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.

Diss. med. dent. Zürich. / Literaturverz.

Objectives: Systemic antibiotics are generally recognized as providing a beneficial impact in treatment of both aggressive and chronic periodontitis. Since strains of periodontal pathogens among periodontitis patients may vary in their antibiotic drug resistance, the American Academy of Periodontology recommends antimicrobial susceptibility testing of suspected periodontal pathogens prior to administration of systemic periodontal antibiotic therapy, to reduce the risk of a treatment failure due to pathogen antibiotic resistance. E-test and MIC Test Strip assays are two in vitro antimicrobial susceptibility testing systems employing plastic- and paper-based, respectively, carriers loaded with predefined antibiotic gradients covering 15 two-fold dilutions. To date, no performance evaluations have been carried out comparing the Etest and MIC Test Strip assays in their ability to assess the in vitro antimicrobial susceptibility of periodontal bacterial pathogens. As a result, the purpose of this study was to compare the in vitro performance of E-test and MIC Test Strip assays in assessing minimal inhibitory concentration (MIC) values of four antibiotics frequently utilized in systemic periodontal antibiotic therapy against 11 fresh clinical subgingival isolates of the putative periodontal pathogen, Prevotella intermedia/ nigrescens, and to compare the distribution of P. intermedia/ nigrescens strains identified with interpretative criteria as "susceptible" and "resistant" to each of the four antibiotics using MIC values determined by the two antimicrobial susceptibility testing methods. Methods: Standardized cell suspensions, equivalent to a 2.0 McFarland turbidity standard, were prepared with 11 fresh clinical isolates of P. intermedia/nigrescens, each recovered from the subgingival microbiota of United States chronic periodontitis subjects, and plated onto to the surfaces of culture plates containing enriched Brucella blood agar. After drying, pairs of antibiotic-impregnated, quantitative, gradient diffusion strips from two manufacturers (E-test, bioMérieux, Durham, NC, USA, and MIC Test Strip, Liofilchem s.r.l., Roseto degli Abruzzi, Italy) for amoxicillin, clindamycin, metronidazole, and doxycycline were each placed apart from each other onto the inoculated enriched Brucella blood agar surfaces, so that an antibiotic test strip from each manufacturer was employed per plate against each P. intermedia/ nigrescens clinical isolate for antibiotic susceptibility testing. After 48-72 hours anaerobic jar incubation, individual MIC values for each antibiotic test strip against P. intermedia/nigrescens were read in &mu;g/ml at the point where the edge of the bacterial inhibition ellipse intersected with the antibiotic test strip. MIC50, MIC90, and MIC range were calculated and compared for each of the test antibiotics, with essential agreement (EA) values determined per test antibiotic for the level of outcome agreement between two antimicrobial susceptibility testing methods. In addition, the identification of antibiotic "susceptible" and "resistant" strains among the P. intermedia/nigrescens clinical isolates was determined for each test antibiotic using MIC interpretative criteria from the MIC interpretative standards developed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for gram-negative anaerobic bacteria for amoxicillin, clindamycin, and metronidazole findings, and from the French Society of Microbiology breakpoint values for anaerobic disk diffusion testing for doxycycline data. Results: For amoxicillin, higher MIC50 and MIC90 values against the P. intermedia/ nigrescens strains were found with the MIC Test Strip assay than with E-test strips, resulting in a relatively low EA value of 45.5% between the two susceptibility testing methods. A higher percentage of amoxicillin "resistant" P. intermedia/nigrescens strains (72.7%) were identified by MIC Test Strips as compared to E-test strips (54.5%), although both methods found the same proportion of amoxicillin "susceptible" strains (27.3%). For clindamycin, both susceptibility testing methods provided identical MIC values (EA value = 100%), and exactly the same distributions of "susceptible" and "resistant" strains of P. intermedia/nigrescens. For metronidazole, only very poor agreement (EA value = 9.1%) was found between the two susceptibility testing methods, with MIC Test Strips exhibiting markedly higher MIC50 and MIC90 values against P. intermedia/nigrescens as compared to E-test strips. However, the distribution of "susceptible" and "resistant" P. intermedia/ nigrescens were identical between the two susceptibility testing methods. For doxycycline, relatively good agreement (EA value = 72.7%) was found in MIC concentrations between the two susceptibility testing methods, although generally lower MIC values were associated with MIC Test Strips. In addition, identical distributions of "susceptible" and "resistant" P. intermedia/nigrescens were provided by both susceptibility testing methods. Conclusions: Relative to MIC values measured against periodontal strains of P. intermedia/nigrescens, MIC Test Strips gave higher MIC values with amoxicillin and metronidazole, equal MIC values with clindamycin, and lower MIC values with doxycycline, as compared to MIC values measured with the E-test assay. Relative to the identification of antibiotic "susceptible" periodontal P. intermedia/ nigrescens strains, both susceptibility testing methods provided identical findings, suggesting that both methods appear to be interchangeable for clinical decision making in regard to identification of antibiotic-sensitive strains of periodontal P. intermedia/nigrescens. However, for epidemiologic surveillance of drug susceptibility trends, where exact MIC values are important to track over time, the relatively higher proportion of non-exact MIC differences between the two susceptibility testing methods argues against using them interchangeably. Instead, one or the other method should be used consistently for such studies. Further comparative studies of the E-test and MIC Test Strip assays are indicated using other periodontopathic bacterial species besides P. intermedia/ nigrescens, and to assess the reproducibility of MIC values provided by both in vitro susceptibility testing methods over time. / Oral Biology

Indiana University-Purdue University Indianapolis (IUPUI) / Periodontal disease is the leading cause of tooth loss in adults. Bacterial biofilm on tooth surfaces is the primary initiator of periodontal disease. Various factors contribute to the severity of periodontal disease including the different virulence factors of the bacteria within the biofilm. In the progression of periodontal disease, the microflora evolves from a predominantly Gram positive microbial population to a mainly Gram negative population. Specific gram negative bacteria with pronounced virulence factors have been implicated in the etiology and pathogenesis of periodontal disease, namely Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola which form the red complex of bacteria. The orange complex bacteria become more dominant in the maturation process of dental plaque and act to bridge the early colonizers of plaque with the later more dominant red complex bacterial and consists of such bacteria as Campylobacter showae, Campylobacter rectus, Fusobacterium nucleatum and Prevotella intermedia. Perhaps the most investigated contributing factor is the relationship between smoking and periodontal disease. When examining the association between cigarette smoking and interproximal bone loss, greater bone loss is associated with higher cigarette consumption, longer duration (i.e., pack year history) and higher lifetime exposure. The presence of various virulence factors such as the production of a capsular material, as well as the proteolytic activity of the various periopathodontic bacteria has been associated with the pathogenesis of periodontitis. Even though many different enzymes are produced in large quantities by these periodontal bacteria, trypsin-like enzymes, chymotrypsin-like enzymes and elastase-like enzymes, as well as dipeptidyl peptidase-like enzymes, have been thought to increase the destructive potential of the bacterium and mediate destruction of the periodontal apparatus. More specifically, it is hypothesized that the proteolytic activity of other clinically important periodontal pathogens, such as Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas assacharolyticus, is increased in the presence of nicotine. The purpose of this study was to determine the effects of nicotine on F. nucleatum, P. intermedia and P. assacharolyticus proteolytic activity. Cultures were maintained on anaerobic blood agar plates containing 3% sheep blood. Bacterial cells were harvested from the plates and washed. Washed F. nucleatum, P. intermedia and P. assacharolyticus cells were incubated with 1 mg/ml of nicotine. Bacterial cells not incubated with nicotine were used as positive controls. Secreted enzymatic activity was measured using the synthetic chromogenic substrates glycyl-L-proline-p-nitroanilide (GPPNA), N-succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide (SAAAPNA), N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (SAAPPPNA) and N-α-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) (Sigma-Aldrich Products, St. Louis, MO, USA). Appropriate means and standard deviations were determined for each of the enzymatic activities measured and analysis of variance (ANOVA) was used to compare the groups utilizing a 5% significance level for all comparisons. Results demonstrated that after 60 minutes of incubation of F. nucleatum, P. intermedia and P. assacharolyticus cells with 1 mg/ml of nicotine and the various synthetic substrates, had the following proteolytic activity for GPPNA: 0.83 ± 0.14, 0.72 ± 0.03 and 0.67 ± 0.10, respectively; SAAAPNA: 0.82 ± 0.06, 0.76 ± 0.05 and 0.68 ± 0.08, respectively; SAAPPPNA: 0.90 ± 0.13, 0.85 ± 0.17 and 0.72 ± 0.03, respectively; and BAPNA: 0.81 ± 0.15, 0.74 ± 0.13 and 0.74 ± 0.16, respectively. In conclusion, the results indicate that in the presence of 1 mg/ml of nicotine, the proteolytic activity of F. nucleatum and P. assacharolyticus was increased with all of the synthetic substrates (with statistical significance seen only in the increases with F. nucleatum and GPPNA, SAAAPNA and BAPNA). The proteolytic activity exhibited an increasing trend in activity for P. intermedia with SAAPPPNA and BAPNA but a decreasing trend in activity with GPPNA and SAAAPNA when incubated with 1 mg/ml of nicotine, once again demonstrating no statistical significance for any of the substrates. Therefore, it could be concluded that based on these results nicotine at a concentration of 1 mg/ml may increase the proteolytic activity of periodontal pathogens and thus may increase periodontal disease activity and subsequent periodontal breakdown. Further studies are needed to validate these results utilizing different concentrations of nicotine.

Made available in DSpace on 2016-06-02T19:55:42Z (GMT). No. of bitstreams: 1 6569.pdf: 7813080 bytes, checksum: 5bddbc1ed667ee4456bc01f930d2c15a (MD5) Previous issue date: 2014-11-25 / Financiadora de Estudos e Projetos / study evaluated the production of hydrogen and methane from sugarcane vinasse in anaerobic fluidized bed reactor. Two fluidized bed reactors filled with expanded clay were operated under two different concentrations: R5 (5 gCOD.L-1) and R10 (10 g.CODL-1). During the first stage, glucose was used as the primary carbon source. Along the first step vinasse was added from 0% to 100% of the organic source under HRT of 6 h. In a second step with 100% of sugarcane vinasse, HRT was reduced to 4, 2 and 1 h. In another reactor R15 (15 gCOD.L-1) it was varied the substrate (100% glucose; 50 % glucose/ 50 % vinasse; 100 % vinasse) under HRT of 8 h. All reactors were operated in room temperature and a sludge from the treatment of swine wastewater was used. It was not observed methane production in R15. Hydrogen production rate and hydrogen yield reached, respectively: 0,01 L.h-1.L-1 e 0,10 mmolH2.g-1COD added. In reactors R5 and R10, biogas was formed by H2 and CO2 when glucose was present in the feed. Methane was formed when vinasse became the main substrate. The best operating condition occurred under HRT of 1 h, vinasse 100% at a concentration of 5 gCOD. L-1 with a hydrogen production rate of 0.57 L.h.-1L-1. As regards the yield, the best condition was under HRT of 6 h when the affluent comprised vinasse and glucose (3:1) reaching an yield of 3.07 mmolH2.g-1CODadded. Methane production in acidic conditions showed that the methanogens have adapted to a slightly acidic pH of 4.5. The principal metabolites were ethanol, butyric acid, propionic acid and methanol. Microbial characterization revealed the presence of Prevotella and Megasphaera belonging to the domain Bacteria and Methanobacterium and Methanosphaera belonging to the Archaea domain. / O presente estudo avaliou a produção de hidrogênio e metano a partir de vinhaça de cana-de-açúcar em reator anaeróbio de leito fluidizado. Dois reatores de leito fluidizado preenchidos com argila expandida foram operados sob duas diferentes concentrações de substratos: R5 (5 gDQO.L-1) e R10 (10 gDQO.L-1). Durante a primeira etapa, a glicose foi utilizada como fonte de carbono principal. Em seguida, a vinhaça foi adicionada passando de 0% a 100% da fonte orgânica sob o tempo de detenção hidráulica de 6 h. Numa segunda etapa com 100 % de vinhaça, foi feita a redução do TDH para 4, 2 e 1 h. Um terceiro reator foi empregado com uma concentração maior R15 (15 gDQO.L-1) na qual foi variado o substrato (100% glicose, 50% glicose/50 % vinhaça, 100 % vinhaça) sob o tempo de detenção hidráulica de 8 h. Todos os reatores foram operados em temperatura ambiente e foi utilizado lodo proveniente do tratamento de resíduos da suinocultura. Não foi observada produção de metano no reator R15, apenas H2 e CO2. A produção volumétrica e rendimento de hidrogênio atingiu um máximo de 0,01 L.h-1.L-1 e 0,10 mmolH2.g- 1DQOadicionada na fase de alimentação com vinhaça. Nos reatores R5 e R10 o biogás foi formado por H2 e CO2 quando ainda havia glicose como substrato. Metano foi formado quando a vinhaça se tornou o substrato principal. A melhor condição de operação se deu sob o TDH de 1 h, vinhaça 100 % a uma concentração de 5 gDQO.L-1 quando foi obtida uma produção volumétrica de hidrogênio de 0,57 L.h-1.L-1. No que se refere ao rendimento, a melhor condição se deu sob o TDH de 6 h quando o afluente era constituído por vinhaça e glicose (3:1) e um rendimento de 3,07 mmolH2.g-1DQOadicionada. A produção de metano em condições ácidas mostrou que as metanogênicas consumidoras de hidrogênio se adaptaram ao pH levemente ácido de 4,5. Os principais metabólitos produzidos foram etanol, ácido butírico, ácido propiônico e metanol. A caracterização microbiana revelou a presença de Prevotella e Megasphaera pertencentes ao domínio Bacteria e Methanobacterium e Methanosphaera pertencentes ao domínio Archaea.