Evaluation of MALDI Biotyper Mycobacteria Library v3.0 for Identification of Nontuberculous Mycobacteria

Abstract

e Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has demonstrated its ability to promptly identify nontuberculous mycobacteria using the Mycobacteria Library v2.0. However, some species are particularly difficult to identify reliably using this database, providing a low log(score). In this study, the identification power of an updated Mycobacteria Library (v3.0) has been evaluated. Overall, 109 NTM isolates were analyzed with both databases. The v3.0 database allowed a high-level confidence in the identification [log(score) value, >1.8] of 91.7% of the isolates versus 83.5% with the v2.0 version (P < 0.01). Rapid identification of nontuberculous mycobacteria (NTM) with matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) has outperformed molecular techniques, such as GenoType (Hain Lifescience GmbH, Nehren, Germany), and provides accurate identification that correlates well with 16S rRNA gene sequencing when applied to the most common species of NTM (1-3). The Mycobacteria Library database (Bruker Daltonik GmbH, Bremen, Germany) available so far (v2.0) provided low scores, particularly for NTM belonging to the slow-growing groups (3-5). In the present study, we assessed the power of a new database to identify NTM (i.e., Mycobacteria Library v3.0) using 109 isolates from 26 NTM species (Table 1) and compared the identification scores of version v2.0 and version v3.0.Ninety-nine nonselected NTM isolates from clinical samples and 10 reference strains (Table 1) were collected in the clinical microbiology laboratory from the Hospital Gregorio Marañón (Madrid, Spain) between January 2011 and May 2015. These isolates were routinely identified by 16S rRNA hsp65 sequencing and, in parallel, by MALDI-TOF MS using a Microflex LT benchtop mass spectrometer (Bruker Daltonik) and the Mycobacteria Library v2.0, containing 313 Mycobacterium isolates from 131 species (Bruker Daltonik) (4). Sample preparation was described elsewhere (6). Briefly, colonies of NTM isolates grown on Lowenstein-Jensen medium were harvested into a 1.5-ml Eppendorf tube with 300 l of deionized water and inactivated for 30 min at 95°C under biosafety level 3 conditions. Then, they were centrifuged at maximal speed and subsequently resuspended in 300 l of water and 900 l of absolute ethanol and centrifuged again at 13,000 rpm. The supernatant was discarded, and the pellet was taken to biosafety level 2 conditions in order to disrupt the mycobacteria cell aggregates with silica bead vortexing and extract the bacterial proteins using formic acid and acetonitrile. In the end, 1 l of supernatant was placed onto a steel plate for MALDI-TOF MS analysis. Samples were analyzed in duplicates; the species identification, using the Mycobacteria Library v2.0, and the higher log(score) value result were recorded. For comparison reasons, all of the protein spectra from the 109 NTM isolates were reanalyzed using the new Mycobacteria Library v3.0, containing 853 references from 149 Mycob...

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria

“…In recent years, the technique has also started to be implemented in mycobacterial laboratories due to substantial improvements in sample preparation and the quality of spectral databases. We confirm in this study that MALDI-TOF MS allows a very reliable distinction between M. tuberculosis and NTM species if score thresholds are lowered from Ն2.0 for routine bacterial identifications to Ն1.8 for mycobacteria (30)(31)(32). Using this cutoff value, 90.4% of the species identifications corresponded to gold-standard methods despite the presence of two MBT-ASTRA standard peaks in the mass spectra.…”

“…As it allows pathogen-specific culture and protein extraction protocols (34,35), we first optimized our in-house mycobacterial cell disruption and protein extraction protocol for MALDI-TOF MS analysis. Upon comparison of various described protocols using M. tuberculosis H37Rv cells (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34), we found that in our hands, bead beating in the presence of 1.2 ml of 70% ethanol gave the best output in terms of peak intensity and identification scores (Table S1).…”

“…Unlike most bacteria, mycobacteria require prior inactivation and disruption of the mycolic acid-rich cell wall, both for biosafety reasons and to liberate the cellular protein content. Numerous suitable procedures have been proposed, combining heat-killing, ethanol, sonication, and/or silica-based bead beating preceding the regular formic acid/acetonitrile extraction (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). The reported success rates vary between 55% and 98% correct species-level identifications, but the large variety in strain collections, culture conditions, extraction protocols, hardware, and databases hinder data comparison between publications.…”

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media

“…Identification of mycobacteria has not reached an optimal level of routine testing yet and published studies showed discrepant performances, depending on spectrometer, extraction method, algorithm, library, media, and biomass used for the assays (15)(16)(17)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). In this study, we compared two distinct databases of Vitek-MS, the Saramis v4.12 open database and the new IVD v3.0 closed database.…”

“…It was initially evaluated with mycobacterial intact cells (9)(10)(11)(12), but several investigators worked on improving inactivation and protein extraction (13)(14)(15)(16)(17)(18)(19)(20). Most published studies assessed the performance of MALDI-TOF BioTyper (Bruker Daltonics, Bremen, Germany) (15,17,(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Few of them evaluated Vitek MS (bioMérieux, Marcy l'Etoile, France) (16,17,20,24,25,32).…”