Frequency and expression of the mutacin biosynthesis genes in Streptococcus mutans isolates

Orientador: Reginaldo Bruno GonçalvesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Esta tese, apresentada na forma de 3 artigos, teve por objetivos: (1) analisar a freqüência dos genes de produção de mutacinas I, II, III e IV, em genótipos de S. mutans isolados de indivíduos cárie-ativos e livres de cárie, (2) analisar a freqüência dos genes de produção das mutacinas I, II, III, IV, N, B-Ny 266, 1140 e genes homólogos às bacteriocinas, identificadas em outras espécies bacterianas, em cepas de S. mutans isolados de crianças, bem como detectar a expressão diferencial dos genes identificados, em células de S. mutans crescidas na condição planctônica e séssil, (3) analisar a expressão dos genes de produção das mutacinas I, II e proteínas kinases CiaH, Dgk e ComD, em diferentes fases do crescimento planctônico e séssil. O rastreamento e a freqüência dos genes estruturais de diferentes mutacinas em isolados de S. mutans, foram realizados pela técnica de PCR e a análise da expressão gênica, pela técnica de RT-PCR semi-quantitativa. Os estudos, apresentados nesta tese, demonstraram o papel das mutacinas como um fator de virulência, altamente diversificado entre a espécie S. mutans, e relacionado com o risco de cárie. Este fator de virulência, pode ser regulado por mecanismos quorum-sensing, sendo assim, dependente da condição de crescimento planctônica ou séssil. A regulação da produção de mutacinas, por mecanismos quorum-sensing, pode representar uma vantagem seletiva à espécie produtora, principalmente em ambiente complexo, como o biofilme dental e lesões de cárie. Futuramente, mais estudos serão necessários para identificar novos determinantes genéticos, necessários para a síntese de substâncias semelhantes às mutacinas, bem como, identificar os mecanismos e componentes, que modulam a expressão deste importante fatorde virulência em S. mutansAbstract: This thesis, comprised of 3 manuscripts was designed (1) to analyse the frequency of biosynthesis genes of the mutacins types I, II, III and IV, in S. mutans isolated from caries-affected and caries-free individuals, (2) to analyse the frequency of biosynthesis genes of the mutacins types I, II, III, IV, N, B-Ny 266, 1140 and genes homologues to bacteriocins identified in other bacterial species, in S. mutans isolated from children, in addition, to detect the differential expression of these genes, in S. mutans cells grown in planktonic and sessil conditions, (3) and to analyse the expression of the mutacins I and II production genes and kinase proteins genes (ciaH, dgk e comD), in different phases of the planktonic and sessile growth. The screening and frequency of the mutacins structural genes in S. mutans isolates were realized by PCR technique and the analysis of genetic expression, by RT-PCR semiquantitative method. The studies, presented in this thesis, demonstrate the role of mutacins as a virulence factor, highly diverse among S. mutans, and related to risk of dental caries. The mutacins production may be regulated by quorum-sensing mechanisms and is dependent on planktonic and sessile conditions. The modulation by quorum-sensing mechanisms may represent a selective advantage for producer S. mutans strain, mainly in complex environments as the dental biofilm and caries. Hereafter, more studies will be necessary to identify new genetic determinants for synthesis of mutacin-like substances and elucidate the mechanisms and components that modulate the genetic expression of this important virulence factor in S. mutansDoutoradoMicrobiologia e ImunologiaDoutor em Biologia Buco-Denta

Analise genotipica e mutacinotipagem de Streptococcus mutans isolados de voluntarios carie-ativos e livres de carie

