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Salmonella est la deuxième cause de zoonoses humaines dans l’Union Européenne et représente un enjeu majeur pour la filière porcine. Les sérovars, S. Typhimurium, S. Derby et le variant monophasique de S. Typhimurium sont très prévalents chez le porc et également chez les humains. Les objectifs de ces travaux étaient d’établir la dynamique d’infection chez le porc par Salmonella en condition d’élevage conventionnel et expérimental, d’évaluer le pouvoir colonisateur chez le porc et le pouvoir pathogène chez les humains des trois sérovars. En élevage conventionnel, le suivi sérologique des anticorps anti-Salmonella a permis d’évaluer l’âge moyen de séroconversion à 137 jours et d’identifier un « effet ferme » sur l’âge de séroconversion. Le premier essai en condition expérimentale a mis en évidence que les porcs inoculés avec le variant monophasique de S. Typhimurium excrétaient ce sérovar de façon continue dans les fèces. Aux dates d’autopsies 21, 49 ou 84 jours après inoculation, les salmonelles ont été retrouvées dans les différentes parties de l’intestin, dans les nœuds lymphatiques et à des niveaux très élevés dans les amygdales. Après passage dans le tractus digestif, des profils MLVA différents de celui de la souche inoculée ont été identifiés suggérant que le génome de la souche a évolué. Le deuxième essai visant à comparer le pouvoir colonisateur chez le porc des trois sérovars a montré que la dynamique d’excrétion et de colonisation était similaire quel que soit le sérovar. Cependant, la quantité excrétée était significativement différente ; plus élevée avec le variant monophasique par rapport à S.Typhimurium. Le pouvoir pathogène chez l’homme de 15 souches d’origine porcine appartenant aux 3 sérovars a été évalué in vivo sur un modèle insecte Galleria mellonella, et in vitro sur cellules Caco-2. Les souches se sont révélées être potentiellement virulentes. Sur Caco-2, le variant monophasique avait un pourcentage d’adhésion aux cellules le plus élevé et Derby le plus bas. Différents niveaux de virulence ont été observés entre souches d’un même sérovar. Ce travail a apporté des nouvelles connaissances sur la problématique Salmonella en filière porcine. / Salmonella is the second leading cause of human zoonoses in the European Union and represents a major challenge for the pork industry. Serovars S. Typhimurium, S. Derby and the monophasic variant of S. Typhimurium, implicated in human salmonellosis, are highly prevalent in pigs and also in human. The objectives of this research were to establish the infection dynamics in pigs by Salmonella under conventional and experimental rearing conditions, to evaluate pig colonization ability and pathogenicity in humans of the three serovars. In conventional farms, the serological monitoring of Salmonella antibodies allowed to evaluate the average age of seroconversion at 137 days and to identify a "farm effect" on the age of seroconversion. The first trial under experimental conditions revealed that pigs inoculated with the monophasic variant of S. Typhimurium excreted this serovar continuously in the feces. At autopsy dates 21, 49 or 84 days after inoculation, Salmonella were found in the different parts of the intestine, in the lymph nodes and at very high levels in the tonsils. After passage through the digestive tract, different MLVA profiles from that of the inoculated strain were identified suggesting that the genome of the strain has evolved. The second attempt to compare the colonizing ability in the pigs of the three serovars showed that the dynamics of excretion and colonization were similar regardless of the serovar. However, the amount excreted was significantly different: higher with the monophasic variant compared to S. Typhimurium. The pathogenicity in humans of 15 strains of porcine origin belonging to the 3 serovars was evaluated in vivo on an insect model Galleria mellonella, and in vitro on Caco-2 cells. The strains were found to be potentially virulent. On Caco-2, the monophasic variant had the highest cell adhesion percentage and Derby, the lowest. Different levels of virulence were observed between strains of the same serovar. This work brought new knowledge on the Salmonella issue in the pig sector.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2016-07-20T00:37:17Z No. of bitstreams: 1 Tese_final_ MARCHI.MNG.pdf: 2477043 bytes, checksum: 875c56fb9a49e8316ee438fe52b80636 (MD5) / Made available in DSpace on 2016-07-20T00:37:17Z (GMT). No. of bitstreams: 1 Tese_final_ MARCHI.MNG.pdf: 2477043 bytes, checksum: 875c56fb9a49e8316ee438fe52b80636 (MD5) Previous issue date: 2016-05-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The family Cactaceae has approximately 100 genera and 1500 species predominantly American and distributed by a wide variety of climates and ecosystems. Due to the high singularity in terms of genres and endemic species as well as the potential use of these plants for various purposes, studies on the in vitro propagation and ex situ conservation of these species must be established. Thus, the aim of this study was to understand the anatomical, physiological and molecular changes associated with the propagation and conservation in vitro Discocactus zehntneri subsp boomianus and Stephanocereus leutezlburgii, species endemic cactus of Bahia with ornamental potential. The regulators applied not exceeded the control in the induction of shoots/explant in both species and for S. luetzelburgii combination of cytokinins and auxins hiperidricos increased the percentage of shoots that did not survive after acclimatization. Normal