Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-03-28T13:58:36Z No. of bitstreams: 1 texto completo.pdf: 1220110 bytes, checksum: 803118ec7eccfad2cba1645f25679903 (MD5) / Made available in DSpace on 2017-03-28T13:58:36Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1220110 bytes, checksum: 803118ec7eccfad2cba1645f25679903 (MD5) Previous issue date: 2016-12-19 / A carne de frango consiste em uma matriz ideal para a multiplicação de micro- organismos, e, portanto, é associada a numerosas infecções em humanos. Entre as bactérias que mais geram infecções intestinais, a partir do consumo de produtos avícolas, encontra-se a espécie Campylobacter jejuni. O uso indiscriminado de antibióticos na produção animal, pode contribuir na seleção de bactérias resistentes, que podem ser disseminadas durante a produção ou processamento dos alimentos. Desta forma, são necessárias novas alternativas para o controle desse patógeno, como a utilização de bacteriófagos. O objetivo deste trabalho foi isolar, caracterizar e avaliar o efeito de um coquetel de bacteriófagos de forma individual ou associado ao antibiótico enrofloxacina, no controle de C. jejuni, em frangos de corte. Foram isolados seis bacteriófagos (BC2, BC7, BC10, BC14, BC18 e BC19) a partir de fezes de frango coletadas em duas granjas avícolas na região de Viçosa, no Estado de Minas Gerais. Os bacteriófagos foram avaliados quanto à especificidade frente a diferentes cepas bacterianas. Cinco bacteriófagos foram específicos para C. jejuni, e um (bacteriófago BC14) além de apresentar ação lítico nas cepas de C. jejuni, apresentou atividade lítica frente a Salmonella Typhimuium (ATCC 14028) e Escherichia coli (CDC0111ab). A caracterização morfológica de dois bacteriófagos (BC7 e BC14) por meio de Microscopia Eletrônica de Transmissão (MET), evidenciou que os bacteriófagos apresentam cabeça icosáedrica e cauda de tamanho médio, indicando que provavelmente eles são vírus da ordem Caudovirales, família Myoviridae, e subfamília Eucampyvirinae. O coquetel de bacteriófagos foi afetado maiormente quando exposto a pH 2,5 (redução de 6,90) ciclos logarítmicos, e aos sais biliares nas concentrações de (0,3 e 1,0) %, com redução de (0,37 e 0,51) ciclos logarítmicos, respectivamente. Portanto, os animais devem ser previamente tratados com um antiácido como CaCO 3 , a fim de manter a viabilidade dos bacteriófagos no trato gastrointestinal das aves. A sinergia entre os bacteriófagos e a enrofloxacina, levou a redução de 1,08 ciclos logarítmicos de C. jejuni (B12313), e 1,48 ciclos logarítmicos de C. jejuni (IOC/ATCC 33560) nas fezes dos frangos após cinco dias de tratamento. No conteúdo cecal, a redução de C. jejuni (B12313) com tratamento de coquetel de bacteriófagos foi de 2,58 ciclos logarítmicos, e o tratamento com o antibiótico foi de 1,67 ciclos logarítmicos. Nos frangos infectados com C. jejuni (IOC/ATCC 33560), a redução foi de 2,42 ciclos logarítmicos com o uso de coquetel de bacteriófagos, e de 2,51 ciclos logarítmicos com o antibiótico enrofloxacina. Os bacteriófagos são apresentados como uma ferramenta útil na redução do patógeno C. jejuni, antes e após o abate. Mais investigações devem ser feitas com outros antibióticos, com o objetivo de aumentar o conhecimento de sinergia entre bacteriófagos e antibióticos como terapêutico dentro da granja. Em conteúdo cecal, as análises indicaram atuação mais eficiente do coquetel de bacteriófagos isoladamente. Isso indica que o tratamento com fagos seria eficiente no controle da contaminação da carne durante o abate. / Poultry meat, consist of an ideal matrix for the multiplication of microorganisms and thus is a source of numerous infections in humans. Among the bacteria, which generates more intestinal infection from the consumption of poultry products, is the species Campylobacter jejuni. The indiscriminate use of antibiotics in animal production may contribute to the selection of resistant bacteria, which may be disseminated during food production or processing. Therefore, new alternatives are needed to control this pathogen, such as the use of bacteriophages. The objective of this study was to isolate, characterize and evaluate the effect of a cocktail of bacteriophages individually or associated with the antibiotic enrofloxacin, in the control of C. jejuni, in broilers. Six bacteriophages were isolated (BC2, BC7, BC10, BC14, BC18 and BC19) from chicken feces collected from two poultry farms in the region of Viçosa, in the State of Minas Gerais. The bacteriophages were evaluated for specificity against different bacterial strains. The bacteriophage BC14 besides presenting lytic power over the strains of C. jejuni presented lithic activity against Salmonella Typhimuium (ATCC 14028) and Escherichia coli (CDC0111ab). The morphological characterization of two bacteriophages (BC7 e BC14) by transmission electron microscopy (TEM) showed that bacteriophages had icosahedral head and medium-sized tail indicating that they are probably viruses of the order Caudovirales order and Myoviridae family. The bacteriophage cocktail was mostly affected when exposed to pH 2.5 (reduction of 6.90) log 10 units and bile salt in the concentrations of (0.3 and 1.0) %, with a reduction of (0.37 and 0.51) log 10 units respectively. Therefore, animals should be pretreated with an antacid such as CaCO 3 , in order to maintain the viability of bacteriophages in the gastrointestinal tract of birds. A synergy was observed between bacteriophage and enrofloxacin with a reduction of 1,08 log 10 units of C. jejuni (B12313) and 1.48 log 10 units of C. jejuni (IOC/ATCC 33560) in the feces of the chickens after five days of treatment. In the cecal content, the reduction of C. jejuni (B12313) with treatment of bacteriophage cocktail was 2.58 log 10 units, and treatment with the antibiotic was 1.67 log 10 units. In chickens infected with C. jejuni (IOC / ATCC 33560), the reduction was 2.42 log 10 units with the use of bacteriophage cocktail, and 2.51 log 10 units with the antibiotic enrofloxacin. Bacteriophages are presented as a useful tool in the reduction of the C. jejuni pathogen, before and after slaughter. Further investigations should be made with other antibiotics, with the aim of increasing the knowledge of synergy between bacteriophages and antibiotics as a therapeutic process within the farm. In cecal content, the analyzes indicated a more efficient performance of the bacteriophage cocktail alone. This indicates that phage treatment would be effective in controlling meat contamination during slaughter.

Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias . / Campylobacter jejuni (C. jejuni) y Campylobacter coli (C. coli) son microorganismos comensales en animales productivos y constituyen una de las principales causas de enteritis de transmisión alimentaria. C. jejuni es responsable del 90% de las campylobacteriosis humanas y C. coli cerca del 10%. Ambas especies son aisladas en proporciones similares de la carne de pollo, la principal fuente de infección del ser humano, representando cada una de ellas cerca del 50% de los Campylobacter spp. aislados. Para estas bacterias, la adherencia e invasión a células intestinales son mecanismos fundamentales de patogenicidad. Se han identificado diversos factores de virulencia asociados a estos mecanismos, como también diferencias entre C. jejuni y C. coli en las prevalencias y tamaños de algunos de los genes que los codifican. El propósito del presente trabajo fue caracterizar la capacidad de adherencia e invasión a células intestinales humanas de cepas de C. jejuni y C. coli aisladas de personas y animales productores de alimentos y relacionar esta capacidad con la presencia de siete genes de virulencia (cadF, flaA, racR, dnaJ, virB11, ciaB y pldA). La hipótesis fue que las cepas de C. jejuni tendrían mayor capacidad de adherir e invadir células intestinales humanas que las cepas de C. coli, y que esta capacidad se relacionaría positivamente con la presencia de genes de virulencia. Se emplearon 15 cepas de C. jejuni y 17 de C. coli aisladas desde pacientes humanos, cerdos, bovinos y pollos broiler. La presencia de los genes de virulencia de cada una de las cepas fue caracterizada en un estudio previo mediante la técnica de PCR convencional. Para evaluar la capacidad de adherencia e invasión a células intestinales humanas se realizaron estudios in vitro empleando la línea celular T84 de epitelio colónico T84 y midiendo el número de bacterias adheridas luego de una h de infección y las bacterias internalizadas luego de tres. La asociación con los genes