A rapid and simple MALDI-TOF MS lipid profiling method for differentiating <i>Mycobacterium ulcerans</i> from <i>Mycobacterium marinum</i>

<i>Mycobacterium ulcerans</i>, a slow-growing nontuberculous mycobacterium, causes Buruli ulcer, a neglected tropical disease. Distinguishing <i>M. ulcerans</i> from related species, including <i>Mycobacterium marinum</i>, poses challenges with respect to making accurate identifications. In this study, we developed a rapid and simple identification method based on mycobacterial lipid profiles and used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the lipid profiles of <i>M. ulcerans</i> (<i>n</i> = 35) and <i>M. marinum</i> (<i>n</i> = 19) isolates. Bacterial colonies pre-cultured on 2% Ogawa egg slants for 2 months were collected, and total lipids were extracted using an MBT Lipid Xtract kit. Spectra were obtained in negative ion mode using a MALDI Biotyper Sirius system, with ClinProTools v3.0 being used to analyze the spectra based on the application of three algorithms (genetic algorithm [GA], supervised neural network [SNN], and quick classifier [QC)]). Cross-validation was performed using a 20% leave-out set randomly selected from the samples. Models generated using GA, SNN, and QC showed cross-validation values of 100%, 100%, and 97.9%, respectively, and all algorithms achieved 100% recognition capability values. Our findings indicate that MALDI-TOF analysis of lipid profiles can accurately differentiate two mycobacterial species (<i>M. ulcerans</i> and <i>M. marinum</i>) that are difficult to distinguish using conventional protein-targeting methods.IMPORTANCEBuruli ulcer, caused by <i>Mycobacterium ulcerans</i>, is a neglected tropical disease. However, distinguishing <i>M. ulcerans</i> from related species, including <i>Mycobacterium marinum</i>, presents certain challenges. In this study, we demonstrate the utility of a rapid yet simple method for differentiating isolates of these mycobacteria based on their lipid profiles using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This new approach can accurately identify species that are otherwise difficult to distinguish using conventional techniques. This represents a significant diagnostic advance for clinical laboratories, in that it enables a more rapid and precise identification, thereby leading to earlier treatment initiation and more appropriate treatment regimens for infections caused by these bacteria.