Skip to main content

Advertisement

Log in

Analysis of Black Fungal Biofilms Occurring at Domestic Water Taps (I): Compositional Analysis Using Tag-Encoded FLX Amplicon Pyrosequencing

Mycopathologia Aims and scope Submit manuscript

Abstract

Mass growth of dark fungal biofilms on water taps and associated habitats was observed in various German drinking water distribution systems recently. Customers of affected drinking water systems are anxious about potential and unknown health risks. These environments are known to harbour a fungal flora also comprising a variety of fungal opportunists that are well known to cause superficial mycoses in humans (Exophiala equina, Exophiala lecanii-corni) but are not known to establish dark biofilms so far. To gain profound insight on composition of respective biofilms, a metagenomic approach using Tag-Encoded FLX Amplicon Pyrosequencing (TEFAP) of the ribosomal internal transcribed spacer 2 region in comparison with a classical cultivation approach using Sabouraud agar with chloramphenicol and erythritol-chloramphenicol-agar was performed. E. lecanii-corni was found to be the major component in 10 of 13 biofilms analysed independently of the method used. Alternaria sp., E. equina, Fusarium spp. and Ochroconis spp. were also relatively abundant. As expected, TEFAP usually revealed a higher diversity than the cultivation approaches. For example, opportunistic species like Candida albicans or Exophiala dermatitidis were detected in very low amounts. In conclusion, TEFAP turned out to be a promising and powerful tool for the semi-quantitative analysis of fungal biofilms. Referring to relevant literature, potential biological hazards caused by fungi of the dark biofilms can be regarded as low.

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

Access this article

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

Similar content being viewed by others

References

  1. Lian X, de Hoog GS. Indoor wet cells harbour melanized agents of cutaneous infection. Med Mycol. 2010;48:622–8.

    Article  PubMed  CAS  Google Scholar 

  2. Hamada N, Abe N. Physiological characteristics of 13 common fungal species in bathrooms. Mycoscience. 2009;50:421–9.

    Article  Google Scholar 

  3. Hamada N. Comparison of fungi found in bathrooms and sinks. Biocontrol Sci. 2010;15:51–6.

    Article  PubMed  Google Scholar 

  4. Saunte DM, Tarazooie B, Arendrup MC, de Hoog GS. Black yeast-like fungi in skin and nail: it probably matters. Mycoses. 2011;55:161–7.

    PubMed  Google Scholar 

  5. de Hoog GS, Mayser P, Haase G, Horré R, Horrevorts AM. A new species, Phialophora europaea, causing superficial infections in humans. Mycoses. 2000;43:409–16.

    Article  PubMed  Google Scholar 

  6. Nishimura K, Miyaji M, Taguchi H, Tanaka R. Fungi in bathwater and sludge of bathroom drainpipes 1. Frequent isolation of Exophiala species. Mycopathologia. 1987;97:17–23.

    Article  PubMed  CAS  Google Scholar 

  7. Matos T, de Hoog GS, De Boer AG, De Crom I, Haase G. High prevalence of the neurotrope Exophiala dermatitidis and related oligotrophic black yeasts in sauna facilities. Mycoses. 2002;45:373–7.

    Article  PubMed  CAS  Google Scholar 

  8. Zalar P, Novak M, de Hoog GS, Gunde-Cimerman N. Dishwashers—a man-made ecological niche accommodating human opportunistic fungal pathogens. Fungal Biol. 2011;115:997–1007.

    Article  PubMed  CAS  Google Scholar 

  9. Dowd SE, Wolcott RD, Sun Y, McKeehan T, Smith E, Rhoads D. Polymicrobial nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP). PLoS ONE. 2008;3:e3326.

    Article  PubMed  Google Scholar 

  10. Dowd SE, Callaway TR, Wolcott RD, Sun Y, McKeehan T, Hagevoort RG, et al. Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). BMC Microbiol. 2008;8:125–32.