de virulencia se valoró mediante análisis de regresión logística, el que se complementó con el test Kruskal-Wallis para evaluar diferencias en adherencia e invasión entre cepas portadoras y no portadoras de los genes. Los resultados demostraron que tanto las cepas humanas como las aisladas desde animales productores de alimento tienen la capacidad de adherir e invadir células intestinales in vitro y que esta capacidad varía entre las diferentes cepas. Estadísticamente, no se encontraron diferencias en la capacidad de adherencia e invasión entre C. jejuni y C. coli. El análisis Kruskal- Wallis (y test post-hoc Dunn) reveló que las cepas de C. coli portadoras del gen dnaJ tenían una mayor capacidad de invasión que las cepas de C. coli no portadoras del gen y que las cepas C. jejuni portadoras del mismo. Asimismo, con el análisis de regresión logística se encontró una asociación significativa entre la presencia del gen dnaJ y una mayor capacidad invasora en la especie C. coli. Los resultados de este trabajo sugieren que C. jejuni y C. coli tendrían la misma capacidad de adherir e invadir células intestinales humanas y que solo existiría una asociación positiva entre el gen dnaJ y la invasión de cepas de C. coli. / Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are commensals microorganisms of food-producing animals, and they are considered one of the major causes of food-borne enteritis. 90% of human campylobacteriosis is caused by C. jejuni and most of the rest by C. coli. Both species are isolated in similar proportions from chicken meat, the main source of human infection, representing each of them almost the 50% of Campylobacter spp. isolated. The adherence to and invasion of human intestinal epithelial cells are essential mechanisms in Campylobacter pathogenesis. There have been identified several virulence factors related to these mechanisms, besides differences between C. jejuni and C. coli in the prevalence and size of some of the genes that encode them. The aim of this work was studied the adherence to and invasion of human intestinal epithelial cells by C. jejuni and C. coli isolated from humans and foodproducing animals, and to relate those abilities to the presence of seven virulence genes (cadF, flaA, racR, dnaJ, virB11, ciaB y pldA). The hypothesis was that C. jejuni strains would have more ability to adhere to and invade human intestinal epithelial cells than C. coli strains, and those abilities would be associated with the presence of virulence genes. We used 15 C. jejuni strains and 17 C. coli strains isolated from human patients, broiler chickens, swine, and bovines. The presence of virulence genes of each of the strains was determined using PCR before this work. We employed the human colonic epithelial cell line T84 to test in vitro the adherence and invasion abilities, and we checked them after one and three hours of infection to determine the number adhered and internalized bacteria, respectively. To test the association with the virulence genes we used logistic regression, and to evaluate differences of adherence and invasion between strains carrying and non-carrying virulence genes we used the Kruskal-Wallis test. We observed that both Campylobacter isolates from humans and from food-producing animals are capable of adhering to and of invading intestinal epithelial cells in vitro, and there are variations between strains in those abilities. Statistically, no significant differences were detected between C. jejuni and C. coli in their abilities to adhere to and to invade T84 cells. The Kruskal-Wallis test (and post-hoc Dunn test) showed that C. coli strains carrying dnaJ gene invaded more than C. coli strains non-carrying the gene, and more than C. jejuni strains carrying the same gene. Besides, a significant association was detected between the presence of the dnaJ gene and a higher invasion in C. coli strains by logistic regression. Our results suggest that C. jejuni and C. coli would have the same adherence and invasion abilities, and that, statistically, it would exist only a positive association between the dnaJ gene and the invasion ability of C. coli strains. / Financiamiento: Programa de Apoyo Económico de Actividades de Investigación para estudiantes de Magíster en Ciencias Animales Veterinarias.

