Streptococcus sanguinis is the second most common causative agent of bacterial infective endocarditis (IE). Risk of S. sanguinis IE is dependent on pre-disposing damage to the heart valve endothelium, which results in deposition of clotting factors for formation of a sterile thrombus (referred to as vegetation). Despite medical advances, high mortality and morbidity rates persist. Molecular characterization of S. sanguinis virulence determinants may enable development of prevention methods. In a previous screen for S. sanguinis virulence determinants by signature-tagged mutagenesis (STM) an attenuated mutant was identified with a transposon insertion in the nrdD gene, encoding an anaerobic ribonucleotide reductase. Evaluation of this mutant, as well as an nrdD in-frame deletion mutant, JFP27, by a soft-agar growth assay confirmed the anaerobic growth sensitivity of these strains. These studies suggest that an oxygen gradient occurs at the site of infection which selects for expression of anaerobic-specific genes at the nexus of the vegetation. The random STM screen failed to identify any favorable streptococcal surface-exposed prophylactic candidates. It was also apparent that additional genetic tools were required to facilitate the in vivo analyses of mutant strains. As it was desirable to insert antibiotic resistance markers into the chromosome, we identified a chromosomal site for ectopic expression of foreign genes. In vitro and in vivo analyses verified that insertion into this site did not affect important cellular phenotypes. The genetic tools developed facilitated further in vivo screening of S. sanguinis cell wall-associated (Cwa) protein mutants. A directed application of STM was employed for a comprehensive analysis of this surface protein class in the rabbit model of IE. Putative sortases, upon which Cwa proteins are dependent for cell surface localization, were also evaluated. No single S. sanguinis Cwa protein was determined essential for IE by STM screening; however competitiveness for colonization of the infection site was reduced for the mutant lacking expression of sortase A. The studies described here present a progressive picture of S. sanguinis IE, beginning with surface protein-dependent colonization of the vegetation in early IE, that later shifts to a bacterial persistence in situ dependent on condition-specific housekeeping genes, including nrdD.

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-14Bitstream added on 2014-06-13T19:22:47Z : No. of bitstreams: 1 romeiro_rl_dr_sjc.pdf: 889307 bytes, checksum: b97ee7a898e0c8f57f25ac75008f2fc8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo desse trabalho foi analisar in vitro a aderência de Streptococcus sanguinis, Candida albicans e associações destes microrganismos com Streptococcus mutans às superfícies dos implantes dentários tratados com jateamento de fosfato de cálcio, anodização, duplo ataque ácido e os de superfície lisa, com ou sem a prévia incubação em saliva ou plasma sanguíneo. Foram selecionados 120 implantes, sendo 30 de cada superfície para cada microrganismo e associações estudadas. Para análise da aderência, foram preparadas suspensões de microrganismos contendo 106 células/ml em espectrofotômetro. Além disso, cada microrganismo e associações foram divididos em três grupos: em um, o implante foi removido da embalagem e colocado diretamente no caldo, em outro, foi previamente embebido em saliva humana por uma hora e no último em plasma humano, também por uma hora. Os implantes foram acondicionados separadamente em poços de placas de cultura de células contendo caldo sacarosado (placa in vitro) e a suspensão do microrganismo. Após 24h de incubação a 37 °C e 5% de CO2, os implantes foram lavados três vezes durante um minuto em solução salina estéril e colocados em sonicador com 10 ml de salina para dispersão das células aderidas. A seguir, foram realizadas diluições seriadas e semeaduras em meios de cultura específico para cada microrganismo. Após 48h de incubação a 37 °C e 5% de CO2, foi realizada a contagem de unidades formadoras de colônias (UFC/ml) e os dados foram submetidos a análise de variância (ANOVA), teste de tukey, com nível de significância de 5%. Para ilustrar a aderência dos microrganismos, foram selecionados o microrganismo Streptococcus sanguinis, e as associações Streptococcus sanguinis e Candida albicans e Streptococcus sanguinis, Streptococcus mutans... / The aim of this study has been to analyze, in vitro the adherence of Streptococcus sanguinis, Candida albicans and associations of those microorganisms with Streptococcus mutans to the surfaces of dental implants treated with calcium phosphate jetting, anodization, double acid attack and to those of smooth surface, with or without previous saliva or blood plasma incubation. Ten implants from each surface were selected for every studied microorganism and association. In order to analyze the adherence, suspensions of microorganisms