Validación de una técnica de PCR múltiple para la detección de Escherichia coli productor de toxina Shiga Validation of a multiplex PCR for detection of Shiga toxin-producing Escherichia coli

La infección por Escherichia coli productor de toxina Shiga (STEC) es causa de diarrea con o sin sangre, colitis hemorrágica y síndrome urémico hemolítico (SUH) en humanos. El objetivo de este trabajo fue validar una técnica de PCR múltiple para el diagnóstico de STEC basado en la detección de los genes stx1, stx2 y rfbO157. La validación de la técnica se realizó en dos laboratorios independientes, en forma paralela. Se determinó rango de trabajo, selectividad y robustez. Se evaluó el desempeño de la técnica al combinar distintas concentraciones de dos cepas con diferentes factores de virulencia. El rango de trabajo dependió de la cepa analizada, los valores máximos y mínimos fueron 6,6 x 107 y 1,0 x 104 UFC/50 µl. El límite de detección fue de 1,0 x 104 UFC/50 µl y el límite de corte de 1,0 x 105 UFC/50 µl. La robustez fue óptima al modificar diferentes variables. Se obtuvo 100% de inclusividad, exclusividad, precisión analítica, valor predictivo positivo y valor predictivo negativo. No se observó interferencia al combinar distintas concentraciones de los factores de virulencia blanco de la reacción. La técnica validada es una alternativa apropiada para la detección y confirmación de STEC O157 y no-O157 a partir de cultivos bacterianos.Shiga toxin-producing Escherichia coli (STEC) cause non-bloody or bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The aim of the present study was to validate a multiplex PCR for the STEC diagnosis based on the detection of stx1, stx2 and rfbO157 genes. The multiplex PCR validation was carried out in two independent laboratories in a parallel way. Work range, selectivity and robustness were established. The PCR performance was evaluated using different concentrations of two STEC strains harboring different target genes. The work range depended on the strain analyzed, the maximum and the minimum values were 6.6 x 107 and 1.0 x 104 CFU/50 µl. The detection limit was 1.0 x 104 CFU/50 µl and the cut limit 1.0 x 105 CFU/50 ml. A good robustness was observed when different variables were introduced. Inclusivity, exclusivity, positive predictivity, negative predictivity and analytical accuracy were of 100%. Interference was not shown when different concentrations of STEC strains, carrying different genes, were used. The validated technique is an appropriate alternative for detection and confirmation of STEC O157 and non-O157 strains from bacterial cultures

Quantitative risk assessment of listeriosis associated with fermented sausage and dry-cured pork shoulder consumption in Argentina

The risk of acquiring listeriosis from consuming fermented sausages and dry-cured pork shoulder (capicola/bondiola) in Argentina was estimated using quantitative microbiological risk assessment. The model included data about initial prevalence and contamination level of Listeria monocytogenes, product formulation with or without a starter culture (lactic acid bacteria [LAB]), time and temperature during distribution and storage prior to consumption, and consumption patterns. The probability of listeriosis from fermented sausage consumption in at-risk populations was estimated with average values of <10−11 per portion consumed. The main factor associated with such probability was the use of LAB in fermented sausage production, which reduced the risk by at least three orders of magnitude. Meat mass pH during fermentation and water activity (aw) at the end of ripening were the factors having the greatest impact on the probability of listeriosis. Thus, LAB added during production, pH values < 5.1 at the end of fermentation and aw <0.93 during ripening indicated that the environment was not appropriate for L. monocytogenes development, suggesting that fermented sausages had an adequate safety level. On the other hand, the risk of listeriosis from dry-cured pork shoulder consumption was negligible, even in the most susceptible populations. Both aw level and the strict control of process temperature were the main risk factors for acquiring listeriosis. The level of protection given by the current Argentine microbiological criterion for fermented sausages and dry-cured pork shoulder (absence of L. monocytogenes in 25 g of product) would guarantee the safety of these products similarly to the <100 cfu/g cut used in other countries.Fil: Brusa, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Prieto, M.. Dirección Nacional de Instituto de Investigación.Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbrán"; ArgentinaFil: Campos, Carmen Adriana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias. Instituto de Tecnología de Alimentos y Procesos Químicos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Tecnología de Alimentos y Procesos Químicos; ArgentinaFil: Epszteyn, S.. No especifíca;Fil: Cuesta, A.. Instituto Nacional de Tecnología Industrial; ArgentinaFil: Renaud, V.. Instituto Nacional de Tecnología Industrial.; ArgentinaFil: Schembri, G.. Instituto Nacional de Tecnología Industrial.; ArgentinaFil: Vanzini, M.. No especifíca;Fil: Michanie, S.. No especifíca;Fil: Leotta, Gerardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Signorini Porchietto, Marcelo Lisandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigación de la Cadena Láctea. - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea; Argentin

Quantitative risk assessment of listeriosis associated with fermented sausage and dry-cured pork shoulder consumption in Argentina

The risk of acquiring listeriosis from consuming fermented sausages and dry-cured pork shoulder (capicola/bondiola) in Argentina was estimated using quantitative microbiological risk assessment. The model included data about initial prevalence and contamination level of Listeria monocytogenes, product formulation with or without a starter culture (lactic acid bacteria [LAB]), time and temperature during distribution and storage prior to consumption, and consumption patterns. The probability of listeriosis from fermented sausage consumption in at-risk populations was estimated with average values of <10−11 per portion consumed. The main factor associated with such probability was the use of LAB in fermented sausage production, which reduced the risk by at least three orders of magnitude. Meat mass pH during fermentation and water activity (aw) at the end of ripening were the factors having the greatest impact on the probability of listeriosis. Thus, LAB added during production, pH values < 5.1 at the end of fermentation and aw <0.93 during ripening indicated that the environment was not appropriate for L. monocytogenes development, suggesting that fermented sausages had an adequate safety level. On the other hand, the risk of listeriosis from dry-cured pork shoulder consumption was negligible, even in the most susceptible populations. Both aw level and the strict control of process temperature were the main risk factors for acquiring listeriosis. The level of protection given by the current Argentine microbiological criterion for fermented sausages and dry-cured pork shoulder (absence of L. monocytogenes in 25 g of product) would guarantee the safety of these products similarly to the <100 cfu/g cut used in other countries.EEA RafaelaFil: Brusa, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina.Fil: Prieto, Monica. Instituto Nacional de Enfermedades Infecciosas (INEI); ArgentinaFil: Campos, Carmen A. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Tecnología de Alimentos y Procesos Químicos. Universidad de Buenos Aires. Instituto de Tecnología de Alimentos y Procesos Químicos; ArgentinaFil: Epszteyn, Sergio. Buenos Aires. Dirección General de Higiene y Seguridad Alimentaria. Laboratorio de Investigación y Monitoreo; ArgentinaFil: Cuesta, Alicia. Instituto Nacional de Tecnología Industrial; ArgentinaFil: Renaud, Viviana. Instituto Nacional de Tecnología Industrial. Gerencia Operativa de Servicios Industriales. Subgerencia Operativa de Alimentos. Departamento de Tecnología de Proceso Industrial; ArgentinaFil: Schembri, Gustavo. Instituto Nacional de Tecnología Industrial. Gerencia Operativa de Servicios Industriales. Subgerencia Operativa de Alimentos. Departamento de Tecnología de Proceso Industrial; ArgentinaFil: Vanzini, Mónica. Campo Del Tesoro; ArgentinaFil: Michanie, S. Consultora de Empresas en Inocuidad de Alimentos; ArgentinaFil: Leotta, Gerardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Signorini, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin