Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / George C. Stewart / Fusobacterium necrophorum, a gram negative anaerobe and an opportunistic pathogen, causes necrotic infections in humans and animals. Two subspecies of F. necrophorum, subsp. necrophorum and subsp. funduliforme are described. Leukotoxin (Lkt), a secreted protein encoded by a tricistronic operon (lktBAC), is the major virulence factor of F. necrophorum. The concentration of Lkt produced by subsp. necrophorum is higher than that of subsp. funduliforme. Quantitative-PCR was used to determine the relative expression of lktA by the two subspecies of bovine origin. The mRNA transcript of lktA was detectable in early-log phase of growth in subsp. necrophorum, whereas in subsp. funduliforme, the lktA transcript was detected only in the mid-log phase. Q-PCR analysis revealed that subsp. necrophorum had 20-fold more lktA transcript than subsp. funduliforme. The amount of lktA transcript declined by late-log phase in both subspecies; but lktA mRNA levels in subsp. necrophorum was 8-fold higher than in subsp. funduliforme. Leukotoxin protein stability assays showed the Lkt to be stable in both subspecies despite the decrease in the concentration of the protein during late-log phase. The subspecies identity of human F. necrophorum strains and whether they possess lktA and leukotoxin activity are not known. Human clinical isolates (n = 4) of F. necrophorum were identified as subsp. funduliforme based on 16S rRNA sequence and absence of hemagglutinin gene. Four human strains had the lkt promoter, lktB, and lktC similar to that of subsp. funduliforme. One strain had full length lktA, while other three strains exhibited considerable heterogeneity. All four strains secreted Lkt that was toxic to human leukocytes. Fusobacterium equinum, formerly F. necrophorum, is a newly recognized species. It is associated with infections of the respiratory tract in horses. Little is known about the virulence factors of F. equinum. Southern hybridization revealed that F. equinum strains had lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The toxicity of culture supernatants of isolates to equine leukocytes was variable. Our data indicate that F. equinum isolates possess lktA gene and exhibit leukotoxin activity. The importance of leukotoxin as a virulence factor in human and equine fusobacterial infections needs to be investigated.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-13T12:33:03Z No. of bitstreams: 1 jobalvesdesouzafilho.pdf: 5002481 bytes, checksum: 64848c1386c2e939797e2ac86f5eddd4 (MD5) / 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) / 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 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / 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. / 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.

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. / 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.

Indiana University-Purdue University Indianapolis (IUPUI) / 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.

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.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Sanjeev K. Narayanan / Fusobacterium necrophorum is a Gram-negative, anaerobic, and rod-shaped to pleomorphic bacterium. It is frequently associated with necrotic infections of animals and humans. It is a major bovine pathogen and causes hepatic abscesses, foot rot, and necrotic laryngitis (calf-diphtheria). Liver abscesses in feedlot cattle and foot rot in beef and dairy cattle are of significant economic importance to the cattle industry. Fusobacterium necrophorum is classified into two subspecies, subsp. necrophorum and subsp. funduliforme. The subsp. necrophorum is more virulent and isolated more frequently from bovine hepatic abscesses than subsp. funduliforme. Outer membrane proteins (OMPs) of Gram-negative bacteria play an important role in their adhesion to host eukaryotic cells and hence, help in the establishment of infection and disease. Our objectives were to characterize OMPs of the two subspecies of F. necrophorum and assess their role in adhesion to bovine cells. Electrophoretic separation of extracted OMPs of subsp. necrophorum showed a total of 19 bands. Four bands of 38, 40, 60 and 74 kDa were more prominent than others. The OMPs of subsp. funduliforme showed a total of 20 proteins bands, of which, five were prominent (37.5, 58, 70, 140 and 150 kDa). The 40 kDa band was prominent in subsp. necrophorum while 37.5 kDa band was prominent in subsp. funduliforme. The human strains of F. necrophorum subsp. funduliforme had more heterogeneous banding patterns than the bovine strains of subsp. funduliforme. The role of OMPs in adhesion was studied using bovine endothelial cell line (EJG cells). A significant decrease in the attachment of subsp. necrophorum and subsp. funduliforme to bovine endothelial cell line (EJG cells) was observed when the cell line was preincubated with the OMPs of each subspecies. Treatment of the bacterial cells with trypsin also decreased their binding. In addition, when each subspecies was incubated with the polyclonal antibody produced against their OMPs before adding them to endothelial cells, there was a significant reduction in the bacterial attachment and the inhibition was subspecies specific. A 40 kDa OMP of subsp. necrophorum was identified that binds to the bovine endothelial cells with high affinity. The protein when preincubated with the endothelial cells, lead to a significant decrease in the bacterial binding to the endothelial cells. The N-terminal sequencing of the protein indicated similarity to FomA, an outer membrane protein of Fusobacterium nucleatum, an oral pathogen of humans. In summary, OMPs of F. necrophorum subsp. necrophorum and subsp. funduliforme differ from each other and they play a significant role in binding to bovine endothelial cells. We identified a 40 kDa OMP in subsp. necrophorum that binds to the bovine endothelial cells with high affinity and have a potential role as adhesin.

