Naomichi Yamamoto

Human Occupancy as a Source of Indoor Airborne Bacteria

Exposure to specific airborne bacteria indoors is linked to infectious and noninfectious adverse health outcomes. However, the sources and origins of bacteria suspended in indoor air are not well understood. This study presents evidence for elevated concentrations of indoor airborne bacteria due to human occupancy, and investigates the sources of these bacteria. Samples were collected in a university classroom while occupied and when vacant. The total particle mass concentration, bacterial genome concentration, and bacterial phylogenetic populations were characterized in indoor, outdoor, and ventilation duct supply air, as well as in the dust of ventilation system filters and in floor dust. Occupancy increased the total aerosol mass and bacterial genome concentration in indoor air PM10 and PM2.5 size fractions, with an increase of nearly two orders of magnitude in airborne bacterial genome concentration in PM10. On a per mass basis, floor dust was enriched in bacterial genomes compared to airborne particles. Quantitative comparisons between bacterial populations in indoor air and potential sources suggest that resuspended floor dust is an important contributor to bacterial aerosol populations during occupancy. Experiments that controlled for resuspension from the floor implies that direct human shedding may also significantly impact the concentration of indoor airborne particles. The high content of bacteria specific to the skin, nostrils, and hair of humans found in indoor air and in floor dust indicates that floors are an important reservoir of human-associated bacteria, and that the direct particle shedding of desquamated skin cells and their subsequent resuspension strongly influenced the airborne bacteria population structure in this human-occupied environment. Inhalation exposure to microbes shed by other current or previous human occupants may occur in communal indoor environments.

Size-resolved emission rates of airborne bacteria and fungi in an occupied classroom

The role of human occupancy as a source of indoor biological aerosols is poorly understood. Size-resolved concentrations of total and biological particles in indoor air were quantified in a classroom under occupied and vacant conditions. Per-occupant emission rates were estimated through a mass-balance modeling approach, and the microbial diversity of indoor and outdoor air during occupancy was determined via rDNA gene sequence analysis. Significant increases of total particle mass and bacterial genome concentrations were observed during the occupied period compared to the vacant case. These increases varied in magnitude with the particle size and ranged from 3 to 68 times for total mass, 12–2700 times for bacterial genomes, and 1.5–5.2 times for fungal genomes. Emission rates per person-hour because of occupancy were 31 mg, 37 × 106 genome copies, and 7.3 × 106 genome copies for total particle mass, bacteria, and fungi, respectively. Of the bacterial emissions, ∼18% are from taxa that are closely associated with the human skin microbiome. This analysis provides size-resolved, per person-hour emission rates for these biological particles and illustrates the extent to which being in an occupied room results in exposure to bacteria that are associated with previous or current human occupants. Practical Implications Presented here are the first size-resolved, per person emission rate estimates of bacterial and fungal genomes for a common occupied indoor space. The marked differences observed between total particle and bacterial size distributions suggest that size-dependent aerosol models that use total particles as a surrogate for microbial particles incorrectly assess the fate of and human exposure to airborne bacteria. The strong signal of human microbiota in airborne particulate matter in an occupied setting demonstrates that the aerosol route can be a source of exposure to microorganisms emitted from the skin, hair, nostrils, and mouths of other occupants.

Accuracy, Precision, and Method Detection Limits of Quantitative PCR for Airborne Bacteria and Fungi

ABSTRACT Real-time quantitative PCR (qPCR) for rapid and specific enumeration of microbial agents is finding increased use in aerosol science. The goal of this study was to determine qPCR accuracy, precision, and method detection limits (MDLs) within the context of indoor and ambient aerosol samples. Escherichia coli and Bacillus atrophaeus vegetative bacterial cells and Aspergillus fumigatus fungal spores loaded onto aerosol filters were considered. Efficiencies associated with recovery of DNA from aerosol filters were low, and excluding these efficiencies in quantitative analysis led to underestimating the true aerosol concentration by 10 to 24 times. Precision near detection limits ranged from a 28% to 79% coefficient of variation (COV) for the three test organisms, and the majority of this variation was due to instrument repeatability. Depending on the organism and sampling filter material, precision results suggest that qPCR is useful for determining dissimilarity between two samples only if the true differences are greater than 1.3 to 3.2 times (95% confidence level at n = 7 replicates). For MDLs, qPCR was able to produce a positive response with 99% confidence from the DNA of five B. atrophaeus cells and less than one A. fumigatus spore. Overall MDL values that included sample processing efficiencies ranged from 2,000 to 3,000 B. atrophaeus cells per filter and 10 to 25 A. fumigatus spores per filter. Applying the concepts of accuracy, precision, and MDL to qPCR aerosol measurements demonstrates that sample processing efficiencies must be accounted for in order to accurately estimate bioaerosol exposure, provides guidance on the necessary statistical rigor required to understand significant differences among separate aerosol samples, and prevents undetected (i.e., nonquantifiable) values for true aerosol concentrations that may be significant.