shoots of this species have 100% survival in ex vitro conditions. The callus formation was enhanced by adding auxin to the nutrient medium for the two species analyzed and calluses with potential for in vitro morphogenesis were obtained. The proteomic analysis revealed the marker proteins differential expression of somatic embryogenesis in two strains of S. luetetzelburgii calluses. The protocol for cryopreservation D. zehntneri shoots was not effective, however the seed storage either in liquid nitrogen conditions as for ultrafreezer maintained physiological quality after storage for 360 and 180 days, respectively. Osmotic agents were effective in inhibiting in vitro growth of the species for a year without loss of viability. / A fam?lia Cactaceae apresenta cerca de 100 g?neros e 1500 esp?cies predominantemente americanas e distribu?das por uma ampla diversidade de climas e ecossistemas. Devido ? alta singularidade em termos de g?neros e esp?cies end?micas bem como o uso potencial destas plantas para diversas finalidades, estudos acerca da propaga??o in vitro e conserva??o ex situ dessas esp?cies devem ser estabelecidos. Desta forma, o objetivo geral deste trabalho foi compreender as mudan?as anat?micas, fisiol?gicas e moleculares associadas ? propaga??o e conserva??o in vitro de Discocactus zehntneri subsp boomianus e Stephanocereus leutezlburgii, esp?cies de cact?ceas end?micas da Bahia com potencial ornamental. Os reguladores aplicados n?o superaram o controle na indu??o de brotos/explante em ambas as esp?cies e para S. luetzelburgii a combina??o de citocininas e auxinas aumentaram o percentual de brotos hiperidricos que n?o sobreviveram ap?s a aclimatiza??o. Brotos normais dessa esp?cie apresentaram 100% de sobreviv?ncia em condi??es ex vitro. A calog?nese foi potencializada pela adi??o de auxinas ao meio nutritivo para as duas esp?cies analisadas e calos com potencial para morfog?nese in vitro foram obtidos. A an?lise prote?mica revelou a express?o diferencial de prote?nas marcadoras da embriog?nese som?tica em duas linhagens de calos de S. luetetzelburgii. O protocolo para criopreserva??o de brotos de D. zehntneri n?o foi eficiente, no entanto a armazenamento de sementes tanto em condi??es de nitrog?nio l?quido quanto em ultrafreezer mantiveram a qualidade fisiol?gica ap?s o armazenamento por 360 e 180 dias, respectivamente. Os agentes osm?ticos foram eficientes em inibir o crescimento in vitro da esp?cie por um ano sem perda na viabilidade.

Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa tend to be geographically scattered, such as GES-2, which partially compromises the efficacy of imipenem. The G170N mutation, ascribed to a CC to AA base pair substitution on positions 493-494 of the blaGES-2 coding region, distinguishes this ESBL from blaGES-1 and the blaIBC-type genes, making it an ideal target for developing a novel sequence-specific, peptide nucleic acid (PNA)-based, multiplex-PCR detection method. Utilizing two primer pairs in conjunction with a PNA probe, this novel method delivered accurate identification of blaGES-2 compared to standard PCR and gene sequencing techniques, when tested against one hundred (n = 100) P. aeruginosa clinical isolates as well as previously published, well-described control strains. This method has the potential to be used in large-scale, cost-effective screening programmes for specific or geographically restricted ESBLs. To date, in addition to being only described in South Africa, GES-2 is notoriously difficult to identify in P. aeruginosa, using standard methodology. A real-time PCR method using the LightCycler™ was compared to a two-step nested-PCR assay for the detection of blaGES and blaIBC genes from one hundred P. aeruginosa clinical isolates collected over a four-year period from two teaching hospitals in Pretoria, South Africa. Real-time PCR amplification was monitored through hybridisation of fluorescently labelled probes followed by melting curve analysis to detect the relevant G170N mutation occurring in the omega loop region of blaGES-2. Nested-PCR products were subjected to automated DNA sequencing and compared to melting point (Tm) analyses results obtained from the LightCycler assay. Real time and nested-PCR assays detected a blaIBC gene product from 83 and 88 clinical isolates respectively, with the LightCycler thus exhibiting a sensitivity of 94.3% compared to the nested-PCR assay. Comparison of Tm and gene sequencing data however revealed 100% specificity for sequence specific detection of blaGES-2 with the LightCycler. One clinical isolate was found to harbour a blaGES-1 gene, making this the first report of this specific ESBL from South Africa. Selective antibiotic pressure has recently been implicated as a possible driving force behind point mutations observed in blaGES–type genes. This part of the study subjected two well-characterized clinical isolates with class 1 integron-borne blaGES-type genes to five days incubation in the presence of sub-inhibitory concentrations of 15 different antibiotics, including beta-lactams, aminoglycosides and quinolones. Restriction enzyme analysis and DNA sequencing of blaGES-1, blaGES-2 and their immediate upstream genetic environments failed to demonstrate any changes compared to non-exposed controls. Short-term exposure to a sub-inhibitory level of a single antimicrobial agent is thus unlikely to select significant mutations in these beta-lactamase genes or their regulatory mechanisms. / Thesis (PhD (Medical Microbiology))--University of Pretoria, 2004. / Medical Microbiology / unrestricted

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 5 uppsatser.

