HILIC-IM-MS for Simultaneous Lipid and Metabolite Profiling of Bacteria.

Although MALDI-ToF platforms for microbial identifications have found great success in clinical microbiology, the sole use of protein fingerprints for the discrimination of closely related species, strain-level identifications, and detection of antimicrobial resistance remains a challenge for the technology. Several alternative mass spectrometry-based methods have been proposed to address the shortcomings of the protein-centric approach, including MALDI-ToF methods for fatty acid/lipid profiling and LC-MS profiling of metabolites. However, the molecular diversity of microbial pathogens suggests that no single "ome" will be sufficient for the accurate and sensitive identification of strain- and susceptibility-level profiling of bacteria. Here, we describe the development of an alternative approach to microorganism profiling that relies upon both metabolites and lipids rather than a single class of biomolecule. Single-phase extractions based on butanol, acetonitrile, and water (the BAW method) were evaluated for the recovery of lipids and metabolites from Gram-positive and -negative microorganisms. We found that BAW extraction solutions containing 45% butanol provided optimal recovery of both molecular classes in a single extraction. The single-phase extraction method was coupled to hydrophilic interaction liquid chromatography (HILIC) and ion mobility-mass spectrometry (IM-MS) to resolve similar-mass metabolites and lipids in three dimensions and provide multiple points of evidence for feature annotation in the absence of tandem mass spectrometry. We demonstrate that the combined use of metabolites and lipids can be used to differentiate microorganisms to the species- and strain-level for four of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa) using data from a single ionization mode. These results present promising, early stage evidence for the use of multiomic signatures for the identification of microorganisms by liquid chromatography, ion mobility, and mass spectrometry that, upon further development, may improve upon the level of identification provided by current methods.

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.

Revealing Fatty Acid Heterogeneity in Staphylococcal Lipids with Isotope Labeling and RPLC-IM-MS.

Up to 80% of the fatty acids in Staphylococcus aureus membrane lipids are branched, rather than straight-chain, fatty acids. The branched fatty acids (BCFAs) may have either an even or odd number of carbons, and the branch position may be at the penultimate carbon (iso) or the antepenultimate (anteiso) carbon of the tail. This results in two sets of isomeric fatty acid species with the same number of carbons that cannot be resolved by mass spectrometry. The isomer/isobar challenge is further complicated when the mixture of BCFAs and straight-chain fatty acids (SCFAs) are esterified into diacylated lipids such as the phosphatidylglycerol (PG) species of the S. aureus membrane. No conventional chromatographic method has been able to resolve diacylated lipids containing mixtures of SCFAs, anteiso-odd, iso-odd, and iso-even BCFAs. A major hurdle to method development in this area is the lack of relevant analytical standards for lipids containing BCFA isomers. The diversity of the S. aureus lipidome and its naturally high levels of BCFAs present an opportunity to explore the potential of resolving diacylated lipids containing BCFAs and SFCAs. Using our knowledge of lipid and fatty acid biosynthesis in S. aureus, we have used a stable-isotope-labeling strategy to develop and validate a 30 min C18 reversed-phase liquid chromatography method combined with traveling-wave ion mobility-mass spectrometry to provide resolution of diacylated lipids based on the number of BCFAs that they contain.

Recent applications of mass spectrometry in bacterial lipidomics.

The popularity of mass spectrometry-based lipidomics has soared in the past decade. While the majority of the lipidomics work is being performed in mammalian and other eukaryotic systems, there is also a growing rise in the exploration of bacterial lipidomics. The lipids found in bacteria can be substantially different from those in eukaryotic systems, but they are equally important for maintaining the structure of the bacteria and providing protection from the surrounding environment. In this article, recent applications of lipidomics in combination with molecular biology and applications in microbial strain identification and antibiotic susceptibility are highlighted. The authors' perspectives on current challenges facing the field and future directions are also provided.