Fungicidal and inhibitory efficacy of cinnamon and lemongrass essential oils on Candida albicans biofilm established on acrylic resin: An in vitro study

Statement of problemIt is unclear whether cinnamon and lemongrass essential oils can effectively reduce the Candida-biofilm frequently formed on dental devices made from heat-polymerized polymethyl methacrylate (PMMA) resin that contributes to the development of mild oropharyngeal as well as life-threatening candidiasis in patients wearing the devices.PurposeThe purpose of this in vitro study was to determine the efficacy of cinnamon and lemongrass essential oils in eradicating Candida albicans biofilm on heat-polymerized PMMA specimens and to determine whether they retard the formation of fungal biofilm.Material and methodsThe antifungal effect of cinnamon and lemongrass essential oils was determined by using agar disk diffusion and broth microdilution methods to obtain minimum inhibitory concentrations. The mature C albicans biofilm (48 hours) was pre-established on PMMA specimens before being individually treated with various concentrations (½, 1, 2, 4, 8, 16 times minimum inhibitory concentration) of each tested oil for different exposure times (1, 2, 4, 8, and 24 hours). In another experiment, fungal biofilm was established on the PMMA specimens that were primed individually with various concentrations of the tested oils for different times. The 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)-reduction assay was used to quantitate biofilm viability in both experiments. Statistical analysis was performed by using the 1-sample Kolmogorov-Smirnov test and 2-way ANOVA followed by the Tukey multiple comparison test (α=.05).ResultsMinimum inhibitory concentration values of cinnamon and lemongrass essential oils against planktonic C albicans were 0.1 μL/mL (0.01% v/v) and 0.4 μL/mL (0.04% v/v). At 8 times the minimum inhibitory concentration, cinnamon oil (0.8 μL/mL or 0.08% v/v) and lemongrass oil (3.2 μL/mL or 0.32% v/v) eradicated the pre-established fungal biofilm by 99.0% in an exposure time of 1 hour. In contrast, high concentrations of 8 and 16 times the minimum inhibitory concentration of cinnamon oil (0.8 μL/mL or 0.08% v/v) and lemongrass oil (6.4 μL/mL or 0.64% v/v) coated on PMMA specimens for 24 hours were only able to inhibit the formation of fungal biofilm by approximately 70.0%.ConclusionsCinnamon and lemongrass essential oils can eliminate pre-established C albicans biofilm and restrain the formation of fungal biofilm on heat-polymerized PMMA specimens. Both effects of the tested essential oils depended on dose and exposure or priming time.     

Relationships between Solobacterium moorei and Prevotella intermedia in subgingival microbiota of periodontitis patients with halitosis: A preliminary study using qPCR

ObjectiveSolobacterium moorei is suggested to be associated with the production of volatile sulphur compounds (VSCs) and can be found in subgingival plaques of deep periodontal pockets. We examined whether this bacterium’s count was reduced in periodontitis patients with halitosis following non-surgical periodontal treatment, while the bacterial count of Prevotella intermedia was measured simultaneously as a control.Material & methodsThis clinical study included 20 adults with chronic periodontitis who complained of halitosis. The bacterial relationship in the subgingival plaque sample was measured after 8 weeks post-treatment, including the probing pocket depth (PPD). Quantitative real-time PCR (qPCR) was used to measure the proportion of S. moorei, while the concentrations of H₂S and CH₃SH were determined using oral ChromaTM.ResultsThe presence of S. moorei was consistently observed in participants with periodontitis before and after non-surgical periodontal treatment and consistent showed a significantly lower proportion compared with P. intermedia. Solobacterium moorei showed a strong positive correlation with H₂S and CH₃SH concentrations, but a negative correlation with deep periodontal pocket measurements. Conversely, reduced P. intermedia may be more associated with a deep pocket, independent of the concentration of CH₃SH.ConclusionThe study data showed that the proportion of S. moorei in the subgingival biofilm can be related to halitosis in periodontitis patients.     

