NMR hydrophilic metabolomic analysis of bacterial resistance pathways using multivalent quaternary ammonium antimicrobials in Escherichia coli and Bacillus cereus exposed to DABCO and mannose functionalized dendrimers

Abstract

Novel antibiotics developed using a new scaffold are needed to combat the rising tide of antibiotic resistant bacteria. Multivalent antibiotics are a relatively new approach that have the potential to greatly increase the efficacy of antibiotics while making it difficult for bacteria to develop resistance. Dendrimers are an attractive framework for the multivalent presentation of antibacterial moieties. Quaternary ammonium compounds (QAC) are a positively charged class of membrane disruptors that are attracted to the large negative charge on phospholipid membranes. Nuclear magnetic resonance (NMR) metabolomics is a quantitative method used for comparison of metabolic profiles of wild type and mutated bacterial samples, enabling the study of bacterial response to antimicrobials. Proton (1 H) NMR hydrophilic metabolomics was used to study gram-negative and gram-positive bacteria upon exposure to 1,4-diazabicyclo-2,2,2-octane (DABCO) with a 16-carbon chain tethered onto a mannose functionalized poly(amidoamine) (PAMAM) dendrimer (denoted as DABCOMD), a membrane disrupting multivalent QAC. Stock Escherichia coli (E. coli) (denoted as wild type) and DABCOMD mutated E. coli (denoted as mutants) were collected in the mid log and stationary phases. The same procedures were used for Bacillus cereus (B. cereus) as for E. coli samples (denoted as unchallenged), except that a DABCOMD challenged sample set was added (denoted as challenged). The challenged sample set procedures were identical to the unchallenged, except DABCOMD was included at 33 % of the MIC value in the growth media for growth curve acquisition and sample collection. The greatest differences observed between the metabolic profiles of the wild type and mutated E. coli samples and between the challenged and unchallenged B. cereus samples were in energy-associated metabolites and membrane-related pathways. The mutants in all sample types were associated with higher levels of spent energy molecules (including AMP and NAD+) and peptidoglycan related compounds (including N-acetylglucosamine). Overall, more changes were observed for B. cereus (gram-positive), especially in challenged mutant B. cereus samples, than for E. coli (gram-negative) samples. Since DABCOMD is a positively charged multivalent membrane disruptor, both B. cereus and E. coli mutated to garner protection by altering their peptidoglycan layer composition, which is energetically costly.