Memoria para optar al Título Profesional de Médico Veterinario / Salmonella spp. es el agente etiológico de la salmonelosis, enfermedad entérica transmitida principalmente por los alimentos. Este patógeno está ampliamente difundido en el medio ambiente y anualmente infecta a millones de animales y personas en todo el mundo. Salmonella es capaz de establecerse en un estado de biopelícula, el que se caracteriza por ser una comunidad de células de una o varias especies protegidas por sustancias poliméricas extracelulares (EPS) autogeneradas y fijadas a una superficie inerte o un tejido vivo. Las biopelículas son de gran importancia pues permiten la persistencia de los patógenos tanto dentro del hospedero como en el medio ambiente. El objetivo de este trabajo fue caracterizar la formación de biopelículas de los diferentes serotipos de S. enterica aisladas de distintos hospederos en Chile. Se cuantificó la formación de biopelículas por medio del método colorimétrico de titulación en placa y a través del coeficiente de correlación de Pearson's se estableció si las características de expresión fenotípica de las biopelículas tenían relación con la capacidad de supervivencia a condiciones de estrés, de las cepas en estado planctónico. Como resultado no se encontró una asociación significativa entre hospedero, serotipos y formación de biopelícula. Las propiedades de supervivencia a condiciones de estrés de las cepas de S. enterica ser. Enteritidis en estado de vida libre fueron independientes de la formación de biopelículas. Junto a esto, se estudió el fenotipo expresados por las cepas en placas de agar. Para observar la producción de fimbrias curli y celulosa se usaron placas de agar con tinción de Rojo Congo y Azul de Comassie, mientras que para determinar la expresión de ácido colánico se utilizaron placas de agar con tinción de Calcofluor. Este análisis evidencio que el morfotipo BDAR (café, seco y rugoso) que expresa fimbiras curli pero no celulosa es ampliamente expresado por los serotipos S. Agona (83%), S. Havana (60%), S. Infantis (57%) y S. Heidelberg (83%). Esto podría indicar que este morfotipo, relativamente poco estudiado, puede estar siendo expresado ampliamente por las cepas de Salmonella no Enteritidis en el medio ambiente. vi Mientras que los serotipos S. Agona, S. Anatum, S. Enteritidis, S. Havana, S. Heidelberg y S. Infantis resultaron positivos a ácido colánico. Lo que les otorgaría a estas cepas una mayor capacidad para formar biopelículas sobre células epiteliales. Palabras claves: Salmonella, Serotipo, Biopelícula, Morfotipo, Sustancias poliméricas extracelulares (EPS). / Fondo de Apoyo a la Investigación Universidad Autónoma 2013

Memoria para optar al Título Profesional de Médico Veterinario / Durante la última década la presencia humana en la Antártica se ha incrementado en forma progresiva, debido al desarrollo de la industria del turismo y a la gran cantidad de asentamientos humanos que existen en la actualidad. Este fenómeno, coincide con las altas tasas de infección registradas en los últimos años y con los recientes aislamientos de microbios que infectan humanos, presentes en animales silvestres que habitan en la Antártica y zonas sub-antárticas, algunos de los cuales, han mostrado resistencia a antibióticos. Esto ha llevado a varios investigadores a pensar que el efecto antropogénico podría estar influyendo sobre estos territorios. En esta investigación se intentó detectar la presencia de cepas de Salmonella enterica resistente a antibióticos en pingüinos Papúa (Pygoscelis papua) que habitan la Antártica Chilena. Para esto, se tomaron 200 muestras fecales de cuatro zonas de la Península Antártica con diferente grado de exposición a la presencia humana. De estas muestras, se aislaron cuatro cepas de S. enterica, todas ellas provenientes de las muestras tomadas en la base General Bernardo O’Higgins. Las cuatro cepas fueron resistentes al menos a tres agentes antimicrobianos, siendo una de ellas multirresistente, mostrando resistencia contra β-lactámicos, fluoroquinolonas y contra la asociación de Sulfametoxazol más Trimetoprim. Los resultados de esta investigación sugieren que los asentamientos humanos estarían afectando negativamente el medioambiente antártico, alterando la flora intestinal de la vida silvestre con la presencia de patógenos zoonóticos resistentes a antibióticos

