Construction and phenotypic characterization of Streptococcus mutans mutant strains of genes related to virulence

Orientadores: Pedro Luiz Rosalen, Gilson César Nobre FrancoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: O fato de Streptococcus mutans conseguir emergir como microrganismo dominante no biofilme dental mesmo sob condições de estresse faz com que ele seja alvo de múltiplos interesses não apenas para a saúde oral, mas também para a saúde sistêmica. O sistema proteolítico Clp (Protease caseinolítica dependente de ATP) detém o papel central na tolerância desse microrganismo frente ao estresse ambiental, por desempenhar um importante papel na homeostase celular e controlar a estabilidade de proteínas reguladoras. Além disso, observou-se que a proteína Spx serve de substrato para esse sistema Clp e por isso foi feita uma detalhada caracterização fenotípica de Spx, regulador global, capaz de regular a transcrição de muitos genes conhecidamente relacionados ao estresse oxidativo em S. mutans e outros genes ainda hipotéticos. Assim, o objetivo deste trabalho foi construir e caracterizar fenotípicamente, in vitro e in vivo, cepas mutantes de S. mutans UA159 com deleção de genes hipotéticos, que apresentaram expressão alterada nas mutantes dos genes spxA1 e spxA1spxA2. Foram construídas as mutantes dos genes selecionados: smu143c smu144c, smu247, smu248, smu540, smu569, smu570, smu929, smu1296, smu1497 e smu1685, hipotéticos e regulados por Spx. Foi feita a análise transcricional dos genes selecionados frente ao estresse oxidativo através de PCR quantitativo (RT-qPCR). As mutantes construídas foram caracterizadas fenotipicamente por meio da avaliação da sua susceptibilidade, sobrevivência e crescimento sob condições de estresse ácido, oxidativo, em presença de ferro e outros metais. Em acréscimo, foi feita uma avaliação quanto à capacidade de formação de biofilme em meio com sacarose e esses genes foram avaliados por meio de ensaio de transcrição in vitro de forma a confirmar a sua regulação direta ou indereta por Spx . As cepas selecionadas por apresentarem fenótipo que justificasse relação com estresse oxidativo foram também investigadas quanto a capacidade de colonização em modelo animal sujeito a alto desafio cariogênico. Além disso, foi pesquisada a capacidade de cepas mutantes de Spx em causar lesões cariosas em ratos e seu papel regulatório como ativador de competência, na capacidade de formar mutacinas, biofilmes e glucanos a partir de sacarose. Além disso, a cepa mutante smu1784c, também regulada por Spx, foi submetida a uma caracterização inicial em busca de fenótipos que justifiquem sua relação com o gene eep de Enterococcus faecalis ou com estresse oxidativo em S. mutans. Com este trabalho, por meio de uma caracterização fenotípica detalhadas de novos genes regulados por Spx pôde-se relacioná-los ao estresse oxidativo em S. mutans, além de que verificou-se relação desses genes e de Spx com a homeostase de ferro. Pôde-se confirmar o papel de Spx na patogênese da doença cárie e virulência do S. mutans uma vez que as mutantes dos genes spxA1 e spxA2 reduziram a formação de cárie em modelo animal, mostraram-se menos competentes e spxA1 mostrou-se incapaz de produzir mutacinas. E o gene smu1784c, também regulado por Spx, mostrou-se envolvido com respostas ao estresse ácido e oxidativo e na virulência de S. mutans, uma vez que a cepa mutante mostrou-se atenuada em modelo de Galleria mellonella e capaz de reduzir os parâmetros bioquímicos relacionados à formação de biofilme. Portanto, esses achados confirmam e elucidam o papel regulador de Spx, possibilitando a identificação de novos alvos terepêuticos para a prevenção/tratamento de doenças envolvendo este microrganismoAbstract: Streptococcus mutans is abble to emerge as dominant flora in biofilms even during stress conditions and has been studied in dentistry and medicine. The S. mutans Clp proteolytic system (Caseinolytic protease ATP-dependent) holds a central role in stress tolerance by controlling cellular homeostasis and stability of regulatory proteins. Besides, it was shown that Spx is substrate of Clp what justified a detailed phenotypic characterization of Spx, a global regulator able to regulate transcription of well-known genes related to oxidative stress and others hypothetical genes. Thus, the goal of this study was to construct and phenotypically characterize, in vitro and in vivo, S. mutans knockout mutants of hypothetical genes that showed altered expression in the ?spxA1 and ?spxA1/sxpA2 strains. The mutants of the following genes, regulated by Spx and strong candidates to be related with survival of S. mutans in stress conditions were constructed: smu143c smu144c, smu247, smu248, smu540, smu569, smu570, smu929, smu1296, smu1497 and smu1685. RT-qPCR was used to evaluate the responses of Spx-regulated genes during oxidative stress in the parental and ?spx strains. The mutant strains were characterized by evaluation of its susceptibility, survival and growth under oxidative and acid stresses conditions, as well as in presence of iron and other metals. In addition, their ability to form biofilm in presence of sucrose was evaluated. These genes were also assessed through in vitro transcription assay to confirm their direct regulation by Spx. Selected strains that showed phenotypes associated with oxidative stress management were also evaluated for their ability to colonize using an animal model subjected to high cariogenic challenges. Besides, Spx mutant strains were evaluated for their ability to cause dental caries using an animal model and their regulatory role as competency activator and mutacin, biofilm and glucan sinthesizer. Moreover, mutant for gene smu1784c, also regulated by Spx, was characterized to find phenotypes that justified its relation with eep in Enterococcus faecalis or with oxidative stress in S. mutans. The detailed phenotipic characterization of novel Spx regulated genes lead to their involvment with oxidative stress in S. mutans. Furthermore, these genes and Spx were associated with iron homeostoasis. The role of Spx in caries pathogenesis was confirmed once spxA1 and spxA2 genes were able to reduce caries score in an animal model. Also, smu1784c gene, regulated by Spx, showed involvement with acid and oxidative stresses responses, and virulence of S. mutans once the mutant strain of this gene showed attenuated behavior in Galleria mellonella model. The mutant ?smu1784c was also able to reduce biochemical parameters related with biofilm formation. Therefore, these findings support the regulatory role of Spx enabling the identification of new therapeutic targets usefull for prevention and treatment of S. mutans related diseasesDoutoradoFarmacologia, Anestesiologia e TerapeuticaDoutora em Odontologia2012/02278-3, 2014/03816-4FAPES

