Otimização de protocolos de extração de metabólitos de Brucella abortus e estudo metabolômico untarget de seus biovares

Abstract

An exploratory study to compare and find alterations in the metabolism among biovars of the bacterium Brucella abortus was carried out. An untargeted metabolomics approach was the tool used to understand the mechanisms involved at the molecular level due to the genetic condition. Two high resolution analytical platforms (GC-QTOF-MS and UHPLC-Q-Orbitrap) were used for chemical analyses. Some procedures of the metabolomics study workflow were optimized and the research was divided into three steps. The first step was dedicated to the development of the protocol for the preparation and collection of bacterial samples from Brucella abortus. The optimized protocol defined colony growth time, biomass to be collected, washing and biomass decantation time, and bacterial inactivation for collection. In the second step, extraction methods were evaluated for the two analytical platforms, selecting the extraction solutions composed by: methanol/water/chloroform for GC-QTOF-MS and acetonitrile/isopropanol/water for UHPLC-Q-Orbitrap. Analytical stability was evaluated using quality controls and internal standards, triphenylmethane for GC-QTOF-MS and p-fluoro-DL-phenylalanine and sulfadiazine for UHPLC-Q-Orbitrap. In the third step, the interpretation of results and biological association were performed. The XCMS Online and MetaboAnalyst platforms were used for data processing and statistical treatment (uni and multivariate analysis), respectively. The results obtained in both analytical platforms showed that there are differences at the metabolic level between the Brucella abortus biovars and that the altered and identified metabolites are related to the bacterial cell membrane. These results are very promising for the development of preventive, diagnostic and therapeutic technologies, since the virulence of the Brucella abortus bacteria is related to the cell membrane.