Analysis of Fungal Flora in Indoor Dust by Ribosomal DNA Sequence Analysis, Quantitative PCR, and Culture

Abstract

In recent years increasing attention has been given to the potential health effects of fungal exposure in indoor environments. We used large-scale sequencing of the fungal internal transcribed spacer region (ITS) of nuclear ribosomal DNA to describe the mycoflora of two office buildings over the four seasons. DNA sequencing was complemented by cultivation, ergosterol determination, and quantitative PCR analyses. Sequences of 1,339 clones were clustered into 394 nonredundant fungal operational taxonomical units containing sequences from 18 fungal subclasses. The observed flora differed markedly from that recovered by cultivation, the major differences being the near absence of several typical indoor mold genera such as Penicillium and Aspergillus spp. and a high prevalence of basidiomycetes in clone libraries. A total of 55% of the total diversity constituted of unidentifiable ITS sequences, some of which may represent novel fungal species. Dominant species were Cladosporium cladosporioides and C. herbarum, Cryptococcus victoriae, Leptosphaerulina americana and L. chartarum, Aureobasidium pullulans, Thekopsora areolata, Phaeococcomyces nigricans, Macrophoma sp., and several Malassezia species. Seasonal differences were observed for community composition, with ascomycetous molds and basidiomycetous yeasts predominating in the winter and spring and Agaricomycetidae basidiomycetes predominating in the fall. The comparison of methods suggested that the cloning, cultivation, and quantitative PCR methods complemented each other, generating a more comprehensive picture of fungal flora than any of the methods would give alone. The current restrictions of the methods are discussed.

Diversity and dynamics of airborne fungi in São Luis, State of Maranhão, Brazil

“…In Mexico City, Penicillium is also the second most common genus 15 . However, in studies performed in other countries, such as France, the USA, Chile and Cuba, Cladosporium has stood out as the most prevalent genus [16][17][18] .…”

What do we learn from cultures in the omics age? High-throughput sequencing and cultivation of leaf-inhabiting endophytes from beech (Fagus sylvatica L.) revealed complementary community composition but similar correlations with local habitat conditions

“…Recently, HTS was rated superior over cultivation, given its ability to detect more obligate, slow growing and rare fungi (Al-Sadi et al 2015, Oono et al 2015. Whereas the general lack of congruence between mycobiomes generated by cultivation and HTS can be safely postulated nowadays (Pitkaranta et al 2008), much less is known about environmental correlations of these differing data sets.…”

Targeted Inspection of Environmental Mycological Load for Mitigation of Indoor Mold toward Improved Public Health

“…Using qPCR has provided valuable information on the occurrence of the most common indoor fungi and this method shows great promise for the rapid provision of quantitative data on the occurrence of the studied organisms [82]. However, apart from cloning and sequencing of amplified fungal DNA, or in some cases DGGE followed by sequencing, these methods do not produce detailed information on previously unknown or unexpected taxa [24]. A comparison of methods has suggested that the cloning, cultivation and qPCR methods complement each other, generating a more comprehensive picture of fungal flora than any of the methods used alone [24].…”

“…These can include EI and long-term sampling, e.g. of settled dust or pumped air, and use of total fungal analyses, such as for ergosterol, beta 1-3 glucan or total molds by the CAMERANEA method [21][22][23] or fungal DNA analysis via quantitative or qualitative polymerase chain reaction (PCR) [21][22][23][24].…”

“…Environmental inspections (EIs) of mycological load have been suggested using a range of fungal-detection techniques, e.g. open Petri dish (OPD) versus active air sampling [19,20] and qualitative identification and quantitative monitoring of diverse fungal species and their mycotoxins [21][22][23][24].…”