Three leaves of errata inserted before title page. / Bibliography: leaves 150-181. / xi, 181 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / 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. / Thesis (Ph.D.)--University of Adelaide, Dept. of Dentistry, 1998

Fusobacterium are Gram-negative anaerobic bacteria that colonize a variety of eukaryotes including cattle and humans. In humans, Fusobacterium coordinates the central architecture of the oral biofilm by expressing an abundance of outer membrane adhesins that mediate bridging between early and late colonizing bacteria. While Fusobacterium are mostly considered commensal microorganisms, they can also become an opportunistic pathogen that spreads throughout the human body and promote the development of oral and extra-oral infections and diseases including colorectal cancer. Importantly for this work, many Fusobacterium species and strains are recalcitrant to genetic manipulation, the majority of which has led to hindrance in the study of their biology, molecular mechanisms, and pathogenesis. The genetic intractability of Fusobacterium is an obstacle for the development of future treatments for diseases associated with these anaerobic bacteria. Therefore, the creation of tools to enhance genome editing in target species is crucial to understand the molecular mechanisms driving Fusobacterium infections. This dissertation exploits innate and adaptive defense systems present in Fusobacterium for their use as molecular tools for genome editing. Accordingly, we first investigated restriction-modification systems with a focus on the role of DNA methyltransferases and endonucleases in host defense and genetic recalcitrance in several strains of Fusobacterium through bioinformatic and biochemical approaches. Altogether, over 15 DNA methyltransferases were characterized. Most notably, we identified and characterized two type II DNA methyltransferases that are capable of methylating plasmid DNA by treating with purified enzymes in-vitro and coexpression approaches in Escherichia coli strains, enabling an statiscally improved transformation efficiency via electroporation in F. nucleatum. Also contained in this dissertation is the first detailed description of CRISPR-Cas adaptive immunity systems present in Fusobacterium strains. Most of the discovered CRISPR-Cas systems in Fusobacterium belong to Class 1 systems. Nonetheless we identified Type II-A and Type VI-C Class 2 systems. The discovery of Cas9 and Cas13c effectors respectively from these systems will be crucial in the development of a new generation of genome-editing tools in Fusobacterium. The studies included in this dissertation provide the framework for overcoming Fusobacterium genetic recalcitrance by the implementation of host mimicking techniques. By utilizing restriction-modification system enzymes and the adaptive immunity CRISPR-Cas systems, we will gain a better understanding of how Fusobacterium modulates infections and diseases, and ultimately explore the potential of novel therapeutic treatments. / Doctor of Philosophy / The oral cavity has one of the most diverse and largest microbial populations, where microorganisms are capable of colonizing hard surfaces of the teeth and the soft tissues of the oral mucosa. A fundamental member of the oral microbiome is Fusobacterium, a Gram-negative bacterium which coordinates the oral biofilm formation by interacting with other microorganisms. In recent studies, Fusobacterium has been associated with oral and extra-oral infections and diseases including periodontitis, preterm birth, Lemiere syndrome, inflammatory bowel disease and colorectal cancer. Importantly, many Fusobacterium species and strains are challenging to study due to their inability to uptake exogenous DNA and lack of genetic tools, which has hindered the study of their biology, molecular mechanisms and pathogenesis. The challenges in the genetic manipulation of Fusobacterium present a significant obstacle for the development of future treatments for diseases associated with these bacteria. Therefore, the creation of tools to expand bacterial transformation of exogenous DNA and genome editing to more than just one Fusobacterium species is crucial to understand how Fusobacterium is causing these infections. This dissertation explores the presence and utilization of defense systems, which defend bacteria from phage attack, as an alternative to improve Fusobacterium genetics. Accordingly, we first studied a set of over 15 enzymes that recognize a specific DNA pattern and add a methyl group (DNA methyltransferases) to specific nucleotides in several strains of Fusobacterium. We discovered that two of these enzymes improve Fusobacterium's ability of importing and genomically incorporating exogenous DNA after an electric discharge permeabilizes the bacterial membrane. Furthermore, for the first time we have described the composition of CRISPR-Cas bacterial defense systems, that detect invading DNA from viruses and provide protection to Fusobacterium strains. These systems have previously been successfully used as genetic tools to achieve genome editing. Thus, their further characterization is warranted to create novel molecular tools in Fusobacterium. Altogether, these discoveries will lead to a better comprehension of Fusobacterium biology in infections and diseases, while exploring novel therapeutic strategies.