The antimicrobial activity of alpha-bisabolol and tea tree oil against Solobacterium moorei,a Gram-positive bacterium associated with halitosis

ObjectiveTo investigate the antimicrobial effect of alpha-bisabolol and tea tree oil alone and in combination against the halitosis-associated Gram-positive bacillus Solobacterium moorei.DesignThe inhibitory activity of alpha-bisabolol and tea tree oil against the reference strain S. moorei CCUG39336 and four clinical S. moorei isolates was investigated by a direct exposure test. Additionally, the ability of alpha-bisabolol to increase the sensitivity of S. moorei was tested by pretreating the bacteria with sublethal concentrations prior to the administration of tea tree oil.ResultsA dose-dependent killing was observed for the antimicrobial agents in a direct exposure test with the reference strain S. moorei CCUG39336. Concentrations of ≥0.5% tea tree oil caused decreases in viability of >5 log colony forming units/ml even after short incubation periods, while bacterial viability was less affected by alpha-bisabolol. The combination of 0.1% alpha-bisabolol plus 0.05% tea tree oil showed a synergistic effect on S. moorei strain CCUG39336 and on two of the four clinical S. moorei isolates tested. However, incubation of S. moorei with a sublethal concentration of 0.1% alpha-bisabolol for three days prior to the administration of 0.05% tea tree oil did not enhance the antibacterial effect of tea tree oil.ConclusionHalitosis-associated bacterium S. moorei is susceptible to the antimicrobial agents tea tree oil and alpha-bisabolol, suggesting that these compounds might be beneficial in oral healthcare products.     

Efficacy of citronella and cinnamon essential oils on Candida albicans biofilms

Introduction: The discovery of new antimicrobials derived from plants could aid in the management of biofilm-associated infections, including denture-induced stomatitis (DS). DS is an oral infection caused by Candida biofilms on the surfaces of poorly cleansed dentures. Effective treatment of DS requires the use of an appropriate denture cleanser and preferably one that exhibits antimicrobial properties.

Objective: This study aimed to evaluate the anti-Candida and anti-biofilm efficacy of two essential plant oils from Cymbopogon winterianus (citronella) and Cinnamon cassia (cinnamon).

Materials and methods: Minimum Inhibitory Concentrations (MICs) and Minimum Fungicidal Concentrations (MFCs) were determined by broth microdilution, whilst anti-biofilm activity was measured against mature (cultured for 72?h) biofilms on acrylic surfaces. Candida cell viability was assessed immediately (0?h) after treatment (T₀) and 48?h after biofilm re-growth (T₄₈). Biofilm structure was determined using Scanning Electron Microscopy (SEM) at T₀ and T₄₈.

Results: The respective MICs of cinnamon and citronella oils were 65 and 250?μg/ml and these were also the MFC values. For anti-biofilm efficacy, both oils significantly (p?0. However, at T₄₈, there was no difference between treated and untreated biofilms.

Conclusions: It is concluded that citronella and cinnamon essential oils have potential for daily anti-candidal denture cleansing.     

Inhibitory effects of tea catechin epigallocatechin-3-gallate against biofilms formed from Streptococcus mutans and a probiotic lactobacillus strain

ObjectiveEffects of tea catechin epigallocatechin-3-gallate (EGCG) against biofilm formation by Streptococcus mutans and probiotic Lactobacillus casei in Yakult^® (LcY) were examined.DesignBiofilms were formed by S. mutans alone (Sm) and co-culture of S. mutans and LcY (Sm + LcY) in the absence or presence of EGCG. The biomass of biofilms, which were sonicated or not, was measured by the crystal violet assay. Biofilm morphology was observed by scanning electron microscopy. Bacterial viability and extracellular polysaccharides were determined by SYTO9/propidium iodide and dextran-conjugated fluorescein staining, respectively, and confocal microscopy. Gene expression of glucosyltransferase was determined by quantitative polymerase chain reaction.ResultsWhile 250 μg/ml EGCG significantly decreased the biomass and acid production of Sm biofilms, 500 μg/ml EGCG was required to inhibit Sm + LcY biofilm formation and acid production. EGCG decreased the amount of live bacteria present in both Sm and Sm + LcY biofilms. The level of dead bacteria in Sm + LcY biofilms was higher than in Sm biofilms when formed in the presence of 250 μg/ml EGCG. EGCG decreased levels of extracellular polysaccharides in Sm and Sm + LcY biofilms. The extent of biofilm removal by sonication was not different between Sm and Sm+LcY biofilms formed in the absence or presence of 62.5 or 125 μg/ml EGCG. The level of Sm gtfB and gtfD expression in Sm + LcY biofilms was higher than those in the Sm biofilms when formed in the presence of EGCG at 250 μg/ml.ConclusionThe results indicated that LcY might interfere the inhibitory effects of EGCG against biofilm formation by S. mutans.     

