Prion Strain Discrimination Based on Rapid In Vivo Amplification and Analysis by the Cell Panel Assay

Prion strain identification has been hitherto achieved using time-consuming incubation time determinations in one or more mouse lines and elaborate neuropathological assessment. In the present work, we make a detailed study of the properties of PrP-overproducing Tga20 mice. We show that in these mice the four prion strains examined are rapidly and faithfully amplified and can subsequently be discriminated by a cell-based procedure, the Cell Panel Assay

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Amplificación cíclica de priones generación de priones infecciosos in vitro: estudio del comportamiento biológico de los priones y su detección ultrasensible en sangre

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 10-02-200

Biofilm formation by Listeria monocytogenes. Resistance to industrial biocides and crossresponse caused by adaptation to benzalkonium chloride.

Introducción. Características biológicas:L. monocytogenes es un bacilo Gram positivo, anaerobio facultativo, móvil a temperaturas inferiores a 25 ºC (Seeliger and Jones, 1986) y altamente resistente en condiciones de estrés: pHs ácidos, baja aw, bajas concentraciones de O2 y baja temperatura (Ross et al., 2000, Kathariou, 2002). Todo ello contribuye a su ubicuidad (Cox et al. 1989, Ivanek et al. 2006) y a su condición de bacteria patógena, causante de listeriosis. Patogeneidad: está asociada a un grupo de riesgo constituido por mujeres embarazadas, individuos de avanzada edad e inmunodeprimidos. A pesar de su ubicuidad, la incidencia anual de listeriosis es de 0.3 casos al año por cada 100.000 habitantes, baja si la comparamos con otras infecciones trasmitidas por alimentos (EFSA 2006). Lo que contrasta con su elevada tasa de mortandad (20-30%), haciéndola especialmente relevante. Incidencia en alimentos: Las últimas inspecciones realizadas en Europa mostraron una mayor incidencia en productos de la pesca listos para el consumo, en los cuales se detectaron una mayor proporción de muestras contaminadas por encima de 100 UFC/g (2.4%) (EFSA, 2009), sobre todo durante y después del procesado (Cox et al., 1989; Hu et al., 2006; Samelis and Metaxopoulos, 1999, Autio et al., 1999, Miettinen et al., 1999; Norton et al., 2001; Rørvik et al., 1995; Vogel et al., 2001a, Wulff et al., 2006). Control: Dada la importancia del este patógeno en el ámbito alimentario, se han desarrollado diferentes estrategias para el control de Listeria monocytogenes a diferentes niveles, desde la consideración de medidas para evitar su aparición mediante la implementación del sistema de prerrequisitos y análisis de riesgo de los puntos críticos de control (HACCP), hasta el diseño de estrategias de conservación que aseguren el control de L. monocytogenes, pasando además por los esfuerzos realizados en la aplicación de protocolos de limpieza y desinfección efectivos. Justificación: Listeria monocytogenes es uno de los patógenos más importantes transmitidos por los alimentos, ya que tiene un alta incidencia en diferentes tipos de productos alimenticios, incluidos los productos de la pesca, y puede causar enfermedades graves e incluso la muerte en personas susceptibles. En términos generales, este trabajo se justifica por la necesidad de reducir el riesgo de la presencia de L. monocytogenes en los alimentos. Dentro de este objetivo global tres razones principales apoyar la adecuación de los objetivos específicos planteados en esta tesis doctoral:1. Que la formación de la biopelículas es una de las principales estrategias de persistir para L. monocytogenes en las plantas de procesado de alimentos. La persistencia implica un mayor riesgo de contaminación cruzada de los alimentos por biotransferencia (contacto, aerosoles). 2. Que la persistencia de biopelículas de L. monocytogenes en la industria alimentaria puede estar relacionada con la resistencia a estímulos externos físicos o químicos, incluidos los biocidas. 3. Que la transferencia de las células resistentes a biocidas procedentes de biopelículas al alimento puede implicar respuestas cruzadas a las técnicas de conservación. El estudio de estos aspectos resultará útil para prevenir la persistencia de los biofilms formados por L. monocytogenes en los alimentos y el medio ambiente y por lo tanto para mejorar el control de este patógeno bacteriano. Además, los sistemas experimentales se desarrollaron con el objetivo de simular condiciones industriales realistas, no tenidas en cuenta en muchos estudios previos. Las biopelículas se formaron por tanto, en condiciones experimentales que simulan condiciones que se encuentran en las plantas de procesado de mejillón. El uso de los mejillones y de las aguas de mejillón como sistemas experimentales se justifica por el hecho de que España, especialmente Galicia, es el principal productor de mejillón Europeo. En segundo lugar, fueron empleadas en este trabajo biopelículas maduras de Listeria monocytogenes y mixtas asociada con Pseudomona putida para evaluar la actividad de desinfectantes, dado que en trabajos previos recogidos en bibliografía se utilizaron comúnmente como sistemas experimentales células planctónicas y biofilms inmaduros. Por último, la complejidad estructural de las biopelículas se midió en términos de la resistencia a los biocidas en lugar del número de células. Objetivos, resultados y conclusiones de la tesis: Los principales objetivos resultados y conclusiones de esta tesis han sido: 1. Comparar la cinética de adhesión de L. monocytogenes (cepas CECT 5873, CECT 936, CECT 911 y CECT 4032, que representa los serotipos 1/2a, 1/2b, 1/2c y 4b, respectivamente) en polipropileno y acero inoxidable bajo dos condiciones de contaminación de la superficie (con y sin película acondicionamiento) simulando las encontradas en plantas de procesado de mejillón. Esto permitió seleccionar para los estudios posteriores los escenarios que dan lugar a las condiciones de mayor riesgo. Se observó que la cinética inicial de adherencia de diferentes cepas de L. monocytogenes en los diferentes escenarios depende de las condiciones medioambientales y nutricionales, de la cepa, de las propiedades físico-químicas de las superficies y de las interacciones que pueden establecerse entre estos factores. Hemos demostrado una mayor adherencia inicial de L. monocytogenes en polipropileno que en acero inoxidable y que, si bien la película acondicionamiento (PA) aumenta la adhesión inicial durante las primeras horas en las condiciones ensayadas, una fuerte disminución en el número de células adheridas a las 24 horas en algunos casos experimentales demuestran que PA podría llegar a plastificarse gradualmente con el tiempo y tiende a desprenderse. Además, cuando empleamos una menor concentración de nutrientes aumenta significativamente el nivel máximo de adherencia en L. monocytogenes en 3 de cada 4 casos ensayados. Por último, los datos experimentales se ajustan de forma satisfactoria a los modelos de tipo logístico lo cual ha demostrado ser una herramienta útil para efectos comparativos, que permite discriminar mejor el efecto de las variables independientes y obtener conclusiones más exactas.TÍTULO DEL PROYECTO: Estudio de las condiciones de formación de biopelículas de Listeria monocytogenes sobre su resistencia al envasado en atmósferas modificadas en mejillón vivo y cocido. ENTIDAD FINANCIADORA: XUNTA DE GALICIA PGIDIT 06 TAL 40201 PR TÍTULO DEL PROYECTO: Estudio de las condiciones de formación de biopelículas de Listeria monocytogenes sobre su resistencia al envasado en atmósferas modificadas en mejillón vivo y cocido. ENTIDAD FINANCIADORA: CICYT- AGL2006-01483/ALIPeer reviewe