Despite its high isolation frequency in Australian chickens, S. II Sofia is rarely associated with animals or human salmonellosis as this serovar is avirulent in nature. The reason for its persistence and avirulence is unknown as very few studies have been conducted on the epidemiology and pathogenicity of this strain. This study details the various experimental methods utilised to investigate the genetic relatedness and molecular mechanisms involved in S. II Sofia pathogenesis. Using PFGE and Rep-PCR, the Australian S. II Sofia isolates were found to show limited genetic diversity and probably share a clonal relationship. A majority of the S. II Sofia isolates were not geographically restricted with the predominant pattern subtype spread out among the isolates from various states. Distribution and variation of the SPI-associated virulence genes within S. II Sofia was also examined. Based on RFLP and sequence analysis, most of the differences observed in SPI1 to SPI5 of S. II Sofia could be attributed to a loss or gain of restriction cleavage sites within these regions. However, a number of genes in SPI1, SPI2, SPI3 and SPI5 were found to have accumulated changes (mutations, insertions and deletions) that could have affected gene transcription and/or protein translation - these genes have been shown to be involved in different aspects of the virulence process. The avirulence of S. II Sofia is probably not the result of a single genetic change but rather a series of alterations to a large number of its virulence-associated genes. Plasmid-mediated virulence was also assessed in S. II Sofia isolates. Southern hybridisation with probes derived from the virulence plasmid of S. Typhimurium indicated either the total absence of the virulence plasmid or possible presence of a virulence plasmid containing major deletions. Clones were constructed with the missing spv operon using high-copy pCR®2.1 and low-copy pWSK29 plasmids and the adherence, invasion and intracellular survival of the mutant strain was evaluated in vitro. The presence of spvRABCD was shown to have no effect on intracellular survival and replication. Although the cloning of spv with pCR®2.1 was observed to significantly increase invasiveness of S. II Sofia, it was not capable of restoring the invasive ability of S. II Sofia to the level of pathogenic S. Typhimurium 82/6915. On the other hand, the uneven adherence and invasion ability of the other mutant strains appeared to be linked to the presence of pWSK29 and this observation is further supported by RT-PCR analysis of the clones - indicating that perhaps pWSK29 is not a suitable vector for this study. Wild-type S. II Sofia isolates are unlikely to regain full pathogenicity because of the numerous mutations in many important virulence genes: even the chance acquisition of a virulence factor (e.g. spvRABCD) is not sufficient to completely restore S. II Sofia virulence. Therefore, S. II Sofia should not be considered similar to other Salmonella spp. when monitoring Salmonellae in food samples.

RdoA, a serine/threonine kinase, is a member of the Cpx regulon, a stress response pathway, in Salmonella enterica serovar Typhimurium. Phenotypic characterization of rdoA null mutants suggested that RdoA kinase activity affects a wide range of cell functions, which could be the result of both direct and indirect phosphorylation of targets. In a search for RdoA’s target(s), the phosphoproteome profile of wild-type and rdoA null S. enterica was examined through phosphoprotein enrichment followed by 2-dimensional gel electrophoresis coupled with phospho-specific fluorescent stains and western blots using phospho-specific antibodies. Three different phosphoprotein enrichment protocols, all based on metal-ion affinity chromatography, were compared for yield and phosphoprotein specificity to determine which would be the most suitable for S. enterica. This study showed that the Phostag Enrich Phosphoprotein kit (PerkinElmer) gave the highest yield, the majority of which were phosphoproteins. These studies also showed that western blots using phospho-specific antibodies were more sensitive than phosphoprotein-specific fluorescent stain ProQ Diamond in detecting phosphoproteins. The phosphoproteome profile of S. typhimurium cells grown under Cpx activating conditions included phosphoproteins involved in the heat shock response, cellular metabolism and protein synthesis. This work also identified changes in the phosphoproteome that were dependent upon the presence or absence of RdoA. Phosphoproteins that showed a significant change in phosphorylation were identified by mass spectroscopy using peptide mass fingerprinting. Proteins identified included protein foldases (DnaK and GroEL), proteins involved in metabolism (glycerol kinase, enolase and E1 subunit of the pyruvate dehydrogenase complex), and in protein synthesis (elongation factor-Tu). These proteins may be phosphorylated in an RdoA-dependent manner to allow normal cell functioning under envelope stress. Several proteins unlikely to be phosphoproteins were also RdoA-dependent. SrgA, encoded on the virulence plasmid, is a disulfide oxidoreductase specific for the PEF fimbriae that was shown to be repressed by RdoA. This work also showed that integration host factor, previously suggested to be an RdoA target, was not affected in terms of expression or phosphorylation by RdoA. The several RdoA-dependent changes in protein expression levels and phosphorylation that were identified contribute to the elucidation of RdoA’s role in the envelope stress response and provided further insight in determining RdoA target(s). / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-11-06 10:51:01.253

