Thesis--University of Helsinki, 1999. / Includes bibliographical references.

Thesis--University of Helsinki, 1999. / Includes bibliographical references.

Prevotella intermedia e Prevotella nigrescens são espécies comumente associadas Porphyromonas gingivalis. os objetivos foram verificar a co-agregação entre cepas de P. intermedia, P. nigrescens e P. gingivalis; quantificar a biomassa, avaliar a proporção dos microrganismos nos biofilmes mistos; verificar a interferência do co-cultivo pela técnica de dois compartimentos, avaliar os biofilmes em ensaios de Hibridização In Situ (FISH) e verificar o papel dos genes PINA0102 e PIN0398 de P. intermedia no biofilme. Assim, 9 isolados clínicos de P. intermedia e 5 de P. nigrescens, as cepas padrão P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277, os mutantes Pi17D0398 e Pi17D0102 foram avaliados em variados consórcios. Os resultados mostraram que as interações investigadas são cepa específicas. / Prevotella intermedia and Prevotella nigrescens are commonly associated with periodontal diseases. The goals of this study were to determine the co-aggregation of strains of P. intermedia, P. nigrescens and P. gingivalis; to quantify the biomass of these associations, to evaluate the ratio of these microorganisms in heterotypic biofilms, to verify the modulation of biofilms in co-culture using a trans-well system; to evaluate the structure of the biofilms by Fluorescent Hybridization In Situ (FISH) and to determine the role of genes PINA0102 and PIN0398 of P. intermedia in the modulation of its biofilm. Therefore, 9 clinical isolates of P. intermedia, 5 of P. nigrescens, type strains P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277 and mutant strains Pi17D0398 and Pi17D0102 were grown in consortia of two strains. Our data demonstrate that the associations of P. intermedia, P. nigrescens and P. gingivalis are strain-specific.

Prevotella intermedia e Prevotella nigrescens são espécies comumente associadas Porphyromonas gingivalis. os objetivos foram verificar a co-agregação entre cepas de P. intermedia, P. nigrescens e P. gingivalis; quantificar a biomassa, avaliar a proporção dos microrganismos nos biofilmes mistos; verificar a interferência do co-cultivo pela técnica de dois compartimentos, avaliar os biofilmes em ensaios de Hibridização In Situ (FISH) e verificar o papel dos genes PINA0102 e PIN0398 de P. intermedia no biofilme. Assim, 9 isolados clínicos de P. intermedia e 5 de P. nigrescens, as cepas padrão P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277, os mutantes Pi17D0398 e Pi17D0102 foram avaliados em variados consórcios. Os resultados mostraram que as interações investigadas são cepa específicas. / Prevotella intermedia and Prevotella nigrescens are commonly associated with periodontal diseases. The goals of this study were to determine the co-aggregation of strains of P. intermedia, P. nigrescens and P. gingivalis; to quantify the biomass of these associations, to evaluate the ratio of these microorganisms in heterotypic biofilms, to verify the modulation of biofilms in co-culture using a trans-well system; to evaluate the structure of the biofilms by Fluorescent Hybridization In Situ (FISH) and to determine the role of genes PINA0102 and PIN0398 of P. intermedia in the modulation of its biofilm. Therefore, 9 clinical isolates of P. intermedia, 5 of P. nigrescens, type strains P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277 and mutant strains Pi17D0398 and Pi17D0102 were grown in consortia of two strains. Our data demonstrate that the associations of P. intermedia, P. nigrescens and P. gingivalis are strain-specific.

Prevotella intermedia/nigrescens group bacteria are Gram-negative, non-motile, anaerobic rods abundant in the subgingival microbiome of human periodontitis patients, and relatively sparse in persons with periodontal health. P. intermedia/nigrescens may be inadequately suppressed in periodontal pockets with conventional mechanical-surgical forms of periodontal therapy. Therefore, short-term systemic antibiotic therapy is often recommended in the treatment of recalcitrant (refractory) severe periodontitis patients where high numbers of P. intermedia/nigrescens persist in the subgingival microbiota. Limited available data suggests that the antibiotic sensitivity profile of periodontal P. intermedia/nigrescens has changed over time among severe periodontitis patients in the United States, with increasing levels of antibiotic resistance reported. These findings have potentially important clinical implications for dental professionals and their severe periodontitis patients which need further confirmation and clarification. To further expand knowledge on this clinically relevant issue, the purpose of the present study was to determine and compare over a 10-year period (2011 versus 2021) the prevalence of in vitro resistance of periodontal P. intermedia/nigrescens to the antibiotics amoxicillin, metronidazole, clindamycin, and doxycycline. / Oral Biology

