Molecular Profiling of Indoor Microbial Communities in Moisture Damaged

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Molecular proﬁ ling of indoor microbial communiƟ es in moisture damaged and non-damaged buildings

Miia Pitkäranta

Division of General Microbiology Faculty of Biological and Environmental Sciences University of Helsinki and DNA Sequencing and Genomics Laboratory InsƟ tute of Biotechnology University of Helsinki and Graduate School in Environmental Health (SYTYKE)

Academic DissertaƟ on in General Microbiology

To be presented for public examinaƟ on with the permission of the Faculty of Biological and Environmental Sciences of the University of Helsinki, in lecture hall Paatsama in the Animal Hospital building of University of Helsinki, on 20.1.2012 at 12 o’clock noon. Supervisors Docent Petri Auvinen Institute of Biotechnology University of Helsinki Helsinki, Finland

Docent Helena Rintala Department of Environmental Health National Institute for Health and Welfare Kuopio, Finland

Professor Martin Romantschuk Department of Environmental Sciences University of Helsinki Lahti, Finland

Reviewers Professor Malcolm Richardson School of Medicine University of Manchester Manchester, UK

Professor Kaarina Sivonen Department of Applied Microbiology and Chemistry University of Helsinki Helsinki, Finland

Opponent Associate Professor James Scott Dalla Lana School of Public Health University of Toronto Toronto, Canada

Custos Professor Jouko Rikkinen Department of Biosciences University of Helsinki Helsinki, Finland

Layout: Tinde Pä ivä rinta Cover: Wordle.net ISBN 978-952-10-7568-1 (paperback) ISBN 978-952-10-7569-8 (PDF) ISSN 1799-7372 http://ethesis.helsinki.fi Unigrafi a, Helsinki 2012

CONTENTS

LIST OF ORIGINAL ARTICLES ABSTRACT TIIVISTELMÄ (Abstract in Finnish) ABBREVIATIONS 1 INTRODUCTION ...... 1 1.1 Th e indoor microbiome and human health ...... 1 1.2 House dust ...... 3 1.3 Moisture damage and indoor microbial communities ...... 14 1.4 Molecular methods in microbial biodiversity studies ...... 16 2 AIMS OF THE STUDY ...... 24 3 MATERIALS AND METHODS ...... 25 3.1 Buildings and samples ...... 25 3.2 Experimental methods ...... 26 4. RESULTS AND DISCUSSION ...... 29 4.1 Microbial diversity in dust (I, II, III) ...... 29 4.2 Variation in microbial community composition ...... 39 4.3 Methodological considerations ...... 45 4.4 Macroarray hybridization method for the enrichment of clone libraries ...... 49 5 CONCLUSIONS ...... 52 ACKNOWLEDGEMENTS ...... 54 REFERENCES ...... 56 APPENDIX ...... 68 LIST OF ORIGINAL ARTICLES The thesis is based on the following articles, which are referred in the text by their Roman numerals.

I Pitkäranta, M., Meklin, T., Hyvärinen, A., Nevalainen, A., Paulin, L., Auvinen, P., Lignell, U. and Rintala, H. 2011. Molecular profi ling of fungal communities in moisture-damaged buildings before and aft er remediation – comparison of culture-dependent and -independent methods. BMC Microbiology 11:235. II Pitkäranta, M., Meklin, T., Hyvärinen, A., Paulin, L., Auvinen, P., Nevalainen, A. and Rinta- la, H. 2008. Analysis of fungal fl ora in indoor dust by ribosomal DNA sequence analysis, quantitative PCR, and culture. Applied and Environmental Microbiology 74:233-244. III Rintala, H., Pitkäranta, M., Toivola, M., Paulin, L. and Nevalainen, A. 2008. Diversity and seasonal dynamics of bacterial community in indoor environment. BMC Microbiology 8:56. IV Hultman, J., Pitkäranta, M., Romantschuk, M., Auvinen, P. and Paulin, L. 2008. Probe-based negative selection for underrepresented phylotypes in large environmental clone libraries. Journal of Microbiological Methods 75:457-463.

Author’s contribution to each publication

I MP participated in study design, did the cloning and sequencing, performed data-analysis and draft ed the manuscript. II MP participated in study design, did the cloning and sequencing, performed data-analysis and draft ed the manuscript. III MP participated in the study design, did the cloning and sequencing and edited the manuscript. HR performed the data analysis and draft ed the manuscript. IV MP participated in the design of the study and hybridization experiments and edited the manuscript. JH participated in the design of the study, did the laboratory experiment and draft ed the manuscript. Th e article is included in the PhD thesis of Jenni Hultman (JH 2009, Department of Ecological and Environmental Sciences, Faculty of Biosciences, University of Helsinki).

Sections of the literature review of this thesis have been used as a part of the following review article: Rintala, H. Pitkäranta, M and Täubel, M. 2012. Microbial communities associated with house dust. Advances in applied microbiology. 78:75-120. ABSTRACT

Epidemiological studies have shown an elevation in the incidence of asthma, allergic symptoms and respiratory infections among people living or working in buildings with moisture and mould problems. Microbial growth is suspected to have a key role, since the severity of microbial contamination and symptoms show a positive correlation, while the removal of contaminated materials relieves the symptoms. However, the cause-and-eff ect relationship has not been well established and knowledge of the causative agents is incomplete. Th e present consensus of indoor microbes relies on culture-based methods. Microbial cultivation and identifi cation is known to provide qualitatively and quantitatively biased results, which is suspected to be one of the reasons behind the oft en inconsistent fi ndings between objectively measured microbiological attributes and health.

In the present study the indoor microbial communities were assessed using culture-independent, DNA based methods. Fungal and bacterial diversity was determined by amplifying and sequencing the nucITS- and16S-gene regions, correspondingly. In addition, the cell equivalent numbers of 69 mould species or groups were determined by quantitative PCR (qPCR). Th e results from molecular analyses were compared with results obtained using traditional plate cultivation for fungi.

Using DNA-based tools, the indoor microbial diversity was found to be consistently higher and taxonomically wider than viable diversity. Th e dominant sequence types of fungi, and also of bacteria were mainly affi liated with well-known microbial species. However, in each building they were accompanied by various rare, uncultivable and unknown species. In both moisture-damaged and undamaged buildings the dominant fungal sequence phylotypes were affi liated with the classes Dothideomycetes (mould-like fi lamentous ascomycetes); Agaricomycetes (mushroom- and polypore-like fi lamentous basidiomycetes); Urediniomycetes (rust-like basidiomycetes); Trem- ellomycetes and the family Malasseziales (both yeast-like basidiomycetes). Th e most probable source for the majority of fungal types was the outdoor environment. In contrast, the dominant bacterial phylotypes in both damaged and undamaged buildings were affi liated with human-associated members within the phyla Actinobacteria and Firmicutes.

Indications of elevated fungal diversity within potentially moisture-damage-associated fungal groups were recorded in two of the damaged buildings, while one of the buildings was characterized by an abundance of members of the Penicillium chrysogenum and P. commune species complexes. However, due to the small sample number and strong normal variation fi rm conclusions concerning the eff ect of moisture damage on the species diversity could not be made. Th e fungal communities in dust samples showed seasonal variation, which refl ected the seasonal fl uctuation of outdoor fungi. Seasonal variation of bacterial communities was less clear but to some extent attributable to the outdoor sources as well. Th e comparison of methods showed that clone library sequencing was a feasible method for describing the total microbial diversity, indicated a moderate quantitative correlation between sequencing and qPCR results and confi rmed that culture based methods give both a qualita- tive and quantitative underestimate of microbial diversity in the indoor environment. However, certain important indoor fungi such as Penicillium spp. were clearly underrepresented in the sequence material, probably due to their physiological and genetic properties. Species specifi c qPCR was a more effi cient and sensitive method for detecting and quantitating individual species than sequencing, but in order to exploit the full advantage of the method in building investigations more information is needed about the microbial species growing on damaged materials.

In the present study, a new method was also developed for enhanced screening of the marker gene clone libraries. Th e suitability of the screening method to diff erent kinds of microbial environments including biowaste compost material and indoor settled dusts was evaluated. Th e usability was found to be restricted to environments that support the growth and subsequent dominance of a small number microbial species, such as compost material. TIIVISTELMÄ (Abstract in Finnish)

Kosteusvaurioiden aiheuttamalla huonolla sisäilmalla tiedetään olevan epidemiologinen yhteys mm. astman, allergisten oireiden ja hengitystieinfektioiden esiintyvyyteen. Mikrobikasvulla epäil- lään olevan tärkeä rooli ilmiön aiheuttajana, sillä havaitun ”home”kasvun laajuuden ja oireiden vakavuuden välillä on positiivinen yhteys ja toisaalta homeisten materiaalien poisto vähentää oireita. Tämänhetkinen tieto oireita aiheuttavista tekijöistä ja oireiden syntymekanismeista on kuitenkin vajavaista. Sisäympäristöjen mikrobilajiston tuntemus perustuu suurelta osin viljely- pohjaisilla menetelmillä saatuun tietoon. Viljelymenetelmien kuitenkin tiedetään antavan laadul- lisesti ja määrällisesti vääristyneen kuvan mikrobistosta, minkä epäillään olevan yhtenä syynä siihen, että sisäympäristöistä mitattujen mikrobistojen ja terveysongelmien välillä ei aina havaita johdonmukaisia yhteyksiä.

Tässä työssä tutkittiin sisäympäristöjen mikrobistoja viljelystä riippumattomin, DNA-pohjaisin menetelmin. Sieni- ja bakteerilajiston kartoittamiseen käytettiin ribosomaalisten DNA-merkki- jaksojen (ITS- ja 16S -geenialueet) monistusta ja sekvensointia. 69 homelajin solumäärät määritet- tiin lisäksi kvantitatiivisella PCR-menetelmällä (qPCR). Saatuja tuloksia verrattiin samoista näyt- teistä viljelymenetelmin saatuihin tuloksiin.

Sisätilojen mikrobidiversiteetin havaittiin olevan DNA-pohjaisin menetelmin merkittävästi viljelymenetelmin todettua monimuotoisempaa ja lajirikkaampaa. Yleisimmät sekvenssityypit olivat peräisin tunnetuista lajeista mutta kaikista tutkituista rakennuksista löydettiin myös uuden- tyyppisiä DNA-merkkisekvenssejä, joista osa saattaa edustaa aiemmin tuntemattomia mikrobila- jeja. Sekä kosteusvaurio- että verrokkirakennuksissa yleisimmät sienten sekvenssityypit vastasivat kaariin Ascomycetes ja Basidiomycetes (kanta- ja kotelosienet) kuuluvien luokkien Dothideo- mycetes, Agaricomycetes, Urediniomycetes ja Tremellomycetes, sekä heimon Malasseziales lajien DNA-sekvenssejä. Ko. ryhmiin lukeutuu home-, lakkisieni-, kääpä-, ruoste- ja hiivalajeja. Suurin osa sienilajistosta oli todennäköisimmin peräisin ulkoympäristöstä. Sitä vastoin bakteerisekvenss- ien enemmistö vastasi ihmisperäisten, pääjaksoihin Actinomycetes ja Firmicutes kuuluvien lajien merkkijaksoja. Mikrobiryhmien esiintymisessä kosteusvaurio- ja verrokkirakennuksissa havaittiin eroja; kahdessa tutkitusta vauriokohteesta havaittiin verrokkia korkeampaa diversiteettiä raken- nusperäisiä lajeja sisältävissä sieniryhmissä, kun taas yhden vauriokohteen sekvenssiaineistossa havaittiin poikkeuksellisen runsaasti Penicillium chrysogenum- ja P. commune –lajiryhmittymiin kuuluvia merkkijaksoja. Pienestä näytemäärästä ja lajiston voimakkaasta normaalivaihtelusta johtuen luotettavia johtopäätöksiä kosteusvaurioiden osuudesta lajiston vaihteluun ei kuitenkaan kyetty tekemään. Sienilajistosta kuvattiin vuodenaikaisvaihtelua, joka vastaa lajiston vaihtelua ulkoympäristössä. Bakteerilajiston vuodenaikaisvaihtelu ei ollut yhtä selkeää, mutta eräiden ryh- mien osalta vaihtelu oli yhdistettävissä ulkoympäristön bakteerikulkeuman vaihteluun.

Menetelmien vertailu osoitti sekvensoinnin toimivuuden kokonaislajiston kuvauksessa, osoitti kohtuullisen kvantitatiivisen korrelaation sekvensoinnin ja qPCR:n antamien tulosten välillä ja vahvisti aiemmat havainnot siitä, että viljelymenetelmä antaa sekä määrällisen että laadullisen aliarvion lajistosta. Toisaalta eräiden merkittävien sisäilmahomeiden kuten Penicillium:in ja sen sukulaisten todettiin olevan aliedustettuja sekvenssiaineistoissa, todennäköisesti lajien fysiologi- sista ja geneettisistä ominaisuuksista johtuen. Lajispesifi sen qPCR:n katsottiin olevan herkkä ja tehokas menetelmä lajiston määrälliseen tutkimiseen, mutta menetelmän hyödyntämiseksi tarvi- taan kattavampaa tietoa kosteusvauriomateriaaleilla esiintyvistä mikrobeista.

Työssä kehitettiin lisäksi menetelmä mikrobilajistojen sekvensointipohjaisessa kartoittamises- sa tarvittavien kloonikirjastojen käsittelyn tehostamiseksi, sekä arvioitiin menetelmän toimi- vuutta komposti- ja huonepölynäytteillä. Menetelmän hyödynnettävyyden todettiin rajoittuvan ympäristöihin joissa olosuhteet suosivat harvojen mikrobilajien voimakasta lisääntymistä ja joissa siten on selkeästi dominoitu mikrobiyhteisörakenne, esimerkkinä kehittynyt kompostimassa. ABBREVIATIONS

ABPA Allergic bronchopulmonary aspergillosis ARDRA Amplifi ed ribosomal DNA restriction analysis DG18 Dichloran-glycerol agar DGGE Denaturing gradient gel electrophoresis DNA Deoxyribonucleic acid EPS Extracellular polysaccharide HDM House dust mite HPLC High-pressure liquid chromatography INSD International nucleotide sequence database LPS Lipopolysaccharide MAC Mycobacterium avium complex MEA Malt extract agar MS-GC Mass spectrometry-gas chromatography MVOC Microbial volatile organic compound nucITS Nuclear internal transcribed spacer (commonly also ITS) ODTS Organic dust toxic syndrome OTU Operational taxonomic unit; here synonymous to “phylotype” PCoA Principal coordinates analysis PCR Polymerase chain reaction qPCR Quantitative polymerase chain reaction rDNA Ribosomal DNA; the genomic region of DNA containing the rRNA coding genes and intervening spacers including nucITS RFLP Restriction fragment length polymorphism RH Relative humidity RNA Ribonucleic acid rRNA Ribosomal RNA RT Respiratory tract RTI Respiratory tract infection sp. Species spp. Species, plural SSCP Single strand -conformation polymorphism SM Storage mite TGGE Temperature gradient gel electrophoresis tRFLP Terminal restriction fragment length polymorphism Introduction

1. INTRODUCTION

1.1 The indoor microbiome and 1.1.1 Building moisture, microbes human health and human illness People in the modern world spend circa A higher prevalence of morbidity, especially in 90% of their time in various indoor environ- respiratory illness has been reported in water ments (Schwab 1992). Thus human expo- damaged, damp and mouldy houses com- sure to microbes concentrates on the species pared to undamaged ones (IOM 2004). Th e present in these environments, in practice, signifi cance of this phenomenon is striking; mainly on food- and indoor airborne bacteria, according to a recent estimate twenty percent fungi and viruses. Environmental microbes of current asthma cases in the United States have both benefi cial and harmful eff ects on – altogether 4,6 million cases – may be attrib- health. Microbial exposure starting at birth utable to residential dampness and mould leads to the development of the commensal (Mudarri and Fisk 2007). In Finland, expo- human microbiome which has an elemen- sure to moisture damaged building condi- tary role on various vital functions, ranging tions is recorded as the most signifi cant indi- from food digestion and pathogen resistance vidual cause of occupational asthma (Piipari to the proper development and maintenance and Keskinen 2005). A recent meta-analysis of immune functions (Jarchum and Pamer of relevant epidemiologic literature by Men- 2011). For example, the exposure to diverse dell et al. (2011) concluded that dampness or micro-organisms is believed to be a critical mould had – globally – consistent positive factor explaining the lower incidence of aller- associations with several allergic and respi- gic diseases in children in farming environ- ratory outcomes. Besides development and ments compared to urban environments (Ege exacerbation of asthma, associated condi- et al. 2011). Microbial health risks of indoor tions included dyspnea and wheeze without environment in turn are oft en associated with asthma diagnosis, cough, allergic rhinitis and low indoor air quality (IAQ) in the presence eczema. Suffi cient evidence of association was of excess moisture and mould contamina- also found for increased occurrence of upper tion in a building (Mendell et al. 2011). Th is respiratory infections and bronchitis (Mendell phenomenon has been of concern at least the et al. 2011). Besides these, there are other con- past 3500 years (Leviticus, ch. 14, v. 33-48) but ditions that have been empirically associated is very acute today. Th e indoor environment with indoor mould exposure, but for which may also serve as a reservoir for pathogens. the epidemiological evidence has been seen The rate of opportunistic infections caused inconclusive (Mendell et al. 2011). Such dis- by a variety of fungal and bacterial species of eases include hypersensitivity pneumonitis low virulence has increased due to the grow- (HP, also known as extrinsic allergic alveolitis) ing proportion of immunocompromised and and the organic toxic dust syndrome (ODTS), chronically ill population (Groll and Walsh which have originally been described as occu- 2001, Liu 2011). pational lung diseases (Husman 1996, IOM 2004, Mendell et al. 2011). HP is an infl ammatory lung illness occurring in susceptible people aft er inhalation of high quantities of

1 Introduction specifi c microbial antigens. ODTS is a non- ly concluded that any quantitative microbial allergic illness associated with occupational measure does not provide a more reliable microbial exposure in agricultural environ- indicator of potential health risks than a care- ments. Besides pulmonary dysfunctions, ful examination of the presence of dampness, these conditions involve non-specifi c symp- water damage, visible mould or mould odor toms such as fever, cough, nausea, fatigue and or a history of water damage in a building. headache, which are also commonly reported Th e poor performance of objectively measur- among occupants of moisture-damaged build- able indicators in epidemiological studies may ings (IOM 2004). be explained by the complex and compound Microbial growth and emissions are nature of indoor exposures, synergistic eff ects hypothesized to play a key role in the develop- of microbial and non-microbial pollutants, ment of building-related illnesses (Figure 1). and the varying extent of the population sus- Th is idea is supported by the fact that micro- ceptibility. Such complexity makes finding bial growth is more or less an inevitable result associations diffi cult even using large data sets of extended wetting of building surfaces, and (Nevalainen and Seuri 2005). observations of “dampness” and “mould” are Besides the complexity of the exposing most easily interpreted as visual and olfactory agent, the microbial exposure assessment has signs of microbial growth. Th e hypothesis is been complicated by the deficiencies in the also supported by the health eff ects of verifi ed traditional methods used to identify and enu- microbial exposure in occupational settings merate microbial agents. Th e major problems mentioned above, as well as by the knowledge relate to selectivity and low resolution of such obtained from toxicological studies; spores, methods. By traditional plate cultivation only fragments and metabolic compounds released species that grow and produce characteristic from several microbial strains isolated from morphological structures in laboratory condi- contaminated building sites have toxic, tions can be identifi ed. Direct microscopy and infl ammatory and immunomodulatory eff ects measurement of proxies for fungal and bacte- on mammalian cells and tissues in vitro and in rial biomass (such as ergosterol, β-D-glucans, vivo (WHO 2009, Mendell et al. 2011). extracellular polysaccharides [EPSs], endotox- Despite the consistent epidemiological in and muramic acid) can reveal also uncul- association between dampness, the causality, turable material, but the capacity to distin- the causative agents and disease mechanisms guish between microbial taxa is more or less are poorly understood. During the last 25 limited (Pasanen 2001). For exposure assess- years, the correlation between the presence of ment, however, an unselective, specifi c iden- moisture damage and microbial attributes (i.e. tifi cation and enumeration of microbes would the how moisture damage alters the indoor be necessary; the potential health-eff ects of microbiomes) and between microbial attri- microbes can be species- or strain-specifi c and butes and adverse health findings (i.e. the independent of cell viability (Flannigan et al. probability that the observed health effects 2011). Th e fast advances in the development occur due to microbial exposure) have been of DNA-based methods for microbial identifi - assessed in tens of studies (as summarized in cation may off er solutions for these problems. e.g. IOM 2004, WHO 2009 and Mendell et al. Th ese methods are further discussed below in 2011). However, based on the currently avail- chapter 1.4. able information, Mendell et al. (2011) recent-

2 Introduction

1.1.2 The indoor environment as a of patients suffering from Mycobacterium reservoir for opportunis c human avium complex (MAC) pulmonary infections. pathogens Th e authors demonstrated that bathtub inlets People with decreased immunocompetence and showerheads in the case homes were may be susceptible to normally harmless envi- commonly colonized by these bacteria, and ronmental microbes. Severe forms of immu- may have served as sources of inoculum for nodefi ciency are caused by cancer radiation- the recurrent infections typical of the patients and chemotherapy, transplantation medica- with MAC infections (Nishiuchi et al. 2009). tion and progressed HIV-infection. Other Moisture damaged buildings in which species susceptible groups include premature infants, capable of producing immunomodulatory trauma patients and people with severe forms mycotoxins occur in parallel with opportunis- of common chronic conditions such as cardio- tic species form an interesting environment vascular disease or diabetes mellitus. While with respect to human health. As mentioned the proportion of populations with increased in the previous chapter, lowered resistance susceptibility is expanding, the diversity of agains upper respiratory infections as well as microbial species associated with infections is an increased prevalence of sequelae diseases also growing; besides well known nosocomial such as otitis media have been associated with agents such as Candida albicans, Staphylococ- mouldy buildings (Mendell et al. 2011). In cus aureus or Pseudomonas aeruginosa, atypi- healthy individuals, building related microbes cal microbes such as various saprotrophic fun- themselves generally establish their harmful gi are emerging as causative agents of oppor- eff ects via allergy or irritation/infl ammation tunistic infections (Groll and Walsh 2001). instead of an invasive infection. However, in Certain fungi such as Aspergillus fumigatus some conditions colonization by a specific are of concern both due to their pathogenic- agent takes place. Th e most signifi cant of such ity as well as their allergenicity in the indoor diseases is probably the allergic bronchopul- environment. monary aspergillosis (ABPA). This chronic Th e information about the potential res- disease involves superfi cial growth of Asper- ervoirs and natural habitats of rarely encoun- gillus (A. fumigatus being the most commonly tered opportunists is oft en scarce, and little is detected species) in RT mucus, which causes known about their occurrence in the normal allergic infl ammation of the epithelia and may living environment (Liu 2011). Recent eff orts lead into lung tissue scarring over time. Th e to map the indoor microbiomes have revealed role of other fungi in related conditions is less that specific indoor niches can maintain well known (Gore 2010). opportunistic microbes; for example, Zalar et al. (2011) found that thermophilic members 1.2 House dust of the genus Exophiala – including species which are increasingly associated with human 1.2.1 Airborne par cles and dust infections - commonly inhabit household sampling dishwashers. Related fungi were also often The major route of human exposure to air- found in other humid indoor environments in borne microbes is via inhalation. However, the study of Lian and de Hoog (2010). Nishiu- direct measurement of airborne particles, chi et al. (2009) in turn investigated the homes especially of microbial ones, has shown to be

3 Introduction

Figure 1. Summary of the hypothesized relationship between water damage, microbial growth and adverse health eff ects. “Building dampness and mould” are known to be associated with adverse health eff ects in building users (right side of the fi gure). Th e left side of the fi gure elucidates the complexity of the involved agents and exposure dynamics. Water is the limiting factor for microbial growth in the indoor environment. Micro-organisms are ubiquitous in the environment and their growth generally initiates within days on wetted building materials. Saprotrophic bacteria, fungi and protists (e.g. amoebas) proliferate on building materials and numbers of Arthropods (eg. mites and insects) may also increase. Th e species diversity depends on the inoculum, substrate type and microbial succession takes place if water is available under a long period (Flannigan and Miller 2011).

4 Introduction

Microbial growth may produce spores, hyphae, yeast- and bacterial cells as well as fragmented cell material (Green et al. 2005); mites contribute to the dispersal of fungal spores (Colloff 2009) while amoebas may aid in the dispersal of bacteria (Yli-Pirilä et al. 2006). Microbial material may contain bioactive compounds such as allergens and microbial toxins (Brasel et al. 2005a, Green et al. 2006, Polizzi et al. 2009, Täubel et al. 2011). Th e amounts and types of produced substances vary depending on the species and growth conditions, including substrate, water availability and co-occurring species (Murtoniemi et al. 2002 and 2005, Nielsen et al. 2003, Hirvonen et al. 2005). Microbial volatile- and particle-bound compounds may become airborne and spread along air currents to the living space (Górny et al. 2001, Green et al. 2006). Exposure to microbial compounds takes place mainly by inhalation, but to lesser extent also by skin contact and inges- tion of dust (infants) (WHO 2009, Roberts et al. 2009). Part of the inhaled particles is deposited in the airways and small particles with diameter under 2.5 μm may end up in the alveoli (Górny 1999). Microbial compounds that become into contact with epithelial and immune cells may launch infl ammatory allergic or non-allergic signaling or have toxic or immunosuppressive eff ects (WHO 2009). Putative candidates for the causative agents include eg. EPS, endotoxin, allergenic enzymes and other proteins, microbial toxins and volatile metabolites (WHO 2009). Immunomodulatory and toxic eff ects of these compounds may be responsible for the observed symptoms and allergic sensitization alone or in combination with other environmental pollutants such as tobacco smoke, traffi c exhausts or non-microbial chemical emissions released from water aff ected building materials or other sources. Moreover, simultaneous exposure to multiple agents may have synergistic eff ects (WHO 2009). Typically only a part of equally exposed population develops symptoms. Th is variation in susceptibility may be attributed to various mechanisms such as individual diff erences in the capacity to tolerate, degrade and metabolize harmful substances (Wu et al. 2010) and genetic variation in the tendency to develop allergic responses in antigen contact (Kelada et al. 2003). Th e diagram shows that the connection between excess moisture and ill health eff ects is complex and dependent on the realization of several independent phenomena: suffi cient microbial growth needs to occur; harmful substances need to be produced and they need to be emitted and spread to the living environment in suffi cient quantities; susceptible people need to be exposed to them and the duration or frequency of exposure needs to be suffi cient to launch the symptoms.

problematic (Pasanen 2001). Airborne con- building diffi cult. Indoor levels usually refl ect centrations of microbes show signifi cant spa- those of outdoor air, where diurnal variation, tial and temporal variability (Verhoeff et al. and variation due to meteorological condi- 1990 and 1992, Nevalainen et al. 1992, Law tions are considerable (Li and Kendrick 1995a, et al. 2001), which makes determining the 1995b, 1995c). Human activity levels aff ect the “representative airborne microbial level” of a resuspension of settled particles and move-

5 Introduction ment of airborne particles from room to room bial levels in a building (Hyvärinen et al. (Law et al. 2001, Ferro et al. 2004). Moreover, 2001). activities such as opening of a cellar door, Th e collection of settled, once airborne cleaning, cooking and handling fi rewood and dust is an alternative to air sampling (Flan- entrance of people and pets from outdoors nigan 1997, Dillon et al. 1999). Dust can be may raise microbial levels significantly in a allowed to accumulate on surfaces for several temporary manner (Lehtonen et al. 1993). weeks or months prior to collection. Th e long Changes in ventilation, whether mechanical collection period acts as a buff er against vari- or natural, aff ect the air movements and trans- able airborne concentrations and makes dust portation of particles and volatiles within the a long term, time-integrate sample of airborne building, and may also alter the routes and material (Portnoy et al. 2004, Egeghy et al. quality of intake air. For example, the use of 2005). Dust samples have been used in the equipments such as central vacuum cleaners, assessment of indoor exposure to environ- local exhaust fans for cloth dryers and bath- mental pollutants such as heavy metals, pesti- rooms, and even furnaces that underpres- cides, phthalates and other chemicals (Roberts surise the room space may lead into tempo- et al. 2009), as well as to biologicals such as rary periods of altered entrance of replacing mites and pet allergens (Roberts et al. 2009, air. In the absence of proper intake air ducts, Colloff 2009). the intake air may infi ltrate trough the build- Viable microbes have been measured ing envelope from contaminated structures from dust in numerous studies (Verhoeff such as crawl spaces, funnels or water-dam- 1994a, Verhoeff and Burge 1997, Dillon et aged sites and increase the airborne concen- al. 1999, Chao et al. 2002, Chew et al. 2003, trations of contaminants. In combination Horner et al. 2004, see also Appendix I table). these phenomena cause significant tempo- Yet the results have sometimes been con- ral and spatial variation to indoor airborne cluded to be poorly representative of airborne microbial levels. To overcome this variability microbial levels due to their low correlation and to obtain a representative sample from with short-term air samples (Ren et al. 1999). a building, sampling over long time periods, One problem with dust is the great diffi- preferably days to weeks has been evaluated culty of enumerating fungal spores by direct to be necessary (Hyvärinen et al. 2001). How- microscopy due to the abundant background ever, long-term air sampling has several tech- debris (Pasanen 2001). For this, viable culti- nical limitations. Depending on the used sam- vation has traditionally been the only feasible pling device, overloading of agar plates and method for the identifi cation and enumera- impaction slides, or blocking of fi lter mem- tion of dustborne microbial communities. branes used to collect particles takes place and While the long term accumulation period is limits the collection time. Signifi cant desicca- the main advantage of dust as a sample type, it tion and subsequent loss of viability of micro- is also its weakness when measuring analytes bial particles is a problem in extended forced with varying stabilities. If signifi cant degra- air sampling (Wang et al. 2001). If short-term dation or inactivation of the measured sub- samples are used, multiple samples are needed stance takes place during the collection, the to gain adequate representativeness of micro- results can be expected to be underestimates of the “fresh” airborne concentrations. Th is is

6 Introduction a valid concern in the case of cultivation, since sition of dust varies depending of building the persistence of microbial species in indoor type and use, and major particle sources. For conditions varies signifi cantly (Sussman 1968, example, the composition of offi ce dust dif- Verhoeff et al. 1994a). Contrasting to cultiva- fers from home dust, while dust in apartments tion methods, direct DNA-based microbial diff ers from that in houses, and further, dust detection methods do not suffer from this in rural houses differs from dust in urban problem since they are independent of cell houses (Macher 2001, Chew et al. 2003, Møl- viability. Microbial DNA also persists well in have et al. 2007, Pakarinen et al. 2008, Ege et indoor conditions (Fierer et al. 2010, Lauber al. 2011). Th e study of Mølhave et al. (2007) et al. 2010). exemplifi es the crude content of offi ce dust. A Th e methods used to collect settled dust vary large composite sample of 11 kg of fl oor dust greatly between studies, ranging from vacu- was collected from seven large Danish offi ce um-collection from carpets, chairs, mattresses buildings (area 12 751 m2). Th e organic frac- of smooth horizontal surfaces to sieving the tion was 33%, total concentration of micro- fi ne dust fraction directly from the dust bag of organisms 130.000 ± 20.000 CFU g-1 and the residential vacuum cleaner (Macher 2001). concentration of viable fungi 71.000 ± 10.000 The diversity of sample types undermines CFU g-1. Th e dust also contained human and between-study comparisons, since different animal skin fragments, hairs, paper and textile dust types are prone to refl ect diff erent aspects fi bers, glass wool, wood and metal particles, as of indoor exposures. For example, mattress well as unknown organic and inorganic par- and chair dust may be heavily contributed by ticles. Th e size fraction of < 10 μm, i.e. the size human microbial fl ora (Täubel et al. 2009), class of most fungal and bacterial spores/cells, while fl oor dust content may be signifi cantly accounted for < 1.5 % of total mass. aff ected by coarse debris, which has not neces- House dust components of microbial ori- sarily been airborne (Lewis et al. 1999). Dust gin may include viable and non-viable intact collected from fl oors is most commonly used fungal conidia, spores and spore clumps; in large epidemiological studies due to its ease fragments of spores, hyphae, sclerotia, lichen and low costs of collection, yet dust on elevat- soredia and fruiting bodies; and bacte- ed surfaces could be considered to represent rial cells, endospores and fragmented cells better the inhalable fraction of airborne dust. (Piecková et al. 2004, Green et al. 2006, Ale- Sampling devices, for example electrostatic nius et al. 2009). Th e size and shape of intact collectors, have recently been developed for fungal spores varies from tiny, round to ovoid standardized sampling of settled dust (Noss 2 - 5 μm conidia of Aspergillus, Penicillium et al. 2008), and airborne dust (Nilsson et al. and other trichocomaceous moulds to large, 2004). With such collectors the sampling time oblong ≥ 50μm conidia of Alternaria and and area/volume can be standardized. Helminthosporium. Fungal fragments vary in size from sub-micrometer (< 1 μm) particles 1.2.2 Microbiological composi on of to larger hyphal parts of the length of tens to dust hundreds micrometers (Górny 2004, Green et al. 2006). Microbial particles carry many Dust is a complex mixture of organic and structural compounds that can be measured inorganic material. In general, the compo- as crude proxies for microbial biomass; such

7 Introduction include ergosterol and (1→3)-beta-D-glucan dust. Th e main sources of indoor microbes are (for fungi) and lipopolysaccharide (LPS, or the outdoor environment (soil, decompos- endotoxin) and N-acetylmuramic acid (for ing plant litter and the phylloplane [surfaces gram negative and –positive bacteria, corre- and tissues of living plants]), humans, pets, spondingly). Enzymes and other proteins are house plants and raw or spoiled materials like also present, as well as non-volatile metabolic vegetables, fruit, mouldy bread and fi rewood products such as microbial toxins and volatile (Hunter et al. 1988, Lehtonen et al., 1993 organic compounds (MVOCs) like alcohols, Wouters et al. 2000, Scott 2001, Glushakova terpenes and aldehydes, whose production is et al. 2004, Aydogdu et al. 2010). Th e trans- usually restricted to few individual fungal spe- fer of microbial particles from outdoors takes cies or strains (Korpi et al. 1997, Górny 2004, place by airborne transmission through open Cho et al. 2005, Green et al. 2006, Bloom et al. doors and windows, ventilation ducts and 2009, Täubel et al. 2011). leakages in the building envelope. Microbes are also carried indoors along with soil, plant 1.2.3 Development and dynamics of debris and other particles attached to shoe microbial popula ons in dust soles, clothes and pet fur (Pasanen et al. 1989, Viable microbial populations in dust may be Lehtonen et al. 1993, Law et al. 2001). In addi- either of autochthonous or allochthonous tion to these “background” microbial sources, nature or a combination of these two (Bron- a potentially very signifi cant indoor contribu- swijk 1981). An autochthonous population (a tor can be active microbial proliferation in true population) develops by active growth building surfaces and constructions (Green et and proliferation on site. Allochthonous al. 2003). It is notable that each of the major populations (pseudopopulations), in contrast, natural fungal habitats (food, phylloplane, develop by mechanisms other than prolifera- soil) harbours species that may actively pro- tion, i.e. by passive accumulation of particles liferate on indoor materials and fi nishes in the from the surroundings. For example, air-cir- presence of excess moisture. Th us it may be culating fi ltering appliances like house hold diffi cult to distinguish between the contribu- vacuum cleaners and HVAC systems without tion of “normal” microbial sources vs. inap- an appropriate high-effi ciency particulate air propriate mould growth due to water damage (HEPA) fi ltration may shape indoor microbial in building (WHO 2009, Lawton et al. 1998, assemblages by ineffi cient removal and even Fahlgren et al. 2010). enrichment of small spores and fragments which may pass through the fi lters and return Deposition and resuspension. Dust acts as into the indoor air (Scott et al. 2004, Cheong both a sink and a source for airborne particles. 2005). Viable communities may be further Yet, the common fi nding that the microbes in shaped by diff erential longevity of individual indoor air resemble more those in outdoor air species, which results in enrichment, and pro- than those in dust, suggests that resuspension portional increase of persistent species (Scott is partial (Ren et al. 1999, Chew et al. 2003, et al. 1999). Shelton et al. 2002). Size and shape variation among microbial particles contributes to their Sources. Bacteria and fungi are ubiquitous in differential dispersal, deposition and resus- the outdoor air, indoor air and also in settled pension in indoor spaces. In practise, due to

8 Introduction ventilation and human activities indoor air is over decades (Sussman 1968). Properties asso- in continuous movement and deposition and ciated with extended spore longevity include resuspension happen all the time in parallel. spherical cell form, thick cell walls, melanin Smaller particles tend to mix more effi ciently and other pigments in cell walls, low spore to the entire room space and stay longer air- water content, and high trehalose content borne compared to bigger particles (Carlile et (Sussman 1968, Carlile et al. 2001). A majority al. 2001, Li et al. 2005, Oberoi et al. 2010). On of the fungal types most commonly cultivated the other hand, fi ne particles (<2.5 μm) have from dust samples persist very long times in been reported to resuspend less effi ciently by indoor conditions. For example, the spores human activity than larger particles (Th atch- of Aspergillus, Penicillium and Fusarium spp. er and Layton 1995, Chen and Hildemann may retain their viability over ten years or lon- 2009). Th e sample type and sampling location ger. Much shorter survival times have been may have an eff ect on the observed dustborne reported for others, e.g. 2-6 months for many microbiomes. Ren et al. (1999) found more basidiospores and only days or even hours for small-spored species such as Aspergillus and some plant pathogens (Sussman 1968). As Penicillium, and less large-spored species such for common phylloplane fungi, apart from as Mucor, Wallemia and Alternaria in indoor Aureobasidium pullulans they survive much air than in vacuumed fl oor dust. Baudisch et shorter times compared to eg. Penicillium spp. al. (2009) in turn reported signifi cantly higher (Scott 2001, Baudisch et al. 2009). Flannigan concentrations of viable Penicillium, Aspergil- and Miller (2011) suggested that the relatively lus and Eurotium in dust collected from top of high abundance of Aureobasidium pullulans, shelves than in fl oor dust. Alternaria alternata and Epicoccum nigrum in indoor vs. outdoor air compared to Clado- Dormancy and longevity. In long-term sam- sporium cladosporioides, C. herbarum and C. ple types such as settled house dust the diff er- sphaerospermum could be explained by supe- ential longevity of deposited organisms may rior longevity of these species in indoor con- greatly aff ect the culture-based measure of the ditions (Flannigan and Miller 2011). sample’s microbial content. In some studies, the viable fungal composition of dust sample Microbial growth in house dust. Active has been suspected to be signifi cantly associ- microbial proliferation, i.e. the development ated with its age (Baudisch et al. 2009). Th e of autochthonous populations in dust can major environmental factors that affect the be expected to alter the microbial commu- survival of fungal propagules include temper- nity content significantly from the original ature, relative humidity (RH), radiation and proportions of microbes in the settled mate- predation by other organisms such as mites. rial. Using such dust as a representative of the In indoor conditions the longevity of fun- microbial load of larger indoor spaces would gal propagules varies greatly between species in turn lead into severely biased view of the and also between diff erent spore- and particle microbiological status of the building. types within one species. In general, hyphal Deposited viable microbial particles may fragments lose their viability, i.e. the ability to germinate, grow and proliferate if minimum start new growth fi rst. Dispersal spores persist requirements for temperature, substrate avail- longer, and survival structures may stay viable ability and water activity are met. Th e pres-

9 Introduction ence of organic debris in house dust gener- microbial proliferation may take place also in ally provides suffi cient nutrients for growth “dry” room areas in principally safe and stable of saprotrophic fungi and bacteria and the indoor humidity levels due to a local increase limiting factor for growth becomes the avail- in RH (Harriman 2011). Th e water activity of ability of water (Korpi et al. 1997). Dust is dust or other material may increase signifi - usually hygroscopic and its water activity [aw] cantly if the surface temperature is lower than reaches equilibrium with the RH of the air in that of the surrounding air. Condensation of the surrounding microenvironment. In the water is an extreme example of this phenom- room temperature the minimum aw required enon, but water activities suffi cient to support by the most xerophilic fungi is ca. 0.70-0.80 microbial growth do not require condensation (Grant et al. 1989, Flannigan and Miller 2011). to occur. For example a local decrease in tem- Fungal species capable of slow proliferation in perature by 5°C from 20 to 15°C increases the such conditions include certain members of RH near the cool surface from 60% to 80%, Eurotiomycetes like Eurotium repens, Asper- a level suffi cient to support slow but steady gillus penicilloides, Penicillium chrysogenum, P. growth of xerophilic fungi. Such conditions brevicompactum and a few other species, e.g. may prevail nearby leaking building corners Wallemia sebi (Lustgraaf 1977, Hay et al. 1992, and seals, cold bridges and insuffi ciently insu- Kalliokoski et al. 1996). Most of the mould lated outer layers of the building envelope. types common in outdoor air, for example Locally, another signifi cant source of moisture Cladosporium and Alternaria are unable to concerns upholstered furniture, especially proliferate in normal indoor conditions but mattresses. Th e regular use of this furniture need a liquid water source or near 100% RH may lead into signifi cant uptake of moisture to grow (Grant et al. 1989). Bacteria require generated by the users, and subsequently into considerably higher water activities, at least microbial proliferation in the bound dust 0.90-0.95 for growth (Brown et al. 1976). (van Reenen-Hoekstra et al. 1993, Beguin et In dry dust the microbial metabolic al. 1995). Mattress dust is oft en collected for activity and growth are negligible and the the evaluation of personal microbial exposure resulting populations develop through alloch- in home environment, but it must be remem- thonous processes. However, there are indi- bered that due to the potential local amplifi ca- cations that local conditions able to support tion of adapted microbes this sample type may some level of fungal proliferation may regular- emphasize fl ora distinct from the remaining ly develop even in normal houses. Xerophilic building. fungi have been shown to form unnoticeable Few laboratory studies report the eff ects microcolonies on temporarily wetted surfaces of elongated storage in slightly elevated RH on (Pasanen et al. 1992). Microcolonies provide real-life dust microbial communities. Korpi et spores into the surroundings and may explain al. (1997) observed signifi cant proliferation the low but stable levels of xerophilic fungi of fungi in house dust aft er 25 days of incu- commonly measured indoors in cool climates bation in 84-86% RH. Th e proliferation rates during winter when the outdoor air is not a differed markedly between species, as over source of these species (Pasanen et al. 1992). thousand-fold amplification was measured Besides bathrooms and other living areas with for viable Aspergillus but less than hundred- obvious occasional moisture burden, indoor fold increases for most other fungi. Incuba-

10 Introduction tion in 75% RH has also been shown to sig- Aspergillus, while other fungi, mainly Alter- nifi cantly alter the community composition; a naria and Cladosporium were reported to ten-week incubation of dust in 75% RH in the dominate in outdoor air. Signifi cantly higher room temperature was reported to result in counts of fungi and bacteria were found in old a ten-fold increase in the levels of xerophilic and used furniture materials compared to new Aspergillus, Penicillium and Wallemia sebi. In ones, which the authors reported to indicate contrast, Aureobasidium, Fusarium, Geotri- active growth and sporulation in and on the chum, Monilia and Mucor, which were present stuffi ng over time (Swaebly and Christensen in fresh dust, were lost within four weeks of 1952). Th e authors noted that the fungal levels incubation. Aft er ten weeks the fungal diver- and types in house dust oft en diff ered signifi - sity had dropped signifi cantly in all analysed cantly from those in both indoor and outdoor samples and mainly xerophilic taxa Aspergil- air. They also reported significant temporal lus penicilloides, Penicillium brevicompactum fl uctuation in the indoor air mould levels dur- and W. sebi could be isolated (Hay et al. 1992). ing the day and associated the variation with Even in conditions that do not support fun- human activities. These early findings con- gal proliferation, the mere survival times of cerning the viable fungal levels; the prevalent microbes aff ect the dust viable composition, indoor and outdoor taxa; the strong variation as described in the previous chapter. These on fungi in indoor air, and the tendency of reports show that dust may not be a stable res- indoor materials to accumulate fungal spores ervoir for viable fungi, but instead, the com- over time have since been verifi ed by numer- munities may be severely affected by local ous other studies (Flannigan and Miller 2011). conditions over time. Major indoor taxa. 1.2.4 Fungal diversity in house dust Table 1 lists the dominant viable microbial History. Th e fi rst studies on fungal levels and genera in indoor air and dust samples accord- diversity in house dust date back to the 1940s ing to the literature. Appendix 1 table gives a and 1950s. By that time, the major outdoor detailed list of the fungal species commonly air spora had already been characterized and isolated from house and offi ce dust samples associations between fungi and allergic diseas- using culture methods. Based on a myriad es such as asthma symptoms and rhinitis had number of culture-dependent studies (see been detected (for an early review, see Morrow footnote in Appendix 1 for references) sup- and Lowe 1943). Th e early studies explored ported by a so far limited number of culture- the occurrence of fungi in pillows, furniture independent reports (Amend et al. 2010a, stuffi ngs and -covers as well as house dust in Noris et al. 2011) the indoor environment is relation to asthmatic reactions and allergic dominated by a restricted number of globally sensitization (Conant et al. 1936, Flood 1931, occurring fungal taxa. Wallace et al. 1950, Swaebly and Christensen Circa twenty fungal genera and about 1952). Among the fi rst studies to explore dusts 200 individual fungal species are commonly collected from the indoor environment, Swae- isolated from dust by standard cultivation bly and Christensen (1952) reported viable methods (Table 1). Sterile isolates (“mycelia fungal levels of 1x104 - 5x105 cfu g-1 of house sterilia” or “non-sporulating isolates”) are also dust, consisting mainly of Penicillium and commonly cultivated from dust samples, and

11 Introduction

Table 1. Fungal and bacterial genera commonly isolated from indoor samples using culture-based methods

Fungia Bacteriab Filamentous, Ascomycota Actinobacteria (gram+) Acremonium Fusarium Arthrobacter 1, 2, 3 Mycobacterium 2, 3 Alternaria Pithomyces * Corynebacterium 1, 2, 3, 4 Nocardia3, 6 Aspergillus Penicillium Kocuria3, 5 Rhodococcus1, 3 Aureobasidium Phoma Micrococcus1, 2, 3, 4, 5, 6 Streptomyces1, 3 Chaetomium Scopulariopsis Firmicutes (gram+) Cladosporium Trichoderma Aerococcus2, 3, 5 Staphylococcus1, 2, 3, 4, 5, 6 Eurotium Ulocladium Bacillus1, 2, 3, 4 Stomatococcus5, 6 Filamentous, Basidiomycota Enterococcus3, 4, 5 Streptococcus2, 3, 4, 6 Wallemia α-Proteobacteria (gram-) Filamentous, Mucoromycotina** Agrobacterium2, 3 Mucor Rhizopus γ-Proteobacteria (gram-) Yeasts, Asco- and Basidiomycota Acinetobacter 1, 2, 3, 4, 5, 6 Klebsiella 1, 2 Candida Saccharomyces Aeromonas2, 3 Moraxella1, 2, 3, 4, 6 Cryptococcus Sporobolomyces Chryseomonas 1, 3 Pantoea1, 3, 5 Rhodotorula Enterobacter1, 2, 3, 5, 6 Pseudomonas 2, 3, 4, 5, 6 Erwinia3, 5 Serratia3, 5 Flavimonas3, 5 a) for references, see footnote in Appendix 1. b) references: 1) Andersson et al. 1999, 2) Flannigan et al. 1999, 3) Górny et al. 2002a, 4) Fleischer et al. 2003 5) Bouillard et al. 2005, 6) Aydogdu et al. 2010. Genera occurring in two or more of the listed studies are included in the table. *Syn. Leptosphaerulina. **previously: phylum Zygomycetes; Hibbett et al. 2007. form a signifi cant proportion of total counts ium, yeasts) may be accentuated compared to in many studies (Beguin et al. 1999, Chao the ambient air (Hyvärinen et al. 1993, Ren et al. 2002, Hicks et al. 2005). Sterile isolates et al.1999, Chao et al. 2002, Chew et al. 2003, consist of both ascomycetous and basidiomy- Horner et al. 2004). cetous colonies that do not form characteristic spore-forming structures on culture and are 1.2.5 Bacterial diversity in house thus morphologically unidentifi able. Species dust common in outdoor and ambient air (Clado- Bacterial genera commonly isolated from sporium, Alternaria, Epicoccum) are prevalent indoor air and dust samples in a selection also in dust, but, depending on the collection of culture-based studies are listed in Tabl e site, taxa with extended longevity (Aureoba- 1. Contrasting to fungi, the major source of sidium, Eurotium, Penicillium, yeasts), large bacteria in indoor environment is humans; spore/particle size (Mucor, Alternaria) and/or the human normal flora constitutes mainly outdoor soil/debris origin (Penicillium, Fusar- of bacteria, which cover all human body sur-

