Rapid identification of clinical mycobacterial strains by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) A.Ingebretsen1-2, A.Myhre2, I.Szpinda2, T. Tønjum2-3, Department of Infection Prevention1, Department of Microbiology2, Oslo University Hospital, Center for Molecular Biology and Neuroscience3, University of Oslo, Oslo University Hospital, Oslo, Norway Introduction: Results: Mycobacterial identification is based on several methods: conventional biochemical tests that We included 39 mycobacterial species identified by sequencing the 16 S rRNA although many of these require several weeks for accurate identification, and molecular tools that are now routinely were not included in the MALDI Biotyper 3.0 software. In the end we identified 61 different strains in used. These techniques are expensive and time-consuming. Over the last few years 17 mycobacterial species by MALDI TOF MS. The identification scores ranged from 1.4 to 2.6. matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI TOF MS) Due to poor results identifying M. tuberculosis complex against the MALDI Biotyper 3.0 database we has been employed as a primary identification method for the identification of microorganisms decided to run additional 12 clinical strains for a total of 21 clinical strains of M. tuberculosis against all in many clinical diagnostic laboratories. Here we seek to optimize the use of MALDI-TOF MS in MSPs including our supplementary database. The identification scores for all M.tuberculosis rapid identification of clinical mycobacterial strains including Mycobacterium tuberculosis in a ranged from 2.1 to 2.6. clinical laboratory setting. Compared to sequencing results all strains were identified at species level although we could not distinguish M. chelonae, M. abscessus ssp. abscessus and M. abscessus ssp. bolletii from each other. We did distinguish M.bovis from M.tuberculosis but we regarded this finding as random. Fig. 2 MALDI-TOF MS spectrum of Mycobacterium tuberculosis Inactivation Growth in MGIT Sample preparation and protein extraction Fig. 3 MALDI Biotyper 3.0 analysis Mycobacterium tuberculosis Data evaluation identification/classification Acquisition of MALDI-TOF MS spectra Fig 1: General workflow for identifying mycobacteria by MALDI-TOF MS. Materials and methods: Species No. of strains with the following (No. of strains tested) highest score Mycobacteria isolates <1.5 1.5-1.7 1.7-2.0 >2.0 Clinical strains from 39 mycobacterial species and species complexes were included in the Mycobacterium avium (9) 3 6 study. The mycobacteria were grown on Middlebrook 7H10 medium and/or liquid Mycobacterium asiaticum (2) 1 1 mycobacterium growth indicator tube [MGIT] medium. The strains were identified by Mycobacterium celatum (2) 1 1 sequencing of 16S rRNA according to standard procedures. Mycobacterium chelonae/abscessus (6)* 4 2 Mycobacterium fortuitum (4) 4 Mycobacteria inactivation Mycobacterium gordonae (1) 1 Clinical isolates of Mycobacterium tuberculosis were used in five different inactivation Mycobacterium kansasii (4) 2 2 procedures including boiling in water, boiling in acetonitril and ethanol inactivation. The Mycobacterium kumamotonense (1) 1 isolates were incubated 2 x 42 days after inactivation and no growth were registered. Ethanol Mycobacterium malmoense (12) 5 7 inactivation became the procedure of choice both due to simplicity and quality of spectra. Mycobacterium nonchromogenicum (1) 1 Mycobacterium peregrinum (2) 2 Optimized protocol for identification of Mycobacterium sp. by MALDI TOF MS. Mycobacterium simiae (4) 4 Cell material was harvested by centrifugation using 1ml broth from the MGIT tube. The pellet Mycobacterium tuberculosis complex (9)** 6 3 was washed twice with water before ethanol (70%) was added and the cells were left for Mycobacterium xenopi (4) 3 1 inactivation in 10 minutes. After centrifugation the pellet was dried. We then added 50 μl Mycobacterium tuberculosis complex (21)*** 21 acetonitrile (100%), mixed thoroughly and a small amount of 0.5mm silica beads was added. * 2 M.chelonae, 3 M.abscessus ssp.abscessus and 1 M.abscessus ssp.bolletii Using the MagNA Lyser (Roche) we then beated the cells twice adding 50 μl formic acid (70%) **8 M.tuberculosis and 1 M.bovis between the beatings. After centrifugation 1μl of the supernatant was spotted on the MALDI ***20 M.tuberculosis and 1 M.bovis target plate and air dried. Matrix (α-cyano-4-hyroxycinnamic acid) was added on the dried spot and left to dry at room temperature. Spectra were acquired using a MALDI TOF Microflex LT mass spectrometer (Bruker Daltonics, Bremen, Germany) at default settings for identification Conclusions: of microorganisms. The spectrum generated was analyzed by MALDI Biotyper 3.0 software MALDI-TOF MS represents a rapid, feasible and inexpensive system for identification (Bruker Daltonics) and compared with the results from 16S rRNA gene sequence analysis. of mycobacterium species from liquid culture media in clinical laboratories. Constructing a supplementary database of Mycobacterium tuberculosis. The present procedure using silica beads produces good spectra for identification of We constructed a supplementary database using 11 well characterized clinical Mycobacterium mycobacterial species. tuberculosis strains. The database was constructed as recommended by the manufacturer of Constructing a supplementary database of mycobacterial species in the the MALDI Biotyper software (Bruker Daltonics). MALDI Biotyper database enhance identification of mycobacterial species. .