Mapping of Lipidome Profile in Drug-Resistant Clinical Isolates of Mycobacterium tuberculosis Through Quali-Quantitative Liquid Chromatography-Mass Spectrometry Identifies Signature Lipids

The complex lipid composition of Mycobacterium tuberculosis (MTB) plays a pivotal role in pathogenesis, immune evasion, and antimicrobial resistance. The continued surge in drug-resistant strains underscores the need for a deeper understanding of the molecular architecture underlying MTB pathogenesis and drug resistance. In this study, a comparative lipidomics approach was applied to explore the resistance-associated lipid alterations in drug-sensitive (DS), drug-resistant (DR), multidrug-resistant (MDR), and pre-extensively drug-resistant (PXDR) MTB clinical isolates. Lipids derived from whole bacilli (TL) and cell wall (CWL) extracts were separately analyzed using untargeted and targeted lipidomics approaches. Untargeted analysis revealed the abundance of fatty acyls, glycerolipids, glycerophospholipids, prenol lipids, polyketides, and saccharolipids, with distinct phenotypes and compartment-specific distributions. Notably, CWL extracts showed clearer resistance-associated separations relative to TL extracts. Targeted profiling further demonstrated enrichment of glycerolipids and PC and LPC lipids among drug-resistant isolates. Biomarker analysis identified discriminative lipid species in both extracts, albeit with greater discriminatory power in CWL. Overall, these findings elucidate coordinated and compartment-associated lipid alterations at the species level in different MTB clinical isolates. This will help to provide a valuable source for screening diagnostic biosignatures and intersecting biosynthetic pathways of mycobacterial lipids in the evolution of drug resistance in MTB for therapeutic interventions.