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.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 155-166).

Thèse (M.Sc.)--Université Laval, 2003. / Bibliogr.: f. [87]-103. Publié aussi en version électronique.

Masters of Science / Background: Infection with HIV results in gradual loss of immunologic functions, especially those mediated by CD4+T helper cells with consequent impairment of the immune response leading to severe manifestations of periodontal disease. The lower the CD4+T lymphocyte cell count or the higher the level of immunosuppression, the higher the incidence of periodontal disease in those patients will be. Putative periodontopathic bacteria namely Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, commonly referred to as "red complex", and many other bacterial species have been implicated in the initiation and progression of periodontal disease. Objective: The present study tests the association between different CD4+T lymphocyte levels and "red complex" pathogens using BANA, in HIV-positive patients with chronic inflammatory periodontal disease (CIPD). Methods: 120 HIV-positive patients from the infectious disease clinic at Tygerberg hospital participated in the study with a mean age of 33.3 years. The CD4+T lymphocyte counts were obtained from patient's medical records. The six Ramjford teeth were used for evaluating periodontal clinical parameters such as plaque index, gingival index, periodontal probing depth and clinical attachment loss. Subgingival plaque samples were collected and analyzed by the enzymatic BANA test for the detection of the "red complex". Results: The CD4+T lymphocyte mean level was 293.43cells/mm3. Statistically significant associations were found between CD4+T cell counts and probing depth (p= 0.0434) and clinical attachment loss (p= 0.0268). Significant associations were found between BANA with all the clinical indices (p= <0.05). However no association was found between CD4+T cell counts and BANA. Conclusion: HIV-positive patients show a high prevalence of "red complex" pathogens subgingivally. Immunosuppression seems to favour the colonization of these species, resulting in periodontal disease manifestations.

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2016-09-15T16:06:05Z No. of bitstreams: 1 Dissertação_ICS_ Soraya Castro Trindade.pdf: 1012276 bytes, checksum: e039010222f6877bd5937655b190d8e2 (MD5) / Made available in DSpace on 2016-09-15T16:06:05Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Soraya Castro Trindade.pdf: 1012276 bytes, checksum: e039010222f6877bd5937655b190d8e2 (MD5) / A periodontite é uma doença inflamatória bucal causada por agentes multifatoriais intrínsecos e extrínsecos, incluindo bactérias Gram-negativas, como Porphyromonas gingivalis. Este estudo foi realizado para avaliar os níveis séricos de IgA, IgG e subclasses de IgG reativos a Porphyromonas gingivalis ATCC33277. Foram examinados 89 pacientes, divididos em quatro grupos: 29 com periodontite crônica (PC), 12 com periodontite agressiva (PA), 22 com gengivite ou periodontite leve (GP), e 26 com periosonto clinicamente sadio (PS). A resposta imune humoral foi avaliada por testes de ELISA, para verificar os níveis séricos de IgG, IgG1, IgG2, IgG3, IgG4 e IgA reativos ao extrato sonicado bruto de P. gingivalis ATCC 33277 e à fração IV obtida por cromatografia. Os níveis de IgA, IgG (p<0,01), IgG2, IgG3 and IgG4 à fração IV foram maiores no grupo PC que no grupo SP. O grupo PC apresentou níveis mais altos de IgG and IgG4 reativas a ambos os antígenos que o grupo GP e níveis mais altos de IgG and IgG4 reativas ao extrato sonicado bruto que o grupo PA. Foram encontradas diferenças estatisticamente significantes nos níveis de IgG reativa às duas preparações de Pg (p<0,01), IgG2 reativa à fração IV (p<0,01), IgG3 reativa à fração IV (p<0,05) e IgG4 reativa ao extrato bruto e à fração IV (p<0,05) entre os grupos PA e SP. Os níveis de IgG1 ao extrato sonicado bruto foram maiores no grupo GP que no grupo AP (p<0,05). Os resultados sugerem que o ELISA indireto para a detecção de IgG total e IgG4 reativas aos dois antígenos e IgG3 reativa à fração IV permitiram as melhores condições de discriminação entre os grupos, com a IgG4 reativa à fração IV apresentando o melhor desempenho. A produção de anticorpos na periodontite agressiva não apresentou um padrão definido em relação aos isotipos estudados.

