Salmonella Enteritidis es actualmente el serovar de Salmonella más prevalente a nivel mundial. Mediante el análisis bioinformático de los genomas de distintos serovares de Salmonella identificamos una región genómica que se encuentra presente en S. Enteritidis, así como en S. Gallinarum y S. Dublin (serovares filogenéticamente cercanos a S. Enteritidis). Esta región corresponde a una Isla de Patogenicidad, la cual denominamos IG-asnT. Resultados obtenidos en nuestro laboratorio sugieren que varias de las proteínas codificadas en IG-asnT de S. Enteritidis participarían en la colonización de órganos internos de ratón en un modelo de virulencia in vivo. Entre ellas se encuentran proteínas estructurales de un pilus tipo IV (SEN1977), una proteína involucrada en movilización de plasmidios (SEN1980) y TlpA (TIR-like protein A), la cual presenta un dominio con homología a proteínas que participan en respuestas inflamatorias de células del sistema inmune. Debido a que la capacidad de Salmonella para sobrevivir en los macrófagos de su hospedero es un factor indispensable para su diseminación sistémica y la posterior colonización de órganos blanco, en este proyecto nos propusimos determinar si la isla IG-asnT codifica productos génicos que cumplen un papel en la interacción de S. Enteritidis con macrófagos murinos y definir si al menos uno de ellos participa en la producción y/o secreción de la proteína TlpA. Para esto, se estudió la capacidad de varias mutantes de genes presentes en IG-asnT para sobrevivir al interior de macrófagos murinos, de las cuales las mutantes SEN1977, SEN1980 y tlpA presentaron una capacidad disminuida de supervivencia en estos macrófagos en comparación a la cepa silvestre. Por otra parte, nuestros resultados indican que tlpA participa en el proceso que lleva a la muerte celular de los macrófagos infectados con S. Enteritidis. Pudimos determinar que TlpA se produce en distintas condiciones de cultivo in vitro, además de expresarse al interior de los macrófagos infectados. El resto de los genes presentes en la isla no participan en la expresión ni en la producción de la proteína TlpA en las condiciones estudiadas. Estos resultados entregan nueva evidencia sobre factores de virulencia de S. Enteritidis involucrados en su adaptación a células hospederas, que podrían explicar en parte su prevalencia sobre otros serovares.

Made available in DSpace on 2014-06-11T19:30:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-17Bitstream added on 2014-06-13T19:39:28Z : No. of bitstreams: 1 soares_vm_me_botfmvz.pdf: 335980 bytes, checksum: 7710ea8d0868af2d34c7f2b28ddc1cb9 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O objetivo do presente estudo foi avaliar o processo de disseminação de Salmonella Enteritidis via diferentes superfícies de corte (madeira, plástico com triclosan, vidro e aço inoxidável), a partir de pele de frango contaminada (5 log UFC/g), para alimentos prontos para o consumo, bem como o efeito de diferentes protocolos de higienização destas superfícies no controle da contaminação. Os seguintes protocolos foram simulados: protocolo 1, nenhuma limpeza foi realizada depois do manuseio da pele contaminada; protocolo 2, as superfícies foram rinsadas em água corrente, protocolo 3 as superfícies foram limpas com detergente e esfregação mecânica; protocolo 4, o protocolo 3 foi aplicado e as superfícies submetidas a sanitização com hipoclorito. A recuperação do patógeno foi possível em todas as superfícies no protocolo 1, com contagens variando de 1,90 a 2,80 log, bem como nos tomates nelas manuseados. Observou-se uma redução no número de células de S. Enteritidis nos protocolos subseqüentes, tanto nas superfícies como nos tomates, chegando a níveis não detectáveis no protocolo 4. As taxas de transferência da pele para superfícies e tomates variaram de 0,09 a 10,1%. Das superfícies avaliadas, a madeira foi a mais difícil de higienizar e o aço inoxidável a mais fácil. O protocolo 4 foi o que proporcionou a maior de redução de contaminação cruzada e proteção à saúde do consumidor / The aim of this research was to evaluate the spreading process of Salmonella Enteritidis on different cutting boards (wood, triclosan incorporated plastic, glass and stainless steel), from contaminated poultry skin (5 log CFU/g) to food ready to eat, as well the effect of different cleaning protocols on these surfaces to contamination control. The following scenarios were simulated: scenario 1, no cleaning was performed after handling the contaminated skin; scenario 2, the surfaces were rinsed in water, scenario 3 the surfaces were cleaned with detergent and mechanical scrubbing; scenario 4, protocol 3 was applied and the surfaces submitted to sanitization with sodium hypochlorite. The recovery of the pathogen was possible in all surfaces in scenario 1, with scores ranging from 1.90 to 2.80 log, as well in the tomatoes handled on them. There was a reduction in the number of S. Enteritidis cells in subsequent scenarios, on the surfaces and in tomatoes, reaching undetectable levels in scenario 4. The transfer rates of skin surfaces and tomatoes ranged from 0.09 to 10.1%. About the surfaces assessed, the wood was the most difficult to sanitize and stainless steel easier. Scenario 4 was the one that provided the largest reduction of cross-contamination and protect consumer health

