Antimicrobial and synergistic effects of lemongrass and geranium essential oils against Streptococcus mutans,Staphylococcus aureus,and Candida spp.

BACKGROUND The oral cavity harbors more than 700 species of bacteria,which play crucial roles in the development of various oral diseases including caries,endodontic infection,periodontal infection,and diverse oral diseases.AIM To investigate the antimicrobial action of Cymbopogon Schoenanthus and Pelargonium graveolens essential oils against Streptococcus mutans,Staphylococcus aureus,Candida albicans,Ca.dubliniensis,and Ca.krusei.METHODS Minimum microbicidal concentration was determined following Clinical and Laboratory Standards Institute documents.The synergistic antimicrobial activity was evaluated using the Broth microdilution checkerboard method,and the antibiofilm activity was evaluated with the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay.Data were analyzed by one-way analysis of variance followed by the Tukey post-hoc test(P≤0.05).RESULTS C.schoenanthus and P.graveolens essential oils were as effective as 0.12%chlorhexidine against S.mutans and St.aureus monotypic biofilms after 24 h.After 24 h P.graveolens essential oil at 0.25%was more effective than the nystatin group,and C.schoenanthus essential oil at 0.25%was as effective as the nystatin group.CONCLUSION C.schoenanthus and P.graveolens essential oils are effective against S.mutans,St.aureus,Ca.albicans,Ca.dubliniensis,and Ca.krusei at different concentrations after 5 min and 24 h.

The Antibacterial Activities of Copper Oxide Nanoparticles Synthesized Using Laser Ablation in Different Surfactants against Streptococcus mutans

Copper oxide nanoparticles(CuO NPs)were synthesised with laser ablation of a copper sheet immersed in deionized water(DW),cetrimonium bromide(CTAB),and sodium dodecyl sulphate(SDS),respectively.The target was irradiated with a pulsed Nd:YAG laser at 1064 nm,600 mJ,a pulse duration of 10 ns,and a repetition rate of 5 Hz.The CuO NPs colloidal were analyzed using UV–Vis spectroscopy,the Fourier transform infrared(FTIR)spectrometer,zeta potential(ZP),X-ray diffraction(XRD),transmission electron microscope(TEM)and field emission scanning electron microscopy(FESEM).The absorption spectra of CuO NPs colloidal showed peaks at 214,215 and 220 nm and low-intensity peaks at 645,650 and 680 nm for SDS,CTAB and DW,respectively.CuO NPs’colloidal results are(−21.6,1.2,and 80 mV)for negatively,neutrally,and positively charged SDS,DW,and CTAB,respectively.The XRD pattern of the NPs revealed the presence of CuO phase planes(110)(111),(20-2)and(11-1).The TEM images revealed nearly spherical NPs,with sizes ranging from 10–90,10–50,and 10–210 nm for CuO NPs mixed with DW,SDS and CTAB,respectively.FESEM images of all the synthesized samples illustrate the formation of spherical nanostructure and large particles are observable.The CuO NPs were tested for antibacterial activity against Streptococcus mutans by using the well diffusion method.In this method,CuO NPs prepared in DW at a concentration of 200μg/mL showed a greater inhibition zone against Streptococcus mutans.

Influences of trans-trans farnesol,a membrane-targeting sesquiterpenoid,on Streptococcus mutans physiology and survival within mixed-species oral biofilms