Characterization of volatile sulfur compound production by Solobacterium moorei

Objective

Solobacterium moorei is a Gram positive bacterium that has been specifically associated with halitosis. The aim of this study was to characterize volatile sulfur compound (VSC) production by S. moorei.

Methods

S. moorei was either grown or incubated in the presence of various supplements prior to determining VSC production with a Halimeter sulfide monitor. The effect of exogenous proteases or glycosidase inhibitors on VSC production by S. moorei was examined.

Results

We first showed that S. moorei can convert cysteine into hydrogen sulfide. The capacity of S. moorei to produce VSCs from serum, saliva, and mucin was dependent on the presence of an exogenous source of proteases such as pancreatic trypsin or Porphyromonas gingivalis gingipains. VSC production from mucin was inhibited by the presence of a β-galactosidase inhibitor, thus suggesting that deglycosylation of mucin by S. moorei is critical for VSC production.

Conclusion

Our study suggests that S. moorei can be a major source of malodorous compounds in halitosis by producing VSCs through a process involving the β-galactosidase activity of the bacterium and an exogenous source of proteases.     

Novel pit and fissure sealant containing nano-CaF2 and dimethylaminohexadecyl methacrylate with double benefits of fluoride release and antibacterial function

ObjectivePit and fissure sealants with antibacterial and remineralization properties have broad application prospects in caries prevention. The objectives of this study were to: (1) develop a novel pit and fissure sealant containing CaF₂ nanoparticles (nCaF₂) and dimethylaminohexadecyl methacrylate (DMAHDM); and (2) investigate the effects of nCaF₂ and DMAHDM on biofilm response and fluoride (F) ion release for the first time.MethodsHelioseal F was used as a control. Bioactive sealants were formulated with DMAHDM and nCaF₂. Flow properties, enamel shear bond strength, hardness and F ion releases were measured. Streptococcus mutans (S. mutans) biofilms were grown on sealants. Biofilm metabolic activity, lactic acid production, colony-forming units (CFU), and pH of biofilm culture medium were measured.ResultsAdding 5% DMAHDM and 20% nCaF₂ did not reduce the paste flow and enamel bond strength, compared to control (p < 0.05). Hardness of sealants with 20% nCaF₂ and DMAHDM was higher than control (p < 0.05). The F ion release from 20% nCaF₂ was much higher than that of commercial control (p < 0.05). The sealant with DMAHDM reduced the S. mutans biofilm CFU by 4 logs. The pH in biofilm medium of the new bioactive sealant was much higher (pH 6.8) than that of commercial sealant (pH 4.66) (p < 0.05).SignificanceThe new bioactive pit and fissure sealant with nCaF₂ and DMAHDM achieved high fluoride release and strong antibacterial performance. This novel fluoride-releasing and antibacterial sealant is promising to inhibit caries and promote the remineralizaton of enamel and dentin.     

Antimicrobial activity of Streptococcus salivarius K12 on bacteria involved in oral malodour

ObjectiveTo investigate the antimicrobial activity of the bacteriocin-producing strain Streptococcus salivarius K12 against several bacteria involved in halitosis.DesignThe inhibitory activity of S. salivarius K12 against Solobacterium moorei CCUG39336, four clinical S. moorei isolates, Atopobium parvulum ATCC33793 and Eubacterium sulci ATCC35585 was examined by a deferred antagonism test. Eubacterium saburreum ATCC33271 and Parvimonas micra ATCC33270, which have been tested in previous studies, served as positive controls, and the Gram-negative strain Bacteroides fragilis ZIB2800 served as a negative control. Additionally, the occurrence of resistance in S. moorei CCUG39336 to S. salivarius K12 was analysed by either direct plating or by passage of S. moorei CCUG39336 on chloroform-inactived S. salivarius K12-containing agar plates.ResultsS. salivarius K12 suppressed the growth of all Gram-positive bacteria tested, but the extent to which the bacteria were inhibited varied. E. sulci ATCC35585 was the most sensitive strain, while all five S. moorei isolates were inhibited to a lesser extent. Natural resistance seems to be very low in S. moorei CCUG39336, and there was only a slight decrease in sensitivity after exposure to S. salivarius K12 over 10 passages.ConclusionOur studies demonstrate that S. salivarius K12 has antimicrobial activity against bacteria involved in halitosis. This strain might be an interesting and valuable candidate for the development of an antimicrobial therapy for halitosis.     

