Dissertations / Theses on the topic 'Fusobacterium nucleatum'

Foram determinadas as atividades proteolíticas do bacilo anaeróbio Fusobacterium nucleatum, presente em várias infecções oral e sistêmica humana. As reações foram otimizadas quanto a pH, temperatura, tempo de reação, volume da fonte de enzima e concentração de substrato. Foram utilizados os substratos sintéticos b-naftilamida (Cys-Na, Ser-Na, Leu-Na, Glu-Na, Lys-Na e BANA), o carboxi-L-tirosina p-nitrofeniléster (CTN) e o substrato natural azoalbumina. Reação do sobrenadante ocorreu apenas com o substrato Glu-Na. Nas células vivas, as reações ocorreram com Cys-Na, Ser-Na e Glu-Na; e com as células lisadas, com os substratos Glu-Na, Leu-Na e CTN. O pH ótimo variou de 6,0 a 7,5 em todas as reações, com excessão para o CTN (pH 13). A temperatura ótima oscilou entre 30 e 40°C. O tempo ótimo de reação foi 60 minutos, exceto para as células vivas com o Glu-Na (40 min), células lisadas (20 min) e o substrato CTN (80 min). Não houve atividade com a azoalbumina. A atividade enzimática foi avaliada por vários inibidores de protease, sendo detectada a presença de metalo, serina, cisteína e aspartato proteases.<br>The proteolytic activities of the anaerobe bacillus Fusobacterium nucleatum present in several oral and systemic human infections were determined. The reactions were optimized in their pH, temperature, reaction time, enzyme source and substrate volume. The synthetic substrates b-?naphthylamides (Cys-Na, Ser-Na, Leu-Na, Glu-Na, Lys-Na and BANA), carboxi-L-tyrosine p-nitrophenylester (CTN), and natural substrate azoalbumin were used. With supernatants reaction occurred only with Glu-Na. In the living cells, reacted with Cys-Na, Ser-Na and Glu-Na; and lysate cells with Glu-Na, Leu-Na and CTN. Optimal pH ranged from 6.0 to 7.5 in all the reaction, except for CTN (pH 13). Optimal temperature oscillated between 30 and 40ºC. The optimal reaction time was 60 minutes, except for Glu-Na with living cells (40 min), lysate cells (20 min), and CTN substrate (80 min). There was no activity with azoalbumin. The enzyme activity by several protease inhibitors was assessed and the presence of metallo, serine, cysteine and aspartato protease was detected.

Fusobacterium nucleatum é uma espécie bacteriana Gram-negativa, anaeróbia estrita e uma das espécies frequentemente encontradas nas infecções primárias do sistema de canais radiculares. Esta espécie tem grande importância na formação de biofilmes por ser uma ponte de união entre espécies que não são capazes de interagir. Os micro-organismos constituintes de biofilmes trocam material genético, aumentando a tolerancia dos mesmos e é quase impossível um isolado clínico ter os genes totalmente iguais à cepa padrão de coleções de cultura como da ATCC (American Type Culture Colection). O presente estudo investigou a espécie bacteriana anaeróbia Fusobacterium nucleatum isolada de canais radiculares, comparando-a com sua cepa de referência. Foi feito a comparação da suscetibilidade microbiana in vitro por meio de cultura microbiológica pelo método do E-test, com as cepas em crescimento planctônico e em biofilme. Também foi definido um protocolo de Purificação de RNA para esta espécie em ambas as condições de crescimento. As cepas clínicas de F. nucelatum foram isoladas por meio da cultura microbiológica de 23 pacientes que apresentavam dentes com infecção primária e periodontite apical visível em radiografia. Foi feito isolamento e identificação da espécie por série bioquímica com testes comerciais (Sistema Api, Bio-Meriéux, França) e PCR convencional, sendo no total 4 isolados clínicos investigados. Foi verificada a suscetibilidade antimicrobiana dos seguintes antibióticos: Amoxicilina, Amoxicilina + ácido clavulânico, Ampicilina, Azitromicina, Clindamicina, Eritromicina e Metronidazol. O protocolo para purificação de RNA foi feito com microesferas de zircônia, dispositivo bead beater, kit comercial RNeasy (Qiagen) e transcrição para DNA complementar (cDNA). A qualidade da purificação foi testada quanto a sua capacidade de amplificação pela reação em cadeia da polimerase em tempo real (qPCR) utilizando primer para o gene 16s RNA específico para F. nucelatum. Todas as cepas testadas foram 100% suscetíveis a Amoxicilina + ácido clavulânico, Ampicilina, Azitromicina, Clindamicina e Metronidazol. Ambos os tipos de crescimento bacteriano demonstraram resistência somente à eritromicina. O protocolo proposto para a purificação do RNA de F. nucelatum dos isolados em crescimento planctônico e em biofilme foi eficaz. A média do rendimento do RNA das amostras para as bactérias em crescimento planctônico foi de 514,2 ng/&#x3BC;L (DP ± 397,7) e para as amostras em biofilme foi de 377,1 ng/&#x3BC;L (DP± 144,1). Os valores encontrados sugerem uma boa qualidade de RNA, livre de contaminação por proteínas. Todas as cepas de Fusobacterium nucleatum isoladas de canais radiculares, assim como a cepa ATCC foram suscetíveis aos antibióticos testados, com exceção do antibiótico eritromicina em ambos os tipos de crescimento bacteriano. As bactérias em biofilme apresentaram aumento na tolerância frente aos agentes antimicrobianos, com diferença estatística. O protocolo estabelecido para a purificação do RNA de cepas de Fusobacterium nucleatum cultivadas em fase planctônica e em biofilmes teve êxito com amplificação por qPCR.<br>Fusobacterium nucleatum is a Gram-negative bacterial species, strict anaerobic and one of the species often found in primary infection of the root canal system. This species has great importance in biofilm formation to be a union bridge between species which are not able to act alone. The constituent microorganisms of the biofilm exchange each tother genetic material, increasing the strength of them. It is almost impossible for a clinical isolate have genes totally equal to a standard strain, such as strains of culture collections like ATCC (American Type Culture Collection). The present study investigated anaerobic bacterial species Fusobacterium nucleatum isolated from root canals, comparing them to the ATCC strain. The microbial in vitro susceptibility of biofilm and planktonic growth of the strains was compared by means of microbiological culture and the E-test method, with the antibiotics Amoxicillin, Amoxicillin + clavulanic acid, Ampicillin, Azithromycin, Clindamycin, Eritromycin and Metronidazole.. Also, a RNA Purification protocol for the strains under the same growth conditions was defined. Clinical isolates were obtained by microbiological samples of patients with teeth with pulp necrosis and apical periodontitis visible on radiographs. The species isolation and identification were performed using commercial biochemical tests (Sistema Api, Bio-Meriéux, France) and conventional PCR, obtaining four clinical isolates. The protocol for RNA purification was done with zirconia beads, bead beater device and commercial kit RNeasy (Qiagen) and transcribed into complementary DNA (cDNA). The quality of purification was tested for its ability of amplification by real time polymerase chain reaction (qPCR) using primer for the gene 16s RNA specific for F. nucleatum. All tested trains were 100% susceptible to amoxicillin + clavulanic acid, ampicillin, azithromycin, clindamycin and metronidazole. Both types of bacterial growth showed resistance to Erythromycin. Bacteria in biofilm showed a decrease in susceptibility to all antibiotics, but without statistical difference. The protocol proposed for the purification of RNA of F. nucleatum was effective, in the planktonic and the biofilm growth. The average yield of RNA samples for bacteria in planktonic growth was 514.2 ng /&#x3BC;L (SD ± 397.7) and the samples in biofilm was 377.1 ng / &#x3BC;L (SD ± 144.1). These found values suggest a good quality of RNA, free of protein contamination. The established protocol for the purification of the RNA of the Fusobacterium nucleatum strains, grown in biofilm and planktonic phase, had successfully amplified by qPCR.

Fusobacterium nucleatum is a Gram-negative bacterium that serves as a bridging organism in polymicrobial biofilms within the oral cavity. Although the bacterium is abundant in healthy gingival tissue, recent studies have found that F. nucleatum is associated with a wide-spectrum of human diseases which include periodontal disease, preterm birth, endocarditis, colorectal cancer, and pancreatic cancer. Previous studies of F. nucleatum virulence have uncovered two surface adhesins, Fap2 and FadA, that interact with the surface of human cells; however, the study of new virulence factors was previously limited as there was no gene deletion system available to functionally analyze F. nucleatum proteins. Interestingly, F. nucleatum has a diverse landscape of structurally unique surface adhesins called Type 5c secreted trimeric autotransporter adhesins (TAAs), which are a family of proteins that are historically known for their contributions to bacterial pathogenesis. This dissertation encompasses the use of recombinant protein expression systems and newly developed gene deletion technology to provide a foundational understanding of the contribution of Type 5c secreted proteins in F. nucleatum pathogenesis. Our results show that the presence of TAAs on the surface of F. nucleatum contribute to the bacterium's ability to bind and invade human cells, establishing the need to characterize other F. nucleatum surface proteins. Additionally, our studies analyzed the proinflammatory landscape induced by F. nucleatum through the identification of specific cytokines that are being secreted during in vitro infections of human cells. Cytokine signaling is a critical aspect of the host cell immune response as it promotes the recruitment of immune cells to the site of infection for efficient clearance of bacterial pathogens. While it has been well established that F. nucleatum modulates the secretion of IL-8, our studies identified that the bacterium also promotes the secretion of CXCL1, which is an important signaling protein that promotes tumor metastases. Overall, the work provided in this dissertation has delivered the initial characterization of TAAs in F. nucleatum virulence, a framework for future studies of Type 5c secreted proteins in Fusobacterium pathogenesis, and the role of Fap2 and FadA in promoting pro-inflammatory and pro-metastatic signaling from colorectal cancer cells.<br>Master of Science in Life Sciences