abstract: Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e., oral pathogen Porphyromonas gingivalis and enterobacterial Escherichia coli. P. gingivalis is a major causative agent of periodontal disease as well as systemic illnesses, like cardiovascular disease. A microarray study found that the putative PorY-PorX TCR system controls the secretion and maturation of virulence factors, as well as loci involved in the PorSS secretion system, which secretes proteinases, i.e., gingipains, responsible for periodontal disease. Proteomic analysis (SILAC) was used to improve the microarray data, reverse-transcription PCR to verify the proteomic data, and primer extension assay to determine the promoter regions of specific PorX regulated loci. I was able to characterize multiple genetic loci regulated by this TCR system, many of which play an essential role in hemagglutination and host-cell adhesion, and likely contribute to virulence in this bacterium. Enteric Gram-negative bacteria must withstand many host defenses such as digestive enzymes, low pH, and antimicrobial peptides (AMPs). The CpxR-CpxA TCR system of E. coli has been extensively characterized and shown to be required for protection against AMPs. Most recently, this TCR system has been shown to up-regulate the rfe-rff operon which encodes genes involved in the production of enterobacterial common antigen (ECA), and confers protection against a variety of AMPs. In this study, I utilized primer extension and DNase I footprinting to determine how CpxR regulates the ECA operon. My findings suggest that CpxR modulates transcription by directly binding to the rfe promoter. Multiple genetic and biochemical approaches were used to demonstrate that specific TCR systems contribute to regulation of virulence factors and resistance to host defenses in P. gingivalis and E. coli, respectively. Understanding these genetic circuits provides insight into strategies for pathogenesis and resistance to host defenses in Gram negative bacterial pathogens. Finally, these data provide compelling potential molecular targets for therapeutics to treat P. gingivalis and E. coli infections. / Dissertation/Thesis / M.S. Biology 2013

Porphyromonas gingivalis é um bacilo anaeróbio Gram negativo envolvido com o início e progressão de doenças periodontais. É considerado um colonizador tardio ou secundário do biofilme oral capaz de aderir a células de Streptococcus gordonii, um colonizador inicial do biofilme dental. Este estudo se propôs a comparar a expressão de genes de amostras de P. gingivalis isoladas de diferentes condições periodontais usando Differential Display (DD) Reverse Transcription PCR, durante a formação de biofilme misto com S. gordonii. O perfil de expressão gênica de células em biofilme (saúde e periodontite) foi comparado, assim como com células planctônicas pareadas. Bandas diferencialmente expressas nas diferentes condições foram clonadas e seqüenciadas, seguida de identificação dos genes em bancos de dados. A confirmação da expressão diferencial dos genes detectados foi realizada através de PCR em Tempo Real. Desses genes, alguns se relacionam com fatores de virulência, outros com obtenção de energia além de genes relacionados com proteínas de membrana e de transporte. / Porphyromonas gingivalis is a Gram negative anaerobic rod involved with the beginning and progression of periodontal diseases P. gingivalis is a late or secondary colonizer of oral biofilms and adhere to cells of Streptococcus gordonii, an early colonizer of dental plaque. This study aim to compare the gene expression of P. gingivalis strains isolated from different periodontal conditions using Differential Display (DD) Reverse Transcription PCR, in a mixed biofilm with S. gordonii. The gene expression profile was determined with biofilm cells (health and disease) and also with their planktonic samples partners. Bands differentially expressed were cloned, sequenciated and analyzed in gene data bank. Differential expression was confirmed by Real Time PCR. Some of the confirmed genes are related to virulence factors, energy metabolism and transport and membrane proteins.