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/μL (DP ± 397,7) e para as amostras em biofilme foi de 377,1 ng/μL (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. / 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 /μL (SD ± 397.7) and the samples in biofilm was 377.1 ng / μL (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.

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. / 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 / Coorientadora: Liliana Scorzoni / Banca: Cristiane Yumi Koga Ito / Banca: Victor Angelo Martins Montalli / 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) / 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) / Mestre

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. / 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.

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. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339503 / Thesis (Ph.D.) - University of Adelaide, Dental School, 2008

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. / 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.

Master of Science / Department of Biomedical Sciences / Sanjeev K. Narayanan / Fusobacterium necrophorum is an anaerobic Gram-negative non spore forming rod shaped bacteria that is a normal inhabitant of the alimentary tract of humans and animals. Two subspecies of F. necrophorum have been recognized- subspecies necrophorum and subspecies funduliforme. Subspecies necrophorum is an opportunistic pathogen in animals causing diseases such as bovine hepatic abscesses and sheep foot rot while as subspecies funduliforme is linked with human oral and hepatic infections such as sore throats, Lemierre’s syndrome and hepatic abscesses. The pathogenic mechanisms of F. necrophorum are complex and are not well understood or defined. Several virulence factors such as leukotoxin, haemolysin, haemagglutinin and adhesin have been described. One of the most important factors in F. necrophorum bacterial pathogenesis is the adhesion of the bacteria to the host cell. The adhesion of the bacteria to the host cell helps it colonize the host tissue and this is followed by intracellular multiplication with dissemination to other tissues, which could ultimately lead to septicemia and death. Bacteria use adhesins which are proteins found in the outer membrane which help them bind with host receptors and this helps with the adhesion of the bacteria to the host cell. Not much is known about F. necrophorum adhesins. Here, we describe and characterize a novel adhesin.

The tumor microenvironment is a complex community consisting of neoplastic cells, surrounding stromal cells, a broad array of immune cells, and a microbiota. By sheer numbers, the microbiota has its greatest manifestation in colorectal cancer (CRC) because the colon contains up to 100 trillion bacteria, outnumbering human cells by a factor of 10 and encoding a gene-content that is 100-fold larger than that of the human genome. Indeed, previous studies using germ-free mice in a variety of genetic backgrounds have demonstrated that the microbiota can impact colorectal tumorigenesis. In addition, specific strains of enterotoxigenic bacteria have been shown to promote colitis-associated cancer in mice. Here, we explore the composition of the tissue-associated microbiota in human CRC and evaluate the role of tumor-enriched microbes in potentiating colorectal tumorigenesis in mice. Advances in DNA sequencing technology have fueled a renaissance in the microbiome field. Deep sequencing metagenomics enables rapid, culture-independent characterization of a microbial community. We present PathSeq, a highly scalable software tool that performs computational subtraction on high-throughput sequencing data to identify nonhuman nucleic acids. PathSeq makes it possible to analyze sequence datasets as large as human whole-genomes for the purpose of metagenomics and also to discover previously unsequenced microorganisms. We used PathSeq to characterize the composition of the microbiota in human CRCusing whole-genome sequencing on nine tumor/normal pairs and 16S rDNA sequencing on an additional 95 pairs. The genus Fusobacterium was highly enriched in tumors, while the Bacteroidetes and Firmicutes phyla were depleted.We show that in the \(Apc^{Min/+}\) mouse model of intestinal tumorigenesis, Fusobacterium nucleatum increases tumor multiplicity, selectively recruits tumor-infiltrating myeloid cells, and is associated with a pro-inflammatory expression signature that is shared with human fusobacteria-positive colorectal carcinomas. We find that Fusobacterium spp. are enriched in human colonic adenomas relative to surrounding tissues and fusobacterial abundance is increased in stool samples from patients with colorectal adenomas and carcinomas, compared to healthy subjects. Collectively, these data support that fusobacteria may be involved in early stages of intestinal tumorigenesis and, through recruitment of tumor-infiltrating immune cells, may generate a pro-inflammatory tissue microenvironment conducive to colorectal neoplasia progression.