Orientador: Edir Nepomuceno da Silva / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-07-22T23:04:37Z (GMT). No. of bitstreams: 1 Oliveira_JairVicentede_D.pdf: 4139067 bytes, checksum: 3827fa3c2dfeb20f2093e4f0f981e9bc (MD5) Previous issue date: 1997 / Resumo: Considerando os contaminantes da casca do ovo e o emprego deste em alta escala na indústria de alimentos, o ovo passa a ser uma matéria prima que oferece riscos a saúde do consumidor, e riscos econômicos ao produtor e ao setor industrial. Com o objetivo de aumentar a eficiência na limpeza e sanificação dos ovos, utilizamos, isoladamente, hipoclorito de sódio nas concentrações de 5,8, 50 e 100 mg/L de cloro ativo, um composto de amônio quatemário (cloreto de alquil dimetil benzil amônio) concentrado a 50% nas diluições 1/500 e 1/1000 e um composto de amônio quatemário comercial contendo 20% do ingrediente ativo em suspensão alcalina, em concentrações de acordo com as recomendações do fabricante. Também foi utilizado um ácido acético nas concentrações de 2,0% e 3,0%. Todos os sanificantes foram aplicados por 15 e 30 segundos, na temperatura de 25°C e 40°C, e comparados com o emprego da água sem sanificantes. Realizou se a contagem global de bactérias mesófilas e psicrotróficas aeróbias, bolores, leveduras, coliformes totais e fecais antes da aplicação dos sanificantes (para acompanhamento dos contaminantes oriundos da granja) e após a sanificação compreendendo o período de estocagem de sete e quatorze dias ( para avaliação da sanificação). Em outro experimento usamos uma cepa de Salmonella enteritidis ácido nalidixico resistente a 100µg/ml, aplicando-se o mesmo procedimento com a finalidade de melhor avaliar os sanificantes. Na prática da aplicação em laboratório, encontramos que a microbiota da casca do ovo foi reduzida de 2 a 3 ciclos logarítmicos quando usado os compostos de amônio quatemário (CAQ). O CAQ na concentração de 50% e diluído 1/500, 1/1000 quando empregado sobre a casca do ovo contaminado previamente com uma população de 104 UFC/ml de S. enteritidis acido nalidixico resistente, propiciou uma redução total da bactéria, o mesmo ocorrendo para o hipoclorito na concentração de 100 mg/L. Para os demais produtos não houve redução da Salmonella enteritidis. Este procedimento demonstrou que a sanificação ajuda na conservação dos ovos e aumenta a sua vida de prateleira. Por isso é importante a monitoração da água empregada na lavagem da casca dos ovos, ressaltando dessa forma a importância do uso de sanificantes na prática rotineira das granjas. Uma contribuição para a legislação brasileira quanto a lavagem e emprego de sanificantes na higienização da casca dos ovos comerciais pode ser alcançada com a elaboração de um roteiro prático e especificando as condições de uso dos sanificantes / Abstract: Considering the high level of contamination on the egg shell and its high level of use on an industrial scale, eggs have become an important health risk to the consumer and a source of economic loss to the producer and the industrial sector. The objective of this work was to increase the efficiency of cleaning and sanitation using chlorinated compounds (sodium hypochlorite) in concentrations of 5.8, 50 and 100 mg/L active chlorine, a 50% quatemary ammonium compound (alkyldimethylbenzyl ammonium chloride) in dilutions of 1/500 and 1/1000, a commercial quatemary ammonium compound containing 20% of the active ingredient in alkaline solution at concentrations recommended by the manufactures and an organic acid (acetic acid) in concentrations of 2% and 3%. All the sanitizing agents were tested using 2 variables: time (15 and 30 seconds) and temperature (25°C and 40°C), and were compared with the use of water with no sanitizing agent. In this experiment total counts of mesophilic and psycrophilic aerobic bacteria, molds, yeasts, coliforms and fecal colifoms, were carried out both before applications (to follow contamination from the hatchery laying house) and after 7 and 14 days of storage in order to evaluate the sanitation process. In another experiment the same treatment was applied to a nalidixic acid resistant (100 µg/m1) strain of Salmonella enteritidis in order to better analyse the sanitizing agents. In the laboratory a decrease in egg shell microbial flora of 2-3 logarithmic cycles was observed when quatemary ammonium compounds (QAC) were used. The use of 500/0 QAC diluted by factors of 1/500 and 1/1000 completely eliminated a 104 CFU/ml population of nalidixic acid resistent Salmonella enteritides, this also ocurring with the use of 100 mg/L sodium hipochlorite but not with the other sanitizing agents. This method showed the influence of sanitation in the shelf life of eggs and the importance of analysing the waters used to wash the eggs, emphasizing the routine use of sanitizing agents in laying houses. A contribution to the brazilian legislation related to the washing and use of sanitizing agents in the cleaning of commercial eggs could be made by producing a routine for this process and the necessary concentration of the sanitizing agents / Doutorado / Doutor em Tecnologia de Alimentos

A high frequency chromosomal gene replacement method of general utility was developed for Salmonella enteritidis. This method uses a segregation-deficient temperature-sensitive replicon, pHSG415, as a carrier of the recombinant gene of interest. It allows for site-specific replacement of chromosomal genes without the need for antibiotic resistance markers in the recombinant genes or the use of specific bacterial strains. This gene replacement strategy was used to investigate the foreign antigen-carrying potential of SefA and AgfA, the major fimbrin subunit proteins of Salmonella SEF14 and SEF17 fimbriae. On the basis of epitope mapping and structural predictions, ten different sites within each fimbrin protein were selected for replacement with PT3, an immunoprotective T-cell epitope from the gp63 protein of Leishmania major. PCR-generated sefA and agfA fimbrin genes containing the 48 bp DNA fragment encoding PT3 were used to replace the native fimbrin genes in the chromosome. PCR and DNA sequence analysis confirmed that 10–20% of potential clones contained the corresponding chimeric fimbrin gene. Fimbrial expression and assembly in the chimeric S. enteritidis strains was analyzed by Congo red binding, Western blotting and immunoelectron microscopy using immune serum raised to whole SEF14, whole SEF17 or PT3 peptide. Remarkably, all ten AgfA chimeric fimbrin proteins were expressed under normal conditions and eight were effectively assembled into external SEF17 fimbrial fibers. In contrast, none of the chimeric SefA proteins were expressed and no assembled SEF14 fimbriae were detected. This represents the first fimbrial epitope replacement system in the Salmonellae and the first chimeric fimbrin genes to be reconstituted into a wild-type genetic background. Results are presented from a preliminary vaccine trial in which BALB/c mice were immunized with a PT3-expressing S. enteritidis strain and challenged with virulent L. major friedlin. This model represents a promising “organelle” expression system for epitope display with broad applications as subunit or attenuated vaccines. / Graduate

Magíster en Bioquímica área de Especialización en Bioquímica de Proteínas y Biotecnología / Memoria para optar al Título de Bioquímico / El género Salmonella agrupa a más de 2500 serovares, muchos de los cuales son patógenos humanos y animales. El serovar Dublin afecta principalmente al ganado bovino pudiendo llegar a infectar a seres humanos por el consumo de alimentos contaminados. Salmonella presenta diversos mecanismos que le permiten ingresar y colonizar sistémicamente a sus hospederos. Algunos de estos mecanismos se relacionan con la capacidad de invadir el epitelio intestinal y sobrevivir al interior de las células del sistema inmune del hospedero, principalmente macrófagos. En ambos procesos los sistemas de secreción de proteínas juegan un papel fundamental. El Sistema de Secreción Tipo VI (T6SS) es el sistema de secreción más recientemente descrito en bacterias Gram negativo. Este sistema se encuentra codificado en el genoma de cerca del 25% de las bacterias secuenciadas y su importancia en las relaciones interbacterianas y en la relación bacteria-hospedero lo han convertido en un blanco de estudio de gran interés. Previamente, hemos identificado la presencia de 5 T6SS filogenéticamente distintos y distribuidos diferencialmente en representantes del género Salmonella. S. Dublin posee dos T6SS codificados en las islas de patogenicidad SPI-6 y SPI-19. El objetivo de este proyecto fue determinar si los T6SS codificados en SPI-6 y SPI-19 se expresan al interior de macrófagos murinos y si cumplen un papel en los procesos de invasión y supervivencia intracelular de S. Dublin en estas células. Para esto, se evaluó la expresión intracelular y translocación de un componente fundamental del sistema (VgrG) y se compararon mutantes de los T6SS de SPI-6 y SPI-19 respecto a la cepa silvestre en su capacidad de invadir y sobrevivir en macrófagos murinos en cultivo in vitro. Para evaluar la expresión del gen vgrG y de la proteína VgrG codificada en cada uno de los T6SS presentes en el genoma de S. Dublin al interior de macrófagos, se construyeron en el cromosoma de la bacteria fusiones transcripcionales y traduccionales a la proteína fluorescente verde y se infectaron macrófagos RAW 264.7 con las cepas que contenían estas fusiones. Mediante microscopía de epifluorescencia se observó la expresión de las fusiones transcripcionales y traduccionales del componente VgrG de ambos T6SS desde tiempos tempranos de infección. Para determinar la translocación de VgrG al citoplasma de macrófagos infectados in vitro, se utilizaron fusiones traduccionales de VgrGSPI-6 y VgrGSPI-19 al reportero TEM-1 construidas en el plasmidio pFlagTEM. La translocación de cada proteína de fusión se determinó mediante un ensayo de fluorescencia acoplado a FRET. Para ello, se infectaron macrófagos RAW 264.7 con las cepas que contenían estas fusiones y posteriormente se analizaron los macrófagos infectados mediante microscopía de epifluorescencia. Se observó la translocación de VgrGSPI-19 a tiempos tempranos de infección y la translocación de VgrGSPI-6 a tiempos tardíos de infección. Sin embargo, la baja frecuencia de los eventos de translocación observados hace necesario realizar la comprobación del fenómeno mediante metodologías más sensibles. Para determinar si los T6SS codificados en el genoma de S. Dublin participan en los procesos de internalización y supervivencia de la bacteria al interior de macrófagos murinos, se realizaron ensayos de protección a gentamicina. En ellos se comparó la capacidad de ingresar y sobrevivir al interior de macrófagos RAW 264.7 de la cepa silvestre y cepas mutantes de los T6SS o del componente ClpV. Los resultados obtenidos en este trabajo demostraron que ninguno de los T6SS contribuye a la invasión y supervivencia intracelular de S. Dublin en macrófagos murinos. En conclusión, los T6SS codificados en las islas de patogenicidad SPI-6 y SPI-19 de S. Dublin se expresan al interior de macrófagos murinos, pero no participan en la capacidad de la bacteria para invadir y sobrevivir al interior de éstos. / The Salmonella genus includes over 2500 serovars, many of which are human and animal pathogens. Although the serovar Dublin affects mainly livestock, it constitutes a threat to humans who consume contaminated foods. Salmonella has several mechanisms to colonize its hosts systemically. These mechanisms allow it to invade the intestinal epithelium and survive within immune cells, mainly macrophages. Of note, protein secretion systems play a central role in these processes. The Type VI Secretion System (T6SS) is the most recently described protein secretion system in Gram-negative bacteria. This secretion system is present in about 25% of all bacterial genomes and its putative role in inter-bacteria and bacteria-host relationships makes it an important target for scientific research. Previously we have identified the presence of 5 phylogenetically diverse T6SS distributed differentially in the Salmonella genus. S. Dublin carries two of these systems codified in the pathogenicity islands SPI-6 and SPI-19. The aim of this thesis was to determine if the T6SSs codified in SPI-6 and SPI-19 are expressed in and translocated to the cytosol of murine macrophages, and if they play a role in the internalization and survival of S. Dublin inside these cells. To achieve this, intracellular expression and translocation of a core component of T6SSs (VgrG) was analyzed and T6SS mutants were compared with the parental strain in their ability to invade and survive within murine macrophages in vitro. To asses the expression of the vgrG gene and VgrG protein codified in each S. Dublin T6SS within murine macrophages, transcriptional and translational fusions to the green fluorescent protein were constructed and macrophages RAW 264.7 were infected using the strains bearing these fusions. Using epifluorescence microscopy, expression was observed for the transcriptional and translational fusions of the VgrG component of both T6SSs. To determine the translocation of VgrG to the cytosol of infected macrophages in vitro, translational fusions of VgrGSPI-6 and VgrGSPI-19 to the TEM-1 reporter were constructed in plasmid pFlagTEM. Translocation of each protein was determined using a FRET-coupled fluorescence assay. To achieve this, RAW 264.7 macrophages were infected using strains bearing these fusions and subsequent analysis of the infected macrophages was carried out by epifluorescence microscopy. VgrGSPI-19 translocation was observed shortly after infection and VgrGSPI-6 translocation was observed several hours after infection. Noteworthy, due to the low frequency of the translocation events observed it is necessary to confirm this observation by means of more sensitive methods. To determine if the T6SSs codified in the S. Dublin genome play a role in the internalization and survival processes of the bacteria within murine macrophages, gentamicin protection assays where conducted. In these experiments, the ability to enter and survive in RAW 264.7 macrophages in vitro was compared between T6SS and ClpV mutant strains and the parental strain. The results of this work show that none of the T6SSs contribute to the ability of S. Dublin to invade and survive in murine macrophages. In conclusion, the T6SS encoded in pathogenicity islands SPI-6 and SPI-19 of S. Dublin are expressed in murine macrophages, but these do not play a role in the invasion or intracellular survival of the bacteria in these cells. / Fondecyt

Magíster en Bioquímica área de Especialización en Bioquímica de Proteínas Recombinantes / Memoria de título de bioquímico / Los sistemas de secreción tipo VI (T6SS) corresponden a un mecanismo de interacción célula-célula ampliamente distribuido entre bacterias Gram negativo. Si bien inicialmente al T6SS se le atribuyó un papel en la virulencia de los microorganismos, estudios posteriores dieron cuenta de su versatilidad, indicando que el sistema también toma parte en relaciones mutualistas o comensales entre bacterias y eucariontes, además de relaciones de competencia interbacteriana. Salmonella Typhimurium codifica un T6SS en la isla de patogenicidad SPI-6 (T6SSSPI-6), sin embargo el rol que cumple en la patogénesis de Salmonella aún no ha sido aclarado. Resultados obtenidos en nuestro laboratorio indican que mutantes de este sistema presentan una menor colonización de órganos internos, tanto en ratones BALB/c como en pollos White Leghorn infectados oralmente. Considerando que los componentes celulares del sistema inmune son la principal puerta de entrada de Salmonella para el desarrollo de la infección sistémica, se planteó como hipótesis de este trabajo que “el Sistema de Secreción Tipo VI codificado en la isla genómica SPI-6 de Salmonella enterica serovar Typhimurium se expresa en el interior de macrófagos de origen murino y aviar, favoreciendo la supervivencia bacteriana en estas células”. Para probar esta hipótesis el objetivo fue evidenciar la expresión, funcionalidad y contribución del T6SS durante la interacción de S. Typhimurium con macrófagos murinos y aviares. Para determinar la expresión del T6SS durante la infección de macrófagos, se construyó el vector pLZ01 que permitio la generación de fusiones transcripcionales y traduccionales a la proteína fluorescente verde (GFP) en Salmonella, mediante recombinación homóloga de productos de PCR. De esta manera, se fusionaron componentes estructurales del T6SSSPI-6 (VgrG, Hcp-1, Hcp-2) a GFP y se evaluó su transcripción y traducción en ensayos de infección in vitro mediante microscopía de epifluorescencia. Por otra parte, para determinar si el sistema es translocado al citoplasma de macrófagos durante la infección de S. Typhimurium, se estudió la translocación de una fusión traduccional de VgrG a la β-lactamasa TEM1, construida en el plasmidio pFlagTEM1. La translocación de las fusiones fue determinada mediante un ensayo de pérdida de FRET de la cefalosporina CCF2, observado mediante microscopía de epifluorescencia y cuantificado mediante fluorometría. Finalmente, para determinar la contribución del T6SS en los procesos de internalización y supervivencia en macrófagos se realizaron ensayos de protección con gentamicina. En ellos se comparó la capacidad de la cepa silvestre para invadir y sobrevivir en el interior de macrófagos, versus mutantes que carecen de todo el T6SSSPI-6 o poseen un T6SSSPI-6 no funcional debido a la mutación de clpV, ATPasa esencial para este sistema. Todos los experimentos se realizaron en líneas de macrófagos murinos (RAW264.7) y aviares (HD11), utilizando cepas derivadas de S. Typhimurium 14028s. Los resultados mostraron que ninguno de los componentes estructurales estudiados (VgrG, Hcp-1, Hcp-2) del T6SSSPI-6 de S. Typhimurium se transcribe y traduce en el medio de cultivo celular, sin embargo su transcripción y traducción es gatillada al infectar tanto macrófagos murinos como aviares. A pesar de observar la transcripción y traducción de VgrG, no se detectó su translocación al citoplasma de las células infectadas. Contrariamente a lo esperado, se observó que la presencia del T6SSSPI-6 no contribuye a la supervivencia en el interior de macrófagos murinos o aviares, pero sí tendría una implicancia en la etapa de internalización de Salmonella, puesto que al utilizar mutantes con un T6SSSPI-6 no funcional se observó un fenotipo de mayor internalización en ambos modelos celulares. Estos resultados permiten aceptar una parte de la hipótesis planteada, ya que el Sistema de Secreción Tipo VI codificado en la isla genómica SPI-6 de Salmonella enterica serovar Typhimurium se expresa en el interior de macrófagos de origen murino y aviar, y rechazar una segunda parte de la hipótesis, pues este sistema no tendría un rol en la supervivencia bacteriana en estas células. No obstante, el aumento en la capacidad de internalización de mutantes del T6SS indica que el sistema tendría un rol durante la infección de los macrófagos. / Type VI Secretion Systems (T6SS) correspond to a widely distributed cell-cell interaction mechanism in Gram-negative bacteria. Although initially the T6SS was attributed a role in the virulence of microorganisms, subsequent studies realized its versatility, indicating that this system also takes part in comensal or mutualistic relationships between bacteria and eukaryotes, as well as interbacterial competition. Salmonella Typhimurium encodes a T6SS in the pathogenicity island SPI-6 (T6SSSPI-6), however the role of this island in the pathogenesis of Salmonella has not been clarified. Results obtained in our laboratory indicate that mutants of this system generate a phenotype of reduced colonization of internal organs, both in orally infected BALB/c mice and White Leghorn chicken. Because the initial contact of Salmonella with cellular components of the immune system is the main gateway for the development of systemic infection of Salmonella, the objective of this work was to determine the expression, functionality and contribution of the T6SS during S. Typhimurium interaction with murine and avian macrophages. The vector pLZ01was built to determine the expression of the T6SS during infection of macrophages. This plasmid enables the generation of transcriptional and translational fusions to the green fluorescent protein (GFP) reporter in Salmonella by homologous recombination of PCR products. In this way, structural components of the T6SSSPI-6 (VgrG, Hcp-1, Hcp-2) were merged to GFP and their transcription and translation were assessed by in vitro infection assays and epifluorescence microscopy. On the other hand, to determine whether the system is translocated to the cytoplasm of macrophages during infection of S. Typhimurium, translocation of VgrG was studied using a translational fusion of VgrG to the β-lactamase TEM1, built in the pFlagTEM1 plasmid. The translocation of the β-lactamase fusion was determined by processing of the CCF2/AM fluorescence substrate, detected by epifluorescence microscopy and quantified using fluorometry. Finally, gentamicin protection assays were performed to determine the contribution of the T6SS in the processes of internalization and survival in macrophages. In these experiments, invasion and survive inside macrophages at the wild type strain was compared to a deletion mutant of the T6SS gene cluster and a mutant on the clpV gene, which encodes the ATPase essential for the functioning of the system, All experiments were carried out in murine (RAW264.7) and avian (HD11) macrophage cell-lines, using strains derived from the sequenced wild-type S. Typhimurium 14028s strain. The results showed that none of the studied structural components (VgrG, Hcp-1, Hcp-2) of T6SSSPI-6 of S. Typhimurium are produced in cell culture media, but their transcription and translation are triggered when murine or avian macrophages are infected. Despite observing transcription and translation of VgrG, translocation of this protein into the cytoplasm of infected cells could not be detected. Contrary to expectations, it was observed that the presence of the T6SSSPI-6 did not contribute to Salmonella survival within murine or avian macrophages. However, internalization experiments showed that non-functional T6SSSPI-6 mutants showed a greater uptake into both cellular models. These results indicate that the T6SSSPI-6 of S. Typhimurium is expressed during infection of murine and avian macrophages (the first part of the hypothesis is true), however it did not have an impact on the ability of S. Typhimurium to survive inside murine or avian macrophages (the second part of the hypothesis is false). However, the increase in the internalization of the T6SS mutants suggests a novel role for the T6SS during infection of macrophages. / Fondecyt

Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias / La enfermedad infecciosa causada por Salmonella spp., se denomina salmonelosis, que es una de las enfermedades transmitidas por los alimentos más comunes. El aislamiento e identificación de Salmonella se basa el esquema de Kaufmann-White, y está compuesto por reacciones serológicas que usan anticuerpos contra las aglutininas de LPS, que se demora varios días para obtener los resultados. En este trabajo se ha implementado el método de genotipificación por PCR múltiple de 46 aislados de S. enterica aisladas de cerdos desde 5 planteles comerciales. Este método consiste en dos reacciones de cinco pares de partidores y una reacción de dos pares, basada en seis loci genéticos de S. enterica serovar Typhimurium y cuatro loci de S. enterica serovar Typhi. Como resultado, se determinaron 20 genotipos distintos que se relacionaron al origen de las cepas. Además se determinó la frecuencia relativa de los genes de virulencia spvC (54%); pagK (96%); sirA (96%); gipA(63%); SEN1417(37%); prot6e(0%) y pefA(80%) formando 12 perfiles genéticos y la frecuencia de genes asociados a resistencia antimicrobiana tetA(85%); tetB(7%); tetG(0%); blaPSE-1(0%); blaTEM(72%); blaCMY(0%); aadB(0%) y aacC(0%), constituyendo en total de 6 perfiles. Se construyó la matriz de datos para determinar la diversidad de estas cepas en base a perfiles genéticos asociados a virulencia y resistencia antimicrobiana, en donde se determinó 22 combinaciones o virulotipos distintos, los cuales se agruparon en 2 clústeres relacionados principalmente al origen de las muestras, confirmando así la hipótesis de circulación de múltiples cepas al interior de estos planteles, lo que representa una amenaza para el estatus sanitario de los animales y para la salud pública por su efecto en la inocuidad alimentaria / The infectious disease caused by Salmonella spp., is called salmonellosis, which is a disease transmitted by the most common foods. The isolation and identification of Salmonella is based on the Kaufmann-White scheme, and is composed of serological reactions using antibodies against LPS agglutinins, which lasts several days to obtain the results. In this work, the PCR-based genotyping method has been implemented to analyze 46 S. enterica isolates from pigs belonging to 5 commercial establishments. This method consists of two reactions of five pairs of primers and a two-pair reaction, based on six genetic loci of S. enterica serovar Typhimurium and four loci of S. enterica serovar Typhi. As a result, 20 different genotypes were determined that maintained great closeness between them and their origin. In addition, the relative frequency of virulence genes was determined spvC (54%); pagK (96%); sirA (96%); gipA (63%); SEN1417 (37%); prot6e (0%) and pefA (80%) forming 12 genetic profiles and the frequency of genes associated with antimicrobial resistance tetA (85%); tetB (7%); tetG (0%); blaPSE-1 (0%); blaTEM (72%); blaCMY (0%); aadB (0%) and aacC (0%), constituting a total of 6 profiles. The data matrix was constructed to determine the diversity of these strains based on genetic profiles associated with virulence and antimicrobial resistance, where 22 different combinations or virulotypes were determined, which were grouped into 2 clusters related mainly to the origin of the samples. These results confirm the working hypothesis of circulation of multiple strains within these farms, which represents a threat to the sanitary status of animals and public health due to its effect on food safety

<p>Poznavanje i razumevanje adhezivne sposobnosti i formiranja biofilma patogenih bakterija, kao i njihovog odnosa prema faktorima koji mogu stimulisati ili inhibirati razvoj biofilma, je od fundamentalnog značaja za iznalaženje mera za njihovu efikasnu prevenciju i eliminaciju.<br />Imajući u vidu navedenu činjenicu kao i da je Salmonella enterica serotip Enteritidis epidemiolo&scaron;ki najfrekventniji serotip cilj ovog istraživanja je bio da se ispita: sposobnost različitih sojeva Salmonella Enteritidis izolovanih iz kliničkog materijala, hrane za životinje i odabranog referentnog soja da formiraju biofilm, adherentnost na povr&scaron;ine od stakla i nerđajućeg čelika, sposobnost preživljavanja odabranih biofilm produkujućih sojeva kao i mogućnost primene konfokalne laserske i skening elektronske mikroskopije u vizuelizaciji trodimenzionalne strukture biofilma.<br />Određivanjem morfotipa kolonija na Kongo red agaru na temperaturi inkubiranja od 25&deg;C među testiranim izolatima detektovana su tri morfotipa RDAR (red, dry and rough), BDAR (brown dry and rough) i SAW (smooth and white). Polovina testiranih izolata je eksprimirala RDAR morfotip. Izolati koji su eksprimirali karakterističan morfotip na ovoj temperaturi su formirali na vazduh tečnost međufazi isti tip pelikule.<br />Uporednom analizom rezultata primenjenih skrining testova za utvrđivanje sposobnosti formiranja biofilma pri temperaturi inkubiranja od 25&deg;C ustanovljena je korelacija između pojave određenog morfotipa na Kongo crvenom agaru i sposobnosti formiranja biofilma u kristal violet i pelikula testu. Međutim, sa povećanjem temperature inkubiranja na 37oC, ova korelacija nije ustanovljena, sa izuzetkom izolata SE8.<br />Svi testirani izolati su pokazali sposobnost adherencije na povr&scaron;inu stakla i nerđajućeg čelika, ali u različitoj meri. Na sposobnost adherencije povoljniji uticaj je imala temperatura inkubiranja od 25&deg;C (p&lt;0,05), sa izuzetkom izolata SE3 (p&gt;0,05). Između stepena adherencije izolata na povr&scaron;ine stakla i nerđajućeg čelika nisu ustanovljene statistički značajne razlike (p&gt;0,05).<br />Praćenjem stope preživljavanja tokom 28 dana u uslovima isu&scaron;ivanja evidentirana je znatno veća stopa preživljavanja ćelija izolata RDAR morfotipa u odnosu na stopu preživljavanja ćelija BDAR morfotipa (p&lt;0,05). Praćenjem stope preživljavanja tokom 90 dana u uslovima povremene dostupnosti hranljivih materija, zabeležena je veća stopa preživljavanja u odnosu na stopu preživljavanja u uslovima isu&scaron;ivanja. U uslovima povremene dostupnosti hranljivih materija nakon devedeset dana ispitivanja kod obe grupe izolata procenat vijabilnih ćelija je iznosio vi&scaron;e od 50%.<br />Primenjenim mikroskopskim tehnikama (CLSM i SEM) omogućena je detaljna vizualizacija formiranih biofilmova. Na modelu izolata SERDAR morfotipa, ustanovljeno je da se formiranje biofilma pod primenjenim eksperimentalnim uslovima, odvija u tri faze: 1) inicijalna adhezija za povr&scaron;inu i formiranje manjih ćelijskih agregata (24h); 2) formiranje većih ćelijskih agregata uz produkciju EPS (48h); 3) sazrevanje biofilma uz značajnu produkciju EPS &scaron;to omogućava formiranje stabilne trodimenzionalne strukture biofilma (96h).<br />Nasuprot karakteristikama koje bakterije pokazuju tokom rasta u medijumima koji obiluju hranljivim materijama, bakterije u biofilmovima pokazuju drugačije osobine u pogledu ekspresije gena i karakteristika rasta. Zahvaljujući ovim razlikama, bakterije u biofilmovima pokazuju povećanu rezistenciju na antibiotike i dezinficijense, zbog čega se konstantno razvijaju nove kontrolne strategije u cilju iznalaženja potencijalnih biolo&scaron;kih re&scaron;enja koja pored različitih enzima, faga, antimikrobnih jedinjenja proizvedenih od strane mikroorganizama uključuju i antimkrobna jedinjenja biljnog porekla kao &scaron;to su biljni ekstrakti, etarska ulja i različiti začini. Stoga, je u okviru drugog segmenta ovog istraživanja ispitivan hemijski sastav etarskih ulja, antimikrobni efekat etarskih ulja (O. heracleoticum , O. vulgare , Th. vulgaris i Th. serpyllum) i pojedinačnih komponenti etarskog ulja (karvakrola i timola) na bujonske kulture testiranih sojeva Salmonella Enteritidis kao i uticaj odabranih koncentracija etarskih ulja na inicijalnu adheziju i već formirani biofilm odabranih sojeva Salmonella Enteritidis.<br />Etarska ulja je karakterisao visok zbirni udeo glavnih fenolnih komponenti karvakrola i timola: O. heracleoticum (71,6%), O. vulgare (63,6%), Th. vulgaris (59,77%), Th. serpyllum (40,04%). Etarska ulja su ispoljila antimikrobni efekat sledećim redosledom: O. heracleoticum &gt;O. vugare =Th. vulgaris &gt;Th. serpyllum. Antimikrobni efekat etarskih ulja je bio direktno srazmeran zbiru fenolnih komponenti (karvakrola i timola) u etarskom ulju. U odgovoru na tretman etarskim uljima između izolata S. Enteritidis nisu ustanovljene razlike.<br />Etarska ulja, karvakrol i timol su pokazali inhibitorni efekat na inicijalnu adheziju i posledično na formiranje biofilma testiranih izolata S. Enteritidis na dozno zavisan način. Upoređivanjem uticaja etarskih ulja na inhibiciju inicijalne adhezije i metabolitičke aktivnosti ćelija između izolata RDAR i BDAR morfotipa ustanovljene razlike nisu bile statistički značajne (p&gt;0,05).<br />Ispitivanjem uticaja etarskih ulja, karvakrola i timola na ukupnu biomasu biofilma i metabolitičku aktivnost ćelija dokazano je da etarska ulja u primenjenim koncentracijama ispoljavaju uticaj na redukciju ukupne biomase formiranog biofilma i metaboličke aktivnosti bakterijskih ćelija na dozno zavisan način u funkciji vremena. Znatno veća efikasnost primenjenih tretmana je pokazana u slučaju njihove primene na biofilmove formirane od strane izolata BDAR morfotipa (p&lt;0,05).</p> / <p><span style="font-size:10px;">Knowledge and understanding ability of the pathogenic bacteria that adhere to surface and form biofilm, as well as their relationship between these abilities and factors that stimulate or inhibit biofilm development, are essential to develop strategies for their prevention and elimination.<br />Considering also the fact that Salmonella enterica serotype Enteritidis has been epidemiologically the most frequently found serotype, the aims of this study were to evaluate: biofilm forming ability of several Salmonella Enteritidis strains isolated from clinical material, feed and selected control strain, their ability to adhere to glass and stainless steel surfaces, survival of selected biofilm-producing strains, as well as the possibility of applying confocal laser scanning (CLSM) and scanning electron microscopy (SEM) for visualization of biofilm three-dimensional structure.<br />Determination of colony morphotype on Congo red agar at incubation temperature of 25&deg;C revealed that among all tested isolates three morphotypes were detected: RDAR (red, dry and rough), BDAR (brown dry and rough) and SAW (smooth and white). Half of all tested isolates expressed RDAR morphotype. All isolates that expressed specific morphotype at this incubation temperature also formed the corresponding type of pellicle at air-liquid interface.<br />Comparing the results of the applied assays was ascertained the correlation between specific morphotype on Congo red agar and biofilm forming ability in Cristal violet and pellicle tests, at incubation temperature of 25&ordm;C. In the case of assays conducted at 37&deg;C, this correlation was not established, except for the isolate SE8.<br />All tested isolates showed varying degree of the ability to adhere to glass and stainless steel surfaces. Incubation temperature of 25&ordm;C had more favorable effect on the adherence, with the exception of isolate SE3 (p&gt;0.05). There were no statistically significant differences between adherence ability of all isolates to glass and stainless steel surfaces (p&gt;0.05).<br />Accompaniment of the survival rate during 28 days in the conditions of desiccation, the significantly higher survival rate was</span> <span style="font-size:10px;">obtained for RDAR than BDAR morphotype isolates (p&lt;0.05). Accompaniment of the survival rate during 90 days in the conditions of occasional availability of nutrients, it was detected the higher survival rate than in condition of desiccation. Under these conditions, after 90 days, there were more than 50% of viable cells among both groups of isolates.<br />Applied microscopic techniques (CLSM and SEM) provided detailed visualization of formed biofilms. On model of SERDAR morphotype isolate, it was established that biofilm formation under this experimental conditions has three phases: 1) initial adhesion to the surface and formation of small cell aggregates (24h); 2) formation of large cell aggregates followed with production of extracellular polymer substance (EPS) (48h); 3) maturation of biofilm followed with significant EPS production, which allows formation of stabile three dimensional structure of the biofilm (96h).<br />Contrary to characteristics that bacteria expressed during their growth in the nutrient media, bacteria in biofilms show different properties in terms of genes expression and growth characteristics. Due to these differences, bacteria in biofilms showed higher resistance to antibiotics and disinfectants. For these reasons are being constantly developed new potential biological control strategies that aim at finding the potential biological solutions that besides different enzymes, phages, antimicrobial compounds produced by microorganisms, also include antimicrobial compounds of plant origin, such as extracts, essential oils and different spices.<br />Therefore, the other segment of this research was investigation of the chemical composition and antimicrobial properties of different essential oils (O. heracleoticum, O. vulgare, Th. vulgaris and Th. serpyllum) and their components (carvacrol and thymol), against broth cultures of Salmonella Enteritidis. Also, selected concentrations of essential oils were tested against initial adhesion and preformed biofilm of selected Salmonella Enteritidis isolates.<br />Essential oils were characterized by high amount of phenol compounds carvacrol and thymol: O. heracleoticum (71.6%), O. vulgare (63.6%), Th. vulgaris (59.77%) and Th. serpyllum (40.04%). Essential oils showed antimicrobial potential as follows: O. heracleoticum &gt; O. vugare = Th. vulgaris &gt; Th. serpyllum. Antimicrobial effect was directly proportional to the total content of phenolic components (carvacrol and thymol) in essential oil. Between responses of different S. Enteritidis isolates to essential oil treatment, there was no significant difference.<br />Essential oils, carvacrol and thymol demonstrated inhibitory effect on initial adhesion and consequently, on biofilm formation of S. Enteritidis isolates, in a dose-dependent manner. Comparing influence of essential oil on the inhibition of initial cell adhesion and metabolic activity of cells RDAR and BDAR morphotype, no statistically significant differences were established (p&gt;0.05).<br />Examination of the influence of essential oils, carvacrol and thymol on total biomass of preformed biofilms and metabolic activity of cells, it was revealed that essential oils in applied concentrations cause reduction of total biomass of preformed biofilm and metabolic activity</span> <span style="font-size:10px;">of bacterial cells in a time and dose dependent manner. Applied treatments demonstrated significantly higher efficiency on BDAR morphotype biofilms (p&lt;0.05).</span></p>

