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Stool collection devices minimizing the exposure of gut bacteria to oxygen are critical for the standardization of further microbiota-based studies, analysis and developments. The aim of this work was to evidence that keeping anaerobiosis has a deep impact on the viability and diversity of the fecal microbiota that is recovered in the laboratory. Recovering certain microbial populations, such as obligate anaerobic bacteria, is particularly critical if the purpose of the study is to envisage personalized therapeutic purposes, such as autologous Fecal Microbiota Transplant. In this study the same fecal specimens were sampled in conventional stool containers and GutAlive, a disposable device that minimizes exposure of the gut microbiota to oxygen. Samples from five healthy donors were analysed and 150 differential colonies were recovered and identified by 16S rRNA gene sequencing. Globally, GutAlive maintained extremely oxygen sensitive (EOS) populations that were lost in conventional stool containers, and thus viability of species such as as Akkermansia muciniphila, Faecalibacterium prausnitzii and a novel member of the Clostridiales order was kept. These obligate anaerobes were not recovered using the conventional stool collection device. In conclusion, the use of GutAlive for stool collection and transport optimized the viability and recovery of EOS bacteria in the lab by diminishing oxygen toxicity.

Bifidobacteria are mutualistic intestinal bacteria, and their presence in the human gut has been associated with health-promoting activities. The presence of antibiotic resistance genes in this genus is controversial, since, although bifidobacteria are nonpathogenic microorganisms, they could serve as reservoirs of resistance determinants for intestinal pathogens. However, until now, few antibiotic resistance determinants have been functionally characterized in this genus. In this work, we show that Bifidobacterium breve CECT7263 displays atypical resistance to erythromycin and clindamycin. In order to delimit the genomic region responsible for the observed resistance phenotype, a library of genomic DNA was constructed and a fragment of 5.8 kb containing a gene homologous to rRNA methylase genes was able to confer erythromycin resistance in Escherichia coli. This genomic region seems to be very uncommon, and homologs of the gene have been detected in only one strain of Bifidobacterium longum and two other strains of B. breve. In this context, analysis of shotgun metagenomics data sets revealed that the gene is also uncommon in the microbiomes of adults and infants. The structural gene and its upstream region were cloned into a B. breve-sensitive strain, which became resistant after acquiring the genetic material. In vitro conjugation experiments did not allow us to detect gene transfer to other recipients. Nevertheless, prediction of genes potentially acquired through horizontal gene transfer events revealed that the gene is located in a putative genomic island. IMPORTANCE Bifidobacterium breve is a very common human intestinal bacterium. Often described as a pioneer microorganism in the establishment of early-life intestinal microbiota, its presence has been associated with several beneficial effects for the host, including immune stimulation and protection against infections. Therefore, some strains of this species are considered probiotics. In relation to this, b

Biogenic amines (BA) are nitrogenous compounds with important biological activities. However, due to the metabolism of certain bacteria, BA can accumulate in high concentrations in food and its consumption constitutes a toxicological threat. Cheese is one of the foods that higher concentrations of BA can accumulate, being lactic acid bacteria (LAB) the main BA-producers. Consequently, most of the methods proposed for reducing BA in food, which are based on reducing the presence of BA-producing bacteria, have not only failed to completely solve the problem, they also affect non-BA producing LAB that are essential in the development of the organoleptic characteristics of cheese. Therefore, new and more specific solutions are needed. Bacteriophages have arisen, in recent years, as a promising antimicrobial weapon both in clinical and food safety applications. They present some properties that made them ideal biotechnological tools against food spoilage microorganisms such as BA-producing bacteria: they are extremely host-specific, are food-grade, have a long shelf life, are able to resist the environmental stresses encountered during food processing, are relatively cheap and easy to isolate and propagate, and since they are self-replicating and self-limiting, low dosages can be employed since they multiply only, and as long as the host is present. In this communication, we present the isolation and further characterization of dairy phages against Enterococcus faecalis, species mainly responsible of the accumulation of tyramine and putrescine in cheese. Among them, we have chosen phage Q69 for an in deep characterization and to perform a proof-of-concept challenge to reduce the presence of tyramine in a cheese experimental model. The addition of Q69 reduced the concentration of tyramine below its toxic threshold limit.

Las Aminas Biógenas (AB) son compuestos nitrogenados presentes en todos los organismos y que poseen actividad biológica. Sin embargo, debido a la acción de ciertos microorganismos, las AB se pueden acumular en altas concentraciones en los alimentos y su consumo provoca una serie de efectos tóxicos (hipertensión, migrañas, taquicardia, etc).Debido a ello, la EFSA recomienda reducir la presencia de AB en alimentos. El queso es uno de los alimentos con mayor riesgo de acumular AB en altas concentraciones, siendo las AB más frecuentes la tiramina, la histamina y la putrescina. Los principales responsables de la acumulación en quesos de estas AB son bacterias del ácido láctico. Entre ellas, destaca Enterococcus faecalis, responsable de la producción y acumulación de tiramina y putrescina. Las medidas tecnológicas más efectivas para la reducción de AB en alimentos, también afectan a las bacterias lácticas necesarias para llevar a cabo la fermentación y el desarrollo de las propiedades organolépticas típicas del producto final. Debido a ello, es necesario el desarrollo de estrategias nuevas para reducir de forma específica la presencia de bacterias productoras de aminas biógenas. Como consecuencia del aumento de las resistencias a los antibióticos en bacterias patógenas, los bacteriófagos han resurgido como una posible herramienta en la lucha frente a las enfermedades infecciosas, debido a su capacidad bactericida y a su gran especificidad. Estas características han hecho que también se estén utilizando para la eliminación de patógenos en alimentos. Sin embargo, su utilización como agentes de biocontrol de microorganismos alterantes, como es el caso de la BAL productoras de aminas biógenas, ha sido poco explorada. Recientemente, hemos demostrado en quesos experimentales el potencial de Q69, un bacteriófago de E. faecalis aislado y caracterizado en nuestro laboratorio, para reducir la acumulación de tiramina por debajo de los niveles que se consideran peligrosos para el con

[Introducción] Actualmente existe muy poca información acerca del efecto de la congelación sobre la viabilidad de la microbiota fecal, y hasta la fecha, no se ha realizado ninguna investigación para determinar las condiciones y compuestos más adecuados para criopreservar y maximizar la viabilidad microbiana durante la conservación de esta microbiota. La preservación de muestras complejas de microbiota puede ser de utilidad para recuperar microorganismos de interés, así como ahondar en estudios de microbiota intestinal mediante el empleo de modelos in vitro e in vivo sin necesidad de depender de muestras fecales frescas. [Objetivos] En el presente trabajo se analizó el papel de distintos criopreservantes y condiciones de congelación en muestras de microbiota intestinal extraída de heces, evaluándose el mantenimiento de la viabilidad y recuperación de la microbiota en su conjunto, así como de distintas poblaciones representativas a los 3 meses de su preservación. [Materiales y Métodos] A partir de 5 muestras de heces de donantes voluntarios sanos se obtuvo la microbiota fecal mediante un procedimiento de separación en gradiente de densidad por ultracentrifugación previamente descrito (Hevia y cols., 2015). Las muestras, procesadas en una estación de trabajo en anaerobiosis, se alicuotaron y se guardaron congeladas bajo 4 condiciones experimentales: A) en presencia de glicerol al 15% a -20°C; B) con glicerol al 20% a -80°C; C) con sacarosa al 10% a -80°C y D) con polietilenglicol (PEG) al 20% a -196°C. Se evaluó la viabilidad de la microbiota extraída antes y después de ser preservada mediante recuentos en placa en distintos medios, microscopia de fluorescencia (kit ¿Live/Dead BacLight¿) y mediante qPCR usando monoazida de propidio (PMA) para la detección de células no dañadas. [Resultados] Al comparar de forma global las cuatro condiciones de preservación se observó una diferencia significativa entre las distintas condiciones en cuanto a la reducción de los recuentos

The probiotic properties of Bifidobacterium breve CECT7263, as well as its safety, have been the focus of in several studies since 2008, including the sequencing of its genome in 2012. This study aims to complete the available genomic data to deepen the knowledge of some phenotypic characteristics of this strain.

Bifidobacteria administered in fermented dairy products have to overcome the acidic pH found in the stomach during the gastrointestinal transit to be able to reach/colonize the lower parts of the intestine. The mechanisms underlying acid response and adaptation in Bifidobacterium longum NCIMB8809 and its acidpH-resistant mutant B. longum 8809dpH were studied. Comparison of protein maps allowed us to identify nine different proteins whose production largely changed in the mutant strain. Furthermore, the production of 47 proteins was modulated by pH in one or both strains. Adaptation and response to low pH involved changes in the glycolytic flux and in the ability to regulate the internal pH. These changes were accompanied by a higher content of ammonium in the cytoplasm. Remarkably, key proteins of the biosynthetic routes of cysteine and methionine were highly overrepresented. These results prompted us to perform a genomic and transcriptomic study in order to clarify the genetic mechanism behind the acquired acid resistance phenotype. Full genome sequencing and a transcriptomic analysis (microarray and RNAseq) allowed us to conclude that: i) a deletion of a large fragment of DNA in the mutant strain could be partially responsible for the acid resistance and ii) genes involved in the sulphur amino acid metabolism (metE, metB and cysD) were constitutively overexpressed in the mutant. Thus, the acquired acid resistance phenotype of B. longum 8809dpH could be related to a different cysteine and methionine metabolism in this strain.

A better understanding of the interactions among intestinal microbes is needed to decipher the complex cross talk that takes place within the human gut. Bacteroides and Bifidobacterium genera are among the most relevant intestinal bacteria, and it has been previously reported that coculturing of these 2 microorganisms affects their survival. Therefore, coculturing of Bifidobacterium longum NB667 and Bacteroides fragilis DSMZ2151 was performed with the aim of unravelling the mechanisms involved in their interaction. To this end, we applied proteomic (2D-DIGE) analyses, and by chromatographic techniques we quantified the bacterial metabolites produced during coincubation. Coculture stimulated the growth of B. longum, retarding that of B. fragilis, with concomitant changes in the production of some proteins and metabolites of both bacteria. The combined culture promoted upregulation of the bifidobacterial pyruvate kinase and downregulation of the Bacteroides phosphoenolpyruvate carboxykinase - 2 enzymes involved in the catabolism of carbohydrates. Moreover, B. fragilis FKBP-type peptidyl-prolyl cis-trans isomerase, a protein with chaperone-like activity, was found to be overproduced in coculture, suggesting the induction of a stress response in this microorganism. This study provides mechanistic data to deepen our understanding of the interaction between Bacteroides and Bifidobacterium intestinal populations. Copyright NRC Research Press

En los ultimos años han aumentado las evidencias sobre la relación existente entre la microbiota intestinal y la salud. No se conoce con certeza en qué consiste esta microbiota saludable pero se sabe que un desbalance de la misma está relacionado con ciertas enfermedades. Esto, junto con el aumento de evidencias científicas sobre su eficacia, ha incrementado el uso de probióticos para intentar equilibrar la microbiota. Las bacterias probióticas de mayor uso pertenecen a los géneros Lactobacillus y Bifidobacterium. Entre las bifidobacterias destaca la utilización comercial de la especie Bifidobacterium animalis subsp. lactis. Resulta de gran importancia profundizar en el conocimiento del efecto de la interacción hospedador ¿ probiótico para lo que se pueden utilizar modelos de líneas celulares intestinales. En el caso del probiótico es recomendable trabajar con aquella fracción bacteriana que ha estado intimamente unida a la célula eucariota, que será la que, en teoría, ejerza el mayor efecto. El porcentaje de adhesión a la célula eucariota así como la facilidad para separar la población adherida, tanto de la bacteria no adherida como de la línea celular, son claves para abordar estudios transcriptomicos. Por ello, el objetivo del presente trabajo fue optimizar un protocolo de extracción del RNA de la población de cepas de B. animalis subsp. lactis, productoras de distintos exopolisacáridos (EPS), adherida a la línea celular de colon HT29. Este hecho nos permitió estudiar la expresión génica diferencial entre dos cepas isogénicas que únicamente difieren en el tipo de EPS que producen, los cuales inducen respuestas inmunes diferentes

Fermented foods are among the food products more often complained of having caused episodes of biogenic amines (BA) poisoning. Concerning milk-based fermented foods, cheese is the main product likely to contain potentially harmful levels of BA, specially tyramine, histamine, and putrescine. Prompted by the increasing awareness of the risks related to dietary uptake of high biogenic amine loads, in this review we report all those elaboration and processing technological aspects affecting BA biosynthesis and accumulation in dairy foods. Improved knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in milk products. Synthesis of BA is possible only when three conditions converge: (i) availability of the substrate amino acids; (ii) presence of microorganisms with the appropriate catabolic pathway activated; and (iii) environmental conditions favorable to the decarboxylation activity. These conditions depend on several factors such as milk treatment (pasteurization), use of starter cultures, NaCl concentration, time, and temperature of ripening and preservation, pH, temperature, or post-ripening technological processes, which will be discussed in this chapter.

Bacteroides is among the most abundant microorganism inhabiting the human intestine. They are saccharolytic bacteria able to use dietary or host-derived glycans as energy sources. Some Bacteroides fragilis strains contribute to the maturation of the immune system but it is also an opportunistic pathogen. The intestine is the habitat of most Bifidobacterium species, some of whose strains are considered probiotics. Bifidobacteria can synthesize exopolysaccharides (EPS), which are complex carbohydrates that may be available in the intestinal environment. We studied the metabolism of B. fragilis when an EPS preparation from bifidobacteria was added to the growth medium compared to its behavior with added glucose. 2D-DIGE coupled with the identification by MALDI-TOF/TOF evidenced proteins that were differentially produced when EPS was added. The results were supported by RT qPCR gene expression analysis. The intracellular and extracellular pattern of certain amino acids, the redox balance and the a-glucosidase activity were differently affected in EPS with respect to glucose. These results allowed us to hypothesize that three general main events, namely the activation of amino acids catabolism, enhancement of the transketolase reaction from the pentose-phosphate cycle, and activation of the succinate-propionate pathway, promote a shift of bacterial metabolism rendering more reducing power and optimizing the energetic yield in the form of ATP when Bacteroides grow with added EPS. Our results expand the knowledge about the capacity of B. fragilis for adapting to complex carbohydrates and amino acids present in the intestinal environment. © 2015 Rios-covian, Sanchez, Salazar, Martinez, Redruello, Gueimonde and De los Reyes-Gavilan.

Exopolysaccharides (EPS) are extracellular carbohydrate polymers synthesized by a large variety of bacteria. Their physiological functions have been extensively studied, but many of their roles have not yet been elucidated. We have sequenced the genomes of two isogenic strains of Bifidobacterium animalis subsp. lactis that differ in their EPS-producing phenotype. The original strain displays a nonmucoid appearance, and the mutant derived thereof has acquired a mucoid phenotype. The sequence analysis of their genomes revealed a nonsynonymous mutation in the gene Balat_1410, putatively involved in the elongation of the EPS chain. By comparing a strain from which this gene had been deleted with strains containing the wild-type and mutated genes, we were able to show that each strain displays different cell surface characteristics. The mucoid EPS synthesized by the strain harboring the mutation in Balat_1410 provided higher resistance to gastrointestinal conditions and increased the capability for adhesion to human enterocytes. In addition, the cytokine profiles of human peripheral blood mononuclear cells and ex vivo colon tissues suggest that the mucoid strain could have higher anti-inflammatory activity. Our findings provide relevant data on the function of Balat_1410 and reveal that the mucoid phenotype is able to alter some of the most relevant functional properties of the cells.

Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirusinduced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23. © 2015, American Society for Microbiology.

We here report a 3.02-Mbp annotated draft assembly of the Lactobacillus casei 5b genome. The sequence of this biogenic amine-degrading dairy isolate may help identify the mechanisms involved in the catabolism of biogenic amines and perhaps shed light on ways to reduce the presence of these toxic compounds in food.

Autoaggregation in lactic acid bacteria is directly related to the production of certain extracellular proteins, notably, aggregation- promoting factors (APFs). Production of aggregation-promoting factors confers beneficial traits to probiotic-producing strains, contributing to their fitness for the intestinal environment. Furthermore, coaggregation with pathogens has been proposed to be a beneficial mechanism in probiotic lactic acid bacteria. This mechanism would limit attachment of the pathogen to the gut mucosa, favoring its removal by the human immune system. In the present paper, we have characterized a novel aggregation- promoting factor in Lactobacillus plantarum. A mutant with a knockout of the D1 gene showed loss of its autoaggregative phenotype and a decreased ability to bind to mucin, indicating an adhesion role of this protein. In addition, heterologous production of the D1 protein or an internal fragment of the protein, characterized by its abundance in serine/threonine, strongly induced autoaggregation in Lactococcus lactis. This result strongly suggested that this internal fragment is responsible for the bioactivity of D1 as an APF. To our knowledge, this is the first report on a gene coding for an aggregation-promoting factor in Lb. plantarum. ©2013, American Society for Microbiology.

El Instituto de productos lácteos del CSIC en Asturias organiza “Planeta Queso”, un ciclo de actividades alrededor de este lácteo, sus bacterias y microorganismos y su composición.

Microorganisms have been empirically used since ancestral times to produce fermented dairy products from milk. In the actual dair y industry, milk is subjected to large scale fermentation processes that involve microorgan isms mostly belonging to the Lactic Acid Bacteria (LAB) group. Bacteriophages that in fect LAB have been claimed as one of the principal sources of fermentation failure (spo ilage or delay) on the manufacture of many dairy products (Brüssow et al., 1998; Josephsen & Neve, 1998; Garneau & Moineau, 2011). Some estimates assume that virulent phages are the primary direct responsible of the largest-economic loss of dairy factories, since th ey affect negatively up to the 10% of all milk fermentations (Moineau & Levesque, 2005).

Tyramine and histamine are the biogenic amines (BAs) most commonly found in cheese, in which they appear as a result of the microbial enzymatic decarboxylation of tyrosine and histidine respectively. Given their toxic effects, their presence in high concentrations in foods should be avoided. In this work, samples of three cheeses (Zamorano, Cabrales and Emmental) with long ripening periods, and that often have high BA concentrations, were screened for the presence of BA-degrading lactic acid bacteria (LAB). Seventeen isolates were found that were able to degrade tyramine and histamine in broth culture. All 17 isolates were identified by 16S rRNA sequencing as belonging to . Lactobacillus casei. They were typed by plasmid S1-PFGE and genomic macrorestriction-PFGE analysis. Two strains (. L. casei 4a and 5b) associated with high degradation rates for both BAs were selected to test how this ability might affect histamine and tyramine accumulation in a Cabrales-like mini-cheese manufacturing model. The quantification of BAs and the monitoring of the strains' growth over ripening were undertaken by RP-HPLC and qPCR respectively. Both strains were found to reduce histamine and tyramine accumulation. These two strains might be suitable for use as adjunct cultures for reducing the presence of BAs in cheese. © 2012 Elsevier B.V.

Fermented foods are within the food products more often complained of having caused biogenic amines poisoning. Concerning milk-based fermented foods, cheese is the main product likely to contain significant levels of biogenic amines, specially tyramine, histamine and putrescine. Prompted by the increasing awareness of the risks related to dietary uptake of high biogenic amine loads, in this review we report about cheese elaboration and processing technological aspects affecting biogenic amines levels. Synthesis of biogenic amines is possible only when three conditions converge: i) availability of the substrate amino acids; ii) presence of microorganisms with the appropriate catabolic pathway activated; and iii) environmental conditions favorable to the decarboxilation activity. These conditions depend on several factors such as milk treatment (pasteurization), use of starter cultures, NaCl concentration, time and temperature of ripening and preservation, pH, which will be discussed in this chapter.

In order to investigate the identity, abundance and toxicity of a distinctive Ostreopsis species present in the southern-central coast of Cuba, Ostreopsis field populations were examined during January-September of 2010. The morphological and partial LSU phylogenetic data suggested the presence of O. lenticularis, although a very close species like O. labens could not be discarded. Results indicated the presence of palytoxin-like molecules in a natural extract at concentration of 0.12 pg/cell. Ostreopsis populations were very stable through the course of the study, and a maximum of 7.9 × 104 cells/g of macroalgae fresh weight was recorded on June.

A series of expression cassettes which mediate secretion or surface display of antibody fragments was stably integrated in the chromosome of Lactobacillus paracasei. L. paracasei producing surface-anchored variable domain of llama heavy chain (VHH) (ARP1) directed against rotavirus showed efficient binding to rotavirus and protection in the mouse model of rotavirus infection. © 2011, American Society for Microbiology.

The use of real time quantitative PCR (qPCR) has recently been extended to food science. The literature has mainly focused on its use in ensuring food safety. However, it offers a number of advantages with respect to the quantification of non-pathogenic food spoilage microorganisms. Indeed, qPCR may have a promising future in improving the quality of food products. The present review examines the use of qPCR in this area, the basis of the technique, the requirements that must be met for optimal qPCR assays to be performed, and the advantages it offers over other techniques. © 2011 Elsevier Ltd.

Oligonucleótidos sintéticos para la detección de enterobacterias lactosa positivo. La presente invención pertenece al grupo de las herramientas de control de contaminación fecal en agua y alimentos. Especialmente, la presente invención se refiere a una pareja de oligonucleótidos sintéticos cebadores para la amplificación genética del gen lacZ así como a un método cuantitativo para la detección de enterobacterias en productos lácteos.

