Marcel Erhard

CBS Fungal Biodiversity Centre - Royal Netherlands Academy of Arts and Sciences, Utrecht, Utrecht, Netherlands

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Publications (35)122.75 Total impact

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    ABSTRACT: Altogether 285 dermatophyte isolates of 21 different species - including both Trichophyton rubrum and T. interdigitale, but also eight additional Trichophyton species, Microsporum canis and seven other Microsporum species, as well as Epidermophyton floccosum and Arthroderma spp. - were analyzed using Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and the AnagnosTec 'SARAMIS' (Spectral Archiving and Microbial Identification System) software. In addition, sequence analysis of the internal transcribed spacer (ITS) of the ribosomal DNA was performed for a high number of the tested strains. Sufficient agreement was found between the results obtained with standard identification methods and those with the MALDI-TOF MS for species identification of dermatophytes. A mass spectra database was constructed which contained the species identifications of all 285 isolates. The results were confirmed for 164 of the isolates by sequence analysis of the internal transcribed spacer (ITS) of the ribosomal DNA. Statistical analysis of all 285 dermatophyte strains showed that conventional identification matched the results of MALDI-TOF MS for 78.2% of the isolates tested. In the case of the 164 isolates for which the identifications were confirmed by PCR, the results of their conventional diagnosis and MALDI-TOF MS were in agreement for only 68.9 % (113 of 164 strains) of the test isolates. In contrast, there was agreement of 99.3 % or 98.8 % in the identifications obtained with PCR and MALDI-TOF MS techniques (283/285 or 162/164). The two exceptions were isolates that proved to be T. violaceum which could not be identified by the MALDI-TOF MS technique. In conclusion, the MALDI-TOF mass spectroscopy represents a fast and very specific method for species differentiation of dermatophytes grown in culture.
    Medical mycology: official publication of the International Society for Human and Animal Mycology 05/2012; · 2.13 Impact Factor
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    ABSTRACT: Clinical microbiology is a conservative laboratory exercise where base technologies introduced in the 19th century remained essentially unaltered. High-tech mass spectrometry (MS) has changed that. Within a few years following its adaptation to microbiological diagnostics, MS has been introduced, embraced, and broadly accepted by clinical microbiology laboratories throughout the world as an innovative tool for definitive bacterial species identification. Herein, we review the current state of the art with respect to this exciting new technology and discuss potential future applications.
    Journal of clinical microbiology 02/2012; 50(5):1513-7. · 4.16 Impact Factor
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    ABSTRACT: Some members of the genus Candida are among the most common human fungal pathogens and cause serious diseases especially in immunocompromised people. A yeast was isolated from a blood culture from an immunocompromised cancer patient who suffered from acute pneumonia. The growth characteristics of the yeast on CHROMagar Candida were similar to those of Candida tropicalis, whereas the API ID 32C system identified the yeast as Candida silvicola. On the basis of the nucleotide divergence in the D1/D2 domain of the 26S nuclear rRNA (nrRNA) gene, as well as the internal transcribed spacer (ITS) domain of the nrRNA gene region, a new species, Candida pseudoaaseri sp. nov. with type strain VK065094 (CBS 11170(T)), which was found to be closely related to Candida aaseri, is proposed. While C. aaseri strains were susceptible to all tested antifungals, the new species is resistant to flucytosine and may also be distinguished from C. aaseri by its ability to assimilate l-rhamnose, whereas its colony morphology on CHROMagar Candida may be helpful for differentiation.