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Over the past few years, there has been increasing evidence of the effect of the oral health over the general health of individuals, supported by a series of biological and epidemiological studies that show a relation between the mouth and many diseases, including cardiovascular diseases. Structural deficiencies and functional abnormalities of heart valves represent an important cause of cardiovascular morbidity and mortality in Brazil, and a few defects have been recently associated with infectious agents. The aim of this study was to identify cariogenic and periodontopathogenic bacteria in dental plaque, saliva and heart valves, without clinical endocarditis, of patients with heart valve diseases, and correlate these findings with the oral health status of the patients. Oral exams using the DMTF (decayed, missing and filled teeth) and PSR (Periodontal Screening and Recording) indexes to evaluate caries and periodontal disease, respectively, were performed. Samples of supragingival and subgingival dental plaque, saliva and cardiac valves were evaluated, through Real Time Polymerase Chain Reaction, for the presence of DNA of Streptococcus mutans (S. mutans), Prevotella intermedia (P. intermedia), Porphyromonas gingivalis (P. gingivalis) and Treponema denticola (T. denticola). A total of 114 samples were collected from 42 patients with a mean age of 55.6  13.8 years. The average number of missing teeth due to caries was 23.52  9.41 teeth per patient, and according to the highest score of periodontal disease observed for each patient, excluding edentulous patients (44.0%), periodontal pockets over 4mm (43.4%) and dental calculus (34.7%) were detected in a higher number of patients. The molecular analysis of the oral samples revealed high frequency of S. mutans and P. intermedia in supragingival dental plaques, subgingival dental plaques and saliva of dentate and edentulous patients (variation 60.0% - 100.0%), while P. gingivalis and T. denticola were detected in a smaller number of oral samples (variation 17.6% - 64.0%). The microorganism most frequently detected in heart valve samples was the S. mutans (89.3%), followed by P. intermedia (19.1%), P. gingivalis (4.2%) e T. denticola (2.1%). Significant difference was observed between the frequency of P. intermedia, P. gingivalis and T. denticola in the heart valve and dental plaque, as oposed to S. mutans. The identification of oral bacteria, especially S. mutans, in heart valves of patients with a previous history of dental caries and gingivitis/periodontitis suggests the possible involvement of these pathogens in the etiopathogenesis of heart valve diseases. / Atualmente, cada vez mais se tem evidÃncias do efeito da condiÃÃo oral na saÃde geral dos indivÃduos, atravÃs de uma sÃrie de estudos epidemiolÃgicos e biolÃgicos que mostram uma relaÃÃo entre a boca e diversas doenÃas, incluindo as doenÃas cardiovasculares. Desordens estruturais e nas funÃÃes das vÃlvulas cardÃacas representam uma importante causa de morbidade e mortalidade cardiovascular no Brasil, sendo alguns processos, como a estenose aÃrtica degenerativa, mais recentemente associados a agentes infecciosos. O objetivo desta pesquisa foi identificar bactÃrias cariogÃnicas e periodontopatogÃnicas na placa dental, saliva e vÃlvulas cardÃacas, sem endocardite clÃnica, de pacientes com doenÃa valvar, correlacionando esses achados à condiÃÃo bucal dos indivÃduos. AvaliaÃÃo, quanto Ãs doenÃas cÃrie e periodontal, foi realizada, atravÃs dos Ãndices CPO-D (Dentes Permanentes Cariados, Perdidos e Obturados) e PSR (Registro Periodontal Simplificado), respectivamente. Amostras de placa dental supragengival, subgengival, saliva e vÃlvula cardÃaca foram coletadas para investigaÃÃo da presenÃa de DNA, atravÃs de PCR (ReaÃÃo em Cadeia de Polimerase) em tempo real, de Streptococcus mutans (S. mutans), Prevotella intermedia (P. intermedia), Porphyromonas gingivalis (P. gingivalis) e Treponema denticola (T. denticola). Um total de 114 amostras foi coletado de 42 pacientes com mÃdia de idade de 55.6  13.8 anos. A mÃdia de dentes perdidos devido à cÃrie, por paciente, foi em torno de 23.52  9.41 e, segundo o maior grau de doenÃa periodontal observado no indivÃduo, excluindo-se os pacientes desdentados totais (44.0%), bolsa superior a 4 mm (43.4%) e o cÃlculo dental (34.7%) esteve presente em um maior nÃmero