Magister Scientiae - MSc / South Africa

Campylobacter jejuni infection is one of the leading causes of gastroenteritis in humans worldwide. During colonization of the gastrointestinal tract, C. jejuni will be unavoidably exposed to reactive oxygen species (ROS) produced by the host immune system and other intestinal microbiota. Identification of defenses against ROS is therefore important for understanding how Campylobacter survives this environmental stress during infection. Construction of isogenic deletion mutants into genes encoding potential oxidative stress defense systems followed by phenotypic screening revealed genes important for oxidant defense within C. jejuni. Surprisingly, genes involved in motility were found to play an indirect role in resistance to oxidative stress. Deletion of the flagellar motor apparatus genes, motAB, resulted in increased sensitivity towards superoxide which could be restored by fumarate supplementation or tandem deletion of motAB with ccoQ (cytochrome c oxidase). This finding suggested that disruption of the proton gradient across the inner membrane resulted in increased superoxide production in non-motile flagellar mutants. Phenotypic screening of the mutant library also identified a novel gene (cj1386) specifically involved in hydrogen peroxide defense within the cell. Hydrogen peroxide detoxification within living organisms is predominantly carried out by catalase enzymes. Interestingly, cj1386 is located directly downstream from katA (catalase) in the C. jejuni genome and it was found that a ∆cj1386 mutant had reduced catalase activity relative to wild-type C. jejuni. Immunoprecipitation of KatA from ∆cj1386 revealed a significant reduction in hemin content associated with KatA suggesting a role for cj1386 in hemin trafficking to KatA. Hemin binding experiments with purified Cj1386 demonstrated the ability of Cj1386 to bind hemin with a 1:1 hemin-to-protein binding ratio. Furthermore, co-immunoprecipitation experiments revealed an interaction between KatA and Cj1386. Mutagenesis of conserved amino acids in Cj1386 demonstrated that tyrosine 57 plays an important role in hemin affinity and is required for proper hemin content of KatA within the cell. Overall, this work provides a global characterization of key oxidant defenses within C. jejuni and provides one of the first studies investigating hemin trafficking to KatA.

Campylobacter organisms are the most commonly reported bacterial causes of foodborne infection in the world, with Campylobacter jejuni and Campylobacter coli responsible for over 99% of reported infections. Traditionally, Campylobacter species detection is an arduous process, requiring a special incubation environment as well as specific growth media for an extended growth period. The development of a rapid and reliable diagnostic tool for the detection of Campylobacter species would be a valuable aid to the medical diagnostic decision process, especially to rule out Campylobacter infection during the enteric pre-surgical time period. Improved patient outcomes would result if this rapid assay could reduce the number of enteric surgeries. Assays performed during this dissertation project have demonstrated that both SYBR® green and hydrolysis probe assays targeting an 84 nucleotide portion of cadF, a fibronectin-binding gene of Campylobacter jejuni and Campylobacter coli, were able to detect from 101 to 108 copies of organism from stool specimens, did not detect nonspecific targets, and exhibited a coefficient of variation (CV) of 1.1% or less. Analytical validation of sensitivity, specificity and precision, successfully performed in these studies, warrants additional clinical validation of these assays.

Mononuclear molybdenum enzymes catalyze a variety of reactions that are essential in the cycling of nitrogen, carbon, arsenic, and sulfur. For decades, the structure and function of these crucial enzymes have been investigated to develop a fundamental knowledge for this vast family of enzymes and the chemistries they catalyze. The dimethyl sulfoxide reductase (DMSOR) family is the most diverse family of molybdoenzymes and, the members of this family catalyze a myriad of reactions that are important in microbial life processes. Periplasmic nitrate reductase (Nap) is an important member of the DMSO reductase family that catalyzes the reduction of nitrate to nitrite, and yet the physiological role of Nap is not completely clear. Enzymes in this family can transform multiple substrates; however, quantitative information about the substrate preference is sparse and more importantly, the reasons for the substrate selectivity are not clear. Substrate specificity is proposed to be tuned by the ligands coordinating the molybdenum atom in the active site. As such, periplasmic nitrate reductase is utilized as a vehicle to understand the substrate preference and delineate the mechanistic underpinning of these differences. To this end, NapA from <i>Campylobacter jejuni </i>has been heterologously overexpressed, and a series of variants, where the molybdenum-coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic and biochemical properties of these variants will be discussed and compared with those of the native enzyme, providing quantitative information to understand the function.

