La Desinfección basada en bacteriófagos como herramienta de biocontrol de Salmonella en alimentos

Descripció del recurs: el 01 setembre 2012Salmonella 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

Genomics of three new bacteriophages useful in the biocontrol of Salmonella

Non-typhoid Salmonella is the principal pathogen related to food-borne diseases throughout the world. Widespread antibiotic resistance has adversely affected human health and has encouraged the search for alternative antimicrobial agents. The advances in bacteriophage therapy highlight their use in controlling a broad spectrum of food-borne pathogens. One requirement for the use of bacteriophages as antibacterials is the characterization of their genomes. In this work, complete genome sequencing and molecular analyses were carried out for three new virulent Salmonella-specific bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) able to infect a broad range of Salmonella strains. Sequence analysis of the genomes of UAB_Phi20, UAB_Phi78, and UAB_Phi87 bacteriophages did not evidence the presence of known virulence-associated and antibiotic resistance genes, and potential immunoreactive food allergens. The UAB_Phi20 genome comprised 41,809 base pairs with 80 open reading frames (ORFs); 24 of them with assigned function. Genome sequence showed a high homology of UAB_Phi20 with Salmonella bacteriophage P22 and other P22likeviruses genus of the Podoviridae family, including ST64T and ST104. The DNA of UAB_Phi78 contained 44,110 bp including direct terminal repeats (DTR) of 179 bp and 58 putative ORFs were predicted and 20 were assigned function. This bacteriophage was assigned to the SP6likeviruses genus of the Podoviridae family based on its high similarity not only with SP6 but also with the K1-5, K1E, and K1F bacteriophages, all of which infect Escherichia coli. The UAB_Phi87 genome sequence consisted of 87,669 bp with terminal direct repeats of 608 bp; although 148 ORFs were identified, putative functions could be assigned to only 29 of them. Sequence comparisons revealed the mosaic structure of UAB_Phi87 and its high similarity with bacteriophages Felix O1 and wV8 of E. coli with respect to genetic content and functional organization. Phylogenetic analysis of large terminase subunits confirms their packaging strategies and grouping to the different phage genus type. All these studies are necessary for the development and the use of an efficient cocktail with commercial applications in bacteriophage therapy against Salmonella

La desinfección basada en bacteriófagos como herramienta de biocontrol de Salmonella en alimentos

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

Broncopneumonia causada por Arcanobacterium pyogenes em Veado Campeiro (Ozoteceros bezearticus)

Arcanobacterium pyogenes   é uma bactéria comensal das membranas mucosas do trato respiratório e genital de várias espécies de animais domésticos e selvagens. Porém, em decorrência de estresse ou traumatismos, A. pyogenes pode se tornar um patógeno oportunista, associado a infecções supurativas. Estresse é um importante fator de risco para doenças em todos os animais, mas especialmente em animais silvestres não habituados ao contato humano. Um veado-campeiro (Ozoteceros bezearticus) criado em área conservacionista, foi encontrado prostado e demonstrando dificuldades respiratórias. Este animal foi encaminhado ao Hospital de Clínica Veterinária - CAV/UDESC, onde recebeu tratamento, mas, dois dias após, morreu. O exame microscópico dos pulmões revelou áreas focalmente extensas de necrose associada a infiltrado moderado de polimorfonucleares, circundados por tecido fibroso. De acordo com a necropsia, cultivo microbiológico e seqüenciamento de DNA, o agente envolvido foi classificado como A. pyogenes, caracterizando o primeiro relato de Broncopneumonia causada por A. pyogenes em Veado-Campeiro (Ozoteceros bezearticus) no Brasil

Composiciones que comprenden bacteriófagos líticos de Salmonella UAB_Phi20, UAB_Phi78 y UAB_ Phi87 y usos de los mismos

Una composicion de bacteriofagos para la prevencion de enfermedades infecciosas provocadas por Salmonella que comprende al menos uno de los bacteriofagos liticos de Salmonella UAB _Phi20, UAB_Phi78 y UAB_Phi87 y/o partes o productos de los mismos

Caracterização epidemiológica, molecular e perfil de resistência aos antimicrobianos de Escherichia coli isoladas de criatórios suínos do sul do Brasil Epidemiology, molecular characterization and resistance to antimicrobials of Escherichia coli isolated from South-Brazilian pig herds

A colibacilose é a enfermidade entérica de maior impacto na suinocultura, sendo ocasionada por cepas enterotoxigênicas de Escherichia coli. Quarenta isolados clínicos de suínos com diarréia e 13 isolados ambientais foram analisados quanto ao perfil genotípico, relação genética e resistência antimicrobiana. O gene que codifica para a toxina Stb foi identificado em 50% dos isolados clínicos, seguido por Sta e Lt, com 35%. Dentre os fatores de adesinas pesquisados, a F18 foi encontrada em 27,5% das amostras. A técnica de ERIC-PCR utilizada para caracterização epidemiológica dos isolados, não demonstrou poder discriminatório esperado, e apesar de permitir a separação dos isolados em grupos, estes não evidenciaram grupos relacionados aos fatores de virulência. No teste de susceptibilidade antimicrobiana a maior resistência foi observada à tetraciclina, em 88,6%. O índice de resistência múltipla aos antimicrobianos (IRMA), variou entre 0 a 0,69.<br>Colibacillosis is an enteric disease with a major impact to the swine industry and is caused by enterotoxigenic strains of Escherichia coli. Forty clinical isolates from pigs with diarrhea and 13 environmental isolates were analysed regarding their genotypic profile, genetic relationship and antibiotic resistance. The most prevalent gene was Stb, identified in 50% of the isolates from clinical cases, and Sta and Lt were detected in 35% of them. Among the adesine factors investigated, F18 was found in 27.5% of the E. coli strains. The ERIC-PCR technique used for epidemiological characterization of the isolates did not show the expected discriminatory power. However, the test allowed separation of the isolates in groups, but did not evidence groups related to virulence factors. In the susceptibility test, the highest values for resistance were to tetracycline, in 88.6%. The index of multiple resistance to antimicrobials varied from 0 to 0.69