Made available in DSpace on 2014-06-12T23:02:15Z (GMT). No. of bitstreams: 2 arquivo969_1.pdf: 1672515 bytes, checksum: 57a925ef24be8c7dd06dd31514a160d4 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O presente trabalho teve por objetivo avaliar a presença dos periodontopatógenos que formam o complexo vermelho (Tannerella forsythia (Tf), Porphyromonas gingivalis (Pg) e Treponema denticola (Td)) e o Aggregatibacter actinomycetemcomitans (Aa) em pacientes portadores de periodontite crônica. A amostra foi constituída de 29 pacientes com diagnóstico clínico e radiográfico de periodontite crônica de acordo com os critérios da AAP (2000). Todos os dentes foram sondados em seis sítios para registro de profundidade, perda de inserção clínica e sangramento após sondagem. As amostras para análise microbiológica foram coletadas dos 4 sítios com maior profundidade de sondagem para cada paciente, totalizando 116 amostras. Estas amostras foram processadas através da técnica de PCR convencional e foram observados os seguintes resultados: 46,6% apresentaram resultado positivo para a bactéria Pg; 41,4% para Tf; 33,6% para Td e 27,6% para Aa. Não se verificou associação significante entre a presença dos periodontopatógenos e as variáveis faixa etária, sexo e sangramento à sondagem. Para a bactéria Pg verificou-se associação significante (p<0,05) com a variável placa visível, e a presença das bactérias Pg e Tf foi mais prevalente (p < 0,05) em bolsas periodontais &#8805; 8 mm. Nos sítios com profundidade &#61619; 8 mm foram observadas com maior freqüência as combinações Pg + Tf (23,2%) e Pg + Tf + Td (20,0%). Foram estatisticamente significantes (p < 0,05) as associações entre a presença simultânea das bactérias Aa + Pg, Aa+ Tf, Pg + Tf e entre Tf + Td. Concluiu-se que as bactérias analisadas, principalmente as do complexo vermelho, estiveram fortemente relacionadas com a periodontite crônica, e que as bactérias Pg e Tf foram mais frequentes em bolsas periodontais profundas

Porphyromonas gingivalis produces proteases capable of degrading cytokines, host heme proteins, and some antimicrobial peptides. In this work, I show that P. gingivalis culture supernatants fully or partially degrade human neutrophil peptide alpha-defensins and human beta-defensins after 30 minutes. This observation suggests that proteases from P. gingivalis degrade defensins and this activity could abrogate defensin-related innate immune functions.

Die ökologische Beziehung parodontopathogener Mikroorganismen ist für die komplexe Ätiologie und Progression der Parodontitis von großer Bedeutung. Daher galt es in vorliegender Studie die Vorhersagbarkeit für klinisch stabile Ergebnisse nach Scaling und Root Planing anhand von zwölf Mikroorganismen des subgingivalen Biofilms zu evaluieren. Derjenige Marker sollte herausgefiltert werden, der eine Vorhersage auf das Therapieergebnis nach SRP liefert. Anhand der Ergebnisse konnte Porphyromonas gingivalis als wichtigster prognostischer Marker dieser Studie ausfindig gemacht werden. Ein positiver Nachweis auf das Vorkommen der Spezies vor Scaling und Root Planing sowie drei Monate danach, könnte eine Prognose für das Therapieergebnis liefern. Hierbei ist es wichtig, dass das Vorhandensein von Leitpathogenen aufgedeckt wird.:1. Einleitung 1.1 Epidemiologie und Ätiopathogenese 1.2 Parodontitis und parodontopathogene Mikroorganismen 1.3 Parodontaltherapie 2. Aufgabenstellung / Ziel der Studie 3. Material und Methoden 3.1 Durchführung klinischer Befunderhebung 3.2 Patientenauswahl und Studiendesign 3.3 Mikrobiologische Analyse 3.4 Gruppenzuordnung 3.5 Statistische Auswertung 4. Ergebnisse 5. Diskussion 6. Zusammenfassung der Arbeit 7. Literaturverzeichnis 8. Anlagen 9. Erklärung über die eigenständige Abfassung 10. Lebenslauf 11. Publikationen 12. Danksagung