Antimicrobial effect of Korean propolis against the mutans streptococci isolated from Korean

The aim of this study was to determine the optimal concentration of Korean propolis against clinical isolates of mutans streptococci (MS) from Koreans. The antimicrobial activity was evaluated using the minimum inhibitory concentration (MIC) and time-kill curves against mutans streptococci. The MIC(90) values of propolis for MS were 35 μg/ml. Propolis had a bacteriostatic effect on Streptococcus mutans ATCC 25175(T) and bactericidal effects on Streptococcus sobrinus ATCC 33478(T) at > 2 × MIC (70 μg/ml). These results suggest that the propolis can be used in the development of oral hygiene products for the prevention of dental caries.     

Isolation and characterization of the mutans streptococci from the dental plaques in Koreans

Mutans streptococci have been implicated as cariogenic bacteria in dental caries because they can produce high levels of dental caries-causing lactic acid and extracellular polysaccharide. The aim of this study was to isolate and characterize the mutans streptococci from the dental plaque obtained from Koreans. The dental plaque samples were collected from the anterior and molar teeth of both jaws in 155 subjects (aged 2 to 33.2 years, average age 13.7+/-4.7 years). The samples were diluted by 100-fold in 1x PBS and plated on mitis-salivarius bacitracin (MSB) agar plates. The mutans streptococci grown on MSB plates were screened by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dextranase gene (dex). The mutans streptococci were identified at the species level using a 16S rDNA sequencing comparison method. The biochemical tests were carried out to biotype the mutans streptococci. Ninety-five strains of the mutans streptococci out of 358 colonies, which were derived from 141 subjects, were isolated. Of them, 77 strains and 18 strains were Streptococcus mutans and Streptococcus sobrinus, respectively. The biotyping data showed that 62, 1, 20, 10, and 2 strains were biotypes I, II, IV, V and variant, respectively. Of the two strains of variant biotype, one strains was similar to biotype IV except that it was positive to the arginine hydrolysis test. We considered this one strain a new biotype, and classified it as biotype VII. In conclusion, S. mutans and its biotype I was most frequently isolated in Korean dental plaque. The mutans streptococci strains isolated in this study might be useful for the study of the pathogenesis and the prevention of dental caries.     

Effect of a mouthrinse containing Malva sylvestris on the viability and activity of microcosm biofilm and on enamel demineralization compared to known antimicrobials mouthrinses

The purpose of this study was to evaluate the antimicrobial (anti-biofilm) and anti-caries (enamel demineralization prevention) effects of Malva sylvestris (Malvatricin^® Plus) compared with known antimicrobial mouthrinses. Microcosm biofilm was produced on enamel, using inoculum from pooled human saliva mixed with McBain saliva (0.2% sucrose) for 14 days. The biofilm was treated with mouthrinses for 1 min day^?1. Oral-B^® Complete, Listerine^® Zero and Malvatricin^® Plus had the greatest effect on the reduction of biofilm viability (p < 0.0001). On the other hand, lactic acid production was reduced significantly with PerioGard^®, Noplak^® Max and Listerine^® Zero compared with the control (p < 0.0001). No significant differences were found among the mouthrinses with respect to the colony-forming unit counting (total microorganisms, total streptococci, mutans streptococci and lactobacilli) and extracellular polysaccharide production. Enamel demineralization was reduced significantly with PerioGard^®, Noplak^® Max and Malvatricin^® Plus compared with the control (p < 0.0001). Malva sylvestris has a comparable anti-caries effect to chlorhexidine mouthrinses.     

Rapid Identification of Mutans Streptococcal Species

Mutans streptococci are considered the predominant pathogens in dental caries. Three methods, i.e. dot blot hybridization analysis, PCR analysis and SDS-blue dextran-PAGE, were examined for identifying mutans streptococcal species. In dot blot hybridization, DNA probe derived from the dextranase gene (dexA) of Streptococcus mutans hybridized with different intensities under the condition of low stringency against each species of mutans streptococci although the dexA probe was specific for S. mutans under the condition of high stringency. Oligonucleotide primers for polymerase chain reaction (PCR) were designed on the basis of the dexA DNA sequence. The primers amplified species-specific PCR products in the reference species (15 strains of 5 species) of mutans streptococci. An electrophoretic profile of dextranases from the mutans streptococci on SDS-blue dextran-PAGE also showed species-specific behavior. These results suggest that the three identification methods examined here are useful for distinguishing the species of mutans streptococci and also indicate that PCR analysis is suitable for simple, rapid and reliable identification of mutans streptococcal species.     