Master of Science / Department of Animal Sciences and Industry / Evan C. Titgemeyer / Seven ruminally cannulated heifers approximately 225 kg initial BW were used in a 7 × 4 Youden square design to determine the effects of different levels of limonene on ruminal Fusobacterium necrophorum populations. Treatments included: 1) control, 2) limonene at10 mg/kg diet DM, 3) limonene at 20 mg/kg diet DM, 4) limonene at 40 mg/kg diet DM, 5) limonene at 80 mg/kg diet DM, 6) CRINA-L (a blend of essential oil components) at 180 mg/kg diet DM, 7) tylosin at 12 mg/kg diet DM. Each period included 11 d with 10 d washouts between periods. Samples were collected on d 0 (before treatment initiation), 4, 7, and 10 for measuring F. necrophorum by most probable number (MPN) method using selective culture medium. Results indicate that CRINA-L (P = 0.52) and tylosin (P = 0.19) did not affect ruminal F. necrophorum populations. Limonene linearly decreased (P = 0.03) F. necrophorum populations, and the optimal dietary concentration for limonene was 40 mg/kg DM. Limonene did not affect ruminal degradation rate of lysine, NH3 concentration, or VFA profiles in ruminal fluid. Limonene was useful for reducing ruminal concentrations of F. necrophorum. It may have potential to control liver abscesses, although further research will be needed to assess the effect of limonene under feedlot conditions.

Orientador: Ana Elizabete Silva / Coorientador: David J. Hughes / Banca: Marcelo Lima Ribeiro / Banca: Rui Manuel Vieira Reis / Banca: Débora Aparecida Pires de Campos Zuccari / Banca: Marilia De Freitas Calmon Saiki / 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... / 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 ... / Doutor

Magister Chirurgiae Dentium (MChD) / There is an absence of both dental services and systematic planning to meet the oral health needs of the Black* population ~f greater Cape Town. Little epidemiological data exists upon which such planning can be based. This study describes the prevalence and treatment need related to tooth decay and periodontal disease ofaXhosa-speaking* squatter community on the outskirts of Cape Town. An age and sex stratified sample of 290 adults attending the SACLA clinic in Crossroads were examined. Examiner variability was measured by a percentage intra-examiner agreement for the DMFT of 95% and for the CPITN 84%. Cohen's kappa statistic, for tooth-specific caries detection errors was k = 0.877. The mean DMFT was 11.8 and varied little with sex or age below 55 years. After this age, the DMFT climbs steeply due largely to the rapid increase in the M value (missing teeth). The results show that for every tooth needing to be extracted, two teeth per subject required a restoration. Only three subjects already had some restorations. Periodontal health was reflected by a high prevalence of calculus (TN2 = 99%; MNS = 5.2) for the whole sample. Deep pockets were detected in 13% of those aged between 15 and 29 years, but only at a relatively low intensity (MNS = 0.1). This prevalence reached a high 60% for those aged between 45 and 64 years (MNS = 1.7). All subjects require oral hygiene instruction and gross scaling in at least four sextants, according to CPITN criteria. In conclus~on it is noted that there is a shortage of relevant epidemiological information necessary to the planning of oral health services to improve the oral health of the Xhosa-speaking community in the Western Cape. Caries prevalence rates are already high in young adults and a high tooth mortality rate and an absence of fillings, suggests that extraction is the only form of treatment made available to this community. The absence of appropriate prevention strategies such as water fluoridation is reflected in these results. The existence of small amounts of severe periodontal disease in young adults is of concern. The high prevalence of mild (and preventable) periodontal disease, seems to reflect a low awareness of the condition and/or a lack of resources to control it. It is no coincidence that such poor oral health was observed in this, a poor, peri-urban squatter community. This study, serves as a sad reminder of the maldistribution of oral health and socia-economic resources in South Africa. The socia-economic and political character of this community is reflected by the epidemiological picture of oral health observed in the study. It is clear that further data must be collected, especially a clear assessment of community-expressed needs. Active planning must take place urgently to integrate oral health with Primary Health Care to rectify the serious misuse and maldistribution of oral health resources required to improve the oral health of this population.

