Matrix-assisted Laser Desorption Ionization Biotyper Research Articles

Comparison of the New VITEK MS PRIME System with the Matrix-Assisted Laser Desorption Ionization Biotyper Microflex LT for the Identification of Microorganisms.

Matrix-assisted laser desorption ionization (MALDI) is the cornerstone of bacterial identification. The performance of a new MALDI time-of-flight mass spectrometry VITEK MS PRIME (VMS-P) system was compared with that of the MALDI Biotyper Microflex LT (MBT) system, which is routinely used in our laboratory. Sixteen bacterial and yeast reference strains cultured in 20 different media were analyzed over 10 consecutive rounds using both systems. Bacterial and yeast isolates from the routine workflow were processed using both systems. Microcolonies were identified after a 4-hour agar subculture from positive blood culture bottles, without extraction. To determine the repeatability based on the reference strains, 1,190 spots were processed using each system. Correct identification was achieved for 94.0% (MBT) and 98.4% (VMS-P; P<0.01) of spots. Among these, 83.0% (MBT) and 100.0% (VMS-P) were identified with a high degree of confidence. For 1,214 spots from routine isolates, species identification was achieved for 90.0% (MBT) and 91.4% (VMS-P; P=0.26) of spots. For 69.8% (MBT) and 87.4% (VMS-P) of the spots, identification was achieved with a high degree-of-confidence score. When identification was performed using both systems, the agreement between them was 97.9%. The identification of microcolonies from positive blood culture bottles was achieved for 55.5% (MBT) and 70.2% (VMS-P; P=0.01) of spots. The MBT and VMS-P systems perform similarly in routine daily practice. The new VMS-P system shows high repeatability, better confidence scores for identification, and promising ability to identify microcolonies.

Rapid Antifungal Susceptibility Testing of Yeasts and Molds by MALDI-TOF MS: A Systematic Review and Meta-Analysis.

Due to the growing burden of fungal infections and a recent rise in antifungal resistance, antifungal susceptibility testing (AFST) is of increasing importance. The common methods of AFST have turnaround times of 24 to 48 h, and the available rapid methods are limited by applicability, cost-efficiency or accuracy. Given the urgency of adequate antifungal treatment in invasive mycoses, the need for the rapid and reliable detection of resistance is evident. In this systematic review and meta-analysis, we evaluated the diagnostic accuracy of AFST based on matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Twelve studies were reviewed, and data for the comparative analysis of their accuracy and methodology were systematically extracted. Compared to broth dilution as the gold standard, MALDI-TOF MS-based AFST reached a pooled sensitivity and specificity of 91% (95% Confidence Interval [CI], 84% to 96%) and 95% (95% CI, 90% to 98%), respectively. A comparative analysis showed that the sensitivity was higher for the semi-quantitative matrix-assisted laser desorption ionization Biotyper antibiotic susceptibility test rapid assay (MBT ASTRA) technique (96%) than for the correlate composite index (CCI) approach (85%), which is based on spectrum changes. Turnaround times below eight hours reached better diagnostic values than longer incubation periods, qualifying MALDI-TOF MS-based AFST as a rapid and accurate method for the detection of antifungal resistance.

Rapid glycosyl-inositol-phospho-ceramide fingerprint from filamentous fungal pathogens using the MALDI Biotyper Sirius system.

Glycosyl-inositol-phospho-ceramides (GIPCs) or glycosylphosphatidylinositol-anchored fungal polysaccharides are known to be major lipids in plant and fungal plasma membranes and to play an important role in stress adaption. However, their analysis remains challenging due to the several steps involved for their extractions and purifications prior to mass spectrometric analysis. To address this challenge, we developed a rapid and sensitive method to identify GIPCs from the four common fungal plant pathogens Botrytis cinerea, Fusarium graminearium, Neurospora crassa and Ustilago maydis. Fungal plant pathogens were cultured, harvested, heat-inactivated and washed three times with double-distilled water. Intact fungi were deposited on a matrix-assisted laser desorption ionization (MALDI) target plate, mixed with the matrix consisting of a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid solubilized at 10 mg/mL in chloroform-methanol (9:1 v/v) and analyzed using a Bruker MALDI Biotyper Sirius system in the linear negative ion mode. Mass spectra were acquired from m/z 700 to 2000. MALDI time-of-flight (TOF) mass spectrometric analysis of cultured fungi showed clear signature of GIPCs in B. cinerea, F. graminearium, N. crassa and U. maydis. We have demonstrated that routine MALDI-TOF in the linear negative ion mode combined with an apolar solvent system to solubilize the matrix is applicable to the detection of filamentous fungal GIPCs.