    Journal of clinical microbiology 12/2011; 49(12):4195-202. · 4.16 Impact Factor
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    ABSTRACT: Species identification of yeasts is based on biochemical (e.g. API ID 32 C®, bioMérieux) and molecular biological approaches. As an alternative to DNA-dependent methods, mass spectral analysis based identification of micro-organisms has become increasingly recognized. In a number of studies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied for the rapid classification and identification of micro-organisms. In this study, the applicability of MALDI-TOF MS for identifying yeasts isolated from dermatological patients was analysed and compared with the results from the API ID 32 C® system. Furthermore, sequencing the internal transcribed spacer (ITS) regions of the ribosomal DNA was employed as reference method. Candida (C.) albicans was isolated in 41.9% of all cases, C. parapsilosis in 20.3%, C. glabrata in 10.8%, and C. krusei in 6, 8.1%. Rarely isolated yeasts were Candida colliculosa, famata, guilliermondii, lusitaniae, and tropicalis as well as Geotrichum candidum, Rhodotorula mucilaginosa and Trichosporon mucoides. The MALDI TOF results were equal to the results gained by ITS sequence analysis in 94%, whereas API ID 32 C® provided the correct diagnosis in 84.3% (of all cases). This lower identification rate is mostly referable to frequent misidentifications of C. krusei as C. inconspicua/norvegensis,Candida tropicalis, or Geotrichum capitatum. In contrast, all C. krusei strains were correctly identified by MALDI TOF MS. In conclusion, species identification by MALDI-TOF MS was proven to be consistent with ITS sequence analysis; the technique has a resolving power comparatively as high as ITS sequence analysis.
    Mycoses 08/2011; 55(3):276-80. · 1.28 Impact Factor
  • Mycoses 05/2011; 54(6):e870-6. · 1.28 Impact Factor
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    ABSTRACT: Gram-positive anaerobic cocci (GPAC) are part of the commensal microbiota of humans and are a phylogenetically heterogeneous group of organisms. To evaluate the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of GPAC, a database was constructed, using reference strains of commonly encountered GPAC and clinical isolates of which the sequence of the 16S rRNA gene was determined. Subsequently, the database was validated by identifying 107 clinical isolates of GPAC. Results were compared with the identifications obtained by 16S sequencing or fluorescent in situ hybridization (FISH). Strains belonging to the same species grouped together, in most cases, by MALDI-TOF MS analyses. Strains with sequence similarities less than 98% to their closest relatives, formed clusters distinct from recognized species in the MALDI-TOF MS dendrogram and, therefore could not be identified. These strains probably represent new species. Only three clinical isolates (2 strains of Finegoldia magna and 1 strain of Anaerococcus vaginalis) could not be identified. For all the other GPAC strains (96/107), reliable identifications were obtained. Therefore, we concluded that MALDI-TOF MS is an excellent tool for the identification of phylogenetically heterogeneous groups of micro-organisms such as GPAC.
    Systematic and Applied Microbiology 02/2011; 34(1):58-62. · 3.29 Impact Factor
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    ABSTRACT: The Streptococcus bovis/equinus complex is a heterogeneous group within the group D streptococci with important clinical relevance regarding infective endocarditis, sepsis and colon carcinoma. The taxonomic identification of species and sub-species of this complex, by the standard methods remains difficult. In the present study, we compared the cluster analysis of 88 strains of species of the S. bovis/equinus complex by sequence analysis of the manganese-dependent superoxide dismutase gene (sodA) and by Matrix Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). We observed a high congruence of strain grouping by MALDI-TOF MS in comparison with sodA sequence analyses, demonstrating the accuracy and reliability of MALDI-TOF MS in comparison to DNA sequence-based method. By generating mass spectra for each species and sub-species, we were able to discriminate all members of the S. bovis/equinus complex. Furthermore, we demonstrated reliable identifications to the species level by MALDI-TOF MS, independently of cultivation conditions.
    Systematic and Applied Microbiology 02/2011; 34(1):52-7. · 3.29 Impact Factor
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    ABSTRACT: Immediately after the first MALDI-TOF instrument became available, microbiologists have been investigating its potential as a platform for high throughput identification of microorganisms. A remarkable finding very early in these investigations was that despite the dynamic nature of the bacterial cell, components of the mass spectral profile were sufficiently stable and remained unchanged in spite of changes in environmental parameters. Further, mass spectral pattern have been found to be taxon specific and thus, consequently, numerous methods were reported that purported to provide an alternative to current identification systems. The SARAMIS system described herein sequentially extracts core stable mass ions from analyses of multiple individual strains of a particular species to yield a ‘SuperSpectrum’, a list of biomarkers that are weighted according to their specificity from family to (sub)species levels. This approach has been used successfully to identify microorganisms from diverse phylogenetic lines of bacteria and fungi with considerable success. The protocol described has evolved of over years of experimental work to yield a robust system that can readily be applied for routine microbiological identification in a clinical diagnostic laboratory.
