Does evidence support measuring spore counts to identify dampness or mold in buildings? A literature review.

BACKGROUND: To identify dampness or mold (D/M) in buildings, investigators generally inspect for observable D/M indicators, the presence of which justifies remediation. Investigators may also use microbiological measurement and interpretation strategies with uncertain scientific support. OBJECTIVE: We assessed available evidence supporting uses of spore counts, the microbiological measurement most commonly used to assess D/M. METHODS: We reviewed published studies assessing relationships between spore counts and observable D/M, across buildings with different observable D/M levels. RESULTS: Penicillium/Aspergillus counts were consistently elevated in damp vs. reference (dry or outdoor) locations. Total spore counts provided a weaker, less consistent signal. The most detailed published analysis could distinguish groups of damp homes but not individual damp homes. SIGNIFICANCE: Evidence did not validate current interpretations of spore count data for identifying single damp homes. Thus, such interpretations rest primarily on professional judgment. An additional series of informative but ineligible articles demonstrated an unconventional, more powerful "statistically based" comparison of multiple indoor vs. outdoor spore counts for identifying elevated indoor spores (and assumed D/M). Findings suggest that validation of enhanced spore trap approaches, including more samples indoors and outdoors plus statistically based comparisons of specific fungal groups, may allow evidence-based microbial identification of probable dampness in individual buildings.

Microbial growth and volatile organic compound (VOC) emissions from carpet and drywall under elevated relative humidity conditions.

BACKGROUND: Microbes can grow in indoor environments if moisture is available, and we need an improved understanding of how this growth contributes to emissions of microbial volatile organic compounds (mVOCs). The goal of this study was to measure how moisture levels, building material type, collection site, and microbial species composition impact microbial growth and emissions of mVOCs. We subjected two common building materials, drywall, and carpet, to treatments with varying moisture availability and measured microbial communities and mVOC emissions. RESULTS: Fungal growth occurred in samples at >75% equilibrium relative humidity (ERH) for carpet with dust and >85% ERH for inoculated painted drywall. In addition to incubated relative humidity level, dust sample collection site (adonis p=0.001) and material type (drywall, carpet, adonis p=0.001) drove fungal and bacterial species composition. Increased relative humidity was associated with decreased microbial species diversity in samples of carpet with dust (adonis p= 0.005). Abundant volatile organic compounds (VOCs) that accounted for >1% emissions were likely released from building materials and the dust itself. However, certain mVOCs were associated with microbial growth from carpet with dust such as C10H16H+ (monoterpenes) and C2H6SH+ (dimethyl sulfide and ethanethiol). CO2 production from samples of carpet with dust at 95% ERH averaged 5.92 mg hr-1 kg-1, while the average for carpet without dust at 95% ERH was 2.55 mg hr-1 kg-1. CONCLUSION: Microbial growth and mVOC emissions occur at lower relative humidity in carpet and floor dust compared to drywall, which has important implications for human exposure. Even under elevated relative humidity conditions, the VOC emissions profile is dominated by non-microbial VOCs, although potential mVOCs may dominate odor production. Video Abstract.

Microbial exposures in moisture-damaged schools and associations with respiratory symptoms in students: A multi-country environmental exposure study.

Moisture-damaged buildings are associated with respiratory symptoms and underlying diseases among building occupants, but the causative agent(s) remain a mystery. We first identified specific fungal and bacterial taxa in classrooms with moisture damage in Finnish and Dutch primary schools. We then investigated associations of the identified moisture damage indicators with respiratory symptoms in more than 2700 students. Finally, we explored whether exposure to specific taxa within the indoor microbiota may explain the association between moisture damage and respiratory health. Schools were assessed for moisture damage through detailed inspections, and the microbial composition of settled dust in electrostatic dustfall collectors was determined using marker-gene analysis. In Finland, there were several positive associations between particular microbial indicators (diversity, richness, individual taxa) and a respiratory symptom score, while in the Netherlands, the associations tended to be mostly inverse and statistically non-significant. In Finland, abundance of the Sphingomonas bacterial genus and endotoxin levels partially explained the associations between moisture damage and symptom score. A few microbial taxa explained part of the associations with health, but overall, the observed associations between damage-associated individual taxa and respiratory health were limited.

Associations of observed home dampness and mold with the fungal and bacterial dust microbiomes.

