of glucose. The CLSM images revealed that the luxS mutant strain tends to aggregate into distinct clusters and relatively dense structures, whereas the wild‐type strain appears confluent and more evenly distributed. All genes examined were up‐regulated in the biofilms formed by the luxS mutant strain. The quorum‐sensing luxS gene can affect E. faecalis biofilm formation.     

Hydrophobicity of graphene as a driving force for inhibiting biofilm formation of pathogenic bacteria and fungi

Objective

To evaluate the surface and wettability characteristics and the microbial biofilm interaction of graphene coating on titanium.

Nano-calcium phosphate and dimethylaminohexadecyl methacrylate adhesive for dentin remineralization in a biofilm-challenged environment

ObjectiveDentin remineralization at the bonded interface would protect it from external risk factors, therefore, would enhance the longevity of restoration and combat secondary caries. Dental biofilm, as one of the critical biological factors in caries formation, should not be neglected in the assessment of caries preventive agents. In this work, the remineralization effectiveness of demineralized human dentin in a multi-species dental biofilm environment via an adhesive containing nanoparticles of amorphous calcium phosphate (NACP) and dimethylaminohexadecyl methacrylate (DMAHDM) was investigated.MethodsDentin demineralization was promoted by subjecting samples to a three-species acidic biofilm containing Streptococcus mutans, Streptococcus sanguinis, Streptococcus gordonii for 24 h. Samples were divided into a control group, a DMAHDM adhesive group, an NACP group, and an NACP + DMAHDM adhesive group. A bonded model containing a control-bonded group, a DMAHDM-bonded group, an NACP-bonded group, and an NACP + DMAHDM-bonded group was also included in this study. All samples were subjected to a remineralization protocol consisting of 4-h exposure per 24-h period in brain heart infusion broth plus 1% sucrose (BHIS) followed by immersion in artificial saliva for the remaining period. The pH of BHIS after 4-h immersion was measured every other day. After 14 days, the biofilm was assessed for colony-forming unit (CFU) count, lactic acid production, live/dead staining, and calcium and phosphate content. The mineral changes in the demineralized dentin samples were analyzed by transverse microradiography.ResultsThe in vitro experiment results showed that the NACP + DMAHDM adhesive effectively achieved acid neutralization, decreased biofilm colony-forming unit (CFU) count, decreased biofilm lactic acid production, and increased biofilm calcium and phosphate content. The NACP + DMAHDM adhesive group had higher remineralization value than the NACP or DMAHDM alone adhesive group.SignificanceThe NACP + DMAHDM adhesive was effective in remineralizing dentin lesion in a biofilm model. It is promising to use NACP + DMAHDM adhesive to protect bonded interface, inhibit secondary caries, and prolong the longevity of restoration.     

Effects of S. mutans gene-modification and antibacterial monomer dimethylaminohexadecyl methacrylate on biofilm growth and acid production

ObjectivesAntibacterial quaternary ammonium monomers (QAMs) are used in resins. The rnc gene in Streptococcus mutans (S. mutans) plays a key role in resisting antibiotics. The objectives of this study were to investigate for the first time: (1) the effects of rnc deletion on S. mutans biofilms and acid production; (2) the combined effects of rnc deletion with dimethylaminohexadecyl methacrylate (DMAHDM) on biofilm-inhibition efficacy.MethodsParent S. mutans strain UA159 (ATCC 700610) and the rnc-deleted S. mutans were used. Bacterial growth, minimum inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) were measured to analyze the bacterial susceptibility of the parent and rnc-deleted S. mutans against DMAHDM, with the gold-standard chlorhexidine (CHX) as control. Biofilm biomass, polysaccharide and lactic acid production were measured.ResultsThe drug-susceptibility of the rnc-deleted S. mutans to DMAHDM or CHX was 2-fold higher than parent S. mutans. The drug-susceptibility did not increase after 10 passages (p < 0.05). Deleting the rnc gene increased the biofilm susceptibility to DMAHDM or CHX by 2-fold. The rnc-deletion in S. mutans reduced biofilm biomass, polysaccharide and lactic acid production, even at no drugs. DMAHDM was nearly 40 % more potent than the gold-standard CHX. The combination of rnc deletion + DMAHDM treatment achieved the greatest reduction in biofilm biomass, polysaccharide synthesis, and lactic acid production.SignificanceGene modification by deleting the rnc in S. mutans reduced the biofilm growth and acid production, and the rnc deletion + DMAHDM method showed the greatest biofilm-inhibition efficacy, for the first time. The dual strategy of antibacterial monomer + bacterial gene modification shows great potential to control biofilms and inhibit caries.     

