Rapid identification and classification of Mycobacterium spp. using whole-cell protein barcodes with matrix assisted laser desorption ionization time of flight mass spectrometry in comparison with multigene phylogenetic analysis.
ABSTRACT The need of quick diagnostics and increasing number of bacterial species isolated necessitate development of a rapid and effective phenotypic identification method. Mass spectrometry (MS) profiling of whole cell proteins has potential to satisfy the requirements. The genus Mycobacterium contains more than 154 species that are taxonomically very close and require use of multiple genes including 16S rDNA for phylogenetic identification and classification. Six strains of five Mycobacterium species were selected as model bacteria in the present study because of their 16S rDNA similarity (98.4-99.8%) and the high similarity of the concatenated 16S rDNA, rpoB and hsp65 gene sequences (95.9-99.9%), requiring high identification resolution. The classification of the six strains by MALDI TOF MS protein barcodes was consistent with, but at much higher resolution than, that of the multi-locus sequence analysis of using 16S rDNA, rpoB and hsp65. The species were well differentiated using MALDI TOF MS and MALDI BioTyper™ software after quick preparation of whole-cell proteins. Several proteins were selected as diagnostic markers for species confirmation. An integration of MALDI TOF MS, MALDI BioTyper™ software and diagnostic protein fragments provides a robust phenotypic approach for bacterial identification and classification.
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ABSTRACT: Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) represents a simple reliable approach for rapid bacterial identification based on specific peptide/protein fingerprints. However, cell wall characteristics of mycobacterial species, and their well known stability, complicate MALDI-TOF MS profiling analysis. In our present study we tested two recently published protocols for inactivation and disruption of mycobacteria and we also examined the influence of different culture conditions (four culture media and five cultivation times) on mass spectral quality and the discriminatory power of the method. We found a significant influence of sample pre-treatment method and culture medium on species identification and differentiation for a total of 10 strains belonging to Mycobacterium phlei and Mycobacterium smegmatis. Optimum culture conditions yielding the highest identification success rate against the BioTyper database (Bruker Daltonics), and permitting the possibility of automatic acquisition of mass spectra were found to be distinct for the two mycobacterial species examined. Similarly, individual changes in growth conditions had diverse effects on the two species. For these reasons, thorough control over cultivation conditions should always be employed to maximize the performance and discriminatory power of MALDI-TOF MS profiling, and cultivation conditions must be optimized separately for individual groups of mycobacterial species/strains. This article is protected by copyright. All rights reserved.FEMS Microbiology Letters 02/2014; · 2.05 Impact Factor
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ABSTRACT: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique for the rapid and high-throughput identification of microorganisms. There remains a dearth of studies in which a large number of pathogenic microorganisms from a particular country or region are utilized for systematic analyses. In this study, peptide mass reference spectra (PMRS) were constructed and evaluated from numerous human pathogens (a total of 1019 strains from 94 species), including enteric (46 species), respiratory (21 species), zoonotic (17 species), and nosocomial pathogens (10 species), using a MALDI-TOF MS Biotyper system (MBS). The PMRS for 380 strains of 52 species were new contributions to the original reference database (ORD). Compared with the ORD, the new reference database (NRD) allowed for 28.2% (from 71.5% to 99.7%) and 42.3% (from 51.3% to 93.6%) improvements in identification at the genus and species levels, respectively. Misidentification rates were 91.7% and 57.1% lower with the NRD than with the ORD for genus and species identification, respectively. Eight genera and 25 species were misidentified. For genera and species that are challenging to accurately identify, identification results must be manually determined and adjusted in accordance with the database parameters. Through augmentation, the MBS demonstrated a high identification accuracy and specificity for human pathogenic microorganisms. This study sought to provide theoretical guidance for using PMRS databases in various fields, such as clinical diagnosis and treatment, disease control, quality assurance, and food safety inspection.PLoS ONE 01/2014; 9(9):e106312. · 3.53 Impact Factor
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ABSTRACT: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled to the original Biotyper database has a poor ability to identify Helicobacter pylori. Furthemore, the existing typing methods for H. pylori have no obvious correlation with the virulence and pathogenicity of H. pylori in East Asia. In this study, MALDI-TOF MS Biotyper system (MBS) was used to identify and type H. pylori. In addition, label-free and bioinformatics techniques were used to reveal the protein components of different types of H. pylori. A total of 56H. pylori isolates were added to the original reference database. For the 92H. pylori strains validated, the identification efficiency at the species level was improved from 3 (3.2%) to 82 (89.1%) strains. A new ribotyping method for H. pylori based on peptide mass fingerprinting was developed. For P1 and P2 type H. pylori, respectively, 7 specific peaks at m/z 4320, 5202, 5246, 5268, 6066, 6941, and 7128 and 2 specific peaks at m/z 5246 and 6941 were found. Between P1 and P2 type strains, 62 proteins were significantly differenct. A total of 206 proteins for H. pylori identification and typing were identified, of which 110 were located on the inner cell membrane and 103 were located in the cytoplasm. The major classifications of these proteins were ribosomal proteins (15.5%) and enzymes (29.6%). MBS is suitable for the identification and typing of variable bacteria such as H. pylori, particularly if further super reference spectra are constructed.Biological significanceHelicobacter pylori (H. pylori) possesses very high genetic variability. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled to the original Biotyper system (MBS) has a poor ability to identify Helicobacter pylori isolated from China. The identification capabilities of MBS for highly variable bacterial species remain to be established. In other side, Scholars of East Asia and Western dispute the theory that there are obvious correlations between cagA and gastric cancer. The existing typing methods for H. pylori based on cagA gene have no obvious correlation with the virulence and pathogenicity of H. pylori in East Asia. In light of this phenomenon of Asian enigma, we suppose that there are other genes beyond cagA that are correlated with the virulence of H. pylori. Here, we improved the original database using numerous H. pylori isolated from different countries and raised the identification capability of MBS from 3.2% to 89.1%. A new ribotyping method for H.pylori based on peptide mass fingerprinting was developed. Furthermore, the protein protein components of H.pylori identification and typing were revealed. These findings thus provide a new way for H.pylori identification, typing and the research of pathogenic mechanism.Journal of proteomics 01/2013; · 5.07 Impact Factor