The objective of this analysis was to examine and compare quantitative metrics of observed dampness and mold, including visible mold and moisture damage, and fungal and bacterial microbiomes. In-home visits were conducted at age 7 for children enrolled in the Cincinnati Childhood Allergy and Air Pollution Study. Trained study staff evaluated the primary residence and measured total areas of visible moisture and mold damage in the home. Floor dust was collected and archived. Archived dust samples collected from each home (n = 178) were extracted and analyzed using bacterial (16S rRNA gene) and fungal (internal transcribed spacer region) sequencing. Fungi were also divided into moisture requirement categories of xerophiles, mesophiles, and hydrophiles. Data analyses used Spearman's correlation, Kruskal-Wallis, Permanova, DESeq, and negative binomial regression models. Comparing high moisture or mold damage to no damage, five fungal species and two bacterial species had higher concentrations (absolute abundance) and six fungal species and three bacterial species had lower concentrations. Hydrophilic and mesophilic fungi showed significant dose-related increases with increasing moisture damage and mold damage, respectively. When comparing alpha or beta diversity of fungi and bacteria across mold and moisture damage levels, no significant associations or differences were found. Mold and moisture damage did not affect diversity of fungal and bacterial microbiomes. Instead, both kinds of damage were associated with changes in species composition of both bacterial and fungal microbiomes, indicating that fungal and bacterial communities in the home might be influenced by one another as well as by mold or moisture in the home.

Fungal Signature of Moisture Damage in Buildings: Identification by Targeted and Untargeted Approaches with Mycobiome Data.

Identifying microbial indicators of damp and moldy buildings remains a challenge at the intersection of microbiology, building science, and public health. Sixty homes in New York City were assessed for moisture-related damage, and three types of dust samples were collected for microbiological analysis. We applied four approaches for detecting fungal signatures of moisture damage in these buildings. Two novel targeted approaches selected specific taxa, identified by a priori hypotheses, from the broad mycobiome as detected with amplicon sequencing. We investigated whether (i) hydrophilic fungi (i.e., requiring high moisture) or (ii) fungi previously reported as indicating damp homes would be more abundant in water-damaged rooms/homes than in nondamaged rooms/homes. Two untargeted approaches compared water-damaged to non-water-damaged homes for (i) differences between indoor and outdoor fungal populations or (ii) differences in the presence or relative abundance of particular fungal taxa. Strong relationships with damage indicators were found for some targeted fungal groups in some sampling types, although not always in the hypothesized direction. For example, for vacuum samples, hydrophilic fungi had significantly higher relative abundance in water-damaged homes, but mesophilic fungi, unexpectedly, had significantly lower relative abundance in homes with visible mold. Untargeted approaches identified no microbial community metrics correlated with water damage variables but did identify specific taxa with at least weak positive links to water-damaged homes. These results, although showing a complex relationship between moisture damage and microbial communities, suggest that targeting particular fungi offers a potential route toward identifying a fungal signature of moisture damage in buildings.IMPORTANCE Living or working in damp or moldy buildings increases the risk of many adverse health effects, including asthma and other respiratory diseases. To date, however, the particular environmental exposure(s) from water-damaged buildings that causes the health effects have not been identified. Likewise, a consistent quantitative measurement that would indicate whether a building is water damaged or poses a health risk to occupants has not been found. In this work, we tried to develop analytical tools that would find a microbial signal of moisture damage amid the noisy background of microorganisms in buildings. The most successful approach taken here focused on particular groups of fungi-those considered likely to grow in damp indoor environments-and their associations with observed moisture damage. With further replication and refinement, this hypothesis-based strategy may be effective in finding still-elusive relationships between building damage and microbiomes.

Indoor bacterial microbiota and development of asthma by 10.5 years of age.