Campylobacter jejuni is the leading bacterial cause of gastrointestinal diarrheal disease in humans worldwide. This zoonotic pathogen has a complex epidemiology due to its presence in many different host organisms. The overall aim of this thesis was to explore the role of amoebae of the genus Acanthamoeba as an intermediate host and vector for survival and dissemination of C. jejuni. Earlier studies have shown that C. jejuni can enter, survive and replicate within Acanthamoebae spp. In this thesis, I have shown that C. jejuni actively invades Acanthamoeba polyphaga. Once inside, C. jejuni could survive within the amoebae by avoiding localization to degradative lysosomes. We also found that A. polyphaga could protect C. jejuni in acid environments with pH levels far below the range in which the bacterium normally survives. Furthermore, low pH triggered C. jejuni motility and invasion of A. polyphaga. In an applied study I found that A. polyphaga also could increase the survival of C. jejuni in milk and juice both at room temperature and at +4ºC, but not during heating to recommended pasteurization temperatures. In the last study we found that forty environmental C. jejuni isolates with low bacterial concentrations could be successfully enriched using the Acanthamoeba-Campylobacter coculture (ACC) method. Molecular genetic analysis using multilocus sequence typing (MLST) and sequencing of the flaA gene, showed no genetic changes during coculture. The results of this thesis have increased our knowledge on the mechanisms behind C. jejuni invasion and intracellular survival in amoebae of the genus Acanthamoeba. By protecting C. jejuni from acid environments, Acanthamoebae could serve as important reservoirs for C. jejuni e.g. during acid sanitation of chicken stables and possibly as vectors during passage through the stomach of host animals. Furthermore, Acanthamoeba spp. could serve as a vehicle and reservoir introducing and protecting C. jejuni in beverages such as milk and juice. Validation of the ACC method suggests that it is robust and could be used even in outbreak investigations where genetic fingerprints are compared between isolates. In conclusion, Acanthamoeba spp. are good candidates for being natural hosts and vectors of C. jejuni.

Le génome de la levure de boulanger Saccharomyces cerevisiae a évolué à partir d'un ancêtre chez lequel une profonde décompaction du génome s'est produite à la suite de la perte de la méthylation de la lysine 9 de l'histone H3, il y a environ 300 millions d'années. Il a été proposé que cette décompaction du génome a entraîné une capacité accrue des levures à évoluer par des mécanismes impliquant des taux de recombinaison méiotique et de mutation exceptionnellement élevés. La capacité à évoluer accrue qui en résulte pourrait avoir permis des adaptations uniques, qui en ont fait un eucaryote modèle idéal et un outil biotechnologique. Dans cette thèse, je présenterai deux exemples de la façon dont les adaptations locales et globales du génome se reflètent dans les changements des propriétés mécaniques de la chromatine qui, à leur tour, indiquent un phénomène de séparation de phase causée par les modifications post-traductionnelles des histones et des changements dans les taux d'échange des histones. Dans un premier manuscrit, je présente des preuves d'un mécanisme par lequel la relocalisation du locus INO1, gène actif répondant à la déplétion en inositol, du nucléoplasme vers l'enveloppe nucléaire, augmente la vitesse d'adaptation et la robustesse métabolique aux ressources fluctuantes, en augmentant le transport des ARNm vers le cytosol et leur traduction. La répartition d'INO1 vers l'enveloppe nucléaire est déterminée par une augmentation locale des taux d'échange d'histones, ce qui entraîne sa séparation de phase du nucléoplasme en une phase de faible densité plus proche de la périphérie nucléaire. J'ai quantifié les propriétés mécaniques de la chromatine du locus du gène dans les états réprimé et actif en analysant le déplacement de 128 sites LacO fusionnés au gène liant LacI-GFP en calculant diffèrent paramètres tel que la constante de ressort effective et le rayons de confinement du locus. De plus, j'ai mesuré l'amplitude et le taux d'expansion en fonction du temps du réseau LacO et j'ai observé une diminution significative du locus à l'état actif, ce qui est cohérent avec le comportement de ressort entropique de la chromatine décompactée. J'ai montré que les séquences d'éléments en cis dans le promoteur du locus, essentielles à la séparation de phase, sont des sites de liaison pour les complexes de remodelage de la chromatine effectuant l'acétylation des histones. Ces modifications de la chromatine entraînent une augmentation des taux d'échanges des sous-unités des complexes d'histones, et une séparation de phase locale de la chromatine. Enfin, je présente