Skip to main content
SpringerLink
Log in

Identification of Stachybotrys spp. by MALDI-TOF mass spectrometry

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

An Erratum to this article was published on 09 September 2016

Abstract

Stachybotrys (S.) spp. are omnipresent cellulolytic molds. Some species are highly toxic owing to their ability to synthesize various secondary metabolites such as macrocyclic trichothecenes or hemolysins. The reliable identification of Stachybotrys at species level is currently limited to genome-based identification. This study aimed to establish a fast and reliable MALDI-TOF MS identification method by optimizing the pre-analytical steps for protein extraction for subsequent generation of high-quality fingerprint mass spectra. Eight reference strains of the American Type Culture Collection and the Technical University of Denmark were cultivated in triplicate (biological repetitions) for 2 days in malt extract broth. The mycelia (1.5 ml) were first washed with 75 % ethanol and an additional washing step with dimethyl sulfoxide (10 %) was added to remove unspecific low weight masses. Furthermore, mycelia were broken with roughened glass beads in formic acid (70 %) and acetonitrile. The method was successfully applied to a total of 45 isolates of Stachybotrys originating from three different habitats (indoor, feed, and food samples; n = 15 each): Twenty-seven isolates of S. chartarum and 18 isolates of S. chlorohalonata could be identified by MALDI-TOF MS. The data obtained exactly matched those obtained by genome-based identification. The mean score values for S. chartarum ranged from 2.509 to 2.739 and from 2.148 to 2.622 for S. chlorohalonata with a very good reproducibility: the relative standard deviations were between 0.3 % and 6.8 %. Thus, MALDI-TOF MS proved to be a fast and reliable alternative to identification of Stachybotrys spp. by nucleotide amplification and sequencing.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Samson RA. Food and indoor fungi. CBS laboratory manual series; no. 2. Utrecht: CBS-KNAW Fungal Biodiversity Centre; 2010.

  2. Hibbett DS, Binder M, Bischoff JF, Blackwell M, Cannon PF, Eriksson OE, et al. A higher-level phylogenetic classification of the fungi. Mycol Res. 2007;111(Pt 5):509–47. doi:10.1016/j.mycres.2007.03.004.

    Article  Google Scholar 

  3. Norvell LL. Fungal nomenclature 1. Mycotaxon. 2011;116(481–490).

  4. The Royal Botanic Gardens Kew MS, Landcare Research-NZ MGaIoM, Chinese Academy of Science SKLoM. Index Fungorum. 2014. http://www.indexfungorum.org. Accessed 29 Jul 2014.

  5. NCBI. National Center for Biotechnology Information. 2014. http://www.ncbi.nlm.nih.gov/. Accessed 29 July 2014.

  6. Hernandez F, Cannon M. Inhibition of protein synthesis in Saccharomyces cerevisiae by the 12,13-epoxytrichothecenes trichodermol, diacetoxyscirpenol and verrucarin A. Reversibility of the effects. J Antibiot. 1982;35(7):875–81.

    Article  CAS  Google Scholar 

  7. Rocha O, Ansari K, Doohan FM. Effects of trichothecene mycotoxins on eukaryotic cells: a review. Food Addit Contam. 2005;22(4):369–78. doi:10.1080/02652030500058403.

    Article  CAS  Google Scholar 

  8. Ueno Y. Mode of action of trichothecenes. Ann Nutr Aliment. 1977;31(4–6):885–900.

    CAS  Google Scholar 

  9. Gareis M. Diagnostischer Zellkulturtest (MTT-Test) für den Nachweis von zytotoxischen Kontaminanten und Rückständen. JVL. 2006;1(4):354–63. doi:10.1007/s00003-006-0058-6.

    CAS  Google Scholar 

  10. Hanelt M, Gareis M, Kollarczik B. Cytotoxicity of mycotoxins evaluated by the MTT-cell culture assay. Mycopathologia. 1994;128(3):167–74. doi:10.1007/BF01138479.

    Article  CAS  Google Scholar 

  11. Hintikka E-L. Stachybotryotoxicosis in horses. In: Wyllie TD, Morehouse LG, editors. Mycotoxic fungi, mycotoxins, mycotoxicosis. An encyclopedia handbook. New York: Dekker; 1977. pp. 181–5.

  12. Nikulin M, Pasanen A-L, Berg S, Hintikka E-L. Stachybotrys atra growth and toxin production in some building materials and fodder under different relative humidities. Appl Environ Microbiol. 1994;81(16):3421–4.

