Abstract
BackgroundThe MALDI (matrix-assisted laser desorption/ionization) Biotyper system for bacterial identification has already been utilized in clinical microbiology laboratories as a successful clinical application of protoemics. However, in cases of Nocardia, mass spectra suitable for MALDI Biotyper identification are often not obtained if such specimens are processed like general bacteria. This problem is related to the insufficiencies in bacterial spectrum databases that preclude accurate specimen identification. Here, we developed a bacterial processing method to improve mass spectra from specimens of the genus Nocardia. In addition, with the new processing method, we constructed a novel in-house bacterial database that combines a commercial database and mass spectra of Nocardia strains from the Department of Clinical Laboratory at Chiba University Hospital (DCLC) and the Medical Mycology Research Center at Chiba University (MMRC).ResultsThe newly developed method (Nocardia Extraction Method at DCLC [NECLC]) based on ethanol-formic acid extraction (EFAE) improved mass spectra obtained from Nocardia specimens. The Nocardia in-house database at Chiba University Hospital (NDCUH) was then successfully validated. In brief, prior to introduction of the NECLC and NDCUH, 10 of 64 (15.6%) clinical isolates were identified at the species level and 16 isolates (25.0%) could only be identified at the genus level. In contrast, after the introduction, 58 isolates (90.6%) were identified at the species level and 6 isolates (9.4%) were identified at the genus level.ConclusionsThe results of this study suggest that MALDI-TOF (time-of-flight) Biotyper system can identify Nocardia accurately in a short time in combination with a simple processing method and an in-house database.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9078-5) contains supplementary material, which is available to authorized users.
Highlights
The Matrix-assisted laser desorption/ionization (MALDI) Biotyper system for bacterial identification has already been utilized in clinical microbiology laboratories as a successful clinical application of protoemics
Comparison of the new method for bacterial extraction (NECLC) approach and conventional extraction methods We proposed a new method for Nocardia extraction (NECLC)
This method is based on the ethanol-formic acid extraction method (EFAE) (Figure 1A), with the addition of silica beads as a component of the hightemperature extraction method (HTEM) (Figure 1B) and use of a 10-minute formic acid extraction time (Figure 1C)
Summary
The MALDI (matrix-assisted laser desorption/ionization) Biotyper system for bacterial identification has already been utilized in clinical microbiology laboratories as a successful clinical application of protoemics. In cases of Nocardia, mass spectra suitable for MALDI Biotyper identification are often not obtained if such specimens are processed like general bacteria. This problem is related to the insufficiencies in bacterial spectrum databases that preclude accurate specimen identification. To distinguish between the genera of actinomycetes, analyses of the taxonomic and physicochemical properties of the organism are necessary, including analyses of mycolic acid and menaquinone and the sugars and amino acids composing the cell wall These physicochemical properties can be used to identify species of the genus Nocardia [5]. As with other bacteria, classification of Nocardia based on the 16S rRNA sequence has been introduced [6] and has simplified species identification, but employing this method in the laboratory remains difficult in routine identification testing
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