Katrin Sparbier

Max von Pettenkofer-Institut, München, Bavaria, Germany

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Publications (25)55.8 Total impact

  • Katrin Sparbier · Sören Schubert · Markus Kostrzewa
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    ABSTRACT: The increasing resistance to antibiotics is an urgent health care problem. Detection of resistant microorganisms is the pre-requisite for initiating an adequate therapy and implementing respective hygiene measures. Depending on the species and the method employed for analysis, the time to result of antibiotic resistance testing ranges between five and 24 h. As MALDI-TOF MS has become an established tool for the fast species identification in microbiological laboratories a time gap between the results of species identification and the information about antibiotic susceptibility arises. Here, we present a semi-quantitative MALDI-TOF MS-based approach for the detection of resistance in different species against different antibiotics.
    No preview · Article · Jan 2016 · Methods
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    ABSTRACT: Antibiotic resistance in Gram-negative microorganisms is an increasing health care problem. The rapid detection of such resistance is crucial for starting an early specific therapy and to enable initiation of the required hygiene measures. With continued emphasis on reducing the cost of laboratory testing, only economical/low-cost approaches have a chance of being implemented. During recent years, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been developed to be a standard method in microbiology laboratories for the rapid and cost-efficient identification of microorganisms. Extending the usage of MALDI-TOF MS in the clinical microbiology laboratory to the area of resistance testing is an attractive option. Quantitative MALDI-TOF MS using an internal standard facilitates the measurement of the quantity of peptides and small proteins within a spectrum. These quantities correlate to the number of microorganisms and therefore to the growth of a microorganism. The comparison of growth in the presence or absence of an antibiotic allows for analysis of the susceptibility behavior of a strain. Here, we describe a novel method and its application in the analysis of 108 Klebsiella sp. isolates. After 1 h of incubation at a meropenem concentration of 8 μg/ml, a sensitivity of 97.3% and a specificity of 93.5% were achieved (compared to Etest results).
    Full-text · Article · Sep 2014 · Journal of Clinical Microbiology
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    ABSTRACT: The identification of pathogens directly from blood cultures by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) can be a valuable tool for improving the treatment of patients with sepsis and bacteremia. However, the increasing incidence of multidrug-resistant Gram-negative bacteria makes it difficult to predict resistance patterns based only on pathogen identification. Most therapy regimens for sepsis caused by Gram-negative rods consist of at least one β-lactam antibiotic. Thus, it would be of great benefit to have an early marker of resistance against these drugs. In the current study, we tested 100 consecutive blood cultures containing Enterobacteriaceae for resistance against 3rd-generation cephalosporins in a MALDI-TOF MS β-lactamase assay. Escherichia coli was also tested for resistance against aminopenicillins. The results of the β-lactamase assay were compared with those of conventional methods. The assay permitted discrimination between E. coli strains that were resistant or susceptible to aminopenicillins with a sensitivity and a specificity of 100%. The same was true for resistance to 3rd-generation cephalosporins in Enterobacteriaceae that constitutively produced class C β-lactamases. Discrimination was more difficult in species expressing class A β-lactamases, as these enzymes can generate false-positive results. Thus, the sensitivity and specificity for this group were 100% and 91.5%, respectively. The test permitted the prediction of resistance within 2.5 h after the blood culture was flagged as positive.
    Full-text · Article · Jan 2014 · Journal of clinical microbiology
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    J S Jung · T Eberl · K Sparbier · C Lange · M Kostrzewa · S Schubert · A Wieser
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    ABSTRACT: With the emergence and growing complexity of bacterial drug resistance, rapid and reliable susceptibility testing has become a topical issue. Therefore, new technologies that assist in predicting the effectiveness of empiric antibiotic therapy are of great interest. Although the use of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid detection of antibiotic resistance is an attractive option, the current methods for MALDI-TOF MS susceptibility testing are restricted to very limited conditions. Here, we describe a technique that may allow for rapid susceptibility testing to an extent that is comparable to phenotypic methods. The test was based on a stable isotope labelling by amino acids in cell culture (SILAC)-like approach. This technique was used to visualise the growth of bacteria in the presence of an antibiotic. Pseudomonas aeruginosa was chosen as the model organism, and strains were incubated in normal medium, medium supplemented with (13)C6-(15) N2-labelled lysine and medium supplemented with labelled lysine and antibiotic. Peak shifts occurring due to the incorporation of the labelled amino acids were detected by MALDI-TOF MS. Three antibiotics with different mechanisms of action, meropenem, tobramycin and ciprofloxacin, were tested. A semi-automated algorithm was created to enable rapid and unbiased data evaluation. With the proposed test, a clear distinction between resistant and susceptible isolates was possible for all three antibiotics. The application of SILAC technology for the detection of antibiotic resistance may contribute to accelerated and reliable susceptibility testing.
