Generation of a Vancomycin-Resistant Enterococcus faecium Clinical Isolate Expressing a (FMN)-Based Fluorescent Protein

[EN] Infections caused by multidrug-resistant (MDR) bacteria, including Vancomycin-Resistant Enterococcus (VRE), have become one of the greatest clinical challenges of the 21st century. Particularly, VRE strains of E. faecium, a natural member of the gastrointestinal consortia, have recently emerged as one of the most problematic cases of MDR nosocomial pathogens. It is widely known that the establishment of high-level intestinal colonization of this bacterium, triggered by antibiotic treatment of the host, potentially leads to bacteremia, endocarditis and surgical wound, urinary tract, and device-related infections in hospitalized patients. However, the unique determinants possessed by these strains, which enable them to benefit from the antibiotic-induced perturbations of the gut microbiota and host intestinal immune defenses, remain to be identified. Essentially, what needs to be revealed is the differential gene expression of E. faecium in its niche at different time points; before and during antibiotic treatment of the host, and very interestingly, in the presence of probiotic bacteria. Differentially expressed genes will highlight how and why E. faecium is able to dominate the gut, as well as how it interacts with its surroundings, possibly pinpointing its potential weaknesses, as well as provide hints on what specific properties should be prerequisites for bacteria to be used as probiotics. Since the global expression intended to be analyzed is that of E. faecium alone, a method to separate this bacteria from the rest of the gut microbiota is required. Failure to do so would result in the study of the gut microbiota’s transcriptome as a whole: metatranscriptomics. In practice, the separation VRE. faecium from the rest of the commensal microbiota could be carried out by means of flow cytometry if the generation of a fluorescent E. faecium was achieved. Additionally, it would serve as a great tool to study a series of facets of VRE faecium colonization which remain virtually unknown. It would enable the monitorization of VRE infection through whole-body imaging of infected mice, fluorescence microscopy analysis of histological cuts to determine its most predominant locations, the use of FbFP as a translational or transcriptional reporter, etc. Consequently, the main objective of this Final Degree Project has been to attempt the generation of a Vancomycin-Resistant Enterococcus faecium clinical isolate (C68) expressing a fluorescent protein whose chromophore can form under the anaerobic conditions found in the guts of animals: (FMN)-based fluorescent protein (FbFP). In order to fulfill the main objective of this work, first, the FbFP gene was synthesized with the appropriate features for its expression in E. faecium and for its cloning. Next, the plasmid construction aimed to confer E. faecium fluorescent properties (pBT2-FbFP), was generated. This was done by inserting the FbFP gene into the pBT2 plasmid. As a previous step to the transformation of E. faecium, pBT2-FbFP was transformed into E.coli DH5a in order more efficiently validate the integrity of the plasmid construction. After validation, E faecium was finally transformed and the corresponding fluorescence analyses were performed.[ES] Las infecciones causadas por bacterias resistentes a múltiples fármacos (MDR), incluyendo el Enterococo resistente a la vancomicina (VRE), se han convertido en uno de los mayores retos clínicos del siglo 21. En particular, las cepas de E. faecium, miembro natural de la microbiota gastrointestinal, suponen actualmente uno de los casos más problemáticos de MDR patógenos nosocomiales. La colonización intestinal con altos niveles de esta bacteria es provocada por el tratamiento con antibióticos y puede producir bacteriemia, endocarditis, infecciones del tracto urinario y de heridas quirúrgicas o relacionadas con dispositivos médicos, en pacientes hospitalizados. Sin embargo, aún no se han identificado los determinantes específicos que les permiten a estas cepas beneficiarse de las alteraciones inducidas por los antibióticos sobre la microbiota y las defensas del sistema inmune intestinal. En esencia, se necesita conocer la expresión diferencial de los genes de E. faecium en su nicho y en diferentes momentos: antes y durante el tratamiento con antibióticos, así como en presencia de bacterias probióticas. Los genes expresados diferencialmente pondrían de relieve cómo y por qué E. faecium es capaz de dominar el intestino o la forma en que interactúa con su entorno, posibilitando la localización de sus debilidades potenciales y mostrando qué propiedades específicas deberían ser requisitos previos de una bacteria para ser utilizada como probiótico. Dado que la expresión global a analizar es la de E. faecium solo, se necesita un método para separar esta bacteria del resto de la microbiota intestinal. No hacerlo, daría lugar al estudio del transcriptoma de la microbiota intestinal en su conjunto: metatranscriptómica. En la práctica, la separación de VRE faecium del resto de la microbiota comensal podría llevarse a cabo por medio de citometría de flujo una vez lograda la generación de un E. faecium fluorescente. Además, constituiría una gran herramienta para el estudio de una serie de facetas de la colonización de VRE faecium que siguen siendo prácticamente desconocidas. Por ejemplo, permitiría la monitorización de la infección por VRE a través de imágenes del cuerpo de ratones infectados, el análisis de microscopía de fluorescencia de cortes histológicos para determinar sus principales localizaciones, el uso de FbFP tanto como reportero transcripcional como de traducción, etc. Por ello, el objetivo principal de este Trabajo de Fin de Grado la generación de un aislado clínico (C68) vancomicina resistente de Enterococcus faecium a través de la expresión de una proteína fluorescente cuyo cromóforo pueda formarse en las condiciones anaerobias de los intestinos de los animales: proteína fluorescente basada en FMN (FbFP). Con el fin de cumplir con el objetivo principal de este trabajo, el gen FbFP fue sintetizado con las características apropiadas para su expresión en E. faecium así como para su clonación. A continuación, se generó la construcción del plásmido destinado a conferir propiedades fluorescentes a E. faecium (pBT2-FbFP). Esto se hizo mediante la inserción del gen FbFP en el plásmido pBT2. Como paso previo a la transformación de E. faecium, pBT2-FbFP se transformó en E. coli DH5a con el fin de validar de manera más eficiente la integridad de la construcción del plásmido. Después de su validación, E. faecium fue transformado y se realizaron los correspondientes análisis de fluorescencia.Pinilla Redondo, R. (2014). Generation of a Vancomycin-Resistant Enterococcus faecium Clinical Isolate Expressing a (FMN)-Based Fluorescent Protein. http://hdl.handle.net/10251/40249.Archivo delegad

Plan de mejora en salud ocupacional para la Alcaldía de Chiquinquirá

El presente trabajo; fue realizado como requisito par a obtener el título de administradores de empresas, basados en lo solicitado en el curso del diplomado de profundización en gerencia del talento humano. La investigación realizada fue de tipo cualitativa y cuantitativa y su desarrollo se describe en cinco capítulos: Inicia con un primer capítulo se planteó el problema relacionado con la salud ocupacional para La Alcaldía de Chiquinquirá, el segundo capítulo contiene las teorías relacionadas con la temática, literatura que sirvió de base para la argumentación y descripción del trabajo, en el capítulo tres, se establecieron los procedimientos utilizados para la indagación de los datos necesarios para el estudio, en consecuencia en el capítulo cuarto se presentan los resultados obtenidos mediante el instrumento utilizado en la recolección de la información y finaliza con el capítulo cinco en el que se realizan las recomendaciones del caso frente a los hallazgos encontrados, luego se plantea la propuesta basada en el Modelo de Nonaka y Takeuchi y por último se entregan las conclusiones a que se llegó producto del desarrollo de estudio y para el área indicada.The present work; It was carried out as a requirement to obtain the title of business administrators, based on what was requested in the course of the Diploma of Deepening  in Human Talent Management. The research carried out was qualitative and quantitative and its development is described in five chapters: Start with a first chapter the problem related to Occupational Health for the Mayor of Chiquinquirá was raised, the second chapter contains the theories related to the topic, literature that served as the basis for the argumentation and description of the work, in chapter three, established the procedures used for the investigation of the data necessary for the study, consequently in the fourth chapter the results obtained through the instrument used in the collection of information are presented and ends with chapter five in which the recommendations of the In the case of the findings found, the proposal based on the Nonaka and Takeuchi Model is then presented and, finally, the conclusions arrived at as a result of the study development and for the indicated area

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

Epidemiology of intra-abdominal infection and sepsis in critically ill patients: “AbSeS”, a multinational observational cohort study and ESICM Trials Group Project

Purpose: To describe the epidemiology of intra-abdominal infection in an international cohort of ICU patients according to a new system that classifies cases according to setting of infection acquisition (community-acquired, early onset hospital-acquired, and late-onset hospital-acquired), anatomical disruption (absent or present with localized or diffuse peritonitis), and severity of disease expression (infection, sepsis, and septic shock). Methods: We performed a multicenter (n = 309), observational, epidemiological study including adult ICU patients diagnosed with intra-abdominal infection. Risk factors for mortality were assessed by logistic regression analysis. Results: The cohort included 2621 patients. Setting of infection acquisition was community-acquired in 31.6%, early onset hospital-acquired in 25%, and late-onset hospital-acquired in 43.4% of patients. Overall prevalence of antimicrobial resistance was 26.3% and difficult-to-treat resistant Gram-negative bacteria 4.3%, with great variation according to geographic region. No difference in prevalence of antimicrobial resistance was observed according to setting of infection acquisition. Overall mortality was 29.1%. Independent risk factors for mortality included late-onset hospital-acquired infection, diffuse peritonitis, sepsis, septic shock, older age, malnutrition, liver failure, congestive heart failure, antimicrobial resistance (either methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum beta-lactamase-producing Gram-negative bacteria, or carbapenem-resistant Gram-negative bacteria) and source control failure evidenced by either the need for surgical revision or persistent inflammation. Conclusion: This multinational, heterogeneous cohort of ICU patients with intra-abdominal infection revealed that setting of infection acquisition, anatomical disruption, and severity of disease expression are disease-specific phenotypic characteristics associated with outcome, irrespective of the type of infection. Antimicrobial resistance is equally common in community-acquired as in hospital-acquired infection