P. gingivalis é um dos principais patógenos da doença periodontal, é encontrado em biofilmes orais com S. gordonii e P. intermedia e em células endoteliais da artéria coronária in vivo. P. gingivalis necessita de ferro em seu metabolismo e pode usar certas proteínas do hospedeiro como fontes deste íon em ambientes limitantes. Assim, este estudo investigou o papel dos genes PGN0741/PG0637 (receptor dependente de TonB) e PGN0531/PG1380 (fvW) de P. gingivalis na formação de biofilme em diferentes concentrações de ferro, em biofilmes mistos com S. gordonii e P. intermedia, e na adesão e invasão de células endoteliais da artéria coronária. Os resultados mostraram divergências no papel dos genes TonB e fvW na formação dos monobiofilmes e mistos e em diferentes concentrações de ferro, demonstrando uma relação cepa-dependente. Na adesão, fvW se mostrou importante para ambas cepas, mas na persistência apenas para P. gingivalis W83. Este trabalho enfatiza, assim, a necessidade do uso de mais de uma cepa de P. gingivalis no estudo do papel de genes em ensaios experimentais. / P. gingivalis is one of the major pathogens of periodontal diseases. It is found in oral biofilms associated with S. gordonii and P. intermedia, and inside of coronary artery endothelial cells in vivo. P. gingivalis requires iron for growth and can exploit iron-carrying proteins of the host as sources in limiting environments. Thus, this work aimed to study the role of genes PGN0741/PG0637 (TonB-dependent receptor) and PGN0531/PG1380 (fvW) of P. gingivalis in biofilm formation under different iron concentrations, in mixed biofilms with S. gordonii and P. intermedia, and in the adhesion and invasion of coronary artery endothelial cells. Our data showed discordance for the role of TonB and fvW in homo- and heterotypic biofilm formation and in different iron concentrations. The relevance of both genes was strain-dependent. Gene fvW was relevant for adhesion to endothelial cells, but only for strain W83 during persistence. Therefore, our study emphasizes the importance of using different strains for a better understanding of the role of genes in experimental assays.

P. gingivalis é um dos principais patógenos da doença periodontal, é encontrado em biofilmes orais com S. gordonii e P. intermedia e em células endoteliais da artéria coronária in vivo. P. gingivalis necessita de ferro em seu metabolismo e pode usar certas proteínas do hospedeiro como fontes deste íon em ambientes limitantes. Assim, este estudo investigou o papel dos genes PGN0741/PG0637 (receptor dependente de TonB) e PGN0531/PG1380 (fvW) de P. gingivalis na formação de biofilme em diferentes concentrações de ferro, em biofilmes mistos com S. gordonii e P. intermedia, e na adesão e invasão de células endoteliais da artéria coronária. Os resultados mostraram divergências no papel dos genes TonB e fvW na formação dos monobiofilmes e mistos e em diferentes concentrações de ferro, demonstrando uma relação cepa-dependente. Na adesão, fvW se mostrou importante para ambas cepas, mas na persistência apenas para P. gingivalis W83. Este trabalho enfatiza, assim, a necessidade do uso de mais de uma cepa de P. gingivalis no estudo do papel de genes em ensaios experimentais. / P. gingivalis is one of the major pathogens of periodontal diseases. It is found in oral biofilms associated with S. gordonii and P. intermedia, and inside of coronary artery endothelial cells in vivo. P. gingivalis requires iron for growth and can exploit iron-carrying proteins of the host as sources in limiting environments. Thus, this work aimed to study the role of genes PGN0741/PG0637 (TonB-dependent receptor) and PGN0531/PG1380 (fvW) of P. gingivalis in biofilm formation under different iron concentrations, in mixed biofilms with S. gordonii and P. intermedia, and in the adhesion and invasion of coronary artery endothelial cells. Our data showed discordance for the role of TonB and fvW in homo- and heterotypic biofilm formation and in different iron concentrations. The relevance of both genes was strain-dependent. Gene fvW was relevant for adhesion to endothelial cells, but only for strain W83 during persistence. Therefore, our study emphasizes the importance of using different strains for a better understanding of the role of genes in experimental assays.

Orientador: Rafael Nobrega Stipp / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-25T14:55:16Z (GMT). No. of bitstreams: 1 Rodrigues_ItaloSartoCarvalho_D.pdf: 947131 bytes, checksum: 718f334861a919e4b10e20f1d03bdfd3 (MD5) Previous issue date: 2014 / Resumo: O biofilme é uma população biológica com um elevado grau de organização, onde os microrganismos presentes formam uma comunidade estruturada, coordenada e funcional. O estudo do comportamento dos biofilmes é fundamental para melhorar as formas de controle, especialmente durante infecções, tais como as doenças periodontais. No primeiro capítulo, foram avaliados os efeitos da aplicação tópica e frequente do digluconato de clorexidina (CLX) em diferentes concentrações na periodontite induzida por ligadura nos primeiros molares de ratos. As ligaduras receberam 10 µl de soluções de CLX à 0,2%, 2%, 10%, 20% ou diluente, de quatro em quatro dias, em um total de quatro aplicações. Após eutanásia, a quantidade de células bacterianas no biofilme formado sobre a ligadura foi estimada por cultura e por PCR quantitativo. A reabsorção óssea foi mensurada em altura e área por fotografia digital e em volume por microtomógrafo. Depois de quatro dias a partir da última aplicação da CLX, as reduções bacterianas mantidas pelos tratamentos com CLX atingiram até 10-6. O grupo que recebeu CLX a 20% teve, em média, logs bacterianos 3,3× menor (p<0.01, Kruskal-Wallis). Não houve diferença estatística entre os grupos em relação à reabsorção óssea por ambos os métodos testados (p>0.05, Kruskal-Wallis), embora 55% dos sítios apresentaram menor reabsorção óssea. No segundo capítulo, foi avaliada a influência de diversas substâncias na formação de biofilme por Prevotella intermedia. Os biofilmes foram formados em placas de 48 poços contendo tratamento de superfície prévio com DNA purificado, hemina, CaCO3, Ca(OH)2, CaCl2, soro, albumina, dextrana, metionina, glicose, glutamina, KCl, complexo vitamínico, cistina ou mucina. O biofilme formado foi corado e quantificado por espectrofotometria. A arquitetura do biofilme foi visualizada por microscopia confocal de fluorescência por varredura laser. O tratamento da superfície com CaCl2 a 1 mg/cm2 permitiu a formação do biofilme em quantidade de 0,3 OD490nm (p<0.01, ANOVA Dunnet), sendo esse valor 10× superior quando a superfície foi tratada com 2,5 mg/cm2 (p<0.01, ANOVA Dunnet). As demais substâncias tiveram pouco ou nenhum impacto sobre a formação do biofilme. A visualização por microscopia confocal revelou uma comunidade estruturada e com vitalidade em toda sua espessura. Conclusões: os dados indicam que a CLX concentrada diminui a carga bacteriana na região da periodontite induzida, que reflete em menor reabsorção óssea apenas em parte das amostras. O pré-revestimento da superfície de crescimento com cálcio promove a formação de biofilme por P. intermedia / Abstract: Biofilms are biological communities with a high degree of organization, in which micro-organisms form structured, coordinated and functional communities. These biological communities are embedded in a self-created extracellular matrix. Biofilm is also associated with a high level of antimicrobial tolerance of the associated organisms. Understanding biofilm behavior is crucial to develop ways for its control during infections, such as periodontal disease. In the first chapter, topical and frequent application of various concentrations of chlorhexidine digluconate (CLX) where evaluated. Periodontitis were induced by ligature on first molars. Then, ligatures were treated with 10 µl of chlorhexidine solutions at 0.2%, 2%, 10%, 20% or diluent, every four days in a total of four applications periods. After euthanasia, bacterial loads on ligatures were estimated by both culture and qPCR. Bone resorption height and area were measured by digital photography and its volume by microtomography. Treated sites had bacterial reductions up to 10-6 cells. Treatment with 20% CLX showed mean of 3.3× lower bacterial levels (p<0.01, Kruskal-Wallis). There was no statistical difference between groups regarding bone resorption (p>0.05, Kruskal-Wallis), although 55% of the treated sites had some lower bone resorption. In the second chapter, substances that may enhance biofilm formation by Prevotella intermedia were investigated. Wells of 48-well plates were coated with DNA, hemin, CaCO3, Ca(OH)2, CaCl2, serum, albumin, dextran, methionine, glucose, glutamine, KCl, vitamin complex, cystine or mucin. Biofilms were grown for 24 h, washed, stained and quantified by spectrophotometry. Biofilm architecture and its viability were visualized by Confocal Laser Scanning Microscopy. Surfaces treated with 1 mg/cm2 of CaCl2 enhanced biofilm amount by 0.3 OD490nm (p<0.01, ANOVA Dunnet), while 2.5 mg/cm2 yielded 10-fold more biofilm mass (p<0.01, ANOVA Dunnet). Other substances had modest or no impact in biofilm mass. Confocal microscopy images showed structured and alive biofilms with no dead areas. Conclusions: concentrated CLX reduces bacterial load, which reflects in lower bone resorption in few sites. Surfaces pre-coated with calcium chloride enhance P. intermedia biofilm formation / Doutorado / Microbiologia e Imunologia / Doutor em Biologia Buco-Dental

Objectives: This investigation adapted the LIVE/DEAD� Baclight[TM] bacterial viability stain for the quantitative determination of bacterial cell viability of the aerotolerant anaerobes Porphyromonas gingivalis ATCC 33277 and Prevotella intermedia ATCC 25611. The Live/Dead stain was used to determine the influence of haem availability on the resistance of P. gingivalis and P. intermedia to the reactive oxygen species (ROS) superoxide anion and hydrogen peroxide and compare the sensitivities between the haem-requiring periodontal bacteria to ROS. Neutrophils use oxidative and non-oxidative killing mechanisms. During phagocytosis, neutrophils kill bacteria via a respiratory burst, producing ROS. P. gingivalis and P. intermedia are oxygen-tolerant gram-negative bacteria found in the gingival crevice. These bacteria express superoxide dismutase (SOD) activity, which extends some protection against superoxide radicals. Methods: Initially, experiments were performed to validate the reliability and accuracy of the fluorogenic Live/Dead stain using Escherichia coli ATCC 10798 (K-12), followed by experiments using P. gingivalis. The Live/Dead stain distinguishes viable:non-viable proportions of bacteria using mixtures of green (SYTO 9) and red (propidium iodide) fluorescent nucleic acid stains respectively. Bacterial cell viability was assessed with fluorescence microscopy and subsequently quantitative measurement using a fluorescence microplate reader (BMG Fluorostar plus Optima). P. gingivalis and P. intermedia colonies were subcultured from frozen cultures, in Tryptic soy broth (TSB) (Difco) and incubated anaerobically for approximately five days. They were further subcultured in pre-reduced TSB, supplemented with menadione 0.5[mu]g/ml (TSB-M) and either 5 [mu]g/ml haemin (Haem 5), 50 [mu]g/ml haemin (Haem 50) or without supplemental haemin (Haem 0). Cultures were grown anaerobically at 37�C to early stationary phase (approximately 48 hours). For experimental purposes, bacteria were harvested, washed and resuspended in 10 mM Tris-buffered saline (pH 7.5) containing peptone (TBS-P) (0.1 mg/ml), with a final adjustment to OD₅₄₀ [approximately equals] 2.0 (which corresponds to 1 x 10⁹ bacteria/ml). Bacterial suspensions were diluted ([approximately equals] 10⁸/ml) into TBS-P containing the fluorogenic viability stain (BacLight, Molecular Probes). Either pyrogallol (0.02 - 2 mM) or hydrogen peroxide (0.01 - 100 mM) was added (except to control tubes); tubes were vortexed for ten seconds and incubated at 37�C. Viability was monitored fluorimetrically for three hours. Results: For both P. gingivalis and P. intermedia, a pyrogallol concentration of 0.2 mM resulted in 80 to 90% cell death; and a hydrogen peroxide concentration of 10 mM killed approximately 80 to 90% of cells. Irrespective of the haem status, no significant difference was determined between the overall maximum rate of killing of P. gingivalis and P. intermedia, in their response to either superoxide or hydrogen peroxide; with the exception that the P. intermedia Haem 0 group was significantly less susceptible to hydrogen peroxide than the P. gingivalis Haem 0 group. For the majority of the experiments, there was no significant difference between final bacterial cell viability in the Haem 0 and Haem 5 cells for both species, after 3 hours exposure to various concentrations of ROS. However, the Haem 50 cells showed a significant increased susceptibility (albeit, a small difference) to both hydrogen peroxide and superoxide. Conclusions: The Live/Dead bacterial viability stain provided a valuable method to monitor "real-time" killing, avoiding the difficulties associated with culture-based methods for assessing viability. Haem availability had no clear influence on the resistance to ROS of either P. gingivalis or P. intermedia Haem 0 and Haem 5 cells. The Haem 50 cells showed a very slight increase in susceptibility to hydrogen peroxide and superoxide. Although P. intermedia may be isolated in significant numbers from healthy gingivae, as well as from periodontally diseased sites, it was no more resistant to ROS than was P. gingivalis, which is associated with periodontal lesions and difficult to cultivate from relatively healthy (more oxygenated) sites. This suggests that resistance to ROS does not contribute to the ecological distinction between these two species. The finding that haem availability did not influence sensitivity implies that these bacteria do not accumulate haem for the purpose of protection from ROS.

Objectives: This investigation adapted the LIVE/DEAD� Baclight[TM] bacterial viability stain for the quantitative determination of bacterial cell viability of the aerotolerant anaerobes Porphyromonas gingivalis ATCC 33277 and Prevotella intermedia ATCC 25611. The Live/Dead stain was used to determine the influence of haem availability on the resistance of P. gingivalis and P. intermedia to the reactive oxygen species (ROS) superoxide anion and hydrogen peroxide and compare the sensitivities between the haem-requiring periodontal bacteria to ROS. Neutrophils use oxidative and non-oxidative killing mechanisms. During phagocytosis, neutrophils kill bacteria via a respiratory burst, producing ROS. P. gingivalis and P. intermedia are oxygen-tolerant gram-negative bacteria found in the gingival crevice. These bacteria express superoxide dismutase (SOD) activity, which extends some protection against superoxide radicals. Methods: Initially, experiments were performed to validate the reliability and accuracy of the fluorogenic Live/Dead stain using Escherichia coli ATCC 10798 (K-12), followed by experiments using P. gingivalis. The Live/Dead stain distinguishes viable:non-viable proportions of bacteria using mixtures of green (SYTO 9) and red (propidium iodide) fluorescent nucleic acid stains respectively. Bacterial cell viability was assessed with fluorescence microscopy and subsequently quantitative measurement using a fluorescence microplate reader (BMG Fluorostar plus Optima). P. gingivalis and P. intermedia colonies were subcultured from frozen cultures, in Tryptic soy broth (TSB) (Difco) and incubated anaerobically for approximately five days. They were further subcultured in pre-reduced TSB, supplemented with menadione 0.5[mu]g/ml (TSB-M) and either 5 [mu]g/ml haemin (Haem 5), 50 [mu]g/ml haemin (Haem 50) or without supplemental haemin (Haem 0). Cultures were grown anaerobically at 37�C to early stationary phase (approximately 48 hours). For experimental purposes, bacteria were harvested, washed and resuspended in 10 mM Tris-buffered saline (pH 7.5) containing peptone (TBS-P) (0.1 mg/ml), with a final adjustment to OD₅₄₀ [approximately equals] 2.0 (which corresponds to 1 x 10⁹ bacteria/ml). Bacterial suspensions were diluted ([approximately equals] 10⁸/ml) into TBS-P containing the fluorogenic viability stain (BacLight, Molecular Probes). Either pyrogallol (0.02 - 2 mM) or hydrogen peroxide (0.01 - 100 mM) was added (except to control tubes); tubes were vortexed for ten seconds and incubated at 37�C. Viability was monitored fluorimetrically for three hours. Results: For both P. gingivalis and P. intermedia, a pyrogallol concentration of 0.2 mM resulted in 80 to 90% cell death; and a hydrogen peroxide concentration of 10 mM killed approximately 80 to 90% of cells. Irrespective of the haem status, no significant difference was determined between the overall maximum rate of killing of P. gingivalis and P. intermedia, in their response to either superoxide or hydrogen peroxide; with the exception that the P. intermedia Haem 0 group was significantly less susceptible to hydrogen peroxide than the P. gingivalis Haem 0 group. For the majority of the experiments, there was no significant difference between final bacterial cell viability in the Haem 0 and Haem 5 cells for both species, after 3 hours exposure to various concentrations of ROS. However, the Haem 50 cells showed a significant increased susceptibility (albeit, a small difference) to both hydrogen peroxide and superoxide. Conclusions: The Live/Dead bacterial viability stain provided a valuable method to monitor "real-time" killing, avoiding the difficulties associated with culture-based methods for assessing viability. Haem availability had no clear influence on the resistance to ROS of either P. gingivalis or P. intermedia Haem 0 and Haem 5 cells. The Haem 50 cells showed a very slight increase in susceptibility to hydrogen peroxide and superoxide. Although P. intermedia may be isolated in significant numbers from healthy gingivae, as well as from periodontally diseased sites, it was no more resistant to ROS than was P. gingivalis, which is associated with periodontal lesions and difficult to cultivate from relatively healthy (more oxygenated) sites. This suggests that resistance to ROS does not contribute to the ecological distinction between these two species. The finding that haem availability did not influence sensitivity implies that these bacteria do not accumulate haem for the purpose of protection from ROS.