Erlangen, Nürnberg, Universiẗat, Diss., 2005. / Enth. 1 Sonderabdr. aus: Cellular microbiology ; 5. 2003. - Beitr. teilw. dt., teilw. engl.

Thesis (Ph. D.)--University of Virginia, 2009. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Erlangen- Nürnberg, Univ., Diss., 2010.

Erlangen-Nürnberg, Univ., Diss., 2010.

Memoria para optar al Título Profesional de Médico Veterinario / El pingüino de Magallanes (Spheniscus magellanicus) es el más abundante del género Spheniscus, vive en Argentina, Chile y las Islas Falkland y es considerado centinela de la vida marina. Salmonella enterica es importante en aves silvestres, ya que se ha descrito la transmisión directa de la bacteria desde éstas a otros animales incluyendo el ser humano. El rol de la actividad humana en la presencia de Salmonella en pingüinos no está clara. Existen pocos estudios sobre Salmonella en pingüinos, siendo los serovares Typhimurium y Enteritidis patógenos para ellos, desconociéndose la presencia de cepas resistentes a los antibióticos en estas aves. El objetivo de este estudio fue determinar la presencia de cepas de S. enterica con resistencia a antimicrobianos en heces de S. magellanicus, de la región de Magallanes y la Antártica Chilena. A partir de 2114 muestras se aislaron 8 cepas pertenecientes a los serovares Agona y Enteritidis, obteniendo una tasa de aislamiento de 0,38%. Aunque todas las cepas resultaron ser sensibles a Cefradina, Cefadroxilo, Enrofloxacino, Amoxicilina + Ácido Clavulánico, Gentamicina y Sulfametoxazol + Trimetoprim, se detectó resistencia a los antibióticos Tetraciclina, Ampicilina, Ceftiofur y Cefotaxima. Estos resultados confirman la presencia de S. enterica en Pingüinos Magallánicos de la Región de Magallanes y la Antártica Chilena, y sugieren que existe un efecto antropogénico por el uso de antibióticos tanto en humanos como en animales domésticos, debido al aumento de la actividad turística y asentamientos humanos (actividad minera y agropecuaria en Seno Otway, y transporte marítimo en el estrecho de Magallanes), incrementando el volumen de desechos en época turística / Financiamiento: Proyecto Fondecyt No. 1110255