bearing 106 viable cells/ml in spectrophotometer were prepared. Additionally, every microorganism and association was divided in three groups: in the first, an implant was removed from its wrap and put right away into the sauce; the second, it was previously drenched into saliva for one hour; and the last one, into plasma, for one hour, as well. The implants were separately placed in culturing plaque wells of cells containing saccharose sauce (in vitro plaque) and the microorganism’s suspension. After 24 hours of incubation time at 37 °C and 5% of CO2, the implants were taken washed three times for a minute in saline sterile solution and put in a sonicator holding 10 ml of saline in order to disperse adherent cells. Then, seriated dilutions were done, and sowing in culture media specific for each of the. After a 48hincubation time at 37°C and 5% of CO2, a counting was carried, of the colony forming units (UFC/ml) and the data were submitted to the ANOVA, Tukey test, at a significance level of 5%. To illustrate the adherence of the microorganisms, some samples were exposed to electronic sweeping microscopy. The results did show great microorganism adherence to the surfaces studied, mainly when associated forming a biofilm. The anodized surface... (Complete abstract click electronic access below)

Submitted by ANA LUIZA DO ROSÁRIO PALMA null (ana.luiza.rp@hotmail.com) on 2017-01-11T17:41:58Z No. of bitstreams: 1 Ana Luiza do Rosário Palma, Biopatologia Bucal.pdf: 3299481 bytes, checksum: 9eb1d5e0b50827ce3d17e38baa851362 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-12T16:46:23Z (GMT) No. of bitstreams: 1 palma_alr_me_sjc.pdf: 3299481 bytes, checksum: 9eb1d5e0b50827ce3d17e38baa851362 (MD5) / Made available in DSpace on 2017-01-12T16:46:23Z (GMT). No. of bitstreams: 1 palma_alr_me_sjc.pdf: 3299481 bytes, checksum: 9eb1d5e0b50827ce3d17e38baa851362 (MD5) Previous issue date: 2016-12-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo foi avaliar as interações entre Candida albicans (ATCC 18804) com Streptococcus mitis (49456) e Streptococcus sanguinis (10556) in vitro e in vivo avaliando-se a possível influência destas associações, na expressão de genes, na filamentação e formação de biofilme de Candida albicans. A formação de biofilme, foi realizado mono e multiespécie em placa de 96 poços por 48 h à 37 ºC com 5% CO2. Os biofilmes foram desagregados e diluídos para semeadura em ágar e após incubação as UFC/mL foram contadas. A filamentação de C. albicans, in vitro foi realizada em placas de 24 poços e in vivo em Galleria mellonella, com análise histológica e contagem de UFC/mL. A avaliação da expressão gênica de ALS1, ALS3, BRC1, CPH1, EFG1 e HWP1, foi realizada por PCR em tempo real utilizando o gene normalizador ACT1. Os resultados da UFC/mL (p < 0.05), demonstrou que o biofilme de C. albicans monoespécie apresentou maior crescimento, quando comparado com o biofilme associado com S. mitis (p = 0,001) ou com S. sanguinis (p = 0,001). A filamentação in vitro demonstrou que a interação com S. mitis inibiu a filamentação de C. albicans (p = 0,0006), entretanto, a interação com S. sanguinis não inibiu (p = 0,1554). Os genes ALS1, ALS3 e HWP1 foram super expressos na interação com S. mitis. A interação com S. sanguinis, promoveu super expressão dos genes ALS3 e HWP1. Os genes BRC1, CPH1 e EFG1 foram super expressos na interação com S. mitis e sub expressos, na interação com S. sanguinis. Não houve diferença estatística nos estudos in vivo de filamentação e UFC/mL. Conclui-se que in vitro, S. mitis e S. sanguinis foram capazes de inibir a formação de biofilme de C. albicans. Assim como a interação com S. mitis inibiu a sua filamentação. A interação com S. mitis parece aumentar o fator de virulência de C. albicans, quanto a expressão dos genes de aderência ALS1, ALS3 e HWP1, bem como na associação com S. sanguinis (ALS3 e HWP1). Os genes de formação de biofilme, BRC1, CPH1 e EFG1, na interação com S. mitis promoveu aumento do fator de virulência. / The objective was to evaluate the interactions between Candida albicans (ATCC 18804) with Streptococcus mitis (49456) and Streptococcus sanguinis (10556) in vitro and in vivo evaluating the possible influence of these associations, in the expression of genes, in the filamentation and biofilm formation of Candida albicans. Biofilm formation was performed mono- and multispecies in 96-well plate for 48 h at 37 ºC with 5% CO2. Biofilms sonicated and diluted for sowing on agar and after incubation the colonies counted to obtain the colony forming units (CFU/mL). The filamentation in vitro was performed in 24-well plate and in vivo Galleria mellonella, with histological and CFU/mL. The evaluation of gene expression ALS1, ALS3, BRC1, CPH1, EFG1 and HWP1 was performed by real-time PCR using the normalizing gene ACT1. The results of CFU/ml (p<0.05), found that C. albicans biofilm monoespécie showed increased growth, as compared to the biofilm associated with S. mitis (p = 0.001) and S. sanguinis (p = 0.001). The filamentation in vitro demonstrated that the interaction with S. mitis inhibited C. albicans filamentation (p = 0.0006), interaction with S. sanguinis could not (p = 0.1554). The ALS1, ALS3, HWP1 gene, were superexpressed in S. mitis interaction. Interaction with S. sanguinis, promoted overexpression of ALS3 and HWP1 genes. The BRC1 genes, CPH1 and EFG1 were super expressed in interaction with S. mitis and sub expressed when there was interaction with S. sanguinis. There was no statistical difference in the in vivo studies of filamentation and CFU/mL. It follows that in vitro, S. mitis and S. sanguinis were able to inhibit the formation of C. albicans biofilms. Like the interaction with S. mitis inhibited its filamentation. The interaction with S. mitis appears to increase the virulence factors of C. albicans. ALS1, ALS3, HWP1 as the expression of adhesion genes, and as well as in association with S. sanguinis (ALS3 and HWP1). Biofilm formation genes, BRC1, CPH1 and EFG1 in interaction with S. mitis promoted increased virulence factor.

Manganese-deficient Streptococcus sanguinis mutants exhibit a dramatic decrease in virulence for infective endocarditis and in aerobic growth in manganese-limited media. Loss of activity of a manganese-dependent, oxygen-dependent ribonucleotide reductase (RNR) could explain the decrease in virulence. When the genes encoding this RNR are deleted, there is no growth of the mutant in aerobic broth culture or in an animal model. Testing the contribution of the aerobic RNR to the phenotype of a manganese transporter mutant, a heterologous class II RNR from Lactobacillus leichmannii called NrdJ that requires B12 rather than manganese as a cofactor was previously introduced into an RNR mutant of S. sanguinis. Aerobic growth was only partially restored. Currently, we sought to improve NrdJ-dependent growth by (i) amending the medium to increase cellular levels of B12; (ii) characterizing a spontaneous mutant of the NrdJ-complemented strain with improved aerobic growth; and (iii) altering this strain through further genetic manipulation.

Um novo monômero, brometo de metacriloiloxidodecilpiridínio (MDPB), com efeito antibacteriano e capacidade de co-polimerizar com outros monômeros, foi apresentado por Imazato, Torii e Tsuchitani (1993). Este estudo avaliou a adesão bacteriana, em 16, 40 e 64 horas, à resina composta contendo ou não o monômero antibacteriano MDPB. A adesão foi testada para Streptococcus sanguinis e Streptococcus sobrinus. Após as amostras terem sido submetidas à incubação, o biofilme foi coletado e a contagem de UFCs foi realizada. Os dados foram comparados pelo método ANOVA complementado por teste de Tukey. Os resultados demonstraram que, para o S. sanguinis, a adesão sobre o MDPB foi significativamente maior (p<0.05) quando comparado ao controle em 16 horas, mas diminuiu significativamente em 40 horas, não apresentando diferenças quando comparado ao controle neste tempo (p<0.01). Para o S. sobrinus, o controle apresentou aumento significativo da adesão bacteriana em 64 horas quando comparado com 16 horas (p<0.01), sendo significativamente maior que para o MDPB em 64 horas (p<0.05). Assim, o estudo mostrou que o MDPB é capaz de inibir a adesão de S. sobrinus sem interferir na adesão do S. sanguinis. Portanto, nas condições deste estudo os resultados sugerem que a incorporação do MDPB a resinas compostas pode ser de importância na prevenção de cáries secundárias favorecendo a adesão de bactérias comensais em detrimento de bactérias com potencial cariogênico. / A new antibacterial monomer, Methacryloyloxydodecylpyridinium bromide (MDPB), with antibacterial property and ability to co-polymerize with other monomers, was introduced by Imazato, Torii and Tsuchitani (1993). This study aimed to analyze the effect of MDPB on bacterial adherence to resin composites containing or not MDPB. Streptococcus sanguinis and Streptococcus sobrinus were used. The biofilms were collected from the samples and the colony forming units (CFUs) were counted after 16, 40 e 64 h of incubation. The data were compared by ANOVA complemented by the Tukey’s test. The results showed that the adhesion of S. sanguinis to MDPB-containing resin composite was significantly higher (p<0.05) than to control samples at 16 h, but significantly diminished at 40 h, reaching values similar to those of control samples (p<0.01). The adherence of S. sobrinus to control samples significantly increased throughout the experimental time (p<0.01) and was considerably higher than to MDPB at 64 h (p<0.05). Thus, the study showed that MDPB is capable of inhibit adhesion of S. sobrinus with no interference on the adhesion of S. sanguinis. Thus, at the conditions of this study we suggest that MDPB incorporated to resin composites could be of importance to prevent secondary caries favoring adhesion of commensal bacteria and impairing adhesion of cariogenic bacteria.

Um novo monômero, brometo de metacriloiloxidodecilpiridínio (MDPB), com efeito antibacteriano e capacidade de co-polimerizar com outros monômeros, foi apresentado por Imazato, Torii e Tsuchitani (1993). Este estudo avaliou a adesão bacteriana, em 16, 40 e 64 horas, à resina composta contendo ou não o monômero antibacteriano MDPB. A adesão foi testada para Streptococcus sanguinis e Streptococcus sobrinus. Após as amostras terem sido submetidas à incubação, o biofilme foi coletado e a contagem de UFCs foi realizada. Os dados foram comparados pelo método ANOVA complementado por teste de Tukey. Os resultados demonstraram que, para o S. sanguinis, a adesão sobre o MDPB foi significativamente maior (p<0.05) quando comparado ao controle em 16 horas, mas diminuiu significativamente em 40 horas, não apresentando diferenças quando comparado ao controle neste tempo (p<0.01). Para o S. sobrinus, o controle apresentou aumento significativo da adesão bacteriana em 64 horas quando comparado com 16 horas (p<0.01), sendo significativamente maior que para o MDPB em 64 horas (p<0.05). Assim, o estudo mostrou que o MDPB é capaz de inibir a adesão de S. sobrinus sem interferir na adesão do S. sanguinis. Portanto, nas condições deste estudo os resultados sugerem que a incorporação do MDPB a resinas compostas pode ser de importância na prevenção de cáries secundárias favorecendo a adesão de bactérias comensais em detrimento de bactérias com potencial cariogênico. / A new antibacterial monomer, Methacryloyloxydodecylpyridinium bromide (MDPB), with antibacterial property and ability to co-polymerize with other monomers, was introduced by Imazato, Torii and Tsuchitani (1993). This study aimed to analyze the effect of MDPB on bacterial adherence to resin composites containing or not MDPB. Streptococcus sanguinis and Streptococcus sobrinus were used. The biofilms were collected from the samples and the colony forming units (CFUs) were counted after 16, 40 e 64 h of incubation. The data were compared by ANOVA complemented by the Tukey’s test. The results showed that the adhesion of S. sanguinis to MDPB-containing resin composite was significantly higher (p<0.05) than to control samples at 16 h, but significantly diminished at 40 h, reaching values similar to those of control samples (p<0.01). The adherence of S. sobrinus to control samples significantly increased throughout the experimental time (p<0.01) and was considerably higher than to MDPB at 64 h (p<0.05). Thus, the study showed that MDPB is capable of inhibit adhesion of S. sobrinus with no interference on the adhesion of S. sanguinis. Thus, at the conditions of this study we suggest that MDPB incorporated to resin composites could be of importance to prevent secondary caries favoring adhesion of commensal bacteria and impairing adhesion of cariogenic bacteria.