Antimicrobial actions of α-mangostin against oral streptococci

The increasing prevalence of dental caries is making it more of a major world health problem. Caries is the direct result of acid production by cariogenic oral bacteria, especially Streptococcus mutans. New and better antimicrobial agents active against cariogenic bacteria are badly needed, especially natural agents derived directly from plants. We have evaluated the inhibitory actions of α-mangostin, a xanthone purified from ethanolic extracts of the tropical plant Garcinia mangostana L., by repeated silica gel chromatography. α-Mangostin was found to be a potent inhibitor of acid production by S. mutans UA159, active against membrane enzymes, including the F(H+)-ATPase and the phosphoenolpyruvate - sugar phosphotransferase system. α-Mangostin also inhibited the glycolytic enzymes aldolase, glyceraldehyde-3-phosphate dehydrogenase, and lactic dehydrogenase. Glycolysis by intact cells in suspensions or biofilms was inhibited by α-mangostin at concentrations of 12 and 120 μmol·L?1, respectively, in a pH-dependent manner, with greater potency at lower pH values. Other targets for inhibition by α-mangostin included (i) malolactic fermentation, involved in alkali production from malate, and (ii) NADH oxidase, the major respiratory enzyme for S. mutans. The overall conclusion is that α-mangostin is a multitarget inhibitor of mutans streptococci and may be useful as an anticaries agent.     

白及提取物抗变异链球菌致龋效果的研究

目的研究白及提取物对变异链球菌粘附、生物膜形成及活性的影响,评价其抗龋效果。方法市售白及95%乙醇浸提;纸片法、打孔法测定直接抑菌作用;液体稀释法检测MIC;结晶紫法研究亚抑菌浓度提取物对变异链球菌粘附能力及生物膜总量的影响;采用荧光显微镜和激光共聚焦显微镜观察常态牙菌斑生物膜生长过程中及药物处理后牙菌斑生物膜中死菌和活菌的构成,研究其对牙菌斑生物膜结构和活性的影响;运用扫描电镜观察白及药液对变异链球菌生物膜的影响。结果白及提取物具有一定的抑菌作用,MIC为16~62 mg/m L;结晶紫法定量研究生物膜结果显示白及药液作用4 h对变异链球菌的粘附均有抑制作用,抑制率为28.63%~60.08%;作用20 h对生物膜总量抑制率达77.08%;白及药液作用20 h,荧光染色显示生物膜活性明显被抑制,抑制率达62.03%;梯度浓度白及药液分别作用20 h后,激光共聚焦显微镜下观察到随着药液浓度增加,绿色的活菌、团块状结构减少,生物膜形成明显被抑制;扫描电镜下可见药液作用后细菌间粘性物质减少。结论高浓度白及提取液对变异链球菌有直接抑菌作用,亚抑菌浓度能抑制其粘附和生物膜的形成,进而具有抗龋作用。     

In vitro antimicrobial activity of Romanian medicinal plants hydroalcoholic extracts on planktonic and adhered cells

The aim of this study was to assess the antibacterial and antifungal potential of some Romanian medicinal plants, arnica--Arnica montana, wormwood--Artemisia absinthium and nettle--Urtica dioica. In order to perform this antimicrobial screening, we obtained the vegetal extracts and we tested them on a series of Gram-positive and Gram-negative bacteria, and also against two fungal strains. The vegetal extracts showed antimicrobial activity preferentially directed against the planktonic fungal and bacterial growth, while the effect against biofilm formation and development was demonstrated only against S. aureus and C. albicans. Our in vitro assays indicate that the studied plant extracts are a significant source of natural alternatives to antimicrobial therapy, thus avoiding antibiotic therapy, the use of which has become excessive in recent years.     