Indiana University-Purdue University Indianapolis (IUPUI) / Mononuclear molybdenum enzymes catalyze a variety of reactions that are essential in the cycling of nitrogen, carbon, arsenic, and sulfur. For decades, the structure and function of these crucial enzymes have been investigated to develop a fundamental knowledge for this vast family of enzymes and the chemistries they catalyze. The dimethyl sulfoxide reductase (DMSOR) family is the most diverse family of molybdoenzymes and, the members of this family catalyze a myriad of reactions that are important in microbial life processes. Periplasmic nitrate reductase (Nap) is an important member of the DMSO reductase family that catalyzes the reduction of nitrate (NO3-) to nitrite (NO2-), and yet the physiological role of Nap is not completely clear. Enzymes in this family can transform multiple substrates; however, quantitative information about the substrate preference is sparse and more importantly, the reasons for the substrate selectivity are not clear. Substrate specificity is proposed to be tuned by the ligands coordinating the molybdenum atom in the active site. As such, periplasmic nitrate reductase is utilized as a vehicle to understand the substrate preference and delineate the mechanistic underpinning of these differences. To this end, NapA from Campylobacter jejuni has been heterologously overexpressed, and a series of variants, where the molybdenum-coordinating cysteine has been replaced with another amino acid, has been produced. The kinetic and biochemical properties of these variants will be discussed and compared with those of the native enzyme, providing quantitative information to understand the function.

Infectious diseases caused by pathogenic microorganisms are one of the largest socioeconomic burdens today. Although infectious diseases have been studied for decades, in numerous cases, the precise mechanisms involved in the multifaceted interaction between pathogen and host continue to be elusive. Thus, it still remains a challenge for researchers worldwide to develop novel strategies to investigate the molecular context of infectious diseases in order to devise preventive or at least anti-infective measures. One of the major drawbacks in trying to obtain in-depth knowledge of how bacterial pathogens elicit disease is the lack of suitable infection models to authentically mimic the disease progression in humans. Numerous studies rely on animal models to emulate the complex temporal interactions between host and pathogen occurring in humans. While they have greatly contributed to shed light on these interactions, they require high maintenance costs, are afflicted with ethical drawbacks, and are not always predictive for the infection outcome in human patients. Alternatively, in-vitro two-dimensional (2D) cell culture systems have served for decades as representatives of human host environments to study infectious diseases. These cell line-based models have been essential in uncovering virulence-determining factors of diverse pathogens as well as host defense mechanisms upon infection. However, they lack the morphological and cellular complexity of intact human tissues, limiting the insights than can be gained from studying host-pathogen interactions in these systems. The focus of this thesis was to establish and innovate intestinal human cell culture models to obtain in-vitro reconstructed three-dimensional (3D) tissue that can faithfully mimic pathogenesis-determining processes of the zoonotic bacterium Campylobacter jejuni (C. jejuni). Generally employed for reconstructive medicine, the field of tissue engineering provides excellent tools to generate organ-specific cell culture models in vitro, realistically recapitulating the distinctive architecture of human tissues. The models employed in this thesis are based on decellularized extracellular matrix (ECM) scaffolds of porcine intestinal origin. Reseeded with intestinal human cells, application of dynamic culture conditions promoted the formation of a highly polarized mucosal epithelium maintained by functional tight and adherens junctions. While most other in-vitro infection systems are limited to a flat monolayer, the tissue models developed in this thesis can display the characteristic 3D villi and crypt structure of human small intestine. First, experimental conditions were established for infection of a previously developed, statically cultivated