Direct matrix-assisted laser desorption ionisation time-of-flight mass spectrometry identification of mycobacteria from colonies

• Published in: European journal of clinical microbiology & infectious diseases Vol. 35; no. 12; pp. 1983 - 1987
• Main Authors: Zingue, D., Flaudrops, C., Drancourt, M.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.12.2016; Springer Nature B.V; Springer Verlag
• Subjects: Acids; Bacteria; Bacteriological Techniques - methods; Biomedical and Life Sciences; Biomedicine; Human health and pathology; Humans; Identification; Infectious diseases; Internal Medicine; Laboratories; Lasers; Life Sciences; Mass spectrometry; Medical Microbiology; Mycobacterium - chemistry; Mycobacterium - classification; Mycobacterium - growth & development; Mycobacterium avium; Mycobacterium tuberculosis; Original Article; Prospective Studies; Proteins; Protocol; Scientific imaging; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Tuberculosis; France; United States > US; Mycobacterium Fortuitum; Identification Score; Mycobacterium Marinum; Direct Deposit; Middlebrook 7H10
• ISSN: 0934-9723; 1435-4373
• DOI: 10.1007/s10096-016-2750-5

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) identification of mycobacteria requires a standard acetonitrile/formic acid pre-MALDI-TOF-MS. We prospectively compared this standard protocol with direct deposit with matrix for the identification of mycobacteria cultured on solid media. We first verified that Mycobacterium tuberculosis was killed after it was mixed with matrix. Then, 111 Mycobacterium isolates previously identified by partial rpo B gene sequencing were tested in parallel by the two protocols. An identification score >1.7 was obtained in 86/111 (77.5 %) isolates after protein extraction versus 97/111 (87.4 %) isolates after direct deposit ( p  = 0.039, Chi-squared test). In a third step, we determined that direct deposit achieved identification for as few as 2.10 4 M. tuberculosis organisms. In a fourth step, we evaluated direct deposit of one colony for 116 solid medium-cultured clinical isolates finally identified as representative of 12 species (63.8 %  M. tuberculosis ). For 114/116 (98.3 %) isolates with an identification score >1.2, the MALDI-TOF-MS identification was in complete agreement with the reference rpo B gene sequencing identification. One isolate with a MALDI-TOF-MS identification score of 1.22 for M. fortuitum was identified as M. avium by partial rpo B gene sequencing. One other isolate with a MALDI-TOF-MS identification score of 1.22 for M. tuberculosis was identified as M. tuberculosis by genotyping. All the original MALDI-TOF-MS spectra reported here have been deposited in a public database. Direct deposit of one colony on a MALDI-TOF-MS plate allows for an accurate identification of mycobacteria for an identification score >1.3.