The ISME Journal (2012) 6, 1801–1811 & 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12 www.nature.com/ismej ORIGINAL ARTICLE Particle-size distributions and seasonal diversity of allergenic and pathogenic fungi in outdoor air Naomichi Yamamoto1,2, Kyle Bibby1, Jing Qian1,4, Denina Hospodsky1, Hamid Rismani-Yazdi1,5, William W Nazaroff3 and Jordan Peccia1 1Department of Chemical and Environmental Engineering, Yale University, New Haven, CT, USA; 2Japan Society for the Promotion of Science, Tokyo, Japan and 3Department of Civil and Environmental Engineering, University of California, Berkeley, CA, USA Fungi are ubiquitous in outdoor air, and their concentration, aerodynamic diameters and taxonomic composition have potentially important implications for human health. Although exposure to fungal allergens is considered a strong risk factor for asthma prevalence and severity, limitations in tracking fungal diversity in air have thus far prevented a clear understanding of their human pathogenic properties. This study used a cascade impactor for sampling, and quantitative real-time PCR plus 454 pyrosequencing for analysis to investigate seasonal, size-resolved fungal commu- nities in outdoor air in an urban setting in the northeastern United States. From the 20 libraries produced with an average of B800 internal transcribed spacer (ITS) sequences (total 15 326 reads), 12 864 and 11 280 sequences were determined to the genus and species levels, respectively, and 558 different genera and 1172 different species were identified, including allergens and infectious pathogens. These analyses revealed strong relationships between fungal aerodynamic diameters and features of taxonomic compositions. The relative abundance of airborne allergenic fungi ranged from 2.8% to 10.7% of total airborne fungal taxa, peaked in the fall, and increased with increasing aerodynamic diameter. Fungi that can cause invasive fungal infections peaked in the spring, comprised 0.1–1.6% of fungal taxa and typically increased in relative abundance with decreasing aerodynamic diameter. Atmospheric fungal ecology is a strong function of aerodynamic diameter, whereby through physical processes, the size influences the diversity of airborne fungi that deposit in human airways and the efficiencies with which specific groups of fungi partition from outdoor air to indoor environments. The ISME Journal (2012) 6, 1801–1811; doi:10.1038/ismej.2012.30; published online 5 April 2012 Subject Category: microbial population and community ecology Keywords: aerodynamic diameter; fungi; 454 pyrosequencing; allergen; bioaerosol Introduction (Crameri et al., 2006). Exposure to fungal allergens is considered to be a strong risk factor for asthma An estimated 28–50 Tg of fungal materials are symptom prevalence and severity, and there is emitted into the earth’s atmosphere each year, and robust evidence to support a relationship between in some regions, fungal particles may constitute elevated environmental fungal spore counts and 420% of the primary emissions of organic aerosol asthma attacks (Crameri et al., 2006; Denning et al., (Elbert et al., 2007; Heald and Spracklen, 2009). 2006). Although less common than allergic disease, Airborne fungal material is an important contributor invasive fungal infections occur in both immuno- to human diseases. In industrialized countries, an competent and immunocompromised hosts estimated 20–30% of the population is affected by (Pitman et al., 2011). Invasive fungal infections from allergic diseases, and at least 3–10% of the general Candidia, Aspergillus and Cryptococcus spp. are population are sensitized to fungal allergens associated with high mortality in immunocompro- mised individuals (Lin et al., 2001). Aerodynamic diameter and species diversity are Correspondence: J Peccia, Department of Chemical and Environ- mental Engineering, Yale University, New Haven, CT 06511, USA. master variables that determine personal exposures E-mail: [email protected] and subsequent human health impacts. For a 4Current address: Department of Civil and Environmental particle of arbitrary shape, size and density, its Engineering, Clarkson University, Potsdam, NY 13699, USA. 5 aerodynamic diameter is uniquely defined as the Current address: Department of Chemical Engineering, Massa- diameter of a sphere of unit density (that is, chusetts Institute of Technology, Cambridge, MA 02139, USA. À 3 Received 21 November 2011; revised 21 February 2012; accepted 1gcm ) that experiences the same gravitational 22 February 2012; published online 5 April 2012 settling velocity. The size of fungal material controls Allergenic and pathogenic fungi in outdoor air N Yamamoto et al 1802 the rate and extent of physical processes that are 3.3–4.7, 