Genome-Scale in silico Reconstruction of the Reactive Oxygen Species (ROS) Generating Metabolism in Pseudomonas putida KT2440 and Study of the role of ROS in Different Metabolic Processes

The understanding of the genotype-phenotype relationship is a fundamental biological question widely studied, but still not understood in all its dimension. The existence of emergent systems' properties largely complicates the lineality of this relationship making it mandatory for the study of such properties to fully understand the biological systems. The robustness, understood as the porperty that allows the systems to maintain their functions despite external and internal perturbations, is a system-level phenomenom ubiquitously observed in living systems (Blanchini and Franco 2011). Metabolic networks can be affected by variables that have the power of modulating it as a whole and, most likely, of influencing the referred-to molecular mechanisms. Those variables, one of whihch being endogenous ROS generating metabolism, must be included in metabolic in silico models to study this robustness. We decided to model endogenous ROS generation in Pseudomonas putida KT2440 genome-scale model. P. putida is a model microorganism in biotechnology and possesses an extremely versatile metabolism, which makes it the perfect candidate to study complex metabolic processes (Belda et al. 2016). The objective of this project is to go deep in the understanding of endogenous ROS metabolism and to study the role of ROS in different metabolic processes. To do so, we built and validated the ROS generating genome-scale model and used it as a tool for two purposes: (1) to analyse, in silico, the main metabolic mechanisms to prevent the cell from ROS damage and (2) to generate hypothesis about the role of ROS in different metabolic processes that we approached experimentaly. As a result of this investigation, we have came up with (1) the theory that the main cellular mechanisms to fight back endogenous ROS generation are fuelled by NADH, and not by the activation of NADPH generating metabolic pathways, as we find in scientific literature (Mailloux, Lemire, and Appanna 2011). (2) We have also found that ROS plays an important role in different molecular mechanisms involving robustness, as the carbon flux deviation to the accumulation of  polyhydroxyalkanoate; and also influenciate the global metabolic regulator gen crc

Regulation of Protein Secretion Systems Mediated by Cyclic Diguanylate in Plant-Interacting Bacteria

The ubiquitous second messenger cyclic diguanylate (c-di-GMP) is involved in the regulation of different processes in bacteria. In phytopathogens, intracellular fluctuations in the concentration of this molecule contribute to the lifestyle switching from a motile and virulent stage to a sessile and biofilm-forming phase. Among the virulence mechanisms used by bacterial pathogens, different specific type secretion systems (TSSs) and the effector proteins that they translocate are included. Some of these TSS are conceived to suppress host immune responses during bacterial colonization. The modulation of the expression of secretion systems components and/or effector proteins can be influenced by c-di-GMP levels at transcriptional, translational, or post-translational levels and can take place directly by binding to specific or global regulators, or via transducer proteins. Different genera of plant-interacting bacteria have been analyzed to shed some light in the implications of c-di-GMP in the regulation of host plant colonization through protein secretion systems. Expression of (1) adhesins secreted by Type 1 secretion systems to bind the host plant in Pectobacterium (formerly Erwinia) and some beneficial Pseudomonas strains; (2) catalytic exoproteins delivered by Type 2 secretion systems to break plant cell wall in Dickeya; (3) effectors secreted by Type 3 secretion systems to suppress plant immunity in Xanthomonas; or (4) the activity of Type 6 secretion systems to export an ATPase in Pseudomonas, are finely tuned by c-di-GMP levels. In this minireview, we summarize the knowledge available about the implications of c-di-GMP in the regulation of protein secretion in different plant-interacting bacteria.Spanish Ministry of Economy, Industry and Competitivity BIO2016-78409-

Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.España, Ministerio de Educación y Ciencia BIO2011-24003España, Ministerio de Educación y Ciencia CSD2007-000

Construcción de una metagenoteca a partir de muestras de una depuradora de aguas de hospital para la obtención de funciones de interés ambiental y biomédico.

Motivación: La demanda de medicamentos, especialmente de los antibióticos, ha ido en aumento en los últimos tiempos. A pesar de que muchos de ellos son biodegradables, a menudo se encuentran como contaminantes de las aguas residuales tratadas, lo cual supone un riesgo ecológico y sanitario ya que es posible la aparición de microorganismos resistentes limitando su efectividad. De aquí surge la necesidad de analizar qué microorganismos pueden ser resistentes, y en concreto qué genes les confieren dicha resistencia, para lo cual se propone construir una metagenoteca a partir de aguas residuales de una depuradora de hospital. En ella la concentración de antibióticos es mayor y existen antibióticos tanto de uso frecuente como exclusivos de hospitales.Métodos: Para la construcción de la metagenoteca se siguió el protocolo estándar, “CopyControlTM Fosmid Library Production Kit protocol (Epicentre)”, con algunas modificaciones [1]. En primer lugar se realizó la extracción del ADN de las muestras de la depuradora, para lo cual se realizaron diversas pruebas utilizando kits comerciales de extracción así como otros métodos descritos [2,3]. Tras la extracción se comprobó que el ADN purificado no se degradara, se seleccionó el tamaño adecuado, determinado por la capacidad de empaquetar del fago lambda (≈40kb), y se repararon los extremos para su clonación. Por otra parte, el fósmido que se empleó para la construcción de la metagenoteca, se digirió con PmlI y desfosforiló con SAP. Para que la metagenoteca sea representativa, han de conseguirse concentraciones muy altas tanto del vector como del inserto. Finalmente, el ADN ligado se empaquetó en extractos del fago lambda que se emplean para infectar células EPI300-T1R. Estas bacterias transformadas con el vector, constituyen la metagenoteca.Resultados: Se han buscado bacterias resistentes a diferentes concentraciones de los antibióticos colistina y ceftriaxona. A las concentraciones probadas por el momento, sólo se ha encontrado resistencia a ceftriaxona. Actualmente se sigue trabajando para construir una nueva metagenoteca y mejorar todo el proceso.Conclusiones: Se han obtenido bacterias resistentes a ceftriaxona en la primera metagenoteca construida, a pesar del bajo número de clones de esta. Esto pone de manifiesto la robustez del sistema para detectar actividades génicas incluso teniendo una baja representación en la metagenoteca de los organismos con dicha actividad presentes en el medio