Antimicrobial activity of essential oils on mutans Streptococci and determination of their antiproliferative effect

Orientadores: Pedro Luiz Rosalen, Marta Cristina Teixeira DuarteDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: O objetivo desse trabalho foi avaliar a atividade antimicrobiana, in vitro, de óleos essenciais e frações dos óleos de melhor atividade, contra microrganismos do grupo mutans em estado planctônico. Além disso, os biofilmes de Streptococcus mutans foram submetidos às frações ativas e os óleos de melhor atividade e frações ativas foram avaliados quanto à sua citotoxicidade e caracterizados quimicamente. Para isso, vinte óleos essenciais (OE) foram obtidos por hidrodestilação a partir de plantas pertencentes ao banco de germoplasmas da Coleção de Plantas Medicinais e Aromáticas (CPMA/CPQBA/UNICAMP). Estes OE foram avaliados quanto à sua atividade antimicrobiana por meio dos ensaios: concentrações inibitória (CIM) e bactericida mínima (CBM) contra Streptococcus mutans UA159. Controles positivo (clorexidina 0,12 %) e negativo (propilenoglicol 6,12 % e 25 %) também foram testados...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digitalAbstract: The aim of this study was to evaluate the in vitro antimicrobial activity of essential oils (EO) and fractions from highest activity EO against planktonic cells of mutans streptococci. Besides, the biofilms formed by this microorganism were submitted to active fractions and the higher activity EO and active fractions were evaluated regarding their citotoxicity and chemically characterized. For this, twenty essentinal oils were obtained from plants of the "Collectio of Medicinal and Aromatic Plants" (CPMA, CPQBA/UNICAMP), germplasm bank by hydrodistillation. These EO were evaluated by antimicrobial assays: minimum inhibitory (MIC) and bactericidal (MBC) concentrations against Streptococcus mutans UA159. Positive (chlorhexidine 0.12%) and negative (propylene glycol 6.12 % and 25%) controls were also tested...Note: The complete abstract is available with the full electronic documentMestradoFarmacologia, Anestesiologia e TerapeuticaMestre em Odontologi

