Screening and isolation of compounds with antimicrobial activity produced by multi-resistant bacteria
Year: 2014
Type: dissertation
Abstract: In the late 20s, after Sir Alexander Fleming discovered the penicillin, a large interest in the search for compounds with antimicrobial activity produced by microorganisms emerged. This event marked the beginning of a new era of antibiotic discovery, where numerous scientist would screen for antibiotics in microorganisms, mostly from the soil. Previous work undertaken at LMB-BioFIG, suggest that ΔphoQ mutation in Stenotrophomonas maltophilia D457 strain is associated to an increased antimicrobial activity. The main focus of this work was to: determine the existence of compounds with antimicrobial activity produced by the aforementioned mutant; determine its spectrum of activity and optimize the culture conditions that are optimal to its production. To answer these questions, a series of biologic susceptibility assays were performed, with both the parental D457 strain and D457ΔphoQ strain. Namely the cross-streak assay, agar-well diffusion assay and drop-diffusion assay. For the later, different culture conditions for the producing strains were tested in order to better understand which conditions favour the compound production, and also, sequential extractions with different organic solvents with different polar characteristics were made to fractionate the obtained batches regarding the polarity of its content, and tested for biologic activity. Finally, in an attempt to unveil the compound of interest, protein quantification, thin layer chromatography and gas chromatography-mass spectrometry analysis were performed on all extracts to better elucidate the nature and structure of the compound. This work revealed that the compound of interest is of non-polar nature produced in low concentrations, around 0.1 g/L. Was only present in the ethyl acetate fraction, with inhibitory activity against the test strains. In neither biologic assay did the parental strain show any inhibitory activity. Additionally, it was revealed by protein quantification assays that the mutant strain secreted almost 3-fold more protein than the parental strain. The results obtained by GC-MS suggest that the compound of interest resembles with the 2,3-dihydroxybenzoic acid, with possible presence of cyclic structures, such as aromatic rings in its molecular structure. However, further analytical procedures must be undertaken to clarify and confirm the structure of the secreted compounds by D457ΔphoQ strain.
Authors: Espírito Santo, Bruno Miguel Prazeres
Language: English
Subfield: Applied Microbiology and Biotechnology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2256361764