Orientador: Marcelo Brocchi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T01:23:26Z (GMT). No. of bitstreams: 1 Pivetta_LucianeBeneditaDuarte_M.pdf: 4595229 bytes, checksum: 95f48747caf7eb8ddce4f6ccacc9d5d4 (MD5) Previous issue date: 2011 / Resumo: Sorovariedades patogênicas de Salmonella enterica constituem um problema de saúde global, que abrange desde gastroenterites até doenças sistêmicas, sendo que ambas podem ser letais. Linhagens atenuadas de S. enterica são promissoras como vacinas vivas, pois são facilmente administradas via oral e capazes de induzir o sistema imune sistêmico e de mucosas do hospedeiro. Além disso, apresentam o potencial de atuar como vacinas multifatoriais, expressando antígenos de outros agentes infecciosos. Neste trabalho, linhagens recombinantes de Salmonella enterica foram construídas e a atenuação da virulência e a estimulação do sistema imune foram avaliadas no modelo murino da salmonelose. As linhagens recombinantes foram desenvolvidas a partir do sistema de recombinação homóloga ?Red, com a deleção de um ou mais genes com efeitos pleiotrópicos na virulência de Salmonella. Após a confirmação da deleção do gene alvo por PCR, as linhagens foram transduzidas com o bacteriófago P22, visando eliminar possíveis problemas com a integridade de lipopolissacarídeos. Posteriormente, caracterizações fenotípicas foram realizadas, por meio de curvas de crescimento in vitro, verificação da capacidade de invasão e sobrevivência no interior de macrófagos e resistência a espécies reativas de oxigênio e nitrogênio. A atenuação da virulência das linhagens foi avaliada in vivo por meio de inoculações orais e intraperitoneais em camundongos BALB/c/AnUnib e a dose letal média também foi estabelecida. Variados níveis de atenuação foram atingidos e dentre as linhagens desenvolvidas, a duplo mutante para os genes hupA e hupB foi a que apresentou os resultados mais promissores. Nos ensaios de proteção e desafio, 100% dos animais vacinados com duas doses da linhagem 'delta'hupA'delta'hupB sobreviveram ao desafio com doses letais da linhagem selvagem. Para a linhagem duplo mutante também foi verificada a capacidade de colonização bacteriana no sangue, placas de Peyer e baço, assim como a produção de anticorpos IgG e IgA. A obtenção de uma linhagem atenuada, porém capaz de induzir com eficácia o sistema imune do hospedeiro é idealmente almejada no desenvolvimento de uma vacina viva. No entanto, atingir esse balanço é um desafio, já que a deleção de genes de virulência de Salmonella acarreta em diferentes níveis de atenuação, mesmo quando os genes são intimamente relacionados. Uma perspectiva futura é o refinamento das linhagens atenuadas por meio da introdução de antígenos heterólogos e de um sistema de expressão regulado, com o estabelecimento de vacinas multifatoriais / Abstract: Pathogenic Salmonella enterica serovars are the cause of a global health problem that ranges gastroenteritis to lethal systemic disease. Attenuated strains of Salmonella enterica are promising as live vaccines because they are orally administrated and capable of inducing a mucosal and systemic immune response in the host. Besides, they can act as multifactorial vaccines delivering antigens from other diseases. In this work, attenuated strains of Salmonella enterica were produced and the attenuation of the virulence and the induction of an immunologic response were evaluated in the murine model of salmonellosis. The recombinant strains were constructed using the ?Red system of homologous recombination deleting one or more genes with pleiotropic effects on Salmonella virulence. After that, a PCR to confirm the deletion of the target gene was made and the strains were transduced with bacteriophage P22 to avoid problems with lipopolysaccharide's integrity. Phenotypic characterization as the in vitro growth curve of the strains, the resistance to ROI and RNI species and the ability of survival inside J774 macrophages cultures were performed. The attenuation of the strains was evaluated in vivo trough oral and intraperitoneal inoculations of the strains into BALB/c/AnUnib mice and the median lethal dose was also established. Many levels of attenuation were reached and among the developed strains, the double mutant strain for the genes hupA and hupB was the one with the best results. In the protection and challenge assays, 100% of the animal vaccinated with two doses of the 'delta'hupA'delta'hupB strain survived to the challenge with lethal doses of the wild type strain. For this strain it was also verified the capacity of colonization into blood, peyer's patches and spleen as well as the production of IgG and IgA antibodies. In the developmente of a live vaccine it is ideal to have a strain that is attenuated but still capable of inducing an immune response in the host. Reaching this balance is a challenge and the deletion of virulence genes in Salmonella entails different levels of attenuation although they are closely related. A future prospect is the improvement of these attenuated strains with the insertion of controlled expression systems and heterologous antigens, creating a multifactorial vaccine / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular