Matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) proteomic profiling of mycobacteria.

Abstract

Mycobacterium tuberculosis still remains a major cause of tuberculosis(TB) and death. In addition, nontuberculous mycobacteria (NTM) play a significant role in the aetiology of tuberculosis-like syndromes. A diagnosis of M. tuberculosis as the causative agent of pulmonary TB using only clinical symptoms may be inadequate and inaccurate in some cases since M.bovis, M.avium, M.kansasii, and M.intracellulare may all produce a pulmonary disease in humans that may be indistinguishable from that caused by M.tuberculosis. For diagnostic purposes, current mycobacterial identification strategies to the species level primarily rely on phenotypic analysis, complex biochemical tests, cultivation on specific growth media and genotype analysis. However, the main disadvantages that are associated with these techniques include extensive processing times (6-12 weeks to confirm a positive identification) and misidentification. Both factors severely hinder the control and management of diseases associated with mycobacteria, particularly tuberculosis. Apart from an increased patient mortality, interpersonal transmission of mycobacterial infections may increase. This scenario will also result in the prescription of inappropriate anti-tubercular treatment regimes. The present study assesses the feasibility of using MALDI-TOF MS proteomic profiling as a rapid and precise technology to identify mycobacteria in Kwazulu-Natal, South Africa which is currently a global epicentre of mycobacterial-associated diseases. In this study a modified EFA protein extraction protocol that incorporates an initial cell envelope delipidation step was formulated and shown to produce mass spectra that were unique and highly reproducible. The spectra were used to create an independent main spectral profile reference library (CMEFAMSP) representing clinically relevant American Type Culture Collection (ATCC) mycobacterial strains. Interestingly, this proof of concept study clearly demonstrates that MALDI-TOF MS-based biotyping of mycobacteria using the CMEFA-MSP reference library correctly identified 11 blind-coded ATCC strains sourced from an autonomous facility to the genus and species level with 100% accuracy.In addition the CMEFA-MSP reference library was employed to differentiate 39 blind-coded clinical mycobacterial isolates. Importantly all CMEFA-derived samples of the 39 clinical mycobacterial isolates displayed log score values of ≥ 2.3 and were correctly identified to the species level. This strongly suggests that MALDITOF MS when used in conjunction with the CMEFA sample preparation protocol has potential as a simple and cost-effective alternative for the unambiguous identification of clinically important mycobacteria. Moreover, individual members of the MTBC and NTM groups used in this study were distinguished from each other.