VAAM-Jahrestagung 2012 18.–21. März in Tübingen

110identification of Staphylococcus aureus along with its virulencecapabilities. Here we describe a hexaplex strategy for a rapid detection ofmethicillin resistance, simultaneously discriminating S. aureus fromcoagulase-negative staphylococci (CoNS) and occurrence of virulencefactors. It targets the nuc (specific for S. aureus), mec A (methicillinresistance determinant), fem A and fem B (S. aureus specific factorsessential for methicillin resistance), Luk S/F PV (encodes for PantonValentine Leukocidin-PVL) and spa (encodes protein A). Validation ofthis strategy was performed using previously characterized clinical isolatesof methicillin susceptible Staphylococcus aureus (MSSA), methicillinresistant Staphylococcus aureus (MRSA) and CoNS from differenthospital facilities. Amplification results were consistent and perfectlyaccurate in accordance to the biochemical and resistance properties of theisolates. This molecular approach renders clinical microbiology a feasible,rapid, simple and reliable technique discriminating MSSA, MRSA andCoNS and provides an early and accurate way of detection, contributing inprevention from widespread dissemination and facilitating antibiotictherapy design.MPP003Optimization of PCR strategy for multilocus sequence analysisof Staphylococcus aureusS.M. Shahid*, A. Khatoon, F. Hussain, M. Ismail, A. AzharThe Karachi Institute of Biotechnology & Genetic Engineering (KIBGE),University of Karachi, Medical Biotechnology, Karachi, PakistanMethicillin-resistant Staphylococcus aureus (MRSA) has been the mostcommon nosocomial pathogen worldwide. It is generally documented asthe most significant due to the burden of diseases it causes and to theevolution and global spread of multidrug-resistant clones. This studydescribes the optimization of PCR assay for the multilocus sequencetyping (MLST) and analysis of housekeeping genes harbored byStaphylococcus aureus isolates. Conditions were optimized for a total ofseven housekeeping genes which are carbamate kinase (arcC), shikimatedehydrogenase (aroE), glycerol kinase (glpF), guanylate kinase (gmk),phosphate acetyltransferase (pta), triosephosphate isomerase (tpi), acetylcoenzyme A acetyltransferase (yqiL) each of which were ~500bp. A totalof 50 human clinical isolates of methicillin-resistant and -sensitiveStaphylococcus aureus were used to validate the method. This assay offerssimple, feasible and specific amplification of multilocus products, whichwould be more precisely and accurately analyzed by direct sequencingMPP004Isothermal Microcalorimetry as a powerful technique forsusceptibility testing and investigation of multidrug resistantorganismsC. OrtmannTA Instruments, Microcalorimetry, Eschborn, GermanyThe need for finding and testing new drugs against multiresistentorganisms is a great challenge for our modern society. With regard to thehigh amounts of antibiotics present in daily life the problem of resistantorganisms will increase in future. Thus, beside improvements in hygieneespecially in hospitals and a conscious usage of antibiotics there is greatneed for developing new drugs and reliable tests to investigate their impacts.Since Isothermal Microcalorimetry (IMC) relies on dissipated heat overtime it is generally applicable to all sorts of organisms. There is hardly anyrestriction to the media either; IMC might be used with all body fluids,with solutions, broths or agars, fluid or solid. Hence, it has proved to be asimple, powerful and convenient technique to record the growth andmetabolism of bacteria, cell cultures and parasites (Braissant et al. 2009).Furthermore it is quicker than established techniques like proportionmethod on plates or blood cultures (e.g. Howell et al. 2012, Buess 2007).In addition the method is nondestructive and provides a real time detectionof the investigated process instead of snapshots which makes it especiallyvaluable for testing the mechanisms of drug effects. It has been shown thatdrugs can diminish the growth of bacteria but there are also drugs that just delaythe onset of growth (e.g. von Ah et al. 2009). To distinguish these two modes ofdrug effect is almost impossible with common techniques. Therefore IMC is apowerful method to determine minimal inhibiting concentrations (MIC) ofdrugs and other toxicological approaches. Actually at standardized conditionsIMC may reveal diagnostic capabilities because the heat flow curves arecharacteristic for most species. In environments with just a few frequentlypresent species like bacteria in a hospital the heat flow curve of a blood samplefor example may reveal the target species.This talk gives a short overview of recent IMC studies in the field ofmicrobiology and drug testing with a focus on human pathogenic organisms. Italso provides some technical aspects of the method and gives an outlook inpossible applications in the future.Braissant O, Wirtz D, Göpfert B & Daniels AU (2010) Use of isothermal microcalorimetry to monitormicrobial activities. FEMS Microbiol Lett 303: 1-8Buess, D (2007) Improved detection of Microorganisms in Blood by Isothermal Microcalorimetry. PhDThesis (Medicine) University of BaselHowell, M.; Wirtz, D.; Daniels, A.U. & Braissant, O. (2012) Application of a Microcalorimetric Method forDetermining Drug Susceptibility inMycobacteriumSpecies. Journal of Clinical Microbiology doi:10.1128/JCM.05556-11von Ah, U.; Wirtz, D. & Daniels, A.U. (2009) Isothermal micro calorimetry - a new method for MICdeterminations: results for 12 antibiotics and reference strains ofE. coliandS. aureus. BMC Microbiology 9:106.MPP005ATP cytotoxicity assay in presence of CyaA and CyaA*preprationsA. Khosravani* 1 , J. Coote 2 , R. Parton 21 Yasouj University of Medical Sciences, Microbiology & Immunology, Yasouj,Islamic Republic of Iran2 Glasgow University, infection and Immunity, Glasgow, United KingdomIntroduction:Adenylate cyclase toxin (CyaA) toxin is an importantvirulence factor ofBordetella pertussis,the causative agent of whoopingcough, and a potential component of acellular pertussis vaccine. Theadenosine triphosphate (ATP) assay is an alternative assay for measuringcytotoxicity as it determines the number of viable cells in a culture basedon quantitation of ATP, by a bioluminescence method, which signals thepresence of metabolically-active cells.Material & Methods: The amount of ATP is directly related to cellnumbers. J774.2 (grown in RPMI 1640 medium), RBL-2H3 and sheepbone marrow mast cells (grown in DMEM medium) were treated withdifferent concentrations of CyaA or CyaA* for 2 h at 37°C. The CellTiter-GloÔ reagent was added to each sample and bioluminescence output wascompared to that of a negative control (untreated) (0% cytotoxicity)preparation.Results:According to this method 50% cytotoxicity for J774.2 cells wascaused by CyaA around 0.02 mg protein/ml but was not achieved byCyaA* up to 1.25 mg protein/ml. Little cell death (