A cavidade bucal é um habitat microbiano complexo que apresenta mais de 500 espécies bacterianas como componentes da microbiota. A saúde periodontal está estabelecida quando há equilíbrio entre os microrganismos patogênicos e o hospedeiro. O digluconato de clorexidina é um dos antimicrobianos bucais mais utilizados, no entanto, essa substância tem sido associada a alguns efeitos colaterais indesejáveis. Os óleos de copaíba e de melaleuca tem sido estudados como importantes fitoterápicos, devido aos seus diversos efeitos, entre eles ação antibacteriana. Partindo-se do princípio de que o óleo copaíba e de melaleuca possuem atividade antimicrobiana e de que não há dados suficientes na literatura utilizando esses fitoterápicos sobre Porphyromonas gingivalis e Fusobacterium nucleatum, foram preparados testes de Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM) das bactérias Fusobacterium nucleatum (ATCC 25586) e Porphyromonas gingivalis (ATCC 3327) frente ao digluconato de clorexidina e aos óleos provindos de Copaifera officinalis e de Melaleuca alternifólia. Realizaram-se ainda testes para determinação de Concentração Subinibitória (CS) e ensaios para determinar a capacidade de autoagregação e coagregação dessas bactérias expostas às concentrações subinibitórias das soluções testadas. Como controles foram utilizados apenas meio de cultura e meio de cultura acrescido de Tween 80. Todos os óleos utilizados tiveram sua composição analisada por cromatografia gasosa acoplada à espectrometria de massa. O óleo de melaleuca, após identificação de sua composição, apresentou, respectivamente, os seguintes constituintes em maiores concentrações: terpin-4-ol, -terpineno, -terpineno, terpinoleno e 1,8-cineol. O óleo de copaíba apresentou como principais constituintes, respectivamente, trans-cariofileno, germacreno B, -humuleno, germacreno D e -copaeno. Os resultados obtidos como CIM para F.nucleatum foram semelhantes à CBM em todas as soluções testadas. Para a bactéria P.gingivalis, todas as soluções testadas inibiram o crescimento bacteriano, contudo, os resultados obtidos durante a determinação da CBM demonstraram que o óleo de copaíba foi bacteriostático. Todas as soluções testadas inibiram o processo de autoagregação e apenas o óleo de copaíba foi eficiente na inibição do processo de coagregação entre F.nucleatum e P.gingivalis. Esses dados sugerem que as três soluções testadas apresentam relevantes mudanças no desenvolvimento normal de P.gingivalis e F.nucleatum, bem como influenciam no processo de autoagregação de F.nucleatum. O óleo de copaíba demonstrou ter uma notável propriedade de inibir o processo de coagregação entre as bactérias testadas neste estudo.<br>The oral cavity is a complex microbial habitat that has more than 500 bacterial species as components of the microbiota. Periodontal health is established when there is equilibrium between pathogens and host. The chlorhexidine digluconate is one of the most commonly used oral antibiotics, however, this substance has been associated with some undesirable side effects. Copaiba and melaleuca oils have been studied as important herbal medicines because of their effects, including antibacterial action. Based on the principle that the copaiba oil and tea tree have an antimicrobial activity and that is no sufficient data in the literature using these herbal medicines against Porphyromonas gingivalis and Fusobacterium nucleatum, Minimum Inhibitory Concentration (MIC) and minimum bactericidal concentration (MBC) tests of Fusobacterium nucleatum (ATCC 25586) and Porphyromonas gingivalis (ATCC 3327) related to chlorhexidine digluconate and oils coming from Copaifera officinalis and Melaleuca alternifolia, were prepared. Assays were performed to determine the subinibitory concentration and the capacity of those bacteria to autoaggregation and coaggregation when exposed to subinibitory concentrations, previously tested. Medium and medium added Tween 80 were used as a control. All oils used had their composition analyzed by gas chromatography-mass spectrometry. The tea tree oil mainly chemical compounds were identified as terpin-4-ol, -terpinen, -terpinen, terpinolene and 1,8-cineole while copaiba oil presented as its main constituents trans-caryophyllene, germacrene B, -humulene, germacrene D and -copaene. The MIC results for F.nucleatum were similar to the CBM data in all solutions. For the bacterium P. gingivalis, all solutions tested inhibited bacterial growth, however, the results obtained during the determination of CBM showed that the copaiba oil was bacteriostatic. All solutions tested inhibited the autoaggregation process but only copaiba oil was effective in inhibiting the coaggregation between F.nucleatum and P. gingivalis. These data suggest that all the solutions tested in this study have relevant changes in the normal development of P.gingivalis and F.nucleatum as well in the influence of the autoaggregation process of F.nucleatum yet Copaiba oil also demonstrated to have remarkable properties to change coaggregation between the bacteria used in this study.

Orientador: Ana Lia Anbinder<br>Coorientadora: Liliana Scorzoni<br>Banca: Cristiane Yumi Koga Ito<br>Banca: Victor Angelo Martins Montalli<br>Resumo: A doença periodontal, afecção crônica inflamatória multifatorial, está entre as principais doenças bucais que afetam a população mundial. Entre as bactérias associadas à periodontite, estão Fusobacterium nucleatum e Aggregactibacter actinomycetemcomitans. Novas terapias adjuntas ao tratamento convencional têm sido propostas para a doença periodontal, entre elas o uso de probióticos. Porém, seu uso não está isento de riscos, e uma alternativa para minimizá-los é inativar os micro-organismos, mantendo suas propriedades benéficas, o que ocorre com os chamados paraprobióticos. Desse modo, são objetivos deste estudo avaliar os efeitos antimicrobianos de L. reuteri vivo, inativado pelo calor e seus produtos sobre F. nucleatum, A. actinomycetemcomitans e sobre as bactérias comensais, Streptococcus mitis e Streptococcus salivarius, além de estudar os efeitos da interação das preparações e periodontopatógenos em modelo de invertebrado. A atividade antimicrobiana in vitro foi avaliada associando-se as bactérias patogênicas ou comensais a L. reuteri vivo, inativado ou sobrenadante. Após interação, as bactérias foram cultivadas em meio seletivo para contagem de unidades formadoras de colônias (UFC). No estudo em Galleria mellonella, após a infecção com as bactérias patogênicas e as preparações de L. reuteri, foi avaliada a curva de sobrevivência e densidade hemocitária. Os dados foram analisados com o teste estatístico apropriado, ao nível de 5%. Após interação bacteriana in vitro, S. sa... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Periodontal disease, a chronic multifactorial inflammatory disease, is among the major oral diseases that affects the worldwide population. Among the bacteria associated with periodontitis are Fusobacterium nucleatum and Aggregactibacter actinomycetemcomitans. New therapies have been proposed for periodontal disease as adjunct to conventional treatment, including the use of probiotics. However, their use isn't risk-free and an alternative to that is the inactivation of the microorganisms, maintaining their beneficial properties, which occurs with the paraprobiotics. Thus, the objective of this study is to evaluate the antimicrobial effects of living and heat-killed L. reuteri, and its products on F. nucleatum, A. actinomycetemcomitans and on the commensal bacteria, Streptococcus mitis and Streptococcus salivarius, as well as to study the interaction of the preparations and periodontopathogens in an invertebrate model. In vitro antimicrobial activity was evaluated by associating the pathogenic or commensal bacteria to live, heat killed or L. reuteri supernatant. After interaction, the bacteria were cultured in a selective medium for colony-forming unit (CFU) count. In the study with Galleria mellonella, after infection with pathogenic bacteria and L. reuteri preparations, the survival curve and hemocyte density were evaluated. The data were analyzed with the appropriate statistical test at the 5% level. After bacterial interaction in vitro, S. salivarius reduced the number of ... (Complete abstract click electronic access below)<br>Mestre

Aggregatibacter actinomycetemcomitans e Fusobacterium nucleatum são microrganismos gram-negativos presentes nos processos inflamatórios e nas diferentes formas da doença periodontal. Ambos formam parte da microbiota residente da cavidade bucal humana podendo levar ao desenvolvimento de infecções endógenas ou exógenas. Neste estudo, as avaliações qualitativa, quantitativa e genotípica de A. actinomycetemcomitans e F. nucleatum isolados de pacientes com gengivite, periodontite crônica e indivíduos sadios foram realizadas. Biofilmes subgengivais de 70 pacientes com gengivite, 75 com periodontite crônica e 95 indivíduos saudáveis foram avaliados. A. actinomycetemcomitans foi isolado em 2 (2,8%) pacientes com gengivite, 4 (5,3%) com periodontite e 5 (5,3%) sadios; e F. nucleatum em 13 (18,6%) pacientes com gengivite, 20 (26,6%) com periodontite crônica e 19 (20%) sadios. Ambos os microrganismos foram isolados em 5 (7,1%) pacientes com gengivite, 9 (12%) com periodontite crônica e 3 (3,15%) sadios. Por PCR, os DNA de A. actinomycetemcomitans foram detectados em 23 (32,8%) pacientes com gengivite, 20 (26,6%) com periodontite crônica e 38 (40%) indivíduos saudáveis; e de F. nucleatum em 17 (24,3%) pacientes com gengivite, 11 (14,6%) com periodontite e 19 (20%) sadios. Em associação esses microrganismos foram detectados em 23 (32,8%) pacientes com gengivite, 40 (53,3%) com periodontite crônica e 17 (17,8%) sadios. A. actinomycetemcomitans isolados de pacientes com gengivite pertenceram aos biotipos I, II, IV, V e X, e aos sorotipos a, c, e e. Em pacientes com periodontite foram encontrados os biotipos II, VI e X, e os sorotipos a, b, e c, sendo o sorotipo c o mais predominante (80%); e em indivíduos sadios os biotipos II e X, e os sorotipos b e c. Os valores quantitativos de A. actinomycetemcomitans para os três grupos analisados variaram em número de cópias de 0 a 1,14 x 108, e de F. nucleatum de 0 a 3,98 x 106. Os resultados obtidos por AP-PCR mostram a heterogeneidade dos isolados de A. actinomycetemcomitans e de F. nucleatum nos diferentes grupos clínicos de pacientes avaliados. Esses resultados comparativos poderão ser levados em consideração pelos clínicos para melhor direcionar o tratamento da doença periodontal, colaborando de forma efetiva para o seu monitoramento.<br>Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are gram-negative microorganisms observed in inflammatory processes and different forms of periodontal disease. Both are part of the human oral resident microbiota which may cause endogenous or exogenous infections. In this study, a qualitative, quantitative and genotypic analysis of A. actinomycetemcomitans and F. nucleatum isolated from patients with gingivitis, chronic periodontitis and healthy subjects were determined. Subgingival biofilms of 70 patients with gingivitis, 75 with chronic periodontitis and 95 healthy subjects were evaluated. A. actinomycetemcomitans was isolated in 2 (2,8%) patients with gingivitis, 4 (5,3%) with periodontitis and 5 (5,3%) healthy individuals; and F. nucleatum in 13 (18,6%) patients with gingivitis, 20 (26,6%) with chronic periodontitis and 19 (20%) healthy. Both microorganisms were identified in 5 (7,1%) patients with gingivitis, 9 (12%) with chronic periodontitis and 3 (3,15%) healthy. By PCR, DNA of A. actinomycetemcomitans were detected in 23 (32,8%) patients with gingivitis, 20 (26,6%) with chronic periodontitis and 38 (40%) healthy individuals; and F. nucleatum 17 (24,3%) patients with gingivitis, 11 (14,6%) with periodontitis and 19 (20%) healthy. In association, microorganisms were detected in 23 (32,8%) patients with gingivitis, 40 (53,3%) with chronic periodontitis and 17 (17,8%) healthy. A. actinomycetemcomitans isolated from patients with gingivitis belonged to biotype I, II, IV, V, X, and serotypes a, c and e. In patients with periodontitis biotypes II, VI and X and serotypes a, b, and c were found and serotype c was the most predominant (80%); and healthy individuals biotypes II and X, and serotypes b and c. Quantitative values for A. actinomycetemcomitans in the three patients groups were ranged from 0 to 1.14 x 108 and F. nucleatum 0 to 3.98 x 106. The results of this study by AP-PCR showed the heterogeneity of A. actinomycetemcomitans and F. nucleatum in the different clinical status. These comparative results can be considered by dentists for the treatment of periodontal disease and its effective monitoring.

Orientador: Ana Cláudia Pavarina<br>Resumo: O objetivo deste estudo foi avaliar a eficácia da terapia fotodinâmica antimicrobiana associada (aPDT) ao metronidazol (MTZ) em biofilmes periodontopatogênicos. Para tal finalidade, foram realizadas as seguintes etapas: (1) determinação do tempo de adesão (24 e 48 horas) e formação de biofilme mono e duo-espécie (3, 5 e 7 dias) de Fusobacterium nucleatum (NCTC 11326) e Porphyromonas gingivalis (ATCC 33277); (2) aplicação da aPDT mediada por PDZ associada ao MTZ em biofilmes mono-espécie de F. nucleatum e P. gingivalis. Foram avaliadas diferentes concentrações do PDZ (50, 75 e 100 mg/L) e dose de luz de 50 J/cm2 (660nm). Após a aplicação da aPDT, os biofilmes foram incubados com diferentes concentrações do MTZ (MIC, 50x MIC e 100x MIC) por 24 horas. Os grupos controles positivos (L-F-) não receberam fotossensibilizador e não foram iluminados. A viabilidade dos microrganismos após os tratamentos foi avaliada por meio da contagem de UFC/ml. Os resultados demonstraram que o período de adesão de 24 horas, seguido de 5 dias de formação de biofilme foi satisfatório para a obtenção de biofilmes maduros mono-espécie. Para F. nucleatum, os resultados demonstraram que aPDT 75 mg/mL associado com MTZ 100x MIC e aPDT 100 mg/L associado com MTZ nas concentrações de 50x MIC e 100x MIC reduziu significativamente o número de UFC/mL, 2,99; 2,9 e 3,94 Log10 respectivamente. Para P. gingivalis, a redução mais significativa de UFC/mL foi obtida quando a associação de aPDT 100 mg/L e MTZ 100x MIC ... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: The aim of this study was to evaluate the efficacy of metronidazole (MTZ) associated antimicrobial photodynamic therapy (aPDT) on periodontopathogenic biofilms. For this purpose, the following steps were performed: (1) determination of adhesion period (24 and 48 hours) and single and duo species biofilm formation (3, 5 and 7 days) of Fusobacterium nucleatum (NCTC 11326) and Porphyromonas gingivalis (ATCC 33277); (2) Photodithazine ® (PDZ)- mediated aPDT in association with MTZ in single-specie biofilms of F. nucleatum and P. gingivalis. Different concentrations of PDZ (50, 75 e 100 mg/L) and light dose of 50 J / cm2 (660nm) were evaluated. After application of aPDT, the biofilms were incubated with different concentrations of MTZ (MIC, 50x MIC and 100x MIC) for 24 hours. Positive control groups (L-F-) received no photosensitizer and were also not illuminated. The viability of the microorganisms after the treatments was evaluated by counting CFU/ml. The results demonstrated that the 24 hours adhesion period followed by 5 days of biofilm formation was satisfactory for obtaining a mature biofilm in single-specie. For F. nucleatum, the results demonstrated that 75 mg/L aPDT associated with MTZ 100x and 100 mg/mL aPDT associated with MTZ at 50x MIC and 100x MIC concentrations significantly reduced the number of CFU/mL, 2.99; 2.9 and 3.94 Log10 respectively. For P. gingivalis, the greatest reduction of CFU/mL was obtained when the association of aPDT 100 mg/L and MTZ 100x MIC was p... (Complete abstract click electronic access below)<br>Doutor