    06/2010: pages 255 - 276; , ISBN: 9780470665497
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    ABSTRACT: Insgesamt sind 12 dermatologisch relevante Microsporum-(M.-)Arten bekannt. Am häufigsten werden M. canis, M.audouinii und M.gypseum nachgewiesen. Wir berichten über eine Infektion mit dem seltenen Erreger M. fulvum am rechten Unterarm. Zur Diagnostik wurden Schuppen von den Hautläsionen gewonnen. Mittels Nativpräparat konnte frühzeitig eine Mykose nachgewiesen werden. Nach kultureller Anzucht wurde zunächst die Diagnose einer Infektion mit dem geophilen Erreger M. gypseum vermutet, mittels ITS-Sequenzierung und Massenspektrometrie konnte schließlich M. fulvum nachgewiesen werden. There are 12 dermatologically relevant Microsporum (M.) species. The most frequent species are M.canis, M.audouinii and M.gypseum. We report an infection of the right forearm with the rare dermatophyte M.fulvum. A KOH examination of scales revealed a tinea corporis. The scales were cultured on Dermasel® agar with the identification of the geophilic dermatophyte M.gypseum. However, ITS sequencing and mass spectrometry revealed M.fulvum as the correct pathogen. Schlüsselwörter Microsporum fulvum - Microsporum gypseum -ITS-Sequenzierung-MALDI-TOF-Massenspekrometrie-Geophil Keywords Microsporum fulvum - Microsporum gypseum -ITS sequencing-MALDI-TOF mass spectrometry-Geophilic
    Der Hautarzt 01/2010; 61(8):694-699. · 0.50 Impact Factor
  • ChemInform 01/2010; 33(17).
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    ABSTRACT: There are 12 dermatologically relevant Microsporum (M.) species. The most frequent species are M. canis, M. audouinii and M. gypseum. We report an infection of the right forearm with the rare dermatophyte M. fulvum. A KOH examination of scales revealed a tinea corporis. The scales were cultured on Dermasel(R) agar with the identification of the geophilic dermatophyte M. gypseum. However, ITS sequencing and mass spectrometry revealed M. fulvum as the correct pathogen.
    Der Hautarzt 09/2009; 61(8):694-9. · 0.50 Impact Factor
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    ABSTRACT: Microcystins (MCs) are cyclic heptapeptides, which are the most abundant toxins produced by cyanobacteria in freshwater. The phytoplankton of many freshwater lakes in Eastern Africa is dominated by cyanobacteria. Less is known, however, on the occurrence of MC producers and the production of MCs. Twelve Ugandan freshwater habitats ranging from mesotrophic to hypertrophic conditions were sampled in May and June of 2004 and April of 2008 and were analyzed for their physicochemical parameters, phytoplankton composition, and MC concentrations. Among the group of the potential MC-producing cyanobacteria, Anabaena (0-10(7) cells ml(-1)) and Microcystis (10(3)-10(7) cells ml(-1)) occurred most frequently and dominated in eutrophic systems. A significant linear relationship (n = 31, r(2) = 0.38, P < 0.001) between the Microcystis cell numbers and MC concentration (1.3-93 fg of MC cell(-1)) was observed. Besides [MeAsp(3), Mdha(7)]-MC-RR, two new MCs, [Asp(3)]-MC-RY and [MeAsp(3)]-MC-RY, were isolated and their constitution was assigned by LC-MS(2). To identify the MC-producing organism in the water samples, (i) the conserved aminotransferase domain part of the mcyE gene that is indicative of MC production was amplified by general primers and cloned and sequenced, and (ii) genus-specific primers were used to amplify the mcyE gene of the genera Microcystis, Anabaena, and Planktothrix. Only mcyE genotypes that are indicative of Microcystis sp. were obtained via the environmental cloning approach (337 bp, 96.1-96.7% similarity to the Microcystis aeruginosa strain PCC7806). Accordingly, only the mcyE primers, which are specific for Microcystis, revealed PCR products. We concluded that Microcystis is the major MC-producer in Ugandan freshwater.
    Environmental Toxicology 07/2009; 25(4):367-80. · 2.71 Impact Factor
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    ABSTRACT: Variations in the mass spectral profiles of multiple housekeeping proteins of 126 strains representing Salmonella enterica subsp. enterica (subspecies I), S. enterica subsp. salamae (subspecies II), S. enterica subsp. arizonae (subspecies IIIa), S. enterica subsp. diarizonae (subspecies IIIb), S. enterica subsp. houtenae (subspecies IV), and S. enterica subsp. indica (subspecies VI), and Salmonella bongori were analyzed to obtain a phylogenetic classification of salmonellae based on whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometric bacterial typing. Sinapinic acid produced highly informative spectra containing a large number of biomarkers and covering a wide molecular mass range (2,000 to 40,000 Da). Genus-, species-, and subspecies-identifying biomarker ions were assigned on the basis of available genome sequence data for Salmonella, and more than 200 biomarker peaks, which corresponded mainly to abundant and highly basic ribosomal or nucleic acid binding proteins, were selected. A detailed comparative analysis of the biomarker profiles of Salmonella strains revealed sequence variations corresponding to single or multiple amino acid changes in multiple housekeeping proteins. The resulting mass spectrometry-based bacterial classification was very comparable to the results of DNA sequence-based methods. A rapid protocol that allowed identification of Salmonella subspecies in minutes was established.
    Applied and environmental microbiology 11/2008; 74(24):7767-78. · 3.69 Impact Factor
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    ABSTRACT: The present study examined the potential of intact-cell matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid identification of Burkholderia cepacia complex (Bcc) bacteria using an Applied Biosystems 4700 Proteomics Analyser. Two software packages were used to analyse mass profiles based on densitometric curves and peak positions. The 75 strains examined, represented the nine established Bcc species and some commonly misidentified species, closely related or biochemically similar to Bcc and relevant in the context of cystic fibrosis microbiology. All Bcc strains clustered together, separated from non-Bcc strains. Within Bcc, most Bcc strains grouped in species specific clusters, except for Burkholderia anthina and Burkholderia pyrrocinia strains which constituted a single cluster. The present study demonstrates that MALDI-TOF MS is a powerful approach for the rapid identification of Bcc bacteria.
    Journal of Microbiological Methods 10/2008; 75(2):279-86. · 2.16 Impact Factor
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    ABSTRACT: Although mycoses are among the most common diseases worldwide, infections with Fusarium spp. occur only rarely. Mostly patients suffering from underlying immune deficiency are infected with this mould, resulting in a considerably decreasing prognosis. In immunocompromised patients, cutaneous manifestations are more often associated with Fusarium sp. than with Candida sp. or Aspergillus sp. We describe one patient with acute lymphoblastic leukaemia, who was first treated with chemotherapy after GMALL protocol 07/03. After relapse, the patient was successfully transplanted in second remission with a human leukocyte antigen (HLA)-matched unrelated peripheral blood stem cell graft. Ten months later, the patient died from respiratory insufficiency and recurrence of leukaemia. Previously, Aspergillus antigen was detected in blood. In the latter course, disseminated papules appeared. One of these was examined histologically and mycologically. Conventional cultural diagnostics led to the diagnosis of a fusariosis, further supported by internal transcribed spacer (ITS) sequencing and matrix assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry, both determining the isolated strain as Fusarium proliferatum, which is a very infrequent pathogen within this genus. Our investigations underline the potential of MALDI-TOF MS based identification of Fusarium species as an innovative, time and cost efficient alternative to ITS sequencing.