Trans-trans farnesol （tt-farnesol） is a bioactive sesquiterpene alcohol commonly found in propolis （a beehive product） and citrus fruits, which disrupts the ability of Streptococcus mutans （S. mutans） to form virulent biofilms. In this study, we investigated whether tt-farnesol affects cell-membrane function, acid production and/or acid tolerance by planktonic cells and biofilms of S. mutans UA159. Furthermore, the influence of the agent on S. mutans gene expression and ability to form biofilms in the presence of other oral bacteria （Streptococcus oralis （S. oralis） 35037 and Actinomyces naeslundii （.4. naeslundil） 12104） was also examined. In general, tt-farnesol （1 mmol-L-1） significantly increased the membrane proton permeability and reduced glycolytie activity of S. mutans in the planktonic state and in biofilms （P〈0.05）. Moreover, topical applications of 1 mmol-L＂l tt-farnesol twice daily （1 min exposure/treatment） reduced biomass accumulation and prevented ecological shifts towards S. mutans dominance within mixed-species biofilms after introduction of 1% sucrose. S. oralis （a non-cariogenie organism） became the major species after treatments with tt-farnesol, whereas vehicle-treated biofilms contained mostly S. mutans （〉90% of total bacterial population）. However, the agent did not affect significantly the expression of S. mutans genes involved in acidogenicity, acid tolerance or polysaccharide synthesis in the treated biofilms. Our data indicate that tt-farnesoi may affect the competi- tiveness of S. mutans in a mixed-species environment by primarily disrupting the membrane function and physiology of this bacterium. This naturally occurring terpenoid could be a potentially useful adjunctive agent to the current anti-biofilm/anti-caries chemotherapeutic strategies.

Targeted Antimicrobial Therapy Against Streptococcus mutans Establishes Protective Non-cariogenic Oral Biofilms and Reduces Subsequent Infection

Aim Dental biofilms are complex communities composed largely of harmless bacteria. Certain pathogenic species including Streptococcus mutans （S. mutans） can become predominant when host factors such as dietary sucrose intake imbalance the biofilm ecology. Current approaches to control S. mutans infection are not pathogen-specific and eliminate the entire oral community along with any protective benefits provided. Here, we tested the hypothesis that removal of S. mutans from the oral community through targeted antimicrobial therapy achieves protection against subsequent S. mutans colonization. Methodology Controlled amounts of S. mutans were mixed with S. mutans-free saliva, grown into biofilms and visualized by antibody staining and cfu quantization. Two specifically-targeted antimicrobial peptides （STAMPs） against S. mutans were tested for their ability to reduce S. mutans biofilm incorporation upon treatment of the inocula. The resulting biofilms were also evaluated for their ability to resist subsequent exogenous S. mutans colonization. Results S. mutans colonization was considerably reduced （9 ± 0.4 fold reduction, P=0.01） when the surface was preoccupied with saliva-derived biofilms. Furthermore, treatment with S. mutans-specific STAMPs yielded S. mutans-deficient biofilms with significant protection against further S. mutans colonization （5 minutes treatment： 38 ± 13 fold reduction P=0.01; 16 hours treatment： 96 ± 28 fold reduction P=0.07）. Conclusion S. mutans infection is reduced by the pre- sence of existing biofilms. Thus maintaining a healthy or ＂normal＂ biofilm through targeted antimicrobial therapy （such as the STAMPs） could represent an effective strategy for the treatment and prevention of S. mutans colonization in the oral cavity and caries progression.

Streptococcus mutans activates the AIM2, NLRP3 and NLRC4 inflammasomes in human THP-1 macrophages

Streptococcus mutans(S. mutans), a major aetiologic agent of dental caries, is involved in systemic diseases, such as bacterial endocarditis, if it enters the bloodstream through temporary bacteraemia. Interleukin(IL)-1β, a proinflammatory cytokine, is related to the host defences against pathogens, and its synthesis, maturation, and secretion are tightly regulated by the activation of the inflammasome, an inflammatory signalling complex. This study examined the signalling mechanism of IL-1β secretion and the inflammasome pathway induced by S. mutans to explain the molecular mechanism through which systemic infection by oral streptococci can occur. After infection of THP-1 cells with S. mutans, the expression of inflammasome components was detected using various methods. S. mutans induced IL-1β secretion via caspase-1 activation, and S. mutans-induced IL-1β secretion required absent in melanoma(AIM2), NLR family pyrin domain-containing 3(NLRP3) and NLR family CARD domain-containing 4(NLRC4)inflammasome activation. In particular, the S. mutans-induced NLRP3 inflammasome was mediated by adenosine triphosphate(ATP) release, potassium depletion and lysosomal damage. Our study provides novel insight into the innate immune response against S. mutans infection.

