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Salmonella enterica subsp. enterica serovar Typhimurium variant 4,[5],12:i:- (so called S. 4,[5],12:i:-) has rapidly become one of the most prevalent serovars in humans in Europe, with clinical cases associated with foodborne from pork products. The mechanisms, genetic basis and biofilms relevance by which S. 4,[5],12:i:- maintains and spreads its presence in pigs remain unclear. In this study, we examined the genetic basis of biofilm production in 78 strains of S. 4,[5],12:i:- (n = 57) and S. typhimurium (n = 21), from human gastroenteritis, food products and asymptomatic pigs. The former showed a lower Specific Biofilm Formation index (SBF) and distant phylogenetic clades, suggesting that the ability to form biofilms is not a crucial adaptation for the S. 4,[5],12:i:- emerging success in pigs. However, using a pan-Genome-Wide Association Study (pan-GWAS) we identified genetic determinants of biofilm formation, revealing 167 common orthologous groups and genes associated with the SBF. The analysis of annotated sequences highlighted specific genetic deletions in three chromosomal regions of S. 4,[5],12:i:- correlating with SBF values: i) the complete fimbrial operon stbABCDE widely recognized as the most critical factor involved in Salmonella adherence; ii) the hxlA, hlxB, and pgiA genes, which expression in S. typhimurium is induced in the tonsils during swine infection, and iii) the entire iroA locus related to the characteristic deletion of the second-phase flagellar genomic region in S. 4,[5],12:i:-. Consequently, we further investigated the role of the iro-genes on biofilm by constructing S. typhimurium deletion mutants in iroBCDE and iroN. While iroBCDE showed no significant impact, iroN clearly contributed to S. typhimurium biofilm formation. In conclusion, the pan-GWAS approach allowed us to uncover complex interactions between genetic and phenotypic factors influencing biofilm formation in S. 4,[5],12:i:- and S. typhimurium.

25 p. : il.-- Bibliogr.: p. 23-25, [ES] DEEPCOV es una red neuronal convolucional capaz de predecir estructuras tridimensionales de proteínas con un alto valor de precisión, utilizando datos de covarianza de las secuencias peptídicas fácilmente obtenibles. La red tiene mejores valores de precisión que las técnicas basadas en Análisis de Emparejamiento Directo demostrando que filtra mejor el ruido evolutivo que tanto afecta a estas técnicas cuando son utilizadas sobre proteínas con pocos homólogos conocidos. En este trabajo se pretende, a través de un entrenamiento adaptado de la red convolucional, conseguir un modelo de DEEPCOV válido para la predicción de estructuras de complejos proteicos. En primer lugar, se desarrolló una herramienta para la colecta y procesamiento de los datos del entrenamiento y que trabaja de forma automática. La herramienta fue capaz de generar 2973 archivos de forma autónoma y es útil para su futuro uso, siendo posible adaptarla con facilidad. En segundo lugar, la red fue entrenada con los archivos generados por la herramienta, obteniendo unos valores de precisión de 18’58%, 15’73% y 19’02% en la predicción de top 1, top 5 y top 10 contactos inter-proteína más cercanos respectivamente. El Valor Predictivo Positivo fue de 40’97%. La baja precisión de la red podría ser explicada por el relativamente bajo número de datos utilizados y la generación de ruido extra en el nuevo procedimiento de generación de datos., [EN] DEEPCOV is a convolutional neural network able to predict tridimensional structures of proteins with high precision values using covariance data of the peptide sequences, which is easily obtainable. The network has higher precision values than the Direct Coupling Analysis based techniques, proving that it is better at filtering the evolutive noise. This noise affects the Direct Coupling Analysis techniques so much when used over proteins with few homologues known. This project meant to, by an adapted training of the network, achieve a DEEPCOV model valid for the prediction of protein complexes’ structures. It took a number of steps to harness this. On first place, a tool for automatic data collection and processing was developed. The tool generated 2973 files for the network’s training autonomously. The tool is also usable in the future since its behavior is adapted easily. On second place, the network was trained with the data generated by the tool, obtaining precision values of 18’58%, 15’73% and 19’02% for the prediction of the top1, top5 and top10 closest intra-protein contacts respectively. The positive predictive value was 40’97%. The low prediction values could be explained with the small amount of data used for the network training, or the introduction of some noise generating steps during the data generation.

