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• W8677036 abstract "Infections caused by multidrug-resistant (MDR) bacteria, including Vancomycin-Resistant Enterococcus (VRE), have become one of the greatest clinical challenges of the 21 st 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 devicerelated 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." @default.
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• W8677036 date "2014-09-25" @default.
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• W8677036 title "Generation of a Vancomycin-Resistant Enterococcus faecium Clinical Isolate Expressing a (FMN)-Based Fluorescent Protein" @default.
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