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Corrigenda attached after the bibliography. Bibliography: leaves 166-206.

Files on accompanying CD-ROM: Appendix III Phages ST64T and ST64B sequences, are in rtf format. Bibliography: leaves 279-324. System requirements for accompanying CD-ROM: IBM or compatible ; Microsoft Word or compatible to read rtf files.

Corrigenda attached after the bibliography. / Bibliography: leaves 166-206. / 206 leaves [6] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Sciences, 2001

Files on accompanying CD-ROM: Appendix III Phages ST64T and ST64B sequences, are in rtf format. / Bibliography: leaves 279-324. / System requirements for accompanying CD-ROM: IBM or compatible ; Microsoft Word or compatible to read rtf files. / xii, 325, [8] leaves, [116] leaves of plates : ill. (some col.) ; 30 cm. + 1 CD-ROM (4 3/4 in.) / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2002?

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.

xiv, 207 leaves : ill., tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1976

Thesis (Ph.D.) -- University of Adelaide, Dept. of Microbiology, 1976.

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

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.