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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.

Biological control is an intriguing alternative to the use of chemical pesticides as it represents a safer, more environmentally friendly approach to managing plant pathogens. Pseudomonas chlororaphis strain PA23 was isolated from soybean root tips and it was found to be an excellent antagonist of sclerotinia stem rot. Our studies have shown that pyrrolnitrin (PRN) is the key metabolite required for S. sclerotiorum inhibition, while phenazine (PHZ) is important for biofilm establishment. For this reason, research efforts were directed towards elucidating the mechanisms governing PA23-mediated antibiotic production. To determine how these compounds were regulated, QS-deficient strains and an rpoS mutant were generated. The QS-deficient strains no longer inhibited the fungal pathogen S. sclerotiorum in vitro and exhibited reduced PRN, PHZ and protease production. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. GacS/GacA, PsrA, RpoS and the PhzI/PhzR QS are members of a complex regulatory hierarchy that influence secondary metabolite production in PA23. An additional system, termed Rsm, was identified, adding yet another layer of complexity to the regulatory network. The Rsm system in PA23 appears to be comprised of a single small non-coding regulatory RNA termed RsmZ, and two RNA binding proteins RsmA and RsmE. We discovered that the expression of rsmZ, rsmA and rsmE all require GacA. In addition, both PsrA and QS were shown to positively regulate rsmZ transcription. For rsmE, GacA may indirectly regulate expression through PsrA, RpoS and QS, as all three regulators control rsmE transcription. Furthermore, we believe that the positive effects of PsrA and QS on rsmE transcription are likely mediated through RpoS as only RpoS show direct activation of rsmE in an E. coli background.

Pseudomonas chlororaphis PA23 inhibits several root pathogens in both the greenhouse and field. A LysR-type transcriptional regulator (LTTR) called PtrA (Pseudomonas transcriptional regulator A) that is essential for Sclerotinia sclerotiorum antifungal activity was discovered through transposon mutagenesis. P. chlororaphis PA23 produces the antibiotics phenazine 1-carboxylic acid, 2-hydroxyphenazine and pyrrolnitrin, and several additional products that contribute to biocontrol. Phenotypic assays and proteomic analysis have revealed that production of these secondary metabolites are markedly reduced in a ptrA mutant. Most LTTRs regulate genes that are upstream of and divergently transcribed from the LTTR locus. A short chain dehydrogenase (scd) gene lies immediately upstream of ptrA in the opposite orientation. Characterization of an scd mutant, however, has revealed no significant changes in antifungal activity compared to wild-type PA23. Gene expression analysis of the ptrA mutant indicates that ptrA may exert its regulatory effects through the Gac-Rsm network, and may be controlling expression of the scd gene. Collectively these findings indicate that PtrA is an essential regulator of PA23 biocontrol and is connected to other regulators involved in fungal antagonism.

Biological control is an intriguing alternative to the use of chemical pesticides as it represents a safer, more environmentally friendly approach to managing plant pathogens. Pseudomonas chlororaphis strain PA23 was isolated from soybean root tips and it was found to be an excellent antagonist of sclerotinia stem rot. Our studies have shown that pyrrolnitrin (PRN) is the key metabolite required for S. sclerotiorum inhibition, while phenazine (PHZ) is important for biofilm establishment. For this reason, research efforts were directed towards elucidating the mechanisms governing PA23-mediated antibiotic production. To determine how these compounds were regulated, QS-deficient strains and an rpoS mutant were generated. The QS-deficient strains no longer inhibited the fungal pathogen S. sclerotiorum in vitro and exhibited reduced PRN, PHZ and protease production. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. GacS/GacA, PsrA, RpoS and the PhzI/PhzR QS are members of a complex regulatory hierarchy that influence secondary metabolite production in PA23. An additional system, termed Rsm, was identified, adding yet another layer of complexity to the regulatory network. The Rsm system in PA23 appears to be comprised of a single small non-coding regulatory RNA termed RsmZ, and two RNA binding proteins RsmA and RsmE. We discovered that the expression of rsmZ, rsmA and rsmE all require GacA. In addition, both PsrA and QS were shown to positively regulate rsmZ transcription. For rsmE, GacA may indirectly regulate expression through PsrA, RpoS and QS, as all three regulators control rsmE transcription. Furthermore, we believe that the positive effects of PsrA and QS on rsmE transcription are likely mediated through RpoS as only RpoS show direct activation of rsmE in an E. coli background.

Two fungal pathogen species, Helminthosporium solani and Pythium ultimum, cause significant economic loss to potato (Solanum tuberosum) growers throughout the world. These pathogens have substantial differences in cellular makeup, pathogenicity, and modes of infection. We studied the efficacy of 82 isolates within the bacterial genus Streptomyces in inhibiting these pathogens under laboratory and greenhouse conditions. Derivatives of Streptomyces have significant implications in medicinal use because of their antibiotic and antifungal properties. Under in-vitro conditions, 25% of Streptomyces isolates inhibited growth of P. ultimum, up to 81%. Ninety-five percent of the Streptomyces isolates inhibited growth of H. solani, with a maximum of 70%. In storage, these findings lead us to believe substantial differences between Streptomyces isolates will allow for some isolates to be effective biological controls at controlling diseases on common pathogens of potatoes.

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