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

## TL;DR

This paper introduces a new NMR-based method to study how antibacterial agents work by analyzing real-time metabolite secretion from bacteria.

## Contribution

The novel contribution is a label-free NMR method called KINEXO for classifying antibacterial mechanisms based on exometabolite secretion kinetics.

## Key findings

- Agents affecting the cell envelope reduce secretion rates but not end concentrations.
- Intracellular-acting agents prolong lag phases and reduce both secretion rates and concentrations.
- KINEXO clusters agents in 3D parameter space according to their mechanism of action.

## Abstract

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 Escherichia coli and Staphylococcus aureus 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.