12 Introduction faces and are shed to the environment alone nifi cant (Noris et al. 2011). Th e dominance of or in fomites (Sciple et al. 1967). Accordingly, gram-negative bacteria, especially of proteo- indoor bacterial levels have been shown to bacterial classes in outdoor air has been dem- be signifi cantly increased by the number of onstrated in several studies (Fierer et al. 2008, occupants (Bischof et al. 2002, Giovannan- Brodie et al. 2007, Fahlgren et al. 2010). gelo et al. 2007). Th e majority of dustborne To our knowledge, the presence or diver- bacteria consists of members of gram-positive sity of Archaea in indoor environments has phyla Actinobacteria and Firmicutes. Gram- not been studied. Since archaea occur in soils, negative bacteria, mainly Proteobacteria are aquatic environments and also in the human also present. Moreover, recent culture-inde- gut, they would probably be found also in the pendent studies have indicated the presence indoor environment using suitable methods. of members of the Bacteroidetes and other In the investigation by Brodie et al. 2007, 307 groups, which occur in lower numbers (Pak- archaeal taxa were detected in urban outdoor arinen et al. 2008, Täubel et al. 2009, Noris et air using DNA-based methods. The major- al. 2011). Actinobacteria, Firmicutes and Bac- ity of the detected arhaea were members of teroidetes dominate on the human skin (Grice Euryarchaeota (Brodie et al. 2007). et al. 2009). Th e recent study by Täubel et al. (2009) confi rmed that the vast majority of 16S 1.2.6 Interac ons between fungi and marker sequences obtained from matress dust mites in the indoor environment corresponded with those of the users’ skin fl o- Moist indoor substrates, especially build- ra. A considerable, yet weaker contribution of ing materials that are constantly wet due to human-associated bacteria was seen in fl oor water damage may maintain diverse ecosys- dusts. Th e dominant human-associated genera tems with various microbes and e.g. mites in dust included Corynebacterium, Propioni- and amoebae. Th e predominant mites found bacterium, Staphylococcus, Lactobacillus and in buildings are house dust mites (HDMs) Streptococcus (Täubel et al. 2009). In addi- and storage mites (SM). Th e dominant HDM tion to humans, pets may be major sources species Dermatophagoides pteronyssinus, or carriers of bacteria indoors; Fujimura et D. farinae and Euroglyphus maynei belong al. (2010) reported a dog-associated increase to the family Pyroglyphidae (“pyroglyphid in bacterial diversity, majority of which was mites”), while most SM species, ie. Glycypha- putatively associated with an increased import gus domesticus, Tyrophagus putrescentieae and of bacteria from outdoors. Acarus siro are members of Glycyphaginae In addition to the inhabitants, outdoor and Acaridae. In addition, tens of other, less air is a significant source of bacteria. The frequent mite species belonging to these and recent study by Noris et al. (2011) indicated other families within the subclass of Acari are that especially gram-negative bacteria in found in the indoor environment (van Asselt house dust were of non-human, putatively 1999). Allergic sensitization to both dust- and outdoor origin. Th is was supported by the fact storage mites is common in both rural and that the proportion of gram-negative bacte- urban environments (Arias-Irigoyen 2007, ria was substantially higher in an unoccupied Pennanen 2002). HDMs, especially D. ptero- test house than in occupied houses, where the nyssinus are oft en considered to be the major amount of gram-positive bacteria was sig- sources of domestic mite allergens due to their

13 Introduction predominance in house dust samples (Arlian 1.3 Moisture damage and indoor et al. 2002). However, in cold climate regions, microbial communiƟ es including Scandinavia, HDMs are rare due to the low indoor air RH. In such environments 1.3.1 Microbial ﬁ ndings associated SMs may be of more importance, especially in with moisture damage the context of water damaged problem build- Th e eff ect of moisture damage on dust- and ings (Warner et al. 1999, Charpin et al. 2010). airborne microbial communities has been HDMs thrive in dark and warm sites assessed in several studies in order to iden- where protein-rich substrate such as human tify probable causative agents for observed dander is available. HDMs are thus the domi- health impacts for research purposes, or to nant mite type found in mattresses and pad- detect useful indicators of moisture problems ded furnitures. Th ey are able to grow in lower for building diagnostics. To summarize, the RH than fungi (RH >55% in RT). In contrast, eff ect of water damage varies greatly. In cul- SM species require RH levels similar to fungi ture-dependent studies, correlations seem to (RH >80%) and thrive in the same microen- be observed more oft en for air than dust sam- vironments. High numbers of storage mites, ples (e.g. Hyvärinen et al. 1993), but culture- especially G. domesticus have been found independent methods have revealed strong from cellars and other home areas with high correlations for dust samples (e.g. Lignell et humidity, as well as from stored foods (Ishii et al. 2008, see chapter 1.4.4). In general, large al. 1979, Mehl 1998, van Asselt 1999). Recent- areas of visible mould usually cause signifi cant ly, mouldy interior wall surfaces colonized changes in viable indoor microbiota and the by Cladosporium, Aspergillus, Ulocladium, airborne microbial levels may clearly correlate Alternaria, Penicillium and Acremonium were with the severity of the damage (Green et al. reported to be commonly co-infested by stor- 2003, Lignell et al. 2008). In contrast, damage age mites (Charpin et al. 2010). Dense mite hidden inside the building cavities may not populations may also develop on e.g. gypsum raise the viable fungal levels notably (Miller board in moist wall cavities, where the mites et al. 2000). In large data sets the mean con- graze on fungal growth, e.g. Stachybotrys centrations of airborne viable fungi are oft en chartarum and Acremonium spp. (Scott 2001). higher in moisture damaged than in undam- Mites are known to feed on fungal mycelium aged buildings (Pasanen et al. 1992, Verhoeff and spores, yet the dietary preferences for dif- et al. 1992, Li and Kendrick 1995a, Garrett ferent fungi may vary between mite species. et al. 1998, Lawton et al. 1998, Hyvärinen Fungal spores are partially digested by mites et al. 2001, Green et al. 2003). However, the and excreted in the mite faeces. Intact spores concentration distributions of damaged and may germinate in the excreted faecal pellet, undamaged buildings are oft en largely over- which provides nutrients for growth even in lapping (Nevalainen et al. 1991, Hyvärinen et the absence of other substrate. Thus mites al. 2001) and in some studies no association contribute to the dispersal and proliferation of between damage and viable fungal concen- indoor fungi (Colloff 2009). trations has been found (Strachan et al. 1990, Chew et al. 2003). Instead, increased diversity and/or changes in the microbial types have been found in some studies (Miller et

14 Introduction al. 2000, Nilsson et al. 2004, Baudisch et al. genera Brevibacterium, Streptomyces, Nocar- 2009). However, in some cases, no detectable dia, Nocardiopsis and Micrococcus in paral- associations between damage and microbial lel material and bioaerosol samples collected concentrations or types have been seen (Ren from moisture damaged buildings. However, et al. 2001, Chew et a. 2003, Piecková et al. Actinobacteria are also common in soil and 2004). Despite the variable fi ndings, detection plant material, and despite their status as indi- of certain species that are seen to occur rarely cator microbes for moisture damage (Samson in normal indoor environment in culture has et al. 1994), their abundance indoors may been concluded to be highly indicative of also represent other sources (Johansson et al. moisture problems in most cases (Samson et 2011). al. 1994). Th e fi rst moisture damage indicator In recent years, increasing attention has list was compiled in an international work- been paid to fragmented spore and vegeta- shop of microbiologists (Samson et al. 1994) tive cell material released from fungi and fi la- and included the following species or genera: mentous actinomycetes growing on indoors Aspergillus fumigatus, A. versicolor, Exophiala, materials (e.g. Górny 2004). Since fragmented Eurotium, Fusarium, Penicillium spp. (e.g. P. material loses its viability faster than intact chrysogenum and P. aurantiogriseum) Phia- spores, it is less effi ciently detected by culti- lophora, Rhodotorula, Stachybotrys, Trichoder- vation and may be largely overlooked. How- ma, Ulocladium and Wallemia. Th is list, some- ever, the mass of released fragments may be times supplemented by additional genera such comparable to that of released spores, and as Botrytis, Chaetomium, Paecilomyces and fragments may outnumber spores by several Rhinocladiella is commonly used as the “fi eld orders of magnitude due to their small size guide“ for evaluating the microbiological sta- (Reponen et al. 2007). Fine fragments of < tus of a building using air sampling. 2.5 m have been shown to be released from Besides fungi, the occurrence of certain microbial growth on indoor materials and to bacteria has been observed to be indicative be present in moisture damaged indoor envi- of moisture problems (Rintala et al. 2004). ronments (Górny et al. 2002b and 2003, Bra- Members of the Actinobacteria, especially sel et al. 2005b, Cho et al. 2005, Reponen et genera Streptomyces, Pseudonocardia and al. 2007). Like spores, fragmented cell mate- Nocardiopsis, but also some Mycobacterium rial may carry toxic and antigenic compounds spp. (see also discussion on bacterial fi ndings and, due to the small size, may be effi ciently in dust), commonly grow on moist building deposited in the human airways (Górny et al. materials (Rintala et al. 2002, Torvinen et al. 2002b, Brasel et al. 2005b, Green et al. 2005, 2006, Schäfer et al. 2009, Suihko et al. 2009). Cho et al. 2005). Fungal fragments have been Many of these genera include known produc- assessed by the measurement of N-acetyl- ers of various bioactive secondary metabolites β-D-glucosaminidase (NAGase) and (1 → and VOCs and are also capable of releasing 3)-β-D-glucans in the fine particle fraction aerosolizeable spores and mycelial fragments (Madsen et al. 2009, Reponen et al. 2007). to the surrounding air (Schöller et al. 2002, Microbial fragments have also been shown Górny et a. 2003) and may thus be of impor- to contain nucleic acids (Madsen et al. 2009), tance with respect to occupant health. Schäfer which makes them feasible targets for PCR et al. (2009) detected matching phylotypes of based detection methods.

15 Introduction

1.3.2 Seasonal varia on and indoor DNA (eg. Edwards et al. 1989, White et al. sampling 1990, Jürgens et al. 1997); the development of In the subarctic climate the effect of out- high-throughput capillary sequencers and up- door microbial sources on indoor micro- scaled pipelines and equipments for process- bial assemblages is at their minimum during ing large DNA clone libraries; and recently, winter due to frozen ground and snow-cover. the advent of highly parallel “next-generation” Th e indoor airborne concentrations of viable sequencing methods. The latter enable the fungi have been shown to be very low then, sequencing of hundreds of thousands of DNA typically between 101 - 102 cfu/m3. Due to low fragments without previous separation by background, unnormal intramural micro- cloning (Margulies et al. 2005). bial sources are most visible and most easily Based on the comparison of direct detected then; thus, restricting air sampling microscopy analysis and cultivation of to winter months is strongly recommended microbes, it has been long known that a vast (Reponen et al. 1992). However, if long term majority of microbial cells are uncultivable samples are collected and analysed using in laboratory conditions (Staley and Konop- culture-independent techniques, microbial ka 1985, Rappe and Giovannoni 2003). Th is material persisting from past seasons may be “uncultivable majority” may include a) dead detected even in winter samples, which may and dormant cells or spores of well-known, undermine the detection of fresher material cultivable species, b) cells of microbial species emitted from intramural sources. Textile-cov- that require specific enrichment and isola- ered furniture are known to accumulate and tion techniques to be detected and c) species maintain even viable microbes over long time whose growth is inhibited by competing spe- periods, but little is known about the persis- cies on culture plates (Amann et al. 1995). tence and seasonality of non-viable microbial Recently, direct DNA-targeting methods that materials in indoor matrices such as dust over circumvent the requirement of monoculture extended time periods. Th e recent study by isolation for characterization have made each Kaarakainen et al. (2009) using qPCR indeed of these uncultivable categories accessible for suggested that diff erential seasonal loads of phylogenetic analysis. Thus it is no wonder fungi are better represented in fl oor than rug that molecular, cultivation-independent tech- dust. niques have become routine tools for microbial community characterization and monitor- 1.4 Molecular methods in ing. Today the list of explored environments microbial biodiversity studies is very long, extending from soils (Fierer et al. 2007, Urich et al. 2008) and plant ecosys- Th e advances in molecular technologies have tems (Neubert et al. 2006) to the atmosphere revolutionized the research of both fungal and (Fröhlich-Nowoisky et al. 2009); and from the bacterial ecology during the last two decades. human skin (Costello et al. 2009) to domestic The key steps have been the establishment environments such as shower curtains, drains of protocols for universal amplifi cation and and even toilet rims (McBain et al. 2003, Kel- sequencing of phylogenetically informa- ley et al. 2004, Egert et al. 2009). tive gene regions, mainly within ribosomal Each of these studies has been effi cient in providing information about the diversity

16 Introduction and structure of microbial communities pres- molecular data (Schadt et al. 2003). Only very ent. In most cases, the observed diversity has recently, the first cultivated member of the greatly exceeded the known viable diversity. clade was described. Based on characteriza- For example, using an RNA-based metatran- tion of the isolates by their morphology and scriptomic approach to assess soil biodiversity multilocus gene sequencing and subsequent Urich et al. (2008) showed that nineteen out clustering with environmental sequences, an of 24 validly described bacterial phyla were entire novel ascomycete class Archaeorhizo- present in a soil sample collected from a single mycetes was proposed (Rosling et al. 2011). site. Fierer et al. (2007) in turn reported that These fungi are slow growing, have incon- the level of fungal diversity in soils was com- spicuous morphology and lack sporulation parable to the bacterial diversity in soil. Th ey in culture but are ubiquitous in various soils. estimated that the total number of fungal phy- Th ese examples show that ecologically signifi - lotypes could exceed 104 in some soil types. cant uncultivable or diffi cult to culture micro- Using PCR and ribosomal DNA sequencing organisms are found from common and non- O’Brien et al. (2005) recorded a high number specifi c habitats such as sea water or soil. (412) of fungal phylotypes in two 1000 m2 As described, molecular techniques uti- forest plots at a single sampling. Th e diversity lizing the direct analysis of phylogenetically is in line with that of an equally sized forest informative sequences from total environ- area previously recorded by a twelve-year mental nucleic acid extracts without prior sampling and identifi cation of 71,222 fruiting microbial isolation have proven to be more bodies (O’Brien et al. 2005). efficient and much less selective methods In addition to elucidating the habitats compared to traditional cultivation methods. and ecological roles of previously cultured However, molecular methods have their own organisms, culture-independent studies have limitations and sources of bias, which can revealed the existence of previously unknown aff ect the results. Th e limitations applicable to phylotypes and even entirely novel microbial the present work are reviewed in chapter 1.4.2. lineages, some of which have been later isolated in culture. The most famous example 1.4.1 Methods for DNA-based may be the alphaproteobacterial group SAR11 microbial community analysis which constitutes up to 50% of the bacterioplankton in open-ocean surface waters and Principles. Th e phylogenetic and functional has been estimated to be the most abundant diversity of microbial communities can be bacterial taxon on Earth (Morris et al. 2002). efficiently assessed through the analysis of Th is wide-spread clade was fi rst detected by amplifi ed marker gene fragments (amplicons) direct 16S rDNA amplifi cation and sequenc- obtained by PCR from total community DNA ing in the beginning of 1990s from the Sar- extracts. The targeted genes can be either gasso Sea (Giovannoni et al. 1990) but the fi rst phylogenetically informative regions of the member of this cluster was not isolated in cul- genome, such as ribosomal genes, or function- ture until a decade later (Rappe et al. 2002). ally (and oft en also phylogenetically) informa- Similarly, a globally distributed root-asso- tive genes that code proteins linked to specifi c ciated fungal clade known as the Soil Clone metabolic functions (Purkhold et al. 2000). Group 1 (SCG1) was long known solely from Universal PCR primers, i.e. primers designed

17 Introduction to amplify the target region from all or most turing gradient gel electrophoresis (TGGE divisions within the kingdom of interest and DGGE), terminal fragment length poly- (fungi/bacteria/archaea) are oft en preferrable morphism (tRFLP) and single-strand con- for total community analysis, yet also group- formational polymorphisms (SSCP) analysis specifi c primer sets can be used (e.g. Gardes off er fast and cost-effi cient ways to perform and Bruns 1993). In practice, universal prim- rough comparisons of microbial communities ers target highly conserved regions fl anking in multiple samples in parallel (Widjojoat- more variable stretches, which in turn contain modjo et al. 1994, Marsh et al. 1999, Muyzer suffi cient amount of phylogenetic information et al. 1999, Anderson et al. 2004). Th e main for identifi cation and classifi cation purposes. drawback of fi ngerprinting methods is their The present consensus regions for phyloge- low detection threshold or, more precisely, netic purposes are the 16S rRNA gene for bac- their low dynamic range. Fingerprinting teria (Edwards et al. 1989, Woese et al. 1990), methods also lack resolution, which may lead and the nuclear Internal Transcribed Spacer into further underestimates of species rich- (ITS or nucITS) region for fungi (White 1990, ness. Due to the lack of resolution, the identity Schoch et al. 2011). of detected DNA fragments (bands or peaks) should also be verifi ed by sequencing if phylo- PCR amplicon analysis. The obtained PCR genetic community data is desired. Fragment amplicons are typically analysed either by extraction from DGGE gels and sequencing is fi ngerprinting- or sequencing techniques, as sensitive to contamination and the quality of discussed below in more detail. Also, specifi c obtained sequences is oft en reduced (Ander- micro- or macroarrays may be set up to enable son et al. 2004). Some of these problems can hybridization-based detection of DNA signa- be overcome by combinatory approaches: tures at species level or at a higher taxonomic for example, parallelization of DGGE and level (Wu et al. 2003, Brodie et al. 2007, Hult- clone library sequencing makes it possible to man et al. 2008, Jakobs et al. 2010). Another affi liate phylogenetic information from long, related solution for amplicon characteriza- good quality sequences to individual DGGE tion is the hierarchical oligonucleotide primer fragments (Liang et al. 2008). Recently, a extension method (HOPE), basically a multi- novel technique that combines HPLC-based plexed minisequencing technique coupled to separation of PCR-amplicons with DNA frac- fluorescent capillary electrophoresis, which tion collection and sequencing was success- enables the semiquantitative detection of mul- fully applied for the characterization of wood tiple target groups from environmental sam- decay fungi (Maurice et al. 2011). Th is meth- ples (Wu and Liu 2007). Probing-based method shows promise in facilitating fragment ods can be very sensitive and fast, but can only sequencing, but it remains to be seen how well detect a limited number of pre-determined this approach works on highly diverse com- species or higher groups, for which specifi c munities. Generally, fi ngerprinting methods probes need to be designed and tested for are suitable for monitoring major changes in specifi city. microbial community composition, but are not ideal for detailed de novo-characterization Fingerprinting methods. Fingerprinting of environmental samples (Brodie et al. 2003). methods, including temperature- and dena-

18 Introduction

Sequencing methods. The full phylogenetic time-consuming and expensive. If the domi- information carried in the amplifi ed marker nant members of a community are known, an genes can be obtained by DNA sequencing. alternative is to design specifi c oligonucleotide Th e analytical sensitivity of sequencing meth- probes for them and remove them from the ods in detecting individual species within amplicon pool by subtractive hybridization. a community depends on the number of However, this method does not provide infor- obtained sequences, and naturally also on the mation about the original abundance of the diversity and structure of the target communi- subtracted sequence types in relation to other ty. For communities with low species richness sequence types. and high evenness a small number of sequences is sufficient to provide coverage over the “Next-gen” sequencing methods. At present, majority of species. In contrast, for the charac- the “next-generation” sequencing methods, terization of diverse communities with uneven especially the 454 pyrosequencing method species distribution more sequences are need- (Margulies et al. 2005) offer an appealing ed. Until the very recently introduced “next alternative to Sanger sequencing and are generation” sequencing methods (see below), becoming increasingly popular. Th e 454 pyro- the PCR amplicon sequencing has been done sequencing method originally suff ered from by cloning the PCR amplifi ed fragments into short (100 bp) sequence read lengths which an E. coli plasmid library. While the contruc- were insuffi cient for phylogenetic purposes. tion of large plasmid clone libraries is relative- Sequencing errors have also been of more con- ly inexpensive, obtaining sequence data from cern in the pyrosequencing method (Kunin et the clones using the common Sanger chemis- al. 2010, Tedersoo et al. 2010). Recent amend- try (Sanger et al. 1977) and capillary electro- ments have signifi cantly improved the usabil- phoresis is costly and time-consuming. Since ity of the methodology in microbial diversity many natural microbial communities have studies. The 2008 update to the “Titanium” long-tailed rank abundance distribution of chemistry yields longer (~400bp) read lengths species (i.e., there are only few abundant spe- and tag-sequence based multiplexing makes cies, but a large number of rare species; Gans it possible to analyse multiple samples in one et al. 2005) obtaining a reasonable coverage run. Improved algorithms for clearing out over the rare species necessitates the repeated the ambiguous sequences have improved the sequencing of the abundant types. To avoid quality of the data (Huse et al. 2010, Quince this, methods for pre-sequencing screening et al. 2011). With the present Roche 454 GS of identical clones have been needed. Finger- FLX System, ca. 100-200 samples can be con- printing methods, mainly amplifi ed ribosomal veniently processed within one sequencing DNA restriction analysis (ARDRA), have been run, producing ca. 5 000 - 10 000 reads of 400 applied to cluster clones into phylotypes prior bp from each sample. While the enormous to sequencing (e.g. Fröhlich-Nowoisky et al. throughput and speed are the clear advances 2009, Fujimura et al. 2010). However, as men- of the method, one disadvantage of pyrose- tioned above, fi ngerprinting methods suff er quencing compared to clone library sequenc- from the lack of resolution, ending up clustering is that the sequenced DNA itself is not ing unrelated sequences together (Sklarz et al. stored for later use in e.g. the design and test- 2009). Moreover, they easily also end up being ing of qPCR or probing methods. Concern-