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-13T12:33:03Z No. of bitstreams: 1 jobalvesdesouzafilho.pdf: 5002481 bytes, checksum: 64848c1386c2e939797e2ac86f5eddd4 (MD5)<br>Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-13T12:46:33Z (GMT) No. of bitstreams: 1 jobalvesdesouzafilho.pdf: 5002481 bytes, checksum: 64848c1386c2e939797e2ac86f5eddd4 (MD5)<br>Made available in DSpace on 2016-09-13T12:46:33Z (GMT). No. of bitstreams: 1 jobalvesdesouzafilho.pdf: 5002481 bytes, checksum: 64848c1386c2e939797e2ac86f5eddd4 (MD5) Previous issue date: 2011-11-04<br>FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais<br>Concentrações subinibitórias de antimicrobianos (CSI), frequentemente decorrentes de antibioticoterapia, podem resultar em alterações na biologia bacteriana, com implicações no seu potencial agressor. Esse efeito tem considerável importância para as bactérias da microbiota residente, em especial para Fusobacterium nucleatum, um dos mais proeminentes anaeróbios residentes em humanos. Os objetivos deste trabalho foram analisar os efeitos de concentrações subinibitórias (CSI) de antimicrobianos em características morfológicas, bioquímicas, fisiológicas e moleculares de F. nucleatum. A partir da linhagem F. nucleatum ATCC 25586 (denominada FnPAR), foram obtidas 14 linhagens selecionadas por 10 cultivos sucessivos em CSI de ampicilina (FnAMP+), ampicilina/sulbactam (FnAMS+), clindamicina (FnCLI+), cloranfenicol (FnCLO+), levofloxacina (FnLEV+), metronidazol (FnMET+) e piperacilina/tazobactam (FnPTZ+) e, subsequente, 10 cultivos na ausência das mesmas drogas. Foram avaliados o perfil de susceptibilidade aos antimicrobianos, a morfologia bacteriana, o perfil bioquímico, a formação de biofilme e o custo de fitness. Também foram realizados genotipagem e análise dos perfis protéicos. Para avaliação global das alterações fenotípicas e genotípicas foram obtidas matrizes de similaridade. O perfil de susceptibilidade mostrou sensibilidade diminuída para a maioria das linhagens derivadas, mesmo após o cultivo sem drogas. Alterações morfológicas e de complexidade celular foram observadas, principalmente nas linhagens cultivadas em CSI de β-lactâmicos (FnAMP+, FnAMS+ e FnPTZ+), que também expressaram capacidade diminuída para formação de biofilme. Contudo, a morfologia regular e a habilidade de formação de biofilme foram retomadas após o cultivo sem droga. As linhagens FnCLI, FnCLO, FnLEV e FnMET não apresentaram alterações morfológicas evidentes, porém, foi observado aumento na formação de biofilme, com destaque para FnCLI+. As linhagens FnMET+ e FnCLI+ apresentaram um alto custo de fitness. Foram observadas alterações no padrão de metabolismo de carboidratos e na atividade de enzimas microbianas. Comparado com a FnPAR, várias proteínas (de 4.5 a 240 kDa) foram positivamente ou negativamente reguladas nas linhagens derivadas. Observou-se polimorfismo no DNA em todas as linhagens derivadas. As matrizes de similaridade não mostraram relações entre o padrão de polimorfismo de DNA e as outras características. Entretanto, existe uma tendência de que as alterações bioquímicas estejam relacionadas com alterações nos perfis protéicos. CSI de antimicrobianos podem, de fato, induzir alterações em F. nucleatum, com reflexo direto na sua biologia. Estes resultados alertam para o risco de antibioticoterapia inadequada, que podem ter sérias implicações para a microbiologia clinica e doenças infecciosas e, ainda, interferir com a relação bactéria-hospedeiro.<br>Subinibitory Concentrations (SIC) of antimicrobials, often arising from antibioticotherapy, may result in alterations in bacterial biology with implications for its potential aggressor. This effect has considerable importance for the bacteria of resident microbiota, especially for Fusobacterium nucleatum, one of the most prominent anaerobes in humans. Our aim was to analyze the effects of antimicrobials SIC in morphological, biochemical, physiological and molecular characteristics of F. nucleatum. From the strain F. nucleatum ATCC 25586 (FnWLD) were obtained 14 strains selected by 10 successive culture on SIC of ampicillin (FnAMP+), ampicillin/sulbactam (FnAMS+), clindamycin (FnCLI+), chloramphenicol (FnCLO+), levofloxacin (FnLEV+), metronidazole (FnMET+) and piperacillin/tazobactam (FnPTZ+) and subsequent 10 cultures in the absence of the same drugs. We evaluated the antimicrobial susceptibility patterns, bacterial morphology, biochemical profile, biofilm formation and the fitness cost. We also performed genotyping and analysis of protein profiles. For the global evaluation of phenotypic and genotypic alterations, similarity matrices were obtained. The antimicrobial susceptibility patterns showed decreased sensitivity to most of derived strains, even after culture without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SIC of β-lactam (FnAMP+, FnAMS+ and FnPTZ+), which also expressed decreased ability to biofilm formation. However, the regular morphology and the ability to biofilm formation were restored after culture without drug. The strains FnCLI, FnCLO, FnLEV and FnMET showed no apparent morphological changes, however, there was an increase in biofilm formation, especially for FnCLI+. The strains FnMET+ and FnCLI+ had a high fitness cost. Changes were observed in the carbohydrate metabolism patterns and activity of microbial enzymes. Compared with the FnWLD, several proteins (from 4.5 to 240 kDa) were positively or negatively regulated in the derived strains. It was observed polymorphism in the DNA in all derived strains. The similarity matrices showed no relationship between the DNA polymorphism patterns and other features. However, there is a tendency that the biochemical changes to be related to alterations in protein profiles. SIC of antimicrobials may, indeed, to induce alterations in F. nucleatum with direct impact on its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for the clinical microbiology and infectious diseases and also to interfere with the bacteria-host relationship.

The aim of this study was evaluate the gene expression of proinflammatory (RANKL, TNF- e IFN-) and regulatory (TGF- e IL-10) cytokines in response to experimental infection in Balb/c mice molars by microorganisms Fusobacterium nucleatum (ATCC 10953) and Enterococcus faecalis (ATCC 19433) in single and bi association. The animals were sacrificed after 10 and 20 days, the periapical tissues were collected and the PCR real time dosed the cytokine expression. It was observed that the single infection with F. nucleatum induced high expression, on the 10º day, of RANKL and TNF- and this response modulation was due to IL-10. E. faecalis caused high expression of IFN- at 20º day, but this modulation in RANKL e TNF-expression was independent of IL-10 e TGF-. The bi association (F. nucleatum e E. faecalis) encouraged high expression of RANKL, TNF- e IFN-, on the 10º day, would be modulated by TGF- increased expression. It can be concluded that, in this model, gene expression of proinflammatory cytokines prevails in earlier periods of periapical changes induction with concomitant decrease in the late periods, and this is due to the regulatory cytokines IL-10 e TGF- modulation, but in a way infection-specific.<br>O objetivo deste estudo foi avaliar a expressão gênica das citocinas pró-inflamatórias (RANKL, TNF- e IFN-) e regulatórias (TGF- e IL-10) em resposta à infecção experimental em molares de camundongos Balb/c com os microrganismos Fusobacterium nucleatum (ATCC 10953) e Enterococcus faecalis (ATCC 19433), em mono ou em bi-associação. Os animais foram sacrificados após 10 e 20 dias, e os tecidos periapicais coletados, dosando-se a presença das citocinas por PCR em tempo real. Observou-se que a mono-infecção com o F. nucleatum induziu, no 10º dia, alta expressão de RANKL e TNF- e que a modulação dessa resposta se deveu à IL-10. O E. faecalis provocou uma alta expressão de IFN- no 20º dia, mas essa modulação observada na expressão do RANKL e TNF- foi independente da IL-10 e TGF-. A bi-associação (F. nucleatum e E. faecalis) estimulou uma alta expressão das citocinas RANKL, TNF- e IFN-, no 10º dia, que seria modulada pela presença aumentada do TGF-. Pode-se concluir que, neste modelo, a expressão gênica das citocinas pró-inflamatórias prevalece nos períodos iniciais de indução das alterações periapicais com concomitante redução no período tardio e isto se deve à modulação promovida pelas citocinas regulatórias IL-10 e TGF-, mas de uma maneira infecção-específica.

Orientador: Ana Elizabete Silva<br>Coorientador: David J. Hughes<br>Banca: Marcelo Lima Ribeiro<br>Banca: Rui Manuel Vieira Reis<br>Banca: Débora Aparecida Pires de Campos Zuccari<br>Banca: Marilia De Freitas Calmon Saiki<br>Resumo: O câncer colorretal (CCR) está associado à patógenos como Fusobacterium nucleatum (Fn), que podem proporcionar um microambiente favorável para a tumorigênese em decorrência de alterações inflamatórias. Visando compreender o efeito de Fn no microambiente de lesões intestinais, avaliou-se a quantificação relativa (RQ) dessa bactéria em amostras de tecido de adenoma colorretal (ACR) e CCR, bem como sua correlação com a expressão de RNAm de mediadores inflamatórios (TLR2, TLR4, NFKB1, TNF, IL1B, IL6 e IL8) e de microRNAs (miRNAs) (miR-21-3p, miR-22-3p, miR-28-5p, miR-34a-5p e miR-135b-5p) envolvidos na resposta inflamatória e carcinogênese. Também delineou-se uma rede de interação miRNA:RNAm para auxiliar na compreensão da participação dos miRNAs no processo carcinogênico. Foram extraídos o DNA e o RNA de 27 amostras de tecido fresco de ACR e 43 de CCR e suas respectivas normais adjacentes. Os níveis de DNA de Fn e de RNAm dos mediadores inflamatórios e miRNAs foram quantificados por PCR quantitativo em tempo real (qPCR). Níveis elevados de Fn foram detectados em ACR (RQ=5,64) e mais acentuadamente em CCR (RQ=8,67). Observou-se expressão elevada do RNAm de TLR4, IL1B, IL8 e miR-135b em ACR, e de TLR2, IL1B, IL6, IL8, miR-34a e miR-135b em CCR em comparação com seus respectivos tecidos normais. Além disso, miR-22 e miR-28 foram encontrados com expressão reduzida em CCR. A expressão de RNAm de IL1B, IL6, IL8 e miR-22 foi positivamente correlacionada com a quantificação de Fn em CCR...<br>Abstract: Colorectal cancer (CRC) is associated with pathogens such as Fusobacterium nucleatum (Fn), which can provide a favorable microenvironment for tumorigenesis due to inflammatory changes. In order to understand the effect of Fn on the microenvironment of intestinal lesions, the relative quantification (RQ) of this bacterium was evaluated in samples of colorectal adenoma tissue (CRA) and CCR, as well as its correlation with the mRNA expression of inflammatory mediators (TLR2, TLR4, NFKB1, TNF, IL1B, IL6 e IL8), and microRNAs (miR-21-3p, miR-22-3p, miR-28-5p, miR-34a-5p e miR-135b-5p) involved in the inflammatory response and carcinogenesis. A miRNA: mRNA interaction network was also delineated to aid in the understanding of miRNA participation in the carcinogenic process. DNA and RNA were extracted from 27 fresh tissue samples of CRA and 43 of CRC and their respective adjacent normal ones. Fn and mRNA levels of the inflammatory mediators and miRNAs were quantified by quantitative real-time PCR (qPCR). Elevated levels of Fn were detected in CRA (RQ=5.64 and more markedly in CRC (RQ=8.67). High mRNA expression of TLR4, IL1B, IL8 and miR-135b in CRA, and of TLR2, IL1B, IL6, IL8, miR-34a and miR-135b in CRC were observed in comparison with their respective normal tissues. In addition, miR-22 and miR-28 were found downregulated in CRC. The mRNA expression of IL1B, IL6, IL8 and miR-22 was positively correlated with the quantification of Fn in CRC. The mRNA expression of miR-135b and ...<br>Doutor

Fusobacterium nucleatum is a Gram-negative, anaerobic bacterium that is a member of the human oral microbiota. Although it is a normal resident of the mouth, it is associated with a number of human diseases including: sepsis, inflammatory bowel disease (IBD), and colorectal cancer (CRC). Despite the important association of F. nucleatum with human health and disease, remarkably little is known about the molecular mechanisms underlying these infections. This knowledge gap can, in part, be attributed to a lack of molecular tools and experimental workflows. Creating the genetic tools to fill this knowledge gap is an imperative undertaking for the future development of treatments for diseases involving F. nucleatum. Previous work in the field has assigned functions to just a handful of Fusobacterium proteins (Fap2, FadA), and only two of those proteins have a well-defined role in the host-pathogen relationship. This dissertation contains work that lays the molecular and genetic foundation for future studies involving F. nucleatum by creating a unique gene deletion system while simultaneously establishing broadly applicable experimental workflows and molecular tools to study initial bacterial attachment and invasion processes crucial to Fusobacterium virulence. Marker-less gene deletions confirm the importance of Fap2 in host-cell attachment and invasion and suggest a lesser role in invasion for FadA, representing a significant revision to the Fusobacterium-host relationship. Also, our system allows for the overexpression and purification of virulence factors directly from Fusobacterium for the first time. This permits us to study aspects of Fusobacterium protein biology that were previously impossible and will provide further insights into the nature of Fusobacterium virulence. A custom suite of molecular tools was also developed to facilitate recombinant expression of these proteins in general laboratory settings using simple E. coli protein expression systems. We have used these new technologies to express and purify a number of potential Fusobacterium virulence factors as detailed in this dissertation. Also contained in this dissertation is the application of these breakthroughs to probe the function of a novel F. nucleatum outer membrane phospholipase, FplA. Phospholipases are important virulence factors in a number of well-studied human pathogens including Pseudomonas aeruginosa and Legionella pneumophila, where they interfere with host cellular signaling processes to increase intracellular bacterial survival. Our data show that FplA is a Class A1 phospholipase (PLA1) with robust catalytic activity capable of binding to and cleaving a number of lipid types. Additionally, we show that it has the ability to bind to important host signaling lipids including phosphatidylinositol 3, 5-bisphosphate and phosphatidylinositol 3, 4, 5-triphosphate. These data suggest FplA may play a role in manipulating the intracellular processes of host cells. Taken together, work in this dissertation provides tools and experimental frameworks for the future study of F. nucleatum pathogenesis while identifying and initially characterizing a new, potentially significant, virulence factor in FplA.<br>Doctor of Philosophy

Bakterier har alltid haft en stor inverkan på mänskligheten. För att diagnostisera bakteriella sjukdomar och behandla dem krävs identifiering av bakterien eller bakteriens relevanta egenskaper. Transportmedium har utvecklats för att hålla bakterierna vid liv från provtagning till analys. Syftet med studien var att utvärdera bakteriers viabilitet i det vätskebaserade mediet Copan Eswab jämfört med kolmedium (Copan swab). Bakterierna som ingick i studien var Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Niesseria gonorrhoeae och Fusobacterium nucleatum. Förutom jämförande mellan medierna genomfördes en jämförelse mellan Eswab i kyl och i rumstemperatur. Resultaten för H. influenzae (n=9) och N. gonorrhoeae (n=9) visade att Eswab gav lika många eller fler överlevande bakterier. Gällande F. nucleatum (n=9) visade resultaten att fler överlevde i Copan swab (Copanpinnar) de första 28 timmarna, men även att bakterien inte klarar mer än 28 timmar i rumstemperatur. Gällande S. pneumoniae (n=9) och C. jejuni (n=9) gav båda opålitliga svar. Ytterligare mätpunkter och studier krävs för att erhålla mer pålitliga resultat gällande hur länge bakterierna överlever i Eswab.<br>Bacteria have always had a great influence on mankind. To diagnose any bacterial disease and treat it it’s necessary to identify the bacteria or any relevant attributes. Different types of specimen transport have been developed to keep the bacteria alive from sampling until the analysis is performed. The purpose of the study was to evaluate the viability of bacteria in the fluid-based media Copan EswabTM compared with charcoal medium (Copan swab). Bacteria included in the study were: Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Niesseria gonorrhoeae and Fusobacterium nucleatum. The study also tried to compare how bacteria survived in Eswab which was refrigerated and in Eswab room temperature. Results for H. influenzae (n=9) and N. gonorrhoeae (n=9) showed that an equal amount or more of the bacteria survived in Eswab. More of F. nucleatum (n=9) survived in Copan swab (Copan swab sticks) for the first 28 hours, additionally they showed that the bacteria won’t survive more than 28 hours in room temperature. Regarding S. pneumoniae (n=9) and C. jejuni (n=9) both displayed unreliable results. Overall more measurements and additional studies are needed for more reliable results.

Dans ce projet de recherche, les propriétés antibactériennes et le potentiel anti-destruction tissulaire des polyphénols du thé vert, plus particulièrement l’épigallocatéchine-gallate (EGCG), ont été évalués. Dans un premier temps, l’effet antibactérien d’un extrait de thé vert et de l’EGCG a été déterminé sur trois bactéries parodontopathogènes d’importance, soit Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans et Fusobacterium nucleatum. Les concentrations minimales inhibitrices se sont avérées être entre 1000 et 62,5 µg/ml. De plus, des effets synergiques et additifs des polyphénols du thé vert ont été observés lorsqu’utilisés en association avec le métronidazole ou la tétracycline, des antibiotiques couramment utilisés en thérapie parodontale. Dans un deuxième temps, un modèle de co-culture constitué de fibroblastes gingivaux intégrés dans un gel de collagène et recouvert de macrophages a été utilisé pour évaluer la capacité des polyphénols du thé vert à inhiber la sécrétion de métalloprotéinases matricielles (MMPs), notamment les MMP-3, MMP-8 et MMP-9. Il a été démontré que les polyphénols du thé vert atténuent la sécrétion des MMPs par le modèle de co-culture. En conclusion, les résultats de cette étude ont apporté des évidences supportant le potentiel des polyphénols du thé vert en vue d’une utilisation préventive et thérapeutique pour le contrôle des maladies parodontales.

La parodontite est une infection polymicrobienne qui entraîne une inflammation du parodonte et provoque une destruction irréversible des tissus de soutien de la dent. Le but de cette étude était d'évaluer les concentrations des cytokines IL-18, IL-1β, IL-10, TNFα ainsi que celles des récepteurs Toll de type 2 (Toll-like receptor 2, TLR2) et 4 (TLR4) solubles dans le fluide créviculaire gingivale (FCG) et la salive d'une cohorte de patients atteints de parodontite. Ces concentrations ont été corrélées avec les paramètres cliniques de la parodontite et la quantité des bactéries Porphyromonas gingivalis, Treponema denticola et Fusobacterium nucleatum retrouvées chez les participants à l'étude. Les résultats obtenus ont révélé que les taux d'IL-18, d'IL-1β et de TLR4 dans le FCG sont plus élevés chez les sujets atteints de parodontite par rapport à ceux exempts de la maladie et que la sévérité de la maladie parodontale est associée à des concentrations élevées d'IL-1β et de TLR4 chez les sujets malades.

El objetivo del presente estudio fue determinar el efecto antibacteriano de la combinación de droga 3Mix y MP y de sus combinaciones alternativas contra Enterococcus faecalis y Fusobacterium nucleatum. Se empleó el método de dilución en caldo para determinar la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM) y método de difusión en agar modificado para determinar el efecto antibacteriano de los vehículos. Se emplearon dos cepas ATCC Enterococcus faecalis y Fusobacterium nucleatum, con la combinación de componentes de la droga 3Mix, 3Mix-Cefaclor(reemplazo de minociclina por cefaclor) y 3Mix-Amoxicilina(reemplazo de minociclina por cefaclor) en las siguientes concentraciones: 25µg/ml; 6,25µg/ml; 1,56µg/ml; 0,39µg/ml; 0,195µg/ml; 0,097µg/ml y macrogol(M), propilenglicol(P) y su asociación. La CIM para 3Mix, 3Mix-cefaclor fue 0,39µg/ml y 0,195µg/ml para 3Mix-Amoxicilina mientras que CBM fue >25µg/ml; 25µg/ml; 6,25µg/ml respectivamente sobre Enterococcus faecalis, para Fusobacterium nucleatum la CIM de 3Mix, 3Mix-Cefaclor fue 0,195µg/ml y ≤0,097µg/ml para 3Mix-Amoxicilina, los cuales coincidieron con CBM. Se obtuvo inhibición de crecimiento bacteriano por parte macrogol y propilenglicol+macrogol, sin embargo propilenglicol no formó halo de inhibición sobre Enterococcus faecalis ni Fusobacterium nucleatum. La Combinación de droga 3Mix, 3Mix-Cefaclor y 3Mix-Amoxicilina presentaron efectos antibacterianos contra Enterococcus faecalis y Fusobacterium nucleatum.<br>Tesis

In der vorliegenden Arbeit sollten die in der veterinärmedizinischen Literatur bisher diskutierten Ursachen für LJD bei Makropoden hinsichtlich ihrer tatsächlichen Bedeutung abgeklärt und die Eignung einer formalininaktivierten, bestandsspezifischen Adsorbatvakzine zur Prophylaxe von LJD getestet werden. Da LJD eine parodontale Erkrankung darstellt, wurden auch die für Entstehung einer humanen Parodontitis prädisponierenden Faktoren mit in die Untersuchung einbezogen. Es wurden Tupferproben zur bakteriologischen Untersuchung von insgesamt 15 gesunden und 11 an LJD erkrankten Kängurus entnommen. Dabei konnten gramnegative Anaerobier bei allen Tieren isoliert werden. Fusobacterium nucleatum wurde in 82% der von an LJD erkrankten und nur in 33% der von gesunden Tieren entnommenen Tupferproben nachgewiesen, womit sich ein signifikanter Zusammenhang (P &lt; 0,05) zwischen diesem Erreger und LJD ergab. Weitere überwiegend bei erkrankten Makropoden nachgewiesene Anaerobier stellten Prevotella oris/oralis (bei 73% der LJD-Fälle und bei 40% der gesunden Tiere) sowie Capnocytophaga spp. (45% vs. 13%) dar. Bacteroides spp. und Porphyromonas gingivalis wurden – wenn auch nur mit 3 bzw. 2 Nachweisen – ausschließlich bei kranken Tieren isoliert. Fusobacterium necrophorum wurde jeweils in 27% der Kängurus gefunden und spielte damit in dieser Studie keine Rolle für die Entstehung von LJD. In Übereinstimmung mit der Literatur konnten Moraxella spp. ausschließlich bei gesunden Makropoden isoliert werden. Vertreter dieser Gattung gehören damit offensichtlich zur normalen Maulflora der Kängurus. Für die Zoos in Halle und Leipzig wurde eine formalininaktivierte, bestandsspezifische Adsorbatvakzine gegen die bei einem an LJD erkrankten Känguru des jeweiligen Bestandes isolierten gramnegativen Anaerobier hergestellt. 7 Tiere (2 Rote Riesenkängurus, 5 Bennettwallabies) des Leipziger Zoos und 6 Bennettkängurus des Zoos in Halle wurden geimpft, wobei Auffrischungsimpfungen nach 4 bzw. 8 Wochen und nach 6 bzw. 12 Monaten erfolgten. Die spezifischen AK gegen das Prüfantigen Fusobacterium necrophorum wurden im SLA bestimmt. Es konnte keine Erhöhung der AK-Titer induziert werden und auch die Todesrate infolge von LJD senkte sich während des Untersuchungszeitraumes von 42 Monaten in den beiden Zoos nicht. Die höchsten AK-Level (1:512 bis 1:2048) ließen sich im Serum von natürlich infizierten und letztendlich tödlich erkrankten Bennettwallabies des Zoos in Hoyerswerda feststellen. Der Nachweis von AK-Titern im Serum von nicht geimpften Jungtieren lässt vermuten, dass AK via Kolostrum oder Dottersackplazenta auf die Jungtiere übertragen werden. Die Untersuchungen hinsichtlich der Fütterung zeigten, dass im Zoo Leipzig eine azidotische Stoffwechsellage induziert wurde, was sich bei den Leipziger Bennettkängurus in einem mit 7,53 signifikant niedrigeren Vormagen-pH-Wert im Vergleich zu den Hallenser und Auer Tieren (8,25 und 8,38) offenbarte. Dies schlug sich auch in erhöhten K-, Cholesterol- und &#61537;-Amylasewerten im Serum der Leipziger Wallabies nieder, womit gezeigt werden konnte, dass sich diese Parameter offenbar auch bei Makropoden zur Diagnostik einer chronischen Azidose eignen. Die Versorgung der Bennettkängurus in Magdeburg und Halle mit Ca und P war zwar nicht ausreichend, spiegelte sich aber nicht in veränderten Blutwerten dieser Mengenelemente wider. Die Aktivität der AP nimmt mit zunehmenden Alter ähnlich wie bei anderen Tierarten ab. Ihre negative Korrelation mit dem Alter der Tiere war dabei hochsignifikant (P &lt; 0,001, r = 0,77 bzw. 0,62). Beim direkten Vergleich gesunder mit an LJD erkrankten Tieren konnte weder eine Störung im Ca/P-Stoffwechsel noch eine Azidose in Verbindung zu LJD gebracht werden. In allen Zoos erfolgte eine Überversorgung mit Vitamin A, wobei die Bedarfswerte für Schaflämmer um das 3,5fache bis 41fache übertroffen wurden. Den Bedarfswerten am nächsten lagen die Versorgungswerte der Bennettkängurus vom TP Aue und der Östlichen Grauen Riesenkängurus vom Zoo Magdeburg, beides Bestände ohne LJD. Die ermittelten Retinolplasmakonzentrationen standen in keiner Beziehung zu den Vitamin-A-Gehalten im Futter, was darauf hindeutet, dass sich Retinolbestimmungen im Blutplasma ebenso wie bei anderen Tierarten nur in extremsten defizitären Situationen zur Einschätzung des Vitamin-A-Status eignen. Ob eine Hypervitaminose A für die Entstehung von LJD tatsächlich eine Rolle spielt, muss in zukünftigen Arbeiten unter Einbeziehung von Retinolesterbestimmungen in der Leber abgeklärt werden. Die Glukosewerte lagen mit 8,57 mmol/l (M. rufus) bzw. 6,51 mmol/l (M. rufogriseus) über den bisher bekannten Werten aus der Literatur. Da die Werte bei an LJD erkrankten Kängurus niedriger waren als bei gesunden Tieren, kann ein Diabetes mellitus als Ursache für LJD ausgeschlossen werden. Weder die Durchsicht von 144 Sektionsprotokollen noch die Bestimmung der Kreatinin- und Harnstoffkonzentration im Serum von an LJD erkrankten Tieren ließen einen Zusammenhang zwischen Erkrankungen der Nieren und LJD erkennen. 30 Tiere verendeten an LJD, wovon 20% auch an den Nieren erkrankt waren. Allerdings wiesen auch 16,7% der anderweitig gestorbenen Kängurus eine Nierenerkrankung auf. Die Serumkonzentrationen von Harnstoff bzw. Kreatinin der an LJD erkrankten Makropoden unterschieden sich nicht von den für die gesunden Roten Riesenkängurus (7,40 mmol bzw. 114 mmol/l) und Bennettwallabies (7,81 mmol/l bzw. 86 mmol/l) ermittelten Werten. Insgesamt 184 Sera von 107 Kängurus wurden auf AK gegen MaHV-1 und MaHV-2 mittels Neutralisationtest geprüft. Während 94,4% bzw. 97,2% der Roten Riesenkängurus serologisch positiv für MaHV-1 bzw. MaHV-2 waren, reagierten von den 71 überprüften Bennettkängurus nur 4 bzw. 3 Tiere positiv. Unter den Wallabies befanden sich auch 21 an LJD erkrankte Tiere, wovon lediglich 2 Tiere gegen MaHV-1 und 1 Tier gegen MaHV-2 eine Serokonversion zeigten. Die AK-Titer der Roten Riesenkängurus ließen keine Unterschiede zwischen gesunden und an LJD leidenden Tieren zu und die entnommenen Serumpaarproben von 5 zum Zeitpunkt der Blutentnahme an LJD leidenden Riesenkängurus zeigten kein einheitliches Verhalten im Sinne einer Serokonversion. Somit ließ sich der Verdacht, dass die Reaktivierung latenter Herpesinfektionen die Ursache für LJD sein könnte, nicht bestätigen. Im Ergebnis der vorliegenden Studie und im Zusammenhang mit den Angaben aus der Literatur stellt sich LJD primär als eine Infektion mit gramnegativen Anaerobiern dar, wovon Fusobacterium nucleatum, Bacteroides spp., Prophyromonas gingivalis und Fusobacterium necrophorum, Biovar A die größte Bedeutung haben dürften. Den Abschluss der Arbeit bilden Empfehlungen für die Haltung von Kängurus in zoologischen Einrichtungen und für die Therapie von LJD. Im Anhang finden sich Röntgenaufnahmen und Photographien von erkrankten und gesunden Makropoden<br>The aim of this thesis was the investigation of the aetiology of Lumpy Jaw Disease (LJD) in macropods concentrating specifically on the causes of the diseases in current veterinary medicine literature and to evaluate the use of a group-specific Al(OH)3-adjuvanted, formalin-inactivated whole-cell vaccine for the control of LJD in kangaroos kept in zoos. LJD is regarded as periodontal disease, therefore the risk factors for the development of human periodontitis were also included in this study. The oral flora from 15 healthy macropods and 11 animals suffering from LJD was isolated. At least one anaerobic gram-negative bacterial species was found in swabs of each macropod. The occurrence of Fusobacterium nucleatum was associated with LJD (P &lt; 0.05) by detecting this bacterium in 82% of the kangaroos suffering from LJD compared to only in 33% of the healthy animals. Prevotella oris/oralis and Capnocytophaga spp. were also predominantly found in diseased animals in comparison with healthy macropods (73% vs. 40% and 45% vs. 13% respectively). Bacteroides spp. and Porphyromonas gingivalis were isolated in only 3 and 2 kangaroos suffering from LJD, respectively. Contrary to previously published studies about LJD Fusobacterium necrophorum was not associated with LJD, as this anaerobe was detected in only 27% of the diseased as well as healthy macropods. Moraxella spp. seem to be a part of the normal oral flora of macropods and was found exclusively in healthy animals. 11 Red-necked Wallabies (Macropus rufogriseus) and 2 Red Kangaroos (Macropus rufus) were immunized with a group-specific Al(OH)3-adjuvanted, formalin-inactivated whole-cell vaccine containing previously in a kangaroo suffering from LJD isolated gramnegative anaerobs. The kangaroos were re-vaccinated after 1, 2, 6 and 12 months. Blood was collected from each animal at the same time. Antibodies were titrated against Fusobacterium necrophorum in an agglutination assay. The vaccine failed to induce increased levels of antibodies as well as to protect wallabies and kangaroos against LJD. As the highest antibody titres were detected in most severely diseased wallabies kept in the Hoyerswerda zoo, the protective role of the humoral immune response in LJD seems to be doubtful. The finding of detectable levels of antibodies in unvaccinated joeys supports the theory, that there is a transmission of antibodies from the mother to the offspring via colostrum or yolk-sac placenta. The diet of the Red-necked Wallabies in one zoo has induced an acidosis: The pH of the forestomach fluid collected by probang was lower in the animals of this zoo (pH = 7.53) than in the wallabies of two other zoos (pH = 8.25 and 8.38, respectively). Potassium, cholesterol and &#61537;-amylase were also higher in the blood of the animals of this zoo in comparison to the wallabies of the two other ones, hence these blood values seem to be helpful for the diagnosis of chronic acidosis in macropods. There was a calcium and phosphor deficiency in the nutrition of the wallabies in two zoos, but the blood concentration of both of these minerals was not changed. The activity of the ALP correlated negative with the age of the Bennett`s Wallabies (P &lt; 0.001, r = -.77 and r = -.62 respectively, depending on the instruments). All of the above mentioned blood values showed no differences between healthy and diseased animals and could so far not support the assumption, that an imbalance in Ca and P metabolism or an acidosis are important factors for LJD. The macropods of all investigated zoos were fed on a diet rich in vitamin A ranging from the 3.5 to the 41fold requirement for lambs. The vitamin A content of the diets for the 2 collections without a history of LJD was the lowest in this study. These results raised the point, that a hypervitaminosis A could be a more predisposing factor for LJD than a vitamin A deficiency. Due to the fact the plasma retinol concentration was independent from the vitamin A content of the diet and so not helpful in diagnosis of a vitamin A deficiency or toxicity, further investigations regarding the role of vitamin A in the aetiopathogenesis of LJD should include measurements of the liver tissue content of retinol esters. The glucose plasma concentration of the healthy Red Kangaroos (8.57 mmol/l) as well as the Red-necked Wallabies (6.51 mmol/l) was higher than previously published values for macropods, but also higher than the results of the diseased animals in this study. Therefore diabetes mellitus can be ruled out as an underlying factor for LJD. The analysis of 144 pathological records showed, that 30 animals died because of LJD, 20% of them and 16.7% of the other 114 macropods had a concurrent kidney disease. The urea and creatinin concentration in serum samples of healthy animals was not higher than the values of diseased animals. In conclusion, these results suggest kidney diseases are not important for the development of LJD. Altogether 184 sera collected from 107 kangaroos were tested for antibodies against MaHV-1 and MaHV-2 using a neutralisation assay. The prevalence of the MaHV-1- as well as MaHV-2-antibodies was high among the Red Kangaroos (94.4% and 97.2% respectively), but low among the Red-necked Wallabies (5.6% and 4.2% respectively). Seroconversion for MaHV-1 was seen in 2 out of 21 wallabies suffering from LJD, only 1 of these animals also had antibodies against MaHV-2. The antibody-titres against both of the macropodid herpes viruses also did not differ between Red Kangaroos with and without LJD, therefore a reactivation of a latent herpesvirus infection does not appear to be causative for LJD. In summary, considering the results of this study and previously published literature LJD is an infectious disease caused by gramnegative anaerobic bacteria with Fusobacterium nucleatum, Bacteroides spp., Porphyromonas gingivalis and Fusobacterium necrophorum subsp. necrophorum being of most significance. Recommendations concerning the keeping of kangaroos in captivity and the management of LJD are listed in the conclusion of this thesis. Some radiographs and photos of diseased and healthy kangaroos are attached