    Google Scholar 

  13. Forgacs J. Stachybotryotoxicosis. In: Kadis S, Ciegler A, Ajl SJ, editors. Fungal toxins. New York, Academic; 1972.

  14. Kriek NPJ, Marasas WFO. Field outbreak of ovine stachybotryotoxicosis in South Africa. In: Ueno Y, editor. Thrichothecenes - chemical, biological and toxicological aspects. Amsterdam: Elsevier; 1983. p. 279–84.

    Google Scholar 

  15. Johanning E, Landsbergis P. Clinical findings related to indoor fungal exposure - review of clinic data of a specialty clinic New York: Eastern New York Occupational and Environmental Health Center, Albany New York; 1999.

  16. Dearborn DG, Smith PG, Dahms BB, Allan TM, Sorenson WG, Montana E, et al. Clinical profile of 30 infants with acute pulmonary hemorrhage in Cleveland. Pediatrics. 2002;110(3):627–37. doi:10.1542/peds.110.3.627.

    Article  Google Scholar 

  17. Vesper SJ, Magnuson ML, Dearborn DG, Yike I, Haugland RA. Initial characterization of the hemolysin stachylysin from Stachybotrys chartarum. Infect Immun. 2001;69(2):912–6. doi:10.1128/IAI.69.2.912-916.2001.

    Article  CAS  Google Scholar 

  18. Biermaier B, Gottschalk C, Schwaiger K, Gareis M. Occurrence of Stachybotrys chartarum chemotype S in dried culinary herbs. Mycotoxin Res. 2015;31(1):23–32. doi:10.1007/s12550-014-0213-3.

    Article  CAS  Google Scholar 

  19. Clark AE, Kaleta EJ, Arora A, Wolk DM. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology. Clin Microbiol Rev. 2013;26(3):547–603. doi:10.1128/CMR.00072-12.

    Article  CAS  Google Scholar 

  20. Mellmann A, Bimet F, Bizet C, Borovskaya AD, Drake RR, Eigner U, et al. High interlaboratory reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based species identification of nonfermenting bacteria. J Clin Microbiol. 2009;47(11):3732–4. doi:10.1128/JCM.00921-09.

    Article  CAS  Google Scholar 

  21. Watkinson S, Gooday GW, Money NP, Carlile MJ. The fungi. London: Academic; 2000.

    Google Scholar 

  22. MALDI. Biotyper protocol guide. 2nd ed. Bruker Daltonics: Bremen; 2014.

    Google Scholar 

  23. Hettick JM, Green BJ, Buskirk AD, Kashon ML, Slaven JE, Janotka E, et al. Discrimination of Penicillium isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting. Rapid Commun Mass Spectrom. 2008;22(16):2555–60. doi:10.1002/rcm.3649.

    Article  CAS  Google Scholar 

  24. Oliveira MM, Santos C, Sampaio P, Romeo O, Almeida-Paes R, Pais C, et al. Development and optimization of a new MALDI-TOF protocol for identification of the Sporothrix species complex. Res Microbiol. 2015;166(2):102–10. doi:10.1016/j.resmic.2014.12.008.

    Article  Google Scholar 

  25. Del Chierico F, Masotti A, Onori M, Fiscarelli E, Mancinelli L, Ricciotti G, et al. MALDI-TOF MS proteomic phenotyping of filamentous and other fungi from clinical origin. J Proteomics. 2012;75(11):3314–30. doi:10.1016/j.jprot.2012.03.048.

    Article  Google Scholar 

  26. Gruenwald M, Rabenstein A, Remesch M, Kuever J. MALDI-TOF mass spectrometry fingerprinting: a diagnostic tool to differentiate dematiaceous fungi Stachybotrys chartarum and Stachybotrys chlorohalonata. J Microbiol Meth. 2015;115:83–8. doi:10.1016/j.mimet.2015.05.025.

    Article  CAS  Google Scholar 

  27. Cruse M, Telerant R, Gallagher T, Lee T, Taylor JW. Cryptic species in Stachybotrys chartarum. Mycologia. 2002;94(5):814–22. doi:10.2307/3761696.

    Article  Google Scholar 

  28. Andersen B, Nielsen KF, Thrane U, Szaro T, Taylor JW, Jarvis BB. Molecular and phenotypic descriptions of Stachybotrys chlorohalonata sp. nov. and two chemotypes of Stachybotrys chartarum found in water-damaged buildings. Mycologia. 2003;95(6):1227–58.

    Article  CAS  Google Scholar 

  29. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Shinsky J, White T, editors. PCR protocols: a guide to methods and applications. London: Academic; 1990. p. 315–22.