Identificação por espectrometria de massa MALDI-TOF de Salmonella spp. e Escherichia coli isolados de carcaças bovinas

RESUMO: O objetivo deste trabalho foi introduzir a técnica de espectrometria de massa com fonte de ionização e dessorção a laser assistida por matriz e analisador de tempo-de-voo (MALDI-TOF) para incrementar o método tradicional microbiológico na detecção de Salmonella spp. e Escherichia coli em carcaças bovinas. Foram avaliadas 270 amostras de 90 carcaças de bovinos. Para isolamento de Salmonella spp. e E. coli, foram utilizadas, respectivamente, as metodologias descritas na ISO 6579:2002 e no Compendium of Methods for the Microbiological Examination of Foods. As análises por MALDI-TOF foram realizadas a partir de isolados cultivados em ágar nutriente ou em caldo triptona de soja, provenientes das amostras com características bioquímicas positivas (n=7), inconclusivas (n=4) e negativas (n=85) para Salmonella spp. e bioquímicas positivas (n=37) e negativas (n=85) para E. coli. Os perfis de massas foram adquiridos com o espectrômetro de massas MALDI-TOF Autoflex III SmartBeam e os espectros brutos foram processados usando o programa MALDI Biotyper (Bruker Daltonics). De acordo com a identificação preliminar, com base na morfologia das colônias e nas reações bioquímicas, sete isolados foram considerados positivos para Salmonella spp. Através do MALDI Biotyper, esses sete isolados foram classificados como pertencentes ao gênero Salmonella e, além disso, identificados como S. enterica. Quatro isolados que apresentaram características fenotípicas não usuais e resultados inconclusivos nos testes bioquímicos para Salmonella foram identificados como pertencentes aos gêneros Citrobacter e Proteus após análise por MALDI. Para E. coli, 37 amostras foram positivas pelos testes bioquímicos da espécie, o que foi confirmado por MALDI Biotyper. A metodologia MALDI-TOF permitiu a rápida confirmação da identidade de Salmonella spp. e E. coli, podendo ser utilizada para detecção desses microrganismos em isolados bacterianos de carcaças bovinas.

Use of MALDI-TOF Mass Spectrometry for the Fast Identification of Gram-Positive Fish Pathogens.

Gram-positive cocci, such as Streptococcus agalactiae, Lactococcus garvieae, Streptococcus iniae, and Streptococcus dysgalactiae subsp. dysgalactiae, are found throughout the world, particularly in outbreaks in farmed fish, and are thus associated with high economic losses, especially in the cultivation of Nile Tilapia. The aim of this study was to evaluate the efficacy of matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) as an alternative for the diagnosis of these pathogens. One hundred and thirty-one isolates from Brazilian outbreaks assisted by the national authority were identified using a MALDI Biotyper from Bruker Daltonics. The results showed an agreement with respect to identification (Kappa = 1) between this technique and 16S ribosomal RNA gene sequencing for S. agalactiae and L. garvieae. However, for S. iniae and S. dysgalactiae subsp. dysgalactiae, perfect agreement was only achieved after the creation of a custom main spectra profile, as well as further comparisons with 16S ribosomal RNA and multilocus sequence analysis. MALDI-TOF MS was shown to be an efficient technology for the identification of these Gram-positive pathogens, yielding a quick and precise diagnosis.

Identification of Nocardia species using matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry

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.

Improvement of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Identification of Clinically Important Candida Species

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is emerging as a rapid and accurate tool for identifying pathogens. MALDI-TOF identification of Candida at the species level remains unsatisfactory because of an incomplete commercial database. Using Bruker Daltonics MALDI Biotyper software, an in-house spectral database library was created with m/z ratios of 2,000 to 20,000 Da from 60 successfully identified clinical isolates of Candida spp. (6 species). It was incorporated into the original database to produce the augmented Bruker-Tianjin database. The augmented database was tested with 347 clinical isolates of 6 species comparing with Bruker database in parallel. The identification rates at the species level were 91.6% (318/347) using the Bruker database and 99.1% (344/347) for the Bruker-Tianjin database. Database expansion and refinement can easily and effectively improve MALDI-TOF MS performance for enhanced clinical pathogen identification.

