Antibacterial Activity of Epigallocatechin-3-gallate (EGCG) Loaded Lipid-chitosan Hybrid Nanoparticle against Planktonic Microorganisms.

Epigallocatechin-3-gallate (EGCG), a polyphenol derived from Green Tea, is one of the sources of natural bioactive compounds which are currently being developed as medicinal ingredients. Besides other biological activities, this natural compound exhibits anti-cariogenic effects. However, EGCG has low physical-chemical stability and poor bioavailability. Thus, the purpose of this study was to develop and characterize lipid-chitosan hybrid nanoparticle with EGCG and to evaluate its in vitro activity against cariogenic planktonic microorganisms. Lipid-chitosan hybrid nanoparticle (LCHNP-EGCG) were prepared by emulsion and sonication method in one step and characterized according to diameter, polydispersity index (PdI), zeta potential (ZP), encapsulation efficiency (EE), mucoadhesion capacity and morphology. Strains of Streptococcus mutans, Streptococcus sobrinus and Lactobacillus casei were treated with LCHNP- EGCG, and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated. LCHNP-EGCG exhibited a size of 217.3 ± 5.1 nm with a low polydispersity index (0.17) and positive zeta potential indicating the presence of chitosan on the lipid nanoparticle surface (+33.7 mV). The LCHNP-EGCG showed a spherical morphology, high stability and a mucoadhesive property due to the presence of chitosan coating. In addition, the EGCG encapsulation efficiency was 96%. A reduction of almost 15-fold in the MIC and MBC against the strains was observed when EGCG was encapsulated in LCHNP, indicating the potential of EGCG encapsulation in lipid-polymer hybrid nanoparticles. Taking the results together, the LCHNP-EGCG could be an interesting system to use in dental care due to their nanometric size, mucoadhesive properties high antibacterial activity against relevant planktonic microorganisms.

Antimicrobial Photodynamic Treatment with Mother Juices and Their Single Compounds as Photosensitizers.

The potent antimicrobial effects of antimicrobial photodynamic therapy (aPDT) with visible light plus water-filtered infrared-A irradiation and natural compounds as photosensitizers (PSs) have recently been demonstrated. The aim of this study was to obtain information on the antimicrobial effects of aPDT with mother juices against typical cariogenic oral Streptococcus pathogens in their planktonic form and determine its eradication potential on total human salivary bacteria from volunteers. Mother juices of pomegranate, bilberry, and chokeberry at different concentrations were used as PSs. The unweighted (absolute) irradiance was 200 mW cm-2, applied five minutes. Planktonic cultures of Streptococcus mutans and Streptococcus sobrinus and total mixed bacteria from pooled saliva of volunteers were treated with aPDT. Up to more than 5 log10 of S. mutans and S. sobrinus were killed by aPDT with 0.4% and 0.8% pomegranate juice, 3% and 50% chokeberry juice, and 12.5% bilberry juice (both strains). Concentrations of at least 25% (pomegranate) and >50% (chokeberry and bilberry) eradicated the mixed bacteria in saliva samples. This pilot study has shown that pomegranate mother juice is superior to the berry juices as a multicomponent PS for killing pathogenic oral bacteria with aPDT.

Multiple biosurfactant production by Aureobasidium pullulans strain YTP6-14 in aqueous and heavy oil layers.

Aureobasidium pullulans YTP6-14 was demonstrated to be an excellent multiple biosurfactant producer utilizing cheap carbon sources available in Thailand, including glycerol and cassava flour hydrolysate. A. pullulans YTP6-14 maximally produced 1.81 g/l biosurfactant in an aqueous layer (BS-AQ) in a medium containing glycerol, and 7.37 or 6.37 g/l biosurfactant in a heavy oil layer (BS-HO) in cassava flour hydrolysate or a glucose containing medium, respectively. Each BS-AQ and BS-HO had critical micelle concentration values of 41.32 mg/l and 13.51 mg/l, and both biosurfactants formed a stable food oil emulsion and reduced the amount of biofilms formed by Streptococcus sobrinus and Streptococcus mutans. BS-AQ and BS-HO were mainly composed of liamocins or exophilins and massoia lactone, respectively.

Tea extracts differentially inhibit Streptococcus mutans and Streptococcus sobrinus biofilm colonization depending on the steeping temperature.