In vitro bactericidal action of a complex of natural cytokins on Streptococcus pyogenes

The data on the antimicrobial in vitro action of a complex of natural cytokins (CNC)--the substance and the preparation "Superlymph"--on virulent museum and clinical macrolid-resistant group A beta-hemolytic streptococci, are presented. CNC suppressed the growth of museum test culture of streptococci in the concentration dependent manner. Moreover, the preparation "Superlymph" proved to be an effective antistreptococcal remedy in its action on erythromycin-resistant group A streptococcal (GAS) strains. The substance of the preparation induced the lysis of GAS cells, grown on lawn plates and in the system of spectrophotometric analysis. The effect thus established could be caused by cationic antimicrobial peptides (protegrins), contained in "Superlymph".     

Effect of oleanolic acid-cyclodextrin inclusion compounds on dental caries by in vitro experiment and rat-caries model.

Earlier work in vitro showed that oleanolic acid (OA) was a potential inhibitor of insoluble glucan (ISG) synthesis from mutans streptococci (MS). In this study, two oleanolic acid-cyclodextrin inclusion compounds (OA-CDs), oleanolic acid-G1-beta-cyclodextrin (OA-G1-beta CD) and oleanolic acid-beta-cyclodextrin (OA-beta CD), were assayed for their effects on ISG synthesis from Streptococcus mutans MT8148R, and on the growth of oral bacteria. OA-beta CD inhibited ISG synthesis by 55.3 and 37.4% at 62.5 and 15.6 micrograms/ml of OA, respectively. Both OA-CDs inhibited the growth of MS, S. sanguis, and S. salivarius at 4 to 8 micrograms/ml of OA. The anticariogenic effect of the OA-beta CD was examined in a rat-caries model. Rats in the infected control groups showed the highest caries score. The infected treatment group B (0.5% OA in diet) showed lower scores than the control group. These results suggest that OA-beta CD is a potential anti-caries agent.     

Inhibition ofStreptococcus mutans and Other Oral streptococci by hop (Humulus lupulus L.) constituents

We report the inhibition of the causative agents of dental caries, Streptococcus mutans and other oral streptococci, by the antimicrobially active ingredients of the hop plant (Humulus lupulus L.). The hop constituents studied were purified beta acid, xanthohumol, isoalpha acid and tetra iso-alpha acid. Cruder hop extracts were also investigated. The antimicrobial activity of these hop constituents was tested against four strainsof Streptococcus mutans as well as one strain each ofStreptococcus sanguis andStreptococcus salivarius and compared to antimicrobial essential oils used in mouthwashes in two independent assay systems. We found that all tested hop constituents inhibited the Streptococci. The minimum inhibitory concentration at pH 7.5 ranged from 2 to 50 μg/ml depending on the microorganism and hop phytochemical tested. Contrary to a previous report, there was no activity enhancement by ascorbic acid over and above the enhancement due to pH lowering. Thére was no resistance development to beta acid after 10 passages in a subinhibitory concentration of this acid. Antimicrobial activity of hop constituents was found to be greater than other plant products such as thymol, nerol, cinnamon oil, oil of clove, menthol and eucalyptol. The possibilities of using hop constituents in mouthwashes are discussed.     

Beta haemolytic streptococci of the Lancefield groups E. P and U:Streptococcus infrequens

The physiological and biochemical characteristics of isolates from swine in Sweden and The Netherlands were compared with those of strains from several culture collections. These characteristics were found to be similar for all three Lancefield groups and they form a well defined pattern distinct from other known streptococcal species. It is suggested that these streptococci be classified together in one species:Streptococcus infrequens.Group and type sera of Group E streptococci have no affinity to Group P and Group U streptococci, and vice versa. None of the sera prepared against group E type strains contains the group antibody. Group P and Group U streptococci have an antigen in common. This common antigen is present in formamide extracts. It is not demonstrable in acid extracts. None of the group P sera tested contains the common antibody. Group P serum has to be considered as a type serum. Group U sera contain the common antibody, and when absorbed with group P cells prove to contain another type antibody, which reacts with extracts of most group U strains.Isolates of all three Lancefield groups were obtained from a variety of pathological conditions in swine.     

