No controle da infecção por Salmonella, a correção de fatores de risco tem sido associada a medidas alternativas como a adição de prebióticos às dietas. O mananoligossacarídeo (MOS) é um carboidrato capaz de adsorver bactérias Gram-negativas que possuem fímbria tipo 1, essencial para a colonização do intestino. O presente estudo teve como objetivo: i. avaliar isolados de Salmonella enterica quanto à presença e expressão de fímbria tipo 1 e adsorção ao MOS; ii. avaliar em suínos o efeito do MOS no índice de infecção e excreção de Salmonella; na microbiota entérica e no título de IgA. Um total de 108 isolados de Salmonella, foi avaliado quanto à amplificação dos genes fimA e fimH, hemaglutinação manose-sensível e adsorção ao MOS. Em todos os isolados ambos os genes foram amplificados, 31 expressaram fímbria tipo 1 e, desses, 74,2% adsorveram fortemente ao MOS. Após, 23 suínos desmamados, alocados em grupo tratamento (com MOS) e controle, foram inoculados, com um isolado de S. Typhimurium capaz de adsorver ao MOS, e acompanhados por 28 dias. A quantidade de Salmonella excretada pelos suínos foi baixa, apresentando tendência de menores medianas no grupo tratamento. Não houve diferença significativa no número de infectados nem nas densidades óticas do ELISA IgG e IgA. As amostras de tonsila apresentaram o maior número de isolamentos. Contagens similares de lactobacilos, enterococos e coliformes foram encontradas nos dois grupos. Concluiu-se que a adição de MOS não impediu a infecção dos suínos por Salmonella, porém houve uma tendência de menor excreção no grupo tratado. / In the control of Salmonella infection, correction of risk factors has been associated with alternative measures such as the addition of prebiotics to diets. Mannanoligosaccharide (MOS) is a complex carbohydrate, able to adsorb Gram-negative bacteria that possess type-1 fimbriae, essential for colonization of the intestine. This study aimed to: i, evaluate isolates of Salmonella enterica, the presence and expression of type-1 fimbriae, and agglutination with MOS; and ii, evaluate in pigs the effect of MOS on the rate of infection and excretion of Salmonella, and enteric microbiota and Salmonella IgA titers. A total of 108 strains of Salmonella sp. isolated from carrier pigs were evaluated for the amplification of fimA and fimH genes, agglutination with MOS and hemagglutination. In all strains tested, amplicons of the expected size were detected, 31 expressing type-1 fimbriae; and of these, 74.2% showed a strong agglutination to MOS. A total of 23 weaned pigs were allotted to a treatment group (with MOS) and a control group, were inoculated with a strain of S. Typhimurium able to adsorb to the MOS, and were evaluated for 28 days. The amount of Salmonella excreted by the pigs was low, showing a trend toward a lower median in the treated group. The ELISA IgG and IgA optical density of both groups showed no significant difference. In our study, the tonsils contributed to the highest rate of Salmonella isolation. The medians of coliforms, enterococci and lactobacilli were similar throughout the experiment in both groups. It was concluded that the addition of MOS did not affect the infection of pigs by Salmonella, although the excretion rate was lower in the treated group.

No controle da infecção por Salmonella, a correção de fatores de risco tem sido associada a medidas alternativas como a adição de prebióticos às dietas. O mananoligossacarídeo (MOS) é um carboidrato capaz de adsorver bactérias Gram-negativas que possuem fímbria tipo 1, essencial para a colonização do intestino. O presente estudo teve como objetivo: i. avaliar isolados de Salmonella enterica quanto à presença e expressão de fímbria tipo 1 e adsorção ao MOS; ii. avaliar em suínos o efeito do MOS no índice de infecção e excreção de Salmonella; na microbiota entérica e no título de IgA. Um total de 108 isolados de Salmonella, foi avaliado quanto à amplificação dos genes fimA e fimH, hemaglutinação manose-sensível e adsorção ao MOS. Em todos os isolados ambos os genes foram amplificados, 31 expressaram fímbria tipo 1 e, desses, 74,2% adsorveram fortemente ao MOS. Após, 23 suínos desmamados, alocados em grupo tratamento (com MOS) e controle, foram inoculados, com um isolado de S. Typhimurium capaz de adsorver ao MOS, e acompanhados por 28 dias. A quantidade de Salmonella excretada pelos suínos foi baixa, apresentando tendência de menores medianas no grupo tratamento. Não houve diferença significativa no número de infectados nem nas densidades óticas do ELISA IgG e IgA. As amostras de tonsila apresentaram o maior número de isolamentos. Contagens similares de lactobacilos, enterococos e coliformes foram encontradas nos dois grupos. Concluiu-se que a adição de MOS não impediu a infecção dos suínos por Salmonella, porém houve uma tendência de menor excreção no grupo tratado. / In the control of Salmonella infection, correction of risk factors has been associated with alternative measures such as the addition of prebiotics to diets. Mannanoligosaccharide (MOS) is a complex carbohydrate, able to adsorb Gram-negative bacteria that possess type-1 fimbriae, essential for colonization of the intestine. This study aimed to: i, evaluate isolates of Salmonella enterica, the presence and expression of type-1 fimbriae, and agglutination with MOS; and ii, evaluate in pigs the effect of MOS on the rate of infection and excretion of Salmonella, and enteric microbiota and Salmonella IgA titers. A total of 108 strains of Salmonella sp. isolated from carrier pigs were evaluated for the amplification of fimA and fimH genes, agglutination with MOS and hemagglutination. In all strains tested, amplicons of the expected size were detected, 31 expressing type-1 fimbriae; and of these, 74.2% showed a strong agglutination to MOS. A total of 23 weaned pigs were allotted to a treatment group (with MOS) and a control group, were inoculated with a strain of S. Typhimurium able to adsorb to the MOS, and were evaluated for 28 days. The amount of Salmonella excreted by the pigs was low, showing a trend toward a lower median in the treated group. The ELISA IgG and IgA optical density of both groups showed no significant difference. In our study, the tonsils contributed to the highest rate of Salmonella isolation. The medians of coliforms, enterococci and lactobacilli were similar throughout the experiment in both groups. It was concluded that the addition of MOS did not affect the infection of pigs by Salmonella, although the excretion rate was lower in the treated group.

No controle da infecção por Salmonella, a correção de fatores de risco tem sido associada a medidas alternativas como a adição de prebióticos às dietas. O mananoligossacarídeo (MOS) é um carboidrato capaz de adsorver bactérias Gram-negativas que possuem fímbria tipo 1, essencial para a colonização do intestino. O presente estudo teve como objetivo: i. avaliar isolados de Salmonella enterica quanto à presença e expressão de fímbria tipo 1 e adsorção ao MOS; ii. avaliar em suínos o efeito do MOS no índice de infecção e excreção de Salmonella; na microbiota entérica e no título de IgA. Um total de 108 isolados de Salmonella, foi avaliado quanto à amplificação dos genes fimA e fimH, hemaglutinação manose-sensível e adsorção ao MOS. Em todos os isolados ambos os genes foram amplificados, 31 expressaram fímbria tipo 1 e, desses, 74,2% adsorveram fortemente ao MOS. Após, 23 suínos desmamados, alocados em grupo tratamento (com MOS) e controle, foram inoculados, com um isolado de S. Typhimurium capaz de adsorver ao MOS, e acompanhados por 28 dias. A quantidade de Salmonella excretada pelos suínos foi baixa, apresentando tendência de menores medianas no grupo tratamento. Não houve diferença significativa no número de infectados nem nas densidades óticas do ELISA IgG e IgA. As amostras de tonsila apresentaram o maior número de isolamentos. Contagens similares de lactobacilos, enterococos e coliformes foram encontradas nos dois grupos. Concluiu-se que a adição de MOS não impediu a infecção dos suínos por Salmonella, porém houve uma tendência de menor excreção no grupo tratado. / In the control of Salmonella infection, correction of risk factors has been associated with alternative measures such as the addition of prebiotics to diets. Mannanoligosaccharide (MOS) is a complex carbohydrate, able to adsorb Gram-negative bacteria that possess type-1 fimbriae, essential for colonization of the intestine. This study aimed to: i, evaluate isolates of Salmonella enterica, the presence and expression of type-1 fimbriae, and agglutination with MOS; and ii, evaluate in pigs the effect of MOS on the rate of infection and excretion of Salmonella, and enteric microbiota and Salmonella IgA titers. A total of 108 strains of Salmonella sp. isolated from carrier pigs were evaluated for the amplification of fimA and fimH genes, agglutination with MOS and hemagglutination. In all strains tested, amplicons of the expected size were detected, 31 expressing type-1 fimbriae; and of these, 74.2% showed a strong agglutination to MOS. A total of 23 weaned pigs were allotted to a treatment group (with MOS) and a control group, were inoculated with a strain of S. Typhimurium able to adsorb to the MOS, and were evaluated for 28 days. The amount of Salmonella excreted by the pigs was low, showing a trend toward a lower median in the treated group. The ELISA IgG and IgA optical density of both groups showed no significant difference. In our study, the tonsils contributed to the highest rate of Salmonella isolation. The medians of coliforms, enterococci and lactobacilli were similar throughout the experiment in both groups. It was concluded that the addition of MOS did not affect the infection of pigs by Salmonella, although the excretion rate was lower in the treated group.

Evalúa la expresión relativa de las citoquinas involucradas en la respuesta inmune Th1 y Th2 en 21 cuyes (Cavia porcellus) inoculados experimentalmente con una cepa aislada de campo de Salmonella Typhimurium a una dosis de 102 UFC/ml vía intraperitoneal y 7 cuyes inoculados con la misma cepa inactivada (grupo control). Se utilizó animales destetados de 15-30 días de edad, sin importar sexo y clínicamente sanos, todos mantenidos en las mismas condiciones en el bioterio de la Facultad de Medicina Veterinaria de la Universidad Nacional Mayor de San Marcos. Se agruparon los cuyes en 4 pozas (A, B, C y D) y cada poza con 7 animales cada uno, además se colocaron 2 cuyes centinelas no inoculados. La toma de muestras de sangre y tejidos se realizó lo días 1, 3, 5, 7, 9, 15 y 30 postinoculación, siendo colectados 3 individuos del grupo tratamiento y 1 del grupo control. El aislamiento de los leucocitos sanguíneos se realizó por la técnica del gradiente de Ficoll y la extracción del ARN total de éstas células mediante el uso de Trizol. El ADNc fue sintetizado a partir de las muestras de ARN y luego se desarrolló la PCR en tiempo real con “primers” específicos para las citoquinas en estudio. La expresión relativa fue determinada por el método comparativo 2-ΔΔCt a fin de evaluar la expresión de los ARNm de las citoquinas analizadas con respecto al calibrador (cuy sano), usando como gen constitutivo de referencia al GAPDH. Las expresiones de los genes de todas las citoquinas mostraron un aumento con respecto a los calibradores y un incremento con mayor intensidad con respecto a las expresiones de los cuyes del grupo control; además, una cinética ascendente con respecto a los días post-inoculación, los primeros días hubo predominio de las expresiones del perfil Th1, posteriormente ambas aumentan con predominio final de las citoquinas de la respuesta inmune Th2. El análisis histopatológico y el aislamiento se realizó para confirmar la infección y los animales mostraron las lesiones características de salmonelosis, siendo el hígado el órgano afectado con mayor frecuencia. / Perú. Ministerio de la Producción. Programa Nacional de Innovación para la Competitividad y Productividad (Innóvate Perú). Fondo para la Innovación, la Ciencia y la Tecnología (FINCyT) / Tesis

Memoria para optar al Título Profesional de Médico Veterinario / En la actualidad la detección de cepas bacterianas resistentes a múltiples agentes antimicrobianos parece ir en aumento y se atribuye principalmente a la inadecuada utilización de ellos. Estos fármacos han permitido la selección de genes de resistencia en bacterias zoonóticas, como Salmonella, los cuales pueden diseminarse a los seres humanos a través de la cadena alimentaria. Además cobra importancia el rol de las aves silvestres como reservorios y agentes diseminadores de genes de resistencia a antibióticos hacia los seres humanos y otros animales. El objetivo de esta Memoria de Título fue comparar los perfiles genéticos de resistencia antimicrobiana de cepas de S. enterica ser. Enteritidis aisladas desde aves silvestres, aves comerciales y seres humanos en Chile, y determinar su relación con el hospedero de origen de estas cepas. Se analizaron 96 cepas en total, de las cuales 33 fueron aisladas desde aves silvestres, 31 de aves comerciales y 32 de seres humanos. Se utilizó la técnica de Reacción en Cadena de la Polimerasa (PCR) para determinar los perfiles de resistencia antimicrobiana. Los genes seleccionados fueron: tet(A), tet(B), tet(G) (resistencia a tetraciclinas), blaPSE-1, blaTEM, blaCMY (resistencia a β-lactámicos), aadB, aacC (resistencia a aminoglicósidos) y además se detectó la presencia de integrones clase 1. Para determinar la asociación de los perfiles genéticos de resistencia con su hospedero de origen se utilizó el programa Infostat®. De las 96 cepas analizadas en esta Memoria, se encontraron 13 perfiles genéticos de resistencia antimicrobiana diferentes, los cuales resultaron estar asociados con el hospedero de origen de las cepas. Adicionalmente, los datos obtenidos fueron analizados con la finalidad de determinar la asociación de genes de resistencia antimicrobiana con hospedero de origen de las cepas, siendo tet(A) el único gen asociado a cepas obtenidas de aves silvestres. Los resultados de esta Memoria proporcionan evidencia sobre los efectos colaterales que provoca la sobreutilización de antimicrobianos en el medio ambiente, impactando indirectamente en la fauna silvestre

Identifica Salmonella Typhimurium y Enteritidis aislada de cuyes mediante la técnica de PCR múltiple. Con ese fin, evalúa un total de 25 cepas obtenidas a partir de órganos de cuyes muertos con signos clínicos y lesiones anatomopatológicas sugerentes de salmonelosis además de pruebas bioquímicas que identifican las cepas como pertenecientes al género Salmonella spp. Analiza dichas cepas mediante la técnica de PCR múltiple con cebadores específicos para los genes invA, fliC y Prot6E correspondientes al género Salmonella, Salmonella Typhimurium y Salmonella Enteritidis, respectivamente. Las 25 cepas evaluadas son identificadas como Salmonella Typhimurium, evidenciando bandas de peso molecular correspondientes a los genes invA (284 pb) y el gen fliC (559 pb). La técnica de PCR es útil para identificar el género Salmonella y el serovar Typhimurium a partir de cuyes aislados con signos clínicos sugerentes de salmonelosis.

Tesis entregada a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al grado de Doctor en Ciencias con Mención Microbiología. / Salmonella Typhimurium is an enteric pathogen able to infect different animal hosts, including humans. In immunocompetent humans, S. Typhimurium mainly causes gastroenteritis, a disease characterized by an inflammatory diarrhoea with massive neutrophil infiltration in the ileum and colon. The infective cycle of Salmonella starts with the ingestion of bacteria that reach the small intestine and invade epithelial cells by its apical face. After crossing the epithelial barrier, bacteria are captured by phagocytic cells of the immune system present in the sub-epithelium, such as macrophages, neutrophils and dendritic cells, being contained within a membrane bound compartment. Here, Salmonella subverts the endocytic route, avoiding the fusion of this compartment with the lysosomes and generating the Salmonella-containing vacuole (SCV). In this compartment, Salmonella is able to survive and replicate. The ability to modify this intracellular niche explains the ability of this pathogen to survive intracellularly. To carry out this process, Salmonella employs two Type Three Secretion Systems (T3SS) and an arsenal of secreted effector proteins in order to take control over the eukaryotic cell. An important aspect of Salmonella’s life cycle that has not been studied in detail is its survival in the environment, where bacteria are exposed to predation by protozoa, and specially by amoebae. These organisms are specialized phagocytes that feed on bacteria and fungi. To address this interaction, we and other groups use amoeba models to characterise the molecular processes involved in the survival of intracellular pathogens within environmental protozoa. Among these model organisms, the social amoeba Dictyostelium discoideum is amenable for molecular analyses in laboratory settings and several tools have been developed in this organism for the study of different aspects of its interaction with bacterial pathogens. Recently, our group described that S. Typhimurium is able to survive intracellularly in the social amoeba Dictyostelium discoideum, and that mutants in genes required for virulence in other infection models present survival defects in this host. Despite of this, the mechanisms that allow the intracellular survival of this pathogen in this kind of organism have not been studied in detail. This Thesis proposed the characterization at the cellular level of this interaction, with a focus on two secreted effector proteins of S. Typhimurium that are directly related to SCV generation and modification in other cell models: SopB and SifA. Our results show that these effectors are needed for intracellular survival of S. Typhimurium in D. discoideum. Furthermore, by means of a combination of microscopy and proteomic analyses we were able to characterise the protein composition of the vacuolar compartment containing Salmonella in this host. Our results show that known markers linked to this compartment in other cell types and the autophagy machinery play a role in the biogenesis of this intracellular niche in D. discoideum. / Salmonella Typhimurium es un patogeno enterico que tiene la capacidad de infectar diversos hospederos animals, incluyendo el ser humano. En individuos inmunocompetentes, S. Typhimurium provoca gastroenteriris, una enfermedad diarreica inflamatoria caracterizada por la masiva infiltracion de neutrofilos en el ileon y el colon. El ciclo infectivo de Salmonella comienza con la ingestion de las bacterias que al llegar al intestino delgado invaden las celulas epiteliales por la cara apical. Luego de cruzar la barrera epitelial, las bacterias son capturadas por las celulas fagociticas del sistema inmune que residen en el sub-epitelio, como macrofagos, neutrofilos y celulas dendriticas, quedando contenida en un compartimento membranoso. En esta etapa, Salmonella interviene la ruta endocitica, evitando la fusion de este compartimento con el lisosoma y generando la vacuola contenedora de Salmonella (Salmonella-containing vacuole: SCV). En este compartimento, Salmonella es capaz de sobrevivir y replicarse. La habilidad de modificar este nicho intracelular explica la habilidad de este patogeno de sobrevivir intracelularmente. Para esto, Salmonella utiliza dos Sistemas de Secrecion Tipo Tres (Type Three Secretion Systems: T3SS) y un arsenal de proteinas efectoras secretadas para tomar control sobre la celula eucarionte. Por otra parte, un importante aspecto del ciclo de vida de Salmonella que no ha sido estudiado en detalle es su supervivencia en el ambiente, donde las bacterias se encuentran expuestas a la depredacion por protozoos y en particular, amebas. Estos organismos son fagocitos profesionales que se alimentan de bacteria y hongos. Recientemente, nuestro grupo describio que S. Typhimurium es capaz de sobrevivir intracelularmente en la ameba social Dictyostelium discoideum y que mutantes en genes requeridos para la virulencia en numerosos modelos de infeccion tambien presentan defectos de supervivencia en este hospedero. A pesar de esto, los mecanismos que le permiten a este patogeno en este tipo de organismo no han sido estudiado en detalle. Para entender esta interaccion, nosotros y otros grupos usamos modelos de ameba a fin de caracterizar los procesos moleculares involucrados en la supervivencia de patogenos intracelulares en el interior de protozoos ambientales. Dentro de estos organismos modelo, la ameba social Dictyostelium discoideum es sencilla para el analisis molecular en condiciones de laboratorio. Por otra parte, numerosas herramientas se han desarrollado en este organismo para el estudio de diversos aspectos de su interaccion con patogenos bacterianos. Esta Tesis propuso caracterizar a nivel cellular esta interaccion, enfocandonos en dos proteinas efectoras secretadas de S. Typhimurium que estan directamente relacionadas a la formacion y modificacion de la SCV en otros modelos celulares: SopB y SifA. Nuestros resultados muestran que estos efectores son necesarios para que S. Typhimurium sobreviva intracelularmente en D. discoideum. Adicionalmente, mediante una combinacion de tecnicas de microscopia y analisis proteomicos pudimos caracterizar la composicion proteica de este compartimento vacuolar que contiene a Salmonella en este hospedero. Nuestros resultados muestran que marcadores asociados a la SCV en otras lineas celulares se encuentran en elcompartimento que se genera en D. discoideum y que la maquinaria de autofagia juega un rol importante en la biogenesis de este nicho intracelular en D. discoideum. / FONDECYT grants 1140754 y 1171844, CONICYT Doctoral Fellowship 21140615. / Enero 2020.

O Brasil é hoje um dos maiores produtores e exportadores de alimento no mundo, destacando-se no cenário de produção de proteína animal como o 1º país exportador em volume de carne de frango, dentre tantas conquistas recentes do agronegócio brasileiro. Essas conquistas foram alcançadas pelo país graças à sua inegável vocação agropecuária, resultado de uma série de fatores, como extensão territorial e áreas agricultáveis, clima, disponibilidade de água e ambiente regulatório favorável à adoção de tecnologias. Dentre as tecnologias existentes e disponíveis que tornam possível um país de vocação agrícola se tornar um dos maiores produtores mundiais de alimentos estão os aditivos zootécnicos melhoradores do desempenho e, dentre eles, os antimicrobianos. Na posição de um dos atores principais no cenário internacional de produção de alimentos, o Brasil vem-se deparando com questões relacionadas à preocupação global com relação à segurança do alimento e aos escândalos de contaminação de alimentos ocorridos em diversos países, inclusive na Europa. A preocupação com a segurança do alimento tem levado os diversos organismos internacionais, dentre eles, a Organização Mundial da Saúde e a Organização Mundial de Saúde Animal a estabelecerem critérios de análise de risco com relação aos principais patógenos envolvidos em surtos de toxinfecções alimentares e ao risco de transmissão de resistência bacteriana através dos alimentos. Sendo assim, o presente estudo foi desenvolvido com base na lista de antimicrobianos criticamente importantes, elaborada em conjunto pela OMS e OIE, na determinação do perfil de resistência destes frente a Salmonella isolada de aves e materiais avícolas em diferentes períodos. Foram utilizadas 100 amostras de Salmonella spp isoladas em granjas no Brasil, sendo 68 amostras colhidas durante o período atual (de 2008 a 2010) e 32 amostras colhidas durante a década de 90 (de 1989 a 1999), as quais passaram por testes de difusão em disco, determinação da concentração inibitória mínima, sorotipificação e determinação clonal por eletroforese em campo pulsado. Com relação às amostras colhidas no período atual (2008 a 2010), evidenciou-se uma redução no perfil de sensibilidade para diferentes antimicrobianos, marcadamente para a classe das cefalosporinas, com 19 (22%) das amostras resistentes e 38 (56%) das amostras apresentando resistência intermediária. Várias das amostras desse período apresentaram multirresistência. Para 32 das amostras isoladas na década de 90, não se evidenciou resistência a nenhum dos antimicrobianos selecionados, embora se tenham encontrado 13 amostras (41%) com perfil de resistência intermediário para o ceftiofur. Ainda, detectaram-se 10 diferentes sorovares de Salmonella com somente um perfil clonal para as amostras antigas e perfil variado para as amostras atuais. Os resultados comprovaram o aumento de resistência aos antimicrobianos criticamente importes em amostras recentes isoladas de aves e material avícola. Tais dados comprovam que cada vez mais a atenção deve ser focada no uso prudente dos antimicrobianos na produção animal. / Brazil is currently one of the largest producers and exporters of food in the world, appearing in the animal protein production ranking as the 1st exporter of poultry, in terms of volume, among many recent achievements of the brazilian agribusiness. These achievements have been made possible due to the country natural agriculture vocation, which is the result of a number of factors, such as land extension and available land for agriculture, climate, water availability and regulatory environment open to technology adoption. Among the existing technologies available that enable a country to become one of the largest producers of food are the feed additives and performance enhancers, including the antibiotics. In the position of one of the key actors in the international food production environment, Brazil has been facing issues related to the global concerns on food safety and the food contamination scandals occurred in several countries, including in Europe. These concerns has led several international organizations, including the World Health Organization and the International Organization of Animal Health to establish criteria for risk analysis in relation to the main pathogens involved in foodborne disease outbreaks and the risk of transmission of resistant bacteria from food. Therefore, this study was developed based on the list of the critically important antimicrobials, developed jointly by WHO and the OIE, in determining the resistance profile of them against Salmonella isolated from poultry and poultry materials in different periods. It have been used 100 samples of Salmonella isolated from poultry farms in Brazil, 68 samples taken during the current period (2008 to 2010) and 32 samples taken during the 90s (1989-1999), which have been tested for antibiotic disk diffusion, minimum inhibitory concentration, serotyping and clone determination by pulsed field gel electrophoresis. In the samples collected in the current period (2008-2010), there was a reduction in susceptibility to different antimicrobials, notably for the class of cephalosporins with 19 (22%) of isolates resistant and 38 (56%) of samples presenting intermediate resistance. Several samples from this period showed multidrug resistance. For 32 of the isolates in the 90s, there was no evidence of resistance to any antimicrobial selected, although it was found 13 samples (41%) with intermediate resistance profile for ceftiofur. It have been identified 10 different Salmonella serovars, among them only one clone profile for the old samples and different clone profiles for the current samples. Results showed increased antimicrobial resistance to the critically important antimicrobials for the more recent isolates from poultry and poultry material. These data demonstrate that more attention should be focused on the prudent use of antimicrobials in animal production.

食物中毒感染是常見但非常重要的全球性公共健康問題。沙門氏鼠傷寒桿菌乃常被分離出來的細菌性病原體之一。隨著實驗室參考菌株LT2的基因組序列於2001年被發表之後，另外9個沙門氏鼠傷寒菌菌株的基因序列均已陸續進行測序。最近，本實驗室亦對十個本地沙門氏鼠傷寒菌臨床分離菌株的基因序列進行了測序。為了為這些基因組序列提供高品質的註釋，我們把預測的基因組提交到質量控制工具GenePRIMP以識別有潛在錯誤或異常的預測基因。本研究針對血液分離菌株78896和糞便分離菌株1047518的GenePRIMP報告進行人工檢查，並對每個菌株超過270個的基因進行了修訂。此外，本研究亦對上述的10個本地菌株進行了功能註釋。註釋項目包括沙門氏菌致病島（SPIs）、致病因子、tRNA和非編碼小分子RNA、噬菌體和CRISPRs結構等基因組及致病元素。 KEGG通路則提供了進一步的功能註釋。 / 本研究同時對本地的血液和糞便分離菌株，連同國外的臨床分離菌株，進行了廣泛的比對，用以識別全身性沙門氏菌感染的潛在遺傳因素。 本研究進行了以下基因分析：（1）多位點序列分型（MLST）；（2）在小鼠全身性感染中涉及的主調控因子和關鍵元素; 及（3）人類腸胃道感染中涉及的基因。然而，這些分析產生只能對全身性沙門氏菌感染提供有限的見解。然而，透過使用RAST註釋系統，我們於其中三個血液分離菌株中發現了一個的額外的螯鐵蛋白aerobactin鐵採集系統。儘管在體外實驗中，這些血液分離菌株並沒有明顯的生長優勢，但實驗結果表明，在缺乏鐵的培養液中，aerobactin基因的表達水平是比較高的。此外，我們亦於其中四個血液分離菌株中，發現負責細胞色素c熟成(ccm)的基因座均被中斷。這可能改變了這些血液分離菌株中細胞色素c的生物合成途徑。這些鐵採集和同化機制的觀察均為未來全身性沙門氏菌感染的研究提供了可能的發展方向。 / 本研究同時識別了用以分別本地及海外的沙門氏鼠傷寒菌菌株的分子標記，並在鮭魚和生菜的接種實驗中，展現了它們分辨本地及海外菌株的能力。然而，在投入實際應用之前，這些標記尚需要進一步的驗證和測試，以便確定快速檢測方法的有效性。 / Foodborne infection is a common but important public health issue worldwide. Salmonella enterica serovar Typhimurium is frequently isolated from outbreaks as one of the common bacterial causative agents. Following the availability of the genome sequence of the reference lab strain LT2 in 2001, nine genomes of S. Typhimurium had been sequenced since then. Recently, genomes of ten local S. Typhimurium clinical isolates have been assembled in our laboratory. In order to provide high quality annotation of these genome sequences, the predicted gene sets were submitted to the quality control tool GenePRIMP (Gene PRediction IMprovement Pipeline) to identify potentially erroneous and abnormal gene calls. The GenePRIMP reports for the local blood isolate 78896 and stool isolate 1047518 were manually inspected and more than 270 genes were amended individually for each isolate. Functional annotation had also been performed for the 10 local isolates. Genomic and virulent elements including Salmonella Pathogenicity Islands (SPIs), virulence factors, tRNAs and small non-coding RNAs, prophage elements and CRISPRs structures had been annotated. The KEGG pathways provided a further means of functional annotation. / The local blood and stool isolates, together with the sequenced foreign clinical isolates, had also been extensively compared to identify potential genetic determinants of Salmonella systemic infection. (1) Multilocus sequence typing (MLST); (2) Alignment of master regulators and key players of systemic infection in mice; and (3) Analyses of the genes responsible for human gastrointestinal tract infection had been performed. However, these analyses yielded limited insights on systemic infection. Alternatively, using subsystems annotation by RAST, an additional aerobactin siderophore iron acquisition system was shown to be prevalent among three of the blood isolates. Despite no obvious growth advantage was offered to the blood isolates in an in vitro experiment, it was demonstrated that expression of the aerobactin genes was higher in iron-depleted culturing medium. In addition, a disrupted cytochrome c maturation (ccm) locus that may alter the cytochrome c biogenesis pathway was also identified in four of the blood isolates. These observations in iron acquisition and assimilation mechanisms suggest their potential in future direction of Salmonella systemic infection studies. / Molecular markers specific to local and foreign S. Typhimurium isolates were also identified and their utility in differentiating local and foreign isolates was demonstrated in a pilot spiking experiment using raw salmon and lettuce. These markers will require further verification and testing prior to actual application in real-world settings in order to examine the validity of the rapid detection method. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheng, Chi Keung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 124-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract of thesis entitled --- p.iii / 摘要 --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xiii / Abbreviations --- p.xiv / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Introduction and Taxonomy --- p.1 / Chapter 1.2 --- Epidemiology of Salmonella Typhimurium infections --- p.2 / Chapter 1.3 --- Pathogenesis of Salmonella Typhimurium infection --- p.4 / Chapter 1.3.1 --- Infection mechanisms --- p.4 / Chapter 1.3.2 --- Salmonella Pathogenicity Islands --- p.6 / Chapter 1.3.3 --- Regulation of virulence --- p.9 / Chapter 1.4 --- Non-typhoid Salmonella (NTS) systemic infection --- p.11 / Chapter 1.4.1 --- Epidemiology of NTS systemic infection --- p.11 / Chapter 1.4.2 --- Salmonella Typhimurium multidrug resistance --- p.12 / Chapter 1.5 --- Salmonella Typhimurium genomics --- p.15 / Chapter 1.5.1 --- Salmonella Typhimurium genome sequencing --- p.15 / Chapter 1.5.2 --- Comparative studies on Salmonella genomes --- p.17 / Chapter 1.6 --- Aims of project --- p.19 / Chapter Chapter 2 --- Curation and detailed annotation of genomes of local Salmonella Typhimurium clinical isolates --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Manual curation of GenePRIMP results --- p.27 / Chapter 2.2.2 --- Salmonella Pathogenicity Islands (SPIs) and virulence factors annotation --- p.29 / Chapter 2.2.3 --- Small RNA and t-RNA annotation --- p.29 / Chapter 2.2.4 --- Phage elements annotation --- p.30 / Chapter 2.2.5 --- CRISPRs annotation --- p.30 / Chapter 2.2.6 --- KEGG annotation --- p.30 / Chapter 2.3 --- Results --- p.32 / Chapter 2.3.1 --- Manual curation of GenePRIMP results --- p.32 / Chapter 2.3.1.1 --- Short genes --- p.35 / Chapter 2.3.1.2 --- Long genes --- p.35 / Chapter 2.3.1.3 --- Unique genes --- p.36 / Chapter 2.3.1.4 --- Overlapped genes --- p.36 / Chapter 2.3.1.5 --- Broken genes --- p.37 / Chapter 2.3.2 --- Salmonella Pathogenicity Islands (SPIs) and virulence factors annotation --- p.37 / Chapter 2.3.2.1 --- Salmonella Pathogenicity Islands (SPIs) annotation --- p.37 / Chapter 2.3.2.2 --- Virulence factors annotation --- p.44 / Chapter 2.3.3 --- Small RNA and t-RNA annotation --- p.44 / Chapter 2.3.4 --- Phage elements annotation --- p.44 / Chapter 2.3.5 --- CRISPRs annotation --- p.50 / Chapter 2.3.6 --- KEGG annotation --- p.51 / Chapter 2.4 --- Discussion --- p.53 / Chapter 2.4.1 --- Manual curation of GenePRIMP results --- p.53 / Chapter 2.4.1.1 --- Gene amendment not required --- p.54 / Chapter 2.4.1.2 --- Genes with boundaries relocated --- p.54 / Chapter 2.4.1.3 --- Genes to be discarded --- p.55 / Chapter 2.4.1.4 --- Gene pairs to be fused --- p.55 / Chapter 2.4.1.5 --- Potential pseudogenes formation --- p.56 / Chapter 2.4.2 --- Salmonella Pathogenicity Islands (SPIs) annotation --- p.57 / Chapter 2.4.3 --- Virulence factors annotation --- p.57 / Chapter 2.4.4 --- Small RNA and t-RNA annotation --- p.58 / Chapter 2.4.5 --- Phage elements annotation --- p.59 / Chapter Chapter 3 --- Identification of genetic determinants of blood invasiveness in local S. Typhimurium clinical isolates --- p.61 / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Materials and Methods --- p.66 / Chapter 3.2.1 --- Multilocus Sequence Typing (MLST) --- p.66 / Chapter 3.2.2 --- Phage elements annotation for foreign isolates --- p.67 / Chapter 3.2.3 --- Alignment of genes inferred to play important roles in NTS systemic --- p.infection67 / Chapter 3.2.4 --- Alignment of genes inferred to involved during infection in the gastrointestinal (GI) tract --- p.68 / Chapter 3.2.5 --- Subsystems assignment using Rapid Annotation using Subsystem Technology (RAST) server --- p.68 / Chapter 3.2.6 --- Growth analysis of local S. Typhimurium clinical isolates in iron-limiting environment --- p.69 / Chapter 3.2.7 --- Reverse transcription and real-time PCR --- p.70 / Chapter 3.2.7.1 --- Primer design and verification --- p.70 / Chapter 3.2.7.2 --- cDNA synthesis and real-time PCR --- p.70 / Chapter 3.3 --- Results --- p.73 / Chapter 3.3.1 --- Multilocus Sequence Typing (MLST) --- p.73 / Chapter 3.3.2 --- Phage elements annotation for foreign isolates --- p.73 / Chapter 3.3.3 --- Alignment of genes inferred to play important roles in NTS systemic infection --- p.74 / Chapter 3.3.4 --- Alignment of genes inferred to involved during infection in the gastrointestinal (GI) tract --- p.79 / Chapter 3.3.4.1 --- Acid tolerance response --- p.79 / Chapter 3.3.4.2 --- Epithelial cells attachment --- p.80 / Chapter 3.3.4.3 --- Epithelial cells invasion --- p.83 / Chapter 3.3.4.4 --- Survival within macrophages --- p.83 / Chapter 3.3.5 --- RAST subsystem analysis --- p.86 / Chapter 3.3.6 --- Growth analysis and aerobactin genes expression --- p.87 / Chapter 3.4 --- Discussion --- p.93 / Chapter Chapter 4 --- Molecular markers identification and testing on selected foodstuff for local S. Typhimurium isolates --- p.97 / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.2 --- Materials and Methods --- p.101 / Chapter 4.2.1 --- Molecular markers identification --- p.101 / Chapter 4.2.2 --- Primer design and verification --- p.101 / Chapter 4.2.3 --- Spiking experiments on selected food samples --- p.103 / Chapter 4.2.4 --- Quantitative TaqMan real-time PCR --- p.103 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- Molecular markers identification --- p.105 / Chapter 4.3.2 --- Spiking experiments and TaqMan real-time PCR --- p.109 / Chapter 4.4 --- Discussion --- p.113 / Chapter 4.4.1 --- Molecular markers identification --- p.113 / Chapter 4.4.2 --- Spiking experiments and TaqMan real-time PCR --- p.114 / Chapter Chapter 5 --- General discussion --- p.116 / Chapter 5.1 --- Manual curation of GenePRIMP results --- p.116 / Chapter 5.2 --- Functional annotation of local S. Typhimurium genomes --- p.118 / Chapter 5.3 --- Systemic infection studies --- p.120 / Chapter 5.4 --- Molecular markers identification and spiking experiments --- p.121 / Chapter 5.5 --- Conclusion and future perspectives --- p.122 / References --- p.124

O Brasil é hoje um dos maiores produtores e exportadores de alimento no mundo, destacando-se no cenário de produção de proteína animal como o 1º país exportador em volume de carne de frango, dentre tantas conquistas recentes do agronegócio brasileiro. Essas conquistas foram alcançadas pelo país graças à sua inegável vocação agropecuária, resultado de uma série de fatores, como extensão territorial e áreas agricultáveis, clima, disponibilidade de água e ambiente regulatório favorável à adoção de tecnologias. Dentre as tecnologias existentes e disponíveis que tornam possível um país de vocação agrícola se tornar um dos maiores produtores mundiais de alimentos estão os aditivos zootécnicos melhoradores do desempenho e, dentre eles, os antimicrobianos. Na posição de um dos atores principais no cenário internacional de produção de alimentos, o Brasil vem-se deparando com questões relacionadas à preocupação global com relação à segurança do alimento e aos escândalos de contaminação de alimentos ocorridos em diversos países, inclusive na Europa. A preocupação com a segurança do alimento tem levado os diversos organismos internacionais, dentre eles, a Organização Mundial da Saúde e a Organização Mundial de Saúde Animal a estabelecerem critérios de análise de risco com relação aos principais patógenos envolvidos em surtos de toxinfecções alimentares e ao risco de transmissão de resistência bacteriana através dos alimentos. Sendo assim, o presente estudo foi desenvolvido com base na lista de antimicrobianos criticamente importantes, elaborada em conjunto pela OMS e OIE, na determinação do perfil de resistência destes frente a Salmonella isolada de aves e materiais avícolas em diferentes períodos. Foram utilizadas 100 amostras de Salmonella spp isoladas em granjas no Brasil, sendo 68 amostras colhidas durante o período atual (de 2008 a 2010) e 32 amostras colhidas durante a década de 90 (de 1989 a 1999), as quais passaram por testes de difusão em disco, determinação da concentração inibitória mínima, sorotipificação e determinação clonal por eletroforese em campo pulsado. Com relação às amostras colhidas no período atual (2008 a 2010), evidenciou-se uma redução no perfil de sensibilidade para diferentes antimicrobianos, marcadamente para a classe das cefalosporinas, com 19 (22%) das amostras resistentes e 38 (56%) das amostras apresentando resistência intermediária. Várias das amostras desse período apresentaram multirresistência. Para 32 das amostras isoladas na década de 90, não se evidenciou resistência a nenhum dos antimicrobianos selecionados, embora se tenham encontrado 13 amostras (41%) com perfil de resistência intermediário para o ceftiofur. Ainda, detectaram-se 10 diferentes sorovares de Salmonella com somente um perfil clonal para as amostras antigas e perfil variado para as amostras atuais. Os resultados comprovaram o aumento de resistência aos antimicrobianos criticamente importes em amostras recentes isoladas de aves e material avícola. Tais dados comprovam que cada vez mais a atenção deve ser focada no uso prudente dos antimicrobianos na produção animal. / Brazil is currently one of the largest producers and exporters of food in the world, appearing in the animal protein production ranking as the 1st exporter of poultry, in terms of volume, among many recent achievements of the brazilian agribusiness. These achievements have been made possible due to the country natural agriculture vocation, which is the result of a number of factors, such as land extension and available land for agriculture, climate, water availability and regulatory environment open to technology adoption. Among the existing technologies available that enable a country to become one of the largest producers of food are the feed additives and performance enhancers, including the antibiotics. In the position of one of the key actors in the international food production environment, Brazil has been facing issues related to the global concerns on food safety and the food contamination scandals occurred in several countries, including in Europe. These concerns has led several international organizations, including the World Health Organization and the International Organization of Animal Health to establish criteria for risk analysis in relation to the main pathogens involved in foodborne disease outbreaks and the risk of transmission of resistant bacteria from food. Therefore, this study was developed based on the list of the critically important antimicrobials, developed jointly by WHO and the OIE, in determining the resistance profile of them against Salmonella isolated from poultry and poultry materials in different periods. It have been used 100 samples of Salmonella isolated from poultry farms in Brazil, 68 samples taken during the current period (2008 to 2010) and 32 samples taken during the 90s (1989-1999), which have been tested for antibiotic disk diffusion, minimum inhibitory concentration, serotyping and clone determination by pulsed field gel electrophoresis. In the samples collected in the current period (2008-2010), there was a reduction in susceptibility to different antimicrobials, notably for the class of cephalosporins with 19 (22%) of isolates resistant and 38 (56%) of samples presenting intermediate resistance. Several samples from this period showed multidrug resistance. For 32 of the isolates in the 90s, there was no evidence of resistance to any antimicrobial selected, although it was found 13 samples (41%) with intermediate resistance profile for ceftiofur. It have been identified 10 different Salmonella serovars, among them only one clone profile for the old samples and different clone profiles for the current samples. Results showed increased antimicrobial resistance to the critically important antimicrobials for the more recent isolates from poultry and poultry material. These data demonstrate that more attention should be focused on the prudent use of antimicrobials in animal production.