Role of Two-component System of Streptococcus mutans in the Adaptive Response to the Oral Environment

Biofilm is a colony-like aggregate consisting of microorganisms and polysaccharide (glycocalyx) that are produced by microorganisms and adhere to solid surfaces. In the oral cavity, biofilm, called dental plaque, is known as a virulence factor for dental caries. Biofilm is formed and adapts to various environmental factors in the oral cavity. Streptococcus mutans, a cariogenic bacteria, plays an important role in biofilm formation. In this study, we focused on a bacterial two-component system (TCS) as an environmental adaptation factor in S. mutans. We comprehensively evaluated the involvement of TCS in response to various factors associated with the oral cavity using 14 TCS deletion mutants of S. mutans. Furthermore, we evaluated the expression of each TCS in sucrose-dependent biofilm cells of S. mutans. Our findings suggested that multiple TCS play important roles promoting in resistance to environmental stresses and the formation of biofilm.     

Inhibitory effects of β-caryophyllene on Streptococcus mutans biofilm

ObjectiveThe biofilm of Streptococcus mutans is associated with induction of dental caries. Also, they produce glucan as an extracellular polysaccharide through glucosyltransferases and help the formation of cariogenic biofilm. β-caryophyllene has been used for therapeutic agent in traditional medicine and has antimicrobial activity. The purpose of this study was to investigate the effect of β-caryophyllene on S. mutans biofilm and the expression of biofilm-related factor.DesignThe susceptibility assay of S. mutans for β-caryophyllene was performed to investigate inhibitory concentration for S. mutans growth. To evaluated the effect of β-caryophyllene on S. mutans biofilm, β-caryophyllene was treated on S. mutans in the various concentrations before or after the biofilm formation. Live S. mutans in the biofilm was counted by inoculating on Mitis-salivarius agar plate, and S. mutans biofilm was analyzed by confocal laser scanning microscope after staining bacterial live/dead staining kit. Finally, the expression of glucosyltransferases of S. mutans was investigated by real-time RT-PCR after treating with β-caryophyllene at the non-killing concentration of S. mutans.ResultsThe growth of S. mutans was inhibited by β-caryophyllene in above concentration of 0.078%, S. mutans biofilm was inhibited by β-caryophyllene in above 0.32%. Also, 2.5% of β-caryophyllene showed anti-biofilm activity for S. mutans biofilm. β-caryophyllene reduced the expression of gtf genes at a non-killing concentration for S. mutans. On the basis on these results, β-caryophyllene may have anti-biofilm activity and the inhibitory effect on biofilm related factor.Conclusionsβ-caryophyllene may inhibit cariogenic biofilm and may be a candidate agent for prevention of dental caries.     

Effect of culture media and nutrients on biofilm growth kinetics of laboratory and clinical strains of Enterococcus faecalis

ObjectiveEnterococcus faecalis is a bacterial pathogen that is often associated with endodontic infections. Biofilm formation is a key virulence attribute in the pathogenicity of E. faecalis. In the present study, we comprehensively examined the effect of various culture media and nutrients on the development of E. faecalis biofilms.DesignA reference strain and a clinical isolate of E. faecalis were used in all experiments for comparison. Commonly used liquid culture media with different nutrient compositions were used to support the development of E. faecalis biofilms in a time-dependent assay. E. faecalis biofilms were quantified by colony forming unit (CFU) and crystal violet (CV) assays. Biofilm architecture and cellular viability were evaluated by scanning electron microscopy and confocal laser scanning microscopy.ResultsGrowth kinetics evaluated by CFU and CV assays and by microscopy showed that E. faecalis biofilms reached maturity at 72 h. “Pg broth” (Tryptic Soy Broth with yeast extract, hemen and vitamin K) promoted E. faecalis biofilm formation more than Brain Heart Infusion broth or Tryptic Soy Broth. Addition of 2% glucose enhanced biofilm formation. Thus, it seems that nutrients such as hemen, vitamin K and glucose are important for E. faecalis for the formation of biofilms.ConclusionThe present study demonstrated that nutrient-rich media containing glucose enhances the formation of E. faecalis biofilms, which exhibit maturation at 72 h.     