La salmonelosis es la segunda zoonosis de transmisión alimentaria más reportada en humanos en Europa, siendo la variante monofásica de Salmonella Typhimurium 4 5 12 i el principal serotipo detectado en España 1 La prevalencia creciente que este serotipo ha experimentado desde 2015 en el ganado porcino, podía estar asociada al uso de metales pesados como desinfectantes y/o aditivos alimentarios presentes en los piensos

The use of whole-genome sequencing (WGS) for bacterial characterisation has increased substantially in the last decade. Its high throughput and decreasing cost have led to significant changes in outbreak investigations and surveillance of a wide variety of microbial pathogens. Despite the innumerable advantages of WGS, several drawbacks concerning data analysis and management, as well as a general lack of standardisation, hinder its integration in routine use. In this work, a bioinformatics workflow for (Illumina) WGS data is presented for bacterial characterisation including genome annotation, species identification, serotype prediction, antimicrobial resistance prediction, virulence-related genes and plasmid replicon detection, core-genome-based or single nucleotide polymorphism (SNP)-based phylogenetic clustering and sequence typing. Workflow was tested using a collection of 22 in-house sequences of Salmonella enterica isolates belonging to a local outbreak, coupled with a collection of 182 Salmonella genomes publicly available. No errors were reported during the execution period, and all genomes were analysed. The bioinformatics workflow can be tailored to other pathogens of interest and is freely available for academic and non-profit use as an uploadable file to the Galaxy platform

During a prokaryotic diversity study in Añana Salt Valley, a new Rhodobacteraceae member, designated ASV31T, was isolated from Santa Engracia spring water. It was extremely halotolerant, tolerating up to 23% NaCl, and facultatively alkaliphilic, growing at pH 6.5–9.5 (optimum at 7.0–9.5). The isolate was a Gram-negative, rod-shaped, aerobic and non-motile bacterium that formed beige-to-pink colonies on marine agar. According to a 16S rRNA gene-based phylogenetic analysis, strain ASV31T forms a distinct branch of the family Rhodobacteraceae, with Thioclava pacifica DSM 10166T being its closest type strain (95.3%). This was confirmed with a phylogenomic tree and the values of ANI (73.9%), dDDH (19.3%), AAI (63.5%) and POCP (56.0%), which were below the genus/species level boundary. Additionally, an ability to degrade aromatic compounds and biosynthesise secondary metabolites was suggested by the genome of strain ASV31T. Distinguishing fatty acid profiles and polar lipid content were also observed. The genome size was 3.6 Mbp, with a DNA G+C content of 65.7%. Based on the data obtained, it was considered that strain ASV31T (=CECT 30309T = LMG 32242T) represents a new species of a new genus in the family Rhodobacteraceae, for which the name Anianabacter salinae gen. nov., sp. nov. is proposed.

24 p. : il. -- Bibliogr.: p. 20-24, Salmonella enterica, patógeno conocido desde el siglo XIX, es el principal microorganismo causante de la mayoría de brotes de origen alimentario por detrás de Campylobacter jejuni. Dentro del género Salmonella, uno de los serotipos generalistas más frecuentemente aislados en las muestras clínicas junto con Salmonella Enteritidis, es Salmonella Typhimurium. Ambos serotipos causan principalmente salmonelosis, una enfermedad auto limitante pero que también puede causar la muerte, por lo que dicho serotipo ha requerido un mayor control y seguimiento epidemiológico. A finales del siglo XX, emergieron en España nuevas variantes monofásicas del serotipo Typhimurium, causadas principalmente por la pérdida de uno de los flagelos. Actualmente la variante monofásica S. 4,[5],12:i:- es la que se notifica con más frecuencia después de los serotipos S. Typhimurium y S. Enteritidis, como causante de salmonelosis. Este problema de salud pública nos lleva a la necesidad de analizar la información bibliográfica disponible del serotipo Typhimurium para determinar qué factores de virulencia posee y expresa, los cuales estarán implicados, entre otros en la supervivencia y en la formación de biofilms, capacidad muy significativa a la hora de facilitar la colonización y supervivencia de la bacteria en el intestino humano. La formación de biofilms está fuertemente influenciada por una compleja red reguladora, permitiendo a S. Typhimurium adaptarse a diferentes entornos, por lo que será necesario conocer dicho proceso para posteriormente poder analizar los distintos métodos que permiten determinar la capacidad de formación de biofilms.

Salmonellosis, is one of the most common toxinfection caused by bacteria of the genus Salmonella in developed countries. In general, human salmonellosis is associated with contaminated food. However, contact with animals can be an important transmission route for Salmonella. Turtles have been shown to be reservoirs of different Salmonella serotypes. Considering that these reptiles are very common domestic pets, they can be a risk for public health, especially among children. In this work, a new monophasic serotype with the antigenic formula 4,5,12:b:- has been characterized. Most of the isolates analyzed have been isolated from children under 5 years of age in different regions of Spain. By means of different molecular techniques, it has been confirmed that 4,5,12:b:- isolates are genetically related to the biphasic isolates of Paratyphi B Java and that this serotype is their possible ancestor. The great homogeneity between the studied strains suggests that the origin of the turtles infected by these strains is closely related and that in Spain turtle shops have a common distribution point that contributes to the spread of this pathogen.; Salmonelosia, herrialde garatuetako toxiinfekzio ohikoenetariko bat da, Salmonella generoko bakterioek eragindakoa. Oro har, giza salmonelosia elikagai kutsatuekin lotzen da. Hala ere, animaliekin izandako kontaktua Salmonella generoaren transmisio bide garrantzitsua izan daiteke. Dortokak Salmonella serotipo desberdinen gordailuak direla frogatu da. Narrasti hauek etxeko maskota oso ohikoak direla kontuan izanda, osasun publikorako arriskutsuak izan daitezke, batez ere haurren artean. Horregatik, lan honetan 4,5,12:b:- formula antigenikoa duen serotipo monofasiko berri bat karakterizatu da. Aztertutako isolatu gehienak 5 urtetik beherako haurretan isolatu dira, Espainiako eskualde desberdinetan. Teknika molekular ezberdinen bidez, baieztatu da 4,5,12:b:- isolatuak Paratyphi B Javako iso- latu bifasikoekin genetikoki erlazionatuta

The Añana Salt Valley in Spain is an active continental solar saltern formed 220 million years ago. To date, no fungal genomic studies of continental salterns have been published, although DNA metabarcoding has recently expanded researchers’ ability to study microbial community structures. Accordingly, the aim of this present study was to evaluate fungal diversity using the internal transcribed spacer (ITS) metabarcoding at different locations along the saltern (springs, ponds, and groundwater) to describe the fungal community of this saline environment. A total of 380 fungal genera were detected. The ubiquity of Saccharomyces was observed in the saltern, although other halotolerant and halophilic fungi like Wallemia, Cladosporium, and Trimmatostroma were also detected. Most of the fungi observed in the saltern were saprotrophs. The fungal distribution appeared to be influenced by surrounding conditions, such as the plant and soil contact, cereal fields, and vineyards of this agricultural region.