19 Introduction ing highly parallel pyrosequencing it also is step are semiquantitative in nature; due to an notable that typical pyrosequencing data sets unequal amplifi cation of sequences that dif- cannot be handled with the traditional desk- fer in length, nucleotide composition and/ top tools for sequence analyses but, instead, or GC content, the proportions of amplifi ca- require signifi cant computational power and tion products do not always mirror the origi- effi cient bioinformatics tools (e.g. Schloss et nal species frequencies in the sample (Suzuki al. 2009). and Giovannoni 1996, Polz and Cavanaugh 1998, Amend et al. 2010b). Th ese biases may Quantitative PCR. Quantitative PCR is a pop- aff ect more severely the analysis of fungal ITS ular and highly accurate and reliable method sequences compared to bacterial 16S sequenc- for the quantification of gene copy number es due to the stronger length- and sequence or gene expression (Giulietti et al. 2001). Th e variation of the former (Huber et al. 2009, quantitativity is obtained by monitoring the Amend et al. 2010b). Th e results may also be accumulation of fluorescently labeled PCR affected by primer mismatches and the dif- product in real time and comparing it with a ferential gene copy number (von Wintzinger- standard curve obtained from a series of con- ode et al. 1997). Th e latter concerns especially trol amplifi cations of known amounts of the ribosomal genes, which are located in repeat- target gene. While qPCR is highly sensitive ed operons whose number may vary up to and fast, the number of species targetable in fi ve-to-ten-fold between species in both bac- parallel is limited, and untargeted species are teria and fungi (Herrera et al. 2009, Rastogi not detected. Th e specifi city depends on the et al. 2009). Th us phylotype frequency results used method (e.g. the commonly used Syb- from PCR based analyses cannot be directly rGreen and TaqMan methods diff er in their translated to relative species frequencies, specifi city) and each assay needs to be tested but instead, should only be treated as rough in vitro with target and nontarget sequences. indications of relative phylotype abundances. The detection limit (cells/spores per reac- However, in spite of potential biases, the bias tion) of qPCR varies between sample types introduced by PCR and other steps of molecu- and assayed species due to e.g. the diff eren- lar community analysis has been considered tial extractability of DNA and the presence to be much smaller than the selective bias of PCR inhibitors. Detection limits ranging typical of cultivation-based methods (Prosser from less than one to several hundreds of et al. 2010). cells/spores per reaction have been reported The quality of ribosomal libraries can (Haugland et al. 2004, Kärkkäinen et al. 2010). also be affected by artificially formed DNA sequences (Jumpponen et al. 2007). Such 1.4.2 Limita ons and challenges of artifacts include chimeras and sequencing DNA-based methods in community errors, which are formed during the PCR characteriza on amplifi cation and also to some extent during In the optimal case, sequencing results would the cloning and transformation process (von fully reproduce the original species frequen- Wintzingerode 1997, Th ompson et al. 2002). cies in the analysed sample. However, in Chimeras have been found to constitute sig- practice the results from whole community nifi cant proportions of some environmental analyses incorporating a PCR amplifi cation clone libraries (Jumpponen et al. 2007), and

20 Introduction have aso been found to accumulate in public molecular taxonomy (Hibbett et al. 2007) and DNA databases (Hugenholz and Huber 2003, various phyla have been re-classifi ed and re- Tedersoo et al. 2011). named. The annotation of environmental Furthermore, it is notable that the con- sequences itself is a critical step in the molec- cept of “species” among clonally reproducing ular identifi cation process of microbes, and organisms such as bacteria and many anamor- may easily produce misleading results for var- phic fungi is questionable since “species” can ious reasons. Th us far, the taxonomic annota- no longer be determined as “a group of indi- tion of marker gene sequences has relied on viduals capable of interbreeding” as for sexual- either pairwise queries against nucleotide ly reproducing organisms. Instead, “high level databases using local or global alignment tools of genetic similarity between the individuals” (such as Blast and Fasta; Pearson and Lipman is used as a proxy for conspecifi city. In bac- 1988, Altschul et al. 1990), and subsequent teria, an arbitrary limit of 97% 16S sequence identifi cation of the nearest relative based on similarity is often used to delineate species sequence similarity. Another method, which level operational taxonomical groups (OTUs). is presently available for microbial ribosomal However, when compared with a more reli- genes but not for the intervening hypervari- able determinant, such as the overall DNA able spacer regions or protein coding genes, similarity between two species, it has become is based on a hierarchical Bayesian classifi er, apparent that these two determinants do not which compares the occurrence of 8-mer sub- necessarily correlate well (Stackebrandt et al. sequences between the query sequence and 2006). Also in Fungi the sequence similarity sets of classifi ed sequences (Th e RDP Classifi - threshold is problematic; in some genera, the er; Wang et al. 2007). Th e methods relying on intraspecies variability can be several percent- pairwise similarity searches are severely chal- ages on the nucITS region, while some genera, lenged by the presence of mis-annotated ref- such as Penicillium, harbour clusters of related erence sequences in the International Nucleo- species that contain identical or nearly iden- tide Sequence Database (INSD) (Nilsson et al. tical ITS sequences. Moreover, observations 2006). Moreover, concerning especially the of intragenomic rDNA sequence variation in fungal identifi cation, the substantial variation fungi have been recently made, which may in the intra- and inter-species variability with- lead into infl ated diversity estimates and false in the nucITS region, plus the complex taxon- conclusions of the presence of “cryptic species” omy of Fungi pose some additional challeng- in DNA based microbial community studies es; hundreds of fungal species have multiple (Lindner and Banik 2011). Th e described phe- names for genetically identical morphotypes nomena call for both similarity and phylogeny within species (which may represent the sexu- based annotation of environmental sequences, ally reproducing teleomorhp form of the spe- if both resolution and sensitivity are wanted. cies and/or one or more asexually reproduc- Recently, novel bioinformatics solutions have ing anamorph forms). Parallel names may also become available, which use the identifi cation represent historical synonymes that cannot be of the “lowest common ancestor” among the solved due to the absence of type strains. In blast-hits, and show promise in facilitating the general, fungal taxonomy has gone through dynamic, automated analysis of environmen- major revisions aft er the recent advances in tal sequences (e.g. “the FungalITSPipeline”,

21 Introduction

MEGAN, CLOTU; Nilsson et al. 2009, Huson what ambiguous since the assay quantitativity et al. 2009, Kumar et al. 2011). Furthermore, may be questionable if the target DNA con- recent efforts towards parsing large sets of tains variable regions that amplify unequally publicly available reference sequences into between species, or if the target copy num- curated databases will greatly improve the ber diff ers between species (Haugland et al. reliability of the annotation results (Tedersoo 1999a). et al. 2011). QPCR has been successfully used to elu- cidate the total counts of specifi c fungi and 1.4.3 DNA-based methods in indoor streptomycetes in e.g. indoor dusts (Meklin et microbial community analyses al. 2004, Rintala et al. 2004, Lignell et al. 2008, Molecular methods were first applied in Kaarakainen et al. 2009) and building mate- indoor research in the late 1990’s. In a series of rial samples (Pietarinen et al. 2008), as well as cases, a toxigenic fungal species Stachybotrys to compare mould concentrations in indoor chartarum was associated with severe forms and outdoor air (Meklin et al. 2007). Th e dif- of building related illness (Dearborn et al. ferences between microbial levels in farm- 1999). Culture-based detection of this species ing and non-farming homes have also been was found to be problematic, but it could be studied (Kärkkäinen et al. 2010). In general, enumerated from samples in a sensitive and the studies have indicated higher prevalences reproducible manner using qPCR (Haugland of occurrence for individual species, and 2-3 et al. 1999b). Since then, qPCR assays have orders of magnitude higher total cell concen- been designed for the enumeration of other trations than viable methods, showing that indoor fungi, and qPCR is practically the only viable methods underestimate both diversity DNA based method that has been widely used and concentration of microbes. Using qPCR, in indoor studies. A large set of qPCR assays several studies have also reported elevated has been designed by the US Environmental fungal or bacterial cell counts in dust in asso- Protection Agency (e.g. Haugland et al. 2004, ciation with water damage in buildings, yet Meklin et al. 2004). At present, the assays cov- the species in question have varied between er over 100 mould species commonly encoun- studies. Lignell et al. (2008) reported a signifi - tered in indoor environments (see: http:// cant correlation between the extent of visible www.epa.gov/nerlcwww/moldtech.htm). moisture damage and cell counts of specifi c In addition to fungi, protocols for detecting fungi, including Penicillium brevicompactum, indoor relevant bacteria, including Mycobac- Wallemia sebi and Trichoderma viride group terium tuberculosis and Legionella spp. (Pas- in dust samples. Bellanger et al. (2009) in turn cual et al. 2001), environmental mycobacteria observed signifi cantly higher concentrations (Torvinen et al. 2010) and Streptomyces spp. of Cladosporium sphaerospermum in the air (Rintala et al. 2004) have been published. In and surface samples collected from moisture order to assess the total concentration of fungi damaged homes than reference homes. and bacteria, universal qPCR methods have In their study of homes of infants who been designed (Haugland and Vesper 2002b, had developed a severe, potentially building- Kärkkäinen 2010, Yamamoto 2010, Chemidlin associated adverse lung condition, Vesper Prévost-Bouré et al. 2011). However, the con- at al. (2004) observed several times higher cept of “universal quantitative PCR” is some- counts of several mould species than in con-

22 Introduction trol homes, including Trichoderma viride, tion obtained almost solely from cultivation Aspergillus fumigatus, A. ochraceus and S. studies. Based on information obtained from chartarum. Th e homes of the children were culture-independent studies from other envi- repored to be severely water damaged (Etzel ronments, it is possible that signifi cant indoor et al. 1998, Dearborn et al. 1999). Meklin et species exist, which have not been identifi ed as al. (2004) studied a larger set of homes with such due to their poor culturability. Th e exis- or without moisture damage in the same geo- tence of such species can be assessed by study- graphic region, and found elevated concentra- ing indoor samples in detail using unselective tions of some of the fungal species observed DNA based methods such as direct sequenc- by Vesper et al. (2004), yet in the diff erences ing approaches. between damaged and undamaged houses were less profound than those observed by 1.4.4 Biomarker analyses used in Vesper et al. Based on the results obtained indoor microbial studies by Vesper et al. (2004), a qPCR-based index, Several structural compounds typical of bac- the “Environmental Relative Moldiness terial and fungal cells (“biomarkers”) have Index”, (ERMI) was developed to describe the been used as proxies for microbial biomass in indoor fungal burden from putatively indoor- the assessment of indoor microbial exposure contamination indicating fungal taxa for US (Pasanen 2001). Biomarkers for fungi include homes (Vesper et al. 2007). The index was ergosterol and (1-3)-β-D-glucan the main designed to document increases in both the component of fungal cell membrane and -wall diversity and the concentrations of “moisture (Pasanen 2001, Gehring et al. 2007). As for indicator” taxa. However, despite the elegant bacteria, lipopolysaccharide (LPS), the main design of the index, the correlation between component of gram-negative cell wall is a ERMI and the extent of moisture damage common biomakrker for this bacterial group, (Reponen et al. 2010) and between ERMI and while the measurement of N-acetylmuramic various health eff ects (Mendell 2011) remains acid is used to evaluate the abundance of unclear. Recently, however, Reponen and col- gram-positive bacteria (Sebastian and Lars- leagues (2011) reported a signifi cant positive son 2003). Especially LPS and (1-3)-β-D- association between elevated ERMI values glucan have been seen to refl ect the relevant and the development of asthma. Th ese studies part of indoor microbial exposure since these show that qPCR may be a highly effi cient tool compounds launch infl ammatory responses in for detecting elevated microbial concentra- humans (Pasanen 2001). However, while bio- tions indoors. However, in order to use qPCR marker analyses are suitable tools for a crude in “building diagnostics”, more robust infor- assessment of microbial concentrations, they mation is needed on the sources and local do not diff erentiate between microbial species, occurrence of diff erent microbial species and which is crucial information e.g. in the evalu- their associations with moisture damage. It ation of the sources or potential health eff ects is also notable that the assayed species them- of the studied microbes. selves have been selected based on informa-

23 Aims of the Study

2 AIMS OF THE STUDY

Th e main aims of this study were:

1. To perform a detailed characterization of indoor fungal and bacterial communities in Finnish offi ce buildings using cultivation-independent methodology on long-term samples of settled particulate material. Based on previous studies from other environments, molecular methods can reveal a substantially wider proportion of the total microbial diversity than culture based methods.

2. To characterize the seasonal variation occurring in these communities using molecular methods. Viable concentrations of fungi are known to vary seasonally, which limits the use of culture methods in building investigations during the growth season. However, no such information is available for uncultivable microbial particles.

3. To investigate the variation in molecular communities within and between sets of moisture damaged and undamaged buildings. Moisture damage does not always manifest itself in altered cultivable microbial communities but adverse heath eff ecs are nevertheless expe- rienced. Major changes in total microbial communities typical of damaged buildings were sought. Offi ces were chosen since the eff ect of human activities on microbiomes is smaller in offi ces than e.g. homes.

4. To compare the results from microbial community analysis obtained using culture dependent and culture-independent methods. Th ree methods: (a) a traditional method (plate cultivation), (b) an unselective, semiquantitative DNA-based method (clone library sequencing) and (c) a quantitative, species-specifi c DNA-based method (qPCR) were used and compared.

5. To develop a method for enhanced identifi cation and sequencing of rare phylotypes in environmental clone libraries. Such method would enable faster and more cost-effi cient sequencing of large environmental clone libraries.

24 Materials and Methods

3 MATERIALS AND METHODS

3.1 Buildings and samples a room matching the type of use was sampled in the reference building. Samples represent- For the characterization of indoor microbi- ing each of the four seasons were collected omes, samples were collected from six small over one year from one building pair (II, III), offi ce buildings in three locations in central while the effect of moisture damage renova- Finland (Table 1). Th ree of the targets were tion was followed in two building pairs by moisture- and mould damaged buildings collecting samples pre- and post-remediation and the remaining three buildings were age-, (I). Building material samples were collected building frame-, ventilation type- and usage from the two damaged buildings during their matched, paired controls that were not mois- renovation (I). ture damaged. Long-term samples of settled For the development and testing a macroar- dust were collected from mainly uncarpeted ray hybridization method for enhanced clone fl oors (II, III) or elevated surfaces (I). Dust library screening (IV), microbial communi- samples were collected by vacuuming sever- ties in biowaste compost samples were used al m2 of smooth surfaces twice a week over as study material. Five separate compost sam- ca. 1-2 months in order to gain a representa- ples representing two different composting tive long-term sample of the studied space. scales (pilot and full) and two facility types A room from the complaint area, yet without (drum and tunnel) were collected from two visible signs of mould, in the moisture dam- municipal composting plants. aged buildings was selected for sampling, and

Table 2. Buildings and samples studied for the characterization of indoor microbial communities. Detailed description of the buildings and sampling methods are given in corresponding studies.

Building Frame type, condition Location Samples (n) Study B1 (1, In)1 brick, moisture damaged 3 fl oor dust (4) II, III B2 (2, Re) 1 brick, undamaged 3 fl oor dust (4) II, III B3 (In1) 1 wood, moisture damaged 1 surface dust (2), I building material (7) B4 (Re1) 1 wood, undamaged 1 surface dust (2) I B5 (In2) 1 concrete, moisture 2 surface dust (2), I damaged building material (9) B6 (Re2) 1 concrete, undamaged 2 surface dust (2) I

1Th e name used to refer to the building in the corresponding study/studies.

25 Materials and Methods

3.2 Experimental methods 3.2.2 Development and evalua on of a nega ve macroarray An overview of the methods applied in this hybridiza on method for enhanced study is given in Table 3. A detailed descrip- screening of fungal diversity in tion of the microbial community analysis pro- environmental samples (IV and tocols is shown in Figure 2. unpublished results) The principle of the macroarray method for 3.2.1 Microbial community detecting, enumerating and negative selection characteriza on of abundant phylotypes in clone libraries is Th e microbial communities in dust samples presented in Figure 3. The suitability of this (I, II, III) were determined, enumerated method for the analysis of indoor samples was and compared using culture-dependent and assessed using sequence data obtained from culture-independent methods as described study II as follows: analogous to the protocol in Figure 2. Fungal communities in building presented in study IV, the most abundant phy- material samples (I) were studied using plate lotypes in indoor dust samples were identiﬁ ed cultivation and clone library sequencing as and their summed proportions were calculated described in Figure 2. for each sample and for the combined data. The expected savings in time and expenses were then evaluated based on the estimated reduction in numbers of sequenced clones.

Table 3. An overview on the experimental methods used in this thesis. Detailed descriptions of the methods are given in corresponding studies.

Method Study Fungal community characterization by nucITS clone library sequencing I, II, IV Bacterial community characterization by 16S rRNA gene clone library sequencing III Species-specifi c quantitative PCR I, II Viable fungal community characterization by plate cultivation I, II Fungal biomass measurement by ergosterol analysis I, II Macroarray probe design IV Macroarray hybridisation IV

26 Materials and Methods

Fungal biomass (I, II) Dust samples: Ergosterol analysis (I, II, III) (MS-GC) 1, II

Plate cultivation DNA extraction II DNA extraction 22 (dilution series, MEA, (+Spiking w/ external control DG18 agar)2,II spores 22) PCR II Fungi: nucITS (Fun18Sf+ITS44) Genus-level Bacteria: 16S (pA-pH’5) qPCR identification & (7 and 69 species- or enumeration 3, II group-specific assays, in Clone library construction (TA- studies II and I) 23 cloning, blue-white-screening, Pure cultures of clone culture& storage) II representative Species isolates concentrations Fragment re-amplification + Sequencing nucITS-PCR II (Fun18sF+ITS4/pD’, pE, pF’ 5) “Mold diversity” (no. of positive qPCR assays) nucITS sequencing II Contig assembly 6, Quality check, Chimera check 7

Viable fungal ERMI 24 communities (I, II) Mutiple sequence alignment 8 Total concentration of fungal species (I, II) OTU clustering (nearest neighbor method) 9, 10

Collector’s curves, Ƚ-diversity & Phylogenetic tree construction richness indices 10 Database query (rDNA genes, neighbor-joining (Shannon 15, Simpson 16, ACE 17, (Fasta 11, Blast 12) method 14) Chao1 18)

Manual screen for Phylogenetic community Sample-to-sample synonymous/anamorph names structure comparison: + mis-annotated reference Shared OTUs: sequences Ⱦ-diversity indices (Sørensen 19, Chao-Sørensen 20) Phylogenetic community Taxonomic annotation Molecular fungal and similarity: (Ciardo 13, manual) bacterial communities UniFraq similarity, distance Æ (I, II, III) PCoA, lineage analysis 21

Figure 2. A fl owchart showing the laboratory protocols and bioinformatic methods used in the present study for characterizing microbial communities in dust samples. Methods for analysis of viable fungi and fungal biomass are shown in green. Cultivation-independent methods for community analysis are shown in blue (clone library sequencing-based) and red (qPCR-based). Refer- ences to methods: 1. Sebastian and Larsson 2003. 2. Samson et al. 1994. 3. Samson et al. 1996. 4. White et al. 1990. 5. Edwards et al. 1989. 6. Staden et al. 2000. 7. Huber et al. 2004. 8. Th ompson et al. 1994. 9. Felsenstein 2005. 10. Schloss et al. 2005 and Schloss et al. 2009. 11. Pearson and Lip- man 1988. 12. Altschul et al. 1990. 13. Ciardo et al. 2007. 14. Saitou and Nei 1987. 15. Krebs 1989. 16. Simpson 1949. 17. Chao et al. 1993. 18. Chao 1984. 19. Sørensen 1948. 20. Chao et al. 2005. 21. Lozupone 2006. 22. Brinkman et al. 2003. 23. Haugland et al. 2002a. 24. Vesper et al. 2007.

27 Materials and Methods

Figure 3. Th e principle of the macroarray hybridization method for detecting, enumerating and negative selection of abundant phylotypes in clone libraries. Th e left side (1) of the fi gure shows the clone library sequencing protocol for identifying the abundant phylotypes in the sample. Th e right side (2) describes the protocol for preparing library macroarrays on nylon membranes and the hybridization step with specifi c oligonucleotide probes. Probes are designed for the abundant phylotypes and hybridized on the membrane. Only unhybridized clones are picked for sequencing, which reduces the number of sequenced clones.

28 Results and Discussion

4. RESULTS AND DISCUSSION

Indoor microbial exposure may have impor- ized by means of PCR-amplifi cation, cloning tant implications on health (Mendell et al. and sequencing of full-length nucITS region 2011), but the indoor microbial determinants (fungi) and the 16S Ribosomal RNA gene are not fully understood. Th e aim of this study (bacteria). Based on our previous work on was to gain a deeper understanding of these clone libraries (Pitkäranta et al. 2005) and determinants by using culture-independent the observations by other authors (Wang DNA-based methods to characterize fungal and Wang 1996, Jumpponen et al. 2007), we and bacterial communities. Th e study focused developed an optimized PCR protocol that on a selection of moisture damaged and combines a low PCR cycle number with a undamaged buildings that represent typical high number of replicate reactions to mini- Scandinavian office-type working environ- mize the eff ect of PCR drift and the number of ments. artefactual PCR products, especially chimeric molecules (II). Th e results indicated that this 4.1 Microbial diversity in dust (I, improvement was feasible as only 0.02% (II) II, III) and 0.3% (I) of the sequences resulting from the optimized protocol were chimeric, com- Microbial diversity was assessed by sequenc- pared to 8.7% (II) and ≥30% (Wang and Wang ing microbial ribosomal marker genes from 1996, Pitkäranta et al. 2005, Jumpponen 2007) 16 long-term, composite samples of fl oor (II, using conventional PCR protocol with a high III) and above fl oor level (I) settled dust from thermocycle number. six offi ce buildings. Large surface areas were Aft er parsing the raw data for chimeras, low- sampled in order to avoid sampling bias due quality sequences and non-target sequences, a to spatial heterogeneity of surface dust. Car- total of 2421 fungal nucITS and 776 bacterial pets or textile covered surfaces were not sam- 16S sequences of full-length were obtained pled in order to avoid collecting old particle (Table 4). Apart from one sample (sample no. material. 4 in Table 4), a minimum of 100 fungal, and 76 bacterial sequences were obtained from 4.1.1 Clone libraries and sequence each library. data (I, II, III) Fungal and bacterial communities in dust- and building material samples were character-

29 Results and Discussion

Table 4. Th e sequence material. Number of good quality clone library sequences obtained and used in downstream analyses in studies I-III.

Sample Source (name1) I, II2 III3 Location 1 1 B3 – pre-remediation (In1a) 225 - 2 B4 – pre-remediation (Re1a) 207 - 3 B3 – post-remediation (In1b) 100 - 4 B4 – post-remediation (Re1b)* 26 - Location 2 5 B5 – pre-remediation (In2a) 100 - 6 B6 – pre-remediation (Re2a) 167 - 7 B5 – post-remediation (In2b) 119 - 8 B6 – post-remediation (Re2b) 137 - Location 3 9 B1 – winter (1W/Winter D) 141 102 10 B2 – winter (2W/ Winter R) 174 159 11 B1 – spring (1Sp/Spring D) 180 76 12 B2 – spring (2Sp/Spring R) 152 109 13 B1 – summer (1Su/Summer D) 225 144 14 B2 – summer (2Su /Summer R) 170 82 15 B1 – fall (1F/Fall D) 141 104 16 B2 – fall (2F/Fall R) 157 117 Combined data 2421 776 1 Th e name used to refer to the sample in the corresponding study/studies 2 Full-length fungal nucITS-sequences 3 Full-length bacterial 16S-sequences *Th e clone library was small due to low starting amount of fungal biomass (as indicated by ergosterol analysis).

4.1.2 Microbial diversity and to that found in soil and plant tissue samples richness in dust samples (I, II, III) using the same methodology (O’Brien et al. Our results show a high diversity of fungi and 2005, Neubert et al. 2006). Th e total number bacteria in dust. A total of 606 fungal and 283 of fungal genera identifi ed from the six build- bacterial OTUs were detected using sequenc- ings was 166. This level of diversity is sig- ing. Th e theoretical fungal richness calculated nifi cantly higher than that usually obtained by using nonparametric ACE-estimator was using culture; eg. the study of Verhoeff et al. about 100 to 400 OTUs per composite dust (1994a), which is one of the most thorough sample (Table 5). Th is richness is comparable investigations of culturable house dust fungi,

30 Results and Discussion

Table 5. Observed and estimated microbial community OTU richness in dust samples.