    Experimental Dermatology 07/2008; 17(11):965-71. · 3.58 Impact Factor
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    ABSTRACT: Purpose – The purpose of this paper is to present a novel microbiological lab robot that facilitates high through-put sample preparation for rapid state-of-the-art identification. Design/methodology/approach – Development concentrated on two main points: research initially focused on various methods for picking a micro-organism colony from a petri dish without any medium adhering; and subsequently on completely documenting sample handling with little effort. Findings – A sensorless system for picking micro-organisms from a petri dish was engineered and prototyped. A documented process in the demonstrator demonstrates its usability even for certified clinical operations. Research limitations/implications – The handling of solid phase biological objects is only in its infancy. This research focused on the preparation of samples from micro-organism colonies for MALDI-TOF MS. A specific type of gripper was developed to do this. The handling of other biological objects, e.g. from cell cultures or intermediate stages of tissue engineering, is still a largely open field for future research. Practical implications – New analysis methods often only become accepted when the preparatory processes are also taken into account – highly parallel operations (e.g. MALDI-TOF MS) are particularly impractical for humans and difficult for data handling to manage. Given the specific demands, only an interdisciplinary team can adapt the automation engineering successfully. Originality/value – This paper presents an approach to and implementation of the automation of manual operations in biotechnology. It is intended to encourage health professionals, biologists and engineers to jointly research and interdisciplinarily automate complex operations.
    Industrial Robot 06/2008; 35(4):311-315. · 0.69 Impact Factor
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    ABSTRACT: Anatoxin-a (ANTX) and homoanatoxin-a (HANTX) are low molecular weight neurotoxic secondary amines of 165 and 179 Da, respectively. We applied matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for the detection of ANTX and HANTX directly on lyophilized and fresh filaments of axenic strains of the genus Oscillatoria, using 2,5-dihydroxybenzoic acid as matrix and purified ANTX and HANTX as references. To counteract the span of low molecular mass ions (< m/z 1000) generated by the matrix, we induced the matrix-suppression effect to obtain high quality ANTX/HANTX MALDI signals. MALDI desorption/ionization of the matrix-ANTX and the matrix-HANTX generated protonated molecules [M+H](+) at m/z 166.12322 and 180.1372, respectively. The masses obtained from the analysis of lyophilized filaments of the ANTX-producer Oscillatoria sp. strain PCC 9240 (m/z 166.15) and of fresh filaments of the HANTX-producers Oscillatoria sp. strains PCC 6506 (m/z 180.1375), PCC 9029 (m/z 180.1334) and PCC 10111 (m/z 180.13996) corresponded to the protonated molecular ions of ANTX and HANTX, respectively. Therefore, the application of MALDI-TOF-MS for the detection of cyanobacterial anatoxins in clonal and axenic strains of the cyanobacterial culture collections worldwide may help to assess ANTX/HANTX incidence among cyanobacteria.
    Toxicon 06/2008; 51(7):1308-15. · 2.92 Impact Factor
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    ABSTRACT: Identification of dermatophytes is currently performed based on morphological criteria and is increasingly supported by genomic sequence comparison. The present study evaluates an alternative based on the analysis of clinical fungal isolates by mass spectrometry. Samples originating from skin and nail were characterized morphologically and by sequencing the internal transcribed spacer 1 (ITS1), ITS2 and the 5.8S rDNA regions of the rDNA clusters. In a blind comparative study, samples were analyzed by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF MS). The mass spectra were compared to a database comprising of the spectral data of reference strains by applying the saramis software package. All fungal isolates belonging to the taxa Trichophyton rubrum, T. interdigitale, T. tonsurans, Arthroderma benhamiae and Microsporum canis were correctly identified, irrespective of host origin and pathology. To test the robustness of the approach, four isolates were grown on five different media and analyzed. Although the resulting mass spectra varied in detail, a sufficient number of signals were conserved resulting in data sets exploitable for unequivocal species identification. Taken together, the usually widespread dermatophytes can be identified rapidly and reliably by mass spectrometry. Starting from pure cultures, MALDI-TOF MS analysis uses very simple sample preparation procedures, and a single analysis is performed within minutes. Costs for consumables as well as preparation time are considerably lower than for PCR analysis.