4.7–5.8, 5.8–9.0 and 49.0 mm; New Star important for human exposure. These include Environmental, Roswell, GA, USA) were deployed settling and deposition onto environmental sur- to collect airborne particles on glass fiber substrates faces, deposition in the human respiratory tract, and operated continuously for the ca. 4-week penetration into buildings, resuspension into air, durations specified above. The cutoff diameters long-range transport and the efficiency of engi- described here are the particle diameters having neered removal systems (Thatcher and Layton, 50% collection efficiency. Aerosol samples were 1995; Yeh et al., 1996; Riley et al., 2002; Nazaroff, collected at an air flow rate of 28.3 l min À 1, which 2004; Prospero et al., 2005; Ariya et al., 2009). Large were calibrated twice weekly. After collection, the hyphal fragments and multicellular spores that are filters were stored at À 20 1C until subsequent analyses. 410 mm in size, unicellular spores with diameters of Outdoor temperature, wind speed and precipitation 1–10 mm and fragments o1 mm combine to permit a during the sampling periods are presented in broad size distribution of airborne fungal material. Supplementary Table 1. The size distribution can also be influenced by the attachment of fungal particles to other particulate matter. Overall, the inherent diversity in fungal DNA extraction particle size, coupled with the strong influence of One-eighth section of the loaded glass fiber filter aerodynamic diameter on the fate, transport and was used for DNA extraction. Extraction was source strength of fungal particles, indicate that size performed using the PowerMax Soil DNA Isolation is an important determinant of how taxonomic Kit (Mobio Laboratory, Carlsbad, CA, USA) with the compositions of airborne fungi are assembled in following modifications. The filter section was the atmosphere and in indoor air. enclosed in a 2-ml microcentrifuge tube along with The study reported here investigates the size- the kit’s power beads (1.0 g), kit’s extraction solution resolved, seasonal diversity of fungi in the atmo- (750 ml), 0.1 mm diameter glass beads (300 mg) and sphere at one location in the north eastern United 0.5 mm diameter glass beads (100 mg). The samples States. We use barcoded DNA pyrosequencing were then homogenized for 5 min by a bead beater technology applied to a fungal genetic marker (Model 607; BioSpec Products, Bartlesville, OK, USA). (Amend et al., 2010) to provide in-depth descrip- After bead beating, the extracted nucleic acids were tions of seasonal aerosol fungal diversity collected purified in accordance with the kit protocol and for eight different size ranges between 0.4 mmto purified DNA was eluted with 50 ml of TE buffer 49 mm in aerodynamic diameter. Genus-level and (10 mM Tris-HCl, 1 mM EDTA, pH ¼ 8.0). species analyses were performed to document the size-distributed compositions and abundances of allergenic and pathogenic fungi whereas selected Pyrosequencing of the internal transcribed spacer allergenic and pathogenic fungal species were (ITS) region of rDNA enumerated by quantitative PCR (qPCR) to charac- Sequencing was performed on 20 impactor samples, terize their absolute concentrations and particle- comprising the four seasons, each with five particle- size distributions. Coupling size-resolved aerosol size stages (da4 2.1 mm). Samples with dao2.1 mm sampling with next generation DNA sequencing were not included for the analyses owing to no or approaches allows for an unprecedented description weak PCR amplification. Universal fungal primers of atmospheric fungal ecology. The results provide ITS1F and ITS4 (Larena et al., 1999; Manter and important insights into how fungal aerodynamic Vivanco, 2007) along with adaptor, key and multi- diameter affects taxonomic composition, and plex identifier sequences were used to amplify the improve our understanding of human inhalation ITS region of fungal rDNA (Supplementary Table 2). exposure to atmospheric fungal allergens and The 50 ml reaction mixture contained the template pathogens. DNA (2 ml sample extract), 1 Â PCR Master Mix (Roche Applied Science, Indianapolis, IN, USA) and 0.3 mM of each primer. Cycling conditions were: Materials and methods 95 1C for 5 min of initial denaturation and 35 cycles of 95 1C for 30 s of dissociation, 55 1C for 30 s of Air sampling annealing, 72 1C for 1 min for each cycle extension Air sampling was conducted for four different and 72 1C for 10 min of final extension step. PCR seasons in 2009–2010 on the rooftop of a five-story amplicons were purified and concentrations nor- building in New Haven, CT, USA (41118’29’N malized with SequalPrep Normalization Plate (96 72155’43’W). Sampling for the four seasons occurred well)