En las últimas décadas ha habido grandes avances en el conocimiento de la etiología y la patogenia de las lesiones pulpares y periapicales. Actualmente se sabe que los microorganismos juegan un papel principal en la enfermedad inflamatoria de la pulpa y tejidos periapicales. Antiguamente las técnicas para identificar los microorganismos no eran las mejores, especialmente para identificar los anaerobios, por lo que se creía que la presencia de microorganismos aerobios era mayor que la de anaerobios, ahora gracias a las nuevas técnicas de cultivo e identificación se puede afirmar que la flora en los conductos radiculares es polimicrobiana y que los microorganismos más prevalentes son lo anaerobios. Es por esto que el tratamiento a tiempo y de manera adecuada lleva a la eliminación o disminución de los microorganismos de la cámara pulpar y conductos radiculares infectados, para así poder lograr una limpieza y desinfección que culmine en una evolución satisfactoria. En nuestro país actualmente existe un alto índice de niños que padecen de caries dental y de estas un gran número de piezas dentarias ya presentan lesiones pulpares, una de ella es la necrosis pulpar, por esto en la presente investigación identificamos a uno de los principales causantes de esta, que es el Fusobacterium nucleatum y la sensibilidad que este presenta a algunos de los irrigantes más usados en la práctica odontológica, esto nos ayudará a poder planear un eficaz tratamiento, evitando la perdida de estas piezas dentarias.<br>Tesis

Objetivo: Evaluar in vitro el efecto antibacteriano del extracto metanólico de Uncaria Tomentosa (Uña de Gato) sobre cepas de Staphylococcus aureus (ATCC®25923™) y Fusobacterium nucleatun (ATCC®25586™) Materiales y métodos: El estudio fue de tipo experimental in vitro. Para la actividad antibacteriana se utilizó el método de Difusión en Agar y para determinar la Concentración Mínima Inhibitoria (CMI) se trabajó con el método de dilución en caldo en medio Brain Heart infusión Broth (BHI). Mientras que, la evaluación del efecto citotoxico del extracto se evaluó mediante el ensayo colorímetro del 3-(4.5-dimeltiltiazol-2-yl) -2.5-difeniltetrazolio bromuro (MTT) utilizando la línea celular Madin Darby Canine Kidney (MDCK). Resultados : El extracto metanólico de Uncaria Tomentosa tiene efecto antibacteriano frente a la cepa de Staphylococcus aureus, observándose halos de inhibición de 31.3 ± 2.66 mm. Se utilizó la Clorhexidina al 0.12% como control positivo, con resultados superiores de 59.1 mm ± 3.6 mm. En el caso de Fusobacterium nucleatun, no se evidenció efecto antibacteriano con el extracto metanólico de Uncaria Tomentosa. La CMI del extracto fue de 0.0016 mg/ml frente al Staphylococcus. La viabilidad celular del extracto se mantiene con valores altos (16 000 mg/ml), no se encontró efecto citotóxico. Conclusiones: Con la actividad antimicrobiana del extracto de Uncaria tomentosa, se sugiere seguir con más investigaciones que aporten al conocimiento científico y a partir de ello, se puedan desarrollar nuevos medicamentos antibacterianos en distintas presentaciones y así afrontar los diferentes problemas en la salud oral.<br>Objective: The aim of this study was to evaluate the antibacterial effect of the methanolic extracts of Uncaria tomentosa (Cat´s Claw) againts Staphylococcus aureus (ATCC® 25923™) y Fusobacterium nucleatun (ATCC® 25586™). Material and Methods: The study was of experimental type in vitro. For the antibacterial activity the diffusion method was used in agar and to determine the minimum inhibitory concentration (MIC), the broth dilution method was used in Brain Heart Broth infusion medium (BHI). While the evaluation of the cytotoxic effect of the extract was evaluated by the colorimeter assay of 3-(4,5-dimethythiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) using the Madin Darby Canine Kidney cell line (MDCK). Results: There is an antibacterial effect of the methanolic extract of Uncaria Tomentosa against Staphylococcus aureus, where inhibition halo of 31.3 ± 2.66 mm were observed. Clorhexidine 0.12% was used as a positive control with superior results of 59.1 mm ± 3.6 mm. On the other hand, there was not antibacterial effect against the Fusobacterium nucleatum strains. Then, the MIC of the extract was 0.0016 mg/ml against Staphylococcus aureus. The cell viability of the extract is maintained at high values (16,000 mg/ml), no cytotoxic effect was found. Conclusion: With the antimicrobial activity of the extract of Uncaria tomentosa, it´s suggested to continue with more research that contributes to scientific knowledge and from this, new antibacterial drugs can be developed in different presentations and thus face the different problems in oral health.<br>Tesis

Indiana University-Purdue University Indianapolis (IUPUI)<br>The primary goal of root canal treatment is to eliminate microbes from the root canal system, which is the cause of pulpal and periapical infections. Research shows that after a single visit of chemomechanical debridement microbes continue to remain within the canal system. An interappointment medication step has been advocated to maximize potential elimination of microbes within the root canal system. Previous studies have shown propolis to be antibacterial against common endodontic microbes. Studies have shown trends in different microbes being present in primary verus secondary endodontic infections. The majority of literature has focused on the efficacy of propolis against Enterococcus faecalis, a microbe commonly implicated in secondary endodontic 95 infections. The aim of this study was to demonstrate the efficacy of propolis against Fusobacterium nucleatum, a microbe commonly found in primary endodontic infections. This study aims to demonstrate the efficacy of propolis against a bacterium of primary endodontic infections (F. nucleatum) as well as against microbial biofilm to further support its potential use as a novel intracanal medicament. Dilutions of propolis were added to cultures of F. nucleatum in microtiter plates in a range from 390 μg/ml to 50,000 μg/ml. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and the minimum biofilm inhibitory concentration (MBIC) were determined. The MIC was determined of the total solution (biofilm+planktonic), planktonic, and biofilm (MBIC) after a 48-hour incubation period. The MBIC was determined by fixing biofilm to the wells and using crystal violet staining with spectrophotometry. The MBC was examined by plating solution from each concentration test well and reading the plates after 48 hours of incubation. The results show that the MIC of the total (biofilm+planktonic) appears to occur at a concentration of 6250 μg/ml. The MBIC appears to occur at the concentration of 1562.5 μg/ml. The planktonic results exhibit no significant difference in test and control wells. There was no MBC at any of the test concentrations. The propolis appears to inhibit bacterial growth and biofilm formation but does not appear to be bactericidal at any of the tested concentrations. The results of this study indicate that propolis has an MIC and MBIC when tested in vitro against F. nucleatum, although it does not show an MBC. There appears to be potentially significant interaction of propolis with biofilm as displayed by the lower concentration needed to exibit inhibitory effects on biofilm formation. This information 96 may contribute to the ability to develop a proper concentration of propolis to use in vivo when treating endodontic infections.

Fusobacterium nucleatum is a saccharolytic Gram-negative anaerobic organism belonging to the so-called ‘orange complex’ which is believed to play an important role in the microbial succession associated with the pathogenesis of periodontal disease. Its genome contains niche-specific genes shared with the other inhabitants of dental plaque, which may help to explain its ability to survive and grow in the changing environmental conditions experienced in the gingival sulcus during the progression from health to disease. The pH of the gingival sulcus increases during the development of periodontitis and is thought to occur by the metabolism of nutrients supplied by gingival crevicular fluid. Studies have shown that F. nucleatum is partly responsible for the rise in pH and have concluded that in comparison to other plaque inhabitants, F. nucleatum has the greatest ability to neutralise acidic environments. In common with a number of other oral bacteria, F. nucleatum has also been shown to produce intracellular polyglucose (IP) from simple sugars such as glucose, galactose and fructose. Its response and adaptation to stressful environmental conditions such as pH is unknown. The overall aim of this study was, therefore, to determine how F. nucleatum copes with environmental stresses induced by pH changes. F. nucleatum was grown by continuous culture in a chemically defined medium at a growth rate corresponding to those measured in vivo. The effect on protein expression, and IP synthesis was examined during steady-state growth at high (>7.2<7.8) or low pH (pH 6.4). The present study also investigated the response of F. nucleatum to growth at pH 8.2. It was found that the organism grew as a biofilm and this corresponded with an increase in cellular hydrophobicity and decreased IP levels. Optimal growth pH’s differed between the different sub-species used in this study. In response to pH stress, F. nucleatum changed its amino acid and glucose utilisation and increased IP synthesis at the expense of cell numbers. Pulsing the chemostat with glutamic acid or serine produced an increase in IP synthesis and the pattern of end-products observed was dependent upon the amino acid being fermented. The effect on IP synthesis in response to increased levels of exogenous fermentable amino acids was also compared during concomitant fructose or glucose fermentation. Growth media containing fermentable amino acids and supplemented with fructose produced higher cell numbers and non-detectable levels of IP compared to media containing glucose. The differential expression of cytoplasmic- and cell envelope-proteins induced by changes in pH were identified by two-dimensional gel electrophoresis. The results represent the first proteomic investigation of F. nucleatum. Twenty-two cytoplasmic proteins were found to have altered expression in response to external pH. At low (sub-optimal) pH, proteins associated with the generation of ATP and ammonia were up-regulated, the latter contributing to the alkalinisation of the gingival sulcus. Conversely, neutral to alkaline pH conditions led to the upregulation of enzymes involved in energy storage. The study also identified several proteins associated with iron limitation and fatty acid synthesis which might not otherwise have been identified as part of the pH-dependent response. In response to growth at pH 7.8, 14 cell envelope proteins were identified as having significantly altered expression. Down-regulated proteins included those associated with uptake of C4 di-carboxylates and phosphorus, a potential membrane protease and an enzyme associated with amino acid fermentation. The up-regulation of a transcriptional regulator linked to the repression of sugar metabolism was also reported along with proteins linked to the transport of iron. The periplasmic chaperone, peptidyl prolyl cis trans isomerase, which is responsible for the folding of outer membrane proteins, was also found to be up-regulated. In conclusion, the proteomic investigation of protein expression by F. nucleatum identified gene products which form part of the organism’s coordinated stress response to changes in environmental pH. In addition to these, the physiological based studies also presented help to explain the organism’s persistence during the transition from health to disease in vivo.<br>Thesis (Ph.D.) - University of Adelaide, Dental School, 2008

Fusobacterium nucleatum is a saccharolytic Gram-negative anaerobic organism belonging to the so-called ‘orange complex’ which is believed to play an important role in the microbial succession associated with the pathogenesis of periodontal disease. Its genome contains niche-specific genes shared with the other inhabitants of dental plaque, which may help to explain its ability to survive and grow in the changing environmental conditions experienced in the gingival sulcus during the progression from health to disease. The pH of the gingival sulcus increases during the development of periodontitis and is thought to occur by the metabolism of nutrients supplied by gingival crevicular fluid. Studies have shown that F. nucleatum is partly responsible for the rise in pH and have concluded that in comparison to other plaque inhabitants, F. nucleatum has the greatest ability to neutralise acidic environments. In common with a number of other oral bacteria, F. nucleatum has also been shown to produce intracellular polyglucose (IP) from simple sugars such as glucose, galactose and fructose. Its response and adaptation to stressful environmental conditions such as pH is unknown. The overall aim of this study was, therefore, to determine how F. nucleatum copes with environmental stresses induced by pH changes. F. nucleatum was grown by continuous culture in a chemically defined medium at a growth rate corresponding to those measured in vivo. The effect on protein expression, and IP synthesis was examined during steady-state growth at high (>7.2<7.8) or low pH (pH 6.4). The present study also investigated the response of F. nucleatum to growth at pH 8.2. It was found that the organism grew as a biofilm and this corresponded with an increase in cellular hydrophobicity and decreased IP levels. Optimal growth pH’s differed between the different sub-species used in this study. In response to pH stress, F. nucleatum changed its amino acid and glucose utilisation and increased IP synthesis at the expense of cell numbers. Pulsing the chemostat with glutamic acid or serine produced an increase in IP synthesis and the pattern of end-products observed was dependent upon the amino acid being fermented. The effect on IP synthesis in response to increased levels of exogenous fermentable amino acids was also compared during concomitant fructose or glucose fermentation. Growth media containing fermentable amino acids and supplemented with fructose produced higher cell numbers and non-detectable levels of IP compared to media containing glucose. The differential expression of cytoplasmic- and cell envelope-proteins induced by changes in pH were identified by two-dimensional gel electrophoresis. The results represent the first proteomic investigation of F. nucleatum. Twenty-two cytoplasmic proteins were found to have altered expression in response to external pH. At low (sub-optimal) pH, proteins associated with the generation of ATP and ammonia were up-regulated, the latter contributing to the alkalinisation of the gingival sulcus. Conversely, neutral to alkaline pH conditions led to the upregulation of enzymes involved in energy storage. The study also identified several proteins associated with iron limitation and fatty acid synthesis which might not otherwise have been identified as part of the pH-dependent response. In response to growth at pH 7.8, 14 cell envelope proteins were identified as having significantly altered expression. Down-regulated proteins included those associated with uptake of C4 di-carboxylates and phosphorus, a potential membrane protease and an enzyme associated with amino acid fermentation. The up-regulation of a transcriptional regulator linked to the repression of sugar metabolism was also reported along with proteins linked to the transport of iron. The periplasmic chaperone, peptidyl prolyl cis trans isomerase, which is responsible for the folding of outer membrane proteins, was also found to be up-regulated. In conclusion, the proteomic investigation of protein expression by F. nucleatum identified gene products which form part of the organism’s coordinated stress response to changes in environmental pH. In addition to these, the physiological based studies also presented help to explain the organism’s persistence during the transition from health to disease in vivo.<br>http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339503<br>Thesis (Ph.D.) - University of Adelaide, Dental School, 2008

