<i>Salmonella enterica</i> serovar Enteritidis (<i>S.</i> Enteritidis) is a major cause of gastrointestinal disease in humans worldwide that is mainly associated with the consumption of contaminated poultry meat and eggs. During the course of infection, <i>S.</i> Enteritidis uses two Type 3 Secretion Systems (T3SS), one of which is encoded by <i>Salmonella</i> Pathogenicity Island-1 (SPI-1). SPI-1 plays a major role in the invasion process.<p> In order to study the role of SPI-1 in the colonization of chickens, we constructed deletion mutants affecting either the complete SPI-1 region (40 kb) or <i>invG</i>, a single gene located on this pathogenicity island. The mutants were impaired in the secretion of effector proteins and were less invasive compared to the wild type strain in polarized Caco-2 cells. Similarly, when chicken cecal and small intestinal explants were co-infected with the wild type and ÄSPI-1 mutant strains we found that the ÄSPI-1 mutant strain was less invasive relative to the wild type strain. Oral challenge of 1-week-old chickens with the wild type or ÄSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection, measured as levels of <i>Salmonella</i> in the liver and spleen, was delayed in birds that were challenged with the ÄSPI-1 strain. This demonstrates that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of S. Enteritidis in chickens.<p> Based on the above results, we examined the effect of sera against SPI-1 T3SS components to <i>S.</i> Enteritidis invasion. Anti-SipD serum protected Caco-2 cells against entry of wild type <i>S.</i>Enteritidis, but not against invasion of a mutant strain lacking sipD. On the other hand, sera against InvG, PrgI, SipA, SipC, SopB, SopE and SopE2 did not affect S. Enteritidis entry. To illustrate the specificity of anti-SipD mediated inhibition, SipD specific antibodies were depleted from the serum. Depleted serum restored the invasion of S. Enteritidis, demonstrating that the SipD protein may be an important target in blocking SPI-1 mediated virulence.<p> To determine if SPI-1 T3SS proteins were protective against <i>S.</i> Enteritidis oral challenge, chickens were vaccinated subcutaneously twice at 14 and 28 days of age with PrgI and SipD. The results indicate that these proteins induce strong IgG antibody responses and confer significant protection against infection of the livers in vaccinated birds. In another study, we vaccinated hens with selected SPI-1 T3SS proteins to determine if their progeny could be protected from <i>S.</i> Enteritidis oral challenge. The proteins induced strong antibody responses but did not affect the levels of the challenge strain in the ceca or internal organs of the vaccinates. Taken together, our results establish that <i>S.</i> Enteritidis SPI-1 is an important virulence factor in chickens and that the proteins associated with this T3SS may form components of a subunit vaccine used for protection against colonization by <i>S.</i> Enteritidis in poultry.

Estudis previs duts a terme en el nostre laboratori han caracteritzat estructuralment i fenotípicament el bacteriòfag SE1. Aquest bacteriòfag presenta una elevada freqüència de transducció, capaç d'infectar S. enterica, serovar Enteritidis i serovar Typhimurium. Seguint aquesta línea d'investigació, el present treball s'ha plantejat ampliar la descripció genètica d'aquest bacteriòfag i caracteritzar les diferents funcions del fag SE1 en estat de lisogènia, tals com la conversió lisogènica, la integració i el seu regulador o interruptor genètic .La seqüenciació del genoma del bacteriòfag SE1, a partir d'una genoteca fàgica o per walking directament sobre el genoma, ha permès determinar que té una llargària de 41.941 pb, que es concreta en 67 orf. La comparació amb la base de dades GenBank ha revelat que aquest fag, com d'altres fags lambdoides, és un mosaic genètic resultat de recombinacions i transferències horitzontals amb d'altres bacteriòfags. La seqüència obtinguda va permetre mostrar que la deficiència en l'extrem carboxi terminal periplasmàtic de la proteïna codificada per un dels gens de conversió lisogènica (GtrC) podria ser la causa per la qual el bacteriòfag P22 podria infectar una cèl·lula lisògena per al bacteriòfag SE1.A més, en el present treball s'han determinat les seqüències dels operadors de la regió reguladora que interaccionen amb la proteïna cI de l'interruptor o regulador genètic. A través d'assaigs de retard electroforètic (EMSA) i de footprinting s'ha definit la seqüència consens dels motius d'unió dels operadors a la proteïna cI: AtAN3tTN3TATT.D'altra banda, l'anàlisi de la composició de la unitat transcripcional del gen cI per RT-PCR va permetre determinar que el gen orf23 en formava part. La proteïna Orf23 seria un potencial regulador membre de la superfamília de les ATPases involucrades en la partició genòmica. Mutants defectius en aquest gen presenten un increment de la inducció del cicle lític en presència de mitomicina C, la qual cosa indica que aquesta proteïna podria ser un modulador de la proteïna cI o que podria haver-hi una interacció entre la proteïna Orf23 i la proteïna cI. Aquesta és la primera vegada que es caracteritza una proteïna d'aquesta família d'ATPases en un bacteriòfag que s'integra en el genoma del seu hoste. / Carried out previous studies in our laboratory have characterized structurally and phenotipically the bacteriophage SE1. This bacteriophage presents a high frequency of transduction; it is able to infect S. enterica, to serovar Enteritidis and to serovar Typhimurium. Continuing this line of investigation, in the present work we have considered to extend the genetic description of this bacteriophage and to characterize different functions from phage SE1 in lysogen state, such as the lysogenic conversion, integration and its regulator or genetic switch. The sequencing of the bacteriophage SE1 genome, from its genotec or by walking directly on the genome, has allowed us to determine that it has a 41,041 pb of length, that takes shape in 67 orfs. The comparison with the data base of the GenBank has revealed that this phage, like other lambdoids phages, is a genetic mosaic result of recombination and horizontal transferences with other bacteriophages. The obtained sequence allowed us to show that the deficiency in the periplasmatic carboxi terminal end of the protein (GtrC) codified by one of the genes of lysogenic conversion could be the cause by which the bacteriophage P22 would be able to infect a lysogen cell by bacteriophage SE1. In addition, in the present work, the sequences of operators of regulating region have been determined, which interact with protein cI of the switch or genetic regulator. Through tests of electrophoretic mobility shift assay (EMSA) and footprinting consensus sequence for union of operators to protein cI has been defined: AtAN3tTN3TATT. On the other hand, analysis of the composition of gene cI transcriptional unit by RT-PCR allowed us to determine that the gene orf23 comprised of the same one. The Orf23 protein is a regulating potential member of the superfamily of the ATPases involved in the genomic partition. Defective mutants in this gene present an increase of the induction of the lytic cycle in presence of mitomicina C, this indicates that this protein could be a modulator of the protein cI or that could have an interaction between the Orf23 protein and protein cI. This is the first time that a protein of this family of ATPases is characterized in a bacteriophage which integrates in the genome of its host cell. / Estudios previos llevados a cabo en nuestro laboratorio han caracterizado estructuralmente y fenotípicamente el bacteriófago SE1. Este bacteriófago presenta una elevada frecuencia de transducción, capaz de infectar S. enterica, serovar Enteritidis y serovar Typhimurium. Continuando esta línea de investigación, en el presente trabajo se ha planteado ampliar la descripción genética de este bacteriófago y caracterizar las diferentes funciones del fago SE1 en estado de lisogenia, tales como la conversión lisogénica, la integración y su regulador o interruptor genético.La secuenciación del genoma del bacteriófago SE1, a partir de su genoteca o por walking directamente sobre el genoma, ha permitido determinar que tiene una longitud de 41.041 pb, que se concreta en 67 orfs. La comparación con la base de datos del GenBank ha revelado que dicho fago, como otros fagos lambdoides, es un mosaico genético resultado de recombinaciones y transferencias horizontales con otros bacteriófagos.La secuencia obtenida permitió mostrar que la deficiencia en el extremo carboxi Terminal periplasmático de la proteína codificada por uno de los genes de conversión lisogénica (GtrC) podría ser la causa por la cual el bacteriófago P22 podria infectar una célula lisógena por el bacteriófago SE1.Además, en el presente trabajo se han determinado las secuencias de los operadores de la región reguladora que interaccionan con la proteína cI del interruptor o regulador genético. A través de ensayos de retardo electroforético (EMSA) y de footprinting se ha definido la secuencia consenso de los motivos de unión de los operadores a la proteína cI: AtAN3tTN3TATT.Por otro lado, el análisis de la composición de la unidad transcripcional del gen cI por RT-PCR permitió determinar que el gen orf23 formaba parte de la misma. La proteína Orf23 seria un potencial regulador miembro de la superfamilia de las ATPasas involucradas en la partición genómica. Mutantes defectivos en este gen presentan un aumento de la inducción del ciclo lítico en presencia de mitomicina C, esto indica que esta proteína podría ser un modulador de la proteína cI o que podría haber una interacción entre la proteína Orf23 y la proteína cI. Esta es la primera vez que se caracteriza una proteína de esta familia de ATPasas en un bacteriófago que se integra en el genoma de su hospedador.

No description available.

El objetivo fundamental de la Tesis doctoral, es aportar una alternativa a los antibióticos como promotores de crecimiento animal. En este sentido se evaluaron productos preparados a partir de extractos naturales, especialmente extractos de Rutáceas y ácidos orgánicos.Los objetivos específicos de esta investigación se resumen en:1. Evaluar la actividad antibacteriana y antifúngica in vitro de extractos naturales sobre bacterias y hongos de importancia en producción y sanidad animal, y su actividad al ser adicionados conjuntamente con ácidos orgánicos, determinando la metodología a seguir.2. Evaluación de los parámetros microbiológicos y productivos de cerdo en diferentes estadios de su ciclo de crecimiento, al suministrarles piensos adicionados de extractos naturales mezclados con ácidos orgánicos, como promotores de crecimiento.A partir de los resultados obtenidos en los ensayos in vitro, se observa que el método del Aromatograma en combinación con el de difusión en agar permite evaluar correctamente, la actividad antimicrobiana de los extractos naturales en combinación o no con ácidos orgánicos. Asimismo podemos indicar que por el método de dilución en microplacas se establecen las concentraciones mínimas inhibitorias de los extractos de plantas sobre bacterias y hongos, comparando la potencia entre productos. Podemos indicar que los extractos de Rutáceas evaluados en nuestros experimentos poseen actividad inhibitoria tanto para bacterias como para hongos, observándose una mayor capacidad inhibidora frente a las bacterias Gram positivas que sobre las bacterias Gram negativas ensayadas.Al mezclar extractos de Rutáceas y ácidos orgánicos se observa un claro efecto sinérgico que se manifiesta en un notable incremento de la actividad inhibitoria del producto resultante frente a bacterias y hongos.Cuando el extracto de Rutáceas se adiciona a sustratos como arroz y maíz contaminados con Aspergillus ochraceus productor de ocratoxina A, es capaz de inhibir el crecimiento fúngico y la producción de ocratoxina A, por lo que este extracto puede ser utilizado como un sistema de control implicado en Seguridad Alimentaria.Se ha evidenciado que el extracto de Rutáceas ensayado posee actividad enzimática, comprobando la presencia de: esterasa, lipasa, fosfatasas y N-acetil-?- glucosaminidasa. Al adicionarlo a un cultivo en medio líquido de Escherichia coli o de Salmonella typhimurium, es capaz de modificar el metabolismo de los citados microorganismos, colaborando en la pérdida de viabilidad de las cepas ensayadas.Cuando los extractos de Rutáceas se adicionan a sustratos destinados a alimentación (piensos compuestos, materias primas, entre otros), la concentración eficaz para que sean capaces de controlar y mantener la calidad microbiológica varía como consecuencia de la capacidad de la matriz para facilitar su difusión y de la presencia de sustancias inhibidoras, entre otros factores.Definidas las características inhibitorias de los extractos ensayados in vitro, se procedió al estudio de la actividad in vivo de aquellos que presentaron mejores resultados, pudiendo observarse que su adición a la dieta de cerdos, tanto destetados, como adultos, no disminuye la ingesta diaria de pienso, por el contrario la incrementan y aumenta la ganancia diaria de peso, determinando un mejor índice de conversión de los 21 a los 63 días de crecimiento, al comparar los resultados obtenidos con los registrados en cerdos destetados a los que se les suministraron antibióticos como promotores de crecimiento durante el mismo periodo de tiempo. Asimismo se ha evidenciado el hecho de que suministrar en la dieta de cerdos tanto en destete como en engorde, una mezcla de extractos de Rutáceas constituye una medida correcta, que mejora sus parámetros productivos, presenta efectos positivos sobre la microbiota intestinal ya que disminuye los recuentos de la microbiota entérica e incrementa la presencia de bacterias del ácido láctico y no determina efectos nocivos sobre el animal. / The aim of this Thesis was to contribute to an alternative to antibiotics as growth promoters in farm-animals. In this way, products prepared from natural extracts were evaluated, specially Rutaceae extracts and organic acids.The specific objectives of this investigation are:1. To evaluate in vitro the antibacterial and antifungal activity of natural extracts against bacteria and fungi of importance in production and animal health as well as its activity when added mixed with organic acids.2. Evaluation of microbiological and productive parameters in swine focusing on different stages of its life cycle, when providing them with feed supplemented with mixtures of natural extracts and organic acids.From the in vitro results, it is assessed that the Aromatogram method in combination with the agar diffusion test, it allows to evaluate correctly, the antimicrobial activity of the natural extracts alone or mixed with organic acids. On the other hand we may indicate that using the microplate dilution, the Minimum inhibitory concentration of plant extracts against bacteria and fungi can be established. At the same time, we can point out that the evaluated Rutaceae extracts possesses an inhibitory activity against bacteria and fungi. This activity seems to be greater in front of Gram-positive bacteria than Gram-negative bacteria.The mixture of Rutaceae extracts with organic acids, show a clear synergic effect as demonstrated in our results.The addition of Rutaceae extracts on rice and maize inoculated with a strain of Aspergillus ochraceus producer of ochratoxin A, determines an inhibition of the fungal growth and minimizes ochratoxin A production.The tested Rutaceae extracts, showed enzymatic activity, we detected the presence of: esterase, lipases, phosphatases and N-acetyl-?- glucosaminidase. When these extracts were added in a broth culture of Escherichia coli or Salmonella typhimurium, the metabolism of these bacteria, was modified.The in vivo studies carried out showed that the Rutaceae extracts addition on swine feed (adults and weaned) did not decrease the daily intake. It was also observed that daily weight gain increased so that a better conversion index was obtained (from 21 to 63 growth days), when comparing the results with those in weaned pigs fed which feed supplemented with antibiotics as growth promoters.In conclusion the addition of Rutaceae extracts in feed improves swine productive parameters and it determine positive effects on intestinal microbiota due to the fact that decreases enteric microbiota counts and increases the presence of lactic acid bacteria and it does not determine harmful effects on the animal.

El consumo de jamón curado de pavo en lonchas es elevado en los países de cultura anglosajona. Las industrias que elaboran este producto se encuentran con la situación de que en la Unión Europea no existe una legislación sobre las tolerancias microbiológicas para los productos curados derivados de las carnes de las aves de corral. Esta falta de legislación específica provoca que los países consumidores apliquen sus propias exigencias sanitarias, que pueden ser diferentes según el cliente. Esto comporta un problema para los industriales homologados, ya que algunos clientes exigen que este producto sea conservado a temperaturas de refrigeración durante su vida comercial, ya que lo consideran como carne fresca.Los objetivos que se plantearon para el estudio fueron los siguientes: Conocer la incidencia, el comportamiento y la supervivencia de Salmonella choleraesuis y de Listeria innocua en las pechugas de pavo frescas; durante el proceso de elaboración de pechugas de pavo curadas; durante la vida comercial del producto mantenido a distintas temperaturas de conservación (4º y 20ºC) y con diferentes tratamientos tecnológicos (vacío y altas presiones); así como la influencia de la flora acompañante sobre la supervivencia de estos microorganismos.Para el estudio se elaboraron tres partidas según la fórmula tradicional utilizada por la industria, a las cuales se les inoculó S. choleraesuis y L. innocua. Cada partida fue dividida en cuatro lotes sometidos, cada uno de ellos, a condiciones distintas. Los lotes 1 y 2, tratados al vacío y con altas presiones, respectivamente, fueron conservados a 4ºC. Los lotes 3 y 4, igualmente tratados al vacío y con altas presiones, se conservaron a 20ºC.Una vez finalizado el estudio de las tres partidas anteriores, se elaboró una cuarta partida para comprobar los resultados obtenidos y para conocer y estudiar el efecto de la flora acompañante sobre la supervivencia de L. innocua. Esta partida se elaboró a partir de jamón curado de pavo en lonchas, envasado al vacío en envase comercial, al que se le había inoculado L. innocua. Posteriormente, se dividió la partida en cuatro lotes con las mismas características de conservación y tratamiento tecnológico que las descritas anteriormente.En las muestras de pechugas de pavo frescas pertenecientes a las tres primeras partidas no se detectó la presencia de Listeria ni de Salmonella. Durante la maduración y curado de las pechugas de pavo, la población de L. innocua disminuyó 6 unidades logarítmicas, mientras que S. choleraesuis no fue detectada al final del proceso de maduración.Los recuentos de L. innocua se mantuvieron constantes durante la vida comercial del jamón curado de pavo mantenido a 4ºC, mientras que disminuyeron cuando se conservaron a 20ºC (al séptimo mes el microorganismo no se detectó). El tratamiento con altas presiones (400 MPa / 10 min / 12ºC) no afectó al comportamiento y supervivencia de L. innocua durante la vida comercial del producto.La flora acompañante del jamón de pavo curado influyó en el comportamiento de L. innocua. A 4ºC, la flora acompañante no influyó en L. innocua. Sin embargo, a 20ºC la competencia por los nutrientes y la producción de metabolitos bacterianos afectaron a la supervivencia de L. innocua. / Consumption of sliced cured turkey ham is high in Anglo-Saxon countries. Industries involved in the elaboration of this product found that the European Union legislation does not include microbiological levels for poultry cured. For that reason, the consumer countries apply their own safety measures which can be different from one to another. That fact is a problem for the industries. Some clients demand that this product be storaged at refrigeration temperatures during its commercial life, because they consider it as raw meat.The aim of this study was to know the incidence, the behaviour and the survival of Salmonella choleraesuis and Listeria innocua in raw poultry breasts; along the elaboration procedure of cured turkey breasts; along the shelf life of the product storaged at different temperatures (4º or 20ºC) and with different technological treatments (vacuum or high pressure); and, the influence of another flora also present in the product on the survival of these microorganisms.According to the industry elaboration process, three sets of raw turkey breasts were made and inoculated with S. choleraesuis and L. innocua. Each set was divided in four bactches and storaged at different conditions: Batch 1 (4ºC, vacuum), 2 (4ºC, high pressure), 3 (20ºC, vacuum), and 4 (20ºC, high pressure).When the study of that three sets was finished, a fourth set was elaborated in order to probe the obtained results and, moreover, to know the effect of another flora on the survival of L. innocua. This set was elaborated with sliced cured turkey ham, vacuum packaged in commercial film wrapping, and inoculated with L. innocua. Afterwards, this set was divided in four batches as it has been previously described.Listeria or Salmonella were not detected in raw turkey breast samples. Population of L. innocua decreased 6 logarithmic cycles during the cured period. However, S. choleraesuis was not detected at the end of that period.Counts of L. innocua were constant during the commercial shelf life of the cured turkey ham storaged at 4ºC. However, that counts decreased when the product was storaged at 20ºC (the microorganism was not detected on seventh month). The processing treatment with high pressure (400 MPa / 10 min / 12ºC) did not affect the behaviour and survival of L. innocua along the commercial shelf life of the product.Flora also present in the cured turkey ham showed some influence on the behaviour of L. innocua. At 4ºC, that flora did not affect L. innocua. However, the competence for nutrients and the production of bacterial metabolites affected the survival of L. innocua.

Salmonella enterica es una enterobacteria zoonótica que reside en el intestino de los animales. Normalmente genera infecciones asintomáticas en animales de granja. Las serovariedades no tifoideas causan brotes asociados a alimentos siendo S. Enteritidis y S. Typhimurium las que presentan una mayor prevalencia a nivel mundial. La principal vía de transmisión de este patógeno a humanos se produce a través del consumo de alimentos contaminados de origen animal. Es por ello que, actualmente, se está promoviendo una actuación integrada en todos los niveles de la cadena alimentaria bajo el lema “de la granja a la mesa”. El principal reservorio de S. enterica son las especies aviares y en particular Gallus gallus. Por ello, la Unión Europea (UE) ha publicado una serie de regulaciones para intentar disminuir, sobretodo en Gallus gallus, la prevalencia de las principales serovariedades de Salmonella como S. Enteritidis y S. Typhimurium. No obstante, dado que con el uso de las medidas utilizadas hasta el momento no han permitido llegar al objetivo, se presenta una clara necesidad de disponer de nuevas herramientas para reducir la presencia de Salmonella en todo el sector de producción aviar. Por ello, el objetivo del presente trabajo ha sido aislar, seleccionar y caracterizar fagos de Salmonella, para estudiar su aplicación en Gallus gallus. Para abordar el objetivo propuesto, se aislaron bacteriófagos específicos de Salmonella, seleccionándose aquellos que presentaron mejores perfiles líticos, rango de huésped y cinética de infección sobre S. Typhimurium ATCC 14028 y S. Enteritidis LK5. Tras estosestudios, se escogieron a los fagos UAB_Phi20 y UAB_Phi87 como candidatos para terapia fágica y se procedió a su caracterización biológica y molecular. El estudio de estos parámetros indicó que el fago UAB_Phi20 pertenece a la familia Podoviridae y al grupo de fagos tipo P22, mientras el fago UAB_Phi87 es de la familia Myoviridae y del grupo de fagos tipo Felix O1. Ambos fagos presentaron una buena estabilidad a distintas temperaturas y pH, si bien su título disminuye significativamente a pH 2. El análisis de las secuencias de ambos fagos indicó que ninguno de ellos contiene genes de virulencia conocidos. Asimismo, se demostró in vitro que la acción combinada de ambos fagos, junto con el fago UAB_Phi78, provoca una mayor reducción de la concentración de Salmonella que la de los fagos individuales. La experimentación animal, realizada en un modelo murino y en Gallus gallus, demostró el corto tiempo de residencia de los fagos en el tracto intestinal de los animales y la necesidad de administrar repetidas dosis fágicas para conseguir un efecto significativo en la reducción de este patógeno. Así, se consiguió que el 50% de los ratones sobreviviera a la infección promovida por S. Typhimurium ATCC 14028 al administrar 1010 pfu/animal simultáneamente a la infección y a las 6, 24 y 30 horas después de la infección. Del mismo modo, en pollos infectados con S. Typhimurium y tratados con dos dosis diarias de 1010 pfu/animal el dia previo a la infección y los 4 días siguientes, junto con la readministración del cóctel en los días 6, 8, 10, 13 y 15 posinfección, produjo una reducción de Salmonella de más de 4 logaritmos durante los dos primeros días de la infección y de 1,5 logaritmos al final del experimento. Finalmente, dado que algunas de las problemáticas que se plantean ante el uso de bacteriófagos en terapia fágica son la selección de bacterias resistentes y la generación de una respuesta inmunitaria que bloquee la acción de los fagos, se estudió el impacto de dicho cóctel en la selección de bacterias resistentes en los animales tratados con fagos y se cuantificó su respuesta inmunitaria. / Salmonella enterica is a zoonotic enterobacteria mainly found in the gut of its hosts, which shows an asymptomatic course of infection in farm animals. Nontyphoidal Salmonella serovars cause gastroenteritis, being S. Enteritidis and S. Typhimurium the most prevalent serovars causing food outbreaks worldwide. The main route of transmission of this pathogen to humans occurs by the consumption of contaminated food from animals infected but not detected. For this reason and to assure the control of Salmonella along all the food chain, a global actuation known as “from the farm to the fork” has been stimulated. The main reservoirs of S. enterica are avian species, above all Gallus gallus. For this reason, the European Union (UE) makes efforts trying to decrease the main Salmonella. The main control strategy used until today is the vaccination. However, in some stages of the avian production is not applicable. Thus, it is necessary to dispose of other new measures to reduce Salmonella prevalence. In this context, the aim of this thesis was focused on the isolation, selection and characterization of new Salmonella bacteriophages to study their effectiveness as a control strategy in Gallus gallus. To address this aim, specific Salmonella bacteriophages were isolated, and Those bacteriophages which better lytic abilities and wider host ranges were selected. In this way, bacteriophages UAB_Phi20 and UAB_Phi87 were selected and further characterized. These studies revealed that the bacteriophage UAB_Phi20 is a virulent Salmonella phage from the Podoviridae family closely resembling to P22, while bacteriophage UAB_Phi87 is enclosed in the Myoviridae family closely related to the lytic bacteriophage Felix O1-like. Both phages, UAB_Phi20 and UAB_Phi87, are highly stable under different temperatures and pH. However, at extremely acidic pH values (pH 2) their viability is clearly compromised. The analysis of both genome sequences showed that none of them contain known virulence genes. Likewise, the in vitro infection kinetics of both phages, together with the new phage UAB_Phi78, as a cocktail in liquid cultures of Salmonella improved the reduction levels on the concentration of this pathogen. The efficacy of this new cocktail was assayed in a mice model and in Gallus gallus. In both cases, the results obtained showed that the time of residence of bacteriophages in the intestinal tract of animals was really short and that the administration of several doses to obtain significant effects on the reduction of this pathogen is necessary, improving its effect during the first days after bacterial infection. The approaches herein studied allowed a 50% reduction in mice death promoted by S. Typhimurium when 1010 pfu/ml was administered simultaneously to the infection and at 6, 34 and 30 hours after the bacterial infection. In the same way, in poultry infected with S. Typhimurium and daily treated with two doses of 1010 pfu/ml since the day before of the infection and during the successive 4 days, with single re-administrations of the phage cocktail on days 6, 8, 10, 13 and 15 after the bacterial infection, promoted a Salmonella reduction of over 4 logs during the first days and a reduction of 1.5 logs at the end of the experiment Finally and due the problematic set out on the selection of resistant bacterial and the activation of the immune system promoted by the use of bacteriophages, the impact of the phage cocktail on the selection of resistant bacteria in animals and their immune response was also evaluated.

Salmonella enterica es una de las principales causas de toxiinfección alimentaria en todo el mundo. Su transmisión a humanos generalmente se produce a través de productos de origen animal y, en menor medida, de origen vegetal. En este contexto, el uso de bacteriófagos podría ser una estrategia aceptable dado que los fagos no alteran dichos productos ni comportan riesgos para la salud humana. El objetivo del presente trabajo ha sido aislar y caracterizar nuevos fagos capaces de infectar a las serovariedades S. Typhimurium y S. Enteritidis a partir de muestras fecales de cerdos y estudiar su aplicación a diferentes matrices alimentarias. Para abordar dicho objetivo y mediante un método basado en medios de enriquecimiento y de selección de Salmonella, se aislaron 33 fagos específicos desde muestras de heces de cerdo de granjas españolas entre los años de 2007 y 2009. Posteriormente, se realizó un estudio de su biodiversidad, comparando el perfil de infección y el perfil de restricción, junto a la de 22 fagos obtenidos por nuestro grupo desde muestras de heces de aves (Bardina, 2011). Los resultados obtenidos mostraron que los fagos procedentes de granjas de aves fueron distintos de los de granjas de cerdos. A pesar de ello, ambos grupos fueron capaces de infectar a cepas de S. Typhimurium y S. Enteritidis, independientemente de su origen (aves, cerdos y humanos). De entre todos los fagos de origen porcino aislados, se seleccionó el fago UAB_Phi78 por presentar un elevado rango de huésped y una mayor y más rápida capacidad lítica. Además, una caracterización más profunda reveló que el fago UAB_Phi78 pertenece al género tipo SP6 de la familia Podoviridae. Mostró una elevada estabilidad a diferentes temperaturas y a pH comprendido entre 4 y 9, aunque su infectividad disminuyó a pH 2. El análisis de la secuencia de su genoma no reveló la existencia de genes de virulencia conocidos. Dicho fago se combinó en un cóctel con otros dos fagos (UAB_Phi20 y UAB_Phi87), abordándose su aplicación conjunta como desinfectante sobre distintas matrices alimentarias. Se obtuvieron resultados significativos de reducción de la concentración de Salmonella en todas las matrices estudiadas: piel de cerdo (modelo de presacrificio de los animales), pechuga de pollo (modelo cárnico), huevos frescos (modelo de producto final de origen animal) y en lechuga envasada (modelo de producto final de origen vegetal). El conjunto de los resultados obtenidos demuestran la potencialidad del uso del cóctel desarrollado en la desinfección específica de alimentos, tanto en el producto final como en algunos puntos críticos de su producción. Los resultados permitieron concluir que el uso de dichos bacteriófagos en el biocontrol de Salmonella puede representar una opción efectiva si es utilizada de manera complementaria a las medidas de buenas prácticas de manufactura, ya utilizadas en la industria alimentaria. Palabras clave: Salmonella, bacteriófagos, lechuga, pollo, huevos, piel, biocontrol. / Salmonella enterica is one of the leading causes of foodborne disease worldwide. This pathogen is usually transmitted to humans through animal products. However, in the last years, fresh vegetables are also an important source of transmission of Salmonella. In this context, the use of bacteriophages could be an acceptable strategy due to they do not cause change in food and are safe for human health. The aim of this work was to isolate and characterize new phages capable of infecting S. Typhimurium and S. Enteritidis serovars from swine faecal samples and use a phage cocktail as a disinfectant in different matrices of the food production chain. A method based on the use of a selective enrichment medium for Salmonella allowed the isolation of 33 phages from faecal samples obtained in Spanish swine farms during 2007-2009. Later, we studied the biodiversity of 55 phages from our collection, which include the phages isolated in this work and 22 isolated from Gallus gallus faeces and obtained in another study of our research group (Bardina, 2011), comparing the host range and the restriction profile. The results showed that the phages from poultry farms were different from those from pig farms. In spite of this, both groups of phages were able to infect S. Typhimurium and S. Enteritidis strains, regardless of their origin (poultry, pigs or human). Among all phages isolated from swine samples, UAB_Phi78 was selected due to its high host range, and a high and faster lytic capacity. Afterwards, a deeper characterization revealed that UAB_Phi78 is a SP6-like phage, from Podoviridae family. Furthermore, it was demonstrated its ability to withstand at different temperatures and pH range from 4 to 9, although its infective capacity diminished significantly at pH 2. The analysis of the sequence of its genome did not reveal the existence of known virulence genes. Finally, we composed a cocktail with UAB_Phi78 and another two phages (UAB_Phi20 and UAB_Phi87) and its capacity as disinfectant on different food matrices was studied. In this way, it is noteworthy the significant results of decreasing the concentration of Salmonella obtained in all the matrices artificially contaminated: pig skin (model for pre-slaughter animals), chicken breast (meat model), fresh eggs (model of animal products) and lettuce (model of plant products). Moreover, it was demonstrated the potential of using a phage cocktail specifically developed for food disinfection, both in final product and in some critical points of production. Finally, all these results allow concluding that the use of these bacteriophages in the biocontrol of Salmonella can be an effective option if they are used as a complement to the measures of good manufacturing practices already applied in food industry. Keywords: Salmonella, bacteriophage, lettuce, chicken, eggs, skin, biocontrol.

<i>Salmonella enterica</i> serovar Enteritidis (<i>S.</i> Enteritidis) is a major cause of gastrointestinal disease in humans worldwide that is mainly associated with the consumption of contaminated poultry meat and eggs. During the course of infection, <i>S.</i> Enteritidis uses two Type 3 Secretion Systems (T3SS), one of which is encoded by <i>Salmonella</i> Pathogenicity Island-1 (SPI-1). SPI-1 plays a major role in the invasion process.<p> In order to study the role of SPI-1 in the colonization of chickens, we constructed deletion mutants affecting either the complete SPI-1 region (40 kb) or <i>invG</i>, a single gene located on this pathogenicity island. The mutants were impaired in the secretion of effector proteins and were less invasive compared to the wild type strain in polarized Caco-2 cells. Similarly, when chicken cecal and small intestinal explants were co-infected with the wild type and ÄSPI-1 mutant strains we found that the ÄSPI-1 mutant strain was less invasive relative to the wild type strain. Oral challenge of 1-week-old chickens with the wild type or ÄSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection, measured as levels of <i>Salmonella</i> in the liver and spleen, was delayed in birds that were challenged with the ÄSPI-1 strain. This demonstrates that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of S. Enteritidis in chickens.<p> Based on the above results, we examined the effect of sera against SPI-1 T3SS components to <i>S.</i> Enteritidis invasion. Anti-SipD serum protected Caco-2 cells against entry of wild type <i>S.</i>Enteritidis, but not against invasion of a mutant strain lacking sipD. On the other hand, sera against InvG, PrgI, SipA, SipC, SopB, SopE and SopE2 did not affect S. Enteritidis entry. To illustrate the specificity of anti-SipD mediated inhibition, SipD specific antibodies were depleted from the serum. Depleted serum restored the invasion of S. Enteritidis, demonstrating that the SipD protein may be an important target in blocking SPI-1 mediated virulence.<p> To determine if SPI-1 T3SS proteins were protective against <i>S.</i> Enteritidis oral challenge, chickens were vaccinated subcutaneously twice at 14 and 28 days of age with PrgI and SipD. The results indicate that these proteins induce strong IgG antibody responses and confer significant protection against infection of the livers in vaccinated birds. In another study, we vaccinated hens with selected SPI-1 T3SS proteins to determine if their progeny could be protected from <i>S.</i> Enteritidis oral challenge. The proteins induced strong antibody responses but did not affect the levels of the challenge strain in the ceca or internal organs of the vaccinates. Taken together, our results establish that <i>S.</i> Enteritidis SPI-1 is an important virulence factor in chickens and that the proteins associated with this T3SS may form components of a subunit vaccine used for protection against colonization by <i>S.</i> Enteritidis in poultry.

Salmonellosis is one of the most important bacterial food borne illnesses worldwide. Among the many Salmonella serotypes, Typhimurium is the most commonly implicated serotype in human disease in the United States. A major source of infection for humans is consumption of chicken or egg products that have been contaminated with S. Typhimurium. The breadth of knowledge regarding colonization and persistence factors in the chicken is small when compared to our knowledge of factors that are important for these processes in other species used in Salmonella research, such as cattle and mice. Defining the factors important for these processes in the chick is the first step in decreasing the transmission of Salmonella between animal and human hosts. In this work, we developed a chicken model to identify and study intestinal colonization and persistence factors of Salmonella enterica serovar Typhimurium. We studied the degree of enteric and systemic colonization of wild type S. Typhimurium ATCC14028, one of the most widely studied Typhimurium isolates, in White Leghorn chicks and in Salmonella-resistant CBA/J mice during infection. Furthermore, we determined the distribution of wild type S. Typhimurium and a SPI-1 mutant (invA) during competitive infection in the cecum of 1-week-old chicks and 8-week-old mice. Cell associated, intracellular and luminal distributions of these strains in the cecum were analyzed as total counts in each compartment and also as a competitive index. Localization of S. Typhimurium ATCC14028 and the role of SPI-1 in colonization are well studied in murine models of infection, but comparative infection in chicks with the same strain has not been undertaken previously. We show that the cecal contents are the major site for recovery of S. Typhimurium in the cecum of 1-week-old chicks and Salmonella-resistant mice. We also show that while SPI-1 is important for successful infection in the murine model, it is important only for cell association in the cecum of 1-week-old chicks. Finally, we found that in chicks infected at 1 week of age, bacterial counts in the feces do not reflect those seen in the cecum as they do in mice.

This study evaluated the thermal drying process commonly used by small and very small beef jerky operations in Texas. It was intended to determine the impact of relative humidity on the production of beef jerky and to provide documentation to beef jerky producers to support their Hazard Analysis and Critical Control Point programs. This project was divided into two phases: Phase I provided a low level of relative humidity (15-25%), whereas Phase II provided a high level (100%) for 25% of the cooking cycle. Both phases consisted of three trials, each representing one of the treatments (n=18) applied to the samples. The first treatment served as the control group and included samples that were non-inoculated, while the other two treatments included inoculations of samples with a bovine fecal slurry and rifampicin-resistant Salmonella Typhimurium. Each of the three treatments for both phases was analyzed for reduction of microbial levels in addition to temperature and product composition. Once the two phases had been completed and all data were analyzed, it was concluded that there was not a statistical difference between the level of reduction for Aerobic Plate Counts, coliforms, Escherichia coli and Salmonella provided by Phase I with low humidity and Phase II with high humidity. Both levels of humidity provided similar levels of reduction within each trial, suggesting that the level of humidity does not have a great impact on the level of microbial reduction achieved. However, this study did not provide the adequate level of initial inoculation levels to support the required 6.5 log reduction stated in 9 CFR 318.7. Inoculation levels were lower than 6.5 logs for all three treatments in both phases, resulting in lower levels of overall reduction. Therefore, based upon the information provided by this study, it cannot be concluded that a low level of humidity will achieve a 6.5 log reduction as mandated in 9 CFR 318.17.