Molecular profiling of fungal communities in moisture damaged buildings before and after remediation - a comparison of culture-dependent and culture-independent methods

Abstract

BackgroundIndoor microbial contamination due to excess moisture is an important contributor to human illness in both residential and occupational settings. However, the census of microorganisms in the indoor environment is limited by the use of selective, culture-based detection techniques. By using clone library sequencing of full-length internal transcribed spacer region combined with quantitative polymerase chain reaction (qPCR) for 69 fungal species or assay groups and cultivation, we have been able to generate a more comprehensive description of the total indoor mycoflora. Using this suite of methods, we assessed the impact of moisture damage on the fungal community composition of settled dust and building material samples (n = 8 and 16, correspondingly). Water-damaged buildings (n = 2) were examined pre- and post- remediation, and compared with undamaged reference buildings (n = 2).ResultsCulture-dependent and independent methods were consistent in the dominant fungal taxa in dust, but sequencing revealed a five to ten times higher diversity at the genus level than culture or qPCR. Previously unknown, verified fungal phylotypes were detected in dust, accounting for 12% of all diversity. Fungal diversity, especially within classes Dothideomycetes and Agaricomycetes tended to be higher in the water damaged buildings. Fungal phylotypes detected in building materials were present in dust samples, but their proportion of total fungi was similar for damaged and reference buildings. The quantitative correlation between clone library phylotype frequencies and qPCR counts was moderate (r = 0.59, p < 0.01).ConclusionsWe examined a small number of target buildings and found indications of elevated fungal diversity associated with water damage. Some of the fungi in dust were attributable to building growth, but more information on the material-associated communities is needed in order to understand the dynamics of microbial communities between building structures and dust. The sequencing-based method proved indispensable for describing the true fungal diversity in indoor environments. However, making conclusions concerning the effect of building conditions on building mycobiota using this methodology was complicated by the wide natural diversity in the dust samples, the incomplete knowledge of material-associated fungi fungi and the semiquantitative nature of sequencing based methods.

Microbial diversity in bioaerosol samples causing ODTS compared to reference bioaerosol samples as measured using Illumina sequencing and MALDI-TOF

“…Furthermore we measure activities of enzymes in dust samples. Considerably more diverse fungal as well as bacterial flora have been revealed when using culture-independent methods compared to culture-dependent methods Pitkaranta et al, 2011). Therefore, next generation sequencing (NGS) has also been employed to characterize the diversity of the microbial community in our aerosol samples.…”

Evaluation of sampling methods for toxicological testing of indoor air particulate matter

“…These approaches include collecting dust in cardboard boxes (Hyvärinen et al, 2006a), in dustfall collectors (Würtz et al, 2005), onto electrostatic cloths (Noss et al, 2008) or in Petri dishes (Adams et al, 2015). Passively settled dust is also part of the house dust reservoir that can be sampled by vacuuming directly from carpets, furniture or floors into filters, tubes or nylon sampling socks (Arbes et al, 2005; Casas et al, 2013; Leppänen et al, 2014; Pitkäranta et al, 2011). …”

Bedroom Allergen Exposure Beyond House Dust Mites

“…Various indirect measures, including spore counts, have been used for the assessment; however, spore counts may not reflect allergen content accurately as allergen content in spores may vary and allergens are also present in fungal components other than intact spores, including hyphal fragments [134]. New assessment techniques, including DNA-based characterization methods, have been developed, but their use remains limited [135, 136••, 137]. …”