This study aimed to evaluate the effects of tea extracts on oral biofilm colonization depending on steeping temperature. S. mutans and S. sobrinus were cultured and treated with green or black tea extracts prepared under different steeping conditions. Biofilm formation, glucosyltransferase (GTF) levels, bacterial growth, and acidogenicity were evaluated. Biofilms were also assessed by gas chromatography-mass spectrometry and confocal laser scanning microscopy. All extracts with hot steeping showed higher inhibitory effects on biofilm formation and cell viability and lower GTF levels compared with those with cold steeping (p < 0.05). Hot steeping significantly reduced bacterial growth (p < 0.05) and maintained the pH. Catechins were only identified from hot steeping extracts. Within the limits of this study, extracts with cold steeping showed lower inhibitory effects on oral biofilms. The different effects between steeping extracts may be attributed to the difference in catechins released from tea extracts under the different steep conditions.

Antibacterial activity of hinokitiol against both antibiotic-resistant and -susceptible pathogenic bacteria that predominate in the oral cavity and upper airways.

Hinokitiol, a component of the essential oil isolated from Cupressaceae, possesses antibacterial and antifungal activities and has been used in oral care products. In this study, the antibacterial activities of hinokitiol toward various oral, nasal and nasopharyngeal pathogenic bacteria, including Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, methicillin-resistant and -susceptible Staphylococcus aureus, antibiotic-resistant and -susceptible Streptococcus pneumoniae, and Streptococcus pyogenes were examined. Growth of all these bacterial strains was significantly inhibited by hinokitiol, minimal inhibitory concentrations of hinokitiol against S. mutans, S. sobrinus, P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, methicillin-resistant S. aureus, methicillin-susceptible S. aureus, antibiotic-resistant S. pneumoniae isolates, antibiotic-susceptible S. pneumoniae, and S. pyogenes being 0.3, 1.0, 1.0, 30, 0.5, 50, 50, 30, 0.3-1.0, 0.5, and 0.3 µg/mL, respectively. Additionally, with the exception of P. gingivalis, hinokitiol exerted bactericidal effects against all bacterial strains 1 hr after exposure. Hinokitiol did not display any significant cytotoxicity toward the human gingival epithelial cell line Ca9-22, pharyngeal epithelial cell line Detroit 562, human umbilical vein endothelial cells, or human gingival fibroblasts, with the exception of treatment with 500 µg/mL hinokitiol, which decreased numbers of viable Ca9-22 cells and gingival fibroblasts by 13% and 12%, respectively. These results suggest that hinokitiol exhibits antibacterial activity against a broad spectrum of pathogenic bacteria and has low cytotoxicity towards human epithelial cells.

Inhibitory Effects of Lingonberry Extract on Oral Streptococcal Biofilm Formation and Bioactivity.

Phenolic compounds in fruits such as cranberries have been shown to promote a number of biological activities. The purpose of this study was to investigate the effects of polyphenolic compound-containing lingonberry extract on oral streptococci and compare them with the known anti-cariogenic activity of cranberries. Water-soluble and polyphenol-rich fractions (Fractions I and II, respectively) were isolated from cranberries and lingonberries. The effects of those fractions on the biofilm formation ability and bioactivity of Streptococcus mutans MT8148R, Streptococcus sobrinus 6715, and Streptococcus sanguinis ATCC 10556 were then evaluated. Cranberry or lingonberry Fraction II (at 0.5-1 mg/ml) significantly reduced biofilm formation by S. mutans, S. sobrinus, and S. sanguinis. In contrast, cranberry or lingonberry Fraction I (at 0.5-2 mg/ml) increased biofilm formation by S. mutans and S. sobrinus, but not by S. sanguinis. Fractions I and II (at 1-2 mg/ml) also reduced the bioactivity of S. mutans, while Fraction II (at 0.5 mg/ml) enhanced the bioactivity of all tested strains. The results revealed that lingonberries contained a larger amount of polyphenol than cranberries and that they showed almost the same level of activity against the biofilm formation ability and bioactivity of oral streptococci. This indicates that polyphenol-rich lingonberry fraction offers a promising natural food derivative for prevention of dental caries.

Novel antibacterial mouthguard material manufactured using silver-nanoparticle-embedded ethylene-vinyl acetate copolymer masterbatch.

The purpose of the present study was to develop an antibacterial mouthguard (MG) material using a masterbatch of silvernanoparticle-embedded ethylene-vinyl acetate (EVA) copolymers. In order to verify that the testing material was clinically applicable as an antibacterial MG material, we conducted an antibacterial test, a shock absorption test, and analysis of in vitro silver release. The colony-forming activity of Streptococcus sobrinus, Porphyromonas gingivalis, and Escherichia coli were significantly inhibited on the testing materials compared with the commercial EVA sheet (p<0.05). The shock absorption capability of the testing material was not significantly different from that of the commercial EVA sheet. Cumulative silver release (in pure water) from the testing materials were infinitesimal after soaking for 20 days, which implied that there could be no harm in wearing the MG during exercise. These results showed that this testing material could be clinically applicable as an antibacterial MG material.

Composition and activity against oral pathogens of the essential oil of Melampodium divaricatum (Rich.) DC.

The chemical composition of the essential oil isolated from the aerial parts of Melampodium divaricatum (Rich.) DC. (Asteraceae) was characterized by GC-FID and GC/MS analyses. (E)-Caryophyllene (56.0%), germacrene D (12.7%), and bicyclogermacrene (9.2%) were identified as the major oil components. The antimicrobial activity of the oil against seven standard strains of oral pathogens from the American Type Culture Collection (ATCC) was evaluated by determining minimum inhibitory concentrations (MICs) using the microdilution method. MIC Values below 100 µg/ml were obtained against Streptococcus sobrinus (90 µg/ml), Lactobacillus casei (30 µg/ml), S. mutans (20 µg/ml), and S. mitis (18 µg/ml). In contrast, the MIC values of the major oil compound (E)-caryophyllene were higher than 400 µg/ml against all pathogens, suggesting that the activity of the oil might depend on minor oil components and/or on synergistic effects. The M. divaricatum essential oil is a promising agent to include in anticariogenic oral rinse formulations for the control of oral pathogens.

Chemical and botanical characterization of Chilean propolis and biological activity on cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus.

Propolis is a non-toxic natural substance with multiple pharmacological properties including anti-cancer, antioxidant, fungicidal, antibacterial, antiviral, and anti-inflammatory among others. The aim of this study was to determine the chemical and botanical characterization of Chilean propolis samples and to evaluate their biological activity against the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. Twenty propolis samples were obtained from beekeeping producers from the central and southern regions of Chile. The botanical profile was determined by palynological analysis. Total phenolic contents were determined using colorimetric assays. Reverse phase HPLC and HPLC-MS were used to determine the chemical composition. The minimum inhibitory concentration (MIC) was determined on S. mutans and S. sobrinus. All propolis samples were dominated by structures from native plant species. The characterization by HPLC/MS, evidenced the presence of quercetin, myricetin, kaempferol, rutine, pinocembrin, coumaric acid, caffeic acid and caffeic acid phenethyl ester, that have already been described in these propolis with conventional HPLC. Although all propolis samples inhibited the mutans streptococci growth, it was observed a wide spectrum of action (MIC 0.90 to 8.22 µg mL(-1)). Given that results it becomes increasingly evident the need of standardization procedures, where we combine both the determination of botanical and the chemical characterization of the extracts. Research conducted to date, describes a promising effectiveness of propolis in the prevention of caries and other diseases of the oral cavity, making it necessary to develop studies to identify and understand the therapeutic targets or mechanisms of molecular action of the various compounds present on them.

Antimicrobial activity of the synthetic peptide Lys-a1 against oral streptococci.

The peptide LYS-[TRP(6)]-Hy-A1 (Lys-a1) is a synthetic derivative of the peptide Hy-A1, initially isolated from the frog species Hypsiboas albopunctatus. According to previous research, it is a molecule with broad antimicrobial activity. The objective of this study was to evaluate the antimicrobial activity of the synthetic peptide Lys-a1 (KIFGAIWPLALGALKNLIK-NH2) on the planktonic and biofilm growth of oral bacteria. The methods used to evaluate antimicrobial activity include the following: determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in microtiter plates for growth in suspension and quantification of biomass by crystal violet staining and counting of colony forming units for biofilm growth. The microorganisms Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis, Streptococcus salivarius, Streptococcus mutans and Streptococcus sobrinus were grown in Brain Heart Infusion broth at 37°C under atmospheric pressure with 10% CO2. The peptide was solubilized in 0.1% acetic acid (v/v) at various concentrations (500-1.9 µg mL(-1)). Chlorhexidine gluconate 0.12% was used as the positive control, and BHI culture medium was used as the negative control. The tested peptide demonstrated a remarkable antimicrobial effect, inhibiting the planktonic and biofilm growth of all strains tested, even at low concentrations. Thus, the peptide Lys-a1 is an important source for potential antimicrobial agents, especially for the control and prevention of microbial biofilms, which is one of the most important factors in cariogenic processes.