Apolar bioactive fraction of melipona scutellaris geopropolis on streptococcus mutans biofilm

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThe aim of this study was to evaluate the influence of the bioactive nonpolar fraction of geopropolis on Streptococcus mutans biofilm. The ethanolic extract of Melipona scutellaris geopropolis was subjected to a liquid-liquid partition, thus obtaining the bioactive hexane fraction (HF) possessing antimicrobial activity. The effects of HF on S. mutans UA159 biofilms generated on saliva-coated hydroxyapatite discs were analyzed by inhibition of formation, killing assay, and glycolytic pH-drop assays. Furthermore, biofilms treated with vehicle control and HF were analyzed by scanning electron microscopy (SEM). HF at 250  μ g/mL and 400  μ g/mL caused 38% and 53% reduction in the biomass of biofilm, respectively, when compared to vehicle control (P 0.05). In conclusion, the bioactive HF of geopropolis was promising to control the S. mutans biofilm formation, without affecting the microbial population but interfering with its structure by reducing the biochemical content of biofilm matrix.The aim of this study was to evaluate the influence of the bioactive nonpolar fraction of geopropolis on Streptococcus mutans biofilm. The ethanolic extract of Melipona scutellaris geopropolis was subjected to a liquid-liquid partition, thus obtaining the bioactive hexane fraction (HF) possessing antimicrobial activity. The effects of HF on S. mutans UA159 biofilms generated on saliva-coated hydroxyapatite discs were analyzed by inhibition of formation, killing assay, and glycolytic pH-drop assays. Furthermore, biofilms treated with vehicle control and HF were analyzed by scanning electron microscopy (SEM). HF at 250 μg/mL and 400 μg/mL caused 38% and 53% reduction in the biomass of biofilm, respectively, when compared to vehicle control (P  0.05). In conclusion, the bioactive HF of geopropolis was promising to control the S. mutans biofilm formation, without affecting the microbial population but interfering with its structure by reducing the biochemical content of biofilm matrix2013FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2009/12354-6; 2010/20214-7; 2013/10879-

Apolar Bioactive Fraction of Melipona scutellaris

The aim of this study was to evaluate the influence of the bioactive nonpolar fraction of geopropolis on Streptococcus mutans biofilm. The ethanolic extract of Melipona scutellaris geopropolis was subjected to a liquid-liquid partition, thus obtaining the bioactive hexane fraction (HF) possessing antimicrobial activity. The effects of HF on S. mutans UA159 biofilms generated on saliva-coated hydroxyapatite discs were analyzed by inhibition of formation, killing assay, and glycolytic pH-drop assays. Furthermore, biofilms treated with vehicle control and HF were analyzed by scanning electron microscopy (SEM). HF at 250 μg/mL and 400 μg/mL caused 38% and 53% reduction in the biomass of biofilm, respectively, when compared to vehicle control (P0.05). In conclusion, the bioactive HF of geopropolis was promising to control the S. mutans biofilm formation, without affecting the microbial population but interfering with its structure by reducing the biochemical content of biofilm matrix

Antimicrobial and antiproliferative activities of stingless bee Melipona scutellaris geopropolis