Alanine racemase is essential for the growth and interspecies competitiveness of Streptococcus mutans

D-alanine （D-Ala） is an essential amino acid that has a key role in bacterial unique enzyme that interconverts L-alanine and D-alanine in most bacteria, antimicrobial drug development. Streptococcus mutans is a major causative cell wall synthesis. Alanine racemase （Air） is a making this enzyme a potential target for factor of dental caries. The factors involved in the survival, virulence and interspecies interactions of S. mutans could be exploited as potential targets for caries control. The current study aimed to investigate the physiological role of Air in S. mutans. We constructed air mutant strain of S. mutans and evaluated its phenotypic traits and interspecies competitiveness compared with the wild-type strain. We found that air deletion was lethal to S. mutans. A minimal supplement of D-Ala （150 pg.mL- 1） was required for the optimal growth of the air mutant. The depletion of D-alanine in the growth medium resulted in cell wall perforation and cell lysis in the air mutant strain. We also determined the compromised competitiveness of the air mutant strain relative to the wild-type S. mutans against other oral streptococci （S. sanguinis or S. gordonil~, demonstrated using either conditioned medium assays or dual-species fluorescent in situ hybridization analysis. Given the importance and necessity of air to the growth and competitiveness of S. mutans, Air may represent a promising target to modulate the cariogenicity of oral biofilms and to benefit the management of dental caries.

The photodynamic therapy on Streptococcus mutans biofilms using erythrosine and dental halogen curing unit

The purpose of our study was to evaluate the effect of photodynamic therapy （PDT）, using erythrosine as a photosensitizing agent and a dental halogen curing unit as a light source, on Streptococcus mutans in a biofilm phase. The S. mutans biofilms were formed in a 24-well cell culture cluster. Test groups consisted of biofilms divided into four groups： group 1： no photosensitizer or light irradiation treatment （control group）; group 2： photosensitizer treatment alone; group 3： light irradiation alone; group 4： photosensitizer treatment and light irradiation. After treatments, the numbers of colony-forming unit （CFU） were counted and samples were examined by confocal laser scanning fluorescence microscopy （CLSM）. Only group 4 （combined treatment） resulted in significant increases in cell death, with rates of 75% and 55% after 8 h of incubation, and 74% and 42% at 12 h, for biofilms formed in brain-heart infusion （BHI） broth supplemented with 0% or O. 1% sucrose, respectively. Therefore, PDT of S. mutans biofilms using a combination of erythrosine and a dental halogen curing unit, both widely used in dental clinics, resulted in a significant increase in cell death. The PDT effects are decreased in biofilms that form in the presence of sucrose.

Antibacterial activity of nanostructured Ti-45S5 bioglass-Ag composite against Streptococcus mutans and Staphylococcus aureus

Mechanical alloying and annealing at 1150 °C for 2 h under an argon atmosphere were used to prepare Ti-45S5 bioglass nanocomposites. Ti-45S5 bioglass material was chemically modified by silver. The antibacterial activity of Ti-10% 45S5 bioglass nanocomposite containing silver against Streptococcus mutans and Staphylococcus aureus was studied. Nanocomposites were characterized by X-ray diffraction, scanning electron microscopy equipped with an electron energy dispersive spectrometer and transmission electron microscopy to evaluate phase composition, crystal structure and grain size. In vitro bacterial adhesion study indicated a significantly reduced number of Streptococcus mutans and Staphylococcus aureus on the bulk nanostructured Ti-45S5 bioglass-Ag plate surface in comparison to that on microcrystalline Ti plate surface. Nanostructured Ti-based biomaterials can be considered to be the future generation of dental implants.