Quantitation of M antigen in Lancefield extracts of group A streptococci type 12 using electro-immuno assay

Anti-type 12 serum incorporated in agarose-polyethylene glycol gel in a concentration of 1.5% (vol/vol) was found to enable a distinct "rocket" precipitate in electro-immuno assay using hot hydrochloric acid extract of type 12 group A streptococci. This precipitate was removed by trypsin treatment of the extract and on addition of anti-M12 typing serum but not of five other typing sera to the extract. The streptococcal component responsible for this precipitate was eluted from a CM-cellulose ion exchange column at pH 6.5. These findings demonstrated that the precipitate was caused by the M12 antigen. Crossed immuno-electrophoresis of hot hydrochloric acid extracts of three different type 12 group A streptococci showed that the electrophoretic mobility of the M12 antigens was similar in the three extracts. A linear correlation was obtained between the concentration of the M12-antigen and the height of the precipitate obtained in the electro-immuno assay using different dilutions of a standard type 12 extract. M12 antigen could thus be quantitated by the electro-immuno assay. In quantitation experiments, uniformly prepared extracts of five randomly selected, freshly-isolated type 12 strains were found to contain from 130 to 1850% of M12 antigen, respectively (expressed in % of the content of the standard type 12 extract).     

Heart cross-reactive antigens of mutans streptococci share epitopes with group A streptococci and myosin

Monoclonal antibodies (mAb) generated by immunization of mice with membranes of Streptococcus pyogenes Manfredo (M type 5) were tested for their reactivity with sodium dodecyl sulfate extracts of Streptococcus mutans GS5, Streptococcus rattus BHT (formerly S. mutans, serotype b), and S. mutans Ingbritt 175 in the Western blot. The reaction of the sodium dodecyl sulfate-extracted proteins of whole S. mutans, serotype b), and S. rattus with the mAb revealed that the shared cross-reactive antigens were near m.w. 62,000 to 67,000. Several higher m.w. proteins in S. mutans Ingbritt 175 reacted with the mAb but were not observed in the other two strains of streptococci. Cytoplasmic membranes of S. rattus BHT reacted with these mAb in the enzyme-linked immunosorbent assay and Western blot. The most reactive BHT membrane component detected by these mAb was a 62-kDa polypeptide that was similar in m.w. to the membrane component recognized by these antibodies in purified S. pyogenes membranes. The reactivity of the mAb with BHT membranes in the enzyme-linked immunosorbent assay was absorbed by rabbit skeletal muscle myosin. The patterns of reactivity observed in the BHT membrane immunoblots in this study closely resemble those previously obtained using polyclonal rabbit anti-human heart sera.     

New serotype of mutans streptococci isolated from pig oral cavity

Gram-positive streptococcal mutans-like strains, but with clearly different colony formation than S. orisuis on Mitis Salivarius agar, were isolated from the pig oral cavity and identified by 16S rRNA sequencing, G+C content, DNA-DNA homology and extensive biochemical and serological testing. The phenotypic data showed that the strains were similar to S. orisuis except for susceptibility to bacitracin. DNA-DNA homology between the isolates and S. orisuis was 72∼81%. However, serological data showed that they have a different sero-specific antigen from S. orisuis and other mutans streptococci. A new serotype, designated p, strains are classified in a serovar of S. orisuis, one of mutans streptococci.     

