Use of stable isotope combined with intact cell lipidomic by routine MALDI mass spectrometry analysis for rapid drug susceptibility assay in mycobacteria.

Rationale: Rapid, accurate, and easy-to-perform diagnostic assays are required to address the current need for the diagnosis of resistant pathogens. That is particularly the case for mycobacteria, such as the human pathogen Mycobacterium tuberculosis, which requires up to 2 weeks for the determination of the drug susceptibility profile using the conventional broth microdilution method. To address this challenge, we investigated the incorporation of deuterium, the stable isotope of hydrogen, into lipids as a read out of the drug susceptibility profile.

Methods: Deuterium is incorporated into newly synthesized proteins or lipids in place of hydrogen as bacterial cells grow, increasing the mass of the macromolecules, which can then be observed via matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). As proof-of-concept, we used the non-pathogenic Mycobacterium smegmatis mc155 strain, which is susceptible to the aminoglycoside antibiotic kanamycin, and M. smegmatis mc155 containing the empty vector pVV16, which is kanamycin-resistant. Bacteria were incubated in a culture medium containing 50% of deuterium oxide (DO) and either 1 or 2 times the minimal inhibitory concentration (MIC) of kanamycin. Lipids were then analyzed using the MBT lipid Xtract matrix combined with routine MALDI mass spectrometry in the positive ion mode to evaluate the changes in the lipid profile.

Results: Using this approach, we were able to distinguish susceptible from resistant bacteria in less than 5 h, a process that would take 72 h using the conventional broth microdilution method.

Conclusions: We therefore propose a solution for the rapid determination of drug susceptibility profiles using a phenotypic assay combining DO stable isotope labelling and lipid analysis by routine MALDI mass spectrometry.