Sample Fungal communities Bacterial communities Study (no. / namea) b c d b c d Sobs SACE Shannon (H’) Sobs SACE Shannon (H’) 1 / B3_Pre 98 220 4.06 - - - 2 / B4_Pre 45 103 2.22 - - - 3 / B3_Post 62 142 3.94 - - - 4 / B4_Post 21 67 2.97 - - - I 5 / B5_Pre 37 77 2.73 - - - 6 / B6_Pre 48 93 2.95 - - - 7 / B5_Post 42 167 2.68 - - - 8 / B6_Post 75 298 3.88 - - - 9 / B1_Wi 50 109 3.44 30 147 1.88 10 / B2_Wi 47 79 3.45 53 282 3.18 11 / B1_Sp 70 295 3.48 59 228 3.96 12 / B2_Sp 61 194 3.58 65 188 3.85 II, III 13 / B1_Su 81 327 3.51 60 176 3.47 14 / B2_Su 69 333 3.52 45 223 3.28 15 / B1_Fa 91 418 4.30 66 305 3.88 16 / B2_Fa 92 393 4.24 55 171 3.27 a Sample name abbreviations: Pre: pre-remediation sample, Post: post-remediation sample, wi: winter-time b sample, Sp: spring-time sample, Su: summer-time sample, Fa: fall-time sample. Sobs: number of observed c OTUs (species level phylotypes); SACE: number of estimated OTUs using the ACE richness estimator (Chao et al. 1993); d H’: Shannon biodiversity index, a measure of community richness and evenness, where a higher value indicates a higher diversity (Krebs 1989). yielded 54 genera from 60 buildings using ten yeasts would be possible by a combined mor- diff erent cultivation protocols in parallel. Our phological and biochemical approach but not eff orts to characterize the viable communities by microscopy alone (Freydiere et al. 2001). indicated that the genus-level diversity was Based on a comparison of Shannon diversity 1-13 genera per sample (I, II), i.e. typical of indices and the estimated total numbers of culturable studies assessing house dusts (e.g. OTUs using the nonparametric ACE estima- Piecková et al. 2004). Th ese results support- tor, the level of fungal diversity in dust sam- ed the hypothesis that the cultivable meth- ples collected from elevated surfaces ( Table 5, ods reveal a minority of the total diversity in samples 1-8) seemed to be similar to that mea- house dust samples. It must be noted, how- sured in fl oor dusts during the same season, ever, that the results obtained by cultivation i.e. winter (Table 5, samples 9-10). However, and by sequencing are not fully comparable; the variation between samples was signifi cant identifi cation of e.g. yeasts on viable cultures and the number of analysed samples is too low was not attempted, and very rarely occurring to make conclusions concerning diversity dif- genera were not identified and enumerated ferences between the sample types. using cultivation. Phenotypic identifi cation of

31 Results and Discussion

Th e numbers of observed and estimat- fully specifi c. Th e latter was the case with our ed fungal phylotypes in the seasonal study primer set (II), which explains plant sequenc- (II) leveled with those reported by Fröhlich- es in our fungal ITS libraries. As for bacteria, Nowoisky et al. in 84 outdoor air fi lter samples universal bacterial primers may amplify plant collected at a single location in the Central DNA located in chloroplasts because of their Europe over a period of one year (Fröhlich- cyanobacterial origin and orthologous ribo- Nowoisky et al. 2009). Th ere were similarities somal genes. Both nuclear rDNA operons and also in the community structures between chloroplast DNAs are present in plant cells in the two data sets, for example the diversity of high numbers. In the present study, the plant decomposing agaric species was substantial sequences could be affiliated to the source and increased towards the fall in both studies. species by pairwise comparison of sequences In addition to our observations on the multi- against public nucleotide databases, which tude of other outdoor taxa in indoor samples, gave additional information on the types of this similarity shows that long-term indoor biological particles in dust. The plant par- dust samples may reflect relatively well the ticles had originated from alimentary plants, seasonally fl uctuating fungal diversity from house plants and tobacco as well as from trees outdoor sources. Th e details of the commu- and grasses growing outside the buildings. nity composition are presented below. Sequences from Betula alba (birch) and Acer The level of bacterial diversity in dust platanoides (norway maple) were most domi- samples was similar to that observed for fungi nant. Plants were detected equally from fl oor- (Table 5, samples 9-16, III), apart from the and above-fl oor level samples. eTh presence in summer and fall samples (samples 13-16) in the latter sample type suggests that the plant which the fungal diversity was higher than material either entered the building airborne bacterial. Similar bacterial diversity estimates or aerosolized and resuspended from fl oors. have been found by others from surface and Th is suggests that a) the biological diversity of fi lter dusts (Noris et al. 2011) and from mat- airborne dust, even wintertime offi ce dust, is tress and floor dusts (Täubel et al. 2009). very wide, and b) the analysis of nucITS DNA Based on the observations on the accumula- sequences might be a feasible method for tion of theoretical total OTU number (by ACE species specifi c assessment of environmental estimator) as a function of sampled clones, it exposure to plant particles. was estimated that the total number of bacterial species in dust was roughly 500 per build- 4.1.3 The fungal community ing per year (III). composi on in dust (I, II) In addition to microbial sequences, con- Overview. Sixteen composite dust samples siderable amounts of plant-borne sequences representing six buildings were collected and were present in both fungal and bacterial their fungal content was analysed by nucITS libraries. Fungal and plant chromosomes con- clone library sequencing. The vast majority tain orthologous rDNA operons with simi- of fungal phylotypes were affi liated with two lar gene organization (Alvarez and Wendel phyla: Basidiomycota and Ascomycota (Figure 2003) and plant nucITS region may amplify 4, A). Th e most diverse phylum was Basidio- with universal fungal primers if plant DNA mycota (58% of OTUs), while Ascomycota is abundantly present and the primers are not was the most abundant phylum in clone num-

32 Results and Discussion bers, accounting for 57% of sequences. Within ciniomycetes were dominant. Th e details of Ascomycota, the classes Dothideomycetes, the fungal community fi ndings are described Eurotiomycetes and Leotiomycetes formed below. Th e species identifi ed in the course of the majority of both diversity (OTUs) and this study (non-singletons) are also listed in clones (Figure 4, B). Within Basidiomycota, parallel with the fungal species identifi ed from the classes Agaricomycetes, Tremellomyce- dust samples in previous studies (Appendix I). tes, Basidiomycetes incertae sedis and Puc- Th e full lists of detected phylotypes, their fre-

&. %.

" '-. " / ),. ""

(. '. &. %. " " *. " " +. ',. " " %%. "$ %'. " %). ##"

(. &. &. " ). " -. "$ "" )(. &(. " !"

Figure 4. Th e taxonomic diversity of dust-borne fungi observed by sequencing: relative proportions of diff erent phyla (A), diff erent classes of Ascomycota (B), and diff erent classes of Basidio- mycota (C). Large circles (A-C) depict the relative OTU richness in each group. Small circles (a-c) depict the relative abundances of nucITS clones. Combined results for all dust samples in studies I and II are shown.

33 Results and Discussion quency of occurrence across samples, annota- (see the Appendix 1 table). Most of the species tion details and INDS identifi cation numbers detected in indoor dust samples here for the are listed in the supplementary materials of fi rst time were not BSL-classifi ed. Two spe- the studies I, II and III (published online). cies of Trichosporon, T. jirovecii and T. mucoi- The present distribution of diversity des (or T. dermatis) were detected. Th ese are among fungal classes largely corresponded classifi ed to BSL-2 due to their ability cause with that observed by Amend et al. (2010a) serious infections in immunocompromised in 31 fl oor dust samples collected from vari- patients. Th ey are, however, oft en members ous geographical locations in the temperate of normal skin fl ora and may also derive from regions in the northern hemisphere. How- other habitats, e.g. soil (Liu 2011). As can be ever, some major diff erences were seen; most seen from the Appendix 1 table, BSL-1 clas- importantly, Agaricomycetes and Ustilagi- sifi ed species are commonly present in dust. nomycetes formed major groups in our data, Th eir occurrence as causative agents of oppor- but were minor constituents in the data set of tunistic infections may relate to the thermo- Amend et al. In contrast, the relative diversity tolerant nature of many of the fungi, but also within Sordariomycetes and Eurotiomycetes to the overall abundance in the human liv- was higher in their material than ours. Similar ing environment. An interesting fi nding was to the results by Amend et al. (2010a), Fujimu- the almost entire lack of Aspergillus spp. in ra and colleagues (2010) who studied micro- our sequence material. In contrast, several bial diversity in urban house dusts, did not species of Aspergillus were detected to occur detect Agaricomycetes or Ustilaginomycetes in house dust in concentrations of 103-105 among the dominant phylotypes. Th e higher CE/g. Among these was A. fumigatus, which abundance of these outdoor-related taxa in is classified as BSL-2 organism. Aspergillus our material is most probably explained by the spp. occurred mainly in one of the locations, fact that the studied buildings were located equally in both the moisture damaged index nearby large forest areas, which are abundant building and the undamaged reference build- sources of both Agaricomycetes (largely cap ing, and probably originated from outdoor air. fungi) and Ustilaginomycetes (rusts). In our study, the buidings were relatively small and Normal variation. The sample-to-sample squat (1-2 fl oors) and thus the outdoor borne variation in molecular fungal assemblages material may have transported indoors more observed in wintertime dust samples was efficiently than in the other studies. These assessed using the data from the undam- results agree with previous observations in aged reference buildings (Figure 5). Th e most that the vegetation in the surrounding envi- prevalent and also dominant classes were ronment and well as the design and usage of Dothideomycetes, Tremellomycetes, Basidi- the studied buildings contribute signifi cantly omycetes incertae sedis, Agaricomycetes and to the indoor microbiomes (Li and Kendrick Saccharomycetes. Most classes were present 1995c). in all or all but one samples studied, but the Th e proportion of pathogenic and oppro- relative abundances of diff erent classes varied tunistic fungi was low in the sequence mate- markedly between samples. Saccharomyces rial. We evaluated the BSL-classifi cations of cerevisiae (Saccharomycetes) and Cladospo- the species affi liations of non-singleton OTUs rium sphaerospermum (Dothideomycetes)

34 Results and Discussion occurred in high numbers in individual build- larity with Phaeotheca fi ssurella, and OTU311 ings but otherwise the proportion of individu- shared 96% similarity with Macrophoma sp. al species was ≤ 10 Together the abundant phylotypes accounted for 51 % of all ascomycete clones. Apart form Filamentous ascomycetes. Filamentous asco- the unknown types, most of the abundant spe- mycetes usually form the majority of culti- cies are typical fi ndings in dust samples (see vable fungi in dust (Verhoeff et al. 1994a and Appendix 1). Th e unknown OTU related to 1994b, Koch 2000, Chao et al. 2002, Scott Ph. fi ssurella (OTU423; INSD id. FR682166) 2001, Chew et al. 2003, Piecková et al. 2004, may be of signifi cance since it was present in Horner et al. 2004, Park et al. 2008, Hicks et nearly all samples collected form the water al. 2005). In the present material OTUs affi li- damaged buildings, sometimes in consider- ated with fi lamentous ascomycetes accounted able abundance, but was largely absent from for ca. 48% of the total clones. Th e dominant the reference buildings (I, II). Th e pattern of filamentous ascomycete phylotypes in our occurrence may be coincidential but may also material were affiliated with (in the order refer to moisture-damage-related- or other of abundance) Cladosporium herbarum, C. indoor sources for this species. While the clos- sphaerospermum, C. cladosporioides, Penicil- est relative Ph. fi ssurella is a phytopathogen, lium chrysogenum, Aureobasidium pullulans, another member of the genus, an oligotro- Leptosphaerulina (Pithomyces) chartarum, phic dematiaceous species Ph. triangularis has L. trifolii, Sclerotinia sclerotiorum, P. com- been isolated from HVAC humidifi er walls in mune and two unknown members of the class offi ce buildings (de Hoog et al. 1997). How- Dothideomycetes; OTU423 shared 86% simi- ever, since the sequence similarity between the

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Figure 5. Th e relative abundances of fungal classes and their variation in dust samples collected from undamaged reference buildings (n=5). Th e average percentage clone frequencies (grey bars), minimum and maximum frequencies (error bars) and median values (black rhombuses) are shown. *Other includes: Agaricostilbomycetes, Arthoniomycetes, Cystobasidiomycetes, Exobasi- diomycetes, Orbiliomycetes, Pezizomycetes, Taphrinomycetes, Ustilaginomycetes, Wallemiomy- cetes, Zygomycetes incertae sedis, unclassifi ed fungi.

35 Results and Discussion unknown phylotype and its known relatives Filamentous basidiomycetes. The propor- was low, the potential ecological similarity tion of OTUs affiliated with filamentous between the species can only be guessed. basidiomycetes (mainly classes Agaricomy- Other phylotypes were present in small cetes and Pucciniomycetes) was ca. 20% of numbers (<1% of total clones each) but their all clones. Individual OTUs representing the diversity was stunning; ca. 200 rare OTUs class Agaricomycetes were present in low were affiliated with filamentous ascomycete numbers; the most abundant types were iden- species. These covered both cultivated and tified as Hypholoma capnoides, Armillaria previously uncultivated species. In study I, borealis, Coprinus stercoreus, Botryobasidium previously unknown, verified phylotypes subcoronatum and Antrodia sitchensis, which accounted for over 10% of all diversity. An are common mushrooms, decomposers and interesting cluster of uncultured ascomyce- polypores outdoors in Scandinavia. Each of tes whose class affi liation could not be deter- these phylotypes accounted for 0.5-1% of total mined, occurred in several samples in the clone numbers, and other OTUs were pres- study (Cluster #3 in Figure 3 of study I; see ent in lower frequencies. Despite the rarity of Additional Table S1 of study I for details). individual OTUs, the summed proportion of Some of the sequences in the group shared Agaricomycete clones was substantial in some high similarities with uncultured fungi detect- clone libraries. Few studies have explored ed in other environments but the cluster the numbers and frequency of occurrence seems to lack cultivated, identifi ed representa- of basidiomycete spores and cell material in tives. Another interesting cluster (Cluster #1 indoor environment. Basidiospores are oft en in Figure 3 of study I) which occurred almost present in outdoor air, and are also known to exclusively in one of the damaged buildings, be transported to indoor air, as observed by contained several unknown phylotypes which e.g. Li and Kendrick using direct microscopy clustered with sequences of Exophiala xenobi- (Li and Kendrick 1995c), but cannot be iden- otica, Rhinocladiella atrovirens and Cladophia- tified and enumerated using culture-based lophora minutissima isolated from the build- methods. On conventional growth media, ing materials in study I. basidiomycetes are easily overgrown by asco- Our results demonstrate the dominance mycetous and zygomycetous moulds. Due to of previously known cultivable mould species their inconspicuous morphology, basidiomy- in dust. However, these species are accompa- cetes are usually included in colony counts of nied by a high diversity of rarer species, some “sterile isolates” if present on culture plates. of which represent unknown, uncultured and/ Our results suggest that cell material of basid- or unsequenced fungi. Sterile, unidentifi able iomycetous origin may be more abundant in isolates are common in viable studies (Beguin dust than previously considered, especially in et al. 1999, Chao et al. 2002, Hicks et al. the autumn, refl ecting the abundancy of this 2005), and have been associated with moisture group outdoors (see results below concern- damage and/or respiratory symptoms in some ing seasonal variation of dust fungi). Recently, studies (Strachan et al. 1990). Th us, the yet basidiomycetous species diversity was shown unidentifi ed species may be of interest with to outnumber that of ascomycetes in out- respect to building related health eff ects and door air, especially in the summer and fall should be studied in more detail. (Fröhlich-Nowoisky et al. 2008). Amend et al.

36 Results and Discussion

(2010a) reported a lower proportion (ca. 5%) may contribute to ergosterol concentrations, of fi lamentous basidiomycete OTUs in their since ergosterol is the major sterol of Malas- house dust samples collected from various sezia cell membrane (Gerla and Scheinfield geographical regions (Amend et al. 2010a). 2008). In contrast, glucan assays may not be aff ected, since unlike many other fungi, the Yeasts. Yeasts-like species are found from main cell wall carbohydrate of Malassezia has both Ascomycota and Basidiomycota. They been observed to be (1→6)-β-d-glucan instead are rarely identified in indoor cultivation- of the (1→3)-β-d-glucan typically targeted based studies, but may be classified as red, in biomarker assays (Kruppa et al. 2009). In dark and white/unpigmented types, or may addition to the known Malassezia spp., an be identified on special culture media and unidentified member of the Malasseziales by using biochemical tests (Freydiere et al. (BF-OTU429, INDS id. FR682172) was preva- 2001). Yeasts are more prevalent in dust than lent in our material (I, II). Th e OTU shares in indoor air samples and occur oft en in con- low (ca. 80%) similarity with any cultivated centrations similar or higher than fi lamentous species, but the identical phylotype has been fungi. In our material, yeasts were prevalent repeatedly encountered in molecular studies and yeast clones were dominant in some sam- assessing soil and oceanic habitats in various ples. Together yeasts accounted for 28% of all geographic locations; including Singapore, clones. Basidiomycetous yeasts were domi- the island of Reunion, Czech Republic, China nant; 76% of total yeast-like clones were affi li- and Hawaii (INDS id. HQ436049, JF691131 ated with this phylum. Members of the class and GU327510, GU941385 and EU915323, Tremellomycetes (mainly Cryptococcus victo- correspondingly). Contrasting to the human- riae, C. wieringae, C. magnus, C. friedmannii, associated members of the Malasseziales in C. carnescens, Mrakia gelida and M. frigida) our data, the phylotype BF-OTU429 occurred accounted for almost 40% of all yeasts clones. with a location- rather than a building spe- Th e second important group of yeasts was of cifi c pattern. Based on these observations, the Malassezia spp. (class Basidiomycetes incertae natural habitat and source of this potentially sedis, 43% of yeasts clones); M. restricta was novel species was the outdoor environment clearly the most common affi liation but also rather than the human occupants. M. sympodialis, M. slooffi ae, M. globosa and OTUs affi liated with yeasts of Microbot- M. japonica were detected. Malassezia spp. ryomycetes, another basidiomycetous class colonize the skin of healthy human individu- (incl. Rhodotorula mucilaginosa, Sporobolomy- als, but several species are also associated with ces ruberrimus, R. slooffi ae, R. glutinis, R. pinic- clinical skin conditions such as seborrheic ola and several related but unidentifi ed yeasts) dermatitis (dandruff ) andpityriasis versicolor were present in dusts with a lower prevalence. (Gupta et al. 2001). Th ese results suggest that The ascomycetous yeasts were solely mem- in addition to contributing signifi cantly to the bers of the class Saccharomycetes, dominant indoor bacterial assemblages (see below), the species being Saccharomyces cerevisiae and human occupants may be signifi cant sources Debaryomyces hansenii. Th ese accounted for of indoor fungal material as well. Th is may be 18% and 2% of all yeast clones, correspond- of importance in e.g. personal exposure stud- ingly. Few clones of Candida albicans and ies using fungal biomarkers; Malassezia spp. Pichia spp. were also present.

37 Results and Discussion

Apart from the abundance of Malassezia, 4.1.4 The bacterial community a species that does not grow well on common composi on in house dust (III) laboratory conditions, our results concerning Bacterial communities were followed over major yeast genera were in agreement with one year in two buildings (III). Th roughout previous culture-based studies. In the exten- the year, the communities were dominated by sive survey of 60 homes in Th e Netherlands gram-positive bacteria, especially members by Verhoeff et al. (1994a) reported R. minuta, of the Firmicutes and Actinobacteria (Figure R. mucilaginosa, Cryptococcus albidus and C. 6). Th e dominant phylotypes were present in laurentii as the dominant yeasts. Glushakova both buildings and were affi liated with spe- et al. (2004) detected several members of Can- cies associated with human skin and mucosa; dida, Cryptococcus, Pichia and Rhodotorula Staphylococcus epidermidis, Propionibacterium in house dust. In that study, the majority of acnes, Lactococcus lactis, Corynebacterium sp., indoor yeasts were shown to originate from Streptococcus thermophilus, Lactobacillus sp., indoor plants and pot soil. Here, based on Streptococcus mitis, S. parasanguis and Gemel- the high number of phylotypes occurring in la morbillorum (in the order of abundance) low frequencies in our samples, their elevated were present in over half of the samples and diversity and abundance during spring and together accounted for ca. 40% of total clones. summer (see below), most Cryptococcus spp. Serratia fonticola, a soil/water-borne member and also other yeasts including Rhodotorula of the Enterobacteriaceae, occurred in high spp. probably originated mainly from outdoor numbers in one sample. All dominant phylo- sources. types, and the majority of minor types shared

70 60

50 U 40

30 No. of OT of No.

20 Ǥ

0 Winter I Spring I Summer I Fall I

70 60 Deinococcus-Thermus 50 Bacteroidetes U 40 Gammaproteobacteria Betaproteobacteria 30

No. of OT Alphaproteobacteria Firmicutes 20 Ǥ Actinobacteria 10

0 Winter R Spring R Summer R Fall R

Figure 6. Bacterial community composition in dust (III). Seasonal variation of bacterial diversity on class level in two buildings (A and B) is shown.

38 a high (>97%) sequence similarity to known sonal trends followed the outdoor fungal suc- bacterial species. cession: • A diversification of decomposing and 4.2 VariaƟ on in microbial mycorrhizal basidiomycetes (class Agari- community composiƟ on comycetes) towards fall. • A high abundance and somewhat elevat- 4.2.1 Seasonal varia on (II, III) ed diversity of several phylloplane spe- Fungi (II). Seasonal variation of indoor fun- cies, including Cladosporium cladospo- gal communities was analysed from floor rioides, C. herbarum, Epicoccum nigrum, dust samples collected from two neighboring Aureobasidium pullulans and Lept- buildings with similar structure but diff erent osphaerulina (Pithomyces) chartarum and moisture damage status (II). Dust was col- related species (class Dothideomycetes) as lected solely from hard fl oor and above floor well as Cryptococcus spp. (class Tremello- surfaces, since carpets and textile-covered mycetes) in the spring and summer. surfaces can retain microbes and thus may not • An abundance of rusts (Pucciniomycetes) refl ect the seasonal load but material accumu- in the spring, summer and fall samples. lated over longer time periods. The main seasonal trends of fungi are Interestingly, one group of clear indoor ori- visible in Figure 7 and Figure 8, which show gin also showed seasonal occurrence; human the seasonal variation in diversity on class skin-associated yeasts (Malassezia spp., class level and examples of clone frequencies of Basidiomycetes incertae sedis) were most selected genera or groups expressing clear diverse and abundant in the cold seasons (fall seasonal pattern. Th e overall diversity of fungi and winter). Th eir abundance may relate to increased markedly from the winter towards low indoor RH, which oft en causes drying of the fall (Figure 7, Table 5). Th e clearest sea- the skin and more excessive scaling of skin material.

,++ % ,++7 4+ #!% 4+7 3+ !% 3+7 % 2+ 2+7 % 1+ 7 % 1+7 0+ %' 0+7 /+ $ % /+7 ' % .+ .+7 % -+ %' -+7 ,+ #% ,+7 + %% +7 ,-,-,-,- ( ,-,-,-,-, - , - , - , - ## # ## #

Figur e 7. Seasonal variation in the OTU diversity (left ) and the relative abundancies of fungal classes (right) in two buildings (B1-2). *Other includes: Agaricostilbomycetes, Arthoniomycetes, Cystobasidiomycetes, Exobasidiomycetes, Orbiliomycetes, Pezizomycetes, Taphrinomycetes, Usti- laginomycetes, Wallemiomycetes, Zygomycetes incertae sedis, unclassifi ed fungi (II).

39 Results and Discussion

'!(

&!( %!( $!( #!( "!( Percentage of clones

Figur e 8. Seasonal variation in the relative abundancies of selected fungal genera. Th e average values of the two buildings (B1 and B2) were used (II).

