Figure 1: Simplified workflow applied in the non-targeted metabolomics study. Three well-isolated colonies from each strain are selected from agar plates containing MH medium and cultivated in overnight cultures. Shake flasks containing MH broth are inoculated w/ overnight culture. Samples are collected with fast filtration and quenched during exponential phase. The extracted samples are subjected to lyophilization before being resuspended in a ACN:H2O mix and analysed with high resolution LC-MS. Figure created with BioRender.Antimicrobial resistance, AMR, has been flagged by the World Health Organization, WHO, as one of the greatest threats towards global public health. In addition, as the demand for new antibiotics is greater than the development rate of new drugs, WHO has now declared that we have entered the post-antibiotic era. We wish to tackle the rapidly increasing challenge with a non-targeted multi-omics approach, focusing primarily on metabolomic profile of clinically isolated E. coli strains.
The metabolome reflects variations and interactions in the environment and between protein and gene expression, rendering metabolomics the ‘omics field closest related to phenotype. Metabolomics data can reveal important initial responses when the bacteria is subjected to antibiotic stress and which adaptions are essential to maintain AMR mechanisms. A confident screening of the metabolic profile is achievable through HRMS analysis and provides insight into fluctuations in the abundance of metabolites when conducting drug treatment. This information contributes to a greater understanding of AMR mechanisms, but more importantly, can also aid to the development of much needed drug targeted treatments.
We focus on the endometabolome, which comprises metabolites present in the bacterial cell. Information acquired through the visualization of the endometabolome from sensitive and resistant clinical E. coli strains will be further subjected to lipidomics analysis. E. coli strains are subjected to adaptive laboratory evolution and endometabolomic E. coli samples are obtained through fast filtration and subjected to liquid nitrogen to quench the metabolome. Efficient sampling and quenching are the pivotal steps in any metabolomics experiment due to the rapid turn-over rates of metabolites. Lipid samples are collected by applying a modified Folch extraction method. The collected samples are analysed with state-of-the-art HR-LCMS, Thermo Scientific Orbitrap Id-X. To optimize coverage of metabolites, analysis is performed in reverse phase in both positive and negative mode and hydrophilic interaction chromatography (HILIC), also in both positive and negative mode.