VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

an un-inoculated reference cell, probably due to formation of a biofilm onthe bottom of the measurement cell. Biofilm quantification 48 hours after theonset of the experiment corroborated a strong biofilm formation in themeasurement cell (10 8 cells / cm 2 ).Following the encouraging data presented here, future work will includetechnical refinement of the sensor prototype and more experimental dataacquisition to improve the correlation between biofilm formation andchanges in the acoustic signals.[1] Flemming, H.-C. (2003): Role and levels of real-time monitoring for successful anti-foulingstrategies - an overview. Water Sci. Technol. 47: 1-8; [2] Janknecht, P. and L. F. Melo (2003): Onlinebiofilm monitoring. Rev. Environ. Sci. Biotechnol. 2: 269-283.[3] Lindner, G. et al (2009): Detection of coatings and measurement of coating thickness on technicalsubstrates using surface acoustic waves in a waveguide configuration. SENSOR 2009 Proceedings,Vol. I, ISBN 978-3-9810993-4-8.NTP012MALDI-TOF Mass Spectrometry as a Diagnostic Toolfor Identification of Important Veterinary StreptococcusSpeciesA.A. Hassan 1 , A. Rothkamp 1 , M. Hijazin 2 , C. Lämmler 2 , T. Eisenberg 3 ,M. Zschöck 3 , M. Timke* 4 , M. Kostrzewa 41 DG-Animal Health Service, Deventer, Germany2 Institute of Pharmacology and Toxicology, Justus-Liebig-University,Gießen, Germany3 Hessian State Office Laboratory, Gießen, Germany4 Bruker Daltonik GmbH, Bremen, GermanyStreptococcus species and subspecies are known to be associated withinfectious diseases of cattle, pigs, sheep, birds, horses, dogs, fish and aquaticmammals. The identification of Streptococcus species traditionally relies onthe determination of biochemical properties, haemolytic reaction on bloodagar and on serological grouping by use of Lancefield antisera. SeveralStreptococcus species are biochemically and serologically almostindistinguishable. During the last few years the use of matrix-assisted laserdesorption ionization-time of flight mass spectrometry (MALDI-TOF MS)technique as a diagnostic tool for the identification of bacterial pathogensbecame more frequent. This technique allows the identification ofmicroorganisms as a result of protein fingerprint analysis. In this study weused the MALDI Biotyper system (Bruker Daltonik) for identification anddifferentiation of 44 different Streptococcus field isolates and 17Streptococcus reference strains. Field isolates were previously identifiedwith biochemical and haemolytical tests and on serological properties. Thebacteria used in this study included S. agalactiae, S. canis, S. dysgalactiae,S. uberis, S. parauberis, S. equi subsp. equi; S. equi subsp.zooepidepidemicus. A total of 56 (91.8%) isolates were identified to specieslevel and five (9.2%) isolates from species S. iniae were not reliablyidentified. This was due to a lack of S. iniae in the database at this time. Thisspecies will be added to the database for further improvement of the system.Differentiation between S. equi subsp. equi and S. equi subsp.zooepidepidemicus is possible by MALDI-TOF MS, however, more strainshave to be analysed for a validation of this finding. MALDI-TOF techniqueis a promising tool for identification of Streptococcus species and might helpto clarify the streptococcal infections in different animals.NTP013A classification method for Enterococcus faecalis afterstress using maldi-tof mass spectrometry and subsequentmultivariate data analysisB. Kühl*, S.-M. Marten, Y. Bischoff, G. Brenner-Weiss, U. ObstInstitute of Functional Interfaces, Karlsruhe Institute of Technology (KIT),Eggenstein-Leopoldshafen, GermanyAs demonstrated before, MALDI-ToF/MS in combination with multivariatedata analysis represent a powerful tool for mass spectrometric patternrecognition of biological samples. We use this technique to classify theviable but not culturable (VBNC) stage as a survival state of bacteria causedby starvation and cold as well as their reactivation for cultivability incomparison to cells during exponential growth phase.In this study Enterococcus faecalis was selected as model organism. Thegenerated „molecular fingerprint” spectra were subjected to multivariatedata analysis without targeting single bacterial molecules or moleculestructures and were compared to the corresponding growth curve afterwards.E. faecalis was kept in dormancy state for 42 days and reactivated byincubation in BHI media at 37 °C at 150 rpm. After 3 h and 6 h,respectively, an aliquot of bacterial suspension was analysed byMALDI/Tof-MS. As a control, bacteria in the exponential growth phasewere analysed. For an effective data analysis a multivariate approach usinghierarchical cluster analysis and principle component analysis was applied inorder to classify each state in comparison to each other. The massspectrometric results were compared with those obtained by CFU (colonyforming units) and Live/Dead staining.NTP014Time resolved protein-based stable isotope probing(Protein-SIP) analysis allows quantification of inducedproteins in substrate shift experimentsM. Taubert 1 , N. Jehmlich 1,2 , C. Vogt 3 , H.H. Richnow 3 , F. Schmidt 1,2 , M. vonBergen 1 , J. Seifert* 11 Department of Proteomics, Helmholtz Center for Environmental Research(USZ), Leipzig, Germany2 Functional Genomics, Ernst-Moritz-Arndt-University, Greifswald,Germany3 Department of Isotope Biogeochemistry, Helmholtz Center forEnvironmental Research (USZ), Leipzig, GermanyThe detection of induced proteins after introduction of specific substrates inculture is of high interest for a comparative description of microorganismsgrowing under different conditions. In the past, protein expression changeswere detected by the use of 35 S-methionine incorporation and the subsequentdetection of labeled proteins by autoradiography. Later 35 S amino acidlabeling was used in 2-DE studies which allowed a direct comparison of theprotein pattern and the protein spot intensities. Other ways to detectquantitative changes in the proteome employ labeling with isotopicallylabeled amino acids (SILAC) [1].In this study protein-based stable isotope probing (Protein-SIP) [2] is usedfor a fast and reliable detection of differentially expressed proteins in asubstrate shift experiment. Stable isotope probing (SIP) is an establishedmethod in microbial ecology to identify metabolic key players in microbialcommunities using substrates labeled with stable isotopes, e.g. 13 C [3-5].Besides the applicability for ecological studies Protein-SIP is now used tostudy protein expression of single cultures. Therefore, Pseudomonas putidaML2 cells pre-cultured on 12 C-acetate and 12 C-benzene, respectively, wereincubated with 13 C-benzene as a stable-isotope labeled substrate.Protein samples from early to stationary growth phase were separated byone-dimensional gel electrophoresis (1-DE), subsequently trypticallydigested and analyzed by UPLC Orbitrap MS/MS measurements. Identifiedpeptides from proteins involved in aerobic benzene degradation as well asfrom house-keeping proteins were chosen to calculate the labeling ratio(proportion of labeled protein on total protein) at different times. Acomparison of parameters from a nonlinear regression analysis of thecalculated data enabled a clear differentiation between induced (proteinsfrom lower degradation pathway, e.g. catechol 1,2-dioxygenase) andconstitutively (proteins from upper degradation pathway, e.g. benzene 1,2-dioxygenase) expressed proteins.Thus, Protein-SIP has proven to be a valuable tool for quantitative analysisof induced proteins in substrate shift experiments.[1] Ong, S. E.et al (2002): Stable isotope labeling by amino acids in cell culture, SILAC, as a simpleand accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376-386.[2] Jehmlich, N. et al (2010): Protein-stable isotope probing (Protein-SIP). Nature Protocols. 5 (12),1957-1966.[3] Jehmlich, N. (2008): Protein-based stable isotope probing (Protein-SIP) reveals active specieswithin anoxic mixed cultures. ISME J. 2, 1122-1133.[4] Neufeld, J. D. et al (2007): Methodological considerations for the use of stable isotope probing inmicrobial ecology. Microbiol. Ecol. 53, 435-442.[5] Dumont, M. G. and J. C. Murrell (2005): Stable isotope probing - linking microbial identity tofunction. Nat. Rev. Microbiol. 3, 499-504.NTP015Rapid identification of cyclic depsipeptides from microorganismsby means of mass spectrometric techniquesT.L.H. Pham* 1 , I. Zaspel 2 , E. Krause 31 Institute for Ecology, University of Technology, Berlin, Germany2 Federal Research Institute for Rural Areas, Forestry and Fisheries (vTI,Institute of Forest Genetics, Waldsieversdorf, Germany3 Mass Spectrometry, Leibniz Institute for Molecular Pharmacology (FMP),Berlin, GermanyRapid screening of secondary metabolites from micro-organisms can becarried out successfully by means of a combination of separation andspektrum | Tagungsband 2011