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Abstract
Bacterial membrane lipids play a vital role in antibiotic resistance by affecting the integrity and function of bacterial cell membranes. Mass spectrometry-based lipidomics is a powerful tool for assessing the lipid profiles of various bacterial strains. However, profiling multiple bacterial strains requires high-throughput sample generation. Traditional chloroform/methanol-based lipid extraction once considered the gold standard, hampers high-throughput lipidomics because of the challenge of separating the lipid-containing layer from the aqueous phase. Although monophasic extraction methods, such as butanol/methanol extraction (BUME), exist, they are primarily designed for mammalian cells and lack proven effectiveness in bacterial lipidomics.First, we describe the development and optimization of a single-phase extraction method that uses methanol, acetonitrile, and water(MAW)- the MAW method for S. aureus. This newly developed method was compared to the gold-standard Bligh and Dyer (B&D) extraction in terms of simplicity, lipid recovery, sample quantity, and high-throughput capability. Secondly, we applied the scaled-down MAW method to differentiate between various gram-positive and gram-negative bacteria that are genetically related. Multivariate statistics revealed distinct lipid profiles and quorum sensors, emphasizing the robustness of the MAW extraction technique. Lastly, we explored how the microbial conditions of bacteria contribute to phospholipid synthesis in the Chagas disease vector, Rhodinius prolixus. Our findings suggest that this avenue holds promise for combating the spread of Chagas disease.