A rapid single-phase extraction for polar staphylococcal lipids

The lipid membrane is gaining appreciation as a critical factor in the emergence of antibiotic resistance, both for antibiotics that target lipid synthesis or the membrane directly and for cell-wall-targeting antibiotics. The methods used to study the emergence of antibiotic resistance in vitro can generate a large number of samples that may be low in volume and in cell density. As in eukaryotic/mammalian lipidomics, two-phase liquid-liquid extractions are the most commonly used approach to recover lipids from bacteria. The need to separate the lipid layer is cumbersome for high-throughput applications and can be a source of poor reproducibility or contaminant introduction. While several single-phase extractions have been proposed for serum, tissue, and eukaryotic cells, there have been far fewer efforts to adapt or develop such methods for bacteria lipidomics. Here, we describe a simple, single-phase lipid extraction method based on methanol, acetonitrile, and water-the MAW method. The merits of the MAW method are evaluated against the Bligh & Dyer (B&D) method for the recovery of the major membrane lipids (phosphatidylglycerols, diglycosyldiacylglycerols, and lysyl-phosphatidylglycerols) in the Gram-positive pathogen Staphylococcus aureus. We demonstrate that the MAW method achieves recoveries that are comparable to that of the B&D extraction (≥ 85% for PG 15:0/d₇-18:1). The benefits of the MAW method enable the detection of lipids from lower amounts of bacteria than the B&D method (0.57 vs 0.74 McFarlands for PG 32:0, respectively) and is easily scaled down to microplate volumes to facilitate high-throughput studies of bacterial lipids.