Foodborne pathogens are a threat to public health worldwide. Because many consumers prefer natural compounds to synthetic additives, research on safe plant-derived compounds with antimicrobial activity against foodborne pathogens is vital. The aim of this investigation was to evaluate the antimicrobial activities of plant essential oils (oregano, cinnamon, lemongrass), their active components (carvacrol, trans-cinnamaldehyde, citral) and plant-extracts such as green tea, apple skin extract, black and decaffeinated black tea, grapes seed and pomace extracts against foodborne bacteria. Salmonella enterica serotype Typhimurium DT104, and serotype Newport, were selected conducting an antibiotic screening on 23 Salmonella isolates using seven antibiotics to determine antibiotic resistance. Listeria monocytogenes (strain 101M; beef and pork sausage isolate; resistant to antimicrobials in past investigations) was included to represent gram-positive bacteria. Escherichia coli O157:H7 virulent isolates (932- apple juice isolate; ATCC 35150- human isolate; F4637- sprouts isolate; used as a cocktail) were selected after conducting a Multiplex PCR over nine E. coli O157:H7 isolates to detect shiga-toxin 1 and 2 genes. All antimicrobials were evaluated in vitro in phosphate buffered saline. In general, all pathogens were more susceptible to essential oils and their active components, than powder extracts. The most active antimicrobials from each category were directly applied on foods. The activity of oregano oil (0.5%) and green tea (3%) was evaluated against S. Typhimurium on chicken and S. Newport on tomatoes and sprouts, and the results showed that oregano oil was more effective. In addition, baby spinach leaf samples inoculated with green fluorescent protein labeled S. Newport were examined under confocal scanning laser microscope before and after antimicrobial treatments. Antimicrobial experiments against L. monocytogenes on sprouts, ham and bologna, carvacrol at 0.5% and grape seed extract at 3% were used and carvacrol showed better activity. Antimicrobial activity against E. coli O157:H7 was tested on romaine lettuce, spinach and ground beef using oregano oil at 0.5% and green tea at 3%. Both compounds were effective showing no recovery of E. coli O157:H7 from lettuce and spinach; however, was not reduced in ground beef. Antimicrobial plant compounds have the potential for reducing foodborne pathogenic bacteria on/in various foods.

Thesis (MSc Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Listeria monocytogenes is a foodborne pathogen that has been isolated from a variety of food sources. It is the cause of the food-borne disease, listeriosis that shows symptoms such as meningitis, encephalitis and abortion. Different strains of L. monocytogenes exist and not all are thought to be pathogenic to humans. The aim of this study was to evaluate and compare conventional methods, culturing on selective (Oxford agar) and chromogenic (RAPID’L.mono agar) media, as well as speciesspecific and multiplex polymerase chain reaction (PCR) methods for the detection and identification of 94 L. monocytogenes isolates from various areas in an avocado processing facility, as well as the final product. To achieve a better understanding of the genetic diversity of the confirmed L. monocytogenes strains isolated from the avocado facility, two subtyping techniques, PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE), were employed. All of the isolates were identified as Listeria species on both Oxford and RAPID’L.mono agar. On the RAPID’L.mono agar, 76 of the 94 isolates produced colonies typical of L. monocytogenes, with the remaining 18 showing colonies typical of L. innocua (n=13) and L. ivanovii (n=5). The species-specific PCR successfully amplified a 730 base pairs region of the hly gene of 80 of the 94 isolates. For the same 80 isolates the multiplex PCR successfully amplified 800, 517 and 238 base pair (bp) fragments of the inlA, inlC and inlJ genes, respectively. The remaining 14 isolates included the 13 isolates identified as L. innocua, as well as an isolate identified as L. monocytogenes on RAPID’L.mono. The results obtained on the Oxford agar showed a 100 % positive correlation when compared to the PCR results in identifying Listeria species, while the RAPID’L.mono had a 4 % false negative result in identifying L. monocytogenes compared to the PCR results. Sixty-four of the confirmed L. monocytogenes isolates were subtyped using PCRRFLP and PFGE. For the PCR-RFLP analysis, a 733 bp fragment of the inlA gene was successfully amplified for all of the isolates, followed by digestion with the restriction enzymes, AluI and Tsp509I. AluI produced three different banding patterns and Tsp509I produced two different banding patterns. Subtyping of the isolates using PFGE was carried out by macrorestriction of the genomic DNA with ApaI and AscI. The restriction fragments were resolved by PFGE and the fingerprints were classified into four clusters. In the combined analyses, cluster I contained forty-eight isolates (n=48), cluster II 1 isolate (n=1), cluster III fifteen isolates (n=15) and cluster IV 1 isolate (n=1). The PCR-RFLP results had a 98 % correlation with the PFGE results. The results of this study indicated inconsistencies between the results obtained by conventional and molecular detection methods for the identification of L. monocytogenes. Species-specific and multiplex PCR, however, proved useful to accurately detect and identify L. monocytogenes in a shorter period of time and could replace the use of conventional agar during identification. Both PCR-RFLP and PFGE proved useful in the subtyping of L. monocytogenes isolates with the PCR-RFLP being less expensive and results obtainable in a shorter period of time. / AFRIKAANSE OPSOMMING: Listeria monocytogenes is ‘n patogeen afkomstig van voedsel wat uit ‘n verskeidenheid voedselbronne geisoleer kan word. Dit is die oorsaak van die voedsel afkomstigde siekte, listeriosis met simptome soos harsingvliesontsteking, ensefalitis en aborsie. ‘n Verskeidenheid L. monocytogenes stamme bestaan, maar nie almal word as patogenies beskou nie. Die doel van hierdie studie was om konvensionele metodes, naamlik mikrobiologiese kweking op selektiewe (Oxford agar) en chromatografiese (RAPID’L.mono agar) media, sowel as spesies-spesifieke en multipleks polimerase ketting reaksie (PKR) metodes te evalueer en vergelyk vir die deteksie en identifikasie van 94 L. monocytogenes isolate geisoleer vanuit verskeie areas in ‘n avokado prosesseringsfasiliteit sowel as die finale produk. Om ‘n beter begrip van die genetiese diversiteit van die isolate wat as L. monocytogenes bevestig is te verkry, is twee subtiperingstegnieke, PKR-restriksiefragmentlengte polimorfisme (PKR-RFLP) en pulsveld jel-elektroforese (PVJE) toegepas. Beide Oxford en RAPID’L.mono agar het al die isolate as Listeria spesies geidentifiseer. Op die RAPID’L.mono agar het 76 van die 94 isolate kolonies tipies van L. monocytogenes gevorm, 13 kolonies was tipies van L. innocua (n=13) en vyf kolonies tipies van L. ivanovii (n=5). Die spesies-spesifieke PKR het ‘n 730 basis paar (bp) streek van die hly geen suksesvol geamplifiseer vir 80 van die 94 isolate. Die multipleks PKR het 800, 517 en 238 bp fragmente van die inlA, inlC and inlJ gene onderskeidelik, vir dieselfde 80 isolate suksesvol geamplifiseer. Die oorblywende 14 isolate het die 13 isolate wat as L. innocua geïdentifiseer is en die een isolaat wat as L. monocytogenes op RAPID’L.mono geïdentifiseer is ingesluit. Resultate verkry met die Oxford agar het 100 % ooreengestem met die PKR resultate vir die identifikasie van Listeria spesies. Die RAPID’L.mono het ‘n 4 % vals negatiewe resultaat gelewer in vergelyking met die PKR resultate. Vier-en-sestig van die bevestigde L. monocytogenes isolate is gesubtipeer deur PKR-RFLP en PVJE. Tydens die PKR-RFLP analise is ‘n 733 bp fragment van die inlA geen suksesvol geamplifiseer, gevolg deur vertering met die restriksie-ensieme, AluI and Tsp509I. AluI het drie verskillende bandpatrone opgelewer en Tsp509I twee verskillende bandpatrone. Subtipering deur PVJE is uitgevoer deur makro-restriksie van die genomiese DNA met ApaI en AscI. Die restriksie fragmente is geskei deur PVJE en die vingerafdrukke is in vier groepe geklassifiseer. Groep I het 48 isolate (n=48), groep II 1 isolaat (n=1), groep III 15 isolate (n=15) en groep IV 1 isolaat (n=1) gehad tydens die gekombineerde analise. Die PKR-RFLP resultate het 98 % ooreengestem met die van die PVJE. Die resultate van hierdie studie het teenstrydighede tussen die resultate van konvensionele en molekulêre deteksie metodes opgelewer vir die identifikasie van L. monocytogenes. Die spesies-spesifieke en multipleks PKR het egter beide goed te pas gekom vir die akkurate deteksie en identifikasie van L. monocytogenes en kan heel moontlik die gebruik van konvensionele agar tydens identifikasie vervang. Beide PKRRFLP en PVJE was nuttig vir die subtipering van L. monocytogenes isolate. PKR-RFLP is egter ‘n goedkoper tegniek en die resultate is in ‘n korter tydsperiode beskikbaar.

<div>Fresh produce has been the leading source of foodborne illness outbreaks in the US, surpassing typical pathogen carriers such as meat, dairy, and seafood. Among the fresh produce popular to the consumers, cantaloupe and sprouts are mostly susceptible to pathogen contaminations and outbreaks. However, it has been a challenge to address the key factor in the contamination - the biofilms formed by pathogens are highly resistant to conventional washing and cleaning procedures. For cantaloupe, the net-like and porous surface forms a barrier for washing. For sprouts, the fragile texture of seedlings prevents aggressive cleaning operation and biofilm removal.</div><div><br></div><div>In this study, innovative interventions were developed to improve microbial safety of fresh produce, using cantaloupe and alfalfa sprouts as models. For cantaloupe, abrasive brushing was designed to remove pathogen biofilm from cantaloupe. Our research found pathogens could form biofilm at cantaloupe rind surface as the residence time of pathogens increased. Biofilm formed on cantaloupe rind was imaged by cryo-scanning electron microscopy (cryo-SEM), and its resistance to sodium hypochlorite and lauroyl arginate ethyl (LAE) was confirmed. Furthermore, abrasive brushing with peroxyacetic acid (PAA) could effectively remove biofilm formed at cantaloupe rind. The efficacy of this novel cleaning technique was highly desirable, which could achieve 3 log reduction in pathogen population. Mechanism of abrasive brushing to remove biofilm at cantaloupe rind surface was also proposed. Conceivably, brushing with diatomaceous earth (DE) and PAA could be an innovative and cost-effective method to remove pathogen biofilm from cantaloupe rind.</div><div><br></div><div>For alfalfa sprouts, since most of the outbreaks are linked to the sprouting seeds, seed disinfection treatments are considered to be the most effective method to improve microbial safety of sprouts. In this study, a newly developed alginate-based, antimicrobial seed coating treatment was evaluated for its efficacy to reduce foodborne pathogens from alfalfa seeds and sprouts. The calcium alginate coating in the presence of 2.5% lactic acid (CA-LA coating) reduced foodborne pathogens inoculated on alfalfa seeds to an undetectable level on day 1 during 28 day-seed storage, while chlorine (20,000 ppm) or lactic acid (2.5%) treatment took longer time to reach the same level. With sprouts, CA-LA coating resulted in > 2.5 log reduction for pathogen cells. In contrast, log reduction was < 0.6 for either chlorine (20,000 ppm) or lactic acid (2.5%) treatment. In general, this study indicated the effect of calcium alginate coating on reducing bacterial load of alfalfa seeds and sprouts, however, the germination rate of treated seeds was compromised due to the addition of lactic acid in the seed coating. Further study is needed to select antimicrobial compounds with minimum impact on germination rate of seeds.</div><div><br></div>

No description available.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2017-03-14T17:41:15Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação Darla Silveira Volcan Maia.pdf: 1194381 bytes, checksum: b604e15daa48092534ea10a3647416c8 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-03-17T22:15:15Z (GMT) No. of bitstreams: 2 Dissertação Darla Silveira Volcan Maia.pdf: 1194381 bytes, checksum: b604e15daa48092534ea10a3647416c8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-17T22:15:15Z (GMT). No. of bitstreams: 2 Dissertação Darla Silveira Volcan Maia.pdf: 1194381 bytes, checksum: b604e15daa48092534ea10a3647416c8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-01 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / A demanda por alimentos livres de conservantes químicos sintéticos tem aumentado. Nos últimos anos, vários estudos foram realizados a fim de se obter compostos antimicrobianos naturais. Alguns estudos foram realizados com frutas nativas do Brasil, no entanto, não existem estudos avaliando o potencial antibacteriano do butiá. O objetivo deste estudo foi prospectar a atividade antibacteriana de extratos de butiá (Butia odorata) com diferentes polaridades e caracterizar quimicamente o extracto com a melhor atividade. Um extrato hexânico e um metanólico de butiá foram avaliados quanto à sua atividade antibacteriana contra três bactérias Gram-positivas (Listeria monocytogenes, Staphylococcus aureus e Bacillus cereus) e três bactérias Gram-negativas (Salmonella Typhimurium, Escherichia coli O157:H7 e Pseudomonas aeruginosa), pelo método de difusão em ágar, Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM). Ambos extratos apresentaram atividade antibacteriana, entretanto, o extrato hexânico (EHB) apresentou desempenho superior, portanto realizou-se a caracterização química por CG-MS deste extrato. Interessantemente, o EHB apresentou maior atividade contra bactérias Gram-negativas, sendo E. coli O157:H7, a mais sensível (CBM 5 µL.mL-1 ). Entre as bactérias Gram-positivas, S. aureus (CBM 20 μL.mL-1 ) foi a mais sensível. Os fitoesteróis representaram 51% do EHB, sendo gama-sitosterol o composto predominante, constituindo 22% do extrato. / Demand for synthetic chemical preservative free foods has increased. In recent years several studies have been conducted in order to obtain natural antimicrobial compounds. Some studies were performed with native fruit from Brazil, however there are no studies evaluating the potential antibacterial of jelly palm fruits (butiá). The objective of the study was exploring the antibacterial activity of butiá (Butia odorata) extracts with different polarities and chemically characterize the extract with the best activity. A hexane and a methanol extract of butiá were evaluated for their antibacterial activity against three Grampositive (Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus) and three Gram-negative bacteria (Salmonella Typhimurium, Escherichia coli O157:H7, and Pseudomonas aeruginosa) by the agar diffusion method, Minimal Inhibitory Concentration (MIC), Minimal Bactericidal Concentration (MBC). Both extracts showed antibacterial activity, however of hexane extract (BHE) showed superior performance, therefore the chemical characterization by CG-MS for this extract. Interestingly, the BHE higher activity against Gram-negative bacteria, and E. coli O157:H7 was the most sensitive (MBC 5 μL.mL-1 ). Of the Gram-positive bacteria, S. aureus (MBC 20 μL.mL-1 ) was the most sensitive. Phytosterols represented 51% of the BHE and gamma-sitosterol was the predominant compound constituting 22% of the extract.

No description available.

The consumption of seafood in the United States has increased rapidly in recent years due to high quality protein and health benefits of seafood. Seafood can be a carrier for bacteria normally distributed in the marine environment and, in some cases, can be contaminated by human pathogens. Therefore, there is a potential health risk if seafood is consumed raw or undercooked. However, information regarding prevalence of foodborne pathogens in retail seafood products and the ability of pathogens to survive in the products during refrigerated and frozen storage is limited. The objective of this study was to generate such information for a better understanding of distribution of foodborne pathogens in seafood products and provide data which might be used for risk assessment of foodborne infection associated with seafood consumption. A total of 45 seafood products were collected from local retail stores and analyzed for aerobic plate counts (APC) and psychrotrophic bacterial counts (PBC) as well as presence of foodborne pathogens, including Escherichia coli O157:H7, Salmonella, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio vulnificus according to procedures described in the U.S. Food and Drug and Administration Bacteriological Analytical Manual (BAM). Presumptive isolates for each foodborne pathogen were further characterized by biochemical reactions using commercial identification kits and confirmed with polymerase chain reaction (PCR) assay. The samples had bacterial populations ranging from 1.90 to 6.11 CFU/g for APC and from 2.00 to 6.78 CFU/g for PBC. According to the microbiological criteria of International Commission on Microbiological Specifications for Foods (ICMSF), all 45 samples were considered acceptable quality (APC < 10⁷ CFU/g, E. coli < 3 MPN/g) with most samples (93.3%) being good quality (APC < 5 × 10⁵ CFU/g, E. coli < 3 MPN/g). No E. coli O157:H7, Salmonella, S. aureus, V. parahaemolyticus, and V. vulnificus was detected in any samples. Two previously frozen shrimp products (4.4%) were confirmed to carry L. monocytogenes. Studies of growth and survival of L. monocytogenes (3 strains), S. aureus (2 strains), and Salmonella (2 serovars) in raw yellowfin tuna meat stored at 5 - 7 °C for 14 days revealed that L. monocytogenes had the ability to multiply in the tuna meat during refrigerated storage while populations of S. aureus and Salmonella were reduced by 1 to 2 log CFU/g after 14 days at 5 - 7 °C. Studies of holding raw yellowfin tuna meat contaminated with L. monocytogenes, S. aureus, and Salmonella at -18 ± 2 °C for 12 weeks observed that all three pathogens, except Salmonella Newport, in tuna samples survived the frozen storage with less than 2- log of reductions in the populations over 12 weeks of storage. No viable cell of Salmonella Newport was detected in samples after 42 days storage at -18 °C. Raw seafood can be a carrier of foodborne pathogens, particularly L. monocytogenes, and many foodborne pathogens can survive in frozen products for several months. Consumption of raw or undercooked seafood products may lead to human infection if the products are contaminated with pathogens. Therefore, sanitation standard operating procedure (SSOP), good manufacturing practice (GMP) and hazards analysis and critical control points (HACCPs) programs shall all be implemented in the seafood industry to prevent seafood products from being contaminated with foodborne pathogens during handling and processing. Moreover, proper storage of raw seafood products and avoiding cross-contamination during handling at the retail levels also helps to minimize risk of human infection associated with ready-to-eat products. / Graduation date: 2013

Micromachined biosensors are used in chemical and biological applications. A biosensor which uses mass based transduction is called a mass sensor. Mass sensors are used to detect extremely small mass of biomolecules such as proteins, viruses or even parts of DNA in the range of femtograms (10-15 gm) to zeptograms (10−21 gm). Highly effective and reliable microcantilevers are used for detecting the mass of biomolecules using either static deflection or dynamic resonant peak shifts. The main objective of our work is to investigate the effect of elasticity of the attached mass on the shift in the resonant frequency and examine the validity of the rigid mass assumption used in the literature. The natural frequencies of a resonator are either found by solving the governing differential equation or approximately using Rayleigh-Ritz method. The mass of a body, attached to a resonator beam is determined using resonant frequency shift method. In our study, we derive an analytical expression for ‘δm’ based on the shift in frequency ‘δf’ that accounts for the elasticity of the added mass and the location of the mass on the beam. We study the simplest model to incorporate these effects where the added mass is itself modeled as a single degree of freedom spring-mass system. The entire system is represented as a 2-DOF lumped model of cantilever and the attached elastic mass. The natural frequencies are obtained using eigenvalue analysis. We study the mass estimation of Escherichia Coli (E. Coli), a food borne pathogen, using experimental results reported in the literature. We treat E.Coli as an elastic mass and model it as a single degree of freedom system to account for its elasticity. We use the elastic model as well as the rigid mass model to check the results available in the literature and point out the difference that results in mass estimation using the two models. To demonstrate the effect of elasticity on mass sensing using the resonant peak shift technique, we conduct mesoscale experiments. Since the fundamental principle does not depend on any phenomenon exclusively dependent on micro scales, the mesoscale experiments are justified. For this purpose, an experimental set-up with metallic cantilevers and flexible rubber strands as attached masses are used. We also use our experimental set-up to study the effect of positional inaccuracy of the added mass (rigid) in the computation of its mass from the shift in the resonance frequency. The results obtained show that elasticity of the added mass as well as its position on the resonator affect the computed mass but this effect is dependent on the relative stiffness and mass of the resonator and the added mass. We also observe the limitations of the experiments in carrying out studies over the desired range of parameters. We also create a computational model of the system and carry out simulations to explore a larger range of parameter values. In particular, we create an FEM model of our system in ANSYS, and carry out modal analysis for the cantilever beam resonator with and without the added mass, varying the relative stiffness and mass of the two components (the cantilever beam and the added mass). We compare the results of shift in the resonant frequency with those obtained from the rigid mass model. The results show the effect of elasticity clearly in certain ranges of relative stiffness and mass.

La presencia de microorganismos patógenos en alimentos es uno de los problemas esenciales en salud pública, y las enfermedades producidas por los mismos es una de las causas más importantes de enfermedad. Por tanto, la aplicación de controles microbiológicos dentro de los programas de aseguramiento de la calidad es una premisa para minimizar el riesgo de infección de los consumidores. Los métodos microbiológicos clásicos requieren, en general, el uso de pre-enriquecimientos no-selectivos,enriquecimientos selectivos, aislamiento en medios selectivos y la confirmación posterior usando pruebas basadas en la morfología, bioquímica y serología propias de cada uno de los microorganismos objeto de estudio. Por lo tanto, estos métodos son laboriosos, requieren un largo proceso para obtener resultados definitivos y, además, no siempre pueden realizarse. Para solucionar estos inconvenientes se han desarrollado diversas metodologías alternativas para la detección identificación y cuantificación de microorganismos patógenos de origen alimentario, entre las que destacan los métodosinmunológicos y moleculares. En esta última categoría, la técnica basada en la reacción en cadena de la polimerasa (PCR) se ha convertido en la técnica diagnóstica más popular en microbiología, y recientemente, la introducción de una mejora de ésta, la PCR a tiempo real, ha producido una segunda revolución en la metodología diagnóstica molecular, como pude observarse por el número creciente de publicaciones científicas y la aparición continua de nuevos kits comerciales. La PCR a tiempo real es unatécnica altamente sensible -detección de hasta una molécula- que permite la cuantificación exacta de secuencias de ADN específicas de microorganismos patógenos de origen alimentario. Además, otras ventajas que favorecen su implantación potencial en laboratorios de análisis de alimentos son su rapidez, sencillez y el formato en tubo cerrado que puede evitar contaminaciones post-PCR y favorece la automatización y un alto rendimiento. En este trabajo se han desarrollado técnicas moleculares (PCR y NASBA) sensibles y fiables para la detección, identificación y cuantificación de bacterias patogénicas de origen alimentario (Listeria spp., Mycobacterium avium subsp. paratuberculosis y Salmonella spp.). En concreto, se han diseñado y optimizado métodos basados en la técnica de PCR a tiempo real para cada uno de estos agentes: L. monocytogenes, L. innocua, Listeria spp. M. avium subsp. paratuberculosis, y también se ha optimizado yevaluado en diferentes centros un método previamente desarrollado para Salmonella spp. Además, se ha diseñado y optimizado un método basado en la técnica NASBA para la detección específica de M. avium subsp. paratuberculosis. También se evaluó la aplicación potencial de la técnica NASBA para la detección específica de formas viables de este microorganismo. Todos los métodos presentaron una especificidad del 100 % con una sensibilidad adecuada para su aplicación potencial a muestras reales de alimentos. Además, se han desarrollado y evaluado procedimientos de preparación de las muestras en productos cárnicos, productos pesqueros, leche y agua. De esta manera se han desarrollado métodos basados en la PCR a tiempo real totalmente específicos y altamente sensibles para la determinación cuantitativa de L. monocytogenes en productoscárnicos y en salmón y productos derivados como el salmón ahumado y de M. avium subsp. paratuberculosis en muestras de agua y leche. Además este último método ha sido también aplicado para evaluar la presencia de este microorganismo en el intestino de pacientes con la enfermedad de Crohn's, a partir de biopsias obtenidas de colonoscopia de voluntarios afectados.En conclusión, este estudio presenta ensayos moleculares selectivos y sensibles para la detección de patógenos en alimentos (Listeria spp., Mycobacterium avium subsp. paratuberculosis) y para una rápida e inambigua identificación de Salmonella spp. La exactitud relativa de los ensayos ha sido excelente, si se comparan con los métodos microbiológicos de referencia y pueden serusados para la cuantificación de tanto ADN genómico como de suspensiones celulares. Por otro lado, la combinación con tratamientos de preamplificación ha resultado ser de gran eficiencia para el análisis de las bacterias objeto de estudio. Por tanto, pueden constituir una estrategia útil para la detección rápida y sensible de patógenos en alimentos y deberían ser una herramienta adicional al rango de herramientas diagnósticas disponibles para el estudio de patógenos de origen alimentario. / The presence of pathogens in foods is among the most serious public health concerns, and the diseases produced by them are a major cause of morbidity. Consequently, the application of microbiological control within the quality assessment programs in the food industry is a premise to minimize the risk of infection for the consumer. Classical microbiological methods involve, in general, the use of a non-selective pre-enrichment, selective enrichment, isolation on selective media, and subsequent confirmation using morphological, biochemical and/or serological tests. Thus, they are laborious, time consuming and not always reliable (e.g. in viable but non-culturable VBNC forms). A number of alternative, rapid and sensitive methods for the detection, identification and quantification of foodborne pathogens have been developed to overcome these drawbacks. PCR has become the most popular microbiological diagnostic method, and recently, the introduction of a development of this technique, RTi-PCR, has produced a second revolution in the molecular diagnostic methodology in microbiology. RTi-PCR is highly sensitive and specific. Moreover, it allows accurate quantification of the bacterial target DNA. Main advantages of RTi-PCR for its application in diagnostic laboratories include quickness, simplicity, the closed-tube format that avoids risks of carryover contaminations and the possibility of high throughput and automation.In this work, specific, sensitive and reliable analytical methods based on molecular techniques (PCR and NASBA) were developed for the detection, identification and quantification of foodborne pathogens (Listeria spp., Mycobacterium avium subsp. paratuberculosis and Salmonella spp.). Real-time PCR based methods were designed and optimised for each one of these target bacteria: L. monocytogenes, L. innocua, Listeria spp. M. avium subsp. paratuberculosis, and also a real-time PCR basedmethod previously described for Salmonella spp. was optimised and multicenter evaluated. In addition, an NASBA-based method was designed and optimised for the specific detection of M. avium subsp. paratuberculosis. The potential application of the NASBA technique for specific detection of viable M. avium subsp. paratuberculosis cells was also evaluated.All the amplification-based methods were 100 % specific and the sensitivity achieved proved to be fully suitable for further application in real food samples. Furthermore, specific pre-amplification procedures were developed and evaluated on meatproducts, seafood products, milk and water samples. Thus, fully specific and highly sensitive real-time PCR-based methods were developed for quantitative detection of L. monocytogenes on meat and meat products and on salmon and cold smoked salmon products; and for quantitative detection of M. avium subsp. paratuberculosis on water and milk samples. The M. avium subsp. paratuberculosis-specific real-time PCR-based method was also applied to evaluate the presence of this bacterium in the bowelof Crohn's disease patients using colonic biopsy specimens form affected and unaffected volunteers. In addition, fully specific and highly sensitive real-time NASBA-based methods were developed for detection of M. avium subsp. paratuberculosis on water and milk samples.In conclusion, this study reports selective and sensitive amplification-based assays for the quantitative detection of foodborne pathogens (Listeria spp., Mycobacterium avium subsp. paratuberculosis and) and for a quick and unambiguously identification of Salmonella spp. The assays had an excellent relative accuracy compared to microbiological reference methods and can be used for quantification of genomic DNA and also cell suspensions. Besides, in combination with sample pre-amplification treatments,they work with high efficiency for the quantitative analysis of the target bacteria. Thus, they could be a useful strategy for a quick and sensitive detection of foodborne pathogens in food products and which should be a useful addition to the range of diagnostic tools available for the study of these pathogens.

No description available.