Role of sortase in Streptococcus mutans under the effect of nicotine

Streptococcus mutans is a common Gram-positive bacterium and plays a significant role in dental caries. Tobacco and/or nicotine have documented effects on S. mutans growth and colonization. Sortase A is used by many Gram-positive bacteria, including S. mutans, to facilitate the insertion of certain cell surface proteins, containing an LPXTGX motif such as antigen 1/11. This study examined the effect of nicotine on the function of sortase A to control the physiology and growth of S. mutans using wild-type S. mutans NG8, and its isogenic sortase-defective and -complemented strains. Briefly, the strains were treated with increasing amounts of nicotine in planktonic growth, biofilm metabolism, and sucrose-induced and saliva-induced antigen I/ll-dependent biofilm formation assays. The strains exhibited no significant differences with different concentrations of nicotine in planktonic growth assays. However, they had significantly increased （P~〈 0.05） biofilm metabolic activity （2- to 3-fold increase） as the concentration of nicotine increased. Furthermore, the sortase-defective strain was more sensitive metabolically to nicotine than the wild-type or sortase-complemented strains. All strains had significantly increased sucrose-induced biofilm formation （2- to 3-fold increase） as a result of increasing concentrations of nicotine. However, the sortase-defective strain was not able to make as much sucrose- and saliva-induced biofilm as the wild-type NG8 did with increasing nicotine concentrations. These results indicated that nicotine increased metabolic activity and sucrose-induced biofilm formation. The saliva-induced biofilm formation assay and qPCR data suggested that antigen 1/11 was upregulated with nicotine but biofilm was not able to be formed as much as wild-type NG8 without functional sortase A.

In silico identification of potential inhibitors targeting Streptococcus mutans sortase A

Dental caries is one of the most common chronic diseases and is caused by acid fermentation of bacteria adhered to the teeth. Streptococcus mutans （S. mutans） utilizes sortase A （SrtA） to anchor surface proteins to the cell wall and forms a biofilm to facilitate its adhesion to the tooth surface. Some plant natural products, especially several flavonoids, are effective inhibitors of SrtA. However, given the limited number of inhibitors and the development of drug resistance, the discovery of new inhibitors is urgent. Here, the high-throughput virtual screening approach was performed to identify new potential inhibitors of S. mutans SrtA. Two libraries were used for screening, and nine compounds that had the lowest scores were chosen for further molecular dynamics simulation, binding free energy analysis and absorption, distribution, metabolism, excretion and toxicity （ADMET） properties analysis. The results revealed that several similar compounds composed of benzofuran, thiadiazole and pyrrole, which exhibited good affinities and appropriate pharmacokinetic In addition, the carbonyl of these compounds can have a strategy for microbial infection disease therapy. parameters, were potential inhibitors to impede the catalysis of SrtA. key role in the inhibition mechanism. These findings can provide a new

Quaternary ammonium-induced multidrug tolerant Streptococcus mutans persisters elevate cariogenic virulence in vitro

Dental caries are the most prevalent chronic infections in the oral cavity, and Streptococcus mutans acts as the main cariogenic bacterial species. Antibacterial quaternary ammonium compounds （QAs） have been developed to preveFnt or treat dental caries. However, there is no report on the tolerance of S. mutans to QAs. In this study, we investigated the development of S. mutans persistence induced by a novel dental caries defensive agent, dimethylaminododecyl methacrylate （DMADDM）, for the first time. Typical biphasic killing kinetics for persisters were observed in both S. mutans planktonic and biofilm cultures challenged by DMADDM at concentrations of 20 and 200 pg.mL- z respectively. The persisters tolerated six other antibiotics with different antibacterial mechanisms, while only daptomycin and vancomycin could slightly reduce the persister numbers in planktonic cultures. The distribution of persisters in DMADDM-treated biofilms was similar to that in the untreated control, except that the total biomass and biofilm height were significantly reduced. A higher exopolysaccharides （EPS）：bacteria ratio was observed in DMADDM-treated biofilms. Persisters in biofilms significantly upregulated gtf gene expression, indicating an increase in the bacteria＇s ability to produce EPS and an elevated capability of cariogenic virulence. Carbon source metabolism was significantly reduced, as related metabolic genes were all downregulated in persisters. Concentrations of 0.1 mM, 1 mM and 10 mM of extra glucose significantly reduced the number of persisters both in planktonic and biofilm conditions. The formation of non- inheritable and multidrug tolerant persisters induced by DMADDM suggested that drug tolerance and new persistent eradication strategies should be considered for oral antibacterial agents.

