High-resolution mass spectrometry-based metabolomics coupled to high-throughput screening of FDA approved drugs against MRSA

Resistance to antibiotics poses a serious threat to public health. Investigation of the mechanism of action of novel agents plays a key role to combat antibiotic resistance.

In this study, high-resolution mass spectrometry (HRMS)-based targeted and untargeted metabolomics coupled to high throughput screening of FDA-approved drugs against Methicillin-Resistant Staphylococcus Aureus (MRSA) presented an efficient route to reveal novel activities of FDA approved drugs which provide a potentially great shortened path to clinical applications.

An amine targeted approach based on Marfey’s reagent is compared with an untargeted approach for the identification of potential biomarkers of the mechanism of action (MoA) of some antibiotics against S. aureus.

This can be correlated to pathway analysis to investigate the MoA and antibiotic resistance.
In this study, unmetabolized (UM) and post metabolized (PM) (hepatic microsomal metabolism) FDA-approved compounds were screened in the presence and absence of low concentration (1/4 MIC) of ceftobiprole to determine probable synergistic combinations.

Performed checkerboard assays (developed isobologram) where antibiotic fractional inhibitory concentration (FIC) was determined against S. aureus for combinations of ceftobiprole with drugs selected from FDA library screening based on their intrinsic-activity merit (Floxuridine, Rifaximine, Cloxacillin, and Gemcitabine).

Growth curves for cultures in the presence of sub-MIC antibiotics were essentially indistinguishable from uninhibited cultures prior to sample collection. Therefore, acute exposure of supra-MIC antibiotics (4X MICs) were individually treated to an exponentially growing S. aureus culture to induce growth inhibition.

Untargeted Metabolomics:  3-dimensional Principal Component Analysis and Partial Least Squares generated distinctive metabolite profiles under different antibiotic treatments when compared from untreated control groups and groups treated with different antibiotics.  Metabolite annotation was done for several potential biomarkers involved in the pathogenicity and resistance mechanism of S. aureus.

Targeted Metabolomics: To enable the identification of modified amines and stoichiometry of modification, a stable heavy atom isotope-labeled Marfey’s atom was developed. Peak lists from the derivatization of samples with both light (L-Mar) and heavy (H-Mar) readily allowed modified amines to be identified. Significant features are identified by Random Forest Algorithm are matched against the METLIN tandem mass spectrometry database which provides an efficient means of metabolomic profiling that facilitates features alignment and identification. Amine targeted metabolomic approach has so far identified 20 distinct mono adducts, 10 diadducts, and a few potential tri-adducts in S. aureus. We are currently working to establish their MoA through pathway analysis.

In this comparative metabolomics study, complex metabolic changes were investigated in response to antibiotic treatment in S. aureus. This study set up significant targets for the future investigation of drugs having antibiotic potential.