High speed imaging of biofilm removal from a dental implant model using ultrasonic cavitation

ObjectivesCurrent instruments cannot clean in between dental implant threads and effectively remove biofilm from the rough implant surface without damaging it. Cavitation bubbles have the potential to disrupt biofilms. The aim of this study was to see how biofilms can be disrupted using non-contact cavitation from an ultrasonic scaler, imaged inside a restricted implant pocket model using high speed imaging.MethodsStreptococcus sanguinis biofilm was grown for 7 days on dental implants. The implants were placed inside a custom made restricted pocket model and immersed inside a water tank. An ultrasonic scaler tip was placed 0.5 mm away from the implant surface and operated at medium power or high power for 2 s. The biofilm removal process was imaged using a high speed camera operating at 500 fps. Image analysis was used to calculate the amount of biofilm removed from the high speed images. Scanning electron microscopy was done to visualize the implant surface after cleaning.ResultsCavitation was able to remove biofilm from dental implants. More biofilm was removed at high power. Scanning electron microscopy showed that the implant surface was clean at the points where the cavitation was most intense. High speed imaging showed biofilm removal underneath implant threads, in areas next to the ultrasonic scaler tip.SignificanceA high speed imaging protocol has been developed to visualize and quantify biofilm removal from dental implants in vitro. Cavitation bubbles from dental ultrasonic scalers are able to successfully disrupt biofilm in between implant threads.     

Comparative effect of a stannous fluoride toothpaste and a sodium fluoride toothpaste on a multispecies biofilm

ObjectivesThis paper aimed to compare the mode of action of a stannous fluoride-containing toothpaste with a conventional sodium fluoride-containing toothpaste on anti-biofilm properties.MethodsA three-species biofilm model that consists of Streptococcus mutans, Streptococcus sanguinis and Porphyromonas gingivalis was established to compare the anti-biofilm properties of a stannous fluoride-containing toothpaste (CPH), a conventional sodium fluoride-containing toothpaste (CCP) and a negative control (PBS). The 48 h biofilms were subjected to two-minute episodes of treatment with test agents twice a day for 5 consecutive days. Crystal violet staining and XTT assays were used to evaluate the biomass and viability of the treated biofilm. Live/dead staining and bacteria/extracellular polysaccharides (EPS) double-staining were used to visualize the biofilm structure and to quantify microbial/extracellular components of the treated biofilms. Species-specific fluorescent in situ hybridization and quantitative polymerase chain reaction (qPCR) were used to analyze microbial composition of the biofilms after treatment.ResultsThe biomass and viability of the biofilms were significantly reduced after CPH toothpaste treatment. The inhibitory effect was further confirmed by the live/dead staining. The EPS amounts of the three-species biofilm were significantly reduced by CCP and CPH treatments, and CPH toothpaste demonstrated significant inhibition on EPS production. More importantly, CPH toothpaste significantly suppressed S. mutans and P. gingvalis, and enriched S. sanguinis in the three-species biofilm. In all experiments CPH had a significantly greater effect than CCP (p < 0.05) and CCP had a greater effect than PBS (p < 0.05).ConclusionsStannous fluoride-containing toothpaste not only showed better inhibitory effect against oral microbial biofilm, but was also able to modulate microbial composition within multi-species biofilm compared with conventional sodium fluoride-containing toothpaste.     