Particle-size distributions and seasonal diversity of allergenic and pathogenic fungi in outdoor air.

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 communities in outdoor air in an urban setting in the northeastern United States. From the 20 libraries produced with an average of ∼800 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.

Residential air exchange rates in three major US metropolitan areas: results from the Relationship Among Indoor, Outdoor, and Personal Air Study 1999–2001

UNLABELLED We report approximately 500 indoor-outdoor air exchange rate (AER) calculations based on measurements conducted in residences in three US metropolitan areas in 1999-2001: Elizabeth, New Jersey; Houston, Texas; and Los Angeles County, California. Overall, a median AER across these urban areas and seasons was 0.71 air changes per hour (ACH, or per hour; n = 509) while median AERs measured in California (n = 182), New Jersey (n = 163), and Texas (n = 164) were 0.87, 0.88, and 0.47 ACH, respectively. In Texas, the measured AERs were lower in the summer cooling season (median = 0.37 ACH) than in the winter heating season (median = 0.63 ACH), likely because of the reported use of room air conditioners as Houston is typically hot and humid during the summer. The measured AERs in California were higher in summer (median = 1.13 ACH) than in winter (median = 0.61 ACH). Because the summer cooling season in Los Angeles County is less humid than in New Jersey or Texas, natural ventilation through open windows and screened doors likely increased measured AER in California study homes. In New Jersey, AER were similar across heating and cooling seasons, although the median AER was relatively lower during the spring. PRACTICAL IMPLICATIONS Adequate ventilation or air exchange rate (AER) for an indoor environment is important for human health and comfort, and relevant to building design and energy conservation and efficiency considerations. However, residential AER data, especially measured by more accurate non-toxic tracer gas methodologies, are at present quite limited worldwide, and are insufficient to represent the variations across regions and seasons within and between homes, including apartments and condominiums in more densely populated urban areas. The present paper presents quantitative and qualitative data to characterize residential AERs in three US urban areas with different climate attributes.

Characterizing airborne fungal and bacterial concentrations and emission rates in six occupied children's classrooms

UNLABELLED Baseline information on size-resolved bacterial, fungal, and particulate matter (PM) indoor air concentrations and emission rates is presented for six school classrooms sampled in four countries. Human occupancy resulted in significantly elevated airborne bacterial (81 times on average), fungal (15 times), and PM mass (nine times) concentrations as compared to vacant conditions. Occupied indoor/outdoor (I/O) ratios consistently exceeded vacant I/O ratios. Regarding size distributions, average room-occupied bacterial, fungal, and PM geometric mean particle sizes were similar to one another while geometric means estimated for bacteria, fungi, and PM mass during vacant sampling were consistently lower than when occupied. Occupancy also resulted in elevated indoor bacterial-to-PM mass-based and number-based ratios above corresponding outdoor levels. Mean emission rates due to human occupancy were 14 million cells/person/h for bacteria, 14 million spore equivalents/person/h for fungi, and 22 mg/person/h for PM mass. Across all locations, indoor emissions contributed 83 ± 27% (bacteria), 66 ± 19% (fungi), and 83 ± 24% (PM mass) of the average indoor air concentrations during occupied times. PRACTICAL IMPLICATIONS An extensive data set of bacterial and fungal size-distributed indoor air concentrations and emission rates is presented. Analysis of these data contributes to an understanding of how indoor bacterial and fungal aerosols are influenced by human occupancy. This work extends beyond prior culture and DNA-based microbiome studies in buildings to include quantitative relationships between size-resolved bacterial and fungal concentrations in indoor air and building parameters such as occupancy, ventilation, and outdoor conditions. The work indicates that occupancy-associated emissions (e.g., via resuspension and shedding) contribute more to both bacterial and fungal indoor air concentrations than do outdoor sources for the occupied classrooms investigated in this study.

Fungal High-throughput Taxonomic Identification tool for use with Next-Generation Sequencing (FHiTINGS)

Improvements in DNA sequencing technology provide unprecedented opportunities to explore fungal diversity, but also present challenges in data analysis due to the large number of sequences generated. Here, we describe an open source software program “FHiTINGS” that utilizes the output of a BLASTn (blastall) search to rapidly identify, classify, and parse internal transcribed spacer (ITS) DNA sequences produced in fungal ecology studies that utilize next‐generation DNA sequencing. This tool was designed for use with 454 pyrosequencing and is also appropriate for use with any sequencing platform that allows for BLAST searches against the indicated ITS database. For each sequence, FHiTINGS uses the lowest common ancestor method (LCA) to produce a single identification from BLAST output results, and then assigns taxonomic ranks from species through kingdom when possible for each sequence based on the Index Fungorum database. The program then sums and sorts this data into tabular form to enable rapid analysis of the sample, including α‐diversity measures or richness. In silico testing demonstrates the time required to analyze and classify 1000 sequences is reduced from over 2 h by manual sorting to <1 min of computational time when using FHiTINGS, and that the classification output from the software is consistent with that derived from manual sorting of the data.