Orientador : Reginaldo Bruno GonçalvesDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Os objetivos deste estudo foram: comparar a atividade de produção de mutacinas, a diversidade fenotípica/genotípica de Streptacoccus mutans de voluntários cárie-ativos e livres de cárie, e determinar/comparar o grau de associação e a capacidade discriminatória da Mutacinotipageme a AP-PCR. Um total de 319 cepas de Smutans isoladas a partir da saliva, placa dental e dorso de língua de 8 voluntários cárie-ativos e 8 livres de cárie foram submetidas à Mutacinotipageme à técnica de AP-PCR. O perfil mutacinogênico foi traçado pela Bacteriocinotipagem, utilizando-se 13 cepas indicadoras, entre elas: S. mutans, S. sabrinus, S mitis, S salivarius, S. sanguinis, S. aralis e S cricetus, e o polimorfismo genético foi detectado através da técnica de AP-PCR com os primers OPA-02 e OPA-13. Cerca de 79,62% das cepas de S mutans (254/319) analisadas produziram mutacinas contra pelo menos uma das 13 indicadoras. Através dos métodos de tipagem empregados, detectou-se que não houve relação entre a diversidade fenotípica e/ou genética de Smutans e atividade de cárie. A utacinotipagem permitiu detectar 55 perfis fenotípicos contra 101 perfis genéticos polimórficos detectados pela AP-PCR, e foi limitada para a análise da heterogeneidade da espécie intra-indivíduo. O grau de associação entre as duas técnicas de agrupamento foi em média de 77,38%, sendo o poder discriminatório da AP-PCR (0,975) superior ao da Mutacinotipagem(0,925). Embora a Mutacinotipagemdemonstre limitações para o estudo da diversidade fenotípica, através dela foi possível demonstrar que o potencial mutacinogênico, de S mutans isolados de indivíduos com cárie, possuíram amplos espectros inibitórios contra as diferentes cepas indicadoras, já os isolados de voluntários livres de cárie apresentaram um perfil mais específico contra S. sanguinis, S. aralis e S mitis, que são colonizadores predominantes nesta população. Teoricamente, inibindo seus competidores potenciais, os S mutans teriam uma colonização bem sucedida, seja pela aquisição da hegemonia nutricional ou de receptores de superficie para sua adesãoAbstract: The objectives of this study were: to compare the activity of mutacin production, the fenotypic/genotypic diversity of S. mutans isolated from caries active and caries free individuals, and to evaluate the association index and the power discriminatory of Mutacin Typing and AP-PCR. In the present study, 319 strains of S. mutans isolated from saliva, dental plaque and tongue dorsum of 8 caries active and 8 caries free voluntareers were submitted to Mutacin Typing and AP-PCR. The mutacinogeny profile was traced, using 13 indicator strains between them: S. mutans, S. sobrinus, S. mitis, S. salivarius, S. sanguinis, S. oralis e S. cricetus and the genetic polymorphism was detected with primers OPA-02 and OPA-13. Mutacin was produced by 79,62% out of 319 isolates (245/319) inhibiting one or more of the 13 indicator strains. No corellation was found between the S. mutans fenotypic/genotypic diversity and caries activity, by the typing methods employed. The Mutacin typing allowed to evidence 55 distinct fenotypic patterns whereas AP-PCR detected 101 distinct patterns genotypic. The Mutacin Typing was limited to analyze S. mutans heterogeneity intra individually. The association index mean between Mutacin Typing and AP-PCR was 77,38%, and the discriminatory power of AP-PCR (0,975) was greater than the one Mutacin Typing (0,925). Bacteriocin Typing was limited to study for fenotypic diversity, however it was possible to demonstrate that S. mutans isolated from caries active group showed higher inhibitory activity than S. mutans isolated from caries free group, and the last presented highest antagonic effect against S. sanguinis, S. oralis and S. mitis (the predominant colonizers of this population). Theorically, inhibiting potencial competitors, S. mutans would have succesful colonization, either by nutricional or adhesion receptor aquisitionsMestradoMicrobiologia e ImunologiaMestre em Biologia Buco-Denta

Phytochemical profile, evaluation of antimicrobial and antioxidant activity in vitro of the hydroalcoholic extract of two species of the genus Cyperus (Cyperaceae)

Several factors contribute to the resistance of some pathogenic microorganisms and this fact requires the search for new therapeutic alternatives. The genus Cyperus (family Cyperaceae) groups species that present chemical compounds of pharmacological interest, mainly with antimicrobial action. Thus, the present work was carried out to investigate the antimicrobial activities, antioxidants and the phytochemical profile of Cyperus articulatus L. and Cyperus iria L. Hydroalcoholic extracts (1:1, v:v) of the aerial and underground parts of these species were used to analyze the total phenol content and to evaluate the in vitro antioxidant activity against the DPPH (2,2-diphenyl-1-picrylhydrazyl). The ethyl acetate and chloroform phases resulting from liquid-liquid partitioning of C. articulatus and C. iria extracts were evaluated in antimicrobial assays and subject to high performance liquid chromatography (HPLC-DAD) analysis. The chromatograms obtained by HPLC-DAD allowed us to identify four compounds: chlorogenic acid, catechin, quercetin, and quercitrin. The hydroalcoholic extracts of C. articulatus and C. iria showed a weak antioxidant activity with IC50 of 395.57 and 321.33 μg/mL (aerial parts), and 1,114.01 and 436.82 μg/mL (underground parts), respectively. Regarding antimicrobial activity, the chloroform phase of C. iria showed the best result at the concentration of only 31.2 µg/mL against the pathogens Candida albicans and Staphylococcus aureus. The ethyl acetate phases of the aerial parts of C. articulatus and C. iria did not show antimicrobial activity