Chemical and botanical characterization of Chilean propolis and biological activity on cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus

Propolis is a non-toxic natural substance with multiple pharmacological properties including anticancer, antioxidant, fungicidal, antibacterial, antiviral, and anti-inflammatory among others. The aim of this study was to determine the chemical and botanical characterization of Chilean propolis samples and to evaluate their biological activity against the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. Twenty propolis samples were obtained from beekeeping producers from the central and southern regions of Chile. The botanical profile was determined by palynological analysis. Total phenolic contents were determined using colorimetric assays. Reverse phase HPLC and HPLC-MS were used to determine the chemical composition. The minimum inhibitory concentration (MIC) was determined on S. mutans and S. sobrinus. All propolis samples were dominated by structures from native plant species. The characterization by HPLC/MS, evidenced the presence of quercetin, myricetin, kaempferol, rutine, pinocembrin, coumaric acid, caffeic acid and caffeic acid phenethyl ester, that have already been described in these propolis with conventional HPLC. Although all propolis samples inhibited the mutans streptococci growth, it was observed a wide spectrum of action (MIC 0.90 to 8.22 µgmL-1). Given that results it becomes increasingly evident the need of standardization procedures, where we combine both the determination of botanical and the chemical characterization of the extracts. Research conducted to date, describes a promising effectiveness of propolis in the prevention of caries and other diseases of the oral cavity, making it necessary to develop studies to identify and understand the therapeutic targets or mechanisms of molecular action of the various compounds present on them.

Antimicrobial effect of linalool and α-terpineol against periodontopathic and cariogenic bacteria.

Linalool and α-terpineol exhibited strong antimicrobial activity against periodontopathic and cariogenic bacteria. However, their concentration should be kept below 0.4 mg/ml if they are to be used as components of toothpaste or gargling solution. Moreover, other compounds with antimicrobial activity against periodontopathic and cariogenic bacteria should be used in combination.

[Effect of lemon essential oil on caries factors of Streptococcus sobrinus].

OBJECTIVE: To investigate the mechanism of lemon peel essential oil (LPE) on the cariogenicity of Streptococcus sobrinus (Ss). METHODS: LPE was extracted by the authors, and the minimum inhibition concentration (MIC) was measured by disc diffusion method. The LPE was used as the experimental group with concentrations ranging from 2.250 g/L to 0.281 g/L prepared with trypticase peptone yeast (TPY) culture medium, and TPY culture medium was used as the control group. Ss at the concentration of 10(8) CFU/ml was added to each group, and cultured for 6, 18, 24, 48 hours. Neson-Somogyi method was used to measure the content of reducing sugar, and glucosyltransferase (GTF) activity. The activity of lactate dehydrogenase (LDH) was measured by lactic acid and pyruvic acid continuous monitoring method. The content of water insoluble glucan (WIG) was measured by anthrone method, and the pH value of the culture solution was detected. The value of pH before the experiment and the time difference was alculated as ΔpH. RESULTS: At the same time point, the activity of GTF and LDH and the concentration of WIG and the value ΔpH decreased gradually with the increase of concentration of LPE. There were significant differences between each experimental group and control group (P < 0.01). The control group had the maximum value, GTF: (6.71 ± 0.61) mIU, LDH: (135.8 ± 1.7) U/L, WIG: (47.15 ± 5.12) mg/L, ΔpH: (2.67 ± 0.01). The highest drug concentration group had the minimum value: GTF: (0.39 ± 0.07) mIU, LDH: (95.0 ± 5.4) U/L, WIG: (2.44 ± 0.38) mg/L, ΔpH: (0.61 ± 0.01). CONCLUSIONS: The LPE below the MIC could still inhibit the GTF, LDH activity and lead to the decrease of WIG and the acid production.

Effect of garlic on bacterial biofilm formation on orthodontic wire.