Another interesting detail was the spo- generally low number of shared OTUs in sam- radic mass occurrence of certain rust species ples samples from successive seasons, and the in dusts; Melampsoridium betulinum (the restricted occurrence of certain species during birch rust) that releases its spores in masses one or two seasons in accordance with their during the late summer and fall, occurred in ecology, it seems that the studied sample type high numbers in summer and fall samples, (fl oor dusts) refl ected well the particle load whereas Thekopsora areolata (the cherry- typical of each season. Furthermore, it was spruce rust) which releases its winter spores estimated that the proportion of old particle earlier in the spring, was observed in spring material in samples collected from hard sur- and summer samples. faces was low. Th us it is unprobable that e.g. Based on the observed numbers of the assessment of winter samples using DNA- shared OTUs between samples, we found that based techniques would be severely aff ected the fungal communities were most similar by material pertaining from the previous sea- in the two studied buildings during the fall, son. Also Kaarakainen et al. (2009) observed even though the diversity was distinguish- in their qPCR based study that outdoor-borne ingly high then. In contrast, lowest similarity taxa such as A. pullulans and Cladosporium between buildings was seen in winter samples. spp. measured from floor (dust bag) dusts The fungal communities also followed the showed clear seasonal variation which was in seasonal succession of outdoor mycota in accordance with their natural occurrence. many aspects. Th ese observations suggest that the eff ect of outdoor environment on indoor Bacteria (III). Little is known about the fungal assemblages was signifi cant, especially seasonal variation of bacterial communi- during the growth season. Similar conclu- ties in house dust. In a study by Reponen et sions have been made by others using culture- al. (1992), indoor air bacterial assemblages dependent (Koch et al. 2000) and culture- showed little seasonal variation whereas independent methods (Amend et al. 2010a). Moschandreas et al. (2003) observed that the However, viable communities in dust samples seasonal variation was residence dependent do not always vary according to season (Ren without clear overall trends. In our material et al. 1999, Horner et al. 2004). Based on the (III), the seasonal variation in bacterial com-

40 Results and Discussion munities was less clear than fungal due to the but changes are not always seen. Neverthe- dominance of human-associated bacterial less, building related symptoms may prevail. phylotypes - largely members of the Actino- In such situations, the exposing agents have bacteria and Firmicutes - that were relatively been suspected to be transferred in non-viable constantly present across seasons. In general, or non-culturable particles, or to be emitted larger diff erences in the bacterial communities sporadically so that short-term samples do not were detected between buildings than between “catch” them. Following this logic, long-term seasons. However, some seasonal trends could samples analysed with a culture-indpendent be seen. Subsequent seasons tended to remind method might better refl ect the actual expo- each other in their phylogenetic community sures, at least if the exposing agents are car- composition. Th e diversity of alpha- and beta- ried in DNA-containg particles. In the pres- proteobacteria varied according to season, ent study, the eff ect of moisture damage on being least diverse during fall and winter the fungal fl ora was studied by comparing the and most diverse during the spring and sum- molecular diversity and community compo- mer. Bacteria of putative outdoor origin (soil, sition in three pairs of damaged (index) and plants) were identifi ed among these groups, reference buildings (studies I and II). The and also within Actinomycetes, including occurrence of bacteria was studied in one members of e.g. Frankiaceae, Nocardiaceae, building pair (III). From the seasonal study Methylobacteriaceae, Rhizobiaceae and Oxa- (II), only winter samples were used to com- lobacteraceae (III). Also house plants are a pare the buildings, since the eff ect of outdoor potential source of soil- and plant-associated sources was obvious in other seasons (see bacteria. However, bacteria from such sources previous chapter). Th e overall level of diver- should be detectable throughout the year, but sity, and diversity within fungal classes was above mentioned families did not occur, or addressed, as well as the occurrence of indi- occurred only sporadically in winter samples, cator fungi (Samson et al. 1994) and fungi which supports their outdoor origin. The known to commonly colonize building mate- dominance of proteobacteria in outdoor air rials (Appendix I). Furthermore, in two of the during the growing season has been observed buildings (B3 and B5), fungi growing on the in several studies (Brodie et al. 2007, Fierer et moisture-damaged sites were identifi ed from al. 2008, Fahlgren et al. 2010). Th ese results material samples collected during renovation, suggest that outdoor sources determine the and the occurrence of these species in the dust indoor bacterial levels and types to some samples was examined. extent, but the eff ect is considerably weaker than for fungi because the eff ect of the indoor Comparison of microbial assemblages in sources (mainly occupants) is significantly damaged and reference buildings. Apart stronger. from a fi nding of high abundance of Penicil- lium spp. in one of the index buildings, no 4.2.2 Moisture damage (I, II, III) obvious indications of the moisture damage’s Mould and moisture problems may dem- eff ect on fungal or bacterial communities were onstrate in elevated levels (Dharmage et al. seen (I, II, III). However, some observations 1999, Klánová et al. 2000) and/or altered types were associated with the building condition. of viable fungi in dust and air (Miller 2000), In two of the index buildings (B1 and B3) an

41 Results and Discussion elevated diversity was observed compared to decay and their spores can occur in indoor the paired reference buildings (B2 and B4); samples. Since basidiomycetes are easily over- the numbers of observed and estimated OTUs grown by ascomycetous moulds, or are left

(Sobs/SACE) were 50/109 and 47/79 for the fi rst unidentifi ed due to lack of specifi c morphol- index-reference pair (B1 and B2, correspond- ogy in culture, their true frequency of occur- ingly), and 98/220 and 45/103 for the second rence on indoor materials and dust and air pair (B3 and B4). samples may be higher than understood thus In the first building pair (B1 and B2, far. Molecular methods have turned out to be study II) fungal community structures were suitable for their identifi cation. A recent study very similar on class level and the diversity using molecular methods indicated that fun- increase in the index building was not clear- gal biodiversity in wood decay turned out to ly affi liated with any individual class. In the be far more signifi cant and diverse than has second pair of buildings (B3 and B4, study hitherto been described (Maurice et al. 2011). I), increased diversity in the index building In that study, several basidiomycetous spe- occurred within several classes, including cies grew in parallel with each other and with Dothideomycetes, Eurotiomycetes, Tremel- ascomycetous mould species on damaged lomycetes and Agaricomycetes, which har- building materials. In our material the phy- bor species capable of growing on indoor lotypes characteristic of the two index build- substrates. However, also classes containing ings (I) were affi liated with Clitocybe subdi- clearly outdoor and human associated fungi, topoda, Gloeophyllum sepiarium, Hypholoma including Lecanoromycetes (lichenized fungi), sublateritium, Serpula lacrymans, Th elephora Pucciniomycetes (rusts) and Basidiomycetes terrestris and Trametes ochracea. Of these, G. incertae sedis (mainly Malassezia skin yeasts) sepiarium and S. lacrymans are known indoor were diversified, indicating that also other wood decay-causing fungi and may well have factors than building growth contributed to originated from the constructions of the the increased fungal diversity in the build- wood-framed index building (I). T. ochracea ing. Such factors may include e.g. diff erences is a common wood-decaying polypore, which in the cleaning routine. In the third pair (B5 to our knowledge has not been reported to be and B6), as already mentioned, high num- building-associated. A relative of H. sublateri- bers of Penicillium chrysogenum and P. com- tium, H. fasciculare, has been detected in fl oor mune occurred in the index building clone boards (Schmidt 2007). Th e remaining species library. However, within the remaining clones, are wood-decomposing, saprotrophic agarics increased diversity in the classes Agaricomy- that produce sporocarps on needle and wood cetes and Dothideomycetes was seen similar litter under conifers and on logs and stumps. to building B3 (I). While the growth of these species in some lay- The high diversity of Agaricomycetes ers of damaged buildings might be possible, in winter samples is interesting, since in the they more probably originated from wood/ seasonal study (II), this class was strongly sea- needle debris carried indoors on shoes. Since sonal occurring in signifi cant numbers only they were detected in dust samples collected in summer and fall samples. Certain basid- solely from elevated surfaces, they obviously iomycetes such as Serpula lacrymans have were resuspended from the fl oor debris and been long known to cause severe indoor wood transferred to the surfaces via the indoor air.

42 Results and Discussion

Table 6. Percentage frequencies of fungal genera in winter-time dust samples from the index and reference buildings. Percentage frequencies of total cfus (cultivation) and clones (sequencing) in a sample are listed. Genera with a frequency of 1% or higher are included (I, II)

Sample Viable Viable taxa (% of total cfus) Molecular taxa (% of total nucITS clones in count (cfu) library)a 1 96 295 Penicillium (61%), Cladosporium Unidentifi ed fi lamentous ascomycetes (15%), yeasts (B3_pre) (9%), non-sporulating (9%), Aureo- (12%), rusts (15%), Cladosporium (8%), Aureobasi- (index) basidium (5%), Verticillium (5%), dium (5%), Leptosphaerulina (4%), Epicoccum (3%), unknown (5%), Sphaeropsidales Phaeothecoidea (3%), Hormonema (2%), Botrytis (4%), Aspergillus (1%), Trichoderma (2%), Phoma (1%), Fusarium (1%), Penicillium (1%) (1%), yeasts (1%) 2 2 500 456 Cladosporium (93%), Acremonium Cladosporium (65%), yeasts (10%), unid. fi l. asco- (B4_pre) (5%), yeasts (2%) myc. (7%), Leptosphaerulina (3%), Hormonema (ref.) (3%), Aureobasidium (2%), Phoma (1%), Ampelomy- ces (1%) 3 5 729 Penicillium (29%), yeasts (29%), Yeasts (40%), Cladosporium (10%), basidiomycetes (B3_post) non-sporulating (29%), Aureobasi- (10%), unid. fi l. ascomyc. (7%), Phoma (6%), Lepto- (index) dium (14%) sphaerulina (4%), Aureobasidium (3%), rusts (2%), Mucor (2%), Penicillium (1%), other 1% 4 136 Eurotium (33%), Alternaria (33%), Yeast (31%), Hormonema (15%), basidiomycetes (B4_post) non-sporulating (33%) (12%), Aureobasidium (8%), unid. fi l. ascomyc. (8%), (ref.) Rhizoctonia (8%), Phaeosphaeria (4%) 5 1 729 729 Penicillium (100%) Penicillium (49%), yeasts (7%), Cladosporium (5%), (B5_pre) Botrytis (4%), unid. fi l. ascomyc. (3%), Aureobasidi- (index) um (3%), basidiomycetes (6%), rusts (3%), Hormo- nema (2%), Leptosphaerulina (2%), Epicoccum (1%), Mycosphaerella (1%) 6 139 963 Penicillium (89%), Acremonium Yeast (63%), Fusarium (8%), unid. fi l. ascomyc. (8%), (B6_pre) (8%), yeasts (2%), non-sporulating Penicilium (3%), Aureobasidium (3%), Phoma (2%), (ref.) (1%) Cladosporium (1%), Botrytis (1%), Hormonema (1%) etc. 1% 7 1 149 099 Penicillium (100%) Yeast (15%), Penicillium (46%), Cladosporium (8%), (B5_post) unid. fi l. ascomyc. (6%), Phoma (3%), Aspergillus (index) (2%), Eurotium (2%), Aureobasidium (1%), Hormo- nema (15), Botrytis etc. 1% 8 270 716 Penicillium (90%), yeasts (3%), non- Yeast (27%), Aureobasidium (15%), Acremonium (B6_post) sporulating (3%), Aureobasidium (9%), unid. fi l. ascomyc. (9%), Cladosporium (6%), (ref.) (2%), unknown (1%) Penicillium (4%), Botrytis (4%), Phoma (1%), Hor- monema (1%), Fusarium (1%), Paecilomyces (1%), Aspergillus (1%), Trichoderma (1%), Alternaria (1%) etc. 1% 9 25 630 Yeasts (32%), Penicillium (18%), Yeasts (63%), unid. fi l. ascomyc. (11%), Clado- (B1_Wi) Sphaeropsidales (14%), Aspergillus sporium (10%), Hypogymnia (3%), Phoma (1%), (13%), Acremonium (5%), non-spor- Aureobasidium (1%), Sclerotinia (1%), Phaeoseptoria ulating (5%), Aureobasidium (3%), (1%), Philophora (1%), Arthrobotrys (1%), Cephalo- Stachybotrys (3%), Exophiala (2%), theca (1%). Paecilomyces (2%), Scopulariopsis (2%), 3 other genera (<0.5% in total) 10 (B2_ 127 990 Yeasts (78%), Penicillium (7%), non- Yeasts (55%), unid. fi l. ascomyc. (14%), Cladospo- Wi) sporulating (5%), Fusarium (4%), rium (7%), Hypogymnia (5%), Capnobotryella (3%), Aspergillus (2%), 7 other genera Aureobasidium (2%), Fusarium (2%), Exophiala (<5% in total) (2%), Pseudocladosporium (2%), basidiomycetes (1%), Melampsoridium (1%), Trimmatostroma (1%), unid. fi l. basidiomyc. (1%) a) For an easier comparison of the cloning and culture results (see below) yeasts are clustered together in this table even though they represent phylogenetically very distant and diverse organisms. Samples 1-8 are from the study I and samples 9-10 from study II.

43 Results and Discussion

For a more convenient comparison of the lotypes were detected either by clone library community structures on higher phylogenetic sequencing or by sequencing cultivated fungal level, OTUs were grouped into genus-level isolates. Among these were species that were entities (Table 6, right column). In two of the very common in most dust samples, such as damaged buildings (B1 and B3), a high pro- C. cladosporioides, C. herbarum, Phoma her- portion of clones were affi liated with “uniden- barum, Leptosphaerulina (syn. Pithomyces) tified filamentous ascomycetes”; the phylo- chartarum, but also species occurring rarely types shared the highest sequence similarity in the clone libraries, including Penicillium with ascomycete species with typically hyphal spp., Trichoderma citrinoviride, T. atroviride growth, but the sequence similarities were too and Rhinocladiella atrovirens. Building-spe- low to allow annotating these phylotypes into cifi c, material-associated taxa accounted for certain genus or species. These occurred in 13/14%, 28/72% and 60/19% of total clones mainly low numbers, but as described in the in index/reference sample pairs. Th e clearly previous chapter, some may have originated elevated percentage in the last index building from indoor growth. was caused by the above-mentioned mass- occurrence of Penicillium spp. The genus Occurrence of indicator microbes and mate- was distinguishingly abundant also in cul- rial-associated fungi in dust samples (I). ture plates from the building. Th us, based on Microbial communities developing on mois- these results, the eff ect of building sources was ture damaged materials are often diverse not better distinguishable using clone library (Andersson et al., 1997; Tuomi et al., 2000, sequencing than by using cultivation. On Hyvärinen et al., 2002; Schmidt 2007); accord- the other hand, since the characterization of ing to literature (Appendix I), a majority of material fungi relied largely on culture (there the fungi common in dust have also been were technical problems in the clone library isolated from building materials. Excluding construction from material samples, see dis- obvious outdoor taxa such as A. pullulans, cussion in study I), it is possible that a larger C. herbarum and C. cladosporioides from the proportion of dust-borne fungi may have been list of material-associated species, it was cal- attributable to building sources than could be culated that in the three pairs of index/ref- verifi ed. erence buildings, material-associated taxa According to the literature, cultivable accounted for 2/6%, 18/63% and 56/18% of bacterial groups associated with moisture total clone in libraries, i.e. the proportion was damaged building materials include mainly higher in the index building in only one of the members of the class Actinobacteria, of which three building pairs. Th e analysis of so called the genera Amycolatopsis, Nocardiopsis, Nocar- indicator taxa (Samson et al. 1994) produced dia, Promicromonospora, Pseudonocardia, Sac- similar results (data not shown). To perform charomonospora, Saccharopolyspora, Strepto- a more exact analysis of the occurrence of myces and have been reported to be the most the material-associated fungi in dust, build- abundant (Suihko et al. 2009, Schäfer et al. ing material samples were collected from the 2010). Moreover, using culture-independent damaged parts of the building during the approach (PCR and clone library sequencing), renovation of the damaged buildings. From Schäfer et al. (2009) detected additional gen- these samples, 45 fungal species-level phy- era in Actinobacteria to occur on materials,

44 Results and Discussion of which Arthrobacter, Jiangella and Nesteren- microbial diversity on materials. For these konia were frequent. Also members of other reasons, material-associated species may have classes, including low-G+C gram-positive actually been present in higher abundance in bacteria (e.g. Laceyella (Th ermoactinomyces) dust samples than could be verifi ed here. In and Bacillus spp. in Firmicutes) and gram- the future, a thorough culture-independent negative bacteria (e.g. Agrobacterium spp.) characterization of fungi and bacteria occur- have been isolated from wet building materi- ring on building materials is needed. After als (Andersson et al. 1997, Schäfer et al. 2009). that, the identified species would be best A range of previously unknown species and quantifi ed from settled dust- or long-term air genera were recently isolated and described samples in the occupied spaces using qPCR, from building materials (Schäfer et al. 2009). which is a truly quantitative method unlike Th e presence of Streptomyces spp. and other clone library sequencing which may both material-associated bacteria listed in the under- and overestimate the relative species above-mentioned studies in the dust samples abundances. From such results, the exposure collected from the index and reference build- to material-associated microbes could be bet- ing was screened in the study III. Some taxa ter evaluated. occurred sporadically on dust samples of both index and reference building but no tenden- 4.3 Methodological cy of occurrence with respect to the damage consideraƟ ons could be seen (III). Bacillus cereus, which has been suspected to be of importance in the 4.3.1 PCR/Clone library indoor environment due to its production representa veness on emetic toxins, and its capability of growth Direct sequencing methods may show a on wet building materials (Andersson et al. biased view of the original template frequen- 2010), was detected only in the index build- cies, yet the estimations of the severity of the ing. problem vary (von Wintzingerode et al. 1997, These results suggested that the effect Huber et al. 2009, Amend et al. 2010b). In of water damage on microbial assemblages our data the relative clone and OTU abun- in dust was not obvious. However, certain dances within most classes seemed to refl ect issues undermined the detection of potential- well the anticipated species abundance dis- ly building related microbes. First, our clone tributions in the environment. For example, library sizes were limited and the abundant- the species richness in Agaricomycetes – the ly present outdoor- and human-associated class harbouring mushrooms and polypo- microbes potentially masked the presence res – followed a clear seasonal pattern being of material-associated species. Second, the very high in the fall. Individual OTUs in this frequencies of certain important, potentially class occurred in low numbers, indicating that material-associated fungal taxa may have these species originated from sporadic, weak been severely underestimated in the sequence or distant sources, which is in agreement with material (see next chapter). Th ird, our knowl- their occurrence in the environment. Within edge of the varieties of potentially material- some other classes, the clone abundance in associated microbes is based on cultivation relation to the OTU richness was high, e.g. for and may thus be an underestimate of the true the classes Pucciniomycetes and Basidiomyce-

45 Results and Discussion tes incertae sedis (Figure 4, C), which harbor bead-beating-based DNA extraction method rusts and human skin yeasts (Malasseziales). (which was applied also in the present study), Th ese species can be expected to accumulate the diff erence in numbers of extracted rDNA in substantial numbers in dust; rusts for their copies was up to three-fold between Asper- massive leaf-surface colonization potential gillus versicolor (lowest) and P. chrysogenum and ubiquitous presence in the immediate (higher), and over twenty-fold between A. ver- environment surrounding the buildings, and sicolor and Alternaria alternata (highest yield/ Malasseziales for their year-round presence in copy number per cell). Using less efficient high numbers on human skin surface (Paulino DNA-extraction methods even larger diff er- et al. 2006). Also the prevalence and abun- ences were seen, e.g. over 300-fold between A. dance of Cladosporium spp. was in line with versicolor and A. alternata using liquid nitro- their prevalence in the environment (e.g. Li gen grinding for DNA extraction (Haugland et and Kendrick 1995 c). al. 1999a). Th ese fi ndings may largely explain There were also some species whose the rarity of Penicillium and other Trichoco- clone abundance did not follow the expected maceae moulds in sequence data sets. abundance in the environment. Such included Another aspect that could explain the for example Penicillium, Eurotium and Asper- low proportion of Penicillium spp. and rela- gillus, which are considered to be the most tives was also observed. Among our data, prevalent and abundant members of viable the nucITS regions of Aspergillus and Penicil- dust communities in most studies, including lium spp. had the highest G+C-content of all those performed in Finland (e.g. Hyvärinen obtained nucITS fragments (Figure 9). The 2002, Flannigan and Miller 2011). Apart from G+C-content of these species was typically one exception (index building B5 with heavy between 56-61%. High G+C-content may contamination by Penicillium chrysogenum reduce both the PCR amplification and the and P. commune), sequences affiliated with sequencing reaction effi ciency due to the for- the above genera were very rare in our mate- mation of stable secondary structures which rial – constituting only 0.7% of total clones. In may not be fully resolved during the denatur- contrast, substantial numbers of Penicillium ation phase of PCR and sequencing reaction and Aspergillus spp. were shown to be pres- (von Wintzingerode et al. 1997). In a multi- ent in several samples using species specifi c template PCR this leads into underrepresenta- qPCR and cultivation. Th is suggests that the tion of such species in the end product. Th e abundance of these common moulds may G+C-content of Penicillium and Aspergillus be severely underestimated by sequencing spp. not represented in our clone libraries methods. As discussed in paper I, this phe- but detected using qPCR in the samples were nomenon has been previously reported in the examined from type strain reference sequenc- literature in various environments (e.g. Hunt es and were observed to posses G+C-contents et al. 2004, O’Brien et al. 2005) but has to our between 56-61% (data not shown). Low- knowledge not been explained. One probable ered detection sensitivity and/or lower clone reason for this discrepancy lies in the diff er- library frequency has also been observed for ential extraction efficiency and rDNA copy low-G+C gram-positive vs. gram-negative number between fungal species. Haugland et bacteria (Korthals et al. 2008) and high-G+C al. (1999a) have demonstrated that using the vs. low C+G-grampositive bacteria (Krogius-

46 Results and Discussion

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ͷͲͲ ΪǦ ͳͲ ͶͲͲ Ͳ

͵ͲͲ ǦͳͲ

ʹͲͲ ǦʹͲ ͳ ʹʹ Ͷ͵ ͸Ͷ ͺͷ ͳͲ͸ ͳʹ͹ ͳͶͺ ͳ͸ͻ ͳͻͲ ʹͳͳ ʹ͵ʹ ʹͷ͵ ʹ͹Ͷ ʹͻͷ ͵ͳ͸ ͵͵͹ ͵ͷͺ ͵͹ͻ ͶͲͲ Ͷʹͳ ͶͶʹ Ͷ͸͵ ͶͺͶ ͷͲͷ ͷʹ͸ ͷͶ͹ ͷ͸ͺ ͷͺͻ Kdh

Figure 9. Th e G+C-contents (red line) and the fragment lengths (blue line) of the obtained nucITS OTU sequences. Th e OTUs are ordered by the G+C-content. Th e Penicillium and Aspergil- lus nucITS regions had high G+C-content (upper red square) but the fragment length was average (lower red square).

Kurikka et al. 2009). The nucITS fragment tion of the representative nucITS sequences lengths were also examined, since fragment using either a simple 99% sequence similar- length may also aff ect the amplifi cation effi - ity threshold for conspecificity (II), or the ciency (von Wintzingerode et al. 1997). With- more fl exible algorithm described by Ciardo in our data, the ITS lengths of Penicillium and et al. (2007), tens of sequence types were Aspergillus spp. represented the average length annotated into higher level groups (genus/ of all OTUs (Figure 9) and fragment length class) instead of species despite high sequence did not correlated with clone frequency (data similarities with fully annotated INSD refer- not shown). ence sequences. Th is was due to equally high Th ese observations indicate that appar- Blast matches with several distinct reference ently, at least in the case of certain fungi, the species. In some cases this was explained by same characteristics that protect the organ- a truly high similarity of the nucITS regions ism against environmental stress and subse- of two or more closely related species. How- quently improve its longevity and dispersal ever, in many cases database sequences from (small, round cells, hardy cell walls and high closely related species had been mis-annotat- G+C-content) seem to undermine the detec- ed, or synonymous names for a single genetic tion of these species by molecular methods in species had been used. Moreover, the phylog- relation to less persistent species. eny of many fungi, including common indoor Affiliating the molecular data reliably moulds such as Cladosporium cladosporioides with fungal species showed to be a challeng- and C. herbarum is unresolved, and the “spe- ing task. Aft er the initial (automated) annota- cies” have been observed to represent species

47 Results and Discussion complexes rather than single, distinguishable wider representation of fungi was obtained entities (Bensch et al. 2011). Moreover, e.g. using clone library sequencing. many Penicillium spp., which closely resemble The relative sensitivities of sequenc- each other in culture are also unresolvable by ing, qPCR and cultivation were assessed in their nucITS sequences. In mentioned situa- study I. Because the true species abundances tions, an automated sequence clustering and in the samples were not known, percent- annotation produces messy results. In general, age sensitivities could not be assigned to the we perceived that a thorough look into the techniques but the methods were only evalu- matching species’ taxonomy, and sometimes ated in relation to each other’s performance. examining the correctness of the nearest ref- QPCR was found to be the most sensitive of erence sequences’ annotations by additional the used techniques; in 92% of cases when a Blast-searches was necessary to ensure the species was detected by sequencing it was also best annotation (I, II). detected by qPCR from the same sample. In contrast, sequencing failed to detect 60% of 4.3.2 Comparison of the methods cases when a species was shown to be present The fungal abundance and diversity results in a sample by qPCR. Th e species not detected obtained using culture, clone library sequenc- by sequencing tended to be those with lower ing and qPCR were compared (I, II). In study cell counts; their median cell equivalent count I qPCR was performed using a set of 69 spe- was 1.4 x 103 CE/g-1 while the median con- cies- or group specifi c assays for common fun- centration of species detected by sequencing gal indoor and indicator species (Samson et al. was 5.9 x 105. Th e Spearman rank correlation 1994). Th e clone library sequencing method between sequencing and qPCR results was provided the most thorough view on fungal 0.59 (p < 0.01), meaning that the clone library diversity in dust; the number of genera identi- frequencies tended to refl ect the original spe- fi ed by sequencing was 140 whereas cultiva- cies abundances in samples yet significant tion yielded 20 genera (Figure 10). Th us, both variation occurred (I). It was observed that taxonomically richer and phylogenetically phylotypes occurring as singletons in clone

Figure 10. Phyla distribution of dust-borne fungal genera observed by cultivation (A) and by nucITS clone library sequencing (B). Number of identifi ed genera is shown (I, II).

48 Results and Discussion libraries were in some cases highly abundant In accordance to our observations concern- in samples as measured by qPCR; their cell ing the underrepresentation of Penicillium in counts could be as high as 105 CE g-1 (I). sequence material, the comparison showed Th e comparison of culture- and qPCR results that the genus was detected by sequencing if is somewhat ambiguous since culture isolates it was clearly the dominant viable genus in the were only named to genus but most qPCR sample. Nevertheless, its proportion of clones assays targeted species. Th e set of qPCR assays was substantial only in samples with very high (69 assays) was also limited and did not cover viable counts (> 1 x 106). It was also seen that all cultivated genera, e.g. Phoma (Sphaeropsi- high viable numbers of Penicillium tended dales) and Verticillium. However, in a majority to cover the presence of other species. The of cases in the study I at least one representa- occurrence of the less abundant species cor- tive of each cultivated genus was detected by related poorly (Table 6). qPCR in the corresponding sample. In such cases, the qPCR-assayed cell counts were ca. 4.4 Macroarray hybridizaƟ on two orders of magnitude higher than the via- method for the enrichment of ble counts. Th is is comparable to the observa- clone libraries tions made by others on the percentage culturability of indoor fungi (Meklin et al. 2004, In study IV a macroarray method for Lignell et al. 2008). In some cases nucITS enhanced screening of clone libraries was sequence analysis of the isolates revealed that developed. Th e method utilizes the detection non-targeted species of the qPCR-targeted and negative selection of abundant fungal genera were present in samples. Such includ- phylotypes in clone libraries by oligonucle- ed Acremonium alternatum, Alternaria citri, otide probing, aft er which the unhybridizing Aspergillus conicus and Wallemia muriae. clones representing rare phylotypes are picked To compare the “big pictures” obtained using for sequencing. Th e method was developed to cultivation and sequencing, fungi identifi ed speed up and lower the costs of sequencing by sequencing were clustered into genus-level environmental clone libraries. Th e principle groups. Yeasts, filamentous basidiomycetes was tested in vitro on fungal communities and unidentifi ed ascomycetes were clustered occurring in composting plants (IV). Th e use- into larger entities (Table 6). V iable commu- fulness of the principle for indoor samples was nities were less diverse than molecular com- subsequently assessed in silico using sequence munities also using this approach, but similar data obtained from indoor dust samples trends were observed in the occurrence of (unpublished data). many taxa, showing correspondence between the methods. Th e dominance of Cladosporium 4.4.1 Study IV – compost fungal or yeasts in a sample was usually detected by communi es both methods. Th e detection of Sphaeropsi- Six oligonucleotide probes were designed for dales (in culture) and Phoma (by sequencing six fungal species that had been identifi ed as – Phoma is the major indoor member of the the dominant members of fungal communi- Sphaeropsidales group) usually correlated, yet ties of composting facilities during the course in some samples it was not detected in culture, of another study (Hultman et al. 2010. The potentially due to overgrowth by Penicillium. hybrizidation method was used for negative

49 Results and Discussion selection of these abundant phylotypes among nal procedure, the six most abundant phylo- 1536 clones gridded to a nylon membrane. types were identifi ed. Abundant phylotypes Positive hybridization was detected for 59% accounted for 26% of total clones (7 - 44% of of clones. The remaining 41% of the origi- clones in individual libraries) (Table 7). Th us, nal clones were identifi ed as rare, and were aft er the probing, an average of 74% of clones subsequently sequenced. The specificity of would have remained left to be characterized the method was calculated from a set of 384 by sequencing. clones, all of which were sequenced to verify Due to the different proportions of their phylotypes. 91% of the hybridization sig- abundant phylotypes, the utility of the prob- nals corresponded to the sequencing result. A ing method differed significantly between false negative hybridization was detected in the compost and dust samples. The advan- the case of 20 clones (5.2% of total) and false tage of screening out abundant phylotypes positive in case of 15 clones (3.9% of total). from compost nucITS libraries was evident, Th ese results demonstrated the effi ciency of as only 41% of clones needed to be sequenced the method for enhanced analysis of fungal after probing. In contrast, such benefit was communities in compost samples. not seen with dust samples, since 74% of the clones belonged to rare or sporadically occur- 4.4.2 Unpublished – dust fungal ring species. Taking onto account the costs communi es of materials and hands-in time required for Clone library sequence data obtained during gridding, hybridization and data-analysis in the course of the study II was used to evaluate the hybridization step, the obtained savings in the feasibility of using the hybridization meth- sequencing (26% of clones) were not seen high od on indoor material. Analogous to the origi- enough to make the inclusion of the hybridization step profi table. Th us the method was not taken into use in the sequencing analysis Table 7. Th e observed diversity and pro- protocol of indoor dust samples. Th is conclu- portion of abundant species in indoor dust sion was further supported our later observa- samples. tions on fungal community structure in other indoor samples (study I), which showed to be Sample Sobs %Ab. at least as diverse as the samples analysed in B1 winter 50 23% study II. B2 winter 47 20% Th e hybridization and sequencing results B1 spring 70 44% suggested that the microbial community B2 spring 61 41% structure diff ered profoundly between the two B1 summer 225 40% environments; compared to the fungal com- B2 summer 170 13% munities of composts, which were dominated B1 fall 141 8% by few species, the assemblages occurring B2 fall 157 7% in indoor dust samples were markedly more Total 345 26% diverse and the role of individual abundant species was less significant. This difference S : number of observed phylotypes in sample. obs was also refl ected in the distinct fungal com- %Ab.: summed percentage frequencies of the six most abundant phylotypes in sample. munity richness estimates for compost and

50 Results and Discussion dust; the estimated number of phylotypes The higher water activity and substrate availability in compost mass supports the fast (SACE) ranged from 9 to 26 for the compost samples (Hultman et al. 2009) but from 79 to growth and dominance of a restricted number as high as 418 for dusts (study II). of adapted species. In contrast, the dry condi- The differential community structure tions in indoor dust make this environment a reﬂ ects the profound differences of compost passive repository of settled material which is mass and indoor dust as microbial habitats. mainly shaped by allochthonous processes.

51 Conclusions

5. CONCLUSIONS

1. We hypothesized that the culture-based methods would reveal only a fraction of the true microbial diversity in indoor dust samples and that a wider diversity of fungi and bacteria would be seen by using DNA based methods. Our results supported this hypothesis; the well-known dominant microbial groups such as outdoor-borne phyllopllane fungi, yeasts and human-skin-associated bacteria were accompanied by a taxonomically wide and diverse array of known and unknown microbes that cannot be readily identifi ed by routine cultivation. Th e number of fungal genera in each analysed sample greatly exceeded the viable diversity. Predominant uncultivated groups included unknown ascomycetes and decomposing agaric fungi, human skin-associated yeasts, and rusts. Th is demonstrates that people are exposed to a diverse array of microbes even in a normal offi ce environment. However, since the sequencing method used here is semiquantitative in nature, the true abundances of the “novel” species are not known, and subsequently their relevance with respect to e.g. human health cannot be evaluated.

2. Viable fungal communities are known to vary according to season, which limits the use of culture methods in building investigations during the growth season. Little was known about the seasonal variation of bacteria, or about the variation of nonviable microbial particles over seasons. We observed that the seasonal variation in the sequence material, which covers both viable and nonviable microbes, was much more prominent than the variation in viable fungal communities. Bacterial communities showed milder seasonal variation due to a stronger contribution of human-associated taxa present throughout the year. Microbial communities were least diverse, and outdoor sources were weakest during winter, while various outdoor- borne microbes crowded other seasons’ samples. Th e results from fungal analysis suggested that the studied sample type (fl oor dust), represents the seasonal load of microbes. Th us, as in culture-based building assessment, winter samples are preferable targets also for the molecular assessment of building sources.

3. We sought for major changes in total microbial communities in association with water damage. Th e eff ect of damage on dust communities was not evident, but instead, other sources were usually seen to dominate in both building types. Certain potentially material-associated species and groups were seen to occur in the damaged buildings, but these were mainly present in low numbers. However, since culture-independent reference data from building-material-associated microbes is scarce, and uncultivable species dominated in dust, our results were not well interpretable with this respect. Also the small sample number and deep normal variation of microbial communities between buildings undermined the assessment of the eff ect of building sources on dust communities. Th ese results call for a) a culture-independent characterization of fungal and bacterial communities growing on building materials, and b) a subsequent assessment of a larger set of indoor bioaerosol samples to characterize the communities in damaged and undamaged buildings.

52 Conclusions

4. Th e comparison of clone library sequencing, qPCR and cultivation turned out to be somewhat irrelevant since the methods measure partly diff erent things and are not fully comparable. However, the three methods largely agreed on the dominant fungal genera in the samples. qPCR was shown to be the most sensitive of the techniques for detecting individual species, yet a vast majority of species present in the samples were not detected due to the lack of a targeting qPCR assay. Th e overall quantitative correlation between qPCR and clone library results for qPCR-targeted species was moderate, but severe underestimating bias was observed in case of individual genera including Penicillium and related genera. Th us, while clone library sequencing is a good method for characterizing previously unknown microbial communities, it is not a suitable method for the quantitative analysis and comparison of samples.

5. In order to make clone library sequencing more effi cient, a method for the negative selection of rare phylotypes was developed. Th is method was seen to be suitable for screening sample types that are heavily dominated by a limited number of species. In contrast, the method was not seen to be feasible for the characterization of highly diverse microbial communities with relatively even species distributions, such as indoor dust samples.

53 Acknowledgements

6. ACKNOWLEDGEMENTS

Th is study was carried out at the DNA Sequencing and Genomics Laboratory of the Institute of Biotechnology, University of Helsinki. Th e present and former heads of the Institute of Biotechnology, Professor Tomi Mäkelä and Professor Mart Saarma, are thanked for giving a home for such an innovative core facility within the Institute. Th e work was fi nancially supported by the Finnish Funding Agency for Technology and Innovation, the Graduate School for Environmental Health and University of Helsinki.

In the thesis project I have had a chance to work and collaborate with the researchers at two groups with very diff erent fi elds of operation; the Environmental Microbiology Unit of the National Institute for Health and Welfare that has a long and succesfull history on indoor air research; and the DNA Sequencing and Genomics Lab, a dynamic unit that has maintained its position as the state-of-the- art technological facility for two decades in the quickly evolving world of genomics research. I owe my gratitude to Professor Aino Nevalainen, Petri Auvinen and Lars Paulin, the heads and hearts of these units, for building up and cherishing such amazing groups.

I want to thank my thesis supervisors Petri Auvinen, Helena Rintala and Professor Martin Romantschuk for the long-term support, trust and helpful discussions at various stages of the project. Petri, you have taught me the value of free and independent way of working, of which I am grateful; I also want to thank you for the vivid morning discussions about science, music, parenthood, pandas etc., even if they oft en made me miss out my morning coff ee. Helena, you have been my primary guide to the indoor air and mould topics since the beginning of my master’s thesis project ten years ago; I am sincerely thankful for this, because I truly fi nd it as “my fi eld”.

Professor Kaarina Sivonen and Professor Malcolm Richardson are acknowledged for careful reviewing and constructive and inspiring comments on the thesis. I am grateful to my co-authors Teija Meklin, Anne Hyvärinen, Ulla Lignell, Mika Toivola and Jenni Hultman for professional and fruitful co-operation during the BioFINE project and while writing the manuscripts. From my heart, I wish to thank the present and former personnel at the THL; Aino, Helena, Anne, Teija and Ulla – thank you for your warm companionship and for familiarizing me with the international indoor air community in various seminars and conferences, from the year 2003 mycotoxin meeting in Saratoga Springs to the 2011 Indoor Air conference in Austin. I also want to thank the brilliant “next-gen” THL researchers Maria Valkonen and Martin Täubel for inspiring discussions on various professional and non-professional issues, as well as for your true friendship.

My sincere thanks go to the present and former personnel of the home lab; Kaisa, Pia, Jenni, Ritu, Päivi, Paula, Panu, Eeva-Marja, Olli-Pekka, Teija, Tuuli, Kirsi, Jarmo, Olli, Harri, Matti, Dario, Ursula, Tuomas, Sini, Suvi, Noora, Riikka, Tuula, Pasi, Mari, Janne, Hannu, Markku and Ari-Matti – thanks for your kind help and advise in the lab, from the colony picking to sorting the fuzzy read lists, from loading the agarose gels to helping with the robotics and from sharing the morning coff ee

54 Acknowledgements

to off ering a shoulder during the tough times. Tiedenaiset, especially Sepi and PKS are thanked for sharing lunch, nail polish, giggles and a very, very bad sense of humor. All this has greatly increased the probability of surviving the last decade without permanent injurys.

I owe my sincere gratitude to my friends and relatives for their presence and support. Maija, your friendship over the years from the pre- to the graduate school has meant a lot to me. I also want to thank Suvi and Leena for our deep-mining discussions on work, life and human mind during the past years. I’m grateful to my parents for the unconditional support for my choices in life, including the present project. Your love and help have been indispensable. Especially, I want to thank my mother and my mother-in-law for the help in childcare during the busy stages of the thesis project, and my father for the interesting discussions about buildings and architecture.

Finally, I cannot tell how much I appreciate the strength, patience and love of my husband, and the joy that my daughters have brought to the life. Kiitos rakkaat.

Helsinki, December 2011

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67 Appendix s-level s-level C 1, 2, 3, 4, 5, 7, 10, 11, 12, 14, 19, 1, 2, 3, 4, 5, 7, 10, 11, 12, 14, 19, 21, 22, 24, 25, 26, (I) 2, 3, 4, 10, 11, 12, 14, 16, 19, 21, 2, 3, 4, 10, 11, 12, 14, 16, 19, 21, 22, 23, 25, 26 1, 2, 3, 4, 5, 7, 10, 11, 12, 14, 18, 1, 2, 3, 4, 5, 7, 10, 11, 12, 14, 18, 20, 21, 23, 24, 25, 26 1, 3, 7, 14, 15, 19, 20, 21, 22, 23, 1, 3, 7, 14, 15, 19, 20, 21, 22, 23, 25 3, 4, 7, 11, 12, 13, 18, 19, 22, 23, 3, 4, 7, 11, 12, 13, 18, 19, 22, 23, 25, 26 1, 2, 3, 4, 5, 7, 11, 12, 13, 14, 15, 1, 2, 3, 4, 5, 7, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 25, 27, 28 1, 2, 4, 7, 11, 12, 15, 18, 19, 22, 23, 24, 25 Reference Reference B BSL1 3, 2, 4, 7, 14, 18, 21, 23, 25, 26 BSL2 BSL1 NC 2, 3, 4, 7, 16, 19, 23, 25 BSL2 BSL1 25, 26 BSL1 BSL1 14, 21, 24, 25 BSL1 25, 26 BSL2 2, 7, 11, 19, 23, 24, 25, 26 BSL1 BSL1BSL1NC 2, 3, 12, 15 1, 2, 3, 7, 11, 15, 18, 22, 21, 25, 26 4, 19, 25 BSL1 2, 3, 4, 7, 18, 19, 23, 24, 25 BSL1 2, 3, 19, 25 Pathog. Pathog. A AF, CA, CO, CT, FI, GB, PA, WO, WP WO, PA, FI, GB, CT, CA, CO, AF, BSL1 AF, CA, CF, CO, FI, GB, UF, W, P, PA, PA, P, W, UF, FI, GB, CO, CA, CF, AF, WP WO, AF, CA, CF, UF, WO UF, CA, CF, AF, CA, CO CF Building material substrate Building material CF CF, I, M, P, UF, W, WP, GB WP, W, UF, I, M, P, CF, BSL1 AF, CA, CO, CT, FI, GB, P, PA, UF, W, W, UF, PA, P, FI, GB, CT, CA, CO, AF, WP WO, M, P, WM, P, NC 2, 3, 7, 23, 25 WP UF, P, GB, CA, CT, AF, CA, CF, GB CA, CF, I I on, on, on, soil WP WO, P, CA, CF, on, soil on, s, soil ff soilon, cereals, CA, P er water, fruits, hay, fruits, hay, er water, fi o on, hay, soil on, hay, CA AF, BSL2 2, 3, 7, 19, 23, 25 on, hay, cereals, soil cereals, on, hay, WO P, CA, CF, AF, our, soil our, s, soil s, soil fl our, soil our, P AF, BSL1 7, 19, 23, 25 ff ff er water, soil er water, er water, soil er water, WP WO, W, M, P, GB, CA, CO, AF, BSL1 2, 3, 4, 7, 13, 16, 22, 23, 25 fl fi fi er, dried plant products, co products, dried plant er, our, stored food, clothes, soil food, stored our, UF WP, CA, G, W, AF, oods, fruit juice, c f soil li air outdoor hay, cereals, dried foods, cereals, soil cereals, dried foods, grains, salted foodstu salted grains, soil products, soy fermented plant material, soil material, plant soil kernels soil material, plant nuts, fl cereals, hay, vegetation, decaying humidi CA, CF BSL1 24, 25, 26 BSL1 (I) outdoor air, humidi air, outdoor soil citrus fruits NC 25, (I) plant material, soil material, plant soil soil cereal, hay, cereal, M, WO CA, CO BSL1 1, 2, 3, 4, 6, 29 BSL1 2, 3, 4, 7, 11, 14, 19, 23, 25 co hay, careal, food, stored NC (I, II) decaying plant material, water, sewage, sewage, water, material, plant decaying soil seeds, cereals, soilhumidi NC co hay, cereal, crops, stored 4, 25 seeds, soil foodstu foodstu soil NCsoil 4, 25 decaying vegeta decaying cereals, Common substrate/habitat ) seeds, co ) material plant NC 2, 4, 25 x murorum) x . Apiospora arundinis (syn . Emericella nidulans . Emericella (teleom genum lamentous fungi) lamentous fi i avipes avus fl fl cations of fungi were included. were fungi of cations Aspergillus restrictus Aspergillus nidulans Aspergillus niger Aspergillus penicilloides Aspergillus ochraceus Aspergillus oryzae Aspergillus glaucus Aspergillus niveus Aspergillus conicus Aspergillus Aspergillus Aspergillus fumigatus Alternaria alternata Alternaria Alternaria citri Alternaria Apiospora montagnei Arthrinium phaeospermum Armillaria borealis Aspergillus candidus Aspergillus clavatus Aspergillus versicolor Acremonium sclero Acremonium strictum Aspergillus terreus Aspergillus unguis Aspergillus ustus Aspergillus wen Acremonium murorum (syn. Gliomas Acremonium murorum (syn. Aspergillus tamarii Acremonium butyri Species ( Hyphomycetes corymbifera) Lichtheimia (syn. Absidia corymbifera Aspergillus sydowii Appendix specie Appendix reporting studies (I, II). Only study the present and literature to according dust from species fungal isolated 1. List of identifi

68 Appendix C 2, 3, 7, 8, 10, 11, 12, 13, 14, 15, 16, 19, 20, 21, 23, 25, 26, 27, 28, (I, II) 1, 2, 3, 7, 10, 11, 19, 23, 25, 26, 27, 28, 20, (I, II) 2, 4, 7, 15, 18, 20, 21, 23, 25, 26, 2, 4, 7, 15, 18, 20, 21, 23, 25, 26, 29 1, 2, 3, 4, 7, 11, 12, 13, 14, 16, 18, 19, 20, 23, 25, 27, (I, II) 1, 2, 3, 4, 7, 12, 16, 20, 23, 25, 26, 27, 28, 29, (I, II) Reference Reference B NC 12, 18, 19, 23, 25, 27, 29, (I, II) NC 2, 3, 20 BSL1 2, 3, 19, 23, 25 NC (I, II) NC 2, 3, 4, 7, 12, 16, 18, 19, 23, 25 NC (I, II) NCBSL1 (II) 4, 25 BSL1 NC (I, II) NC (II) BSL1 BSL1 NC (II) NC 2, 23, 26 NCBSL1 (I) 2, 25 NCNC (II) (I) NC 10, 25 BSL1 25, 26 BSL1 2, 3, 4, 7, 17, 19 BSL1 (II) NCNCNC 2, 3, 4, 7, 21, 23, 25, 27, 29 (II) 23, 25, 26 Pathog. Pathog. NC 7, 19, 25 NCNC (I) 22, 26, (I, II) BSL1 2, 4, 19, 20, 23, 25, 26 NC (II) BSL1 7, 12, 15, 23, 25, 27, 28 BSL2 3, 25 A I, M, P, UF I, M, P, AF, CA, CT, P CA, CT, AF, AF, CA, CO,CT, GB, P, PA, W, WO, WO, W, PA, P, GB, CA, CO,CT, AF, WP CF, M, P, UF, W, WP W, UF, M, P, CF, Building material substrate Building material CF AF, CA, CF, GB, P, WP, WO WP, P, GB, CA, CF, AF, BSL1 CF AF, CA, CF, P, PA, W, WO W, PA, P, CA, CF, AF, CF AF, CA, CO, CT, FI, GB, P FI, GB, CT, CA, CO, AF, CF s s, soil s, soil P GB, CA, CO, CA ff ff ff s, soil er, wood er, er water, soil er water, fi oods, dried meat, cereals, nuts, nuts, cereals, oods, dried meat, dried foods, cereals, nuts, fruit juices, nuts, fruit juices, cereals, dried foods, soil hay, soil seeds, hay, cereals, dried foods, I seeds, cereals, beans, soil seeds, cereals, plant material, foodstu material, plant (rust) phylloplane dead wood plant material, soil material, plant forest soil forest insects (entomopathogenic) forest soil forest dead wood phylloplane ff foodstu cereals, cereals, senescent plant material, soil material, plant senescent cereals, WO UF, PA, P, GB, soil material, plant decaying CT, CO, CA, CF, BSL1 indoor surfaces, bread, soil bread, indoor surfaces, plant material, soil material, plant bakery goods (“bread mould”) (“bread goods bakery I, CF hay, cereals, soil cereals, hay, rot) (white wood forest soil forest soil soil dead wood (white rot) (white dead wood dead wood hay, soil hay, foodstu material, plant foodstu material, plant soil material, wood decaying plant, salmon, leaf li salmon, leaf dead wood, living trees dead wood, important material, plant woody phytopathogen foodstuffs, fruit, vegetables, cereals, cereals, fruit, vegetables, foodstuffs, humidi dried f Common substrate/habitat ) soil cereals, hay, material, plant CA ) soil nia fuckeliana nia C. cochliodes Gibberella Gibberella (teleom. ssima (syn. pes lamentous fungi) lamentous fi Botryo (teleom. herbariorum ) s cinerea s um amstelodami um chevalieri um um um rubrum um um repens um narius pholideus narius narius armillatus narius Euro Euro Euro Epicoccum nigrum Epicoccum Epicoccum purpurascens Epicoccum caricis Eudarluca Cylindrobasidium evolvens Curvularia senegalensis Cor Cordyceps sinensis Cordyceps Cor Coprinus stercoreus Cladophialophora minu Cladosporium cladosporioides Cladosporium herbarum Cladosporium macrocarpum Cladosporium sphaerospermum Coniothyrium fuckelii Coniothyrium Chrysonilia sitophila Chaetomium globosum Chaetomium indicum Chaetomium purpureum Chondrostereum Clitocybe subditopoda Clitocybe Chaetomium funicola Chaetomium Chaetomium aureum Chaetomium Botry Botryobasidium subcoronatum Cerrena unicolor Euro jeanselmei Exophiala Euro Exophiala salmonis Exophiala Flammulina velu avenaceum Fusarium avenacea Species ( Hyphomycetes Aureobasidium pullulans

69 Appendix C 1, 3, 7, 11, 13, 15, 18, 19, 21, 23, 1, 3, 7, 11, 13, 15, 18, 19, 21, 23, 25, 26, 29 Reference Reference 10, 11, 21 B BSL1 1, 2, 10, 19, 23, 25 NC (II) NC (I, II) NC (II) BSL1 (I, II) NCNC (I, II) (I, II) BSL1NC 2, 4, 3, 25 NC 3, 25 8, 11, 18, 19, 20, 23, 25 NC 23, 25 NCBSL1NC (I, II) NC 18, 25 2, 4, 8, 12, 23, 25 (I, II) NC (I) BSL1 1, 11, 23 NC (I, II) BSL1 2, 3, 23, 25 NC (I) NC (I, II) Pathog. Pathog. NC BSL1 25, (I, II) NC (II) NC 2, 3, 25, 26 BSL1 19, 25 A CA, CT, UF CA, CT, CO, PA, WO, WP WO, PA, CO, Building material substrate Building material M M WO W, UF, PA, P, GB, CF, CA, CO, BSL2 W BSL2 2, 18, 23 M CF c, c, s, hay, hay, s, ff er water, soil er water, WO I, CF, BSL2 2, 3, 19, 20, 23, 26 fi c) soil on, cereals, s of plant origin, hay, soil origin, hay, s of plant s, paper I, W BSL1 2, 13, 19, 23, 25 er water, stored foods, plas foods, stored er water, ff ff fi foodstu conifers (needle blight) conifers stored plant produce, hay, soil hay, produce, plant stored soil cereals, I, M, WO grass roots (snow mould) roots grass poaceae (endophy soil material, plant soil birch rust birch alder rust trees, rocks (lichen) rocks trees, soil grass, and decaying live soil grass, and decaying live rust larch-willow decaying wood, plant material plant wood, decaying M dead wood dead wood decaying wood decaying humidi soil foodstu fat-containing compost, soil grain, dead wood foodstu seeds, fruits, soil debris, soil plant woody M W BSL1 BSL1 18, 3, 23, 25, 26 decaying plant material plant decaying plant material, soil material, plant cork, straw cork, soil grass, cereals, fruit, soil cereals, grass, soil material, plant soil material, plant humidi material, plant CF I, W, CF CF NC 2, 3, 4, 19, 23 NC BSL2 25, 26 3, 4, 25, 26, (I, II) stagnant warm water, woody plant plant woody water, warm stagnant co material, needles material, plant forest soilforest soilforest NC NC (II) (II) Common substrate/habitat

) cereals CA, UF NC 2, 7, 23, 25 ) soil material, wood decaying plant, I Gibberella Gibberella (teleom. Sydowia (teleom. (teleom. (teleom. Clonostachys rosea Clonostachys s Exophiala werneckii Exophiala um (syn. i mannii lamentous fungi) lamentous

ﬀ ﬁ dematioides nus ) ) or spinosus Mucor hiemalis Mucor Meria laricis Mucor plumbeus Mucor racemosus Mucor Microdochium nivale Microdochium phragmi circinelloides Mucor mucedo Mucor Muc Melampsoridium betulinum Melampsoridium hiratsukanum Itersonilia perplexans Itersonilia chartarum / Pithomyces Leptosphaerulina trifolii Leptosphaerulina Melampsora caprearum Lecythophora ho Hypogymnia physodes Hypholoma sublateri Panellus sero Panellus Paecilomyces lilacinus Paecilomyces vario Paecilomyces Hypholoma capnoides Fusarium verticillioides Fusarium moniliformis pannorum Geomyces Geotrichum candidum Gibberella fujikuroi Gliocladium roseum Mycosphaerella phacae-frigidae Mycosphaerella Myrothecium roridum Oidiodendrum rhodogenum Species ( Hyphomycetes culmorum Fusarium Fusarium equise Fusarium oxysporum Fusarium solani Fusarium werneckii (syn. Hortaea polyspora Gymnopus dryophilus Gymnopus Hormonema Gymnopus aquosus Gymnopus

70 Appendix C 1, 2, 3, 4, 7, 12, 13, 14, 15, 16, 18, 19, 23, 24, 25, 27, 28, (I) Reference Reference 1, 2, 3, 4, 7, 11, 12, 15, 16, 18, 19, 22, 23, 24, 25, 27, (I) B BSL1 7, 12, 14, 15, 18, 25 NC 1, 3, 4, 7, 18, 22, 23 BSL1 2, 3, 4, 7, 12, 14, 19, 23 NCBSL1 7, 14, 19, 25 7, 12, 22, 26, 25 NC (I) NC 7, 25 NC 1, 7, 12, 18, 22, 23, 25 NC 7, 12, 13, 15, 18, 22, 23, 26, 27 NCNC 4, 13, 26 25, 26 NC 12, 26 NC 1, 2, 7, 12, 15, 18, 22, 23, 25, (I, II) NC 7, 14, 25 BSL1 12, 14, 19, 20, 22, 23, 25 NC 23, 26, 25 NC 2, 22, 25 NC 4, 25 BSL1 NC 2, 3, 23, 25 Pathog. Pathog. A AF, CA, CO, CT, P, UF, GB UF, P, CT, CA, CO, AF, AF, CA AF, CA, GB CA, CF AF, CA, P, W CA, P, CF AF, CA, UF AF, CF Building material substrate Building material AF, CA, GB, P, CO, CT, WO CT, CO, P, CA, GB, AF, CF, W CF, AF, CA, WO AF, CA, UF, CF, GB, W GB, CF, CA, UF, CA, CF, P CA, CF, M AF, CA, GB, P, UF, W UF, P, CA, GB, AF, BSL1 AF, CA, CO, CT, GB, UF, P, WP, PA, W, W, PA, WP, P, UF, GB, CT, CA, CO, AF, WO s, cereals, cereals, s, our, soil our, P GB, I, CF, BSL1 4, 7, 12, 14, 15, 19, 23, 25, 26 ff fl our, soil our, M on, s s, soil ff er water, soil er water, WO NC 23, 25 fi on, foodstu on, on, co on, soilon, cork, M, WO NC 12, 18, 23, 25 on, soil CA, CF AF, NC 7, 25, 26 s, soil nuts, soils, fruits, cereals, CF s, soil P GB, CT, CA, CO, AF, BSL1 7, 12, 15, 18, 19, 22, 23, 25, (I) s, soil CF NC 2, 14, 26 s, paper, soil s, paper, ff ff ff ff ff soiler, CA, P AF, BSL1 2, 3, 7, 12, 25 aying vegeta aying blue cheese, foodstu foodstu dried foods, peanuts, soil dried foods, water, straw, bread, jam, soil bread, straw, water, tropical greenhouse, seeds, cereals, soil products, soil cereals, soil soil citrus fruits, soil soil ff foodstu refrigerated soil seed, cereal crops, soil crops, seed, cereal mouldy citrus fruits, soil lipids, cheese, soil wood, fl pomeaceous fruits, cereals, foodstu soil fruits, soil foodstu decaying vegeta decaying foodstu soil nuts, (soil)cereals, dung, insects, soil materialplant surfacerock materialplant W GB, UF, CA, CO, AF, M WO CA, GB, NC 7, 13, 14, 19, 25 NC NC 7, 12, 16, 23 NC 22, 25 NC NC (II) 8, (I, II) (II) cereals, plant material, soil material, plant cereals, hay, cotton, vegetation, decaying soil cereals, GB CA, CO, AF, BSL1 2, 3, 4, 7, 12, 15, 19, 25, 28 foodstu vegeta decaying pomaceous fruits, (soil) li plant CA AF, NC 1, 7, 12 rocks, trees (shield lichen) trees rocks, soilcheeses, walnuts, CF NC NC (I) 25, 26 decaying vegeta decaying dec Common substrate/habitat ) seeds, humidi grass giata Cadophora fas (syn. i ogriseum orum lamentous fungi) lamentous fi giata giata enicillium simplicissimum Penicillium roquefor Penicillium Penicillium rugulosum Penicillium Penicillium sclero Penicillium P Penicillium olsonii Penicillium oxalicum Penicillium purpurogenum Penicillium Penicillium miczynskii Penicillium Penicillium janthinellum Penicillium Penicillium italicum Penicillium Penicillium janczewskii Penicillium Penicillium crustosum Penicillium Penicillium decumbens Penicillium digitatum Penicillium echinulatum Penicillium expansum Penicillium Penicillium funiculosum Penicillium Penicillium glabrum Penicillium Penicillium implicatum Penicillium Penicillium commune Penicillium Penicillium corylophilum Penicillium Penicillium waksmanii Penicillium Penicillium variabile Penicillium viridicatum Penicillium vulpinum Penicillium incarnata Peniophora nigricans Phaeococcomyces Phaeosphaeria herpotrichoides Phialophora fas Penicillium brevicompactum Penicillium Penicillium chrysogenum Penicillium Penicillium citrinum Penicillium Penicillium solitum Penicillium Penicillium spinulosum Penicillium Species ( Hyphomycetes sulcata Parmelia Penicillium atramentosum Penicillium auran Penicillium Penicillium citreonigrum Penicillium

71 Appendix C 2, 3, 4, 7, 11, 13, 15, 16, 18, 19, 20, 23, 25, 26, 29, (II) Reference Reference B BSL2NC 2, 7, 12, 23, 25 7, 25 NC (I, II) NC 4, 25 BSL1 2, 3, 4, 7, 12, 15, 18, 25, (I) NC 2, 3, 4, 7, 23, 25 NC 26, 29 BSL1 2, 3, 7, 14, 16, 23, 25, 26, 29 NC (II) NC 4, 25 BSL2 1, 3, 4, 7, 25 BSL1 2, 22, 23, 25 BSL2 2, 4, 7, 13, 23, 25, 27, 29 NC 7, 23 NC (II) BSL1 3, 4, 7, 23, 25, 26 Pathog. Pathog. NCNC (I, II) (II) A Building material substrate Building material CA AF, CA, CO, GB, P GB, CA, CO, AF, CF M WP P CA, CT, P, WO, WP, W WP, WO, P, CA, CT, UF WO, CA, GB, AF, CA, CF M, WO AF, CA, WO, GB, P, UF, CF UF, P, GB, CA, WO, AF, s, cereals, cereals, s, ff les, cereals, cereals, les, s, soil er water, soil er water, WP W, UF, P, GB, CA, CF, AF, NC fi er, soil er, t ff foodstu er, er, cereals, hay, soil hay, cereals, er, P CT, origin, soil s of plant ff -spruce rus on,straw, humidi on,straw, o plant material, dung, foodstu material, plant soil cork, material plant dung cherry decaying wood in buildings wood decaying dung bark, dead wood soil plant ma plant plant material, soil material, plant soil ma plant decaying plant material, tex material, plant decaying soil compost, material, plant decaying soil household waste, dead wood dead wooddead wood (soil) material, plant soil material, plant soil material, plant soil material, plant soil material, plant materialplant dead woodsoil materialplant grassescereals, soil fruit, vegetables, decaying P WO, soil P fruit, vegetables, decaying W, houseplants vegetables, pathogen; plant fruit, cereals, and animal products, plant cheese, bu meat, I BSL1 BSL1 NC NC BSL1 NC 3, 25 4, 7, 18, 23, 25, (II) 2, 3, 4, 7, 13, 18, 20, 25, (I, II) (I, II) NC 22, 25, (I, II) (I, II) NC BSL1 BSL1 BSL1 (I, II) (I, II) 2, 4, 25 dead wood NC (II) 2, 3, 4, 19, 20, 23, 24, 25, 28 NC NC (II) NC (II) (I) cereals, material, plant decaying foodstu (II) Common substrate/habitat ) ) )c S. verruculosum S. atra Hypocrea lixii (syn. (syn. R. nigricans, Mucor Mucor R. nigricans, (syn. i orum num lamentous fungi) lamentous fi mbriatum avus fl micola ) fi nia sclero nia Talaromyces Talaromyces Thamnidium elegans areolata Thekopsora (syn. chartarum Stachybotrys Serpula lacrymans Sordaria fi Steccherinum Suillus luteus Syncephalastrum racemosum racemosum Syncephalastrum Scopulariopsis fusca Scopulariopsis Scopulariopsis chartarum Scopulariopsis Scopulariopsis candida Scopulariopsis Trichoderma harzianum (syn. Trichoderma Scopulariopsis brump Scopulariopsis koningii Trichoderma Trichaptum abie Trichaptum Phlebia radiata Species ( Hyphomycetes Phlebiopsis gigantea Phoma eupyrena Phoma exigua Phoma glomerata Phoma herbarum Phoma macrostoma cucumerina Plectosphaerella tuberaster Polyporus candolleana Psathyrella Pyrenophora teres cerealis Rhizoctonia oryzae Rhizopus stolonifer Rhizopus stolonifer Sclero brevicaulis Scopulariopsis Thelephora terrestris viride Trichoderma

72 Appendix C Reference Reference 2, 3, 4, 7, 14, 15, 16, 19, 22, 23, 24, 25, 27, 28 (I, II) B BSL1 2, 3, 23, 27, 28 NCNC (I, II) (I, II) NCNC (I) II) (I, NCBSL1 (I, II) 2, 3, 4, 9 BSL1 BSL1 2, 4, 9, 27, 28, (I, II) NC (II) BSL1 2, 3, 4, 8, 9, 23, 27, 28, 29, (I, II) NCNCNC 8, (I, II) (II) (I, II) NCNC (II) (I, II) BSL1BSL1BSL1 (I, II) BSL1 (I, II) BSL1 8, (I, II) NC (II) (I, II) (I, II) Pathog. Pathog. NC (I, II) BSL1 4, 8, 9, 27, 28, (I) BSL1BSL2 2, 3 4, 9 NC (I) NC NC (II) NC 3, 4 A Building material substrate Building material AF, CA, P AF, sh, hay, hay, sh, c, house plants, c, house plants, s fi salted s, cereals, ff s, soil, epiphy ff c animals, soil ax food, beverages, plants, soil plants, beverages, food, atmosphere soil leaf surface leaf fl forest soil forest soil soil sea water, pot soil, house plants, plant material, material, plant pot soil, house plants, ff foodstu fruits, water, skin, nails human skin human skin human skin human skin soil humans, animals, soil of humans and animals mucosa pigeon droppings pigeon soil soil, plant surfaces, pot soil, house pot soil, house surfaces, soil, plant plants plant material plant material plant material plant surface rock soil foodstu soil ectomycorrhizalectomycorrhizalskin, soilskin, nails, soilskin, soilpot soil, house plants soil material, plant decaying soil material, plant decaying dried foodstu W UF, P, CA, CO, AF, BSL1 7, 11, 13, 19, 20, 22, 23, 25, 27, 28 NC NC BSL1 NC (I, II) BSL2 2, 3, 4, 29 (II) BSL2 (II) 3, 9, 29 BSL2 2, 3 (I) phylloplane soil domes water, plants, soil plants, water, Common substrate/habitat ) s R. Rubra i ulatum s nis lamentous fungi) lamentous ae fi lobasidium capitatum lobasidium Cryptococcus magnus Cryptococcus niccombsii Cryptococcus Cryptococcus oeirensis Cryptococcus wieringae Cryptococcus Cryptococcus terricola Cryptococcus victoriae Cryptococcus fi Cysto Debaryomyces hansenii Debaryomyces Filobasidium unigu Malassezia globosa Malassezia restricta Malassezia sloo ffi Malassezia sympodialis Malassezia Mrakia frigida Candida tropicalis Candida zeylanoides Cryptococcus adeliensis Cryptococcus Cryptococcus albidosimilis Cryptococcus Cryptococcus albidus Cryptococcus Cryptococcus carnescens Cryptococcus dimennae Cryptococcus festucosus Cryptococcus friedmannii Cryptococcus Cryptococcus lauren Cryptococcus Mrakia gelida Rhodotorula glu Rhodotorula Tricholoma lascivum Tricholoma Species ( Hyphomycetes sejunctum Tricholoma cutaneum Trichosporon jirovecii Trichosporon / derma mucoides Trichosporon pullulans Trichosporon Ulocladium botry Ulocladium chartarum sebi Wallemia phylloplana Rhodotorula Yeasts Candida parapsilosis Rhodotorula mucilaginosa (syn. Rhodotorula

73 Appendix

, fer- (2); (25); (25); (25); 1992. minuta (25); P . (25); P . (25); A . (3); Gil- furcatum lusitaniae . exophialae et al. solani

) (4); P . protuberata (anam. Bot- (anam. (25); Conio- (25); Ochro- atus australis

(2); R . variecolor infl

longibrachiatum cervinus

hirsutum (29); A ) (2); C. pseudokoningii (4); Trichothecium (25); S. 2008; 21: Piecková 2008; 21: Piecková sativus

(25); C . proliferatum pelliculosa sphaerica

graminis piluliferum (4). 2009; 7: Flannigan and and 2009; 7: Flannigan (25); P .

atrum

(25); P . Emericella ovoides

(25); T . Aspergillus (2); F . prasadii ; marxianus

botryosum

(2); Candida Leptosphaerulina (25); C . Reference Reference on data on the type strains was was on the type strains on data nivale 1994 b; 29: Wickman (25) (25); Phaeococcomyces luteoalbum B (4); (8); Botryosporium (syn. fumosoroseus ) (25); C . ll surface; WO: wood;wall paper. WP: ll surface; WO: Acremonium (25); Cochliobolus griseofulvum polysporum (21); Nigrospora

2004; 13: Hunter and Bravery 1989; 14: Bravery and 2004; 13: Hunter ythrospila (2); F . (18); Rhodotorula er palustris Pathog. Pathog.

burtonii Kluyveromyces tenuissima

nozdrenkoae crassa

(25); P . (25); Trichosporon anomala

2003; 6: de Hoog et al. 2003; 6: de Hoog (25); P . A . geniculatus (25); Stemphylium

sarmentorum

(25); D . variabilis fulvus A (8) ;

(25); Verticillium merismoides (25); C . (teleom. (teleom. chlorocephalum 1994 a; 28: Verhoeff

tonsurans collec on, and culture Hyphopichia glandicola (25); Pichia kefyr (3); P . parvispora longipes

biseptata

(3); Neurospora (3); Cochliobolus (22); Pestalotiopsis humicola (3); T . 1999; 12: Horner et al. 1999; 12: Horner circinatum

caginis i (9); C . (14); P . (25); Trichoderma asperum carneus

grisea 1999 ; 5: Chew et al. scheri fi (8); Rhizomucor

1995 ; 20: Park et al. 1995 ; 20: Park al. et 2009; 19: Northolt al. et med (8); Botryosphaeria H . Alternaria terrestre (25); F . occiferum ;

fl verrucosum ) (3); Cladosporium fusca

ed unambiguously on the species level, and present in dupletons or higher or in dupletons present the and species level, on ed unambiguously informa on source (25); Building material substrate Building material (29) haemulonii (25); Drechslera frequentans (25); Stachybotrys (2); T . (25); P . graminis

Paecilomyces (25); Wardomyces Urea-formaldehyde foam insulation; W: wa W: insulation; foam Urea-formaldehyde (teleom. (teleom. geniculata

(25); P . sitophila (9); Chaetomium

(3); C . levellei mirabilis fuscoatra ed, GRAS: generally regarded as safe. regarded GRAS: generally ed, (25); (4); Trichocladium

(14); P . (25); Neosartorya atrum pruinosum rugosa microsporus

) (25); F . cinctum 1996; 4: Beguin et al.

(2); Podosphaera roquefortii nellum (25); P . mentagrophytes

te (25); Blumeria

delbruekii Humicola cyclopium

(syn. Monilia s, fruits GRAS 2, 3, 4, (I, II) ) (3); C . (4); Curvularia ﬀ guilliermondii meti fi ramanniana

table. Species encountered in one study included (reference): (reference): included study in one encountered Species table. (25); P . (2); Acrospeira (25); herbarum

2008; 17: Miller 1995; 18: Nilsson 1995; 18: Nilsson 2008; 17: Miller (9); C . sitophila (25); P . (25); Ulocladium

lythri (4); Sporotrichum sclerotiorum 2004; 10: Gravesen 1979; 11: Gravesen et al. 1979; 11: Gravesen 2004; 10: Gravesen

(25); P . (25); Doratomyces hungaricus (2009). NC: not classifi not (2009). NC: pulcherrima

A. 1995; 3: Beguin et al. restrictum ;

salmoneum

atum et al. ) (25); Ophiostoma (25); Torulaspora plant materialplant foodstu fermented NC (I, II) pot soil, house plantsatmospherephylloplane W NC 9, 23 NC NC (I, II) (II) Common substrate/habitat angustata catenulate catenulate infl

Hainesia (9); Pleospora (25); Mortierella (25) (syn. Myrothecium cinctum coprophilum spicatum

lter; CA: carpets; CF: Cotton fabrics; CO: concrete; CT: ceramic tiles; FI: fi ber insulations; G: glues; GB: G: glues; gypsum board; ber insulations; tiles; FI: fi ceramic CT: CO: concrete; fabrics; lter; CA: carpets; Cotton CF: ) (25); Trichophyton irens (25); P . v

(3); uens (9); C . ruber

(25); Chrysonilia herbarum onychis diffl

p://www.mycobank.org), INSD organism isola INSD organism p://www.mycobank.org),

chrysanthemicola

constrictum (25); P .

h parasiticus luzulae

raistrickii (25); Candida A . (syn. Metschnikowia (2); Truncatella ;

(25); Acrodontium ) (4); P . 2011; 2: Beguin et al. subspirale (25); Fusarium 2010; 9: Glushakova et al. 2010; 9: Glushakova (2); Scedosporium canescens

(8); Diplococcium (25); Torula (25) 2009; 23: Samson 2011; 24: Schober 1991; 25: Scott 2001; 26: Siu 1951; 27: Verhoeff 2011; 24: Schober 2001; 26: Siu 1991; 25: Scott 2009; 23: Samson myrti (2); Phoma (25); P . (25); Monascus et al. pilulifera Gliomastix rutilum lambica

) (3); C . pulcherrima (25); Cryptococcus (syn. Gliocladium irens melinii v ed in the present study (I, II) are indicated in bold. Phylotypes identifi Phylotypes in bold. indicated (I, II) are study ed in the present (25); olivacea ae

ﬃ radicicola hyalinospora paradoxus lamentous fungi) lamentous (25); B .

macrospora

ﬁ

(25); A . (syn. Scolecobasidium ochrosalmonium

(9); C . references. the listed on to trachyspermus allii

(2); Penicillium piluliferum

(25); P . (25); T . 1979; 15: Jarvis and Miller 2005; 16: Lignell et Miller al. and 1979; 15: Jarvis humicola

sporulosum (syn. Candida kiliense

(25); A . (2); Tripospermum constricta maltosa Davidiella A. Rollandina et al.

2004; 22: Polizzi et al. 2004; 22: Polizzi Phialophora Botrytis C . roseum ; e species identifi

Building material abbreviations: AF: AHU fi AF: abbreviations: material Building References: 1: Andersen et al. References: Rhodotorula sloo Rhodotorula Species ( Hyphomycetes cerevisiae Saccharomyces Sporobolomyces roseus Sporobolomyces ruberrimus Sporobolomyces pannonicus Udeniomyces Biosafety level class (BSL) according to de Hoog de Hoog to according (BSL) class level Biosafety conis Talaromyces Ishii Ishii Microsphaeropsis T . et al. used in addi roseum (28); Miller 2011; 8: Fujimura 2011; 8: Fujimura Miller For habitat information, Mycobank ( Mycobank information, habitat For ornatus I: indoor environment; M: materials (general); P: paint; PA: plaster; UF: PL: plastic; PA: paint; P: (general); M: materials environment; I: indoor maniella mentans Th A B C Species reported in two or more studies were included in the included were studies more or in two reported Species ryotrichum Eupenicillium (4); clone frequency are included. are frequency clone (25); (25); thyrium 4 marquandii islandicum (2);