NMR Based Real-Time Analysis of Exometabolites Decodes the Mechanism of Action of Antibacterial Molecules, Nanoparticles, and Materials

Understanding the mechanism of action of antimicrobial agents is critical for guiding the development of new drugs to overcome antimicrobial resistance. We present a label-free NMR-based approach to characterize the mechanism of action of antibacterial compounds and materials by the analysis of metabolite secretion kinetics. The method (KINEXO, KINetics of EXOmetabolites) is set up using <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> as representative Gram-negative and Gram-positive model organisms. By monitoring the real-time production of key secreted metabolites (acetate, formate, lactate, ethanol, pyruvate, succinate) in response to antimicrobial treatment and analyzing the secretion kinetics, we can classify the agents' mechanisms of action. We validate KINEXO using agents with well-characterized mechanism of action (kanamycin, ampicillin, irgasan, caprylic acid, graphene-like nanoparticles, and a functionalized silicon material), and we further apply it to silver nanoparticles, whose mechanism of action remains under debate. Agents that perturb the cell envelope reduce secretion rates while maintaining end-point metabolite concentrations with only moderate lag phase extension. In contrast, agents that act on intracellular pathways drastically prolong lag phases and reduce both secretion rates and end-point concentrations. When plotted in 3D parameter space (exometabolite secretion lag time, secretion rate, end-point concentration), antibacterial agents cluster according to their mode of action, offering a mechanistically informative phenotypic readout. This platform provides a generalizable and robust analytical framework for rapid antimicrobial profiling and mechanism-based screening of novel bioactive agents.