intestinal tissue model with C. jejuni. This included successful isolation of bacterial colony forming units (CFUs), measurement of epithelial barrier function, as well as immunohistochemical and histological staining techniques. In this way, it became possible to follow the number of viable bacteria during the infection process as well as their translocation over the polarized epithelium of the tissue model. Upon infection with C. jejuni, disruption of tight and adherens junctions could be observed via confocal microscopy and permeability measurements of the epithelial barrier. Moreover, C. jejuni wildtype-specific colonization and barrier disruption became apparent in addition to niche-dependent bacterial localization within the 3D microarchitecture of the tissue model. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D host environment deviated from those obtained with conventional in-vitro 2D monolayers but mimicked observations made in vivo. Furthermore, a genome-wide screen of a C. jejuni mutant library revealed significant differences for bacterial factors required or dispensable for interactions with unpolarized host cells or the highly prismatic epithelium provided by the intestinal tissue model. Elucidating the role of several previously uncharacterized factors specifically important for efficient colonization of a 3D human environment, promises to be an intriguing task for future research. At the frontline of the defense against invading pathogens is the protective, viscoelastic mucus layer overlying mucosal surfaces along the human gastrointestinal tract (GIT). The development of a mucus-producing 3D tissue model in this thesis was a vital step towards gaining a deeper understanding of the interdependency between bacterial pathogens and host-site specific mucins. The presence of a mucus layer conferred C. jejuni wildtype-specific protection against epithelial barrier disruption by the pathogen and prevented a high bacterial burden during the course of infection. Moreover, results obtained in this thesis provide evidence in vitro that the characteristic corkscrew morphology of C. jejuni indeed grants a distinct advantage in colonizing mucous surfaces. Overall, the results obtained within this thesis highlight the strength of the tissue models to combine crucial features of native human intestine into accessible in-vitro infection models. Translation of these systems into infection research demonstrated their ability to expose in-vivo like infection outcomes. While displaying complex organotypic architecture and highly prismatic cellular morphology, these tissue models still represent an imperfect reflection of human tissue. Future advancements towards inclusion of human primary and immune cells will strive for even more comprehensive model systems exhibiting intricate multicellular networks of in-vivo tissue. Nevertheless, the work presented in this thesis emphasizes the necessity to investigate host-pathogen interactions in infection models authentically mimicking the natural host environment, as they remain among the most vital parts in understanding and counteracting infectious diseases. / In der heutigen Zeit tragen insbesondere durch pathogene Mikroorganismen ausgelöste Infektionskrankheiten zur sozioökonomischen Belastung bei. Obwohl bereits jahrzehntelang an der Entstehung von Infektionskrankheiten geforscht wird, bleiben in zahlreichen Fällen die genauen Mechanismen, welche an den vielfältigen Interaktionen zwischen Pathogen und Wirt beteiligt sind, unbeschrieben. Gerade deshalb bleibt es für Wissenschaftler weltweit eine Herausforderung, neue Strategien zur Untersuchung des molekularen Kontexts von Infektionskrankheiten zu entwickeln, um präventive oder zumindest anti-infektive Maßnahmen ergreifen zu können. In den meisten Fällen ist jedoch das Fehlen geeigneter Infektionsmodelle, mit denen der Krankheitsverlauf im Menschen authentisch nachgestellt werden kann, eines der größten Hindernisse um detailliertes Wissen darüber gewinnen zu können wie bakterielle Pathogene die Krankheit auslösen. Zahlreiche Studien sind dabei auf Tiermodelle angewiesen, um die komplexen zeitlichen Abläufe zwischen Wirt und Pathogen im menschlichen Körper nachzuahmen. Während diese Modelle in hohem Maß dazu beigetragen haben, Aufschluss über diese Abläufe zu geben, sind sie doch sehr kostenintensiv, mit ethischen Bedenken behaftet und können nicht immer die Folgen einer Infektion