After Salmonella Enteritidis and S. Typhimurium, S. 4,[5],12:i:- is the most reported serovar in human clinical cases. During the past 20 years, many tools have been used for its typing and second-phase flagellar deletion characterization. Currently, whole genome sequencing (WGS) and different bioinformatic programs have shown the potential to be more accurate than earlier tools. To assess this potential, we analyzed by WGS and in silico typing a selection of 42 isolates of S. 4,[5],12:i:- and S. Typhimurium with different in vitro characteristics. Comparative analysis showed that SeqSero2 does not differentiate fljB-positive S. 4,[5],12:i:- strains from those of serovar Typhimurium. Our results proved that the strains selected for this work were non-clonal S. 4,[5],12:i:- strains circulating in Spain. Using WGS data, we identified 13 different deletion types of the second-phase flagellar genomic region. Most of the deletions were generated by IS26 insertions, showing orientation-dependent conserved deletion ends. In addition, we detected S. 4,[5],12:i:- strains of the American clonal line that would give rise to the Southern European clone in Spain. Our results suggest that new S. 4,[5],12:i:- strains are continuously emerging from different S. Typhimurium strains via different genetic events, at least in swine products.

After Salmonella Enteritidis and S. Typhimurium, S. 4,[5],12:i:- is the most reported serovar in human clinical cases. During the past 20 years, many tools have been used for its typing and second-phase flagellar deletion characterization. Currently, whole genome sequencing (WGS) and different bioinformatic programs have shown the potential to be more accurate than earlier tools. To assess this potential, we analyzed by WGS and in silico typing a selection of 42 isolates of S. 4,[5],12:i:- and S. Typhimurium with different in vitro characteristics. Comparative analysis showed that SeqSero2 does not differentiate fljB-positive S. 4,[5],12:i:- strains from those of serovar Typhimurium. Our results proved that the strains selected for this work were non-clonal S. 4,[5],12:i:- strains circulating in Spain. Using WGS data, we identified 13 different deletion types of the second-phase flagellar genomic region. Most of the deletions were generated by IS26 insertions, showing orientation-dependent conserved deletion ends. In addition, we detected S. 4,[5],12:i:- strains of the American clonal line that would give rise to the Southern European clone in Spain. Our results suggest that new S. 4,[5],12:i:- strains are continuously emerging from different S. Typhimurium strains via different genetic events, at least in swine products.

Salmonellosis, is one of the most common food-poisoning disease caused by bacteria of the genus Salmonella in developed countries. More than 2,500 serotypes of Salmonella enterica species are described today, but a few serotypes have been re-ceived a harbor epidemiological monitoring. On the first decade of the 21th century, thanks to advances in molecular biology techniques, the Spanish National Center of Microbiology detected an emergence and increase of monophasic variants of Salmo-nella enterica. Typification methods allow us the analysis and characterization of 4 monophasic variants. As a result, it has been possible to determine their evolutionary origin and provide epidemiological markers designed for their detection, monitoring and control to facilitate the work in epidemiology of the health authorities.; Salmonelosia, herrialde garatuetako toxiinfekzio ohikoenetariko bat da, Salmonella generoko bakterioek eragindako elikagaien bidezkoa. Salmonella ente-ricaespeziearen barruan 2.500 serotipo baino gehiago deskribatuta daude gaur egun, baina serotipo gutxi batzuk izan dira jarraipen epidemiologiko sakonagoa jaso dutenak. XXI. mendearen lehenengo hamarkadan, biologia molekularreko tekniken aurrerakun-tzei esker, Salmonella enterica espeziearen aldaera monofasikoen gehikuntza detek-tatu zen Espainiako Mikrobiologiako Zentru Nazionalean. Lan honetan, aldaera mo-nofasiko horietako 4 aztertu eta karakterizatu dira tipikazio metodoen bidez. Ondorioz, posible izan da lau aldaeren eboluzio-jatorri zehaztea eta euren detekziorako, jarraipe-nerako eta kontrolerako diseinatu diren markatzaile epidemiologikoak eskaintzen dira, osasun agintariei epidemiologia esparruan lana erraztearren.