Inflammatory Bowel Disease (IBD) is an umbrella term used to describe a group of chronic, relapsing/remitting disorders of the gastrointestinal tract (GIT). While the precise aetiology of IBD is unknown, it is believed to be a result of the interaction of genetics, the immune system and the enteric microbiota. Thus, the search for potentially pathogenic microbial residents of the GIT is a current research focus. Fusobacterium nucleatum (Fn) is a member of the normal human microflora, including the GIT and has a well-characterized role in periodontitis in the oral setting. We have determined that Fn can be frequently recovered from human intestinal biopsies and furthermore, there is a positive correlation between recovery of Fn and the IBD status of the host. Fn strains from IBD patients were more invasive in vitro than strains from healthy controls and also demonstrated the ability to survive and proliferate inside host cells. Furthermore, while Fn strains from both IBD patients and healthy controls were able to induce expression of the pro-inflammatory cytokine IL-8 in vitro, in comparison to strains from controls, Fn strains from IBD patients resulted in decreased levels of IL-8 protein outside the host cells, suggesting that these strains may utilize sophisticated tactics to promote their survival. Thus, differences in virulence determinants among strains may be key to understanding a potential role for Fn in IBD. Characterization of virulence mechanisms utilized by Fn isolates from IBD patients could define a potentially important aspect of microbe/host interactions in this devastating disease, and indicate future therapeutic targets.<br>Canadian Institutes of Health Research, Canadian Digestive Health Foundation, Crohn's and Colitis Foundation of Canada

Intrauterine infection with the oral commensal anaerobe Fusobacterium nucleatum has been associated with adverse pregnancy outcomes. We have previously established a mouse model to study the mechanism of hematogenous F. nucleatum leading to fetal and neonatal death. Here, we report that Toll-like Receptor 4 (TLR4) from the maternal rather than paternal, and endothelial rather than hematopoietic cells mediate placental inflammation, especially the production of the proinflammatory cytokine interleukin-1 beta. Downstream of TLR4, a spatiotemporal pattern of the transcription factor NF-kB activation was observed spreading from the decidual endothelium to the surrounding spongiotrophoblasts within the first six hours of infection. Maternal TRIF, an adaptor protein downstream of TLR4 pathway, but not NLRP3, a cytosolic signaling receptor that constitutes inflammasome complex, mediated the fetal and neonatal death. In an effort to find a prophylactic preventive method against the detrimental birth outcome induced by F. nucleatum placental infection, omega-3 fatty acids were tested for their anti-inflammatory properties. Omega-3 oil supplementation in pregnant mice inhibited the transcription and release of inflammatory cytokines, prevented fetal and neonatal death, and also suppressed the proliferation of F. nucleatum in the placenta. Moreover, omega-3 supplementation was shown to enhance neutrophil recruitment to the site of infection. However, omega-3 supplementation did not protect the pregnancy from Listeria monocytogenes infection in vivo, despite the in vitro results where inflammation induced by both Gram-negative and Gram-positive bacteria were suppressed by omega-3 fatty acids. This study presents the first direct evidence of maternal, rather than fetal, signal leading to adverse pregnancy outcome, and suggests an exciting therapeutic potential of dietary omega-3 fatty acids.

Fusobacterium nucleatum is a Gram negative anaerobic bacterium, frequently isolated from both healthy and diseased dental plaque and has been implicated in the aetiology of periodontal diseases. Studies have shown that this organism increases in number and proportion in diseased periodontal pockets suggesting that changes in the environment favour the growth of the organism. One of the physico-chemical changes that occurs in the diseased periodontal sulcus is an increased environmental pH, which may be as high as 8.5. In our laboratory, F. nucleatum subspecies polymorphum ATCC 10953 formed mono-culture biofilms at pH 8.2. The formation of biofilms in nature is a survival strategy for bacteria, often associated with altered physiology and increased virulence. The aim of this study was to investigate changes in F. nucleatum protein expression associated with these alkaline induced biofilms. A chemostat was used to produce F. nucleatum planktonic and biofilm cells grown at pH 7.4 and 8.2, respectively. The bacterial cells were separated into cell envelope and cytoplasmic fractions. The soluble proteins were extracted from each fraction and resolved by two-dimensional gel electrophoresis. Fifty five differentially expressed proteins were identified using mass spectrometry and database searching. These proteins were classified according to functional class, including metabolic, transport and stress proteins. One of the most interesting findings was the significant up-regulation of Fusobacterial Outer Membrane Protein A (FomA), a porin that is responsible for the coaggregation between F. nucleatum and periodontopathogens. It has been suggested that this protein may be a target for future vaccination against periodontal diseases. Other proteomic findings showed that transport proteins such as ATP binding cassette (ABC) transporter binding protein was down-regulated while tripartite ATP-independent (TRAP) transporter binding protein was enhanced in the alkaline environment. ABC transporters require ATP hydrolysis for solute transport. In contrast, TRAP transporter is ATP independent and co-transports protons into the cytoplasm, which may help maintain a neutral intracellular pH under alkaline conditions. The regulation of these transport proteins reflected the conservation of energy and maintenance of pH homeostasis in biofilm cells. In addition, F. nucleatum regulated key enzymes in energy-producing pathways. A combination of proteomic and other methods confirmed that biofilm cells increased their glucose uptake and storage as intracellular polyglucose in comparison to their planktonic counterparts. Furthermore, the increased energy requirement of these cells was associated with changes in metabolism resulting in both qualitative and quantitative shifts in acidic end-products. Bacterial biofilm formation can be triggered by adverse environmental conditions and the expression of stress proteins help to protect cellular functions. The stress proteins, GroEL and RecA, were up-regulated in biofilm cells and the expression of these proteins was verified using Western-blotting and quantitative real-time polymerase chain reaction techniques. In conclusion, this investigation successfully identified F. nucleatum proteins that were regulated in response to alkaline conditions, similar to those that are thought to exist in diseased periodontal pockets. The physiological adaptations observed, to the changed ecology, provide evidence at the molecular level supporting the key role of F. nucleatum in the establishment of mature dental plaque.<br>Thesis (Ph.D.) -- University of Adelaide, School of Dentistry, 2010

Three leaves of errata inserted before title page.<br>Bibliography: leaves 150-181.<br>xi, 181 leaves : ill. ; 30 cm.<br>Determines whether growing cells of F. nucleatum ATCC 25586 could actually utilise peptides to provide the energy-yielding amino acid residues. Overall shows the important role of aminopeptidase in the nutrition and survival of F. nucleatum.<br>Thesis (Ph.D.)--University of Adelaide, Dept. of Dentistry, 1998

Dental plaque forms sequentially, with Fusobacterium nucleatum facilitating the adhesion of pathogenic late colonizers. We hypothesize that a single-chain variable fragment (scFv) antibody library will enable the identification of F. nucleatum adhesins and help elucidate the molecular mechanisms of coaggregation between F. nucleatum and other bacteria. A 4X10^8 clones scFv phage display library was created using spleen RNA from a mouse immunized with F. nucleatum. The library was enriched by biopanning against F. nucleatum 6 times and 292 individual clones tested by ELISA reacted strongly to F. nucleatum. Sixty-two of those clones inhibited F. nucleatum coaggregation with Streptococcus sanguinus. Analysis of select clones revealed differences in coaggregation inhibition, recognition of outer membrane proteins, and BstOI restriction pattern. DNA sequencing showed 6 unique scFvs and of them 3 strongly inhibited interaction with 5 Streptococcus species. These scFvs recognize the outer membrane autotransporter protein RadD (Fn1526), as determined by mass spectrometry.<br>Farhan Khan placed second in the International Association for Dental Research/Unilever Hatton Competition in the Senior Basic Science Research Category representing Canada, while presenting the research contained in this dissertation. This international competition took place during the 90th General Session & Exhibition of the International Association for Dental Research in Iguaçu Falls, Brazil in June 2012.