Infectious diseases are the top leading cause of death worldwide. Identifying essential genes, genes indispensable for survival, has been proven indispensable in defining new therapeutic targets against pathogens, major elements of the minimal set genome to be harnessed in synthetic biology, and determinants of evolutionary relationships of phylogenetically distant species. Thus, essentiality studies promise valuable revenues that can decipher much of biological complexities. Taking advantage of the available microbial sequences and the essentiality studies conducted in various microbial models, we proposed a framework for the prediction of essential genes based on our experimentally verified knowledge of the pathways involved in three essential xiv functions: genetic information processing, cell wall biosynthesis, and energy metabolism. We investigated physiological pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG) database and developed a bioinformatics approach to predict essential genes in 13 different microbial species. Our in silico findings matched to a high degree the experimental data derived from essentiality studies conducted on the same microbial models, providing insights about the microbial lifestyles, including energy resources, cell wall structure, and ecological preferences, but not virulence tools and mechanisms. Furthermore, we believe that essential genes have survived the evolutionary purifying selection due to their evolved capacity to re-wire genetic and protein networks in response to any emerging stress. In this sense, an environmental specificity (stress) provides a dominant determinant of an essential gene set. The new challenge was understanding the contribution of the essential genome in S. sanguinis to the coping mechanisms to different clinically relevant stress factors, namely temperature elevation (43oC) and sub-inhibitory concentration of ampicillin, an abundantly prescribed antibiotic for prophylaxis and treatment against S. sanguinis. The current project investigated the transcriptomic and proteomic profiles of essential genes and proteins, using RNA-seq and mass spectrometry respectively, under the impact of the two stressors separately, to elucidate the essential genome and proteome dynamics on a temporal basis and define “pathogenesis signatures” as potential therapeutic targets. We believe that the current findings will help characterize a bacterial model for studying the dynamics of essential genes and assist in designing evidence-based guidelines for drug prescription in clinical practice.

Objetivo: Evaluar in vitro el efecto antibacteriano y citotóxico del extracto metanólico del café peruano y colombiano frente a cepas de Streptococcus mutans (ATCC® 25175) y Streptococcus sanguinis (ATCC® 10556). Métodos: El extracto metanólico de café peruano y colombiano fue preparado utilizando una proporción 1:2 (P/V). Para determinar las propiedades antibacterianas de cada extracto se utilizó el método de difusión en pozo y la concentración mínima inhibitoria (CMI) se determinó mediante el método de microdilución. Como control positivo se utilizo la clorhexidina al 2%. La viabilidad celular y la citotoxicidad de los extractos fueron realizados utilizando el ensayo de reducción de MTT en la línea celular MDCK. Resultados: El extracto metanólico del café colombiano (Coffea) tuvo un mayor efecto antibacteriano (18.9 ± 2.8 mm) en comparación al café peruano (15+2.54mm), mientras que para el grupo de Streptoccocus mutans y para el grupo de Streptoccocus sanguinis el café colombiano y peruano mostraron halos de 18.8 ± 2.77 mm y 14.7± 3.8 mm, respectivamente. La concentración mínima inhibitoria (CMI) para los extractos del café colombiano y peruano fue de 3.1 μg/ml y 1.6 μg/ml para Streptoccocu mutans y 6.3 μg/ml para Streptoccocu sanguinis, respectivamente. El ensayo de MTT muestras que ambos extractos no fueron citotóxicos a altas concentraciones. Conclusiones: Los hallazgos que se encontraron en este estudio muestran que los extractos del colombiano y peruano (Coffea) tienen propiedades antibacterianas efectivas contra cepas de Streptococcus mutans y Streptococcus sanguinis. / Objective: To evaluate in vitro the antibacterial and cytotoxic effect of the methanolic extract of peruvian and colombian coffee against strains of Streptococcus mutans (ATCC® 25175) and Streptococcus sanguinis (ATCC® 10556). Methods: An extract was prepared for each type of coffee, carrying out nine tests independently, chlorhexidine 2% was used as a positive control. The well diffusion method was performed to determine the antibacterial properties of each extract. The minimum inhibitory concentration (MIC) was determined by the microdilusion method and the cytotoxicity was analyzed by the MTT reduction test using a MDCK cell line. Results: The methanolic extract of colombian coffee (Coffea) had a greater antibacterial effect (18.9 ± 2.8 mm) compared to peruvian coffee (15 + 2.54mm) for the group of Streptoccocus mutans and for the group of Streptoccocus sanguinis, colombian coffee and peruvian obtained 18.8 ± 2.77 mm and 14.7 ± 3.8 mm, respectively. The minimum inhibitory concentration (MIC) for colombian and peruvian coffee extracts was 3.1 μg / ml and 1.6 μg / ml for S. mutans and 6.3 μg / ml for S. sanguinis, respectively. These extracts were not cytotoxic at high concentrations. Conclusions: The findings found in this study show that colombian and peruvian extracts (Coffea) have effective antibacterial properties against Streptococcus Mutans and Sanguinis strains. Further study with Coffea is recommended as there is very little research on this. / Tesis