The pathogenesis of Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever in humans, is mainly attributed to the acquisition of horizontally acquired DNA elements. Salmonella pathogenicity islands (SPIs) are indubitably the most important form of horizontally acquired DNA with respect to pathogenesis of this bacterium. The insertion or deletion of any of these transferrable SPIs may have impact on the virulence potential of S. Typhi. In this study, the virulence potential and genetic relatedness of 35 S. Typhi isolates, collected from 2004 to 2013 was determined by identification of SPI and non-SPI virulence factors through a combination of techniques including virulotyping, Whole Genome Sequencing (WGS), and Variable Number of Tandem Repeats (VNTR) profiling. In order to determine the virulence potential of local S. Typhi isolates, 56 virulence related genes were studied by PCR. These genes are located in the core as well as accessory genome (SPIs and plasmid). Major variations among studied virulence determinants were found in case of SPI-7 and SPI-10 associated genes. On the basis of presence of virulence related genes, the studied S. Typhi isolates from Pakistan were clustered into two virulotypes Vi-positive and Vi-negative. Interestingly, SPI-7 and SPI-10 were collectively absent or present in Vinegative and Vi-positive strains, respectively. Two Vi-negative and 11 Vi-positive S. Typhi strains were also analyzed by whole genome sequencing (WGS) and their results supported the PCR results. Genetic diversity was tested by VNTR-based molecular typing. All 35 isolates were clustered into five groups. Overall, all Vi-negative isolates were placed in a single group (T5) whereas Vi-positive isolates were grouped into four types. Vi-negative and Vi-positive isolates were mutually exclusive. This is the first report on the comparative distribution of SPI and non-SPI related virulence genes in Vi-negative and Vi-positive S. Typhi isolates with an impo

224 p., [ES] Salmonella es uno de los patógenos alimentarios más importantes a nivel mundial y causante de enfermedades gastroentéricas en humanos y animales. La presencia de Salmonella en un alimento, sea en la cantidad que sea, se considera peligroso y por lo tanto no será apto para el consumo humano. Por ello, los controles microbiológicos rutinarios en la cadena alimentaria son de vital importancia para no comprometer la salud del consumidor. Los controles se deben realizar a lo largo de todo el proceso de elaboración del alimento, es decir, controlando desde las condiciones implantadas en la granja hasta la calidad del producto final que llegará al consumidor. Tradicionalmente los métodos microbiológicos empleados para detectar patógenos en los alimentos han sido métodos basados en el cultivo. El método tradicional de detección de Salmonella comprende cuatro fases: una primera fase de preenriquecimiento no selectivo, una segunda fase de enriquecimiento selectivo, un paso adicional de aislamiento en medios sólidos selectivos, y una última fase de confirmación mediante pruebas bioquímicas y serológicas. Estos procesos tradicionales de cultivo son laboriosos y alargan la obtención de los resultados hasta una semana. Las técnicas moleculares han sido un gran avance de las cuales la microbiología se ha beneficiado para desarrollar métodos nuevos de detección de patógenos. Entre ellas, las técnicas basadas en la amplificación de los ácidos nucleicos como la PCR (reacción en cadena de la polimerasa) proporcionan una estrategia rápida y sensible para la detección de patógenos. Además, la mejora que ha supuesto la PCR a tiempo real permite obtener los resultados incluso en menos tiempo y evitar el procesado post-PCR que puede provocar contaminación cruzada debido a la manipulación de las muestras. En todo caso, para el desarrollo de métodos basados en PCR es esencial la elección de una diana genética adecuada a cada patógeno que permita diseñar técnicas totalmente específicas d

[EN] Background: Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism of PCR products (PCR-RFLP) are extensively used molecular biology techniques. An exercise for the design and simulation of PCR and PCR-RFLP experiments will be a useful educational tool. Findings: An online PCR and PCR-RFLP exercise has been create that requires users to find the target genes,compare them, design primers, search for restriction endonucleases, and finally to simulate the experiment. Each user of the service is randomly assigned a gene from Escherichia coli; to complete the exercise, users must design an experiment capable of distinguishing among E. coli strains. By applying the experimental procedure to all completely sequenced E. coli, a basic understanding of strain comparison and clustering can also be acquired. Comparison of results obtained in different experiments is also very instructive. Conclusions: The exercise is freely available at http://insilico.ehu.es/edu.