Detection of Species-Specific Lipids by Routine MALDI TOF Mass Spectrometry to Unlock the Challenges of Microbial Identification and Antimicrobial Susceptibility Testing

Abstract

MALDI-TOF mass spectrometry has revolutionized clinical microbiology diagnostics by delivering accurate, fast, and reliable identification of microorganisms. It is conventionally based on the detection of intracellular molecules, mainly ribosomal proteins, for identification at the species-level and/or genus-level. Nevertheless, for some microorganisms (e.g., for mycobacteria) extensive protocols are necessary in order to extract intracellular proteins, and in some cases a protein-based approach cannot provide sufficient evidence to accurately identify the microorganisms within the same genus (e.g., Shigella sp. vs E. coli and the species of the M. tuberculosis complex). Consequently lipids, along with proteins are also molecules of interest. Lipids are ubiquitous, but their structural diversity delivers complementary information to the conventional protein-based clinical microbiology matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) based approaches currently used. Lipid modifications, such as the ones found on lipid A related to polymyxin resistance in Gram-negative pathogens (e.g., phosphoethanolamine and aminoarabinose), not only play a role in the detection of microorganisms by routine MALDI-TOF mass spectrometry but can also be used as a read-out of drug susceptibility. In this review, we will demonstrate that in combination with proteins, lipids are a game-changer in both the rapid detection of pathogens and the determination of their drug susceptibility using routine MALDI-TOF mass spectrometry systems.

Keywords: diagnostics; drug resistance; gram-negative; gram-positive; lipids; matrix-assisted laser desorption ionization; mycobacteria.

Figure 1

Schematic diagram of the proposed workflow for combining lipid and protein MALDI-TOF MS analysis. For protein analysis, a colony is smeared on the target plate and overlaid with matrix. For analysis of lipid A, heat-inactivated microorganisms are submitted to mild-acid hydrolysis and the membranes resulting from this treatment are deposited on an adjacent position on the target plate, followed by matrix addition. Alternatively, for mycobacteria and filamentous fungi, heat-inactivated colonies are directly smeared on the target plate and overlaid with the appropriate matrix. The combined approach can yield species- and subspecies-level microbial identification and detect antibiotic resistance in one analysis.