Assessing allergenic fungi in house dust by floor wipe sampling and quantitative PCR.

Abstract  In the present study, we modified an existing surface wipe sampling method for lead and other heavy metals to create a protocol to collect fungi in floor dust followed by real‐time quantitative PCR (qPCR)‐based detection. We desired minimal inconvenience for participants in residential indoor environmental quality and health studies. Accuracy, precision, and method detection limits (MDLs) were investigated. Overall, MDLs ranged from 0.6 to 25 cell/cm2 on sampled floors. Overall measurement precisions expressed as the coefficient of variation because of sample processing and qPCR ranged 6–63%. Median and maximum fungal concentrations in house dust in study homes in Visalia, Tulare County, California, were 110 and 2500 cell/cm2, respectively, with universal fungal primers (allergenic and nonallergenic species). The field study indicated samplings in multiple seasons were necessary to characterize representative whole‐year fungal concentrations in residential microenvironments. This was because significant temporal variations were observed within study homes. Combined field and laboratory results suggested this modified new wipe sampling method, in conjunction with growth‐independent qPCR, shows potential to improve human exposure and health studies for fungal pathogens and allergens in dust in homes of susceptible, vulnerable population subgroups. Practical Implications Fungi are ubiquitous in indoor and outdoor environments, and many fungi are known to cause allergic reactions and exacerbate asthma attacks. This study established—by modifying an existing—a wipe sampling method to collect fungi in floor dust followed by real‐time quantitative PCR (qPCR)‐based detection methodologies. Results from this combined laboratory and field assessment suggested the methodology’s potential to inform larger human exposure studies for fungal pathogens and allergens in house dust as well as epidemiologic studies of children with asthma and older adults with chronic respiratory diseases.

Indoor emissions as a primary source of airborne allergenic fungal particles in classrooms.

This study quantifies the influence of ventilation and indoor emissions on concentrations and particle sizes of airborne indoor allergenic fungal taxa and further examines geographical variability, each of which may affect personal exposures to allergenic fungi. Quantitative PCR and multiplexed DNA sequencing were employed to count and identify allergenic fungal aerosol particles indoors and outdoors in seven school classrooms in four different countries. Quantitative diversity analysis was combined with building characterization and mass balance modeling to apportion source contributions of indoor allergenic airborne fungal particles. Mass balance calculations indicate that 70% of indoor fungal aerosol particles and 80% of airborne allergenic fungal taxa were associated with indoor emissions; on average, 81% of allergenic fungi from indoor sources originated from occupant-generated emissions. Principal coordinate analysis revealed geographical variations in fungal communities among sites in China, Europe, and North America (p < 0.05, analysis of similarity), demonstrating that geography may also affect personal exposures to allergenic fungi. Indoor emissions including those released with occupancy contribute more substantially to allergenic fungal exposures in classrooms sampled than do outdoor contributions from ventilation. The results suggest that design and maintenance of buildings to control indoor emissions may enable reduced indoor inhalation exposures to fungal allergens.

Optimization of a real-time PCR assay to quantitate airborne fungi collected on a gelatin filter

The present study aimed to optimize a real-time PCR assay to quantitate airborne fungi collected on a gelatin filter. In particular, the study optimized conditions for the DNA extraction and real-time PCR amplification to accurately measure airborne fungal concentrations. First, time of fine bead homogenization to extract the DNA from fungal cells was optimized to maximize the DNA yield and prepare the DNA suitable for sensitive and precise quantification by a subsequent real-time PCR analysis. Second, a condition for the real-time PCR amplification was optimized to successfully amplify and quantitate the extracted fungal DNA. In particular, a dilution ratio of the DNA extracts to be introduced to PCR was optimized to achieve an appropriate balance between mitigating PCR inhibition and securing detection sensitivity. Since concentrations of airborne fungi generally observed in indoor and outdoor environments (i.e., 10(1)-10(4) CFU m(-3)) were found to be near the limit of quantification by the generally-used molecular-based detection technique in conjunction with use of gelatin filters, optimizations of these conditions were found to be crucial. Our preliminary result showed that a culture-based method underestimated concentrations of airborne environmental fungi by 1 to 2 orders of magnitude compared to those characterized by the real-time PCR assay.