Dissertations / Theses on the topic 'Salmonella'

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Foi desenvolvido um ensaio imunoenzimático do tipo ELISA indireto para a detecção de resposta sorológica de aves para Salmonella sorotipos Gallinarum, Pullorum, Enteritidis e Typhimurium. Utilizou-se antígeno solúvel obtido por meio de sonicação de cultura de Salmonella Gallinarum (AgSG), Salmonella Enteritidis cepa aflagelar (AgSE) e Salmonella Typhimuirum cepa aflagelar (AgTM), os conjugados peroxidase e fosfatase alcalina e amostras de soros positivos e negativos de vários sorotipos de salmonelas. Os resultados demonstraram que o AgSG pode ser utilizado diluído a 1:25.000 (peroxidase e fosfatase alcalina). Observou-se que o ELISA contendo S. Gallinarum como antígeno e fosfatase alcalina como enzima, propicia a separação de reações positivas para Gallinarum e Pullorum de Enteritidis. O AgSE pode ser utilizado diluído a 1:10.000 (peroxidase) ou 1:5.000 (fosfatase alcalina). Nestas condições, o ELISA/AgSE detectou resposta sorológica para os sorotipos Enteritidis, Gallinarum e Pullorum. O ELISA com o AgTM demonstrou que o antígeno pode ser diluído a 1:20.000 para ambos os conjugados. O ELISA/AgTM demonstrou reatividade entre salmonelas dos grupos B e D. Todas as amostras de soros testes devem ser analisadas diluídas a 1:1.000. Concluindo, o ELISA mostrou-se um teste útil para identificar aves com reação sorológica contra S. Gallinarum, S. Pullorum, S. Enteritidis e S. Typhimurium, podendo ainda identificar aves com sorologia positiva para S. Gallinarum, S. Pullorum sem que haja reação cruzada com amostras de soro de aves vacinadas ou infectada por S. Enteritidis.
This study was done to assess the enzyme-linked immunosorbent assays (ELISA) for detection chicken serologic response against Salmonella enterica sorotypes Gallinarum, Pullorum, Enteritidis and Typhimurium. The test was performed using soluble proteins from Salmonella Gallinarum strain 9 (AgSG), from non-flagellate Salmonella Enteritidis strain (AgSE) and from not flagellate Salmonella Typhimurium (AgTM) strain as detecting antigen and peroxidase and alkaline phosphatase enzymes, as conjugate. According to the results, the antigen has to be diluted at 1:25.000 (AgSG, peroxidase and alkaline phosphatase). In addition, using alkaline phosphatase enzyme, the assay was helpful to separate positive serological reaction to serotypes Gallinarum and Pullorum from Enteritidis. To the ELISA/AgSE, the antigen has to be diluted at 1:10.000 for peroxidase assay and at 1:5.000 for alkaline phosphatase assay. In this condition, the ELISA/AgSE can detect serological reaction to S. Enteritidis, S. Gallinarum and S. Pullorum. To the ELISA/AgTM the antigen has to be diluted at 1:20.000 to both enzymes. In this condition the ELISA/AgTM showed sensibility but was no possible to separate positive serological reaction to serotype concerning at the group B and group D. In all test, the sample of serum has to be diluted at 1:1.000. Therefore, the ELISA was able to identity reactors birds to Salmonella antigens and also to detect serological response to S. Gallinarum, S. Pullorum antigen with no cross-reaction with serum samples taken from birds either challenged or vaccinated against S. Enteritidis.

Resumo: Foi desenvolvido um ensaio imunoenzimático do tipo ELISA indireto para a detecção de resposta sorológica de aves para Salmonella sorotipos Gallinarum, Pullorum, Enteritidis e Typhimurium. Utilizou-se antígeno solúvel obtido por meio de sonicação de cultura de Salmonella Gallinarum (AgSG), Salmonella Enteritidis cepa aflagelar (AgSE) e Salmonella Typhimuirum cepa aflagelar (AgTM), os conjugados peroxidase e fosfatase alcalina e amostras de soros positivos e negativos de vários sorotipos de salmonelas. Os resultados demonstraram que o AgSG pode ser utilizado diluído a 1:25.000 (peroxidase e fosfatase alcalina). Observou-se que o ELISA contendo S. Gallinarum como antígeno e fosfatase alcalina como enzima, propicia a separação de reações positivas para Gallinarum e Pullorum de Enteritidis. O AgSE pode ser utilizado diluído a 1:10.000 (peroxidase) ou 1:5.000 (fosfatase alcalina). Nestas condições, o ELISA/AgSE detectou resposta sorológica para os sorotipos Enteritidis, Gallinarum e Pullorum. O ELISA com o AgTM demonstrou que o antígeno pode ser diluído a 1:20.000 para ambos os conjugados. O ELISA/AgTM demonstrou reatividade entre salmonelas dos grupos B e D. Todas as amostras de soros testes devem ser analisadas diluídas a 1:1.000. Concluindo, o ELISA mostrou-se um teste útil para identificar aves com reação sorológica contra S. Gallinarum, S. Pullorum, S. Enteritidis e S. Typhimurium, podendo ainda identificar aves com sorologia positiva para S. Gallinarum, S. Pullorum sem que haja reação cruzada com amostras de soro de aves vacinadas ou infectada por S. Enteritidis.
Abstract: This study was done to assess the enzyme-linked immunosorbent assays (ELISA) for detection chicken serologic response against Salmonella enterica sorotypes Gallinarum, Pullorum, Enteritidis and Typhimurium. The test was performed using soluble proteins from Salmonella Gallinarum strain 9 (AgSG), from non-flagellate Salmonella Enteritidis strain (AgSE) and from not flagellate Salmonella Typhimurium (AgTM) strain as detecting antigen and peroxidase and alkaline phosphatase enzymes, as conjugate. According to the results, the antigen has to be diluted at 1:25.000 (AgSG, peroxidase and alkaline phosphatase). In addition, using alkaline phosphatase enzyme, the assay was helpful to separate positive serological reaction to serotypes Gallinarum and Pullorum from Enteritidis. To the ELISA/AgSE, the antigen has to be diluted at 1:10.000 for peroxidase assay and at 1:5.000 for alkaline phosphatase assay. In this condition, the ELISA/AgSE can detect serological reaction to S. Enteritidis, S. Gallinarum and S. Pullorum. To the ELISA/AgTM the antigen has to be diluted at 1:20.000 to both enzymes. In this condition the ELISA/AgTM showed sensibility but was no possible to separate positive serological reaction to serotype concerning at the group B and group D. In all test, the sample of serum has to be diluted at 1:1.000. Therefore, the ELISA was able to identity reactors birds to Salmonella antigens and also to detect serological response to S. Gallinarum, S. Pullorum antigen with no cross-reaction with serum samples taken from birds either challenged or vaccinated against S. Enteritidis.
Orientador: Angelo Berchieri Júnior
Coorientador: Hélio José Montassier
Banca: Raul José Silva Girio
Banca: Fernando Antonio de Ávila
Banca: Paulo Lourenço da Silva
Banca: Ana Maria Iba Kanashiro
Doutor

Salmonella are Gram-negative facultative intracellular pathogens that cross the intestinal barrier, and are taken up by phagocytes where, they can replicate and spread to systemic sites. Salmonella encode two type III secretion systems, Salmonella pathogenicity island 1 and 2 (SPI1 and SPI2), which mediate the translocation of bacterial effectors into the host cell. SPI1 facilitates bacterial uptake into non-phagocytic cells and is involved in forming a special replicative niche, called the Salmonella containing vacuole (SCV). SPI2 is required for maintenance of the SCV, macrophage replication and systemic disease. A comprehensive study of the contribution of individual SPI2 effectors to virulence had not been previously done, and was therefore performed. Strains deficient in specific SPI2 genes were tested for alterations in virulence in a mouse model of typhoid fever, and in epithelial and macrophage cell infections. These experiments showed that many SPI2 effectors are required for replication in macrophages, and that ΔspvB, ΔssaR, and ΔspiC strains were attenuated in mice. Salmonella infection causes many perturbations to the host, including changes in metabolites, specifically arachidonic acid metabolism, which leads to the production of eicosanoids. The effects of Salmonella infection of macrophages on eicosanoids were examined. Salmonella infection increased the expression of prostaglandin synthases, but decreased thromboxane and leukotriene synthases. The SPI2 deletion strains were tested to determine involvement of SPI2 in arachidonic acid metabolism. The SPI2 effectors SseF and SseG, which are largely uncharacterized in macrophage infections, were mainly responsible for the induction of prostaglandins. The effects of prostaglandins on Salmonella infection were studied. It was found that 15-deoxy-Δ12,14-prostaglandin-J2 (15d-PGJ2) significantly reduced Salmonella colonization of macrophages, but not epithelial cells. Furthermore, this occurs independently of SPI1, SPI2, and PPAR-γ. 15d-PGJ2 reduces cytokines and reactive nitrogen species produced by infected macrophages. A role for 15d-PGJ2 in Salmonella infection has not been previously demonstrated. This thesis examines the role of SPI2 in Salmonella virulence and arachidonic acid metabolism.

Thesis (Ph.D.) - University of Glasgow, 2008.
Ph.D. thesis submitted to the Department of Infection and Immunity, Faculty of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

Découverte il y a plus d’un siècle, Salmonella n’a cessé d’intriguer les chercheurs. Sa capacité à résister à de nombreux antibiotiques est de plus en plus préoccupante. La surveillance de ce pathogène repose sur un typage rapide et discriminant de façon à identifier le plus précocement possible les sources alimentaires contaminées. Les méthodes classiques sont longues, lourdes et non automatisables. Comprendre l’émergence et l’évolution des Salmonella est la clé pour éradiquer ce pathogène resté l’une des premières causes de diarrhées bactériennes d’origine alimentaire dans le monde. Au cours des dernières décennies, des progrès spectaculaires ont été menés dans le monde de la microbiologie avec l’arrivée des séquenceurs de paillasse, passant du traitement d’une dizaine à des centaines de millions de séquences. L’accès facilité aux séquences génomiques et aux outils qui leurs sont dédiés sont devenus une nécessité. Les outils actuellement disponibles ne sont pas assez discriminants pour sous-typer S. enterica sérotype Typhimurium (STM), sérotype prédominant de Salmonella. Nous avons voulu lors de ce travail, montrer l’intérêt du séquençage entier du génome, pour l’étude génomique de Salmonella. (1) Après avoir séquencé plus de 300 génomes de STM, nous avons mis au point un outil de sous-typage in silico de ce sérotype, basé sur le polymorphisme des CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). La surveillance à haut débit des salmonelloses a été validée en routine sur plus de 800 génomes. L’étude de la coévolution entre le chromosome (SNPs) et les régions CRISPR ont permis d’établir une nomenclature définissant les différentes populations de STM. (2) L’analyse génomique de 280 souches historiques de STM a montré que les gènes de bêta-lactamase conférant une résistance à l’ampicilline et portés par des plasmides étaient répandus chez STM à la fin des années 1950, bien avant l’utilisation de cet antibiotique. La présence de la pénicilline G dans le milieu agricole où ces composés ont été utilisés en tant que promoteurs de croissance ont pu conduire à la sélection des premières souches résistantes à l’ampicilline. (3) L’étude phylogénétique d’un génome issu du cadavre d’une femme décédée il y a plus de 800 ans, probablement à cause de la fièvre entérique et de 219 génomes historiques et récents des sérotypes Paratyphi C, Choleraesuis et Typhisuis ont montré que leurs génomes étaient très similaires au cours des 4000 dernières années. Ainsi, la combinaison des approches génotypique et phylogénétique ont accru nos connaissances sur l’évolution de ce pathogène.Mots clés : Séquençage entier du génome, surveillance épidémiologique, CRISPR, SNP, résistance antibiotique, phylogénie, évolution
Over a century has passed since the discovery of Salmonella and yet, this pathogen still intrigues researchers. Its ability to withstand many antibiotics is of increasing concern. The monitoring of this pathogen is based on a rapid and discriminatory typing to identify the sources of contaminated food as early as possible. The conventional methods are long, heavy and non-automatable. Understanding the emergence and evolution of Salmonella is the key to eradicate this pathogen, which has remained one of the leading causes of foodborne bacterial diarrhea in the world. During the last decades, spectacular progress has been made in the world of microbiology with the arrival of workbench sequencers, passing from a dozen to hundreds of millions of sequences processed. Facilitated access to numerous genome sequences and dedicated tools are mandatory. Tools currently available are not sufficiently discriminating for the subtype of S. enterica serotype Typhimurium, a predominant serotype of Salmonella. Throughout this study, we showed the interest of whole genome sequencing, a multidisciplinary tool, for the genomic study of Salmonella. (1) After sequencing over 300 S. enterica serotype Typhimurium genomes, we have developed an in silico subtyping tool for this serotype, based on the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) polymorphism. High-throughput microbiological monitoring of salmonellosis has been routinely validated on over 800 genomes. The study of coevolution between the chromosome (SNPs of the core genome) and the two CRISPR regions made it possible to establish a nomenclature defining the different populations of this serotype. (2) Genomic analysis of 280 historical strains of S. enterica serotype Typhimurium showed that plasmids carrying beta-lactamase genes, which confer resistance to ampicillin, were widespread within this serotype in the late 1950s, years before ampicillin was first used for clinical purposes. The presence of penicillin G in the farming environment where these compounds were used as growth promoters, may have led to the selection of the first ampicillin-resistant strains. (3) The phylogenetic study of a genome from the corpse of a young woman who died over 800 years ago, probably due to enteric fever, and 219 historical and recent genomes of the serotypes Paratyphi C, Choleraesuis and Typhisuis have shown, despite the differences in host specificity, that their genomes were very similar over the past 4000 years. Thus, the combination of genotypic and phylogenetic approaches has increased our knowledge of the evolution of this pathogen.Key words: Whole genome sequencing, epidemiological monitoring, CRISPR, SNP, antibiotic resistance, phylogeny, evolution

Of the two currently available typhoid fever vaccines, one is potentially inappropriate for immuno-compromised individuals while the other could be rendered ineffective as the pathogen evolves. This work addresses these shortcomings and explores the possibility of identifying novel antigens for use in a multiple subunit vaccine. S. enterica serovar Typhimurium infection of mice is a well-established animal model of human typhoid fever. Phage display is a method of generating particles that embody a physical link between a given DNA sequence and the polypeptide it encodes. In this work, a phage display library was constructed from random fragments of Salmonella genomic DNA. Hyper-immune serum from infected mice was used for affinity selection of potential antigens from this library in a process called biopanning. Individual clones encoding potential antigens were subjected to further screening alongside a positive and negative control, and 10 were consistently positive. Sequences encoding the potential antigens were then sub-cloned into an expression vector encoding a His tag for affinity purification.  Six of the ten sub-clones could not be induced to express the fusion peptide. Poor induction of the remaining four led to multiple purification steps. The four potential antigens were then tested by Western blot and ELISA alongside the same positive control for antigenicity. It was then found that the positive control in itself was poorly antigenic and the four candidates were not antigens. Possibilities for modification of the original affinity selection process, as well as phage-displayed positive control antigens are discussed.

In an increasingly hygiene concerned society, a major barrier to pet ownership is the perceived role of companion animals in contributing to the risk of exposure to zoonotic bacterial pathogens, such as Salmonella. Manifestations of Salmonella can range from acute gastroenteritis to perfuse enteric fever, in both humans and dogs. Dogs are heavily associated with asymptomatic carriage of Salmonella as the microorganism can persist in the lower intestines of this host which can be then excreted into the environment. Studies in to the asymptomatic carriage of Salmonella in dogs are somewhat dated and there is limited UK data. The current UK carriage rate in dogs was investigated in a randomised dog population and it was revealed that the carriage rate in this population was very low with only one household dog positive for the carriage of Salmonella enterica arizonae (0.2%), out of 490 dogs sampled. Salmonella serotypes share phenotypic and genotypic similarities which are captured in epidemiological typing methods. Therefore, in parallel to the epidemiological investigations, a panel of clinical canine (VLA, UK) and human (Aston University, UK) Salmonella isolates were profiled based on their phenotypic and genotypic characteristics; using API 20E, Biolog Microbial ID System, antibiotic sensitivity testing and PFGE, respectively. Antibiotic sensitivity testing revealed a significant difference between the canine and human isolates with the canine group demonstrating a higher resistance to the panel of antibiotics tested. Further metabolic capabilities of the strains were tested using the Biolog Microbial ID System, which reveal no clear association between the two host groups. However, coupled with Principle Component Analysis two canine isolates were discriminated from the entire population on the basis of a high up-regulation of two carbohydrates. API 20E testing revealed no association between the two host groups. A PFGE harmonised protocol was used to genotypically profile the strains. A dendrogram depicting PFGE profiles of the panel of Salmonella isolates was performed where similarities were calculated by Dice coefficient and represented by UPGMA clustering. Clustering of the profiles from canine isolates and human isolates (HPA, UK) was diverse representing a natural heterogeneity of the genus, additionally, no clear clustering of the isolates was observed between host groups. Clustering was observed with isolates from the same serotype, independent of host origin. Host adaption is a common phenomenon in certain Salmonella serotypes, for example S. Typhi in humans and S. Dublin in cattle. It was of interest to investigate potential host adaptive or restricted strains for canine host by performing adhesion and invasion assays on Dog Intestinal Epithelial Cells (DIECs) (WALTHAM®, UK) and human CaCo-2 (HPA, UK) cell lines. Salmonella arizonae and Enteritidis from an asymptomatic dog and clinical isolate, respectively, demonstrated a significantly high proportion of invasion in DIEC in comparison to human CaCo-2 cells and other tested Salmonella serotypes. This may be suggestive of a potential host restrictive strain as their ability to invade the CaCo-2 cell line was significantly lower than the other serotypes. In conclusion to this thesis the investigations carried out suggest that asymptomatic carriage of Salmonella in UK dogs is low however the microorganism remains as a zoonotic and anthroponotic pathogen based on phenotypic and genotypic characterisation however there may be potential for particular serotype to become host restricted as observed in invasion assays

Magíster en Bioquímica en el área de especialización de Bioquímica Toxicológica y Diagnóstico Molecular
Memoria para optar al Título de Bioquímica
El género Salmonella comprende dos especies, S. enterica y S. bongori, que en conjunto agrupan a más de 2.500 serovares. De éstos, los pertenecientes a S. enterica subespecie enterica son responsables de aproximadamente el 99% de los casos de salmonelosis en animales de sangre caliente. A nivel mundial se producen anualmente millones de casos de salmonelosis en el ser humano y miles de muertes, principalmente en países subdesarrollados. En esta tesis se propuso identificar un conjunto de genes requeridos para la colonización sistémica de un hospedero murino por tres serovares de Salmonella: S. Typhi, S. Typhimurium y S. Enteritidis. Este estudio se realizó mediante un análisis masivo de mutantes bajo selección negativa in vivo. La detección de aquellas mutantes con defectos en la colonización sistémica aguda de ratones BALB/c se realizó mediante hibridaciones comparativas utilizando un microarray genómico de Salmonella. El posterior análisis comparativo de las mutantes bajo selección negativa in vivo en los tres serovares, nos permitió identificar que mutantes en 131 genes serían atenuadas in vivo. Dentro de este grupo identificamos genes codificados en islas de patogenicidad conservadas del género Salmonella, genes necesarios para la biosíntesis de purinas y compuestos aromáticos (aro, pur y gua), genes relacionados con la biosíntesis y modificación del LPS (rfa, rfb) y genes que codifican reguladores globales asociados a patogenicidad (phoP, envZ, rpoN, dam y rsd). Otros genes identificados corresponden a los que codifican el sistema transportador de proteínas Twin-Arginine (tatABC), genes que codifican las diferentes subunidades de una NADH deshidrogenasa (genes nuo); un locus que corresponde a un transportador de péptidos del tipo ABC (sapBF). También pudimos detectar que mutantes en genes involucrados en el transporte de solutos se encuentran bajo selección, como trkH que codifica un transportador de potasio. El sistema de transporte Twin-Arginine corresponde a una de las dos vías de translocación de proteínas hacia el espacio periplasmático en bacterias Gram negativo. La participación de este sistema en la patogenicidad de Salmonella se confirmó mediante ensayos de competencia in vivo entre mutantes definidas del operón y la respectiva cepa silvestre en los tres serovares estudiados. El análisis global de mutantes en tres serovares nos permitió determinar un conjunto de genes comunes necesarios para establecer la colonización sistémica aguda en un hospedero murino. Posteriormente, se confirmó la participación del sistema de transporte de proteínas Tat en la patogenicidad de Salmonella. Los resultados de los ensayos de competencia nos permitieron confirmar la predicción obtenida en el análisis de masivo de mutantes bajo selección negativa in vivo.
The Salmonella genus comprises two species: S. bongori and S. enterica, which can be grouped into more than 2,500 serotypes. Serovars within S. enterica subspecies enterica account for ~99% of all salmonellosis in warm-blooded animals. Worldwide, these organisms are responsible for hundreds of millions of salmonellosis cases and hundreds of thousands of deaths, mainly in underdeveloped countries. In this thesis, we aimed to identify a group of genes required for systemic colonization of a murine host by three Salmonella serotypes: S. Typhi, S. Typhimurium and S. Enteritidis. We used a high-throughput microarray-based screening for mutants with defects in systemic colonization of BALB/c mice. Subsequent comparative analysis of mutants under negative selection in vivo allowed us to identify that mutants in 131 genes are attenuated in the three serotypes under study. Within this group we found genes encoded in some of the pathogenicity islands conserved in the Salmonella genus, genes required for biosynthesis of purines and aromatic compounds (aro, pur and gua), genes related to LPS biosynthesis (rfa and rfb) and genes encoding regulators previously associated with virulence (phoP, envZ, rpoN, dam and rsd). Other genes identified are those encoding the Twin-Arginine transport system (tatABC), genes coding the different subunits of a NADH dehydrogenase (nuo genes) and a locus encoding an ABC peptide transporter (sapBF). We also identified that mutants in genes involved in solute transport (i.e: trkH, that encodes a potassium transporter) are under negative selection in vivo. The Twin-Arginine transport system corresponds to one of the two pathways used by Gram-negative bacteria to translocate proteins to the periplasmatic space. Participation of this system in Salmonella pathogenicity was confirmed in the three serotypes under study by means of in vivo competition assays between targeted mutants of the operon and the corresponding wild-type strains. Overall, the global analysis of mutants under negative selection in vivo in three serotypes of Salmonella allowed us to identify a common group of genes required to establish acute systemic colonization of a murine host. We confirmed the participation of the Tat transport system in the pathogenicity of Salmonella using in vivo competition assays. These results further support the predictions obtained in our global analysis.
Fondecyt

Nous avons décrit la présence de tubules inter-cellulaires [inter-cellular tubules (ICTs)], qui apparaissent entre les deux cellules filles lors de la cytokinèse d’une cellule infectée. Nos données suggèrent que ces structures sont des vestiges de SITs qui connectaient les SCVs initialement présentes dans la cellule mère et qui ont été distribuées dans les cellules filles. Les effecteurs de T3SS2 sont nécessaires à la formation de ces tubules. De plus, nous avons établit une corrélation entre la formation des ICTs et la distribution asymétrique des vacuoles bactériennes entre les cellules filles. Les protéines effectrices de T3SS2 peuvent donc modifier la distribution des bactéries pendant la cytokinèse. Il a été démontré l’existence de différents types de SITs, de composition différentes et qui interagissent avec différents compartiments de la cellule hôte. Pendant la deuxième partie de ma thèse, nous avons caractérisé des tubules dépourvus de protéines de l’hôte mais riches en protéines effectrices [LAMP1-negative tubules (LNT)]. Nous avons montré que les effecteurs de T3SS2 SseF et SseG sont nécessaires à la formation de ces structures. L’inhibition de la formation des LNTs par la suppression de sseF/G est corrélée à un recrutement réduit de LAMP1 sur les SCVs. Cela suggère que la formation des tubules favorise la capture et le transport de LAMP1 vers les SCVs. Nous avons également observé une interaction indépendante de SKIP entre Arl8b et les deux domaines de l’effecteur SifA. En absence de SifA ou de Arl8b, les tubules ont une capacité limitée à capturer les protéines membranaires lysosomales
We describe the presence of inter-cellular tubules (ICTs) that arise between daughter cells during cytokinesis of an infected cell. Our data suggest that these structures are remnants of SITs that connect bacterial vacuoles originally present in the parent cell and that have been distributed between daughters. T3SS-2 effectors are required for the formation of these tubules. Importantly, there is a correlation between the formation of ICTs and the asymmetric distribution of bacterial vacuoles in daughters. Thus, T3SS-2 effector proteins can manipulate the distribution of bacteria during cytokinesis. This may further increase bacterial spreading and the systemic character of the infection. Different kinds of SITs with diverse host protein contents have been characterised, suggesting the capacity of these tubules to interact with different host compartments. In the second part of my thesis, we performed a biochemical and functional characterization of LAMP1-negative tubules (LNT) that are decorated with effector proteins but essentially devoid of host proteins. We show that T3SS2 effectors SseF and SseG are required for the formation of these structures. The inhibition of LNTs formation by deletion of sseF/G is correlated with a reduced recruitment of LAMP1 to the SCVs. It suggests that formation of tubules favours the capture and the transport of LAMP1 towards the SCV to keep vacuole stable. An additional observation added to this study is that there is a SKIP-independent interaction between Arl8b and both domains of SifA. In the absence of SifA or Arl8b, tubules have a limited capacity to capture host membrane proteins from the late endosomal compartments

Salmonella outbreaks associated with orange juices have been reported in the past. Though there have been studies on the internalization of Salmonella into oranges there is inadequate information on the ability of this pathogen to survive on orange surfaces, become internalized, and survive the low pH internal conditions. The objective of this work was to study the survival of Salmonella gaminara and Salmonella agona on oranges obtained from the field and retail outlets and investigate their attachment and internalization potential. These studies showed that oranges obtained from both the field and retail outlets harbored relatively high concentrations of aerobic heterotrophic bacterial populations. There were significant differences in the survival of Salmonella agona and Salmonella gaminara at 4??C, room temperature (25??C) and 37??C. Survival was highest at 37??C and lowest at 4??C for both Salmonella gaminara and Salmonella agona. Salmonella agona and Salmonella gaminara showed significant differences in recovery when the cells were treated with pH 4.0, 7.0 and 9.5 buffers. The internalization studies suggest that a negative temperature differential favors the internalization of Salmonella cells into the fruit. Significant differences in the internalization of Salmonella into field and market oranges were observed with more internalization in the field oranges as compared to the market oranges. These results suggest that to prevent Salmonella contamination of orange juices adequate pre-harvest protection against pathogen contamination and post-harvest cleaning and disinfection strategies need to be employed.

Orientador: Marcelo Brocchi
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Salmonella enterica é uma bactéria Gram-negativa classificada em diferentes sorovariedades que podem causar desde gastroenterites a infecções sistêmicas. A sorovariedade Enteritidis é predominante nos casos de salmonelose em humanos, tendo produtos derivados do frango como principal fonte de infecção. Uma forma de se controlar infecções por Enteritidis é através da vacinação de frangos, uma estratégia já utilizada, porém com limitações, pois estas vacinas muitas vezes são inativadas ou possuem origem de atenuação desconhecida. Outra limitação é a falta de estudos específicos para Enteritidis, pois maior parte dos estudos feitos com S. enterica se baseiam na sorovariedade Typhimurium. Um alvo para a construção de linhagens vacinais são os genes codificadores de Nucleoid Associated Proteins que são proteínas que se ligam ao DNA alterando sua topologia, afetando a transcrição global dos genes. Neste projeto realizamos a construção de mutantes nulos de S. enterica Enteritidis para alguns destes genes com a finalidade de avaliar seu potencial vacinal e papel na patogênese. As linhagens foram testadas no modelo de infecção sistêmica e de inflamação do ceco. No modelo de infecção sistêmica, a linhagem selvagem e ?fis se apresentaram virulentas ou pouco atenuadas enquanto as linhagens ?ihfA e ?ihfB foram atenuadas. Os testes de proteção foram feitos com os dois mutantes atenuados que induziram 100% de proteção. A linhagem selvagem e o mutante pouco atenuado induziram inflamação neste modelo, mas o mutante induziu de forma mais amena. Analises morfométricas futuras irão elucidar com mais clareza o papel deste gene na inflamação. Este projeto teve como principais realizações: (i) a construção de duas linhagens atenuadas, com alto potencial para uso vacinal e (ii) abriu novas possibilidades para o estudo nas NAP's durante a patogênese de diferentes sorovariedades
Abstract: enterica is a Gram-negative bacterium classified in different sorovars which may causes gastroenteritis and systemic infections. The serovar Enteritidis is responsible for most of the cases of salmonellosis in humans and have poultry based products as its main source of infection. Poultry vaccination has been and effective strategy to control Enteritidis infections and it's already applied, but with limitations, because the vaccines sometimes are inactivated or are attenuated by unknown mechanisms. Another limitation is the lack of studies especific to Enteritidis, most of the research related to S. enterica is based on serovar Typhimurium. A target to vaccine strains development are the genes members of the group called Nucleoid Associeted Proteins, which are proteins that bind to DNA changing its topology and affecting global gene transcription. In this project, we constructed S. enterica null mutants to some of these genes aiming the evaluation of their vaccine potential and and role in pathogenesis. The strains were tested with the systemic infection model and cecum inflamation. In the systemic infection model, the wild and ?fis strain were virulent while the ?ihfA and ?ihfB were attenuated. The protection essays were made with attenuated mutants and provided 100% of protection. The wild and ?fis strains lead to inflammation in this model, but the mutant induced a mild inflammation, morfometrics analysis will clarify the role of this gene in inflammation. This project have as main outcomes: (i) the construction of strains, ?ihfA and ?ihfB, with good potential to be used as vaccines; (ii) New possibilities to the role of fis gene during inflammation
Mestrado
Microbiologia
Mestre em Genética e Biologia Molecular

Orientador: Marcelo Brocchi
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O gênero Salmonella spp é formado por bacilos gram-negativos, que podem ser divididos em 3 espécies: S. enterica, S. bongori e S. subterranea. A maioria das sorovariedades patogênicas para o homem está incluída no subgrupo I da espécie S. enterica. A infecção por S. enterica inicia-se com a ingestão de água ou alimentos contaminados. Estes microrganismos são patógenos intracelulares facultativos e, uma vez ingeridos, apresentam a capacidade de aderir e invadir células da mucosa intestinal, preferencialmente células M. Uma vez ultrapassada a mucosa intestinal, S. enterica invade, persiste e prolifera no interior de vacúolos de células do sistema retículo endotelial podendo assim, alcançar diferentes órgãos e tecidos do hospedeiro, causando infecção sistêmica. Sendo assim, linhagens mutantes avirulentas, mas ainda capazes de causar infecção transitória, são boas candidatas a potenciais vacinas vivas orais. Tais mutantes são potenciais carreadores de proteínas heterólogas, compondo, assim, as chamadas vacinas multi-valentes. Que seja de nosso conhecimento, não existem mutantes atenuados de S. enterica desenvolvidos inteiramente no Brasil, havendo necessidade de pagamento de patentes a grupos estrangeiros para sua utilização. Em eucariotos, o DNA cromossômico bacteriano está associado a proteínas (histonas) formando o núcleo, enquanto em procariotos, estas proteínas são denominadas histona-like, formando um nucleóide. Dentre essas proteínas podemos citar a IHF (integration host factor), um heterodímero que controla ou influencia vários processos celulares, como a duplicação e recombinação do DNA, além de regular positiva ou negativamente a expressão de vários genes. Neste estudo, mutantes nulos para os genes himA e himD de IHF foram criados pela técnica de recombinação homóloga mediada pelo sistema ? Red (Datsenko e Wanner, 2000) e testados quanto a atenuação da virulência e capacidade de desencadear resposta imune efetiva e protetora contra a salmonelose murina. Os mutantes também foram caracterizados quanto a diversas características biológicas, como a capacidade de invasão e sobrevivência intracelular, resistência a radicais reativos de oxigênio e nitrogênio, ente outras, sendo os resultados comparados com as respectivas linhagens selvagens. Os mutantes himA e himD de S. enterica foram atenuados e capazes de induzir resposta imune protetora quando desafiados com doses elevadas da linhagem selvagem, indicando que estas linhagens recombinantes são potenciais candidatas a vacinais vivas orais
Abstract: The genus Salmonella sp is formed by gram-negative bacilli, which can be divided into 3 species: S. enterica, S. bongori and S. subterranea. The majority of the serovars pathogenic to humans is included in the subgroup I of the S. enterica species. The infection with S. enterica starts either the ingestion of contaminated water or food. These microorganisms are facultative intracellular pathogens and, once ingested, they have the capacity to adhere and invade cells of the intestinal mucosa, with preference for M cells. Then, S. enterica can invade and proliferate within vacuoles of immune cells, particularly macrophages, achieving different organs and tissues of the host, causing systemic infection. Mutant strains of S. enterica with attenuation of the virulence but that are still able to cause a transient infection, are good candidates for potential live oral vaccines. These mutants are also good carriers of heterologous antigens to cells of the immunological system, been able to induce an effective immunological response. To the best of our knowledge, no mutants of this type were developed in Brazil leading to the needed to pay royalties to foreign groups for their use. In prokaryotes the genomic DNA are associated with a number of proteins, the so called histone-like proteins, with structural and regulatory properties, forming the nucleoid. The IHF (Integration Host Factor) is one of the more abundant histone-like in prokaryotes. IHF is a heterodimeric DNA-binding protein that controls a number of cellular processes, such as DNA duplication and DNA recombination and also modulates the expression of different genes. In this work we constructed recombinant strains of S. enterica mutants for the himA and himD genes that encode for the IHF subunits using the ? Red system (Datsenko and Wanner, 2000) and tested for attenuation and immunogenicity. The mutant strains were also characterized and compared to the parental strains for other biological characteristics such as the capacity to invade and proliferate into eukaryotic cells and to survive to different stress conditions. The S. enterica himA and himD mutant strains were attenuated for virulence and able to induce a protective immunity against the wild type strain of S. enterica indicating that these recombinant strains are candidates to formulate a new live oral vaccine
Mestrado
Ciencias Basicas
Mestre em Clinica Medica

Immunity to Salmonella enterica serovar Typhimurium (STm) is complex and requires both cell mediated and humoral immunity at different stages of infection. In infants in sub-Saharan Africa infection with non-typhoidal Salmonella (NTS), such as STm, can commonly cause fatal invasive disease. Evidence indicates that disease may be preventable by antibody, which makes vaccine development against these devastating infections a promising option. This work has explored the cell-mediated and humoral response to STm and its component antigens, their intrinsic properties, and capacity to act as protective immunogens in a mouse model. In particular, responses to surface exposed structures such as the outer membrane proteins (Omps) and the flagellar protein FliC, which are potent, immunodominant antigens and frequent targets of antibody, that may offer potential as vaccine candidates have been examined. Immunisation with soluble flagellin (sFliC) induces a potent Th2 response. Despite this, immunisation with sFliC results in accelerated clearance of STm after the first week of infection in an antibody independent, but T-bet-regulated manner. This suggests that the Th2 responses to flagellin are flexible since they can promote Th1 mediated clearance of STm. This moderate protection conferred by sFliC contrasts with the potent benefit conferred by porins. These proteins induce, and can mediate protection through a T-independent B1b cell population. In particular, antibody to OmpD is key for this protection. These results suggest that vaccines that induce protective antibody to STm may be more effective than vaccines that induce T cell-mediated protection, since they reduce bacterial numbers at the earliest stages of infection. Lastly, experiments using N. brasiliensis show that infectious history can impact on the host’s ability to control primary STm infection and the efficacy of antibody-mediated protection against infection. These projects further our understanding of the relationship between host and pathogen and the mechanisms used to control infection, but also identify the need to consider the impact of infectious history on the host’s capacity to implement protective immunity.

Salmonella Typhimurium est un pathogène intracellulaire dont la virulence repose sur l'expression de protéines effectrices qui sont transportées dans les cellules hôtes infectées. Dans la cellule cette bactérie réside dans un compartiment appelé SCV (Salmonella-containing vacuole). Au cours de l'infection, la protéine effectrice SifA est transportée du cytosol bactérien à celui de la cellule infectée. Après sa translocation SifA est retrouvée à la surface de la SCV et des tubules. Cette protéine est constituée de deux domaines distincts. Le domaine N-terminal interagit avec la protéine hôte SKIP. Le domaine C-terminal a une structure similaire à d'autres protéines bactériennes possédant une activité d'échange de nucléotide guanine (GEF). Cependant on ignore si le domaine C-terminal contribue aux fonctions de SifA dans la virulence de Salmonella et, si c'est le cas, si il exerce une activité GEF sur une protéine de l'hôte. Nous avons utilisé un modèle de souris invalidée pour SKIP pour montrer que SKIP est un médiateur du rôle de SifA dans la virulence de Salmonella et que SifA à également un rôle qui est indépendant de son interaction avec SKIP. Ce dernier est porté par le domaine C-terminal de SifA. Nous avons montré que ce domaine de SifA se lie à la petite GTPase Arl8b, une protéine lysosomale. Le domaine C-terminal de SifA et Arl8b sont importants pour le recrutement de LAMP1 sur les SCVs et les tubules associés. L'utilisation d'une lignée cellulaire invalidée pour l'expression d'Arl8b a montré une prolifération réduite de Salmonella. Ces résultats nous permettent de proposer un modèle pour le rôle du domaine C-terminal de SifA dans la virulence de Salmonella
The virulence of the intracellular pathogen Salmonella Typhimurium relies on the expression of bacterial effector proteins that are translocated into infected host cells. This bacterium resides and proliferates in a host-cell compartment named the Salmonel-la-containing vacuole (SCV). Following translocation in the infected host cells, the effector protein SifA localizes onto the SCV and SCV-associated membrane tubules. This protein is made of two distinct domains. The SifA N-terminal domain interacts with the host-cell protein SKIP. The SifA C-terminus has a fold similar to other bacterial effector proteins having a guanine nucleotide exchange factor (GEF) activity. Indeed, SifA binds preferentially a GDP-bound form of RhoA but does not stimulate GDP disso-ciation. Therefore it remains unknown whether the SifA C-terminus contributes to the functions of SifA in Salmonella virulence and, if it does, whether it has a GEF activity towards a host protein. We used a model of SKIP knockout mice to show that SKIP mediates susceptibility to Salmonellosis and to establish that SifA also contributes to Salmonella virulence independently of its interaction with SKIP. We next identified that the SifA C-terminal domain supports this contribution. We have further showed that the SifA C-terminus binds the small GTPase Arl8b and that both SifA C-terminus and activated Arl8b are important for the recruitment of LAMP1 on SCVs and associated tubules. Using an Arl8b knock down cell line, we observed that the absence of Arl8b results in a reduced proliferation of wild-type Salmonella. Finally, we proposed a model for the role of the SifA C-terminus in Salmonella virulence

Low-dosage ultraviolet confocal fluorescence microscopy and filipin staining showed that cholesterol accumulated at Salmonella entry foci, and was retained by Salmonella-containing vacuoles (SCVs) following bacterial uptake. This was particularly evident in the previously uncharacterised membrane ruffles induced on cultured fibroblasts, when viewed by scanning electron microscopy and three-dimensional fluorescence rendering. Cellular cholesterol redistribution required bacterial effector delivery directed by the SPI1 encoded TTSS. These results showed that host cholesterol is essential for Salmonella uptake, and indicated the involvement of host cell rafts in the entry process. Purified SipB was identified as a cholesterol binding protein in novel gel filtration assays in vitro. Upon cholesterol depletion of cultured eukaryotic cells, addition of purified SipB was sufficient to restore correct localisation of the raft marker, caveolin-1. Fluorescence microscopy, immunogold electron microscopy and biochemical assays demonstrated that cellular binding of purified SipB was reduced in cholesterol depleted cells compared with untreated cells. Localisation of SipB to rafts was observed in Salmonella infected cells, or cells treated with purified SipB. Enhanced localisation for purified SipB was observed when added to cells in complex with SipC, or following treatment of cells with cytoskeleton-disrupting drugs. In combination, these data indicate that SipB specifically targets hosts rafts during entry. New insights into the uptake of Salmonella and their attached flagella were gained by immunoflourescence microscopy. Flagella are lengthy, rigid, helical structures and their fate, following cell entry, was previously unknown. The flagella filament was observed to adopt novel, uncharacterised conformations within the host cell and whilst further flagella assembly was not apparent, pre-existing flagella exhibited long term intracellular stability. This study addresses the issue of the fate Salmonella flagella after cell entry and the nature of the SCV microenvironment.

This thesis presents two novel immunisation strategies against Salmonella enterica serovar Typhimurium (S. typhimurium) in the mouse model. Firstly, mice immunised with multiple antigens in the form of a DNA vaccine have been shown to develop specific humoral and cellular responses to proteins encoded within the vaccine. Secondly, it has been shown that immunisation with multiple cytosolic antigens (CA) of S. typhimurium SL1344, formulated with the adjuvant dimethyl dioctadecyl ammoniumbromide (DDA), induces strong humoral and cellular responses. These responses have been characterised, and their ability to confer protection on mice challenged with a lethal dose of S. typhimurium SL1344 has been investigated. The aim of DNA vaccination is to induce immune responses to protein antigens expressed in vivo by injection plasmid DNA encoding the antigen sequence. Expression library immunisation (ELI) is a new technique that draws on DNA immunisation and can be developed to identify the key antigens of a pathogen that confer protection. ELI has previously been used in a number of disease models in mice and has been used in this thesis as a means of inducing immune responses against an unspecified subset of bacterial antigens. An expression library (EL) of S. typhimurium SL1344 was constructed using a mammalian expression vector encoding EGFP. The expression of S. typhimurium SL1344 antigens forming fusion proteins with EGFP was visualised as green fluorescence in mouse fibroblast cells in vitro. The EL consisted of 140,000 clones of which 14,000 were used for immunisation. The library was administered to both BALB/c and CBA mice to examine the effect of mouse strain and was administered both intradermally (ID) and intramuscularly (IM) to examine the effect of immunisation route. Analysis by Western blot and ELISA showed that humoral responses were induced in both BALB/c and CBA mice. ID and IM inoculation produced similar results in BALB/c mice. Responses included a significant Thl component as judged by the presence of IgG2a in the serum and the secretion of IFN-γ when T cells from peripheral lymph nodes were stimulated by CA in culture.

Memoria para optar al Título Profesional de Bioquímico
Autorizada por el autor, pero con restricción para ser publicada a texto completo en el Portal de Tesis Electrónicas, hasta diciembre de 2014
Salmonella enterica es un patógeno intracelular Gram negativo capaz de infectar un amplio rango de hospederos. En particular, S. enterica serovar Gallinarum infecta aves de corral causando una enfermedad sistémica que puede provocar la muerte. Una vez que entra al organismo, Salmonella utiliza una serie de factores de virulencia que le permiten sobrevivir al pH ácido del estómago, resistir a sales biliares y péptidos antimicrobiales, invadir y traspasar la barrera epitelial intestinal, sobrevivir y replicarse dentro de macrófagos y diseminarse dentro de los órganos del hospedero, principalmente a nivel del bazo e hígado. Hasta el momento, se desconoce gran parte de los factores de virulencia que utiliza S. Gallinarum para infectar un hospedero. Es por eso que en este trabajo se propuso identificar los genes de S. Gallinarum involucrados en la colonización sistémica en un modelo murino a través de un análisis global de mutantes bajo selección negativa in vivo. Para ello, se utilizó una genoteca de ~48.000 mutantes por inserción del transposón EZ-Tn5, la que se inyectó en ratones BALB/c por vía intraperitoneal. La comparación entre la población de bacterias inyectadas (input) y la población de bacterias recuperadas a partir del bazo de los animales (output) mediante hibridaciones competitivas en un microarray genómico nos permitió obtener una base de datos en la que identificamos 280 mutantes bajo selección negativa in vivo. La lista de mutantes bajo selección negativa incluye genes descritos previamente como necesarios para la virulencia de S. enterica, como los sistemas de secreción tipo III codificados en la SPI-1 y SPI-2, genes que codifican efectores de estos sistemas (sseB, sseE), genes relacionados a la síntesis y modificación del LPS (rfaL, rfaJ, rfbK, rfbM), genes que codifican reguladores globales de la virulencia (phoP, phoQ, ompR, envZ), genes que codifican proteínas de respuesta a estrés (oxyR, rpoE, htrA), entre otros. La lista también incluye genes no reportados previamente como necesarios para la virulencia de S. Gallinarum, pero si para la virulencia de otros serovares de S. enterica, como tatB y tatC que codifican proteínas del sistema Twin-Arginine Transport; y genes con funciones desconocidas como la región génica STM3118 a STM3121 perteneciente a la SPI-13. El sistema Twin-Arginine Transport es un sistema que transporta proteínas plegadas hacia el periplasma de bacterias Gram negativo. Por otra parte, aún se desconoce la función exacta de SPI-13, pero se ha visto que es necesaria para la replicación de S. Typhimurium dentro de macrófagos murinos. A través de ensayos de competencia y complementación in vivo entre la cepa silvestre y las mutantes ΔtatABC o ΔSPI-13, se confirmó la participación de estas regiones génicas en la colonización sistémica de ratones BALB/c. Cabe destacar que la lista de mutantes bajo selección negativa in vivo no incluye ciertos genes previamente descritos como necesarios para la virulencia de S. enterica, como el gen aroA que codifica una proteína involucrada en la síntesis de compuestos aromáticos. Se realizó un ensayo de competencia con una mutante ΔaroA y se determinó que presenta una colonización sistémica deficiente en ratones BALB/c, confirmando la participación de aroA en la virulencia de esta bacteria. Finalmente, mediante este análisis global de mutantes bajo selección negativa in vivo logramos identificar genes de S. Gallinarum requeridos para la colonización sistémica eficiente de un hospedero murino, comprobando de forma independiente la participación del operón tatABC, la isla de patogenicidad SPI-13 y el gen aroA en este proceso. El análisis individual de los genes identificados en esta base de datos permitirá ampliar el conocimiento sobre los mecanismos de patogenicidad de S. Gallinarum
Salmonella is a Gram negative intracelular pathogen able to infect a broad range of hosts. Specifically, S. enterica serovar Gallinarum infects poultry leading to a systemic illness that may cause death. Once Salmonella enters the organism, it uses a variety of virulence factors which allows it to survive in the gastric acid, resist bile salts and antimicrobial peptides, invade and cross the intestinal epithelium, survive and grow within macrophages, and colonize internal organs of the host, mainly spleen and liver. The main virulence factors that S. Gallinarum uses to infect a host remained unknown until know. In this work we proposed to identify genes involved in the systemic colonization of S. Gallinarum in the murine model through a genome-wide screening of mutants under negative selection in vivo. To accomplish this, we used a pool of ~48.000 mutants generated by random insertion of the EZ-Tn5 transposon to inoculate BALB/c mice intraperitoneally. The comparison between the pool of inoculated bacteria (input) and the pool of bacteria recovered from the spleen of the animals (output) through high-throughput microarray-based screening of mutants allowed us to obtain a database of 280 mutants under negative selection in vivo. Within this database we found mutants in several genes known to be required for Salmonella enterica virulence, like those related to the type III secretion system encoded in SPI-1 and SPI-2, genes encoding efectors secreted by these systems (sseB, sseE), genes related to LPS synthesis and modification (rfaL, rfaJ, rfbK, rfbM), genes encoding global virulence regulators (phoP, phoQ, ompR, envZ), and genes encoding proteins involved in response to stress (oxyR, rpoE, htrA), among others. We also found genes not previously reported as required for S. Gallinaum virulence, like tatB and tatC, encoding components of the Twin-Arginine Transport system; and genes with unknown function like the genetic region comprised by STM3118 to STM3121, belonging to SPI-13. The Twin-Arginine Transport system transports a number of folded proteins to the periplasma of Gram-negative bacteria. Besides, the exact function of SPI-13 is still unknown, but has been seen that is necessary for the growth of S. Typhimurium inside murine macrophages. Through in vivo competition and complementation assays between wild type and ΔtatABC or ΔSPI-13 mutants, we were able to confirm the important role of these genes in the systemic colonization of BALB/c mice by S. Gallinarum. Noteworthly, there were genes that we didn’t observe on our database that have been reported as required S. enterica virulence like aroA, a gene involved in the synthesis of aromatic coumpounds. Using an in vivo competition assay we observed a systemic colonization defect for the ΔaroA mutant in BALB/c mice, indicating that this gene is indeed required for S. Gallinarum virulence in this host. Overall, our genome-wide screening of mutants under negative selection in vivo allowed us to identify 280 genes of S. Gallinarum required for the systemic colonization of a murine host. Also, we confirmed the role played by the tatABC operon, SPI-13 and aroA in the systemic colonization by this serovar. The in-depth analysis of the genes identified in our screening will expand our current knowledge on the mechanisms of S. Gallinarum pathogenesis
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

Salmonella Gallinarum (SG) et Salmonella Enteritidis (SE) sont deux sérotypes génétiquement proches. Pourtant, chez la volaille, SG induit une infection systémique létale tandis que SE est responsable d’une infection systémique transitoire et d’un portage intestinal asymptomatique. De plus, SE est un sérotype ubiquiste tandis que SG est spécifique d’hôte. Outre ces différences in vivo, ces deux sérotypes présentent des différences in vitro. Nous avons montré que SG est in vitro moins invasif que SE. Ce phénotype est indépendant de l’origine aviaire ou non-aviaire des cellules en dépit du tropisme de SG pour la volaille. LeT3SS-1, le facteur d’invasion majeur chez Salmonella, est composé d’un appareil de sécrétion et des effecteurs transloqués par celui-ci. Nous avons montré que les composants du T3SS-1 sont autant exprimés chez SG que chez SE et que tous deux possèdent un appareil de sécrétion fonctionnel. Pourtant, l’étude des capacités d’invasion dépendantes du T3SS-1 suggère que ce facteur est impliqué dans le défaut d’invasion de S. Gallinarum. L’analyse des gènes codant les effecteurs transloqués par le T3SS-1 a montré qu’il existe des mutations chez SG pouvant être à l’origine de ce défaut d’invasion
Salmonella Gallinarum and Salmonella Enteritidis are genetically closed. However, whereas SG induces lethal systemic infection in poultry, SE is responsible for transient systemic infection and asymptomatic intestinal carriage. Moreover, SE is ubiquitous whereas SG is poultry specific. These serotypes also present in vitro differences. We have shown that SG is in vitro less invasive than SE whatever the avian or non-avian cell origin, in spite of SG’s tropism for avian species. T3SS-1, the main invasion factor in Salmonella, is composed of a secretion apparatus and its translocated effectors. We have shown that T3SS-1 components a expressed in a similar way by SE and SG and both harbor a functional secretion apparatus. However, study of T3SS-1 dependent invasion abilities suggested that T3SS-1 is involved in SG’s invasion defect. Sequence analysis has revealed that SG possesses several mutations in genes encoding main T3SS-1 effectors, that could explain the low invasive phenotype

La salmonel·losi és una zoonosi causada per soques no adaptades de bacteris del gènere Salmonella. La Salmonella es troba habitualment a l’intestí d’ocells i mamífers sans i pot entrar a la cadena alimentària mitjançant la contaminació de carn, ous i els seus productes derivats. El consum d’aliments o aigua infectada amb Salmonella causa gastroenteritis en humans. L’últim informe de l’European Food Safety Authority indica que aquest bacteri va ser la segona causa de zoonosi en humans a la Unió Europea el 2018. En particular, Salmonella va causar un terç de tots els brots alimentaris (30,7%). Gairebé la meitat d’aquests brots van ser causats per ous i productes derivats (45,6%), i la majoria (70%) van ser causats només per dues serovarietats: Salmonella Enteritidis i Salmonella Typhimurium. L’any 2003 la UE va començar a implementar els Programes Nacionals de Control de Salmonella a tots els estats membres per reduir la prevalença. Aquests plans van consistir en mesures de bioseguretat i eines addicionals com la vacunació, i van aconseguir una reducció important de la prevalença. Les serovarietats no adaptades de Salmonella no causen malaltia clínica en gallines majors de 3 dies. Per tant, l’objectiu principal de les vacunes contra aquestes serovarietats en aus és augmentar la resistència dels animals contra la infecció per tal de reduir la contaminació dels productes derivats. L’objectiu principal d’aquesta tesi va ser determinar la funció protectora d’una soca viva atenuada de Salmonella Typhimurium (sola o combinada amb una soca de Salmonella Enteritidis) contra les infeccions de soques de camp i avaluar la resposta immune associada. Es van realitzar tres estudis en tres èpoques de producció diferents en gallines. En el primer estudi, l’objectiu va ser determinar l’eficàcia d’una primera dosi de vacuna a dia de vida comparant l’administració oral i via aerosol, després d’una infecció als 14 dies de vida. Els animals vacunats via oral van estar parcialment protegits i es va reduir la colonització en òrgans interns, fet que indica la necessitat d’utilitzar dosis addicionals de vacuna per desenvolupar una immunitat més forta contra la Salmonella. La protecció no es va correlacionar amb la resposta humoral o cel·lular. L’administració de vacuna via aerosol va fallar, indicant la importància de la ruta d’administració. En el segon estudi, es va provar l’eficàcia de la vacuna després de 2 dosis (amb infecció experimental a les 16 setmanes de vida) o 3 dosis (infecció experimental a les 35 setmanes de vida). Els dos grups vacunals van reduir l’excreció de Salmonella i la colonització d’òrgans interns. Aquesta resposta immune adaptativa protectora no es va correlacionar amb els nivells d’anticossos (que van ser més alts a l’intestí d’animals no vacunats), però es correlacionà amb un augment general de la població de cèl·lules T CD3+ a l’intestí i amb l’expressió d’IFNγ, fet que indica una possible deriva cap a una resposta immune Th1. Al tercer estudi, es va determinar l’eficàcia de la vacunació durant la cria amb una vacuna combinada de Salmonella Enteritidis i Salmonella Typhimurium al final del període de posta. Els animals vacunats van reduir l’excreció de Salmonella Enteritidis i Salmonella Typhimurium en femtes i van estar protegits contra la colonització d’òrgans interns. La protecció no es va relacionar amb la resposta humoral, però si amb una resposta cel·lular a l’intestí. Els resultats extrets d’aquesta tesi indiquen que les vacunes estudiades podrien ser una eina útil per disminuir la transmissió vertical als ous (reduint la infecció d’òrgans interns) i la transmissió horitzontal a ous i productes avícoles (reduint l’excreció) per atenuar el risc de salmonel·losi en humans.
La salmonelosis es una zoonosis causada por cepas bacterianas no adaptadas del género Salmonella. La Salmonella se encuentra comúnmente en el intestino de aves y mamíferos sanos y puede entrar a la cadena alimentaria a través de la contaminación de carne, huevos y sus productos derivados. El consumo de alimentos o agua contaminada con Salmonella causa gastroenteritis en humanos. El último informe de la European Food Safety Authority indica que esta bacteria fue la segunda causa de zoonosis en humanos en la Unión Europea en 2018. En particular, Salmonella causó un tercio de todos los brotes transmitidos por alimentos (30.7%). De estos brotes, casi la mitad fueron causados por huevos y sus derivados (45,6%), y la mayoría (70%) fueron causados por solo dos serovariedades: Salmonella Enteritidis y Typhimurium. El año 2003, la Unión Europea comenzó a implementar los Programas Nacionales de Control de Salmonella en todos los estados miembros para reducir la prevalencia. Estos planes consistieron en medidas de bioseguridad y herramientas adicionales como la vacunación, y lograron una reducción importante en la prevalencia. Las serovariedades no adaptadas de Salmonella no causan enfermedad clínica en gallinas mayores de 3 días. Por lo tanto, el objetivo principal de las vacunas contra estos serotipos en aves es aumentar la resistencia de los animales contra la infección para reducir la contaminación de los productos derivados. El objetivo principal de esta tesis fue determinar la función protectora de una cepa viva atenuada de Salmonella Typhimurium (sola o combinada con una cepa de Salmonella Enteritidis) contra las infecciones por cepas de campo y evaluar la respuesta inmune asociada. Se realizaron tres estudios en tres tiempos de producción diferentes en gallinas. En el primer estudio, el objetivo era determinar la eficacia de una primera dosis de vacuna a día de vida comparando la administración oral y vía aerosol, tras una posterior infección a los 14 días de vida. Los animales vacunados estuvieron parcialmente protegidos y se redujo la colonización en órganos internos, lo que indica la necesidad de utilizar dosis adicionales de vacuna para desarrollar una inmunidad más fuerte contra la Salmonella. La protección no se correlacionó con la respuesta inmune humoral o celular. La administración de vacuna vía aerosol falló, indicando la importancia de la ruta de administración. En el segundo estudio, se probó la eficacia de la vacuna después de 2 dosis (con infección experimental a las 16 semanas de vida) o 3 dosis (infección experimental a las 35 semanas de vida). Ambos grupos vacunales redujeron la excreción de Salmonella y la colonización de órganos internos. Esta respuesta inmune adaptativa protectora no se correlacionó con los niveles de anticuerpos (que fueron más altos en el intestino de animales no vacunados), pero se correlacionó con un aumento general de la población de células T CD3+ en intestino y con la expresión de IFNγ, lo que indica una deriva hacia una respuesta inmune Th1. En el tercer estudio, se determinó la eficacia de la vacunación durante la cría con una vacuna combinada de Salmonella Enteritidis y Salmonella Typhimurium al final del periodo de puesta. Los animales vacunados redujeron la excreción de Salmonella Enteritidis y Salmonella Typhimurium en heces y estuvieron protegidos contra la colonización de órganos internos. La protección no se relacionó con la respuesta humoral, pero si con una respuesta celular en el intestino incluyendo la infiltración de macrófagos, células T CD4+ y CD8+. Los resultados extraídos de esta tesis indican que las vacunas estudiadas podrían ser una herramienta útil para disminuir la transmisión vertical a los huevos y la transmisión horizontal a huevos y productos avícolas para atenuar el riesgo de salmonelosis en humanos.
"Salmonellosis is a zoonosis caused by non-adapted bacterial strains of the genus Salmonella. Salmonella is commonly found in the intestines of healthy birds and mammals and can enter the food chain through contaminated meat, eggs and their products. Consumption of food or water contaminated with Salmonella causes gastroenteritis in humans. The last report from the European Food Safety Authority indicates that this bacterium was the second cause of human zoonosis in the European Union in 2018. Particularly, Salmonella caused one third of all foodborne outbreaks (30.7%). From these outbreaks almost half were caused by eggs and egg products (45.6%), and most (70%) were caused by only two serovars: Salmonella Enteritidis and Salmonella Typhimurium. In 2003 the EU started to implement the Salmonella National Control Programmes in all member states to reduce Salmonella prevalence in the poultry and pig industry. These plans consisted in measures of biosafety and additional tools like vaccination against Salmonella, which achieved an important reduction in prevalence and the consequent reduction of reported cases of human salmonellosis. Non-adapted serovars do not cause clinical disease in hens older than 3 days. Therefore, the main objective of vaccines against these Salmonella serovars in poultry is to increase resistance of animals against infection in order to reduce the contamination of poultry derived products. The main objective of this thesis was to determine the protective function of a live attenuated Salmonella Typhimurium strain (alone or combined with a Salmonella Enteritidis strain) against field strains infections and to evaluate the associated immune response. Three studies were carried out at three different production times in hens. In the first study the objective was to determine the efficacy of a first vaccine dose at day-old after challenge at 14 days comparing oral and spray administration. The oral vaccination partially protected and reduced colonisation in internal organ, indicating the need of additional vaccine boosters to develop a stronger immunity against Salmonella. The protection was not correlated with humoral or cellular immune response. The spray administration failed, and animals were not protected, indicating the importance of the administration route. In the second study, the efficacy of the vaccine was tested after 2 vaccine doses (challenged at 16 weeks of life) or 3 vaccine doses (challenged at 35 weeks of life). Both vaccine groups had reduction of Salmonella excretion and colonisation of internal organs. This adaptive protective immune response was not correlated with levels of antibodies (which were especially higher in non-vaccinated animals in the intestine), but was correlated with a general increase in CD3+ T cell population in the intestine and a IFNγ; up-regulation, indicating a possible drift to a protective Th1 immune response. In the third study, the efficacy of vaccination during rearing with a combined Salmonella Enteritidis and Salmonella Typhimurium vaccine was determined at the end of the laying period. Vaccinated animals had reduced rates of Salmonella Enteritidis and Salmonella Typhimurium excretion in cloacal swabs and were protected against internal organ colonisation. The protection was not related with humoral response but with a cellular response in the intestine including the infiltration of macrophages, CD4+ and CD8+ T cells. The results extracted from this thesis indicate that the tested vaccines could be a useful tool to decrease vertical transmission to eggs (reducing infection of internal organs) and horizontal transmission to eggs and poultry products (reducing excretion of bacteria) to ultimately minimise the risk of salmonellosis in humans.

Resumo: Dentre as doenças transmitidas por alimentos, a salmonelose é a que mais preocupa os consumidores e produtores de aves, pois na maioria dos casos de toxinfecção por este agente, carne de aves ou derivados estão envolvidos. Devido ao impacto na saúde pública, se faz necessário a busca de ferramentas de controle efetivas deste patógeno na produção de aves. Ao longo dos últimos anos, percebe-se que, sempre quando se estabelece controle para um sorovar, ocorre alternância de prevalência para outro sorovar da mesma espécie. Esse comportamento, aliado a enorme quantidade de sorovares conhecidos, levam a crer que o sucesso no controle desse agente está associado a diversas ações, dentre elas, o uso de vacinas. Para entender melhor o uso de vacinas no controle de Salmonella, o presente trabalho foi dividido em dois capítulos: Capítulo I que apresenta uma revisão bibliográfica sobre "Vacinas como Alternativa de Controle à Salmonella" e no Capítulo II que descreve um experimento científico sobre o "Vacinação com Bacterina de Salmonella Enteritidis e Salmonella Minnesota em Matrizes de Frangos de Corte: Controle da Colonização e Resposta Imune na Progênie Desafiada", onde foi observado, em frangos de 1 dia, que o grupo oriundo de reprodutoras vacinadas com Salmonella Enteritidis (SE) e Salmonella Minnesota (SM) apresentaram maior quantidade de células CD4+ no íleo e CD8+ no ceco. Aos sete dias após o desafio, a progênie destas reprodutoras também apresentaram menor colonização no fígado que frangos oriundos de reprodutoras vacinadas com SE, porém não houve diferença para a colonização do ceco, papo e fígado aos três, 14 e 21 dias após desafio.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A avicultura comercial tem como objetivo obter alta produtividade a baixo custo e oferecer ao consumidor produto de qualidade. Uma bactéria patogênica que tem preocupado o setor avícola nos últimos tempos é a Salmonella. Neste sentido, o presente trabalho objetivou caracterizar seis isolados de Lactobacillus acidophilus em combinação com antimicrobianos na inibição de Salmonella Enteritidis e Salmonella Gallinarum. O experimento foi desenvolvido nas dependências da FCAV/UNESP – Campus de Jaboticabal. Para tanto foram conduzidos quatro estudos, sendo que dois foram dedicados em selecionar uma bactéria probiótica produtora de bacteriocina e avaliar a interação de substâncias antimicrobianas como EDTA, àcido acético e Nisina “in vitro” sobre a capacidade inibitória de Salmonella Enteritidis e Salmonella Gallinarum em diferentes tempos e o terceiro e quarto experimento estudam o potencial de aplicação do probiótico e do antimicrobiano em aves. O isolado de Lactobacillus acidophilus C1, demonstrou ser produtor de bacteriocina e inibir a multiplicação de Salmonella Enteritidis e Salmonella Gallinarum. Dentre os antimicrobianos testados o que apresentou efeito na eliminação de Salmonella Enteritidis foi a combinação de Ácido Acético + nisina e Ácido Acético + Lactobacillus acidophilus C1. O uso do probiótico no estudo em aves reduziu a excreção de Salmonella sp, mas não sua eliminação no trato intestinal das aves, já que é possível constatar sua presença no tratamento controle. A combinação de nisina e ácido acético não levou a redução da multiplicação de Salmonella sp. nas aves. O estudo permitiu verificar que existe um bom potencial de aplicação do probiótico citado, assim como o uso de alguns antimicrobianos na segurança alimentar.
The commercial poultry aims at obtaining high productivity at low cost and offering quality products to the consumer. A pathogenic bacterium which has worried the poultry sector in recent times is Salmonella. Therefore, the present study aimed at characterizing six isolates of Lactobacillus acidophilus in combination with antimicrobials in the inhibition of Salmonella enteritidis and Salmonella gallinarum. The experiment was developed in the dependencies of FCAV/UNESP – Campus of Jaboticabal. In order to do this, four studies were conducted, being two of them dedicated to select a bacteriocin-procucer probiotic bacterium and evaluate the interaction of antimicrobial substances such as EDTA, acetic acid and “in vitro”Nisin on the inhibitory capacity of Salmonella enteritidis and Salmonella gallinarum in different periods and the third and fourth experiment study the potential of application of the probiotic and antimicrobial in poultry. The isolate of Lactobacillus acidophilus C1, showed to be bacteriocin producer and inhibit the multiplication of Salmonella enteritidis and Salmonella gallinarum. Among the antimicrobial tested, the one which presented effect in the elimination of Salmonella enteritidis, was the combination of Acetic Acid + nisin and Acetic Acid + Lactobacillus acidophilus C1. The use of the probiotic in the study of birds reduced the counting of Salmonella sp, but not its elimination in the intestinal tract of the birds, as we can see its presence in the control treatment. The combination of nisin and acetic acid did not show the reduction of the multiplication of Salmonella sp. in birds. The study has shown that there is a good potential of application of the mentioned probiotic, as well as the use of some antimicrobial in food safety.

Enteric fever remains the most common febrile illness in urban Nepal. Some individuals may have recurrent infection and some may even progress to become long term chronic carriers. The aim of this thesis was to investigate the rate and factors leading to relapse with typhoid fever in patients who were enrolled in clinical treatment trials for acute enteric fever. The results show that relapses in enteric fever is a common complication and is more likely to be associated with the treatment antimicrobial, cefixime. Gallbladder carriage of invasive Salmonella is considered fundamental in sustaining enteric fever transmission as humans are the only known natural host. This thesis, therefore, also aimed to investigate the prevalence, characteristics, immunological responses, and mechanism of carriage of invasive Salmonella in the gallbladder by examining bile and tissue obtained from individuals who underwent cholecystectomy in Kathmandu. Data presented here demonstrate that S. Paratyphi A is almost as prevalent as S. Typhi in the gallbladder and that carriage may not be driven by antimicrobial resistance. Gallbladders that contained Salmonella were more likely to show evidence of acute inflammation with extensive neutrophil infiltrate. Chronic carriers were found to have dramatically elevated levels of IgG to O:2 and Vi antigens with high bactericidal activity yet low pro-inflammatory cytokine levels suggesting that Salmonella are stimulating a constant immunological response, in the form of antibody. S. Typhi may be controlling the inflammatory process through the expression of the Vi capsule in the gallbladder. Genome sequencing of S. Typhi isolated from chronic carriers were different from those S. Typhi causing acute disease. These data question the current dogmas surrounding the carriage of S. Typhi in gallbladder and predict a pivotal role of Vi capsule and gallstones in maintaining carriage. Therefore, prospectively identifying these individuals is paramount for rapid local and regional elimination. Furthermore, combining cytokine profiles and antibody levels may be a method of prospectively detecting carriers in the general population.

Orientador: Ruben Pablo Schocken-Iturrino
Banca: Alessandra Aparecida Medeiros
Banca: Ricardo de Albuquerque
Banca: Antonio Carlos Paulillo
Banca: Hélio José Montassier
Resumo: A avicultura comercial tem como objetivo obter alta produtividade a baixo custo e oferecer ao consumidor produto de qualidade. Uma bactéria patogênica que tem preocupado o setor avícola nos últimos tempos é a Salmonella. Neste sentido, o presente trabalho objetivou caracterizar seis isolados de Lactobacillus acidophilus em combinação com antimicrobianos na inibição de Salmonella Enteritidis e Salmonella Gallinarum. O experimento foi desenvolvido nas dependências da FCAV/UNESP - Campus de Jaboticabal. Para tanto foram conduzidos quatro estudos, sendo que dois foram dedicados em selecionar uma bactéria probiótica produtora de bacteriocina e avaliar a interação de substâncias antimicrobianas como EDTA, àcido acético e Nisina "in vitro" sobre a capacidade inibitória de Salmonella Enteritidis e Salmonella Gallinarum em diferentes tempos e o terceiro e quarto experimento estudam o potencial de aplicação do probiótico e do antimicrobiano em aves. O isolado de Lactobacillus acidophilus C1, demonstrou ser produtor de bacteriocina e inibir a multiplicação de Salmonella Enteritidis e Salmonella Gallinarum. Dentre os antimicrobianos testados o que apresentou efeito na eliminação de Salmonella Enteritidis foi a combinação de Ácido Acético + nisina e Ácido Acético + Lactobacillus acidophilus C1. O uso do probiótico no estudo em aves reduziu a excreção de Salmonella sp, mas não sua eliminação no trato intestinal das aves, já que é possível constatar sua presença no tratamento controle. A combinação de nisina e ácido acético não levou a redução da multiplicação de Salmonella sp. nas aves. O estudo permitiu verificar que existe um bom potencial de aplicação do probiótico citado, assim como o uso de alguns antimicrobianos na segurança alimentar.
Abstract: The commercial poultry aims at obtaining high productivity at low cost and offering quality products to the consumer. A pathogenic bacterium which has worried the poultry sector in recent times is Salmonella. Therefore, the present study aimed at characterizing six isolates of Lactobacillus acidophilus in combination with antimicrobials in the inhibition of Salmonella enteritidis and Salmonella gallinarum. The experiment was developed in the dependencies of FCAV/UNESP - Campus of Jaboticabal. In order to do this, four studies were conducted, being two of them dedicated to select a bacteriocin-procucer probiotic bacterium and evaluate the interaction of antimicrobial substances such as EDTA, acetic acid and "in vitro"Nisin on the inhibitory capacity of Salmonella enteritidis and Salmonella gallinarum in different periods and the third and fourth experiment study the potential of application of the probiotic and antimicrobial in poultry. The isolate of Lactobacillus acidophilus C1, showed to be bacteriocin producer and inhibit the multiplication of Salmonella enteritidis and Salmonella gallinarum. Among the antimicrobial tested, the one which presented effect in the elimination of Salmonella enteritidis, was the combination of Acetic Acid + nisin and Acetic Acid + Lactobacillus acidophilus C1. The use of the probiotic in the study of birds reduced the counting of Salmonella sp, but not its elimination in the intestinal tract of the birds, as we can see its presence in the control treatment. The combination of nisin and acetic acid did not show the reduction of the multiplication of Salmonella sp. in birds. The study has shown that there is a good potential of application of the mentioned probiotic, as well as the use of some antimicrobial in food safety.
Doutor

I have investigated the possibility that specific conalbumin (ovotransferrin) iron saturation levels enable less virulent strains of Salmonella to become more virulent. Iron starved cells of two pathogenic Salmonella strains, S. paratyphi B var. java and S. thompson, were cultured in iron limited media at 3 different iron conalbumin saturation levels. Results indicate that strains differ significantly at both low and high iron saturation conalbumin. These differences depict a growth advantage for S. paratyphi B which correlates with reports by the Centers for Disease Control that S. paratyphi B was 3 times more frequent in blood isolates than S. thompson. The ability to use protein bound iron may account for the higher involvement of S. paratyphi B in bacteremia.

Ziel dieser Arbeit war es, die zeit- und kostenintensive Salmonellen-Serotypisierung mit Hilfe von molekularbiologischen Methoden zu komplettieren und nach Möglichkeit zu ersetzen. Es war geplant, verschiedene publizierte, molekularbiologische Nachweise zu prüfen und gegebenenfalls zu erweitern. Hierfür wurde ein Aufbau dreier Multiplex-PCRs (RAJTAK et al., 2011) und ein High Resolution Melting (HRM) (BRATCHIKOV & MAURICAS, 2011) etabliert und anschließend evaluiert. Zudem sollte im Rahmen dieses Projektes eine molekularbiologische Methode zur Unterscheidung von lebenden Impf- und Feldstämmen der Serovare Salmonella Enteritidis und Salmonella Typhimurium entwickelt werden. Deshalb war geplant, die genetischen Grundlagen der phänotypischen Marker zu ergründen und anhand dieser ebenfalls ein High Resolution Melting für die Differenzierung der Impf- und Feldstämme zu entwickeln. Für die Salmonella-Serovar-Differenzierung mittels Multiplex-PCRs (RAJTAK et al., 2011) wurden 210 Stämme verteilt auf 31 Serovare geprüft. Es konnten hier jedoch nur wenige Serovare eindeutig differenziert werden (Salmonella Livingstone, Salmonella Goldcoast, Salmonella Ohio, Salmonella Kentucky, Salmonella Typhimurium, Salmonella-Typhimurium-Variante monophasisch (1,4,[5],12:i:-), Salmonella Subspezies IIIb, Salmonella Enteritidis sowie der IDT-Salmonella-Enteritidis-Impfstamm). Für die übrigen Serovare konnte in Verbindung mit der Gelelektrophorese nur eine grobe Einteilung in insgesamt zehn Gruppen erreicht werden. Diese Gruppen enthielten bis zu zehn Serovare. Zudem konnten sehr wichtige Serovare wie Salmonella Enteritidis und Salmonella Typhimurium nicht endgültig differenziert werden. Für spezielle Fragestellungen, wie die Differenzierung der eindeutig unterscheidbaren Serovare, könnten diese Ergebnisse dennoch nützlich sein. Das High Resolution Melting nach Bratchikov und Mauricas aus dem Jahr 2011 wurde mit guten Ergebnissen evaluiert. Es konnten alle geprüften 100 Proben (zusammengesetzt aus 21 Serovaren) innerhalb eines Laufes mit dem LightCycler® 96 differenziert werden. Für eine zusätzliche Verbesserung dieser Methode, in Form einer genaueren Differenzierung des Serovars Salmonella Typhimurium, sollte das Protokoll um Differenzierungsmöglichkeiten für die Salmonella-Typhimurium-Variante O5-negativ (1,4,12:i:1,2) und die Salmonella-Typhimurium-Variante monophasisch (1,4,[5],12:i:-) erweitert werden. Dies gelang gänzlich für die monophasische Variante (1,4,[5],12:i:-) durch eine Erweiterung der HRM-Analyse mit dem Primerpaar FljB 1,2;1,5 nach Rajtak et al. aus dem Jahr 2011 sowie teilweise für die Variante O5-negativ (1,4,12:i:1,2). In den gesetzlichen Grundlagen, wie der Geflügel-Salmonellen-Verordnung und allen relevanten EU-Verordnungen für Bekämpfung von Salmonellen beim Geflügel, werden alle Varianten als Salmonella Typhimurium klassifiziert und deshalb gleich behandelt. Alle anderen Verordnungen, wie die Rinder-Salmonellose-Verordnung oder die Verordnung für Meldepflichtige Tierkrankheiten, erfassen ohnehin alle Salmonellen-Serovare. Somit ist eine weitere Unterscheidung der Serovare nicht notwendig, bietet aber Möglichkeiten für eine tiefgründigere Diagnostik, falls diese gewünscht ist. Auch ein Nachteil gegenüber der serologischen Differenzierung stellt sich durch die nur teilweise Differenzierungsmöglichkeit der Variante O5-negativ somit nicht. Um die Anwendbarkeit der HRM-Analyse in der Routinediagnostik, insbesondere bei auftreten neuer Serovare, besser beurteilen zu können, muss diese molekularbiologische Methode jedoch zunächst über einen längeren Zeitraum parallel zur klassischen Diagnostik angewandt werden, bevor sie in der Routinediagnostik verwendet werden kann. Mit Hilfe dieser Methode könnte allerdings, bei in etwa gleichem finanziellem Aufwand, ein erheblicher Zeitgewinn bei der Salmonellen-Differenzierung erzielt werden. Voraussetzung hierfür wäre jedoch das Vorhandensein eines HRM-fähigen Gerätes. Die Entwicklung einer molekularbiologischen Methode für die Impfstamm/Feldstamm-Differenzierung konnte ebenfalls mit sehr gutem Ergebnis durchgeführt werden. Es konnten mehrere Punktmutationen, sogenannte single nucleotide polymorphisms (SNPs), auf verschiedenen Genen (rpoB und his) mittels Sequenzierung entdeckt werden. Anschließend wurden HRM-Tests entwickelt, die die SNPs aller vier zugelassenen Salmonella-Lebendimpfstämme eindeutig differenzieren können. Diese Methode wurde auf dem LightCycler® 96 erfolgreich etabliert und die mögliche Durchführung der HRM-Analysen auch auf zwei weiteren Geräten (LightCycler® 480 und Rotor Gene Q) geprüft. Hier zeigte sich, dass der Rotor Gene Q ebenso alle vier Impfstoffe, der LightCycler® 480 hingegen aufgrund einer schlechteren Auflösung nur drei Impfstoffe unterscheiden kann. Die entwickelte Methode bietet eine erhebliche Zeitersparnis, da die derzeitige Unterscheidung anhand der phänotypischen Merkmale eine Inkubation von 18-24 Stunden (LAH-Impfstämme) beziehungsweise 18-48 Stunden (IDT-Impfstämme) benötigt. Die Laufdauer der entwickelten HRM-Analyse hingegen beträgt nur 1,5 Stunden.

A presença de Salmonella em 200 amostras de alimentos crus de origem animal foi investigada empregando-se os dois ensaios imunoenzimáticos rápidos TECRA™ Salmonella VIA e TECRA™ Salmonella UNIQUE (TECRA Diagnostics, Rosewille, NSW, Australia) e o método de cultura convencional empregado rotineiramente no Instituto Adolfo Lutz, São Paulo, SP. Quarenta e cinco amostras (22.5%) foram Salmonella positivas por pelo menos um dos três métodos. O número de amostras positivas de acordo com o método analítico foi 34 (75,6%) para o método de cultura convencional, 29 (64,4%) para TECRA™ Salmonella VIA e 27 (60.0%) para TECRA™ Salmonella UNIQUE. O método de cultura convencional detectou quatro amostras positivas não detectadas por nenhum dos outros dois métodos rápidos. TECRA™ Salmonella UNIQUE detectou sete amostras positivas não detectadas pelos demais métodos. Uma amostra foi positiva apenas pelo método TECRA™ Salmonella VIA. Considerando todos os resultados (positivos e negativos) o teste de qui quadrado de McNemar indicou que as diferenças entre os resultados obtidos pelos métodos rápidos, quando comparados aos obtidos pelo método convencional, não foram estatisticamente significativas (p>0.05).
The presence of Salmonella in 200 raw food samples of animal origin was investigated by means of rapid immunoassays TECRA™ Salmonella VIA and TECRA™ Salmonella UNIQUE (TECRA Diagnostics, Rosewille, NSW, Australia) and the cultural procedure used routinely in Instituto Adolfo Lutz, Sao Paulo, SP. Forty-five samples (22.5%) were Salmonella positive by at least one of the three methods. The number of positive samples according to the analytical method was 34 (75.6%) for the cultural procedure, 29 (64.4%) for TECRA™ Salmonella VIA and 27 (60.0%) for TECRA™ Salmonella UNIQUE. The cultural method detected four positive samples that both rapid methods were unable to detect. TECRA™ Salmonella UNIQUE detected seven positive samples that were not detected by the two other methods. One sample was positive by the TECRA™ Salmonella VIA exclusively. Considering overall results (positive and negative) McNemar\'s chi square tests indicated that the differences between results given by the rapid immunoassays when compared to those of the cultural method were not significant (p>0.05).

La persistance asymptomatique de Salmonella Enteritidis au niveau du tube digestif des volailles est une préoccupation majeure pour la sécurité alimentaire. Les mécanismes de ce portage sont encore mal connus. L'objectif de la thèse était de mettre en évidence les facteurs de l'immunité innée intestinale qui pouvaient être déficients chez les oiseaux fortement porteurs. Pour cela nous avons analysé l'expression différentielle des gènes de médiateurs inflammatoires et anti-infectieux en comparant des lignées de volaille plus ou moins porteuses de salmonelles. Nous avons pu montrer en particulier que la résistance au portage variait selon l'âge des animaux et que l'expression élevée des gènes de cytokines inflammatoires et des défensines était liée à un meilleur contrôle de la colonisation chez l'adulte mais pas chez le jeune. Ces résultats suggèrent que l'efficacité de ces facteurs dans la lutte des oiseaux contre le portage dépend de la maturation du système immunitaire
Asymptomatic persistence of Salmonella Enteritidis in the digestive tract of fowls is a major concern of food safety. The mechanisms involved in this carriage are still unknown. The objective of this thesis was to identify intestinal innate immunity factors which could be deficient in high carrier birds. Therefore we analysed the differential expression of genes of inflammatory and anti-infectious mediators between lines of birds which vary in the level of Salmonella colonisation. We have particularly shown that the resistance to bacterial colonization was related to the age of the animals, and that the high expression of inflammatory cytokine and defensin genes was related to a better control of Salmonella colonisation in adult but not in young birds. These results suggest that the efficiency of these factors in fighting against Salmonella carriage depends on the maturation of the immune system

No Rio Grande do Sul, a Salmonella Enteritidis vem se destacando como o principal microrganismo causador de surtos de doenças transmitidas por alimentos. A capacidade desse sorovar em aderir ao aço inoxidável e ao polietileno, assim como a resistência a desinfetantes comumente utilizados nas indústrias de alimentos foi avaliada e comparada com outros dois sorovares de Salmonella. Para mensurar a aderência bacteriana, corpos de prova de aço inoxidável (2 x 2 x 0,1cm) e de polietileno (2 x 2 x 0,7cm) permaneceram em contato com as culturas bacterianas por 15, 30 e 60 minutos e então ultrasonicados, para a realização das contagens de células aderidas. A resistência bacteriana aos desinfetantes foi avaliada através do teste de suspensão como preconizado pela legislação brasileira. Para desinfecção das superfícies, os corpos de prova permaneceram por 15 minutos em contato com as culturas bacterianas e, após dez minutos de exposição aos desinfetantes, o número de células sobreviventes foi determinado. A aderência bacteriana após os tempos de exposição indicou que a S. Typhimurium aderiu significativamente mais ao aço inoxidável que ao polietileno, enquanto que a S. Bredeney aderiu mais ao polietileno. Entretanto, não houve diferenças significativas nos níveis de aderência do sorovar S. Enteritidis, mesmo que as análises de microscopia eletrônica de varredura e de hidrofobicidade tenham indicado diferenças significativas entre os materiais. Foi observada a produção de bioemulsificante pelos três sorovares de Salmonella, sendo que a S. Enteritidis e a S. Bredeney produziram quantidades bem maiores que a S. Typhimurium. A resistência aos desinfetantes ácido peracético, hipoclorito de sódio e quaternário de amônio, quando avaliados pelo teste de suspensão, demonstraram que, nas concentrações indicadas pelo fabricante, os três compostos foram capazes de inativar os três sorovares de Salmonella. Entretanto, na concentração de 200ppm de hipoclorito de sódio comumente utilizada no Brasil, o sorovar S. Enteritidis foi o mais resistente, uma vez que sobreviveu até 15 minutos de exposição. Nenhum desinfetante conseguiu inativar todas as células aderidas (aproximadamente 5log UFC/cm2) em aço inoxidável e polietileno, exceto o quaternário de amônio que eliminou totalmente a S. Enteritidis do aço inoxidável. Tendo em vista a importância desses microrganismos como patógenos alimentares, cuidados especiais devem ser tomados nos processos de desinfecção e contaminação cruzada por Salmonella.
In Rio Grande do Sul, southern Brazil, Salmonella Enteritidis have been considered the principal microorganism responsible for foodborne disease. The ability of this sorovar in adhering to stainless steel and polyethylene, as well as the resistance to biocides commonly used in food industries was evaluated and compared with other two serovars of Salmonella. To measure the bacterial adherence, coupons of stainless steel (2 x 2 x 0,1cm) and polyethylene (2 x 2 x 0,7cm) remained in contact with the bacterial cultures for periods of 15, 30 e 60 minutes and so that ultrassonicated for count the adherent cells. The bacterial resistance to biocides was evaluated through the suspension test as praised for the Brazilian legislation. For surfaces disinfection, the coupons remained for 15 minutes in contact with bacterial cultures and after ten minutes of exposition to biocides, the surviving cells were determined. Bacterial adherence after 15, 30, and 60 minutes of exposure indicated that S. Typhimurium adhered significantly more to stainless steel than to polyethylene, whereas S. Bredeney adhered more to polyethylene than to stainless steel. However, there was no significative difference in adherence levels of S. Enteritidis, even when analysis of sacanning electronic microscopy has indicated expressive differences between adherence to the materials. The production of bioemulsifier by Salmonella serovars was observed, being that S. Enteritidis and S. Bredeney produced larger amounts than S. Typhimurium. The resistance to peracetic acid, and quaternary ammonium biocides when evaluated using suspension test in the presence of organic matter, demonstrated that in the concentrations indicated by manufacturers, all of the three biocides were able to inactivate the three serovars. However, using 200ppm of sodium hypochlorite, commonly used in Brazil, S. Enteritidis showed to be the most resistant serovar, since it has survived for up to 15 minutes of exposure. None of the biocides inactivate all the cells adhered (approximately 5log CFU/cm2) in stainless steel and polyethylene, except the quaternary ammonium which totally eliminated S. Enteritidis on the stainless steel surface. In view of the importance of these micorrganismos as alimentary pathogens, special cares of disinfection processes and cross-contamination for Salmonella must be taken.

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

Thesis Submitted in Partial Fulfillment for the Requirements to achieve the Degree of PhD in Biochemistry
El genero Salmonella comprende a mas 2,500 serotipos conocidos distribuidos en dos especies: enterica y bongori. Estos serotipos difieren mucho en términos de patogenicidad y especificidad hospedero. Dos serotipos de Salmonella entérica son de especial relevancia: los serotipos Gallinarum y Enteritidis. S. Gallinarum presenta un rango hospedero restringido a aves y causa una severa enfermedad sistémica conocida como tifoidea aviar, la que causa grandes perdidas económicas en la producción aviar en distintas partes del mundo. S. Enteritidis, en cambio, infecta a un amplio rango de hospederos incluyendo humanos, ratones y aves. A diferencia de S. Gallinarum, S. Enteritidis genera una infección subclinica en los pollos, y las aves infectadas pueden convertirse en portadores crónicos, poniendo huevos contaminados por Salmonella. El consumo humano de productos aviares o huevos resulta en un cuadro de gastroenteritis aguda autolimitante, la cual es responsable por ~61% del 1.5 millones de casos de salmonelosis reportados entre los años 1995 y 2008 (WHO Global Foodborne Infections Network Country Databank). Existen pocos trabajos realizados sobre los mecanismos moleculares detrás de la adaptación al hospedero aviar y sobre las implicancias clínicas de las infecciones causadas por los serotipos Enteritidis y Gallinarum, sin embargo evidencia reciente sugiere que estos serotipos poseen factores de virulencia no descritos que pueden ser responsables de estas diferencias. La interación entre las bacterias y sus hospederos es guiada por una comunicación dinámica que busca influenciar la respuesta del hospedero. Dentro de las herramientas utilizadas por las bacterias para influir la respuesta de sus hospederos, las maquinas secretoras que entregan proteínas y toxinas hacia el ambiente intracelular de sus blancos eucariontes son cruciales para la supervivencia y virulencia bacteriana. El Sistema de Secreción Tipo VI (T6SS) es un nuevo mecanismo de translocación de proteínas que existe en la mayoría de bacterias Gram-negativo que se encuentran en contacto íntimo con células eucariontes, incluyendo a aquellas que son patógenos humanos y de plantas. El papel preciso que cumplen estos T6SS todavía es desconocido pero es claro que cumple un papel importante en la virulencia bacteriana. En Salmonella enterica, solo se ha descrito un T6SS el cual esta codificado en la Isla de Patogenicidad 6 de Salmonella (SPI-6). En esta tesis, a través de análisis bioinformaticos y de genomica comparativa se determinó que el genero Salmonella codifica 5 T6SS, distribuidos diferencialmente entre distintos serotipos y con historias evolutivas diferentes. Los nuevos T6SS fueron identificados en islas genómicas designadas SPI-19, SPI-20, SPI-21 and SPI-22. Ademas de la identificación de estas islas, una nueva proteína VgrG “evolucionada” con un dominio del tipo S-Piocina fue identificado en SPI-21. La presencia de este dominio sugirió por primera vez un papel de los T6SS en muerte bacteriana, abriendo un nuevo capitulo en el estudio de T6SS y su papel en relaciones interbacterianas. El T6SS de SPI-19 fue de especial relevancia debido a su amplia distribución dentro de serotipos virulentos de Salmonella y porque análisis bioinformaticos mostraron que mientras Gallinarum codifica un T6SS completo, el serotipo Enteritidis solo codifica para remanentes de este sistema. A pesar de estar estrechamente relacionados, los serotipos Gallinarum y Enteritidis presentan diferencias profundas en su rango de hospederos y patogenicidad. Por lo tanto, es posible especular que la presencia de un T6SS activo esta relacionada de alguna manera con las diferencias en especificidad hospedero y patogenicidad presentada por estos dos serotipos. Para resolver esta hipótesis y determinar la contribución de SPI-19 a la patogenicidad de Salmonella, el objetivo de esta tesis fue determinar si la isla genomica SPI-19 codifica un T6SS funcional que contribuye a la patogenicidad de Gallinarum y Enteritidis en el hospedero aviar. De manera de caracterizar el T6SS de SPI-19, fusiones génicas y de operon fueron construidas y la expresión, producción y secreción de componentes del T6SS fueron evaluadas bajo diferentes condiciones de crecimiento in vitro. El análisis mostro que la mayoría de los componentes se mantienen reprimidos bajo las condiciones analizadas. Infección de macrófagos murinos con una cepa de Gallinarum con una fusión entre el componente estructural/secretado VgrG al reportero GFP, mostró que los componentes del T6SS son preferencialmente producidos al interior de células infectadas. Mutantes por deleción no polares de la isla SPI-19 y componentes específicos del T6SS reveló que este T6SS es necesario para la supervivencia de Salmonella Gallinarum al interior de macrófagos a tiempos tardios de infección. Sin embargo, el T6SS de SPI-19 no pudo ser asociado muerte celular o citotoxicidad de macrófagos inducida por Salmonella. Para determinar la contribución del T6SS de SPI-19 a la patogenicidad de Salmonella, mutantes del T6SS fueron analizadas en ensayos de competencia contra la cepa silvestre de Gallinarum. Infección oral de pollos White Leghorn de cuatro días de edad, reveló que las mutantes del T6SS colonizaron pobremente el ileo, ciego, hígado y bazo comparado a la cepa silvestre. Restitución de SPI-19 a la mutante SPI- 19, utilizando el sistema VEX-Capture, complementó este defecto en colonización. Para analizar el impacto de poseer un T6SS completo en la habilidad de S. Enteritidis para colonizar al hospedero aviar, la SPI-19 de Gallinarum fue transferida a Enteritidis. Experimentos in vivo mostraron que la presencia de una SPI-19 completa aumento significatvamente la habilidad de Enteritidis para colonizar el ileo, hígado y bazo de pollos infectados al dia 1 post-infección. Sin embargo, esa ventaja en la colonización no fue duradera ya que esta cepa mostró un fuerte defecto en la colonización desde el día 3 post-infección hasta el final de los experimentos. Estos resultados sugieren que transferencia de SPI-19 desde S. Gallinarum tiene un impacto negativo en la habilidad de S. Enteritidis para colonizar el hospedero aviar. De esta forma podemos especular que perdida del T6SS de SPI-19 corresponde a un evento patoadaptativo durante la evolución de S. Enteritidis. Del mismo modo, este es el primer trabajo en el que se utiliza el método VEX-Capture para determinar el efecto de la transferencia de islas genómicas en un modelo animal de infección bacteriana. El reciente descubrimiento de Sistemas de Secreción Tipo VI (T6SS) ha abierto un nuevo capitulo en el estudio de la adaptación de Salmonella hacia sus hospederos y el medio ambiente. Si Salmonella codifica T6SS y si aquellos pueden ser considerados eventos evolutivos cuanticos, fueron algunas de las preguntas que el descubrimiento de los T6SS en los genomas bacterianos generó. En esta tesis, hemos expandido el actual conocimiento sobre los T6SS bacterianos y el potencial patogénico de Salmonella mediante: i) la identificación y descripción de 4 nuevas Islas de Patogenicidad de Salmonella (SPI-19, SPI-20, SPI-21 and SPI-22) que codifican para T6SS filogenéticamente distintos, ii) el descubrimiento de una nueva “VgrG” evolucionada que sugirió por primera vez un papel de los T6SS en relaciones interbacterianas, iii) identificando que el T6SS de SPI-19 contribuye a la supervivencia intracelular de Salmonella en macrófagos y iv) determinando que el T6SS de SPI-19 contribuye a la colonización de pollos por S. Gallinarum.
The Salmonella genus includes over 2,500 known serotypes distributed between the two species: enterica and bongori. These serotypes differ greatly in terms of pathogenicity and host specificity. Two Salmonella enterica serotypes are of significant relevance: serotypes Gallinarum and Enteritidis. S. Gallinarum has a host range restricted to birds and causes a severe systemic disease called fowl typhoid, which causes major economic losses in poultry production in several parts of the world. S. Enteritidis on the other hand, infects a broad range of hosts including humans, mice and avian species. In contrast to S. Gallinarum, S. Enteritidis generates a subclinical infection in poultry, and infected hens can become chronic carriers laying Salmonella contaminated eggs. Human consumption of contaminated poultry or egg products results in an acute self-limiting gastroenteritis, being responsible for ~61% of the estimated 1.5 million human salmonellosis cases reported between 1995 and 2008 (WHO Global Foodborne Infections Network Country Databank). There is little work done on the molecular mechanisms behind the differential host-adaptation and clinical outcomes of infections caused by serotypes Enteritidis and Gallinarum in their susceptible hosts, including birds, but recent evidence suggests that these serotypes might possess undescribed virulence factors that may account for these differences. Interaction between bacteria and hosts is guided by a communication/signaling interplay which aims to influence the host response. Among the tools used by bacteria to influence the host response, secretion machines that deliver proteins and toxins into the environment and within eukaryotic target cells are crucial for bacterial virulence and survival. The Type VI Secretion System (T6SS) is a newly described mechanism for protein translocation that exists in most Gram-negative bacteria that come into close contact with eukaryotic cells, including plant and animal pathogens. The precise role and mode of action of T6SS is still unknown, but it is clear that plays an important role in bacterial virulence. In Salmonella enterica, only one T6SS encoded in Salmonella Pathogenicity Island 6 (SPI-6) has been described. In this thesis, through bioinformatics and comparative genomic analyzes it was determined that the genus Salmonella encodes 5 T6SS loci, differentially distributed among different serotypes and with distinct phylogenetic histories. The novel T6SS loci were identified in genomic islands designated SPI-19, SPI-20, SPI-21 and SPI-22. In addition of the identification of these T6SS loci, a novel “evolved” VgrG protein with a S-Type Pyocin containing-domain, was identified in SPI-21. The presence of this protein domain suggested for the first time a role for T6SSs in bacterial killing opening a new chapter in the study of T6SS and its role in inter-bacterial relationships. The SPI-19 T6SS was of significant relevance due to its wide distribution among virulent Salmonella serotypes and because bioinformatics analyzes showed that while Gallinarum encodes a complete T6SS, serotype Enteritidis only encodes for remnants of this system. Despite being closely related, serotypes Gallinarum and Enteritidis present profound differences in their host-range and pathogenicity. Therefore, it is tempting to speculate that the presence of an active T6SS is somehow related to the host-adaptation and pathogenicity differences presented by these serotypes. To resolve this hypothesis and assess the contribution of SPI-19 to Salmonella pathogenicity, the objective of this thesis was to determine whether the SPI-19 genomic island encodes a functional T6SS contributing to the pathogenicity of Gallinarum and Enteritidis in the avian host. In order to characterize the SPI-19 T6SS, gene and operon fusions were constructed and expression, production and secretion of T6SS components were evaluated under different in vitro growth conditions. The analysis showed that most T6SS components remain repressed under the conditions tested. Infection of murine macrophages with a Gallinarum strain harboring the structural/secreted T6SS component VgrG fused to the GFP reporter showed that T6SS components are preferentially produced inside infected cells. Non-polar deletion mutants of the whole SPI-19 and specific T6SS core components revealed that this T6SS was necessary for Salmonella Gallinarum survival within macrophages at late time points after infection. Furthermore, the SPI-19 T6SS function could not be linked to Salmonella-induced cytotoxicity or cell death of infected macrophages. To determine the contribution of SPI-19 T6SS to Salmonella pathogenesis, T6SS mutants were tested in competitive infection assays against the wild-type Gallinarum parental strain. Oral infection of four-day-old White Leghorn chicks revealed that T6SS mutants colonized the ileum, ceca, liver and spleen poorly compared to the wild-type strain. Restitution of SPI-19 to the ΔSPI-19 mutant, using VEX-Capture, complemented this colonization defect. Altogether, the data indicate that SPI-19 and the T6SS encoded therein contributes to macrophage intracellular survival and colonization of chicks infected by S. Gallinarum. To assess the impact of carrying a complete T6SS locus on the ability of S. Enteritidis to colonize the avian host, the SPI-19 from Gallinarum was transferred to Enteritidis. In vivo experiments showed that presence of a complete SPI-19 significantly increased the ability of Enteritidis to colonize the ileum, liver and spleen of infected chicks by day 1 post-infection. This colonization advantage was not lasting however, as this strain presented a strong colonization defect for each organ analyzed from day 3 post infection to the conclusion of the experiment. These results suggest that transfer of SPI-19 from S. Gallinarum has a negative impact on the ability of S. Enteritidis to colonize the avian host. In this context is tempting to speculate that loss of the SPI-19 T6SS corresponds to a pathoadaptative event during S. Enteritidis evolution. In addition, this is the first report of the use of Vex-Capture method to assess the effect of Genomic Island transfer in an animal model of bacterial infection. The recent discovery of Type VI Secretion Systems (T6SS) has opened a new chapter in the study of Salmonella host and environmental adaptation. Whether Salmonella encodes T6SSs and whether they could be considered as quantum leap evolution events are some of the questions that the discovery of T6SS in bacterial genomes generated. In this thesis, we have expanded the current knowledge on bacterial T6SSs and Salmonella virulence potential by: i) the identification and description of 4 novel Salmonella Pathogenicity Islands (SPI-19, SPI-20, SPI-21 and SPI-22) encoding phylogenetically distinct T6SS loci, ii) the discovery of a novel “evolved” VgrG protein, which suggested for the first time a role for T6SSs in interbacterial relationships, iii) identifying that the SPI-19 T6SS contributes to Salmonella intracellular survival in macrophages and iv) determining that the SPI-19 T6SS contributes to chicken colonization by S. Gallinarum.

Salmonella Enteritidis y Salmonella Gallinarum son dos patógenos importantes para el hospedero aviar. El primero causa infecciones silentes en aves de postura, lo que le permite infectar los huevos y tras el consumo de éstos, llegar al hospedero humano. El segundo, causa en el ave un cuadro de gastroenteritis severa y posterior tifoidea aviar, generando graves consecuencias económicas y productivas. Previamente, mediante herramientas bioinformáticas, identificamos una nueva isla de patogenicidad no caracterizada en cuatro serotipos distintos de Salmonella, entre ellos Gallinarum y Enteritidis. Esta fue nombrada isla de patogenicidad 19 de Salmonella (SPI-19), y codificaría para un sistema de secreción tipo VI (T6SS), un sistema de secreción de proteínas el cual se encuentra ampliamente distribuido entre las bacterias Gram negativo. S. Gallinarum posee codificado en la SPI-19 todos los genes esenciales para un T6SS funcional, mientras que S. Enteritidis posee una versión trunca de la isla, y sólo posee algunos genes del T6SS. Este sistema cumpliría una serie de funciones en los procesos patogénicos dependiendo de la bacteria, y hasta el momento se ha descrito su participaciòn en adherencia, invasión, citotoxicidad y proliferación intracelular. Al analizar filogenéticamente el T6SS codificado en SPI-19 se descubrió que se encontraba cercano a los sistemas codificados en bacterias como Vibrio cholerae y Aeromonas hydrophila. En estos patógenos ya se ha descrito un rol del T6SS en la interacción con células del sistema inmune, particularmente en la sobrevida y citotoxicidad en líneas celulares de macrófagos. Debido a que la capacidad de Salmonella para sobrevivir dentro de macrófagos es un proceso fundamental para su diseminación sistémica y colonización de órganos blanco, en esta memoria se propuso determinar el papel que cumple el T6SS codificado en SPI-19 en la interacción con macrófagos murinos. Para esto se estudió la capacidad de sobrevivir al interior de macrófagos RAW 264.7 de mutantes en genes estructurales y efectores del T6SS. A su vez, se analizó la capacidad citotóxica de estas cepas. Los resultados muestran que mutantes de S. Gallinarum presentan una supervivencia menor a tiempos tardíos de infección. En S. Enteritidis la mutante para SPI-19 no presenta una disminución significativa de la sobrevida. En ambos serotipos no se presentan cambios en la citotoxicidad. También se estudió la expresión en la infección de macrófagos del efector VgrG mediante una fusión con el fluoróforo GFP. Esto reveló una rápida expresión de la fusión, lo que sugiere que el contacto bacteria-macrófago gatilla la expresión del efector. Estos resultados demuestran que la isla SPI-19 codifica para un T6SS funcional, que participa en un proceso esencial de la infección por Salmonella.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A Salmonella Pullorum (SP) é muito semelhante à Salmonella Gallinarum (SG), agentes da Pulorose e Tifo aviário, respectivamente, sendo responsáveis por perdas econômicas no setor avícola. São salmonelas de composição antigênica similar e a distinção de ambas tem sido baseada em provas bioquímicas. Entretanto, este procedimento é laborioso e o surgimento de estirpes com comportamento bioquímico atípico tem estimulado a procura por outros métodos de diagnóstico, como os testes moleculares. No presente estudo, analisou-se o método descrito na literatura envolvendo o gene rfbS em conjunto com testes desenvolvidos com base nos genes speC e speF. O gene rfbS codifica a enzima que atua na parede celular de bactérias e tem sido utilizado para diferenciar SP e SG. Com o propósito de padronizar a PCR para ser utilizada na rotina laboratorial, empregou-se dois métodos de extração de DNA e diferentes concentrações de reagentes. Entretanto, tendo em vista a dificuldade de padronização encontrada desde o início, foi concomitantemente desenvolvida a PCR para os genes speC e speF. Estes dois últimos estão relacionados com a produção da enzima ornitina-descarboxilase, a qual é ativa em SP e inativa em SG. Após testes inicias, foi possível padronizar a PCR do gene speC com posterior utilização da técnica de tratamento enzimático com a enzima de restrição Eco RI. Tanto para o gene rfbS quanto para os genes speC e speF, os resultados encontrados permitiram a diferenciação de 13 amostras de SP e 20 de SG isoladas no Brasil e duas cepas ATCC. Sendo assim, as ações sanitárias e a tomada de decisão no campo podem ser conduzidas de maneira rápida e segura.
Salmonella Pullorum (SP) is very similar to Salmonella Gallinarum (SG). They are respectively agents of Pulorosis and Avian Typhoid, both diseases responsible for economic losses to poultry production. Due to the fact that both salmonellas have similar antigenic composition, their differentiation has been based in biochemical tests. However, this fact that this kind of procedure is very troublesome and the occurence of strains showing atypical behavior have stimulated the search for others diagnostic methods, such as molecular tests. In the present study, a method described in the literature using gene rfbS was analyzed together with tests developed based on genes speC and speF. Gene rfbS encodes an enzyme that acts on the cell wall of bacteria and has been used in the differentiation between SP and SG. In order to standardize the PCR procedure to be used in laboratory routine, two methods for DNA extraction were used, as well as different concentrations of the reagents. However, due to the difficulty in the standardization observed from the beginning of the study, a PCR procedure was developed for genes speC and speF. These genes are related to the production of the enzyme ornithine-descarboxilase, active in SP and inactive in SG. After the initial tests, PCR of gene speC was standardized, and later on it was coupled with the enzymatic treatment with restriction enzyme Eco RI. Results obtained for gene rfbS as well as for genes speC and speF enabled differentiation of 13 SP and 20 SG strains isolated in Brazil and two strains ATCC. So, the sanitary actions and the decisions in the livestock can to be conduced with safety and rapidity.