l’analyse de simulations in silico qui montrent que la séparation de phase locale de la chromatine peut être prédite à partir d'un modèle de formation/disruption des interactions multivalentes protéine-protéine et protéine-ADN qui entraîne une diminution de la dynamique de l'ADN. Ces résultats suggèrent un mécanisme général permettant de contrôler la formation rapide des domaines de la chromatine, bien que les processus spécifiques contribuant à la diminution de la dynamique de l'ADN restent à étudier. Dans un second manuscrit, je décris comment nous avons induit la « retro-évolution » de la levure en réintroduisant la méthylation de la lysine 9 de l'histone H3 par l'expression de deux gènes de la levure Schizosaccaromyces pombe Spswi6 et Spclr4. Le mutant résultant présente une augmentation de la compaction de la chromatine, ce qui entraîne une réduction remarquable des taux de mutation et de recombinaison. Ces résultats suggèrent que la perte de la méthylation de la lysine 9 de l'histone H3 pourrait avoir augmenté la capacité à l'évoluer. La stabilité inhabituelle du génome conférée par ces mutations pourrait être utile pour l'ingénierie métabolique de S. cerevisiae, dans laquelle il est difficile de maintenir des gènes exogènes intégrés pour les applications de nombreux processus biotechnologiques courants tels que la production de vin, de bière, de pain et de biocarburants. Ces résultats soulignent l'influence des propriétés physiques d'un génome sur son architecture et sa fonction globales. / The genome of the budding yeast Saccharomyces cerevisiae evolved from an ancestor in which a profound genome decompaction occurred as the result of the loss of histone H3 lysine 9 methylation, approximately 300 million years ago. This decompaction may have resulted in an increased capacity of yeasts to evolve by mechanisms that include unusually high meiotic recombination and mutation rates. Resultant increased evolvability may have enabled unique adaptations, which have made it an ideal model eukaryote and biotechnological tool. In this thesis I will present two examples of how local and global genome adaptations are reflected in changes in the mechanical properties of chromatin. In a first manuscript, I present evidence for a mechanism by which partitioning of the active inositol depletion-responsive gene locus INO1 from nucleoplasm to the nuclear envelope increases the speed of adaptation and metabolic robustness to fluctuating resources, by increasing mRNA transport to the cytosol and their translation. Partitioning of INO1 to the nuclear envelope is driven by a local increase in histone exchange rates, resulting in its phase separation from the nucleoplasm into a low-density phase closer to the nuclear periphery. I quantified the mechanical properties of the gene locus chromatin in repressed and active states by monitoring mean-squared displacement of an array of 128 LacO sites fused to the gene binding LacI-GFP and calculating effective spring constants and radii of confinement of the array. Furthermore, I measured amplitude and rate of time-dependent expansion of the LacO array, and observed a significant decrease for the active-state locus which is consistent with entropic spring behavior of decompacted chromatin. I showed that cis element sequences in the promoter and upstream of the locus that are essential to phase separation are binding sites for chromatin remodeling complexes that perform histone acetylation among other modifications that result in increased histone complex exchange rates, and consequent local chromatin phase separation. Finally, I present analytical simulations that show that local phase separation of chromatin can be predicted from a model of formation/disruption of multivalent protein-protein and protein-DNA interactions that results in decreased DNA dynamics. These results suggest a general mechanism to control rapid formation of chromatin domains, although the specific processes contributing to the decreased DNA dynamics remain to be investigated. In a second manuscript, I describe how we retro-evolutionarily engineered yeast by reintroducing histone H3 lysine 9 methylation through the expression of two genes from the yeast Schizosaccaromyces pombe Spswi6 and Spclr4. This mutant shows an increase in compaction, resulting in remarkable reduced mutation and recombination rates. These results suggest that loss of histone H3 lysine 9 methylation may have increased evolvability. The unusual genome stability imparted by these mutations could be of value to metabolically engineering S. cerevisiae, in which it is difficult to maintain integrated exogenous genes for applications for many common biotechnological processes such as wine, beer, bread, and biofuels production. These results highlight the influence of the physical properties of a genome on its overall architecture and function.