    Full-text · Article · Dec 2013 · European Journal of Clinical Microbiology
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    Markus Kostrzewa · Katrin Sparbier · Thomas Maier · Sören Schubert
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    ABSTRACT: MALDI-TOF mass spectrometry profiling for microorganism detection has already been demonstrated in the 1990s, but has evolved to the first line identification method in many laboratories just during the past five years. While this application of MALDI-TOF MS has proven its broad applicability, accuracy, robustness, and cost-effectiveness it is of particular interest to expand the capabilities of the mass spectrometric platform. Resistance detection is the most desirable further application of MALDI-TOF MS in microbiology, but maybe also the most challenging. Different approaches have been published regarding diverse antibiotic drugs and distinct microorganism classes. The current review shall give an overview about the developments of the recent years and their potential to get transformed in clinical useful assays in the future.
    Full-text · Article · Dec 2013 · PROTEOMICS - CLINICAL APPLICATIONS
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    ABSTRACT: Against the background of increasing numbers of resistant microorganisms, the fast and cost-efficient detection of microbial resistance is an important clinical requirement for optimal therapeutic intervention. Current routine assays take at least 5 h, but in most cases an overnight incubation is necessary to identify resistant isolates. The usage of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) profiling in combination with growth media containing isotopically labeled amino acids facilitates the detection of resistant microorganisms after 3 h or less directly from the profile spectrum. Growing microorganisms incorporate isotopically labeled amino acids, increasing protein masses and thereby leading to mass shifts of their corresponding peaks in the profile spectra. In the presence of antibiotics, only resistant microorganisms are able to grow and to incorporate the labeled amino acids. This leads to a difference in the mass spectra of susceptible and resistant isolates, allowing their differentiation. In the presented study, we demonstrated the applicability of this novel approach for the detection of methicillin-resistant Staphylococcus aureus and tested different bioinformatics approaches for automated data interpretation.
    Full-text · Article · Sep 2013 · Journal of clinical microbiology
  • K. Sparbier · C. Lange · J. Jung · S. Schubert · M. Kostrzewa

    No preview · Conference Paper · Sep 2013

  • No preview · Article · Sep 2013 · International Journal of Medical Microbiology Supplements
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    ABSTRACT: Nosocomial infections involving epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains are a serious problem in many countries. In order to analyze outbreaks, the infectious isolates have to be typed; however, most molecular methods are expensive or labor-intensive. Here, we evaluated matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) of cell extracts for the molecular characterization of S. aureus strains. The peak patterns of 401 MRSA and methicillin-susceptible S. aureus (MSSA) strains, including clinical and laboratory strains, were analyzed. Database searches indicated the peptides that were represented by the corresponding peaks in the spectra. The identities of the peptides were confirmed by the sequencing of mutants, the expression of antisense RNA fragments that resulted in the knockdown of the peptide of interest and the concomitant loss of the signal, or tandem MALDI-TOF MS (MALDI-TOF/TOF MS). It was shown that the signals derive mainly from stress proteins and ribosomal proteins. Peak shifts that differentiate the main S. aureus clonal complexes CC5, CC22, CC8, CC45, CC30, and CC1 correlate to point mutations in the respective genes. Retrospective typing of an MRSA outbreak showed that it is possible to differentiate unrelated MSSA, MRSA, and borderline resistant S. aureus (BORSA) strains isolated from health care workers. In conclusion, this method allows for the detection of the epidemic lineages of S. aureus during species identification by MALDI-TOF MS analysis.
    Full-text · Article · Apr 2013 · Journal of clinical microbiology
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    ABSTRACT: Carboxyterminally elongated and aminoterminally truncated Aβ peptides as well as their pyroglutamate and oxidized derivates are major constituents of human amyloid plaques. The objective of the present study was to characterize aminoterminally truncated or oxidized Aβ38, Aβ40, and Aβ42 peptide species in immunoprecipitated human cerebrospinal fluid (CSF). We invented a novel sequential aminoterminally and carboxyterminally specific immunoprecipitation protocol and used the Aβ-SDS-PAGE/immunoblot for subsequent analysis of CSF Aβ peptide patterns. In the present study, we identified the aminoterminally truncated Aβ peptides 2-40 and 2-42 as well as oxidized forms of Aβ1-38 and Aβ1-42 in CSF. Our protocol allowed the quantification of a pattern of Aβ peptides 1-38(ox), 2-40, and 2-42 in addition to the well known panel of Aβ 1-37, 1-38, 1-39, 1-40, 1-40(ox), and 1-42 in a group of seven patients with peripheral polyneuropathy. In the present approach, we could broaden the range of quantifiable Aβ peptides described in previous studies (i.e., 1-37, 1-38, 1-39, 1-40, 1-40(ox), and 1-42) by Aβ 1-38(ox), 2-40, and 2-42. An exact analysis of CSF Aβ peptides regarding their carboxy- and aminoterminus as well as posttranslational modification seems promising with respect to diagnostic and pathogenic aspects.
    No preview · Article · Apr 2012 · PROTEOMICS - CLINICAL APPLICATIONS
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    K. Sparbier · S. Schubert · U. Weller · C. Boogen · M. Kostrzewa
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    ABSTRACT: Resistance against ss-lactam antibiotics is a growing challenge for managing severe bacterial infections. The rapid and cost efficient determination of ss-lactam resistance is an important prerequisite for the choice of an adequate antibiotic therapy. ss-lactam resistance is mainly based on the expression/overexpression of ss-lactamases destroying the central ss-lactam ring of these drugs by hydrolysis. Hydrolysis corresponds to a mass shift of plus 18 Da which can be easily detected by MALDI-TOF mass spectrometry. Therefore, a MALDI-TOF MS based assay was set up to investigate different enterobacteria for resistance against different ss-lactam antibiotics: Ampicillin, Piperacillin, Cefotaxime, Ceftazidime, Ertapenem, Imipenem and Meropenem. ss-lactamases are enzymes comprising a high turn over rate. Therefore, hydrolysis can already be detected by MALDI-TOF MS after a few hours of incubation of the bacteria to be tested with the respective antibiotic. The comparison of the MS-derived data with the dat
    Full-text · Article · Dec 2011
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    ABSTRACT: Resistance against β-lactam antibiotics is a growing challenge for managing severe bacterial infections. The rapid and cost-efficient determination of β-lactam resistance is an important prerequisite for the choice of an adequate antibiotic therapy. β-Lactam resistance is based mainly on the expression/overexpression of β-lactamases, which destroy the central β-lactam ring of these drugs by hydrolysis. Hydrolysis corresponds to a mass shift of +18 Da, which can be easily detected by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Therefore, a MALDI-TOF MS-based assay was set up to investigate different enterobacteria for resistance against different β-lactam antibiotics: ampicillin, piperacillin, cefotaxime, ceftazidime, ertapenem, imipenem, and meropenem. β-Lactamases are enzymes that have a high turnover rate. Therefore, hydrolysis can be detected by MALDI-TOF MS already after a few hours of incubation of the bacteria to be tested with the given antibiotic. The comparison of the MS-derived data with the data from the routine procedure revealed identical classification of the bacteria according to sensitivity and resistance. The MALDI-TOF MS-based assay delivers the results on the same day. The approved routine procedures require at least an additional overnight incubation.
    Full-text · Article · Dec 2011 · Journal of clinical microbiology
  • K Sparbier · U Weller · C Boogen · M Kostrzewa
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    ABSTRACT: The identification of Salmonella sp. in stool samples usually takes 2 days when employing routine procedures. Fast approaches are necessary in order to shorten the analysis time. The aim of this work was the development of a rapid procedure for the detection of Salmonella sp. from clinical stool samples. Spiked stool samples were cultured in selective selenite enrichment broth. Identifications were directly performed from the liquid broth by the MALDI Biotyper. After the evaluation of this method, the same procedure was applied to clinical samples. Coevally, the samples were streaked on Hektoen agar and single colonies were analyzed by the MALDI Biotyper. For comparison, the liquid broth was plated according to the standard laboratory procedure. A total of 4,847 samples were analyzed for Salmonella sp. In total, 108 Salmonella sp.-positive samples were identified; 66 of these were identified after the streaking of stool samples on Hektoen agar and subsequent MALDI Biotyper analysis of Salmonella sp. suspicious colonies. These and a further 34 samples were detected as Salmonella sp.-positive directly from the selenite enrichment broth on day one. Eight Salmonella sp.-positive samples were not detected before plating of the selenite broth and subsequent MALDI Biotyper analysis on day two. The combination of MALDI Biotyper analysis and selective selenite enrichment broth identification delivers positive results for the majority of the samples already after one day.
