Improved workflow for untargeted metabolomics and NMR analysis of intracellular and extracellular metabolites isolated from Gram positive and Gram negative bacteria

The emergence and spread of antibiotic resistance has become a major global health concern. ESKAPE pathogens, defined by the World Health Organization (WHO), are currently listed as high priority pathogens and many of these species are capable of developing resistance to multiple antibiotics. It has been described that various metabolic processes can influence antibiotic susceptibility. Thus, further research deciphering the global metabolic changes that occur in response to antimicrobial agents may prove to be useful for combatting bacterial infections. Metabolomics and Nuclear Magnetic Resonance (NMR) spectroscopy have proven to be useful for characterising key metabolome fluctuations in bacteria in response to antibiotic stress. However, a number of impediments to current workflows have led to the reduced use of these techniques in microbial research. In this study, we address these limitations and in response have developed a novel 1H NMR-based untargeted metabolomics workflow. This method is suitable for use with various bacterial species, reducing the workload in comparison to previously established workflows. Our protocol is simple and reproducible and allows for the isolation of both intracellular (IC) and extracellular (EC) metabolites simultaneously from both Gram (+) and Gram (-) species. This method has been shown to produce consistent results for the ESKAPE pathogens Escherichia coli, Klebsiella pneumoniae, Enterococcus faecium and Staphylococcus aureus. By using these data as a baseline, future studies involving antibiotic stress conditions can be compared to identify key metabolome differences in each species and to potentially identify targets to combat antimicrobial resistance.