Background\ud Geopropolis is a type of propolis containing resin, wax, and soil, collected by threatened stingless bee species native to tropical countries and used in folk medicine. However, studies concerning the biological activity and chemical composition of geopropolis are scarce. In this study, we evaluated the antimicrobial and antiproliferative activity of the ethanolic extract of geopropolis (EEGP) collected by Melipona scutellaris and its bioactive fraction against important clinical microorganisms as well as their in vitro cytotoxicity and chemical profile.\ud \ud Methods\ud The antimicrobial activity of EEGP and fractions was examined by determining their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against six bacteria strains as well as their ability to inhibit Streptococcus mutans biofilm adherence. Total growth inhibition (TGI) was chosen to assay the antiproliferative activity of EEGP and its bioactive fraction against normal and cancer cell lines. The chemical composition of M. scutellaris geopropolis was identified by reversed-phase high-performance liquid chromatography and gas chromatography–mass spectrometry.\ud \ud Results\ud EEGP significantly inhibited the growth of Staphylococcus aureus strains and S. mutans at low concentrations, and its hexane fraction (HF) presented the highest antibacterial activity. Also, both EEGP and HF inhibited S. mutans biofilm adherence (p < 0.05) and showed selectivity against human cancer cell lines, although only HF demonstrated selectivity at low concentrations. The chemical analyses performed suggest the absence of flavonoids and the presence of benzophenones as geopropolis major compounds.\ud \ud Conclusions\ud The empirical use of this unique type of geopropolis by folk medicine practitioners was confirmed in the present study, since it showed antimicrobial and antiproliferative potential against the cancer cell lines studied. It is possible that the major compounds found in this type of geopropolis are responsible for its properties.The authors are grateful to Mr. José Emídio Borges de Souza for providing the geopropolis samples. This research was supported by FAPESP (#2009/12354-6 and #2010/20214-7)

The Effect of Essential Oils and Bioactive Fractions on Streptococcus mutans

The essential oils (EO) and bioactive fractions (BF) from Aloysia gratissima, Baccharis dracunculifolia, Coriandrum sativum, Cyperus articulatus, and Lippia sidoides were proven to have strong antimicrobial activity on planktonic microorganisms; however, little is known about their effects on the morphology or viability of oral biofilms. Previously, we determined the EO/fractions with the best antimicrobial activity against Streptococcus mutans and Candida spp. In this report, we used a confocal analysis to investigate the effect of these EO and BF on the morphology of S. mutans biofilms (thickness, biovolume, and architecture) and on the metabolic viability of C. albicans biofilms. The analysis of intact treated S. mutans biofilms showed no statistical difference for thickness in all groups compared to the control. However, a significant reduction in the biovolume of extracellular polysaccharides and bacteria was observed for A. gratissima and L. sidoides groups, indicating that these BF disrupt biofilm integrity and may have created porosity in the biofilm. This phenomenon could potentially result in a weakened structure and affect biofilm dynamics. Finally, C. sativum EO drastically affected C. albicans viability when compared to the control. These results highlight the promising antimicrobial activity of these plant species and support future translational research on the treatment of dental caries and oral candidiasis

Modulation of Streptococcus mutans virulence by dental adhesives containing anti-caries agents

The aim of this in vitro study was to analyze the effect of the incorporation of two anti-caries agents into dental adhesives on the reduction of the virulence of Streptococcus mutans and on the adhesion to dentin. Apigenin (1 mM) and tt-Farnesol (5 mM) were added separately and in combination to a self-etch adhesive (CS3 — Clearfil S3 Bond Plus) and to an each-and-rinse adhesive (OPT — OptiBond S). Biofilm of S. mutans was grown on adhesive-coated hydroxyapatite disks for 115 h and bacterial viability, dry-weight, alkali soluble, water soluble, intracellular polysaccharides and protein were quantified. Bond strength and dentin-adhesive interface were performed to analyze the effects of the incorporation on the physical properties and to identify changes in hybrid layer morphology. Addition of Apigenin and Apigenin + tt-Farnesol to CS3, and Apigenin or tt-Farnesol to OPT reduced the dry-weight of S. mutans biofilm. Insoluble polysaccharide decreased with the addition of Apigenin to CS3 and tt-Farnesol to OPT. Intracellular polysaccharide decreased with addition of Apigenin and Apigenin + tt-Farnesol to CS3. No changes in dentin bond strength, resin–dentin interfacial morphology, total amount of protein and soluble polysaccharide were observed with the additions. Biofilms that are less cariogenic around dental restorations could decrease secondary caries formation; in addition, the reduction of virulence of S. mutans without necessarily killing the microorganism is more unlikely to induce antimicrobial resistance.331010841092CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP140698/2013-2; 310522/2015-32013/22823-9; 2014/17543-

Desenvolvimento vocacional no ensino superior: o foco em estudantes do 1º ano

Desenvolvimento vocacional no ensino superior: o foco em estudantes do 1º an

Antimicrobial activity of essential oils against streptococcus mutans and their antiproliferative effects