Effectiveness of Antibiotic Medicaments against Biofilm Formation of Enterococcus faecalis and Porphyromonas gingivalis

IntroductionIn this study we compared the antibacterial effect of triple antibiotic paste (TAP), double antibiotic paste (DAP), and calcium hydroxide [Ca(OH)₂] against Enterococcus faecalis and Porphyromonas gingivalis biofilm.MethodsThe minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and biofilm formation were measured by using microtiter plate methods. The 2 bacteria were treated with different dilutions of TAP, DAP, and Ca(OH)₂ solutions. The turbidities of the bacterial cultures in the microtiter plate were measured by optical density at 490 nm by using a spectrophotometer. Data were analyzed by 2-way analysis of variance (α = 0.05).ResultsFor TAP, the MIC and MBIC values were 0.003 mg/mL for E. faecalis and 0.006 mg/mL for P. gingivalis. The MBC values for TAP were 0.3 mg/mL for both bacteria. The MIC and MBIC values for DAP were 0.001 mg/mL for E. faecalis and P. gingivalis. The MBC values for DAP were 0.14 mg/mL for both bacteria. Biofilm formation of the 2 bacteria was significantly decreased with TAP and DAP at all tested dilutions (P < .0001) compared with control groups; however, TAP and DAP biofilm formations were not significantly different from each other. Ca(OH)₂ significantly decreased bacterial biofilm formation compared with the control, but it was significantly less than TAP and DAP (P < .05).ConclusionsBoth TAP and DAP were more effective than Ca(OH)₂ against E. faecalis and P. gingivalis. DAP can be considered an effective and comparable antibacterial substitute for TAP.     

The effects of orthodontic bonding steps on biofilm formation of Streptococcus mutans in the presence of saliva

Abstract

Objective. To investigate the effects of various orthodontic bonding steps on biofilm formation of Streptococcus mutans in the presence of saliva. Materials and methods: Hydroxyapatite (HA) and orthodontic adhesive (AD) disks were prepared to a uniform size. HA disks were etched with 37% phosphoric acid gel in the etched group (HE). In the primed group (HP), Transbond XT primer was applied to the etched HA surface and light-cured. For biofilm formation, Streptococcus mutans was grown on each specimen in a biofilm medium with either glucose or sucrose in the presence of fluid-phase UWS (F-UWS) or surface adsorbed saliva (S-UWS). The adherent bacteria were quantified by enumeration of the total viable counts of bacteria. Biofilms formed on each surface were examined by scanning electron microscopy. Results. When glucose was used, both F-UWS and S-UWS suppressed biofilm formation of S. mutans. Compared to HA and HE, biofilm formation was significantly inhibited on HP and AD in the presence of glucose. Biofilm-forming patterns that were inhibited by saliva were restored in a sucrose-containing medium. F-UWS promoted biofilm formation on HA and HE, while S-UWS significantly promoted biofilm formation on HP. S. mutans developed biofilm better on HA and HE than on AD when sucrose was used as the sole carbohydrate source. Conclusions. This study suggests that the biofilm development by S. mutans is significantly influenced by the orthodontic bonding procedure. Biofilm formation of S. mutans was inhibited on AD more than other surfaces, irrespective of the presence of saliva or a carbohydrate source.     

Characterization of the clustered regularly interspaced short palindromic repeats sites in Streptococcus mutans isolated from early childhood caries patients

ObjectiveThe aim of this study was to analyze the characteristics of the clustered regularly interspaced short palindromic repeats (CRISPR) sites in 45 clinical Streptococcus mutans strains and their relationship to the clinical manifestations of early childhood caries (ECC).MethodsForty-five S. mutans strains were isolated from the plaque samples taken from sixty-three children. CRISPR sites were sequenced and BLAST was used to compare these sites to those in the CRISPRTarget database. The association between the distribution of CRISPR sites and the manifestation of caries was analyzed by Chi-Square test. Further, biofilm formation (by crystal violet staining) and the synthesis of polysaccharide (by anthrone-sulfuric method) of all clinical isolated S. mutans strains with both CRISPR sites and no CRISPR site were comapared. Finally, acidogenicity and acidurity of two typical strains were determined using pH drop and acid tolerance assays. Biofilm formation and EPS synthesis by two typical strains were compared by 3D CLSM (Confocal Laser Scanning Microscope) assays and the expression of gtf genes were evaluated using qPCR.ResultsWe found that most of the spacers in the clinical S. mutans strains were derived from Streptococcus phages APCM01 and M102. The number of CRISPR sites in these strains was associated with the clinical manifestations of ECC. Moreover, we found that the biofilm formation and EPS synthesis ability of the S. mutans strains with both CRISPR sites was significant improved.ConclusionsAn association was found between the distribution of CRISPR sites and the clinical manifestations of caries. The CRISPR sites might contribute to the cariogenic potential of S. mutans.     

Curcumin reduces Streptococcus mutans biofilm formation by inhibiting sortase A activity

BackgroundSortase A is an enzyme responsible for the covalent attachment of Pac proteins to the cell wall in Streptococcus mutans. It has been shown to play a role in modulating the surface properties and the biofilm formation and influence the cariogenicity of S. mutans. Curcumin, an active ingredient of turmeric, was reported to be an inhibitor for Staphylococcus aureus sortase A. The aim of this study was to investigate the inhibitory ability of curcumin against S. mutans sortase A and the effect of curcumin for biofilm formation.MethodsThe antimicrobial activity of the curcumin to the S. mutans and inhibitory ability of the curcumin against the purified sortase A in vitro were detected. Western-blot and real-time PCR were used to analysis the sortase A mediated Pac protein changes when the S. mutans was cultured with curcumin. The curcumin on the S. mutans biofilm formation was determined by biofilm formation analysis.ResultsCurcumin can inhibit purified S. mutans sortase A with a half-maximal inhibitory concentration (IC₅₀) of (10.2 ± 0.7) μmol/l, which is lower than minimum inhibitory concentration (MIC) of 175 μmol/l. Curcumin (15 μmol/l) was found to release the Pac protein to the supernatant and reduce S. mutans biofilm formation.ConclusionsThese results indicated that curcumin is an S. mutans sortase A inhibitor and has promising anti-caries characteristics through an anti-adhesion-mediated mechanism.     

Clotrimazole and econazole inhibit Streptococcus mutans biofilm and virulence in vitro

Objective: The aim of this study was to determine the inhibitory effect of eight antifungal drugs on S. mutans growth, biofilm formation and virulence factors.MethodsThe actions of antifungal drugs on S. mutans were determined by recovery plates and survival kinetic curves. Biofilms were observed by scanning electron microscopy and the viable cells were recovered on BHI plates, meanwhile biofilms were stained by BacLight live/dead kit to investigate the biofilm viability. Bacteria/extracellular polysaccharides staining assays were performed to determine the EPS production of S. mutans biofilms. Acidogenicity and acidurity of S. mutans were determined using pH drop and acid tolerance assays, and the expression of ldh gene was evaluated using qPCR.ResultsWe found that clotrimazole (CTR) and econazole (ECO) showed antibacterial activities on S. mutans UA159 and S. mutans clinical isolates at 12.5 and 25 mg/L, respectively. CTR and ECO could also inhibit S. mutans biofilm formation and reduce the viability of preformed biofilm. CTR and ECO affected the live/dead ratio and the EPS/bacteria ratio of S. mutans biofilms. CTR and ECO also inhibited the pH drop, lactate acid production, and acid tolerance. The abilities of CTR and ECO to inhibit S. mutans ldh expression were also confirmed.ConclusionsWe found that two antifungal azoles, CTR and ECO, had the abilities to inhibit the growth and biofilm formation of S. mutans and more importantly, they could also inhibit the virulence factors of S. mutans.     

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.     

Antibacterial and mechanical properties of arginine-containing glass ionomer cements

