Objectives:
Rapid detection of bacterial pathogens to species and subspecies level is crucial for appropriate treatment, infection control and public health management. Currently, one of the challenges in clinical microbiology is the discrimination of mycobacterial sub-species within the M. tuberculosis complex (MTBC). Our objective was to evaluate the ability of a biosafe mycobacterial-lipid based approach to identify MTBC cultures and subspecies.

Methods:
A blinded study was performed using 90 mycobacterial clinical isolates strains comprising MTBC strains sub-cultured in Middlebrook 7H11 media supplemented with 10% OADC growth supplement and incubated for up to six weeks at 37°C and using the following 7 reference strains (M. tuberculosis H37Rv, M canettii, M. africanum, M. pinnipedii, M. caprae, M. bovis, M. bovis BCG) grown under the same conditions, in order to set the reference lipid database and test it against the 90 MTBC clinical isolates. Cultured mycobacteria were heat-inactivated and loaded onto the MALDI target followed by addition of the matrix. Acquisition of the data was done using the positive ion mode.

Results:
Based on the identification of clear and defined lipid signatures from the 7 reference strains, the method we have developed is fast (<10 mins) and produced interpretable profiles for all but four isolates, caused by poor ionization giving an n = 86 with interpretable spectra. The sensitivity and specificity of the MALDI-ToF, were 94.4 (95% CI 86.4-98.5) and 94.4 (95% CI 72.7-99.9) respectively. .

Conclusions:
Mycobacterial lipid profiling provides for a means of rapid, safe and accurate discrimination of species within the MTBC.