Biogenic amines (BAs) are organic nitrogenous compounds that occur naturally in small concentrations in most living organisms, in which they have different biological functions. However, they can accumulate in foods due to the decarboxylating activity of certain bacteria. The consumption of food containing large amounts of some BAs can have toxicological consequences. Tyramine is one of the most active and common BAs found in cheeses. This article reports the design of an real-time quantitative PCR (qPCR) qPCR method for the detection and quantification of the microorganisms responsible for tyramine production in cheeses. A good linear relationship was obtained between the quantity of tyramine-producer DNA used in PCR reactions and the Ct (threshold cycle) value. The method was validated using 57 cheese samples in which the tyramine concentration was analysed by HPLC. Tyramine-producing bacteria were detected in different numbers in all samples, although tyramine itself was only detected in 56.1% of the samples. The results suggest that, independent of technological conditions such as pasteurisation or the ripening period, the presence of tyramine-producing bacteria over a threshold limit of 104 cfu g-1 - easily detected by the proposed qPCR method - leads to the accumulation of tyramine beyond recommendable limits. © 2009 Elsevier Ltd. All rights reserved.

The presence of lactose-fermenting Enterobacteriaceae and coliforms is routinely assessed to determine the hygienic quality of water and foods, particularly dairy products. This paper reports the use of lacZ-specific primers in an SYBR green I-based real-time PCR method for the easy and rapid detection of coliforms in dairy products. A large number of bacterial species were assayed to establish the specificity of the method. The sensitivity of the method was assessed using artificially contaminated cheeses. The limit of detection was 1 coliform cell in cheese samples enriched for 8. h in a culture medium. The entire procedure, including sample processing, enrichment, DNA extraction, and real-time PCR amplification, can be completed within 10 to 12. h, making it a single-day assay. © 2010 American Dairy Science Association.

Detección de bacteriófagos que infectan Lactobacillus delbrueckii mediante reacción en cadena de la polimerasa cuantitativa a tiempo real (QRT-PCR) y su uso. La presente invención describe un procedimiento de detección, especialmente en leche, de trazas de bacteriófagos destructivos de la especie Lactobacillus delbrueckii utilizada en fermentaciones lácticas industriales, mediante QRT-PCR. La amplificación se realiza utilizando una única pareja de oligonucleótidos cebadores que bajo unas condiciones adecuadas amplifican un fragmento de doble cadena flanqueado por dichos oligonucleótidos. Estos virus son una de las principales causas de fallo en las fermentaciones lácteas industriales, provocando importantes pérdidas económicas. Este procedimiento permite tomar decisiones tales como destinar la leche contaminada hacia procesos donde no intervenga Lb. delbrueckii sensibles a los fagos detectados o hacia tratamientos de inactivación, así como la desinfección de la planta de producción. La principal ventaja del procedimiento es la rapidez, junto con la especificidad, la sencillez y la sensibilidad.

Las salmonelas no-tifoideas que causan actualmente infecciones en seres humanos son frecuentemente epidémicas y resistentes a los antimicrobianos (R), además de virulentas (V). La evolución de las bacterias en cuanto a la adquisición de determinantes-V y/o -R se debe,fundamentalmente, a la incorporación, a menudo secuencial, de piezas de ADN, cuya combinación origina nuevos elementos genéticos, que quedan inmediatamente sometidos a la selección natural en un ambiente cambiante. Los genes-V y -R pueden estar aislados, formando pequeñas agrupaciones (islotes) y/o en agrupaciones mayores (islas genómicas o de patogenicidad) y pueden ser de localización cromosómica o plasmídica. Los genes-R, una vez seleccionados, pueden mantenerse en las bacterias originarias y su descendencia o bien diseminarse entre bacterias más o menos relacionadas a través de los elementos genéticos móviles (EGMs) entre los que destacan el sistema integrón-casete génica, los transposones, los plásmidos y las islas gen. En la presente tesis se llevaron a cabo estudios de epidemiología molecular en 176 aislamientos de Salmonella enterica pertenecientes a tres serotipos no prevalentes, aunque causantes de alertas epidemiológicas: Hadar, Brandenburg y Ohio. Se determinó el impacto epidemiológico de los mismos, identificando los tipos endémicos y prevalentes en el Principado de Asturias y su posible presencia en otras áreas geográficas. Los subtipos fueron trazados mediante análisis de macrorrestricción genómica-PFGE y genotipos-R y -V. Como ejemplo tenemos que los subtipos prevalentes de Hadar se pueden considerar endémicos en España y uno de ellos causó un brote nacional asociado a la salsa de pollos asados comerciales en 2005. Los estudios de resistencia revelaron un amplio porcentaje de cepas con resistencia múltiple (RM) y una correlación entre ésta y la presencia de EGMs. En los tres serotipos se identificaron plásmidos-R, de tamaño variable (9-300 Kb) y diferentes genotipos-R, la mayoría de los