ObjectiveThe study investigated the effect of incorporating l-arginine (Arg) in a glass ionomer cement (GIC) on its mechanical properties and antibacterial potential.MethodsPre-determined proportions (1%, 2%, and 4% by wt.) of Arg were incorporated in GIC powder; while GIC without Arg served as control. The flexural strength, nanohardness, surface roughness, elemental analysis using SEM-EDX (n = 6) and F/Arg/Ca/Al/Si release in deionized water for 21 days were assessed. The antibacterial potential was evaluated in a multi-species biofilm model with Streptococcus mutans, Streptococcus sanguinis, Streptococcus gordonii, and Lactobacillus acidophilus for 72 h. Real-time qPCR was used to analyse biofilm bacterial concentrations. Propidium monoazide modification of real-time qPCR was performed to quantify viable/dead bacteria. The pH, lactic acid, ADS activity, and H₂O₂ metabolism were measured. Confocal microscopy was used to investigate the biofilm bacterial live/dead cells, density, and thickness.ResultsThere was no difference in flexural strength among the different groups (p > 0.05). No significant difference in nanohardness and surface roughness was observed between 4% Arg + GIC and control (p > 0.05). The 4% Arg + GIC showed significantly higher F/Arg/Al/Si release than the other groups (p < 0.05), reduced total bacterial concentration and growth inhibition of viable S. mutans and S. sanguinis (p < 0.05). Lactic acid formation for 4% Arg + GIC was significantly higher than 1% Arg + GIC (p < 0.05). The spent media pH of 4% Arg + GIC was higher than the other groups (p < 0.05), with proportionately lower ammonia and higher H₂O₂ released (p < 0.05).SignificanceAddition of 4% l-arginine in GIC enhanced its antibacterial activity via a biofilm modulatory effect for microbial homeostasis, with no detrimental effect on its mechanical properties.     

Hydrophilicity of dentin bonding systems influences in vitro Streptococcus mutans biofilm formation

Objective

To evaluate in vitro Streptococcus mutans (S. mutans) biofilm formation on the surface of five light-curing experimental dental bonding systems (DBS) with increasing hydrophilicity. The null hypothesis tested was that resin chemical composition and hydrophilicity does not affect S. mutans biofilm formation.

Methods

Five light-curing versions of experimental resin blends with increasing hydrophilicity were investigated (R₁, R₂, R₃, R₄ and R₅). R₁ and R₂ contained ethoxylated BisGMA/TEGDMA or BisGMA/TEGDMA, respectively, and were very hydrophobic, were representative of pit-and-fissure bonding agents. R₃ was representative of a typical two-step etch-and-rinse adhesive, while R₄ and R₅ were very hydrophilic resins analogous to self-etching adhesives. Twenty-eight disks were prepared for each resin blend. After a 24 h-incubation at 37 °C, a multilayer monospecific biofilm of S. mutans was obtained on the surface of each disk. The adherent biomass was determined using the MTT assay and evaluated morphologically with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM).

Results

R₂ and R₃ surfaces showed the highest biofilm formation while R₁ and R₄ showed a similar intermediate biofilm formation. R₅ was more hydrophilic and acidic and was significantly less colonized than all the other resins. A significant quadratic relationship between biofilm formation and hydrophilicity of the resin blends was found. CLSM and SEM evaluation confirmed MTT assay results.

Conclusions

The null hypothesis was rejected since S. mutans biofilm formation was influenced by hydrophilicity, surface acidity and chemical composition of the experimental resins. Further studies using a bioreactor are needed to confirm the results and clarify the role of the single factors.     

Comparative in vitro investigation of the cariogenic potential of bifidobacteria

ObjectiveThis study aimed to assess the in vitro cariogenic potential of some Bifidobacterium species in comparison with caries-associated bacteria.DesignBifidobacterium lactis, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium dentium, Lactobacillus acidophilus, Lactobacillus casei, Actinomyces israelii, Streptococcus sobrinus and Streptococcus mutans were tested for acidogenicity and aciduricity by measuring the pH of the cultures after growth in glucose and bacterial growth after exposure to acid solutions. Biofilm biomass was determined for each species either alone or associated with S. mutans or S. mutans/S. sobrinus. Enamel hardness was analyzed before and after 7-days biofilm formation using bacterial combinations.ResultsB. animalis and B. longum were the most acidogenic and aciduric strains, comparable to caries-associated bacteria, such as S. mutans and L. casei. All species had a significantly increased biofilm when combined either with S. mutans or with S. mutans/S. sobrinus. The greatest enamel surface loss was produced when B. longum or B. animalis were inoculated with S. mutans, similar to L. casei and S. sobrinus. All strains induced similar enamel demineralization when combined with S. mutans/S. sobrinus, except by B. lactis.ConclusionThe ability to produce acidic environments and to enhance biofilm formation leading to increased demineralization may mean that Bifidobacterium species, especially B. animalis and B. longum, are potentially cariogenic.