Systematic characterization of microbes in several tumors including colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) has revealed the presence of multiple species of intracellular bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in co-culture of host epithelial cells and bacteria, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging oncomicrobe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a Gram-negative, anaerobic bacterium that is normally found within the oral cavity. However, its selective enrichment in CRC and PDAC tumors is correlated with poor clinical outcomes. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum infection of host cancer cells induced robust secretion of select cytokines that increased cancer cell migration, impacted cell seeding, and enhanced immune cell recruitment. In PDAC, we uncovered additional cytokines that were secreted from both normal and cancerous pancreatic cell lines upon infection with F. nucleatum that increased cancer cell proliferation and migration via paracrine and autocrine signaling, notably in the absence of immune cell participation. In order to examine the contribution of a hypoxic TME on infection dynamics, we used a multi-omics approach that combined RNA-seq and ChIP-seq of H3K27ac to determine epigenomic and transcriptomic alterations sustained within hypoxic CRC cells upon infection with F. nucleatum. Our findings revealed that F. nucleatum can subvert host cell recognition in hypoxia and can modulate the expression of multiple cancer-related genes to drive malignant transformation. Insights gained from this research will pave the way for future studies on the impact of the tumor microbiome in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer. / Doctor of Philosophy / Colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) are the second and third leading causes of cancer death in the United States, respectively. Recent systematic characterization of various tumor types revealed the presence of distinct bacteria within tumors. However, there is limited knowledge on how these bacteria colonize tumors, how they survive inside host cells, how they modulate host cell phenotypes, and if their elimination should complement cancer therapy. This is, in part, due to the lack of representative animal models, challenges in developing host cell-microbe co-culture models, and limited resolution of available analytical techniques to study host-microbial interactions. I have addressed these challenges by harnessing multiple technologies from microbiology, genetic engineering, tissue engineering, and microfluidics, in order to investigate the role of an emerging cancer-associated microbe, Fusobacterium nucleatum, in the tumor microenvironment (TME). F. nucleatum is a microbe commonly found within the oral cavity. However, clinical studies revealed that selective enrichment of F. nucleatum in CRC and PDAC tumors significantly correlated with poor prognosis. My work along with collaborators in the Verbridge, Slade, and Lu labs at Virginia Tech has revealed a multifactorial impact of F. nucleatum in influencing cancer progression. First, in CRC, we discovered that F. nucleatum invasion of host cancer cells induced the secretion of select proteins called cytokines that cells use to signal and communicate with each other. These cytokines directly stimulated the cell migration of host cancer cells which is usually associated with increased cancer aggressiveness. In PDAC, F. nucleatum infection induced the secretion of additional cytokines from both cancer cells and normal cells that, in addition to cell migration, impacted the proliferation of cancer cells, another feature of aggressive cancers. F. nucleatum usually thrives in a low oxygen environment that is prevalent in cancer tissue and hence, we examined how a low oxygen environment can influence infection dynamics using sequencing technologies that probe the genomic constitution within cells. Our findings revealed that F. nucleatum can escape recognition in low oxygen environments and can modulate the expression of multiple cancer-related programs within the cell to drive cancer progression. Insights gained from this research will pave the way for future studies on the impact of the tumor-associated microbes in cancer and will identify novel targets for therapy and clinical intervention to control bacteria-induced exacerbation of cancer.