Metabolic activity of Streptococcus mutans biofilms and gene expression during exposure to xylitol and sucrose

The objective of the study was to analyse Streptococcus mutans biofilms grown under different dietary conditions by using multifaceted methodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generated during a period of 24 h in the following media： Schaedler broth as a control medium containing endogenous glucose, Schaedler broth with an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy（CLSM）-based analyses of the microbial vitality, respiratory activity（5-cyano-2,3-ditolyl tetrazolium chloride, CTC） and production of extracellular polysaccharides（EPS） were performed separately in the inner, middle and outer biofilm layers. In addition to the microbiological sample testing, the glucose/sucrose consumption of the biofilm bacteria was quantified, and the expression of glucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutans biofilms, as monitored by the following experimental parameters： culture growth, vitality, CTC activity and EPS production. However,xylitol exposure caused a difference in gene expression compared to the control. Gtf C was upregulated only in the presence of xylitol.Under xylitol exposure, gtf B was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three.Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose was almost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametric measurements showed that xylitol in the presence of glucose caused predominantly genotypic differences but did not induce metabolic differences compared to the control. Thus, the availability of dietary carbohydrates in either a pure or combined form seems to affect the cariogenic potential of S. mutans biofilms.

The synergistic effect of honey and cinnamon against Streptococcus mutans bacteria

Objective: To investigate the effect of Iranian honey, cinnamon and their combination against Streptococcus mutans bacteria.Methods: Nine experimental solutions were examined in this study, including two types of honey(pasteurized and sterilized), two types of cinnamon extract(dissolved in distilled water or dimethyl sulfoxide) and five different mixtures of cinnamon in honey(prepared by admixing 1%–5% w/w of cinnamon extract into 99%–95% w/w of honey, respectively).Meanwhile, each of mentioned agent was considered as the first solution while it was diluted into seven serially two-fold dilutions(from 1:2 to 1:128 v/v).Therefore, eight different concentrations of each agent were tested.The antibacterial tests were performed through blood agar well diffusion method, and the minimum inhibitory concentration(MIC) was determined.Ultimately, the data were subjected to statistical analysis incorporating Two-way ANOVA and Bonferroni post hoc tests(a = 0.01).Results: The highest zone of inhibition was recorded for the mixtures of honey and cinnamon while all the subgroups containing 95%–99% v/v of honey were in the same range(P < 0.01).The MIC for both honey solutions were obtained as 500 mg/mL whereas it was 50 mg/m L for both cinnamon solutions.Moreover, the MIC related to all honey/cinnamon mixtures were 200 mg/mL.Conclusions: A profound synergistic effect of honey and cinnamon was observed against Streptococcus mutans while there was no significant difference among extracts containing 99%–95% v/v of honey admixing with 1%–5% v/v of cinnamon, respectively.

Deactivation of Streptococcus mutans Biofilms on a Tooth Surface Using He Dielectric Barrier Discharge at Atmospheric Pressure

This paper presents a study of the effect of the low temperature atmospheric helium dielectric barrier discharge（DBD） on the Streptococcus mutans biofilms formed on tooth surface. Pig jaws were also treated by plasma to detect if there is any harmful effect on the gingiva.The plasma was characterized by using optical emission spectroscopy.Experimental data indicated that the discharge is very effective in deactivating Streptococcus mutans biofilms.It can destroy them with an average decimal reduction time（D-time） of 19 s and about 98%of thein were killed after a treatment time of 30 s.According to the survival curve kinetic an overall 32 s treatment time would be necessary to perform a complete sterilization.The experimental results presented in this study indicated that the helium dielectric barrier discharge,in plan-parallel electrode configuration,could be a very effective tool for deactivation of oral bacteria and might be a promising technique in various dental clinical applications.

Immunogenicity and Prediction of Epitopic Region of Antigen Ag Ⅰ/Ⅱ and Glucosyltransferase from Streptococcus mutans

The levels of Streptococcus（S.） mutans infections in saliva were evaluated and a comparison for specific antibody levels among children with different levels of S. mutans infection was made. The promising epitopic regions of antigen AgⅠ/ Ⅱ（PAc） and glucosyltransferase（GTF） for potential vaccine targets related to S. mutans adherence were screened. A total of 94 children aged 3–4 years were randomly selected, including 53 caries-negative and 41 caries-positive children. The values of S. mutans and those of salivary total secretory immunoglobulin A（s Ig A）, anti-PAc and anti-Glucan binding domain（anti-GLU） were compared to determine the correlation among them. It was found the level of s-Ig A against specific antigens did not increase with increasing severity of S. mutans infection, and the complete amino acid sequence of PAc and GTFB was analyzed using the DNAStar Protean system for developing specific anti-caries vaccines related to S. mutans adherence. A significantly positive correlation between the amount of S. mutans and children decayed, missing, and filled teeth index was observed. No significant difference was detected in specific s Ig A against PAc or GLU between any two groups. No significant correlation was found between such specific s Ig A and caries index. A total of 16 peptides from PAc as well as 13 peptides from GTFB were chosen for further investigation. S. mutans colonization contributed to early children caries as an important etiological factor. The level of s Ig A against specific antigens did not increase with increasing severity of S. mutans infection in children. The epitopes of PAc and GTF have been screened to develop the peptide-based or protein-based anti-caries vaccines.

Farnesol inhibits development of caries by augmenting oxygen sensitivity and suppressing virulence-associated gene expression in Streptococcus mutans

Streptococcus mutans is a primary etiological agent of dental caries.Farnesol,as a potential antimicrobial agent,inhibits the development of S.mutans biofilm.In this study,we hypothesized that farnesol inhibits caries development in vitro and interferes with biofilm fonnation by regulating virulence-associated gene expression.The inhibitory effects of farnesol to S.mutans biofilms on enamel surfaces were investigated by determining micro-hardness and calcium measurements.Additionally,the morphological changes of S.mutans biofilms were compared using field emission scanning electron microscopy and confocal laser scanning microscopy,and the vitality and oxygen sensitivity of S.mutans biofilms were compared using MTT assays.To investigate the molecular mechanisms of farnesol＇s effects,expressions of possible target genes luxS,brpA,ffh,recA,nth,and smx were analyzed using reverse-transcription polymerase chain reaction（PCR） and quantitative PCR.Farnesol-treated groups exhibited significantly higher micro-hardness on the enamel surface and lower calcium concentration of the supernatants as compared to the-untreated control.Microscopy revealed that a thinner film with less extracellular matrix formed in the farnesol-treated groups.As compared to the-untreated control,farnesol inhibited biofilm formation by 26.4%with500 μmol/L and by 37.1%with 1,000 μmol/L（P〈 0.05）.Last,decreased transcription levels of luxS,brpA,ffh,recA,nth,and smx genes were expressed in farnesol-treated biofilms.In vitro farnesol inhibits caries development and S.mutans biofilm formation.The regulation of luxS,brpA,ffh,recA,nth,and smx genes may contribute to the inhibitory effects of farnesol.

Enhancement of the Photodynamic Therapy Effect on Streptococcus Mutans Biofilm

Biofilm is a community of bacteria, less susceptible to traditions treatments. Although photodynamic therapy （PDT） is a very effective way to microorganism inactivation, in biofilm it is not as efficient as it is in planktonic bacteria cultures. The increment of an element to increase the effectiveness of PDT was our aim. Therefore, this in vitro study evaluates the susceptibility ofa biofilm formed by Streptococcus mutans on metallic surface of orthodontic accessories under the application of PDT with a surfactant. Samples obtained from blades of orthodontic bands （NiCr）, where used as adhesion surface for the biofilm. They were treated with 1 mg/ml of curcumin, with 0.1% of sodium dodecyl sulfate and exposed to 30 J/cm^2 of light （455 nm）. Eight experimental groups were studied, including the positive and negative controls. The results show that the group with PDT and surfactant had a significant decrease （p 〈 0.001） in viability. In this case, the reduction observed was of 5.6 log10 （CFU/ml） in comparison to the control group. We have shown that, even though the biofilm is very tough and complex structure, we are able to promote almost the complete inactivation ofS. mutans in systems similar to an orthodontic treated patient＇s mouth.