In der vorliegenden Arbeit sollten die in der veterinärmedizinischen Literatur bisher diskutierten Ursachen für LJD bei Makropoden hinsichtlich ihrer tatsächlichen Bedeutung abgeklärt und die Eignung einer formalininaktivierten, bestandsspezifischen Adsorbatvakzine zur Prophylaxe von LJD getestet werden. Da LJD eine parodontale Erkrankung darstellt, wurden auch die für Entstehung einer humanen Parodontitis prädisponierenden Faktoren mit in die Untersuchung einbezogen. Es wurden Tupferproben zur bakteriologischen Untersuchung von insgesamt 15 gesunden und 11 an LJD erkrankten Kängurus entnommen. Dabei konnten gramnegative Anaerobier bei allen Tieren isoliert werden. Fusobacterium nucleatum wurde in 82% der von an LJD erkrankten und nur in 33% der von gesunden Tieren entnommenen Tupferproben nachgewiesen, womit sich ein signifikanter Zusammenhang (P < 0,05) zwischen diesem Erreger und LJD ergab. Weitere überwiegend bei erkrankten Makropoden nachgewiesene Anaerobier stellten Prevotella oris/oralis (bei 73% der LJD-Fälle und bei 40% der gesunden Tiere) sowie Capnocytophaga spp. (45% vs. 13%) dar. Bacteroides spp. und Porphyromonas gingivalis wurden – wenn auch nur mit 3 bzw. 2 Nachweisen – ausschließlich bei kranken Tieren isoliert. Fusobacterium necrophorum wurde jeweils in 27% der Kängurus gefunden und spielte damit in dieser Studie keine Rolle für die Entstehung von LJD. In Übereinstimmung mit der Literatur konnten Moraxella spp. ausschließlich bei gesunden Makropoden isoliert werden. Vertreter dieser Gattung gehören damit offensichtlich zur normalen Maulflora der Kängurus. Für die Zoos in Halle und Leipzig wurde eine formalininaktivierte, bestandsspezifische Adsorbatvakzine gegen die bei einem an LJD erkrankten Känguru des jeweiligen Bestandes isolierten gramnegativen Anaerobier hergestellt. 7 Tiere (2 Rote Riesenkängurus, 5 Bennettwallabies) des Leipziger Zoos und 6 Bennettkängurus des Zoos in Halle wurden geimpft, wobei Auffrischungsimpfungen nach 4 bzw. 8 Wochen und nach 6 bzw. 12 Monaten erfolgten. Die spezifischen AK gegen das Prüfantigen Fusobacterium necrophorum wurden im SLA bestimmt. Es konnte keine Erhöhung der AK-Titer induziert werden und auch die Todesrate infolge von LJD senkte sich während des Untersuchungszeitraumes von 42 Monaten in den beiden Zoos nicht. Die höchsten AK-Level (1:512 bis 1:2048) ließen sich im Serum von natürlich infizierten und letztendlich tödlich erkrankten Bennettwallabies des Zoos in Hoyerswerda feststellen. Der Nachweis von AK-Titern im Serum von nicht geimpften Jungtieren lässt vermuten, dass AK via Kolostrum oder Dottersackplazenta auf die Jungtiere übertragen werden. Die Untersuchungen hinsichtlich der Fütterung zeigten, dass im Zoo Leipzig eine azidotische Stoffwechsellage induziert wurde, was sich bei den Leipziger Bennettkängurus in einem mit 7,53 signifikant niedrigeren Vormagen-pH-Wert im Vergleich zu den Hallenser und Auer Tieren (8,25 und 8,38) offenbarte. Dies schlug sich auch in erhöhten K-, Cholesterol- und &#61537;-Amylasewerten im Serum der Leipziger Wallabies nieder, womit gezeigt werden konnte, dass sich diese Parameter offenbar auch bei Makropoden zur Diagnostik einer chronischen Azidose eignen. Die Versorgung der Bennettkängurus in Magdeburg und Halle mit Ca und P war zwar nicht ausreichend, spiegelte sich aber nicht in veränderten Blutwerten dieser Mengenelemente wider. Die Aktivität der AP nimmt mit zunehmenden Alter ähnlich wie bei anderen Tierarten ab. Ihre negative Korrelation mit dem Alter der Tiere war dabei hochsignifikant (P < 0,001, r = 0,77 bzw. 0,62). Beim direkten Vergleich gesunder mit an LJD erkrankten Tieren konnte weder eine Störung im Ca/P-Stoffwechsel noch eine Azidose in Verbindung zu LJD gebracht werden. In allen Zoos erfolgte eine Überversorgung mit Vitamin A, wobei die Bedarfswerte für Schaflämmer um das 3,5fache bis 41fache übertroffen wurden. Den Bedarfswerten am nächsten lagen die Versorgungswerte der Bennettkängurus vom TP Aue und der Östlichen Grauen Riesenkängurus vom Zoo Magdeburg, beides Bestände ohne LJD. Die ermittelten Retinolplasmakonzentrationen standen in keiner Beziehung zu den Vitamin-A-Gehalten im Futter, was darauf hindeutet, dass sich Retinolbestimmungen im Blutplasma ebenso wie bei anderen Tierarten nur in extremsten defizitären Situationen zur Einschätzung des Vitamin-A-Status eignen. Ob eine Hypervitaminose A für die Entstehung von LJD tatsächlich eine Rolle spielt, muss in zukünftigen Arbeiten unter Einbeziehung von Retinolesterbestimmungen in der Leber abgeklärt werden. Die Glukosewerte lagen mit 8,57 mmol/l (M. rufus) bzw. 6,51 mmol/l (M. rufogriseus) über den bisher bekannten Werten aus der Literatur. Da die Werte bei an LJD erkrankten Kängurus niedriger waren als bei gesunden Tieren, kann ein Diabetes mellitus als Ursache für LJD ausgeschlossen werden. Weder die Durchsicht von 144 Sektionsprotokollen noch die Bestimmung der Kreatinin- und Harnstoffkonzentration im Serum von an LJD erkrankten Tieren ließen einen Zusammenhang zwischen Erkrankungen der Nieren und LJD erkennen. 30 Tiere verendeten an LJD, wovon 20% auch an den Nieren erkrankt waren. Allerdings wiesen auch 16,7% der anderweitig gestorbenen Kängurus eine Nierenerkrankung auf. Die Serumkonzentrationen von Harnstoff bzw. Kreatinin der an LJD erkrankten Makropoden unterschieden sich nicht von den für die gesunden Roten Riesenkängurus (7,40 mmol bzw. 114 mmol/l) und Bennettwallabies (7,81 mmol/l bzw. 86 mmol/l) ermittelten Werten. Insgesamt 184 Sera von 107 Kängurus wurden auf AK gegen MaHV-1 und MaHV-2 mittels Neutralisationtest geprüft. Während 94,4% bzw. 97,2% der Roten Riesenkängurus serologisch positiv für MaHV-1 bzw. MaHV-2 waren, reagierten von den 71 überprüften Bennettkängurus nur 4 bzw. 3 Tiere positiv. Unter den Wallabies befanden sich auch 21 an LJD erkrankte Tiere, wovon lediglich 2 Tiere gegen MaHV-1 und 1 Tier gegen MaHV-2 eine Serokonversion zeigten. Die AK-Titer der Roten Riesenkängurus ließen keine Unterschiede zwischen gesunden und an LJD leidenden Tieren zu und die entnommenen Serumpaarproben von 5 zum Zeitpunkt der Blutentnahme an LJD leidenden Riesenkängurus zeigten kein einheitliches Verhalten im Sinne einer Serokonversion. Somit ließ sich der Verdacht, dass die Reaktivierung latenter Herpesinfektionen die Ursache für LJD sein könnte, nicht bestätigen. Im Ergebnis der vorliegenden Studie und im Zusammenhang mit den Angaben aus der Literatur stellt sich LJD primär als eine Infektion mit gramnegativen Anaerobiern dar, wovon Fusobacterium nucleatum, Bacteroides spp., Prophyromonas gingivalis und Fusobacterium necrophorum, Biovar A die größte Bedeutung haben dürften. Den Abschluss der Arbeit bilden Empfehlungen für die Haltung von Kängurus in zoologischen Einrichtungen und für die Therapie von LJD. Im Anhang finden sich Röntgenaufnahmen und Photographien von erkrankten und gesunden Makropoden.<br>The aim of this thesis was the investigation of the aetiology of Lumpy Jaw Disease (LJD) in macropods concentrating specifically on the causes of the diseases in current veterinary medicine literature and to evaluate the use of a group-specific Al(OH)3-adjuvanted, formalin-inactivated whole-cell vaccine for the control of LJD in kangaroos kept in zoos. LJD is regarded as periodontal disease, therefore the risk factors for the development of human periodontitis were also included in this study. The oral flora from 15 healthy macropods and 11 animals suffering from LJD was isolated. At least one anaerobic gram-negative bacterial species was found in swabs of each macropod. The occurrence of Fusobacterium nucleatum was associated with LJD (P < 0.05) by detecting this bacterium in 82% of the kangaroos suffering from LJD compared to only in 33% of the healthy animals. Prevotella oris/oralis and Capnocytophaga spp. were also predominantly found in diseased animals in comparison with healthy macropods (73% vs. 40% and 45% vs. 13% respectively). Bacteroides spp. and Porphyromonas gingivalis were isolated in only 3 and 2 kangaroos suffering from LJD, respectively. Contrary to previously published studies about LJD Fusobacterium necrophorum was not associated with LJD, as this anaerobe was detected in only 27% of the diseased as well as healthy macropods. Moraxella spp. seem to be a part of the normal oral flora of macropods and was found exclusively in healthy animals. 11 Red-necked Wallabies (Macropus rufogriseus) and 2 Red Kangaroos (Macropus rufus) were immunized with a group-specific Al(OH)3-adjuvanted, formalin-inactivated whole-cell vaccine containing previously in a kangaroo suffering from LJD isolated gramnegative anaerobs. The kangaroos were re-vaccinated after 1, 2, 6 and 12 months. Blood was collected from each animal at the same time. Antibodies were titrated against Fusobacterium necrophorum in an agglutination assay. The vaccine failed to induce increased levels of antibodies as well as to protect wallabies and kangaroos against LJD. As the highest antibody titres were detected in most severely diseased wallabies kept in the Hoyerswerda zoo, the protective role of the humoral immune response in LJD seems to be doubtful. The finding of detectable levels of antibodies in unvaccinated joeys supports the theory, that there is a transmission of antibodies from the mother to the offspring via colostrum or yolk-sac placenta. The diet of the Red-necked Wallabies in one zoo has induced an acidosis: The pH of the forestomach fluid collected by probang was lower in the animals of this zoo (pH = 7.53) than in the wallabies of two other zoos (pH = 8.25 and 8.38, respectively). Potassium, cholesterol and &#61537;-amylase were also higher in the blood of the animals of this zoo in comparison to the wallabies of the two other ones, hence these blood values seem to be helpful for the diagnosis of chronic acidosis in macropods. There was a calcium and phosphor deficiency in the nutrition of the wallabies in two zoos, but the blood concentration of both of these minerals was not changed. The activity of the ALP correlated negative with the age of the Bennett`s Wallabies (P < 0.001, r = -.77 and r = -.62 respectively, depending on the instruments). All of the above mentioned blood values showed no differences between healthy and diseased animals and could so far not support the assumption, that an imbalance in Ca and P metabolism or an acidosis are important factors for LJD. The macropods of all investigated zoos were fed on a diet rich in vitamin A ranging from the 3.5 to the 41fold requirement for lambs. The vitamin A content of the diets for the 2 collections without a history of LJD was the lowest in this study. These results raised the point, that a hypervitaminosis A could be a more predisposing factor for LJD than a vitamin A deficiency. Due to the fact the plasma retinol concentration was independent from the vitamin A content of the diet and so not helpful in diagnosis of a vitamin A deficiency or toxicity, further investigations regarding the role of vitamin A in the aetiopathogenesis of LJD should include measurements of the liver tissue content of retinol esters. The glucose plasma concentration of the healthy Red Kangaroos (8.57 mmol/l) as well as the Red-necked Wallabies (6.51 mmol/l) was higher than previously published values for macropods, but also higher than the results of the diseased animals in this study. Therefore diabetes mellitus can be ruled out as an underlying factor for LJD. The analysis of 144 pathological records showed, that 30 animals died because of LJD, 20% of them and 16.7% of the other 114 macropods had a concurrent kidney disease. The urea and creatinin concentration in serum samples of healthy animals was not higher than the values of diseased animals. In conclusion, these results suggest kidney diseases are not important for the development of LJD. Altogether 184 sera collected from 107 kangaroos were tested for antibodies against MaHV-1 and MaHV-2 using a neutralisation assay. The prevalence of the MaHV-1- as well as MaHV-2-antibodies was high among the Red Kangaroos (94.4% and 97.2% respectively), but low among the Red-necked Wallabies (5.6% and 4.2% respectively). Seroconversion for MaHV-1 was seen in 2 out of 21 wallabies suffering from LJD, only 1 of these animals also had antibodies against MaHV-2. The antibody-titres against both of the macropodid herpes viruses also did not differ between Red Kangaroos with and without LJD, therefore a reactivation of a latent herpesvirus infection does not appear to be causative for LJD. In summary, considering the results of this study and previously published literature LJD is an infectious disease caused by gramnegative anaerobic bacteria with Fusobacterium nucleatum, Bacteroides spp., Porphyromonas gingivalis and Fusobacterium necrophorum subsp. necrophorum being of most significance. Recommendations concerning the keeping of kangaroos in captivity and the management of LJD are listed in the conclusion of this thesis. Some radiographs and photos of diseased and healthy kangaroos are attached.

Yes<br>Infection is the major reason for guided tissue regeneration/guided bone regeneration (GTR/GBR) membrane failure in clinical application. In this work, we developed GTR/GBR membranes with localized drug delivery function to prevent infection by electrospinning of poly(ε-caprolactone) (PCL) and gelatin blended with metronidazole (MNA). Acetic acid (HAc) was introduced to improve the miscibility of PCL and gelatin to fabricate homogeneous hybrid nanofiber membranes. The effects of the addition of HAc and the MNA content (0, 1, 5, 10, 20, 30, and 40 wt.% of polymer) on the properties of the membranes were investigated. The membranes showed good mechanical properties, appropriate biodegradation rate and barrier function. The controlled and sustained release of MNA from the membranes significantly prevented the colonization of anaerobic bacteria. Cells could adhere to and proliferate on the membranes without cytotoxicity until the MNA content reached 30%. Subcutaneous implantation in rabbits for 8 months demonstrated that MNA-loaded membranes evoked a less severe inflammatory response depending on the dose of MNA than bare membranes. The biodegradation time of the membranes was appropriate for tissue regeneration. These results indicated the potential for using MNA-loaded PCL/gelatin electrospun membranes as anti-infective GTR/GBR membranes to optimize clinical application of GTR/GBR strategies.