Abstract
ObjectivesBacterial diagnosis of mycobacteria is often challenging because of the variability of the sensitivity and specificity of the assay used, and it can be expensive to perform accurately. Although matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has become the workhorse of clinical laboratories, the current MALDI methodology (which is based on cytosolic protein profiling) for mycobacteria is still challenging due to the number of steps involved (up to seven) and potential biosafety concerns. Knowing that mycobacteria produce surface-exposed species-specific lipids, we here hypothesized that the detection of those molecules could offer a rapid, reproducible and robust method for mycobacterial identification. MethodsWe evaluated the performance of an alternative methodology based on characterized species-specific lipid profiling of intact bacteria, without any sample preparation, by MALDI MS; it uses MALDI-time-of-flight (ToF) MS combined with a specific matrix (super-2,5-dihydroxybenzoic acid solubilized in an apolar solvent system) to analyse lipids of intact heat-inactivated mycobacteria. Cultured mycobacteria are heat-inactivated and loaded directly onto the MALDI target followed by addition of the matrix. Acquisition of the data is done in both positive and negative ion modes. Blinded studies were performed using 273 mycobacterial strains comprising both the Mycobacterium tuberculosis (Mtb) complex and non-tuberculous mycobacteria (NTMs) subcultured in Middlebrook 7H9 media supplemented with 10% OADC (oleic acid/dextrose/catalase) growth supplement and incubated for up to 2 weeks at 37°C. ResultsThe method we have developed is fast (<10 mins) and highly sensitive (<1000 bacteria required); 96.7% of the Mtb complex strains (204/211) were correctly assigned as MTB complex and 91.7% (22/24) NTM species were correctly assigned based only on intact bacteria species-specific lipid profiling by MALDI-ToF MS. ConclusionsIntact bacterial lipid profiling provides a biosafe and unique route for rapid and accurate mycobacterial identification.
Highlights
The study examined the lipidomic profile of the TB isolates by MALDI-ToF mass spectrometry
A good mycobacterial mass spectral signal in negative mode was generated for 235 out of 273 isolates; 38 isolates (14%) could not be assigned to either the Mycobacterium tuberculosis (Mtb) or the nontuberculous mycobacteria (NTM) group, and nine isolates (3%) were misidentified
Identification of Mtb was achieved by the presence of SL-I, which is a negatively charged lipid uniquely found in Mtb [15,16]
Summary
Its broad application for mycobacteria has been challenging due to the fact that sample preparation and handling should happen in biosafety level 3 facilities, and that the mycobacterial wall needs special treatment to allow the molecules of interest to be released for MALDI-ToF proteomic analysis. This is time-consuming and requires a careful extraction step [5]. The mycobacterial wall is composed of 60% lipids such as Cmycoside glycopeptolipid, phenol glycosides, trehalose-containing lipopolysaccharides, sulpholipids, lipoarabinomannan, and mycolic acids [7]. Some lipidsdsuch as sulphoglycolipids (SL-I) and the polyacyltrehaloses (PATs)dare specific for the M. tuberculosis complex; other lipids, such as Cmycoside glycopeptidolipids (GPLs), are found only in nontuberculous mycobacteria (NTM) (e.g. M. avium) [9,10]
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