OBJECTIVE: To examine the effect of garlic extract on the biofilm formation by Streptococcus mutans on orthodontic wire and on glucosyltransferase gene expression. MATERIALS AND METHODS: Growth inhibition of oral bacteria was tested after 50 µL of garlic extract was placed on an agar plate. The minimum inhibitory concentration (MIC) of garlic extract on S mutans growth was first determined. After cultivating streptococci in biofilm medium (BM)-sucrose with garlic extract and orthodontic wire, adenosine triphosphate (ATP) measurement and viable cell counting was performed from the bacteria attached on the wire. Scanning electron microscopy (SEM) analysis of morphology was observed on bacterial cells attached to orthodontic wire. The effect of garlic extract on gene expression was evaluated using quantitative real-time polymerase chain reaction (PCR) of glucosyltransferase. RESULTS: Though garlic extract had a clear antibacterial effect on all microorganisms, it also enhanced S mutans attachment on orthodontic wire. Low concentration of garlic extract also increased glucosyltransferase gene expression of S mutans. CONCLUSIONS: Despite its antibacterial function, garlic extract increases biofilm formation by S mutans to orthodontic wire, likely through upregulation of glucosyltransferase expression. Garlic extract may thus play an important role in increased bacterial attachment to orthodontic wires.

Effects of a composition containing lactoferrin and lactoperoxidase on oral malodor and salivary bacteria: a randomized, double-blind, crossover, placebo-controlled clinical trial.

We report a clinical trial of the effects of test tablets containing bovine lactoferrin and lactoperoxidase on oral malodor and salivary bacteria. Fifteen subjects with volatile sulfur compounds (VSCs) in mouth air above the olfactory threshold (H(2)S >1.5 or CH(3)SH >0.5 ng/10 ml) as detected by gas chromatography were enrolled in the trial. Either a test or a placebo tablet was ingested twice at 1-h intervals in two crossover phases. Mouth air was monitored for VSC levels at the baseline before ingestion of a tablet, 10 min after the first ingestion, 1 h (just before the second ingestion), and 2 h after the first ingestion. Whole saliva was analyzed at the baseline and at 2 h for bacterial numbers. At 10 min, the level of CH(3)SH was significantly lower in the test group (median [interquartile range] = 0.28 [0.00-0.68] ng/10 ml) compared to that in the placebo group (0.73 [0.47-1.00] ng/10 ml; P = 0.011). The median concentration of CH(3)SH in the test group was below the olfactory threshold after 10 min until 2 h, whereas the level in the placebo group was above the threshold during the experimental period. No difference in the numbers of salivary bacteria was detected by culturing or quantitative PCR, but terminal restriction fragment length polymorphism detected one fragment with a significantly lower copy number at 2 h in the test group (mean ± standard error, 4.89 ± 0.11 log(10) copies/10 µl) compared to that in the placebo group (5.38 ± 0.15 log(10) copies/10 µl; P = 0.033). These results indicate a suppressive effect of the test composition on oral malodor and suggest an influence on oral bacteria.

In vitro anticariogenic effects of aerial parts of Potentilla recta and its phytochemical profile.

This study, for the first time, investigated the in vitro inhibitory effects of Potentilla recta extracts and subfractions obtained with solvents of different polarity (aqueous, 50% ethanol, diethyl ether, ethyl acetate and n-butanol) against cariogenic Streptococcus spp. strains. It was found that the tested samples inhibited the growth of oral streptococci. Furthermore, all five P. recta preparations exhibited an inhibitory effect on water-insoluble α-(1→3)-,α-(1→6)-linked glucan (mutan) and artificial dental plaque formation. The ethyl acetate fraction showed the highest antibiofilm activities especially against S. sobrinus GCM 20381, with minimum mutan and biofilm inhibition concentrations of 6.25 and 25 µg/mL, respectively. The phytochemical profile of active constituents in the investigated samples was analysed. The high polyphenolics (total phenol, phenolic acids, tannins, proantocyanidins, flavonoids) content were found. The ethyl acetate fraction showed the highest concentration of total polyphenol content which may correlate with the high cariogenic activity of this subfraction. The results demonstrate that P. recta extracts and subfractions could become useful supplements for pharmaceutical products as new anticariogenic agents in a wide range of oral care products. Further studies are necessary to clarify the precise bioactive constituents of P. recta responsible for the anticariogenic properties.

Antibacterial activity of the essential oil from Rosmarinus officinalis and its major components against oral pathogens.