    Article  PubMed  Google Scholar 

  11. Sun Y, Wolcott RD, Dowd SE. Tag-encoded FLX amplicon pyrosequencing for the elucidation of microbial and functional gene diversity in any environment. Methods Mol Biol. 2011;733:129–41.

    Article  PubMed  CAS  Google Scholar 

  12. Jünemann S, Prior K, Szczepanowski R, Harks I, Ehmke B, Goesmann A, et al. Bacterial community shift in treated periodontitis patients revealed by Ion Torrent 16S rRNA gene amplicon sequencing. PLoS ONE. 2012;7:e41606.

    Article  PubMed  Google Scholar 

  13. Jumpponen A, Jones KL. Massively parallel 454 sequencing indicates hyperdiverse fungal communities in temperate Quercus macrocarpaphyllosphere. New Phytol. 2009;184:438–48.

    Article  PubMed  CAS  Google Scholar 

  14. Buée M, Reich M, Murat C, Morin E, Nilsson RH, Uroz S, et al. 454 Pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity. New Phytol. 2009;184:449–56.

    Article  PubMed  Google Scholar 

  15. Lumini E, Orgiazzi A, Borriello R, Bonfante P, Bianciotto V. Disclosing arbuscular mycorrhizal fungal biodiversity in soil through a land-use gradient using a pyrosequencing approach. Environ Microbiol. 2010;12:2165–79.

    PubMed  CAS  Google Scholar 

  16. Öpik M, Metsis M, Daniell TJ, Zobel M, Moora M. Large-scale parallel 454 sequencing reveals host ecological group specificity of arbuscular mycorrhizal fungi in a boreonemoral forest. New Phytol. 2009;184:424–37.

    Article  PubMed  Google Scholar 

  17. Tedersoo L, Nilsson RH, Abarenkov K, Jairus T, Sadam A, Saar I, et al. 454 Pyrosequencing and Sanger sequencing of tropical mycorrhizal fungi provide similar results but reveal substantial methodological biases. New Phytol. 2010;188:291–301.

    Article  PubMed  CAS  Google Scholar 

  18. Wallander H, Johansson U, Sterkenburg E, Brandström Durling M, Lindahl BD. Production of ectomycorrhizal mycelium peaks during canopy closure in Norway spruce forests. New Phytol. 2010;187:1124–34.

    Article  PubMed  CAS  Google Scholar 

  19. Jumpponen A, Jones KL, Blair J. Vertical distribution of fungal communities in tallgrass prairie soil. Mycologia. 2010;102:1027–41.

    Article  PubMed  Google Scholar 

  20. Rousk J, Bååth E, Brookes PC, Lauber CL, Lozupone C, Caporaso JG, et al. Soil bacterial and fungal communities across a pH gradient in an arable soil. ISME J. 2010;4:1340–51.

    Article  PubMed  Google Scholar 

  21. Amend AS, Seifert KA, Bruns TD. Quantifying microbial communities with 454 pyrosequencing: does read abundance count? Mol Ecol. 2010;19:5555–65.

    Article  PubMed  CAS  Google Scholar 

  22. Ghannoum MA, Jurevic RJ, Mukherjee PK, Cui F, Sikaroodi M, Naqvi A, et al. Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathog. 2010;6:e1000713.

    Article  PubMed  Google Scholar 

  23. Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, et al. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. Proc Natl Acad Sci USA. 2012;109:6241–6.

    Article  PubMed  CAS  Google Scholar 

  24. Haase G, de Hoog GS. Nutritional physiology and selective isolation of Exophiala dermatitidis. Ant Leeuw. 1993;64:17–26.

    Article  Google Scholar 

  25. Kõljalg U, Larsson K, Abarenkov K, Nilsson R, Alexander I, Eberhardt U, et al. UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi. New Phytol. 2005;166:1063–8.

    Article  PubMed  Google Scholar 

  26. Abarenkov K, Nilsson RH, Larsson K-H, Alexander IJ, Eberhardt U, Erland S, et al. The UNITE database for molecular identification of fungi—recent updates and future perspectives. New Phytol. 2010;186:281–5.