Is it really penetration? Locomotion of devitalized Enterococcus faecalis cells within dentinal tubules of bovine teeth

ObjectiveThe aim of the present study was to evaluate the penetration characteristics of devitalized and vital E. faecalis cells into root dentinal tubules.DesignThirteen root canals were incubated with devitalized (4 days, 7 days, 14 days, 28 days) and vital (28 days) E. faecalis strains (streptomycin-resistant strains) after root canal enlargement (size 80, taper 0.02) with 3 % NaOCl solution. The smear layer was intentionally removed with 20 % EDTA before inoculation. Samples were processed for analysis by scanning electron microscopy (SEM) and DAPI (4′,6-diamidino-2-phenylindole) staining. DAPI was conducted for fluorescence microscopic visualization of the bacterial penetration into dentinal tubules. The penetration depth was calculated with the measurement tool of the Axio Vision program (Zeiss, Jena, Germany).ResultsDevitalized E. faecalis strains were able to penetrate into dentinal tubules of the root canal. Apikal penetration depths of the devitalized cells were 100.67 μm ± 26.54 μm after 7 days, 230.67 μm ± 111.5 μm after 14 days and 266.5 μm ± 92.63 μm after 28 days of incubation. The total number and penetration depth of E. faecalis cells was lower compared to a vital suspension of E. faecalis (1002.45 μm) after 28 days. It was noted that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control.ConclusionsIncreased exposure times of devitalized bacteria into root canals lead to an increased number of penetrated dentinal tubules as well as to a deeper penetration.     

Biofilm formation on denture liners in a randomised controlled in situ trial

ObjectivesThis randomised clinical trial assessed how biofilm development and composition is affected by time and denture material type in denture wearers with and without denture stomatitis.MethodsSpecimens of acrylic resin (control) and denture liners (silicone-based or acrylic resin based, depending on the experimental phase) were inserted into the surface intaglio of 30 denture wearers. Biofilm was formed in two phases of 21 days, and counts of viable micro-organisms in the accumulating biofilm were determined after 7, 14 and 21days of biofilm formation. Data were analysed by three-way ANOVA followed by Tukey test to assess differences among health condition (healthy or with denture stomatitis), materials and time point.ResultsNon-albicans Candida species counts were higher in diseased patients with silicone-based denture liners (p = 0.01). Denture stomatitis patients showed higher mutans streptococci counts after 7 days (p = 0.0041).ConclusionsLonger biofilm formation time periods did not result in differences on biofilm composition. The denture liners evaluated in this study accumulate greater amount of biofilm, and therefore their use should be carefully planned.Clinical significanceThe silicone-based denture liner tested should be used cautiously in patients with denture stomatitis as it showed increased non-albicans species counts, known to be difficult to treat.     

De novo synthetic short antimicrobial peptides against cariogenic bacteria

ObjectiveAntimicrobial peptides (AMPs) have shown the ability to inhibit planktonic bacteria and biofilms. The objectives of this study were to de novo design and synthesize a series of cationic, amphipathic α-helical AMPs that would be shorter, less cytotoxic, and more potent than existing AMPs against cariogenic bacteria.DesignThree short AMPs (GH8, GLLWHLLH-NH₂; GH12, GLLWHLLHHLLH-NH₂; and GH16, GLLWHLLHHLLHLLHH-NH₂) were designed, synthesized and characterized structurally. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against eight major cariogenic bacteria were tested to select the most promising peptide. Scanning electron microscopy (SEM) was used to observe the bacterial membrane after treatment with selected peptides. The bactericidal kinetics, effects on biofilm and cytotoxity were further investigated.ResultsOf the three AMPs, GH12 had the most balanced structural parameters and a high content of α-helical structure. GH12 had a MIC of 4.0-8.0 μg/mL and MBC of 8.0-32.0 μg/mL. The corresponding values for the other two AMPs were 2- to 64- fold higher. In time-kill assays, GH12 killed all bacterial strains within 60 min at 4- fold MBC. SEM observed lysis and pore formation of the cytomembrane after treatment with GH12. 8.0 μg/mL GH12 inhibited Streptococcus mutans biofilm formation. Confocal laser scanning microscopy showed that GH12 effectively reduced the biomass of 1-day-old S. mutans biofilm. Cytotoxicity assays indicated that GH12 showed little toxic effect on the viability of human gingival fibroblasts.ConclusionThese results indicate that GH12 shows antimicrobial activity against cariogenic bacteria and biofilms in vitro.