The essential oil of Rosmarinus officinalis L. (rosemary) was obtained by hydro-distillation and analysed by gas chromatography-mass spectrometry. Sixty-two constituents were identified, representing 98.06% of the total oil content. Oxygenated monoterpenes were the predominant components. The rosemary oil was characterized as having prominent (> 5%) contents of camphor (18.9%), verbenone (11.3%), a-pinene (9.6%), beta-myrcene (8.6%), 1,8-cineole (8.0%), and beta-caryophyllene (5.1%). The antimicrobial activity of the oil as well as of its major constituents was tested against the following microorganisms: Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus sobrinus, and Enterococcus faecalis, which are potentially responsible for the formation of dental caries in humans. The microdilution method was used for determination of the minimum inhibitory concentration (MIC) during evaluation of the antibacterial activity. The essential oil displayed low activity against the selected microorganisms. In the present study, the pure major compounds were more active than the essential oil. Among all the microorganisms tested, the pathogen S. mitis was the most susceptible and E. faecalis was the most resistant to the evaluated samples. This is the first report on antimicrobial activity of the major components of rosemary oil against oral pathogens.

Anticariogenic activity of the active fraction from isertia laevis against S. mutans and S. sobrinus: comparison of two extraction methods

Dental caries is considered a multi-factorial, infectious, chronic, localized, post-eruptive, transmissible disease that leads to the destruction of dental hard tissue. The recognition of Streptococcus mutans as the major bacterial species involved in dental caries has led to the implementation of prevention and control measures for eliminating or reducing it in oral cavity. The main goal of research on medicinal plants is the search for substances or compounds with antimicrobial activity. The aim of this study was to evaluate the antimicrobial activity of fractions obtained by two methods from Isertia laevis against S. mutans and S. sobrinus. The plant material was collected in Medina (Colombia), at an elevation of 550 meters above sea level. From the ethanol extract of leaves of I. laevis, fractions were obtained by two methods: extraction by column vacuum chromatography (CVC) and extraction by continuous liquid / liquid partitioning (CLLP). The evaluation of the antimicrobial activity of fractions against S. mutans and S. sobrinus was performed by well diffusion and bioautography assays. From the CVC technique, only the methanol and methanol-dichloromethane fractions showed activity against S. mutans and S. sobrinus, with a minimum inhibitory concentration of 2 mg/well. From the CLLP technique, only the dichloromethane fraction showed activity against both microorganisms, with a minimum inhibitory concentration of 1 mg/well. Compounds C1 and C2 were isolated from the three active fractions, and showed a minimum inhibitory concentration of 0.4 mg/well for S. mutans and S. sobrinus, with zones of inhibition measuring 6.5 and 6.2 mm, respectively. In conclusion: 1) the three active fractions of I. laevis showed activity against S. mutans and S. sobrinus, 2) compounds C1 and C2 were present equally in the three active fractions showing activity against the two bacteria, 3) compounds C1 and C2 may be triterpenoid and/or steroidal saponin structures, and 4) the two extraction methods lead equally to obtaining the active fractions.

La caries dental es considerada una enfermedad infecciosa multifactorial, cronica, localizada, poseruptiva y transmisible que conlleva a la destruccion del tejido dental duro. El claro reconocimiento de Streptococcus mutans como la principal especie bacteriana implicada en caries dental, ha conducido a la implementacion de medidas de prevencion y control para la eliminacion o disminucion de este microorganismo en cavidad oral. El objetivo fundamental de la investigacion en plantas medicinales, es la busqueda de sustancias o compuestos con actividad antimicrobiana para ser utilizadas en el control o prevencion de enfermedades infecciosas. En este sentido, en salud bucal muchas sustancias obtenidas de plantas han mostrado actividad antimicrobiana. El objetivo de este estudio fue evaluar la actividad antimicrobiana de fracciones obtenidas de la planta Isertia laevis mediante dos metodologias contra S. mutans y S. sobrinus. El material vegetal se colecto en el municipio de Medina (Cundinamarca- Colombia) situado a una altura de 550 metros sobre el nivel del mar. A partir del extracto etanolico de hojas de I. laevis se obtuvieron fracciones mediante dos metodologias, extraccion por cromatografia en columna al vacio (CCV) y extraccion por fraccionamiento liquido/liquido continuo (FLLC). La evaluacion de la actividad antimicrobiana de las fracciones frente a S. mutans y S. sobrinus se realizo por el metodo de difusion en pozo y bioautografico. De las fracciones obtenidas por CCV, solamente las fracciones metanol y metanol-diclorometano presentaron actividad antimicrobiana sobre S. mutans y S. sobrinus, con una concentracion minima inhibitoria de 2 mg/pozo. De las fracciones obtenidas por FLLC solamente la fraccion diclorometano presento actividad antimicrobiana sobre S. mutans y S. sobrinus, con una concentracion minima inhibitoria de 1 mg/pozo. De las 3 fracciones activas se aislaron los compuestos C1 y C2, que presentaron una concentracion minima inhibitoria de 0.4 mg/pozo tanto para S. mutans como para S. sobrinus con halos de inhibicion, respectivamente, de 6.5 y 6.2 mm. En conclusion, 1. Las fracciones metanol y diclorometano obtenidas por CCV y la fraccion diclorometano obtenida por FLLC de hojas de I. laevis presentaron actividad antimicrobiana sobre S. mutans y S. sobrinus; 2. Los compuestos C1 y C2 presentes por igual en las tres fracciones activas tuvieron accion inhibitoria sobre las dos bacterias en evaluacion; 3. Las pruebas quimicas cualitativas para los compuestos C1 y C2 indican que posiblemente corresponden a estructuras de saponinas triterpenicas y/o esteroidales; y 4. Las dos metodologias de extraccion conducen por igual a la obtencion de las fracciones activas.

