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...

In recent years, matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has proved a rapid and reliable method for the identification of bacteria and yeasts that have already been isolated. The objective of this study was to evaluate this technology as a routine method for the identification of microorganisms directly from blood culture bottles (BCBs), before isolation, in a large collection of samples. For this purpose, 1000 positive BCBs containing 1085 microorganisms have been analysed by conventional phenotypic methods and by MALDI-TOF MS. Discrepancies have been resolved using molecular methods: the amplification and sequencing of the 16S rRNA gene or the Superoxide Dismutase gene (sodA) for streptococcal isolates. MALDI-TOF predicted a species- or genus-level identification of 81.4% of the analysed microorganisms. The analysis by episode yielded a complete identification of 814 out of 1000 analysed episodes (81.4%). MALDI-TOF identification is available for clinicians within hours of a working shift, as oppose to 18 h later when conventional identification methods are performed. Moreover, although further improvement of sample preparation for polymicrobial BCBs is required, the identification of more than one pathogen in the same BCB provides a valuable indication of unexpected pathogens when their presence may remain undetected in Gram staining. Implementation of MALDI-TOF identification directly from the BCB provides a rapid and reliable identification of the causal pathogen within hours.

Despite the benefits of MALDI-TOF MS technology (Matrix-Assisted Laser Desorption-Ionization Time-Of-Flight Mass Spectrometry) reported worldwide and the continuous improving of the available databases, discrimination between Streptococcus pneumoniae and closely related species within the Streptococcus mitis group (SMG) using this methodology has been suboptimal. However, the accurate identification at the species level of this group of microorganisms is important for the appropriate management of infected patients. In this study, 216 SMG isolates -101 S. pneumoniae and 115 corresponding to 7 non-pneumococcal species within this group- were analyzed. All the isolates had been previously identified by conventional methods (optochin and bile solubility tests) and non-pneumococcal isolates were confirmed by sequence analysis (sodA and plys genes) when required. The isolates were also identified with the MALDI Biotyper 3.1 (Bruker Daltonics, Bremen, Germany) using an updated library containing 6,903 Main Spectra Profiles (MSPs). All the analyzed S. pneumoniae were correctly identified with MALDI-TOF MS at species level using the most updated database and all the non-pneumococcal SMG isolates were also identified at the group level. Several peaks (4,964.32, 6,888.90, and 9,516.46 m/z) have been found to be specific of S. pneumoniae, whilst a different set of peaks have proved to be present only in S. mitis (6,839.07 m/z) and S. oralis (5,297.61, 5822.53, and 6,839.07 m/z). Peak analysis allowed correct species assignment of 101/101 S. pneumoniae isolates (100%) and 102/105 S. mitis/oralis isolates (97.1%). Thus, the implementation of MALDI-TOF MS plus peak analysis for the identification of this group of microorganisms may provide precise species-level information that will allow the early implementation of directed antibiotic therapy.

In this study we evaluated the capacity of MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) to identify clinical mould isolates. We focused on two aspects of MALDI-TOF MS identification: the sample processing and the database. Direct smearing of the sample was compared with a simplified processing consisting of mechanical lysis of the moulds followed by a protein extraction step. Both methods were applied to all isolates and the Filamentous Fungi Library 1.0 (Bruker Daltonics) was used for their identification. This approach allowed the correct species-level identification of 25/34 Fusarium spp. and 10/10 Mucor circinelloides isolates using the simplified sample processing. In addition, 7/34 Fusarium spp. and 1/21 Pseudallescheria/Scedosporium spp. isolates were correctly identified at the genus level. The remaining isolates-60-could not be identified using the commercial database, mainly because of the low number of references for some species and the absence of others. Thus, an in-house library was built with 63 local isolates previously characterized using DNA sequence analysis. Its implementation allowed the accurate identification at the species level of 94 isolates (91.3%) and the remaining nine isolates (8.7%) were correctly identified at the genus level. No misidentifications at genus level were detected. In conclusion, with improvements of both the sample preparation and the feeding of the database, MALDI-TOF MS is a reliable, ready to use method to identify moulds of clinical origin in an accurate, rapid, and cost-effective manner.

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