Construction and in vitro Expression of Streptococcus Mutans Surface Protein Encoding DNA Vaccine

DNA vaccine plasmids were constructed that encoded two highly conservative regions of a surface protein, PAc, from the human major cariogenic bacterium, Streptococcus mutans . Antigen expression was evaluated in vitro by immunohistochemical analysis of human endothelial cells following cationic liposome mediated transient transfection with recombinant plasmid. The results of this study provided a basis for further testing of these recombinant plasmids in primates and for efficacy testing of dental caries DNA vaccines in human volunteers in future.

Streptococcal SspB Peptide Analog Inhibits Saliva-Promoted Adhesion and Biofilm Formation of Streptococcus mutans

Background: Streptococcus gordonii, a pioneer colonizer of dental plaque biofilm, expresses surface protein adhesin SspB by which the bacteria bind to salivary agglutinin (gp340). SspB has extensive homology with PAc, a surface adhesin of Streptococcus mutans. Hence, SspB of S. gordonii competes with PAc of S. mutans for the same niche environment in the salivary pellicles. The aim of this study was to develop anti-adherence agents that enabled us to control cariogenic biofilms by using the streptococcal SspB peptide analog SspB (A4K-A11K). Methods: First, we performed ELISA to determine the S. mutans-saliva interaction and saliva-binding activities of SspB (A4K- A11K). The inhibitory effects of SspB (A4K-A11K) were then evaluated by examining S. mutans adhesion to saliva-coated hydroxyapatite disks (s-HA). To determine peptide interference with biofilm formation, S. mutans biofilms were quantified by counting CFUs on MS agar plates and by measuring the absorbance at 492 nm of safranin-stained biofilms on s-HA. Results: Saliva, particularly salivary gp340 peptide, promoted adherence of S. mutans to polystyrene surfaces. SspB (A4K-A11K) significantly bound to saliva and inhibited the adhesion of S. mutans to s-HA without bactericidal activity. Furthermore, biofilms of S. mutans on s-HA were successfully reduced by pretreatment with SspB (A4K-A11K). Conclusion: SspB (A4K-A11K) peptide competitively blocked S. mutans adhesion to experimental pellicles through SspB-gp340 interaction, thereby inhibiting biofilm formation. These findings will contribute to the control cariogenic biofilms.

Periodontitis and Inflammation: Plasma High Titer Naturally Occurring Anti-Glucan Antibodies Form Immune Complex with Streptococcus mutans Antigen

Atheromatous plaques usually contain antigens of the periodontitis-causing bacteria Streptococcus mutans though molecular mechanism of this incorporation remains unknown. Since vascular adhesion and inflammatory potential of Immune Complexes (IC) are known we investigated the naturally occurring plasma antibodies that recognize major antigens from S. mutans. S. mutans-binding plasma proteins (SMBP) prepared by affinity chromatography on a column of heat-killed S. mutans could recognize α- and β-linked glucose in dextran and yeast respectively but not galactose in glycoproteins. SMBP contained only three proteins, each corresponding in electrophoretic mobility to standard plasma IgG, IgA or IgM. The major positively and negatively charged protein antigens (PSMAg and NSMAg) isolated from S. mutans by electrophoresis and ion exchange chromatography respectively were recognized sugar-reversibly by the anti-β-glucan antibody (ABG) and though less avidly, by the dextran-binding immunoglobulin (DIg) in normal plasma. NSMAg addition resulted in near doubling of IC-bound immunoglobulins in immunoglobulin-rich fraction of plasma. IC isolated from above fraction after NSMAg addition had substantially more IgA and IgM content than total plasma immunoglobulins. IC formation by NSMAg was significantly inhibited by ABG- and DIg-specific sugars or by selective withdrawal of ABG or DIg from plasma. ABG and DIg being relatively high titer plasma antibodies IC formation with them suggested a possible route for vascular adhesion and damage by S. mutans and its antigens. Further, high IgA content of these ICs indicated their susceptibility to tissue uptake through cell surface galectin-1 for which IgA is the lone immunoglobulin ligand.