    Article  PubMed  Google Scholar 

  27. Heinrichs G, de Hoog GS, Haase G. Barcode identifiers as a practical tool for reliable species assignment of medically important black yeast species. J Clin Microbiol. 2012;50:3023–30.

    Article  PubMed  CAS  Google Scholar 

  28. Hageskal G, Lima N, Skaar I. The study of fungi in drinking water. Mycol Res. 2009;113:165–72.

    Article  PubMed  Google Scholar 

  29. Müller FMC, Seidler M, Beauvais A. Aspergillus fumigatus biofilms in the clinical setting. Med Mycol. 2011;49:S96–100.

    Article  PubMed  Google Scholar 

  30. Rooney AP, Ward TJ. Evolution of a large ribosomal RNA multigene family in filamentous fungi: birth and death of a concerted evolution paradigm. Proc Natl Acad Sci USA. 2005;102:5084–9.

    Article  PubMed  CAS  Google Scholar 

  31. Feinstein LM, Sul WJ, Blackwood CB. Assessment of bias associated with incomplete extraction of microbial DNA from soil. Appl Environ Microbiol. 2009;75:5428–33.

    Article  PubMed  CAS  Google Scholar 

  32. DeSantis TZ, Stone CE, Murray SR, Moberg JP, Andersen GL. Rapid quantification and taxonomic classification of environmental DNA from both prokaryotic and eukaryotic origins using a microarray. FEMS Microbiol Lett. 2005;245:271–8.

    Article  PubMed  CAS  Google Scholar 

  33. Engelbrektson A, Kunin V, Wrighton KC, Zvenigorodsky N, Chen F, Ochman H, et al. Experimental factors affecting PCR-based estimates of microbial species richness and evenness. ISME J. 2010;4:642–7.

    Article  PubMed  CAS  Google Scholar 

  34. Polz MF, Cavanaugh CM. Bias in template-to-product ratios in multitemplate PCR. Appl Environ Microbiol. 1998;64:3724–30.

    PubMed  CAS  Google Scholar 

  35. Jumpponen A. Soil fungal communities underneath willow canopies on a primary successional glacier forefront: rDNA sequence results can be affected by primer selection and chimeric data. Microb Ecol. 2007;53:233–46.

    Article  PubMed  CAS  Google Scholar 

  36. Kunin V, Engelbrektson A, Ochman H, Hugenholtz P. Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. Environ Microbiol. 2010;12:118–23.

    Article  PubMed  CAS  Google Scholar 

  37. Kangawa T. Bias and artifacts in multitemplate polymerase chain reactions (PCR). J Biosci Bioeng. 2003;96:317–23.

    Google Scholar 

  38. Gonzalez JM, Portillo MC, Belda-Ferre P, Mira A. Amplification by PCR artificially reduces the proportion of the rare biosphere in microbial communities. PLoS ONE. 2012;7:e29973–83.

    Article  PubMed  CAS  Google Scholar 

  39. de Hoog GS, Vicente VA, Najafzadeh MJ, Harrak MJ, Badali H, Seyedmousavi S. Waterborne Exophiala species causing disease in cold-blooded animals. Persoonia. 2011;27:46–72.

    Article  PubMed  Google Scholar 

  40. Pirnie-Fisker EF, Woertz JR. Degradation of ethanol plant by-products by Exophiala lecanii-corni and Saccharomyces cerevisiae in batch studies. Appl Microbiol Biotechnol. 2006;74:902–10.

    Article  PubMed  Google Scholar 

  41. Woertz JR, Kinney KA, McIntosh NDP, Szaniszlo PJ. Removal of toluene in a vapor-phase bioreactor containing a strain of the dimorphic black yeast Exophiala lecanii-corni. Biotechnol Bioeng. 2001;75:550–8.

    Article  PubMed  CAS  Google Scholar 

  42. Zeng JS, Sutton DA, Fothergill AW, Rinaldi MG, Harrak MJ, de Hoog GS. Spectrum of clinically relevant Exophiala species in the United States. J Clin Microbiol. 2007;45:3713–20.