Zymographic characterization of bacteriolytic enzymes produced by oral streptococci

Zymographic analysis was performed to know the bacteriolytic enzyme profiles of 4% SDS extracts of oral streptococci, Streptococcus mutans, S. sobrinus, S. sanguis, S. mitis and S. salivarius. We investigated the five strains in each species and found that the profile was very similar among strains of the same species except for S. salivarius(the profile was classified into two types). On the other hand, the profile was considerably different among species. Two major bacteriolytic enzymes of S. mutans showing molecular mass of 80 and 100 kDa were found using SDS-boiled S. mutans or S. sobrinus cells as substrate. These bacteriolytic activities were less apparent in the gel containing S. mitis or S. salivarius, and also not detectable in the gel containing S. sanguis. S. sobrinus extract showed only one bacteriolytic band (78 kDa) as strong activity using S. sobrinus cells as substrate. S. sanguis extract showed no bacteriolytic bands using any streptococcal cells. Extracts of either S. mitis or S. salivarius showed weak activity by using respective strains as substrate.     

Inhibition by yeast killer toxin-like antibodies of oral Streptococci adhesion to tooth surfaces in an ex vivo model

BACKGROUND: Monoclonal (KTmAb) and recombinant (KTscFv) anti-idiotypic antibodies, representing the internal image of a yeast killer toxin, proved to be microbicidal in vitro against important eukaryotic and prokaryotic pathogens such as Candida albicans, Pneumocystis carinii, Mycobacterium tuberculosis, Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae, including multidrug-resistant strains. KTmAb and KTscFv exerted a strong therapeutic effect in well-established animal models of candidiasis and pneumocystosis. Streptococcus mutans is the most important etiologic agent of dental caries that might result from the metabolic end products of dental plaque. Effective strategies to reduce the disease potential of dental plaque have considered the possibility of using antibiotics or antibodies against oral streptococci in general and S. mutans in particular. In this study, the activity of KTmAb and KTscFv against S. mutans and the inhibition and reduction by KTmAb of dental colonization by S. mutans and other oral streptococci in an ex vivo model of human teeth were investigated. MATERIALS AND METHODS: KTscFv and KTmAb were used in a conventional colony forming unit (CFU) assay against a serotype C strain of S. mutans, and other oral streptococci (S. intermedius, S. mitis, S. oralis, S. salivarius). An ex vivo model of human teeth submerged in saliva was used to establish KTmAb potential of inhibiting or reducing the adhesion to dental surfaces by S. mutans and other oral streptococci. RESULTS: KTmAb and KTscFv kill in vitro S. mutans and other oral streptococci. KTmAb inhibit colonization of dental surfaces by S. mutans and oral streptococci in the ex vivo model. CONCLUSIONS: Killer antibodies with antibiotic activity or their engineered derivatives may have a potential in the prevention of dental caries in vivo.     

Modifications of hydrophobicity, in vitro adherence and cellular aggregation of Streptococcus mutans by Helichrysum italicum extract

AIMS: The purpose of the present study was to examine whether sublethal concentrations of Helichrysum italicum extract could affect some of the cariogenic properties of Streptococcus mutans. METHODS AND RESULTS: We studied the antibacterial activity of H. italicum (ethanolic extract) against oral streptococci (Strep. mutans ATCC 35668, Strep. salivarius ATCC 13419 and Strep. sanguis ATCC 10556) and its influence on cell-surface hydrophobicity, in vitro sucrose-dependent adherence to glass surface and cellular aggregation of Strep. mutans. The results indicate that all streptococci were susceptible to ethanolic extract with minimum inhibitory concentration (MIC) values of 31.25-62.50 microg x ml(-1). Sub-MIC concentrations of H. italicum (7.81-31.25 microg x ml(-1)) reduced the hydrophobicity and the adherence (almost 90%) to glass surface of Strep. mutans. The aggregation in the presence of dextran T2000 was also affected. CONCLUSION: The inhibitory activity of H. italicum extract on Strep. mutans is worthy of further study. SIGNIFICANCE AND IMPACT OF THE STUDY: There is considerable interest in the use of natural compounds as alternative methods to control undesirable micro-organisms.     

A lacticin 3147 enriched food ingredient reduces Streptococcus mutans isolated from the human oral cavity in saliva

AIMS: To isolate and characterise Streptococcus mutans from Irish saliva samples and to assess their sensitivity to a food-grade preparation of the lantibiotic, lacticin 3147, produced by Lactococcus lactis DPC3147. METHODS AND RESULTS: Saliva samples collected from children with varying oral health status were screened on Mitis Salivarius agar for the presence of pathogenic streptococci. Following selective plating, 16S rDNA sequencing and Pulsed Field Gel Electrophoresis (PFGE), 15 distinct strains of Strep. mutans were identified. These were grouped according to their relative sensitivity to lacticin 3147 which ranged from 0.78 to 6.25%; relative to a sensitive indicator strain, Lactococcus lactis ssp. lactis HP. Inhibition of indicator Strep. mutans strains from sensitive, intermediate and tolerant groupings were assessed in microtitre plate assays with increasing concentrations of lacticin 3147. The concentration of lacticin 3147 required to give 50% growth inhibition correlated with their relative sensitivities (as assayed by well diffusion methodology) and ranged from 1280 to 5120 AU ml(-1). Concentrated preparations of lacticin 3147 caused a rapid killing of Strep. mutans strains in broth. Moreover, in human saliva deliberately spiked with Strep. mutans, the pathogen was eliminated (initial inoculum of 10(5)) in the presence of 40,000 AU ml(-1) of lacticin 3147. Furthermore, a food-grade lacticin 3147 spray dried powder ingredient was assessed for the inhibition of Strep. mutans in human saliva, spiked with a strain of intermediate sensitivity, resulting in up to a 4-log reduction in counts after 20 min. CONCLUSION: A food grade preparation of lacticin 3147 was effective in the inhibition of oral Strep. mutans. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of oral streptococci by food grade preparations of lacticin 3147 may offer novel opportunities for the development of lacticin 3147 as an anti-cariogenic agent particularly in the area of functional foods for the improvement of oral health.     

Xylitol and Erythritol Decrease Adherence of Polysaccharide-Producing Oral Streptococci

Xylitol consumption decreases counts of mutans streptococci. However, the mechanism behind this decrease is not well understood. We studied not only type strains and clinical isolates of mutans streptococci, but also other polysaccharide-forming oral streptococci. Growth inhibition and adherence of cells to a smooth glass surface—reflecting synthesis of water-insoluble polysaccharides were studied in the presence of 2% (0.13 mol/l) and 4% (0.26 mol/l) xylitol. The effect of xylitol was compared to a novel polyol sweetener, erythritol. Except for Streptococcus mutans 10449 and S. sobrinus OMZ 176, the glass surface adhesion of most polysaccharide-forming streptococci was reduced by the presence of both 4% xylitol and erythritol. For the S. mutans and S. sobrinus type strains, the growth inhibition with 4% xylitol and erythritol was 36–77% and for the clinical S. mutans isolates 13–73%. Of the other oral streptococci, only S. sanguinis was inhibited with 4% xylitol (45–55%). For both polyols, the magnitude of the growth inhibition observed was not associated with the magnitude of the decrease in adherence (xylitol: r = −0.18; erythritol: r = 0.49). In conclusion, both xylitol and erythritol can decrease polysaccharide-mediated cell adherence contributing to plaque accumulation through a mechanism not dependent on growth inhibition.     

Development of species-specific primers for detection of Streptococcus mutans in mixed bacterial samples

Streptococcus mutans is the major microbial pathogen associated with dental caries in children. The objectives of this study were to design and evaluate species-specific primers for the identification of S. mutans. Validation of the best primer set, Sm479F/R, was performed using seven S. mutans reference strains, 48 ATCC non-S. mutans strains, 92 S. mutans clinical isolates, DNA samples of S. mutans-Streptococcus sobrinus or S. mutans-Streptococcus sanguinis, and mixed bacterial DNA of saliva samples from 33 18-month-old children. All of the S. mutans samples tested positive, and no PCR products were amplified from members of the other streptococci or nonstreptococci strains examined. The lowest detection level for PCR was 10(-2) ng of S. mutans DNA (c. 4.6 x 10(3) copies) in the test samples. The results of this study suggest that the Sm479F/R primer pair is highly specific and sensitive for identification of S. mutans in either purified or mixed DNA samples.