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

Abstract

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. NMR metabolomics is used to elucidate metabolite differences between wild type and antimicrobially mutated Escherichia coli (E. coli) samples. Proton (1H) 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 C16-DABCO quaternary ammonium endgroups and compared to the metabolic profile of wild type E. coli. 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. The metabolic profile suggests that E. coli change their peptidoglycan composition in order to garner protection from the highly positively charged and multivalent C16-DABCO and mannose functionalized dendrimer.