NMR metabolomic analysis of bacterial resistance pathways using multivalent quaternary ammonium functionalized macromolecules.

Introduction: Multivalent antimicrobial dendrimers are an exciting new system that is being developed to address the growing problem of drug resistant bacteria. Nuclear Magnetic Resonance (NMR) metabolomics is a quantitative and reproducible method for the determination of bacterial response to environmental stressors and for visualization of perturbations to biochemical pathways.

Objectives: NMR metabolomics is used to elucidate metabolite differences between wild type and antimicrobially mutated Escherichia coli (E. coli) samples.

Methods: Proton (H) NMR hydrophilic metabolite analysis was conducted on samples of E. coli after 33 growth cycles of a minimum inhibitory challenge to E. coli by poly(amidoamine) dendrimers functionalized with mannose and with C-DABCO quaternary ammonium endgroups and compared to the metabolic profile of wild type E. coli.

Results: The wild type and mutated E. coli samples were separated into distinct sample sets by hierarchical clustering, principal component analysis (PCA) and sparse partial least squares discriminate analysis (sPLS-DA). Metabolite components of membrane fortification and energy related pathways had a significant p value and fold change between the wild type and mutated E. coli. Amino acids commonly associated with membrane fortification from cationic antimicrobials, such as lysine, were found to have a higher concentration in the mutated E. coli than in the wild type E. coli. N-acetylglucosamine, a major component of peptidoglycan synthesis, was found to have a 25-fold higher concentration in the mid log phase of the mutated E. coli than in the mid log phase of the wild type.

Conclusion: The metabolic profile suggests that E. coli change their peptidoglycan composition in order to garner protection from the highly positively charged and multivalent C-DABCO and mannose functionalized dendrimer.