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Connecting Branched Isomer Distributions and Membrane Characteristics in Daptomycin-Resistant Staphylococcus aureus Using RPLC-IM-MS

Portrait of Christian Freeman, speaker
Christian Freeman
Graduate Student, Department of Chemistry
University of Georgia
iSTEM Building 2, Room 1218
Analytical Seminar

Staphylococcus aureus is a common cause of hospital and community-acquired infections. Although uncommon, resistance to daptomycin is mediated by mutations in genes associated with the regulation, synthesis, or relocation of fatty acids and membrane lipids. These mutations influence physical properties such as cell surface charge, cell membrane fluidity, and daptomycin resistance, however, recent results were unable to provide an in-depth isomeric comparison between daptomycin susceptible and resistant strains. Common lipid analysis techniques result in whole lipid class determination or fatty acyl chain identification, leaving out information on intact lipid isomers.1 Staphylococcus aureus is unique amongst many strains by varying its membrane fluidity using branched fatty acyl chain combinations, providing an avenue for isomer identification. We developed a Reversed-Phase Liquid Chromatography Ion Mobility Mass Spectrometry (RPLC-IM-MS) method for classifying lipid composition of intact branched isomers.2 Through reversed-phase separation, an in-depth overview of abundant isomers determining membrane fluidity is possible, as we further applied this technique for evaluating changes in membrane synthesis caused by external fatty acid supplementation. In addition, membrane physiology was evaluated using a combination of microbiology techniques for confirming membrane fluidity, charge, and cell wall structure for determining the leading factor in daptomycin resistance. 

(1) Santos, I. C.; Smuts, J.; Choi, W.-S.; Kim, Y.; Kim, S. B.; Schug, K. A. Analysis of bacterial FAMEs using gas chromatography – vacuum ultraviolet spectroscopy for the identification and discrimination of bacteria. Talanta 2018, 182, 536-543. DOI: 10.1016/j.talanta.2018.01.074.

(2) Freeman, C.; Hynds, H. M.; Carpenter, J. M.; Appala, K.; Bimpeh, K.; Barbarek, S.; Gatto, C.; Wilkinson, B. J.; Hines, K. M. Revealing Fatty Acid Heterogeneity in Staphylococcal Lipids with Isotope Labeling and RPLC–IM–MS. Journal of the American Society for Mass Spectrometry 2021. DOI: 10.1021/jasms.1c00092.

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