Orientador: Raphael Lucio Andreatti Filho / Coorientador: Adriano Sakai Okamoto / Banca: Alessandre Hataka / Banca: Ana Angelita Sampaio / Resumo: A microbiota intestinal das aves exerce papel fundamental no desenvolvimento de seu potencial produtivo por garantir o bom funcionamento do trato gastroentérico e do sistema imunológico a ele intimamente ligado. Por isso diversos aditivos já foram utilizados para favorecer o desenvolvimento de uma microbiota saudável, entretanto, ainda não temos informações sobre o perfil desejável para ela. Sendo assim, nos propusemos a utilizar a Eletroforese em Gel com Gradiente de Temperatura (TGGE), para avaliar a microbiota do ceco de aves alimentadas com dietas acrescidas de halquinol, ácido caprílico e Lactobacillus, e posteriormente desafiadas com Salmonella Enteritidis (SE). Aos 10, 20, 30 e 42 dias de vida foram quantificadas as imunoglobulinas IgA, IgM, IgY e interleucinas 17 e 8, avaliada a colonização cecal de SE (UFC/mL), observação histopatológica de tonsila cecal, e esses resulatados relacionados com a microbiota cecal avaliada pelo método TGGE. O tratamento com ácido caprílico foi igualmente eficaz ao halquinol na diminuição da colonização por SE, seguidos do grupo tratado com Lactobacillus. As concentrações de Il-8 e IgM demonstraram relação com a maturação e desenvolvimento imune local e sistêmico das aves, porém, não identificamos relação com o aumento da complexidade da microbiota, entre os diferentes trtamentos e nos grupos desafiados. No presente estudo também n"ao foi possível reproduzir a técnica de TGGE / Abstract: The intestinal microbiota of birds plays a fundamental role in the development of their production potential to ensure the proper functioning of a gastrointestinal tract and immune system closely linked to it. So many additives have been used to encourage the growth of a healthy microbiota, however, we still have no information about the desired profile for her. So we set out to use the Gel Electrophoresis with Temperature Gradient (TGGE) to assess the cecal microbiota of birds fed diets plus halquinol, caprylic acid and Lactobacillus, and subsequently challenged with Salmonella Enteritidis (SE). At 10, 20, 30 and 42 days old were quantified immunoglobulins IgA, IgM, IgY and interleukins 17:08 assessed cecal colonization IF (CFU / ml), cecal tonsil histopathological observation, and those related to resulatados cecal microbiota evaluated by TGGE method. The treatment with caprylic acid was equally effective in reducing the halquinol colonization by IF, followed by the group treated with Lactobacillus. IL-8 and IgM concentrations shown maturation and compared with the local and systemic immune development of the poultry, however, we do not identify relationship with the increased complexity of the microbiota between different trtamentos and challenged groups. In this study it was also not possible to reproduce the TGGE / Mestre

Se realizó un estudio con la cepa Salmonella enterica, subespecie entérica, serotipo Enteritidis, var. Danysz lisina negativa, principio activo de un producto comercial usado para el control biológico de roedores. Evaluándose su inocuidad en pollos parrilleros. Se usaron 120 aves de 1 día de edad de la linea Cobb Vantress divididas en 3 grupos, con igual porcentaje de hembras y machos. El grupo A fue alimentado los días 5, 6 y 7 de edad con un alimento comercial que contenía 20% del producto raticida. El grupo B fue inoculado al 8vo día de edad directamente al buche con 1 ml. de 108 UFC de la bacteria. El tercer grupo permaneció como control. Se registraron signos clínicos y mortalidad diaria, peso corporal individual semanal, recuperación de la cepa de Salmonella usada y reapuesta serológica. A partir del quinto día postinoculación y durante una semana, dos aves de cada grupo fueron necropsiadas. Adicionalmente se tomaron muestras de sangre a todos los grupos a los 21, 28 y 35 días post inoculación y en aves sacrificadas, para detectar anticuerpos a Salmonella mediante la prueba de aglutinación en placa. Los resultados del estudio bacteriológico, y de las pruebas serológicas realizadas entre el día 5 a 12 post desafio fueron negativos a Salmonella spp. No se encontraron lesiones macroscópicas compatibles con infección por Salmonella, sin embargo a los 28 y 35 días post desafio las aves del grupo A dieron 5.5% a los 28 días (1/18) y 16.6% a los 35 días de edad (3/18) aves positivas a la prueba de aglutinación para Salmonella spp.. las aves que reaccionaron serológicamente fueron sometidas a una evaluación bacteriológica, se aisló Salmonella spp. tipificada como lisina positiva. Los pesos corporales a la sétima semana no mostraron diferencias significativas entre grupos. / The Salmonella enterica serotipe Enteritidis var Danysz negative lysine strain is used as a component of a product for biological control of rodents. This product is a mixture of grains containing the strain. The present study evaluated whether the strain was innocuous for chickens or not. One hundred and twenty day – old Cobb Vantress broiler chicks (50% each sex) were divided into 3 groups of 40 chicks each. Four chicks per group were separated to verify if they were free from Salmonella spp. The group A was fed with standard broilers food containing 20% of the product for biological rodent control. The group B received 1 ml containing 108 CFU of Salmonella enterica serotipe Enteritidis var Danysz negative lysine by crop gavage at eighth day. The group C was kept as uninoculated control group. Clinical signs, mortality, recovery strain used in the experiment, body weight, and serological response were recorded. Chicks were observed daily for clinical signs and mortality. Bacteriological cultures were made in order to recover the microorganism from liver and spleen. All birds were weighed every week. Two chicks were randomly selected from each treatment group, euthanatized, and necropsied since the fifth day during one week. Serum samples and select tissues (liver and spleen) were collected for bacteriological culture. Additionally, serum samples were collected from each group in the 21st , 28th and 35th days postinoculation. The results of bacteriological work of select tissues (direct and indirect cultures) and the serological study of serum samples tested negative for Salmonella spp. Positive birds to the agglutination test to Salmonella spp. were found only in the group A. It was found 5.5% at 28 days and 16.6% at 35 days of age. The positive birds were submitted to a bacteriological evaluation; in one of them Salmonella spp was isolated and typified by biochemistry as positive lysine. Significant weight differences among groups recorded in the seventh week, were not found.