de pacientes. A anÃlise molecular das amostras bucais revelou alta frequÃncia de S. mutans e P. intermedia nas placas supragengival, subgengival e saliva de pacientes dentados e desdentados (variando entre 60.0% e 100.0%), enquanto que P. gingivalis e T. denticola estiveram presentes em menor nÃmero de amostras bucais (variando entre 17.6% e 64.0%). O micro-organismo mais frequentemente encontrado nas amostras valvares foi o S. mutans (89.3%), seguido da P. intermedia (19.1%), P. gingivalis (4.2%) e T. denticola (2.1%). DiferenÃa significativa foi encontrada entre a presenÃa de P. intermedia, P. gingivalis e T. denticola na vÃlvula e na placa dental, diferentemente do S. mutans. A identificaÃÃo de bactÃrias orais, principalmente S. mutans, em vÃlvulas cardÃacas de pacientes com elevada experiÃncia prÃvia de cÃrie e ocorrÃncia de gengivite/periodontite, sugere o possÃvel envolvimento desses patÃgenos nas doenÃas valvares.

Background &amp; Aims: Celiac disease (CD) is a chronic inflammatory small-bowel enteropathy caused by permanent intolerance to gliadin in wheat gluten, and related proteins in ray and barley. It is disputed whether CD patients tolerate oats. The only treatment of CD is lifelong gluten-free diet (GFD). Only individuals that carry the HLA-DQ2 and/or DQ8 alleles, and eat gluten can develop CD. Dysbiosis in the gut microbiota is a suggested risk factor for CD. T cells in small intestinal mucosa, including intraepithelial lymphocytes (IELs), are known to be important in the pathogenesis of CD. In contrast, the role of intestinal epithelial cells (IECs) is poorly understood. In this thesis we investigated the role of IECs in the immune pathology of CD from duodenal mucosa of children with CD, clinical controls and treated CD. We also investigated the role of CD associated bacteria and oats supplemented GFD on the mucosal immune system. Results: A new CD-associated bacterium, Prevotella jejuni, was isolated and characterized. It is a saccharolytic and proteolytic anaerobe. More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were upregulated in IECs in active CD. In two in vitro models for intestinal epithelium, small intestine enteroids and T84 polarized tight monolayers, we showed that 70% of these genes were upregulated by interferon (IFN)-γ via the IRF1 pathway. IRF1 was also upregulated by the CD-associated bacteria P. jejuni and Actinomyces gravenitzii. IECs expressed the NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin (IL)-18, which induces IFN-γ in IELs. P. jejuni bound the intestinal epithelial cell lines T84, Caco2, HT29, and INT407, while Lachnoanaerobaculum umeaense preferentially bound Caco2. P. jejuni caused decreased transepithelial resistance over tight monolayers, while L. umeaense caused an increase. P. jejuni upregulated mRNAs for the detoxification molecules CYP1A1, CYP1A2, CYP1B1, and TIPARP, the chemokines CX3CL1, CXCL1, and CXCL10, the sialyltranserase ST3GAL4, and the inflammation promoting protein S100A3 in tight monolayers. L. umeaense upregulated the chemokines CCL20 and CXCL10, and down-regulated TLR2. In a randomized, double-blinded intervention trial comparing two study-groups, standard GFD and oat-containing GFD, we found that mRNAs for several immune effector molecules and tight junction proteins were only reduced in patients receiving GFD, but not in a substantial fraction of patients on GFD with oats. The down-regulatory cytokines IL-10 and TGF-β1, the cytotoxicity-activating NK-receptors NKG2C and NKG2E, and the tight junction protein claudin-4 remained elevated in the study group on GFD with oats. Conclusions: IECs are far from inactive in CD. A key factor in the epithelial reaction in CD appears to be over-expression of IRF1 in IECs. Dual activation of IRF1 and IRF1-regulated genes, both directly by P. jejuni and indirectly by IFN-γ via the IL-18-inflammasome, would drastically enhance the inflammatory response and lead to the pathological situation seen in active CD. P. jejuni harms the intestinal epithelium, i.e., it is a likely risk factor for CD, while L. umeaense strengthen barrier function and local immunity, possibly acting as a protective. A fraction of CD patients should avoid oats in the diet. / Doctoral thesis