Automutanolysin disrupts clinical isolates of cariogenic streptococci in biofilms and planktonic cells.

INTRODUCTION: Dental caries remains one of the most common chronic infectious diseases throughout the world. The formation of dental plaque is one of the caries risk factors. As a consequence, the removal of plaque may reduce the incidence of caries development. We identified an autolysin produced by Streptococcus mutans named auto-mutanolysin (Aml). Aml selectively lyses S. mutans and Streptococcus sobrinus. The specificity towards these cariogenic bacteria suggests that Aml may be used to prevent dental caries. Here, with the aim towards therapeutic application, we investigated the lytic activity of Aml against clinical isolates of S. mutans and S. sobrinus using planktonic cells and biofilms. METHODS: Planktonic cell suspensions and biofilms of clinically isolated streptococci were treated with Aml in the absence or the presence of Triton X-100. The lytic activity of Aml was monitored as the change in turbidity. The disruption of biofilms was evaluated by detecting the released DNA by polymerase chain reaction and observing the alteration of optical density of treated biofilms. RESULTS: Triton X-100 enhances the lytic ability of Aml. Using planktonic cells, Aml had various lysis levels against clinical strains. Repeated Aml treatment showed disruption of the biofilm using the representative clinical strains. CONCLUSION: Our study demonstrates that Aml has an ability to lyse planktonic and biofilm cells of clinically isolated mutans streptococci in the presence of Triton X-100. These results suggest the possibility of using Aml as an alternative or additional approach for caries prevention.

Human milk compounds inhibiting adhesion of mutans streptococci to host ligand-coated hydroxyapatite in vitro.

Acquisition of mutans streptococci at an early age is a risk factor for later caries development. Following our recent finding that human milk may inhibit adhesion of Streptococcus mutans the aim of the present study was to identify compounds in human milk preventing adhesion of mutans streptococci to saliva- or gp340-coated hydroxyapatite (s-HA and gp340-HA) using an in vitro model system. Superdex 200 fractions of human milk and purified proteins were screened for binding inhibition of the S. mutans strain Ingbritt. Avid inhibition was seen to both s-HA and gp340-HA for caseins, lactoferrin, IgA and IgG, and moderate inhibition for alpha-lactalbumin and bile salt-stimulated lipase, whereas albumin and lysozyme had no effect. The inhibitory epitope in beta-casein was delineated to its C-terminal LLNQELLNPTHQIYPVTQPLAPVHNPISV stretch by use of synthetic peptides. Similarly, a peptide (SCKFDEYFSQSCA) corresponding to the human lactoferrin stretch that is highly homologous to the previously shown inhibitory stretch of bovine lactoferrin was found to inhibit S. mutans Ingbritt binding. Inhibition by human milk, IgA, and the inhibitory beta-casein peptide was universal among 4 strains of S. mutans (Ingbritt, NG8, LT11, JBP) and 2 strains of S. sobrinus (6715 and OMZ176). IgG inhibited 4, alpha-lactalbumin 3 and lactoferrin 2 of these 6 strains. It was also confirmed that none of the milk components coated on HA mediated S. mutans Ingbritt adhesion, which was consistent with the finding that no milk protein was recognized on Western blots by gp340/DMBT1 monoclonal antibodies.