Bacterial Classification of Fish-Pathogenic Mycobacterium Species by Multigene Phylogenetic Analyses and MALDI Biotyper Identification System

Mycobacterium marinum is difficult to distinguish from other species of Mycobacterium isolated from fish using biochemical methods. Here, we used genetic and proteomic analyses to distinguish three Mycobacterium strains: M. marinum strains MB2 and Europe were isolated from tropical and marine fish in Thailand and Europe, and Mycobacterium sp. 012931 strain was isolated from yellowtail in Japan. In phylogenetic trees based on gyrB, rpoB, and Ag85B genes, Mycobacterium sp. 012931 clustered with M. marinum strains MB2 and Europe, but in trees based on 16S rRNA, hsp65, and Ag85A genes Mycobacterium sp. 012931 did not cluster with the other strains. In proteomic analyses using a Bruker matrix-assisted laser desorption ionization Biotyper, the mass profile of Mycobacterium sp. 012931 differed from the mass profiles of the other two fish M. marinum strains. Therefore, Mycobacterium sp. 012931 is similar to M. marinum but is not the same, suggesting that it could be a subspecies of M. marinum.

Comparison of the MALDI Biotyper System Using Sepsityper Specimen Processing to Routine Microbiological Methods for Identification of Bacteria from Positive Blood Culture Bottles

Bloodstream infections are a leading cause of admissions to hospital intensive care units and carry a high mortality rate. Clinical outcome can be greatly improved by early effective antibiotic therapy; therefore, broad-spectrum antimicrobial therapy is often initiated when there is a clinical suspicion of bloodstream infection. Unfortunately, this method may not always be effective when dealing with inherently resistant organisms and can also result in iatrogenic infection and the development of resistant isolates. Rapid identification of the infecting organism may aid in choosing appropriate antimicrobial therapy, thereby reducing these potential adverse events. We compared the matrix-assisted laser desorption ionization (MALDI) Biotyper system with Sepsityper specimen processing (Bruker Daltonics, Billerica, MA) to routine methods for the identification of microorganisms from 164 positive blood cultures. The MALDI Biotyper/Sepsityper identified 85.5% of bacterial isolates directly from positive monomicrobial blood cultures with 97.6% concordance to genus and 94.1% concordance to species with routine identification methods. Gram-negative isolates were more likely to produce acceptable confidence scores (97.8%) than gram-positive isolates (80.0%); however, genus and species concordance with routine identification methods were similar in both groups. Reanalysis of collected spectra using modified blood culture-specific parameters resulted in an improved overall identification rate for gram-positive bacteria (89.0%). Median times to identification using the MALDI Biotyper/Sepsityper were 23 to 83 h faster than routine methods for gram-positive isolates and 34 to 51 h faster for gram-negative isolates.

Matrix-Assisted Laser Desorption Ionization (MALDI)-Time of Flight Mass Spectrometry- and MALDI Biotyper-Based Identification of Cultured Biphenyl-Metabolizing Bacteria from Contaminated Horseradish Rhizosphere Soil

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.

Improved Identification of Yeast Species Directly from Positive Blood Culture Media by Combining Sepsityper Specimen Processing and Microflex Analysis with the Matrix-Assisted Laser Desorption Ionization Biotyper System

Current methods for identification of yeast from blood cultures may take several days after these microorganisms have been observed by Gram stain smears from positive blood cultures. We explored the use of a matrix-assisted laser desorption ionization (MALDI) Biotyper system in combination with Sepsityper specimen processing and Microflex analysis for improved detection and identification of yeast species directly from positive blood culture specimens demonstrating yeast-like organisms by Gram stain. The limit of detection of yeast species in blood culture medium was determined to be 5.9 × 10(5) CFU, with intra- and interstrain coefficients of variation of 1.8 to 3.6% and 2.9%, respectively. A total of 42 yeast-containing positive blood culture specimens were processed, and the identification results were compared to those obtained by routinely used phenotypic methods. Specimens with discrepant results between the Biotyper and phenotypic methods were identified on the basis of internal transcribed spacer region sequencing. The MALDI Biotyper system correctly identified the 42 specimens to species level, including 28 (66.7%) Candida albicans, 8 (19.0%) Candida parapsilosis, and 5 (11.9%) Candida tropicalis isolates and 1 (2.4%) Cryptococcus neoformans isolate. The entire procedure, from specimen extraction to final result reporting, can be completed within 1 h. Our data indicated that the Sepsityper specimen processing and Microflex analysis by the MALDI Biotyper system provide a rapid and reliable tool for yeast species identification directly from positive blood culture media.