    Experimental Dermatology 05/2008; 17(4):356-61. · 3.58 Impact Factor
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    Martin Welker, Marcel Erhard
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    ABSTRACT: The high metabolic diversity of potentially toxigenic cyanobacteria has been recently demonstrated for several bloom-forming genera. To understand the dynamics of cyanobacterial blooms and fluctuations in toxin content, techniques are required that allow the distinction of clones in field samples on a subspecific taxonomic level. We evaluated the possibility to analyze single filaments of a common toxigenic taxon, Planktothrix rubescens, by comparing peptide profiles obtained from HPLC/MS analyses of bulk biomass to mass spectra of single filaments for 11 clonal strains. For all strains a significant relationship of chromatogram peak area to mass spectrum peak height was found for individual peptides. Individual peptide's response in mass spectrometry compared to HPLC, however, differed, likely attributable to the presence of particular moieties with high proton affinities in the molecules. Thirty-four individual peptides detected in HPLC fractions of all strains could also be detected in the corresponding single filaments. In conclusion, MALDI-TOF MS analysis of single filaments is shown to be an efficient tool for the study of chemotype diversity and dynamics in field studies and laboratory experiments. The good correspondence between peak area and peak height in chromatograms and mass spectra, respectively, is promising with respect to the use of MALDI-TOF MS as a quantitative analytical tool for natural products studies and cyanotoxin research.
    Journal of Mass Spectrometry 09/2007; 42(8):1062-8. · 3.21 Impact Factor
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    ABSTRACT: Nine Corallococcus isolates and three type strains of Corallococcus species were characterized by Intact Cell Mass Spectrometry using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The resulting phenetic clustering was compared to the phylogenetic grouping based upon sequences of two housekeeping genes. The three dendrograms of relatedness resembled each other in that the isolates were highly similar to the type strains of Corallococcus exiguus and Corallococcus coralloides, while Corallococcus macrosporus and Myxococcus xanthus were more distantly related. While certain pairs of organisms were recovered by spectrometry and genes sequence analysis, others were detected by two of the three approaches. The degree of similarity determined by sequence analysis of the two genes was not higher than that revealed by MALDI-TOF analysis. The results show that the spectral profile, consisting of about 25 to 45 masses ranging between 2 and 20 kDa, have indeed taxonomic significance, confirming literature data that ribosomal proteins and certain housekeeping proteins are responsible for the masses obtained. Provided the availability of a database of type strains, MALDI-TOF analysis of unknown strains appears to be a rapid and inexpensive method to taxonomically cluster environmental isolates, expanding the spectrum to strains other than those of medical importance predominantly investigated so far.
    Current Microbiology 03/2005; 50(2):71-7. · 1.52 Impact Factor

Publication Stats

1k Citations
122.75 Total Impact Points

Institutions

  • 2011
    • CBS Fungal Biodiversity Centre - Royal Netherlands Academy of Arts and Sciences
      Utrecht, Utrecht, Netherlands
    • Charité Universitätsmedizin Berlin
      Berlín, Berlin, Germany
  • 2008–2011
    • Universitätsklinikum Jena
      • Klinik für Hautkrankheiten
      Jena, Thuringia, Germany
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 2004–2011
    • Telekom Germany GmbH
      Bonn, North Rhine-Westphalia, Germany
  • 1997–2007
    • Technische Universität Berlin
      • Department of Chemistry
      Berlin, Land Berlin, Germany
  • 2005
    • Leibniz Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
      Brunswyck, Lower Saxony, Germany
  • 2000
    • University of Freiburg
      • Institute of Biology I
      Freiburg, Lower Saxony, Germany