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.
- SourceAvailable from: Ondrej Šedo
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- "This requires special treatment prior to MALDI-TOF MS analysis to allow release of compounds of interest from within the cells. Interestingly, although several authors have clearly demonstrated the potential of MALDI-TOF MS for mycobacterial species identification (Hettick et al., 2004, 2006; Pignone et al., 2006; Lotz et al., 2010; El Kh echine et al., 2011; Saleeb et al., 2011; Shitikov et al., 2011), there is no agreement on bacterial sample preparation methods, that is, whether direct analysis of whole cells (Pignone et al., 2006; Lotz et al., 2010) or analysis of the cell extract (Hettick et al., 2006; Saleeb et al., 2011; Wang et al., 2012) is more "
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; 353(1). DOI:10.1111/1574-6968.12408 · 2.12 Impact Factor
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- "BIOTYPER, Bruker Daltonics, Bremen, Germany or SERAMIS, bioMérieux, Craponne, France).      Although suitable pollen MS databases are not yet available, the imaging experiments were performed with intact pollen grains and data evaluation was carried out using those characteristic (but still widely unknown) peaks that were used for HCA as well.  EXPERIMENTAL Materials For the investigation, both commercially available lyophilized pollen (Populus nigra, Ambrosia trifida, Artemisia absinthium, Sigma, Germany) and fresh pollen acquired from biological samples (Hibiscus syriacus) were utilized. "
ABSTRACT: The fast and univocal identification of different species in mixtures of pollen grains is still a challenge. Apart from microscopic evaluation and Raman spectroscopy, no other techniques are available. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry was applied to the analysis of extracts of single pollen grains and pollen mixtures. Pollen grains were fixed, treated and covered with matrix directly on the MALDI target. Clearly resolved MALDI ion intensity images could be obtained enabling the identification of single pollen grains in a mixture. Our results demonstrate the potential and the suitability of MALDI imaging mass spectrometry as an additional method for the identification of pollen mixtures. CopyrightRapid Communications in Mass Spectrometry 04/2013; 27(8):896-903. DOI:10.1002/rcm.6523 · 2.25 Impact Factor
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ABSTRACT: Abstract Recently, different bacteriological laboratory interventions that decrease reporting time have been developed. These promising new broad-based techniques have merit, based on their ability to identify rapidly many bacteria, organisms difficult to grow or newly emerging strains, as well as their capacity to track disease transmission. The benefit of rapid reporting of identification and/or resistance of bacteria can greatly impact patient outcomes, with an improvement in the use of antibiotics, in the reduction of the emergence of multidrug resistant bacteria and in mortality rates. Different techniques revolve around mass spectrometry (MS) technology: matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), PCR combined with electrospray ionization-mass spectrometry (PCR/ESI-MS), iPLEX MassArray system and other new evolutions combining different techniques. This report emphasizes the (r) evolution of these technologies in clinical microbiology.Clinical Chemistry and Laboratory Medicine 10/2012; DOI:10.1515/cclm-2012-0291 · 2.71 Impact Factor