    Article  PubMed  CAS  Google Scholar 

  43. Nyaoke A, Weber ES, Innis C, Stremme D, Dowd C, Hinckley L, et al. Disseminated phaeohyphomycosis in weedy seadragons (Phyllopteryx taeniolatus) and leafy seadragons (Phycodurus eques) caused by species of Exophiala, including a novel species. J Vet Diagn Invest. 2009;21:69–79.

    Article  PubMed  Google Scholar 

  44. Richards RH, Holliman A, Helgason S. Exophiala salmonis infection in Atlantic salmon Salmo salar L. J Fish Dis. 1978;1:357–68.

    Article  Google Scholar 

  45. Blazer VS, Wolke RE. An Exophiala-like fungus as the cause of a systemic mycosis of marine fish. J Fish Dis. 1979;2:145–52.

    Article  Google Scholar 

  46. Otis EJ, Wolke RE, Blazer VS. Infection of Exophiala salmonis in Atlantic salmon (Salmo salar L.). J Wildl Dis. 1985;21:61–4.

    CAS  Google Scholar 

  47. Porteous NB, Grooters AM, Redding SW, Thompson EH, Rinaldi MG, de Hoog GS, et al. Identification of Exophiala mesophila isolated from treated dental unit waterlines. J Clin Microbiol. 2003;41:3885–9.

    Article  CAS  Google Scholar 

  48. Porteous NB, Redding SW, Thompson EH, Grooters AM, de Hoog GS, Sutton DA. Isolation of an unusual fungus in treated dental unit waterlines. J Am Dent Assoc. 2003;134:853–8.

    Google Scholar 

  49. de Hoog GS, Guarro J, Gené J, Figueras MJ. Atlas of clinical fungi. 3rd ed. Utrecht: Centraalbureau voor Schimmelcultures; 2011.

    Google Scholar 

  50. Ge YP, Lv GX, Shen YN, Li M, Deng SW, de Hoog S, et al. First report of subcutaneous phaeohyphomycosis caused by Ochroconis tshawytschae in an immunocompetent patient. Med Mycol. 2012;50:637–40.

    Article  Google Scholar 

  51. Faisal M, Elsayed E, Fitzgerald SD, Silva V, Mendoza L. Outbreaks of phaeohyphomycosis in the chinook salmon (Oncorhynchus tshawytscha) caused by Phoma herbarum. Mycopathologia. 2007;163:41–8.

    Article  Google Scholar 

  52. Armougom F, Raoult D. Exploring microbial diversity using 16S rRNA high-throughput methods. J Comput Sci Syst Biol. 2009;2:69–92.

    Article  Google Scholar 

  53. Tan XM, Chen XM, Wang CL, Jin XH, Cui JL, Chen J, Guo SX, Zhao LF. Isolation and identification of endophytic fungi in roots of nine Holcoglossum plants (Orchidaceae) collected from Yunnan, Guangxi, and Hainan provinces of China. Curr Microbiol. 2012;64:140–7.

    Article  CAS  Google Scholar 

  54. Heinrichs G, Hübner I, Schmidt CK, de Hoog GS, Haase G. Analysis of black fungal biofilms occurring at domestic water taps (II): potential routes of entry. Mycopathologia. 2013. doi:10.1007/s11046-013-9619-2.

Download references

Acknowledgments

One of the authors (G. Heinrichs) was supported by a research grant from RheinEnergie AG. We also want to thank Manfred Bovi, Institute of Pathology, RWTH Aachen University Hospital for performing the scanning electron microscopy and Kittipan Samerpitak, CBS for kindly checking DNA sequences in case Ochroconis spp.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Gerhard Haase.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Heinrichs, G., Hübner, I., Schmidt, C.K. et al. Analysis of Black Fungal Biofilms Occurring at Domestic Water Taps (I): Compositional Analysis Using Tag-Encoded FLX Amplicon Pyrosequencing. Mycopathologia 175, 387–397 (2013). https://doi.org/10.1007/s11046-013-9618-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11046-013-9618-3

Keywords

Navigation