    No preview · Article · Aug 2011 · European Journal of Clinical Microbiology
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    ABSTRACT: The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI-TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI-TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI-TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.
    Full-text · Article · Mar 2009 · Proteomics
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    ABSTRACT: In routine clinical diagnostics, peptide biomarkers are most commonly quantified using immunological techniques but these methods often lack sensitivity and/or specificity. Hence, quantitative mass spectrometry detection is desirable as an alternative diagnostic tool. To date, quantitative mass spectrometry is mostly based on ESI-MS coupled to LC, requiring highly sophisticated instrumentation and knowledge and is time consuming and expensive. In contrast, MALDI-TOF-MS is a very simple, sensitive and rapid method for the detection of peptide biomarkers. However, the infeasibility of absolute quantification has been a tremendous handicap to the use of MS in stable clinical diagnostics. Here, we describe the development of a technical platform based on ClinProt particles and heavy-isotope internal peptide standards for the fast and reliable preparation of samples. This combines the advantages of MALDI-TOF as a read-out system with absolute quantitation of peptide biomarkers. As a proof-of-concept, this platform was successfully employed for the absolute determination of the concentration of the highly abundant serum peptide des-Ala-Fibrinopeptide A in 45 serum samples from healthy donors. Such technology essentially contributes to the development of a stable MALDI-TOF-MS-based clinical assay.
    No preview · Article · Oct 2007 · PROTEOMICS - CLINICAL APPLICATIONS
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    ABSTRACT: Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins, and automated analysis workflows enabling the detection, identification, and structural characterization of the corresponding peptide modifications. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task. selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids), and isolated proteins were digested with trypsin. Subsequently, the resulting complex mixture of peptides and glycopeptides was subjected to LC-MALDI analysis and database searching. In parallel, a second magnetic bead capturing was performed on the peptide level to separate and analyze by LC-MALDI intact glycopeptides, both peptide sequence and glycan structure. Detection of glycopeptides was achieved by means of a software algorithm that allows extraction and characterization of potential glycopeptide candidates from large LC-MALDI-MS/MS data sets, based on N-glycopeptide-specific fragmentation patterns and characteristic fragment mass peaks, respectively. By means of fast and simple glycospecific capturing applied in conjunction with extensive LC-MALDI-MS/MS analysis and novel data analysis tools, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from the experiments using either magnetic ConA-, LCA-, WGA-, and boronic acid beads, respectively.
    Full-text · Article · Oct 2007 · Journal of biomolecular techniques: JBT
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    ABSTRACT: 2,5-Dihydroxybenzoic acid (DHB) is the matrix of choice for carbohydrate and glycopeptide analysis, but due to the inhomogeneous surface morphology of samples prepared with DHB, it is typically incompatible with automated measurements.
    Full-text · Article · Jan 2007
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    M. Macht · C. Albers · K. Sparbier · A. Asperger · J. Glandorf · H. Thiele
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    ABSTRACT: Proteomic analyses typically produce massive amounts of mass spectrometric data, which are analyzed in an automated way by database search engines for retrieval of peptide sequences and subsequent inference on the corresponding protein sequences. However, this process turned out to be error prone, producing false positives and multiple hits for the same proteins for various reasons.
    Full-text · Article · Jan 2007
  • Katrin Sparbier · Thomas Wenzel · Markus Kostrzewa
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    ABSTRACT: Concanavalin A, boronic acid and Wheat germ agglutinin functionalized magnetic micro-particles were developed to enrich glycosylated peptides and proteins. The bead functionalities were validated according to their specificity by analyses of model proteins. Validated beads were employed for the enrichment of glycosylated human serum proteins. Eluted glycoproteins were digested by trypsin and the resulting peptides were purified by magnetic MB-HIC C8 beads. Each fraction was analyzed by MALDI-TOF MS and single peaks were subjected to MALDI-TOF/TOF MS with the objective to identify the respective proteins by database search. Search results revealed overlapping profiles of known serum glycoproteins.
    No preview · Article · Sep 2006 · Journal of Chromatography B
  • Article: P2-157

    No preview · Article · Jul 2006