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

Abstract

Mycobacterium ulcerans, a slow-growing nontuberculous mycobacterium, causes Buruli ulcer, a neglected tropical disease. Distinguishing M. ulcerans from related species, including Mycobacterium marinum, 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 M. ulcerans (n = 35) and M. marinum (n = 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 (M. ulcerans and M. marinum) that are difficult to distinguish using conventional protein-targeting methods.IMPORTANCEBuruli ulcer, caused by Mycobacterium ulcerans, is a neglected tropical disease. However, distinguishing M. ulcerans from related species, including Mycobacterium marinum, 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.

Keywords: Buruli ulcer; MALDI-TOF MS; Mycobacterium marinum; Mycobacterium ulcerans; lipid profiling; nontuberculous mycobacteria.

Fig 1

Average mass spectra of mycobacteria within the Mycobacterium ulcerans/marinum complex. (A) Comparison between M. marinum (green) and M. ulcerans, including M. ulcerans subsp. shinshuense (red). (B) Comparison between M. ulcerans (green) and M. ulcerans subsp. shinshuense (red). The lipid spectra were acquired in negative ion mode using a MALDI Biotyper Sirius system. The ellipses represent the standard deviation of the class average of the peak areas/intensities or the 95% confidence interval, which is the standard deviation weighted by the reciprocal number of data points. Mu, M. ulcerans; Mu_sh, M. ulcerans subsp. shinshuense; and Mm, M. marinum.

Fig 2

Strain distribution map. (A) Comparison between Mycobacterium marinum (green) and M. ulcerans, including M. ulcerans subsp. shinshuense (red). (B) Comparison between M. ulcerans (green) and M. ulcerans subsp. shinshuense (red). Mu, M. ulcerans; Mu_sh, M. ulcerans subsp. shinshuense; Mm, M. marinum. The x-axis shows the peak area or intensity values with respect to the most relevant peak for distinguishing the two groups (green and red). The y-axis shows the peak area or intensity values with respect to the second or third most relevant peak for distinguishing the two groups.