Chemical and microbiological characterization of tinctures and microcapsules loaded with Brazilian red propolis extract

The aim of this study was to characterize tinctures and microcapsules loaded with an ethanol extract of red propolis through chemical, physicochemical and microbiological assays in order to establish quality control tools for nutraceutical preparations of red propolis. The markers (isoflavonoids, chalcones, pterocarpans, flavones, phenolic acids, terpenes and guttiferones) present in the tinctures A and B were identified and confirmed using LC/ESI/FTMS/Orbitrap. Four compositions (A, B, C and D) were prepared to contain B tincture of the red propolis with some pharmaceutical excipients and submitted to two drying processes, i. e. spray-drying and freeze-drying to obtain microcapsules loaded with the red propolis extract. The tinctures and microcapsules of the red propolis were submitted to the total flavonoid content and antioxidant activity tests. The antibacterial activity and minimum inhibitory concentration (MIC) were tested using Staphylococcus aureus ATCC 25293 and Pseudomonas aeruginosa ATCC 27853 strains. The tinctures and microcapsules presented high flavonoid quantities from 20.50 to 40.79 mg/100 mg of the microcapsules. The antioxidant activity and IC50 were determined for the tinctures A and B (IC50: 6.95 µg/mL and 7.48 µg/mL), the spray-dried microcapsules (IC50: 8.89–15.63 µg/mL) and the freeze-dried microcapsules (IC50: 11.83–23.36 µg/mL). The tinctures and microcapsules were proved to be bioactive against gram-positive and gram-negative bacteria with inhibition halos superior to 10 mm at concentration of 200 µg/mL and MIC values of 135.87–271.74 µg/mL using gram-positive strain and 271.74–543.48 µg/mL using gram-negative strain. The tinctures and microcapsules of the red propolis have a potential application for nutraceutical products

Mechanical and aesthetics compatibility of Brazilian red propolis micellar nanocomposite as a cavity cleaning agent

Abstract Background Propolis is a natural substance produced by bees and is known to have antimicrobial activity. Our aim was to evaluate the antimicrobial effect of micellar nanocomposites loaded with an ethyl acetate extract of Brazilian red propolis as a cavity cleaning agent and its influence on the color and microtensile bond strength (μTBS) of the dentin/resin interface. Methods An ultra-performance liquid chromatography coupled with a diode array detector (UPLC-DAD) assay was used to determine the flavonoids and isoflavones present in an ethyl acetate extract of Brazilian red propolis (EARP) and micellar nanocomposites loaded with EARP (MNRP). The antimicrobial activity of EARP and MNRP was tested against Streptococcus mutans, Lactobacillus acidophilus, and Candida albicans. One of the following experimental treatments was applied to etched dentin (phosphoric acid, 15 s): 5 μL of MNRP (RP3, 0.3%; RP6, 0.6%; or RP1, 1.0% w/v), placebo, and 2% chlorhexidine digluconate. Single Bond adhesive (3 M/ESPE) was applied and a 4-mm-thick resin crown (Z350XT, 3 M/ESPE) was built up. After 24 h, the teeth were sectioned into sticks for the μTBS test and scanning electron microscopy. Spectrophotometry according to the CIE L*a*b* chromatic space was used to evaluate the color. Data were analyzed using one-way ANOVA and the Tukey test or Kruskal-Wallis test and the same test for pairwise comparisons between the means (P < 0.05). Results The UPLC-DAD assay identified the flavonoids liquiritigenin, pinobanksin, pinocembrin, and isoliquiritigenin and the isoflavonoids daidzein, formononetin, and biochanin A in the EARP and micellar nanocomposites. EARP and MNRP presented antimicrobial activity against the cariogenic bacteria Streptococcus mutans and Lactobacillus acidophilus, and for Candida albicans. ΔE values varied from 2.31 to 3.67 (P = 0.457). The mean μTBS for RP1 was significantly lower than for the other groups (P < 0.001). Dentin treated with RP1 showed the shortest resin tags followed by RP6 and RP3. Conclusions The EARP and (MNRP) showed antimicrobial activity for the main agents causing dental caries (Streptococcus mutans and Lactobacillus acidophilus) and for Candida albicans. MNRP at concentrations of 0.3 and 0.6% used as a cavity cleaner do not compromise the aesthetics or μTBS of the dentin/resin interface