Incidence and characterization of Staphylococcus aureus in fishery products marketed in Galicia (Northwest Spain)

11 páginas, 5 figguras, 2 tablasA total of 298 fishery products purchased from retail outlets in Galicia (NW Spain) between January 2008 and May 2009 were analyzed for the presence of Staphylococcus aureus. S. aureus was detected in a significant proportion of products (~ 25%). Incidence was highest in fresh (43%) and frozen products (30%), but it was high in all other categories: salted fish (27%), smoked fish (26%), ready-to-cook products (25%), non-frozen surimis (20%), fish roes (17%) and other ready-to-eat products (10%). A significant proportion of smoked fish, surimis, fish roes and other ready-to-eat products did not comply with legal limits in force. RAPD-PCR of 125 S. aureus isolated from fishery products was carried out using three primers (AP-7, ERIC-2 and S). Isolates displayed 33 fingerprint patterns. Each pattern was attributed to a single bacterial clone. Cluster analysis based on similarity values between RAPD fingerprints did not find relationship between any RAPD pattern and any product category. Isolates were also tested for se genes and susceptibility to a range of antibiotics (cephalothin, clindamycin, chloramphenicol, erythromycin, gentamicin, oxacillin, penicillin G, tetracycline, vancomycin, methicillin, ciprofloxacin and trimethoprim-sulfamethoxazole). Most isolates (88%) were found to be sea positive. Putative enterotoxigenic strains counts reached high risk levels in 17 products. No relationship was found between the presence of se genes and RAPD patterns. All isolates were resistant to penicillin, chloramphenicol and ciprofloxacin, and most to tetracycline (82.4%), but none was methicillin-resistant. A revision of pre-requisite programs leading to improve hygienic practices in handling and processing operations from fishing or farming to retail is recommended to ensure fishery products safety.This work was financially supported by the Xunta de Galicia (PGIDIT 07 TAL 014402PR). Authors DVS and PSI were supported by a research grant (JAE Program) by CSIC. Author JJRH was supported by the Ramón y Cajal Programme (co-financed by the European Social Fund).Peer reviewe

Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes

8 páginas, 5 figuras, 3 tablasIncrease of resistance to the application of benzalkonium chloride (BAC), peracetic acid (PA) and nisin during biofilm formation at 25 C by three strains of Listeria monocytogenes (CECT 911, CECT 4032, CECT 5873 and BAC-adapted CECT 5873) in different scenarios was compared. For this purpose, resistance after 4 and 11-days of biofilm formation was quantified in terms of lethal dose 90% values (LD90), determined according with a dose-response logistic mathematical model. Microscopic analyses after 4 and 11-days of L. monocytogenes biofilm formation were also carried out. Results demonstrated a relation between the microscopic structure and the resistance to the assayed biocides in matured biofilms. The worst cases being biofilms formed by the strain 4032 (in both stainless steel and polypropylene), which showed a complex “cloud-type” structure that correlates with the highest resistance of this strain against the three biocides during biofilm maturation. However, that increase in resistance and complexity appeared not to be dependent on initial bacterial adherence, thus indicating mature biofilms rather than planctonic cells or early-stage biofilms must be considered when disinfection protocols have to be optimized. PA seemed to be the most effective of the three disinfectants used for biofilms. We hypothesized both its high oxidizing capacity and low molecular size could suppose an advantage for its penetration inside the biofilm. We also demonstrated that organic material counteract with the biocides, thus indicating the importance of improving cleaning protocols. Finally, by comparing strains 5873 and 5873 adapted to BAC, several adaptative cross-responses between BAC and nisin or peracetic acid were identified.This work was financed by the MCYT (project AGL2006-01483/ALI) and Xunta of Galicia (PGIDIT06TAL40201PR)Peer reviewe

Comparison between the resistance of benzalkonium chloride-adapted and -nonadapted biofilms of listeria monocytogenes to modified atmosphere packaging and nisin once transferred to mussels

7 páginasBenzalkonium chloride-adapted and -nonadapted Listeria monocytogenes biofilm cells were transferred by contact to cooked or live mussels and packed in rich CO2 and O2, respectively. The viabilities of transferred cells during storage of these packed samples at 2.5°C were compared. In addition, in cooked mussels the combined effect of CO2 and nisin against the survival of L. monocytogenes was also studied by using a first-order factorial design. The results obtained demonstrated that biofilms formed by benzalkonium chloride-adapted L. monocytogenes cells could be more resistant to the application of modified atmospheres rich in CO2 and nisin once they have been transferred to cooked mussels by contact (simulating cross-contamination). This implies an increase in the risk associated with the presence of these cells in food processing plants. Significant empirical equations obtained after 7, 11, and 20 days showed an inhibitory effect of CO2 and nisin against L. monocytogenes. However, a significant positive interaction between both variables highlights an incompatibility between CO2 and nisin at high concentrations. Results also demonstrated that L. monocytogenes could persist after cross-contamination during the processing of live mussels, so L. monocytogenes is of concern as a contaminant in live mussels packaged in high-O2 atmospheres.Peer reviewe

Effects of mussel processing soils on the adherence of listeria monocytogenes to polypropylene and stainless steel

6 páginasA comparative study of adhesion kinetics of L. monocytogenes (strains CECT 5873, CECT 936, CECT 911, and CECT 4032, representing serotypes 1/2a, 1/2b, 1/2c, and 4b, respectively) to polypropylene (PP) and stainless steel (SS) under two surface contamination conditions in plants processing cooked mussel was carried out. The conditions were either (i) contamination of clean surfaces with mussel cooking juice carrying L. monocytogenes or (ii) contamination with L. monocytogenes after soiling with mussel cooking juice, i.e., conditioning film (CF). The kinetics of adhesion were successfully described by a modified logistic model. Adhesion to PP was higher than to SS in all strains, except CECT 5873. Adhesion was initially higher in the presence of CF, but numbers of adherent cells decreased sharply in the late phase of study in three of eight cases as a result of cell detachment. Combinations of strain, surface material, and surface conditioning where adhesion was most enhanced were defined as the worst-case scenarios (CECT 911-PP, 4032-PP-CF, 5873-SS, and 4032-SS-CF). Subsequently, adhesion in worst-case scenarios was compared with a similar contamination event taking place in plants processing live mussels, using intervalvar water of mussel as a food residue matrix. Adhesion levels were higher in intervalvar water than in cooking juice, especially in both cases with no CF; this was attributed to more space available for adhesion or to physicochemical conditions enhancing cells to adherePeer reviewe