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- Expansion of identification capabilities of bacterial isolates by MALDI-ToF MSPublication . Silva, Mariana Branco; Reichardt, Nicole; Mörtelmaier, Christoph; Ferreira, Susana Margarida ParaísoThe present dissertation results from a six-month internship conducted in Ghent, Belgium, at MRM Health NV, as part of the Master's program in Biotechnology at the University of Beira Interior. The primary focus of this internship was to expand company’s database using MALDI-ToF MS as a robust and efficient tool for bacterial identification. Throughout the internship and subsequently, for the development of the internship report, the main objective was defined as integration and active participation within the discovery team. This involved engaging in daily routine tasks, particularly focusing on the identification and creation of Main Spectra Profiles (MSPs) of bacterial isolates using MALDI-ToF MS. Additionally, the 16S rRNA gene sequencing technique was employed for validation, quality assessment, and performance of the identification. Specific microbiological techniques tailored for anaerobic bacteria were also applied, alongside with characterisation techniques utilising Biolog Plates - Phenotype Microarrays (PM), to monitor the growth and metabolic activity of various anaerobic bacteria over time. In the first part, the description of the company is provided, focusing on its role in developing therapeutics by harnessing the full potential of the human intestinal microbiome. The company encompasses various therapeutic programs, including clinical trials, preclinical studies, and discovery programs. The second part concentrated on consolidating and comprehending the theoretical concepts involved in the research project. This segment explores topics, such as the identification and characterization of anaerobic bacteria using diverse techniques. Subsequently, daily tasks necessary for the project's completion are mentioned and described. These tasks primarily involve extending the company's database, entailing the creation of MSPs for different anaerobic bacteria. For validation, the 16S rRNA gene sequencing technique was employed, to enable comparison of results between both techniques for improved anaerobic bacteria identification. Furthermore, the process of characterizing eight (8) anaerobic bacteria using Biolog Plates (PM1 and PM2) - Phenotype Microarrays has also been delineated. By employing MALDI-ToF to add new MSPs of well-characterized bacterial isolates to the company's and Bruker Daltonics' databases, a significant improvement in the identification of anaerobic bacteria at the genus and species levels was observed. The implementation of this system substantially expanded the number of anaerobic bacterial species and genera, which is particularly relevant for species exhibiting high intra-specific variation. However, to maintain the reliability of MALDI-ToF, regular updates of the database are necessary to keep pace with changes in anaerobic taxonomy and its increasing diversity.