Abstract Book of EAVLD2012 - eavld congress 2012

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S4 - K - 01 MALDI-TOF AND OTHER NEW DIAGNOSTIC Markus Kostrzewa Bruker Daltonik GmbH, Bioanalytical development, Bremen, Germany Microorganism identification, FT-IR spectroscopy, Raman spectroscopy, mass spectrometry, MALDI-TOF MS Introduction – the “status quo” Since more than one hundred years, identification of microorganisms mainly is performed by methods investigating their phenotypic characteristics. Gram stain together with microscopy in combination gives a first quick hint about identity. The screening of series of tests for biochemical capabilities is used to generate a pattern which is more or less species specific. Although these tests now have been automated for the detection of the most prevalent microorganisms, the accuracy of biochemical tests is limited, in particular for organisms with few characteristic biochemical features like Gram negative nonfermenting bacteria of anaerobes. Even worse is the situation for rare bacteria, mycobacteria or fungi where mainly labourintensive manual methods like API are applied for identification. Therefore, there is an urgent need for modern automated methods for identification which can be introduced in routine laboratories to shorten time-to-result and decrease the overall costs of the laboratories. Promising approaches appearing in the areas of molecular biology, optical technologies and mass spectrometry have appeared through the recent years and are on the way to revolutionize microbial analysis. Methods based on molecular biology have already been introduced in many laboratories, mainly PCR based tests to screen for dedicated microorganisms, e.g. food pathogens. While having the advantage of high sensitivity and short analysis time one drawback of PCR based tests is that they generally need a pre-assumption which microorganism is searched for, i.e. a dedicated primer design. To apply DNA analysis to broad species identification, PCR typically is coupled with subsequent sequence determination of the amplicon. Sequencing of the 16S rDNA region nowadays is regarded the gold standard for species identification. Nevertheless, 16S rDNA sequencing has never made it to the routine but is mainly a second- or third-line method for cases where other methods have failed. Reasons are the high costs, elaborate sample handling, and PCR-related contamination risk. Further, in quite a number of cases, determination of the 16S ribosomal RNA gene sequence is not sufficient for the discrimination of closely related species and further gene sequences have to be analysed, increasing time to result, costs and labour, significantly. Novel next generation sequencing technologies might change the position of sequencing in future by their enormous throughput capability, but today they are still expensive and labour-intensive research tools. The upcoming alternatives Vibrational spectroscopy Optical technologies have been shown to be powerful for microorganism identification as well as for analyses on the subspecies level. These techniques are based on a molecular fingerprint of the entire biomass, proteins, carbohydrates, lipids, nucleic acids. Analyses are not depending on reagent kits, labels or drugs, and these technologies are applicable to a broad spectrum of organisms. Raman spectroscopy is a non-destructive optical technique, based on scattering of light by molecules, the so called “Raman effect”. The atoms in a molecule move in so called vibrational modes. When light, i.e. a photon, and a molecule interact, some of the energy of the photon can be transferred to the molecule. Thereby, one of the vibrational modes in the molecule is excited. The energy of the scattered photon is reduced by the exact amount of energy received by the molecule. The energy required to excite a molecular vibration is exactly defined and depends on the masses of the atoms involved in the vibration, on the type of chemical bonds between these atoms, on the structure of the molecule and on interactions with its environment and other molecules. The spectrum of the scattered light is used as a pattern to identify microbes. In contrast, Fourier-transformation infrared spectroscopy uses absorption of an infrared light spectrum (not a single wavelength) to create a fingerprint of the whole cell composition. Mainly transmission of the light is used to create the characteristic profile. The first deviation of the transmission profile spectrum is generated and sophisticated mathematical algorithms are used to identify strains by matching against a database or to build relational dendrograms for classification. Restrictions of the optical technologies is mainly that they require a strict standardization of cultivation conditions (time, media, temperature) as these are influencing the cell content and thereby the optical fingerprint. The speed of the analyses is also counteracted by the fact that a pure culture is required (i.e. often no analysis from primary culture can be performed). Mass spectrometry Some technological approaches for identification and further analysis of microorganisms based on mass spectrometry may have the most impact on microbial characterization in the near future. One of these approaches is based on a combination of molecular biology and electrospray time-of-flight mass spectrometry (ESI-TOF MS). In a first step, several target regions of the DNA from the unknown microorganism are amplified with generic primer pairs. This is creating a set of amplification products with specific molecular masses. Subsequently, the PCR products