    Google Scholar 

  30. MALDI. Biotyper 3.1 user manual. Revision 1 ed. Bruker Daltonics: Bremen; 2012.

    Google Scholar 

  31. Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G. MycoBank: an online initiative to launch mycology into the 21st century. Stud Mycol. 2004;50:19–22.

    Google Scholar 

  32. Valentine NB, Wahl JH, Kingsley MT, Wahl KL. Direct surface analysis of fungal species by matrix-assited laser desorption/ionisation mass spectrometry. Rapid Commun Mass Spectrom. 2002;16:1352–7.

    Article  CAS  Google Scholar 

  33. Hettick JM, Green BJ, Buskirk AD, Kashon ML, Slaven JE, Janotka E, et al. Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting. Anal Biochem. 2008;380(2):276–81. doi:10.1016/j.ab.2008.05.051.

    Article  CAS  Google Scholar 

  34. Buskirk AD, Hettick JM, Chipinda I, Law BF, Siegel PD, Slaven JE, et al. Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi. Anal Biochem. 2011;411(1):122–8. doi:10.1016/j.ab.2010.11.025.

    Article  CAS  Google Scholar 

  35. Alshawa K, Beretti JL, Lacroix C, Feuilhade M, Dauphin B, Quesne G, et al. Successful identification of clinical dermatophyte and Neoscytalidium species by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2012;50(7):2277–81. doi:10.1128/JCM.06634-11.

    Article  CAS  Google Scholar 

  36. Andersen B, Nielsen KF, Jarvis BB. Characterization of Stachybotrys from water-damaged buildings based on morphology, growth, and metabolite production. Mycologia. 2002;94(3):392–403.

    Article  CAS  Google Scholar 

  37. Wang Y, Hyde KD, McKenzie EHC, Jiang Y-L, Li D-W, Zhao D-G. Overview of Stachybotrys (Memnoniella) and current species status. Fungal Divers. 2015;71(1):17–83. doi:10.1007/s13225-014-0319-0.

    Article  Google Scholar 

  38. Chen HY, Chen YC. Characterization of intact Penicillium spores by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom. 2005;19(23):3564–8. doi:10.1002/rcm.2229.

    Article  CAS  Google Scholar 

  39. Fenselau C, Demirev PA. Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrom Rev. 2001;20(4):157–71. doi:10.1002/mas.10004.

    Article  CAS  Google Scholar 

  40. Kemptner J, Marchetti-Deschmann M, Mach R, Druzhinina IS, Kubicek CP, Allmaier G. Evaluation of matrix-assisted laser desorption/ionization (MALDI) preparation techniques for surface characterization of intact Fusarium spores by MALDI linear time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 2009;23(6):877–84. doi:10.1002/rcm.3949.

    Article  CAS  Google Scholar 

  41. Schrodl W, Heydel T, Schwartze VU, Hoffmann K, Grosse-Herrenthey A, Walther G, et al. Direct analysis and identification of pathogenic Lichtheimia species by matrix-assisted laser desorption ionization-time of flight analyzer-mediated mass spectrometry. J Clin Microbiol. 2012;50(2):419–27. doi:10.1128/JCM.01070-11.

    Article  Google Scholar 

  42. Schmidt O, Kallow W. Differentiation of indoor wood decay fungi with MALDI-TOF mass spectrometry. Holzforschung. 2005;59(3):374–7. doi:10.1515/hf.2005.062.

    Article  CAS  Google Scholar 

  43. Seyfarth F, Ziemer M, Sayer HG, Burmester A, Erhard M, Welker M, et al. The use of ITS DNA sequence analysis and MALDI-TOF mass spectrometry in diagnosing an infection with Fusarium proliferatum. Exp Dermatol. 2008;17(11):965–71. doi:10.1111/j.1600-0625.2008.00726.x.

    Article  CAS  Google Scholar 

  44. Erhard M, Hipler UC, Burmester A, Brakhage AA, Wostemeyer J. Identification of dermatophyte species causing onychomycosis and tinea pedis by MALDI-TOF mass spectrometry. Exp Dermatol. 2008;17(4):356–61. doi:10.1111/j.1600-0625.2007.00649.x.

    Article  Google Scholar 

  45. Marinach-Patrice C, Lethuillier A, Marly A, Brossas J-Y, Gené J, Symoens F, et al. Use of mass spectrometry to identify clinical Fusarium isolates. Clin Microbiol Infec. 2009;16:634–42.

    Article  Google Scholar 

  46. Tao J, Zhang G, Jiang Z, Cheng Y, Feng J, Chen Z. Detection of pathogenic Verticillium spp. using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 2009;23(23):3647–54. doi:10.1002/rcm.4296.

    Article  CAS  Google Scholar 

  47. De Respinis S, Vogel G, Benagli C, Tonolla M, Petrini O, Samuels GJ. MALDI-TOF MS of Trichoderma: a model system for the identification of microfungi. Mycol Prog. 2009;9(1):79–100. doi:10.1007/s11557-009-0621-5.

    Article  Google Scholar 

  48. Rodrigues P, Santos C, Kozakiewicz Z, Venancio A, Lima N. MALDI-TOF ICMS as a modern approach to identify potential aflatoxigenic fungi. In: Latinamerican Congress of Mycotoxicology and II International Symposium on Fungal and Algal Toxins in Industry. Merida, México; 2010.

  49. Alanio A, Beretti JL, Dauphin B, Mellado E, Quesne G, Lacroix C, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for fast and accurate identification of clinically relevant Aspergillus species. Clin Microbiol Infec. 2011;17(5):750–5. doi:10.1111/j.1469-0691.2010.03323.x.

    Article  CAS  Google Scholar 

  50. Cassagne C, Ranque S, Normand AC, Fourquet P, Thiebault S, Planard C, et al. Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. PLoS One. 2011. doi:10.1371/journal.pone.0028425.

    Google Scholar 

  51. De Carolis E, Posteraro B, Lass-Florl C, Vella A, Florio AR, Torelli R, et al. Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Microbiol Infec. 2012;18(5):475–84. doi:10.1111/j.1469-0691.2011.03599.x.

    Article  Google Scholar 

  52. Horka M, Kubesova A, Salplachta J, Zapletalova E, Horky J, Slais K. Capillary and gel electromigration techniques and MALDI-TOF MS–suitable tools for identification of filamentous fungi. Anal Chim Acta. 2012;716:155–62. doi:10.1016/j.aca.2011.12.032.

    Article  CAS  Google Scholar 

  53. Normand A-C, Cassagne C, Ranque S, L’Ollivier C, Fourquet P, Roesems S, et al. Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi. BMC Microbiol. 2013. doi:10.1186/1471-2180-13-76.

    Google Scholar 

  54. Bernhard M, Zautner AE, Steinmann J, Weig M, Gross U, Bader O. Towards proteomic species barcoding of fungi - an example using Scedosporium/Pseudallescheria complex isolates. Fungal Biol. 2016;120(2):162–5. doi:10.1016/j.funbio.2015.07.001.

    Article  Google Scholar 

Download references

Acknowledgements

We are especially grateful to the Brigitte and Wolfram Gedek foundation for the financial support of this research work. Part of the work was supported by the German Academic Exchange Service (DAAD), project number 56269877.

Author information

Authors and Affiliations

  1. Chair of Food Safety, Department of Veterinary Sciences, LMU Munich, Schoenleutnerstr. 8, 85764, Oberschleissheim, Germany

    Sebastian Ulrich, Barbara Biermaier, Brigitte Sperner, Karin Schwaiger, Manfred Gareis & Christoph Gottschalk

  2. Institute for Medical Microbiology, University Medical Center Goettingen, Kreuzbergring 57, 37075, Goettingen, Germany

    Oliver Bader

  3. Chair of Microbiology and Mycology, Department of Veterinary Sciences, LMU Munich, Veterinaerstr. 13, 80539, Munich, Germany

    Georg Wolf & Reinhard K. Straubinger

  4. Chair for Hygiene and Technology of Milk, Department of Veterinary Sciences, LMU Munich, Schoenleutnerstr. 8, 85764, Oberschleissheim, Germany

    Andrea Didier

Authors
  1. Sebastian Ulrich
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Barbara Biermaier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Oliver Bader
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Georg Wolf
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Reinhard K. Straubinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Andrea Didier
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Brigitte Sperner
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Karin Schwaiger
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Manfred Gareis
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Christoph Gottschalk
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sebastian Ulrich.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest

Additional information

An erratum to this article can be found at http://dx.doi.org/10.1007/s00216-016-9921-1.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ulrich, S., Biermaier, B., Bader, O. et al. Identification of Stachybotrys spp. by MALDI-TOF mass spectrometry. Anal Bioanal Chem 408, 7565–7581 (2016). https://doi.org/10.1007/s00216-016-9800-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-016-9800-9

Keywords

Search

Navigation