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOThis study aimed to evaluate the activity of essential oils (EOs) against Streptococcus mutans biofilm by chemically characterizing their fractions responsible for biological and antiproliferative activity. Twenty EO were obtained by hydrodistillation and submitted to the antimicrobial assay (minimum inhibitory (MIC) and bactericidal (MBC) concentrations) against S. mutans UA159. Thin-layer chromatography and gas chromatography/mass spectrometry were used for phytochemical analyses. EOs were selected according to predetermined criteria and fractionated using dry column; the resulting fractions were assessed by MIC and MBC, selected as active fractions, and evaluated against S. mutans biofilm. Biofilms formed were examined using scanning electron microscopy. Selected EOs and their selected active fractions were evaluated for their antiproliferative activity against keratinocytes and seven human tumor cell lines. MIC and MBC values obtained for EO and their active fractions showed strong antimicrobial activity. Chemical analyses mainly showed the presence of terpenes. The selected active fractions inhibited S. mutans biofilm formation (P < 0.05) did not affect glycolytic pH drop and were inactive against keratinocytes, normal cell line. In conclusion, EO showed activity at low concentrations, and their selected active fractions were also effective against biofilm formed by S. mutans and human tumor cell lines.This study aimed to evaluate the activity of essential oils (EOs) against Streptococcus mutans biofilm by chemically characterizing their fractions responsible for biological and antiproliferative activity. Twenty EO were obtained by hydrodistillation and submitted to the antimicrobial assay (minimum inhibitory (MIC) and bactericidal (MBC) concentrations) against S. mutans UA159. Thin-layer chromatography and gas chromatography/mass spectrometry were used for phytochemical analyses. EOs were selected according to predetermined criteria and fractionated using dry column; the resulting fractions were assessed by MIC and MBC, selected as active fractions, and evaluated against S. mutans biofilm. Biofilms formed were examined using scanning electron microscopy. Selected EOs and their selected active fractions were evaluated for their antiproliferative activity against keratinocytes and seven human tumor cell lines. MIC and MBC values obtained for EO and their active fractions showed strong antimicrobial activity. Chemical analyses mainly showed the presence of terpenes. The selected active fractions inhibited S. mutans biofilm formation (P < 0.05) did not affect glycolytic pH drop and were inactive against keratinocytes, normal cell line. In conclusion, EO showed activity at low concentrations, and their selected active fractions were also effective against biofilm formed by S. mutans and human tumor cell lines2012FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2009/12353-0; 2011/14757-0308644/2011-

Development and Physicochemical Characterization of Eugenia brejoensis Essential Oil-Doped Dental Adhesives with Antimicrobial Action towards Streptococcus mutans

Dental caries is a multifactorial, biofilm-dependent infectious disease that develops when detrimental changes occur in the oral cavity microenvironment. The antimicrobial and antivirulence properties of the essential oil obtained from the leaves of Eugenia brejoensis Mazine (EBEO) have been reported against Gram-positive and Gram-negative bacteria. Herein, the antimicrobial action of EBEO towards Streptococcus mutans is reported, along with the development and characterization of dental adhesives doped with. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EBEO were determined against S. mutans, while its toxicity was analyze using Tenebrio molitor larvae. EBEO (MIC and 10&times;MIC) was incorporated into the Ambar Advanced Polymerization System&reg; (Ambar APS), a two-step total-etch adhesive system (FGM Dental Group), and the antibiofilm action was evaluated. The reflective strength, modulus of elasticity, degree of conversion, and maximum rate of polymerization of each adhesive were also determined. The MIC and MBC values of EBEO against S. mutans were 62.5 &micro;g/mL. The tested concentrations of EBEO were non-toxic to T. molitor larvae. The formation of S. mutans biofilms was significantly inhibited by EBEO and EBEO-coated resin discs (p &lt; 0.05). Importantly, EBEO incorporation did not affect the mechanical and physicochemical properties in relation to oil-free adhesive version. EBEO showed strong antibacterial and antibiofilm activity against S. mutans, no toxicity effect against T. molitor larvae, and did not jeopardize the physical-chemical properties tested