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Abstract
Staphylococcus aureus is a common cause of hospital and community-acquired infections. Over time, antibiotics have been discovered to combat S. aureus infections. However, many strains have developed resistance to most antibiotic classes discovered since the 1940s. Resistance to daptomycin has proved to be one of the most troubling cases for S. aureus treatment. Daptomycin resistance 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. Staphylococcus aureus is unique amongst many strains by varying its membrane fluidity using branched fatty acyl chain combinations, providing an avenue for isomer identification. By investigating fatty acid and lipid biosynthesis in resistant and stressed S. aureus through lipid composition analysis, we aimed to connect gene expression, membrane physiology, and lipid production to cell viability. We achieved this through a Reversed-Phase Liquid Chromatography Ion Mobility Mass Spectrometry (RPLC-IM-MS) method for classifying lipid composition of intact branched isomers. Through reversed-phase separation, an in-depth overview of abundant isomers determining membrane fluidity was possible. We further applied this technique for evaluating changes in membrane synthesis caused by external fatty acid supplementation, plasmid insertion, and transposon mutations. In addition, membrane physiology was evaluated using a combination of microbiology techniques for confirming gene expression, growth patterns, membrane fluidity, charge, and cell wall structure for determining the leading factors in virulence.