The Rhizobial Type 3 Secretion System: The Dr. Jekyll and Mr. Hyde in the Rhizobium–Legume Symbiosis

Rhizobia are soil bacteria that can establish a symbiotic association with legumes. As a result, plant nodules are formed on the roots of the host plants where rhizobia differentiate to bacteroids capable of fixing atmospheric nitrogen into ammonia. This ammonia is transferred to the plant in exchange of a carbon source and an appropriate environment for bacterial survival. This process is subjected to a tight regulation with several checkpoints to allow the progression of the infection or its restriction. The type 3 secretion system (T3SS) is a secretory system that injects proteins, called effectors (T3E), directly into the cytoplasm of the host cell, altering host pathways or suppressing host defense responses. This secretion system is not present in all rhizobia but its role in symbiosis is crucial for some symbiotic associations, showing two possible faces as Dr. Jekyll and Mr. Hyde: it can be completely necessary for the formation of nodules, or it can block nodulation in different legume species/cultivars. In this review, we compile all the information currently available about the effects of different rhizobial effectors on plant symbiotic phenotypes. These phenotypes are diverse and highlight the importance of the T3SS in certain rhizobium–legume symbioses.Ministerio de Ciencia e Innovación PID2019-107634RB-I00Junta de Andalucía P20_00185Universidad de Sevilla FEDER-US 1259948, FEDER-US 125054

NopC is a rhizobium-specific type 3 secretion system effector secreted by sinorhizobium (ensifer) fredii HH103

Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in hostrange determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1-and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103Junta de Andalucía P11-CVI-7050Ministerio de Economía y Competitividad AGL2012-38831Universidad de Sevill

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

Estudio del efecto de auxotrofías y del intercambio de plásmidos pSym en las propiedades simbióticas de Sinorhizobium fredii

En nuestro grupo de investigación disponíamos de varios mutantes de la estirpe HH103 de S. fredii auxótrofos a diversos compuestos, obtenidos mediante inserción del transposón Tn5-B20. La gran mayoría de los auxótrofos para aminoá cidos de S. fredii, inc

Zoé enters the house: biology in architectural training

Los conocimientos en la etapa formativa para la posterior práctica arquitectónica se actualizan rigurosamente por la preparación y autoexigencia del profesorado pero, estructuralmente, un cambio radical de planteamiento acaece con dificultad en la renovación de los Planes de Estudios. Una lógica disciplinar distante de los problemas actuales enfoca conservadoramente la docencia y, por extensión, la profesión y, más allá, la propia definición de la Arquitectura. Durante este siglo XXI, los desafíos sociales, económicos y ambientales no han logrado promover cambios sustanciales en la idea de lo que un arquitecto debe ser para sus sociedades. La actual crisis pandémica aboca a ampliar con conocimiento de biología la formación de arquitectos. El máster en Ciudad y Arquitectura Sostenibles, de la  ETS Arquitectura de Sevilla, enseña desde 2010 a sus estudiantes a proyectar con materia viva y a evaluar sus consecuencias, en una experiencia transdisciplinar que aspira a ser pionera como modelo para futuras actualizaciones de los Planes de Estudio, incluso en Grado.The knowledge in the formative stage for the later architectural practice is rigorously updated due to the preparation and self-demanding of the teaching staff but, structurally, a radical change of approach occurs with difficulty in the renewal of the curricula. A disciplinary logic far removed from the current problems conservatively focuses on teaching and, by extension, on the profession and, beyond that, on the very definition of Architecture. During this 21st century, social, economic and environmental challenges have not managed to promote substantial changes in the idea of what an architect should be for their societies. The current pandemic crisis is leading to the extension of the training of architects with knowledge of biology. Since 2010, the Master in Sustainable City and Architecture at the ETS Architecture in Seville has been teaching its students to design with living matter and to evaluate its consequences, in a transdisciplinary experience that aims to be a pioneer as a model for future curricular updates, including in Bachelor's degrees.Peer Reviewe