im menschlichen Patienten vorhersagen. Seit Jahrzehnten werden daher alternativ in-vitro 2D Zellkultursysteme eingesetzt, um den Verlauf von Infektionskrankheiten zu erforschen, welche die Bedingungen im menschlichen Wirt wiederspiegeln sollen. Diese auf Zelllinien basierenden Modelle sind essentiell in der Entdeckung von Virulenzfaktoren diverser Pathogene, aber auch in der Aufklärung von wirtsspezifischen Abwehrmechanismen. Dennoch fehlt ihnen die morphologische und zelluläre Komplexität von intaktem menschlichen Gewebe. Dadurch sind die Erkenntnisse, die mit diesen Systemen über Infektionsverläufe gewonnen werden können, limitiert. Die vorgelegte Arbeit konzentriert sich auf die Etablierung und Weiterentwicklung intestinaler, humaner Zellkulturmodelle, um dreidimensionales Gewebe in vitro zu rekonstruieren mit dem Ziel, Pathogenese-beeinflussende Prozesse des zoonotischen Bakteriums C. jejuni nachzustellen. Das Fachgebiet der Gewebezüchtung wird üblicherweise für rekonstruktive Medizin eingesetzt und bietet exzellente Mittel zur in-vitro Herstellung organspezifischer Zellkulturmodelle, welche die unverkennbare Mikroarchitektur humanen Gewebes realistisch nachempfinden können. Die in dieser Arbeit verwendeten Modelle basieren auf einem extrazellulären Matrixgerüst, das aus der Dezellularisierung von Schweinedarm gewonnen wurde. Durch die Wiederbesiedelung mit human Kolonzellen und der Kultivierung unter dynamischen Bedingungen entwickelte sich ein hochpolarisiertes mucosales Epithel, das durch funktionale Zell-Zell-Kontakte (tight und adherens junctions) aufrechterhalten wird. Während andere in-vitro Infektionssysteme meist durch die Präsenz einer flachen Zellschicht limitiert werden, entwickelt das in dieser Arbeit eingeführte Gewebemodell die für den menschlichen Dünndarm charakteristische Architektur aus Villi und Krypten. Zunächst wurden experimentelle Bedingungen für die Infektion eines zuvor entwickelten, statisch kultivierten Dünndarmmodells mit C. jejuni etabliert. Dies beinhaltete die erfolgreiche Isolierung koloniebildender Einheiten, die Messung der epithelialen Barrierefunktion, sowie immunhistochemische und histologische Färbetechniken. Dadurch konnte die Anzahl der Bakterien sowie deren Translokalisierung über das polarisierte Epithel während des Infektionsprozesses nachvollzogen werden. Außerdem konnte die Beeinträchtigung von Zell-Zell-Kontakten durch konfokale Mikroskopie und Permeabilitätsmessungen der epithelialen Barriere beobachtet werden. Neben der Bestimmung der Kolonisierungsrate von C. jejuni Isolaten und der dadurch hervorgerufenen spezifischen Zerstörung der epithelialen Barriere konnten die Bakterien auch innerhalb der 3D Mikroarchitektur des Gewebemodells lokalisiert werden. Außerdem konnte im Rahmen der 3D Gewebeumgebung beobachtet werden, dass Pathogenese-relevante Phänotypen von C. jejuni Mutantenstämmen im Vergleich zu konventionellen in-vitro 2D Zellschichten abwichen, diese aber dafür mit den in-vivo gemachten Beobachtungen übereinstimmten. Darüber hinaus wies die genomweite Suche einer C. jejuni Mutantenbibliothek signifikante Unterschiede zwischen bakteriellen Faktoren, die für die Interaktion mit nicht polarisierten Wirtszellen oder dem hochprismatischen Epithel des Gewebemodells bedeutsam oder entbehrlich waren, auf. Die Aufklärung der Funktion einiger bisher nicht charakterisierter Faktoren, die zu einer effizienten Kolonisierung menschlichen Gewebes beitragen, verspricht eine faszinierende Aufgabe für die zukünftige Forschung zu werden. Die vorderste Verteidigungslinie gegen eindringende Pathogene bildet die schützende, viskoelastische Mukusschicht, die mukosale Oberflächen entlang des menschlichen Gastrointestinaltrakts überzieht. Mit der Entwicklung eines mukusproduzierenden Gewebemodells in der hier vorgelegten Arbeit gelang ein entscheidender Schritt zur Erforschung der Wechselbeziehungen zwischen bakteriellen Pathogenen und wirtsspezifischen Muzinen. Während des Infektionsverlaufs wurde das unterliegende Epithel durch die Anwesenheit der Mukusschicht vor der Zerstörung durch die Mikroben geschützt und eine erhöhte bakterielle Belastung verhindert. Darüber hinaus liefern die Resultate dieser Arbeit einen in-vitro Nachweis für den bakteriellen Vorteil einer spiralförmigen Morphologie, um muköse Oberflächen zu besiedeln. Zusammenfassend unterstreicht diese Arbeit das Potential der hier entwickelten Gewebemodelle, entscheidende Eigenschaften des menschlichen Darms in einem leicht zugänglichen in-vitro Infektionsmodell zu vereinigen. Der Einsatz dieser Modelle im Rahmen der Infektionsforschung bewies deren Fähigkeit in-vivo beobachtete Infektionsverläufe widerzuspiegeln. Während diese Infektionsmodelle bereits organotypische Architektur und hochprismatische Zellmorphologie aufweisen, ist ihre Darstellung von menschlichem Gewebe noch nicht perfekt. Durch den Einsatz von humanen Primär- und Immunzellen wird es in Zukunft möglich sein, noch umfassendere Modellsysteme zu entwickeln, die komplexe multizelluläre Netzwerke von in-vivo Geweben aufweisen. Nichtsdestotrotz verdeutlicht die hier vorgelegte Arbeit wie wichtig es ist, die Interaktionen zwischen Wirt und Pathogen innerhalb von Infektionsmodellen zu erforschen, welche die natürliche Wirtsumgebung wiedergeben. Dies spielt eine entscheidende Rolle, um die Entstehung von Infektionskrankheiten nachvollziehen und ihnen entgegenwirken zu können.

Campylobacter spp. infections are an increasing global concern responsible for a significant burden of disease every year. Wildlife and domestic animals are considered important reservoirs, but little is known about host-factors driving pathogen infection dynamics in wild mammal populations. In countries like Botswana, there is significant spatial overlap between humans and wildlife with a large proportion of the population vulnerable to Campylobacter infection, making Botswana an ideal location to study these interactions. This thesis reviews mammalian wildlife species that have been identified as carriers of Campylobacter spp., identifies life-history traits (urban association, trophic level, and sociality) that may be driving Campylobacter infection, and utilizes banded mongoose (Mungos mungo) (n=201) as a study species to illuminate potential Campylobacter spp. transmission at the human-wildlife interface in northern Botswana. Results of the latter study suggest that human-landscapes are critical to C. jejuni infection in banded mongooses, as mongooses utilizing man-made structures as dens had significantly higher levels of C. jejuni than mongooses using natural dens (p=0.019). A similar association was found across all wild mammals with significantly greater number of urban dwelling species positive for C. jejuni than urban avoiders (p = 0.04). Omnivorous and social mammals were significantly associated with C. coli presence (p=0.04 and p<0.00 respectively), but not with C. jejuni indicating there may be important differences in transmission dynamics between Campylobacter species. These results suggest that landscape features and life-history traits can have important influences on Campylobacter species exposure and transmission dynamics in wildlife. / Master of Science / Campylobacter infections are increasing worldwide but we still know little about the true burden of disease in the developing world, and even less about the role of wildlife and environmental reservoirs in human exposure and disease. I reviewed life-history traits (urban association, animal rank on the food chain, and sociality) that might be driving Campylobacter spp. infection in wildlife and investigated interactions between an urbanizing wildlife species, banded mongoose (Mungos mungo), humans, and the environment. Banded mongooses live in close association with humans and infections with C. jejuni were greater among mongooses utilizing man-made structures compared to those using natural dens. Across all wild mammal species tested for Campylobacter spp., mammals associated with urban living were significantly more likely to be positive for C. jejuni than mammals that avoid urban areas. Lowerranking mammals on the food chain and social mammals were associated with presence of C. coli, suggesting life-history rates are playing a role in wild mammal exposures to the pathogen and that these exposures are different for C. coli than C. jejuni. These data suggest that wildlife life-history traits and utilization of human landscapes are important for pathogen presence. In turn, pathogen circulation and transmission in urbanizing wildlife reservoirs may increase human vulnerability to disease, particularly in impoverished populations, where greater environmental exposures are expected. Improvement of waste management and hygiene practices may help reduce transmission between wildlife and humans.

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