are purified with a magnetic bead based system and then injected in liquid phase into the ESI-TOF mass spectrometer. During the ionization process, the two DNA strands of an amplicon are separated. The mass of both DNA strands is measured in the ESI-TOF very accurately. From the combination of the accurate masses of the both pairing strands the composition of the polynucleotides (i.e. the number of A, C, G, and T, respectively) can be determined. The projection of the base compositions of the selected PCR products into a database is used for the identification of an organism. The more PCR products are analysed, the more accurate/discriminative the identification can be. One advantage of this technology is that in parallel to species identification also other characteristics, e.g. resistance genes or virulence factors, can be detected. On the other hand, this approach still relies on a relatively complex (although in parts automated) workflow, and both the instrument as well as consumable costs are significant. The second, currently most successful and promising mass spectrometry technology is matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry (MALDI- TOF MS). In contrast to electrospray ionization the MALDI process (generating the ions measured in the TOF analyser) is performed from a solid sample. For this, the sample is deposited on a target plate, overlaid with the so called matrix, generally an organic acid related to cinnamic acid, which co-crystalizes with the sample. This matrix supports the evaporation of the sample and its ionization after absorption of the energy of a pulsed laser beam. One advantage of this technology is that the sample can be easily prepared offline before measurement and then transported or stored until measurement. For one molecular biology based approach, MALDI-TOF MS is used to detect specific RNA fragments generated by PCR followed by transcription of both DNA strands and the cleavage of the transcripts by RNAses. The exact masses of the fragments can be used as pattern for identification and typing of microorganisms. Alternatively, minisequencing/primer extension of specific regions after PCR amplification, followed by mass determination in a MALDI-TOF MS, has been shown to have some potential for typing or resistance detection. While none of these DNA based mass spectrometry approaches made it to routine yet, MALDI-TOF MS whole cell protein profiling has entered many clinical and veterinary microbiology laboratories, already. This MALDI-TOF fingerprinting is based on the specific pattern of high abundant proteins of microbial cells which can be measured quickly and accurately by MALDI-TOF mass spectrometry. Sample preparation is very simple, fast and cheap: A small amount of a bacterial colony is transferred to a position on a MALDI sample target and smeared on it as a thin film. More robust cells can be broken by a short extraction with
acid and organic solvent. Then a droplet of matrix solution is added. Thereby, the cells are destructed, the protein content gets released and is embedded into the matrix crystals which are forming after solvent evaporation. During the subsequent MALDI- TOF analysis the most abundant proteins, in particular ribosomal proteins, are detected as a characteristic molecular profile (fingerprint). This pattern then is compared with the profiles stored in a database. Identification is done based on highest similarity and a minimum identity score. The whole process, from sample preparation to identification, can be as fast as ten minutes for a single sample/colony, less than one hour for about one hundred samples. For difficult to prepare microorganisms like actinomycetes, fungi, or mycobacteria special protocols are available which allow also their analysis with high success rate. Dependance on cultivation conditions is generally low. Thereby, this is one of the broadest applicable technologies for microbial identification. MALDI-TOF fingerprinting is already used in many clinical and veterinary laboratories, frequently as the first-line routine identification method. Its accuracy has been reported in various publications. Extensive commercial databases are available, but also alternative or supplementary laboratoryspecific libraries can be established. Recent developments in the direction of direct specimen analysis, epidemiology, virulence typing, and resistance determination will make the technology even more valuable for the clinical microbiology. Conclusions New technologies for microorganism identification and characterization are no more on the horizon, they are on the playground. In the near future it can be expected that a combination of molecular and spectroscopic/spectrometric technologies will substitute most of phenotypic/biochemical assays, increasing accuracy, efficiency and speed of the clinical microbiology laboratory.
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2 nd CONGRESSOFTHEEUROPEAN ASSOCIAT
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Welcome Dear colleagues, Welcome to
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worked with many diagnostic compani
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S3 - P - 12 PRELIMINARY VALIDATION
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S4 - P - 01 MOLECULAR CHARACTERIZAT
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List of Abstracts (in order of numb
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S1-P-03 BALLAGI ANDREA S1-P-04 BENI
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S3-P-06 KELLER SELINA S3-P-07 KÖNI
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