BACKGROUND: Early-life indoor bacterial exposure is associated with the risk of asthma, but the roles of specific bacterial genera are poorly understood. OBJECTIVE: We sought to determine whether individual bacterial genera in indoor microbiota predict the development of asthma. METHODS: Dust samples from living rooms were collected at 2 months of age. The dust microbiota was characterized by using Illumina MiSeq sequencing amplicons of the bacterial 16S ribosomal RNA gene. Children (n = 373) were followed up for ever asthma until the age of 10.5 years. RESULTS: Richness was inversely associated with asthma after adjustments (P = .03). The phylogenetic microbiota composition in asthmatics patients' homes was characteristically different from that in nonasthmatic subjects' homes (P = .02, weighted UniFrac, adjusted association, permutational multivariate analysis of variance, PERMANOVA-S). The first 2 axis scores of principal coordinate analysis of the weighted UniFrac distance matrix were inversely associated with asthma. Of 658 genera detected in the dust samples, the relative abundances of 41 genera correlated (r > |0.4|) with one of these axes. Lactococcus genus was a risk factor for asthma (adjusted odds ratio, 1.36 [95% CI, 1.13-1.63] per interquartile range change). The abundance of 12 bacterial genera (mostly from the Actinomycetales order) was associated with lower asthma risk (P < .10), although not independently of each other. The sum relative abundance of these 12 intercorrelated genera was significantly protective and explained the majority of the association of richness with less asthma. CONCLUSION: Our data confirm that phylogenetic differences in the microbiota of infants' homes are associated with subsequent asthma risk and suggest that communities of selected bacteria are more strongly linked to asthma protection than individual bacterial taxa or mere richness.

Author Correction: Farm-like indoor microbiota in non-farm homes protects children from asthma development.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Farm-like indoor microbiota in non-farm homes protects children from asthma development.

Asthma prevalence has increased in epidemic proportions with urbanization, but growing up on traditional farms offers protection even today1. The asthma-protective effect of farms appears to be associated with rich home dust microbiota2,3, which could be used to model a health-promoting indoor microbiome. Here we show by modeling differences in house dust microbiota composition between farm and non-farm homes of Finnish birth cohorts4 that in children who grow up in non-farm homes, asthma risk decreases as the similarity of their home bacterial microbiota composition to that of farm homes increases. The protective microbiota had a low abundance of Streptococcaceae relative to outdoor-associated bacterial taxa. The protective effect was independent of richness and total bacterial load and was associated with reduced proinflammatory cytokine responses against bacterial cell wall components ex vivo. We were able to reproduce these findings in a study among rural German children2 and showed that children living in German non-farm homes with an indoor microbiota more similar to Finnish farm homes have decreased asthma risk. The indoor dust microbiota composition appears to be a definable, reproducible predictor of asthma risk and a potential modifiable target for asthma prevention.

A different suite: The assemblage of distinct fungal communities in water-damaged units of a poorly-maintained public housing building.

Water-damaged housing has been associated with a number of negative health outcomes, principally respiratory disease and asthma. Much of what we know about fungi associated with water-damaged buildings has come from culture-based and immunochemical methods. Few studies have used high-throughput sequencing technologies to assess the impact of water-damage on microbial communities in residential buildings. In this study we used amplicon sequencing and quantitative-PCR to evaluate fungal communities on surfaces and in airborne dust in multiple units of a condemned public housing project located in the San Francisco Bay Area. We recruited 21 households to participate in this study and characterized their apartments as either a unit with visible mold or no visible mold. We sampled airborne fungi from dust settled over a month-long time period from the outdoors, in units with no visible mold, and units with visible mold. In units with visible mold we additionally sampled the visible fungal colonies from bathrooms, kitchens, bedrooms, and living rooms. We found that fungal biomass in settled dust was greater outdoors compared to indoors, but there was no significant difference of fungal biomass in units with visible mold and no visible mold. Interestingly, we found that fungal diversity was reduced in units with visible mold compared to units with no visible mold and the outdoors. Units with visible mold harbored fungal communities distinct from units with no visible mold and the outdoors. Units with visible mold had a greater abundance of taxa within the classes Eurotiomycetes, Saccharomycetes, and Wallemiomycetes. Colonies of fungi collected from units with visible mold were dominated by two Cladosporium species, C. sphaerospermum and C halotolerans. This study demonstrates that high-throughput sequencing of fungi indoors can be a useful strategy for distinguishing distinct microbial exposures in water-damaged homes with visible and nonvisible mold growth, and may provide a microbial means for identifying water damaged housing.

Ten questions concerning the implications of carpet on indoor chemistry and microbiology.

Carpet and rugs currently represent about half of the United States flooring market and offer many benefits as a flooring type. How carpets influence our exposure to both microorganisms and chemicals in indoor environments has important health implications but is not well understood. The goal of this manuscript is to consolidate what is known about how carpet impacts indoor chemistry and microbiology, as well as to identify the important research gaps that remain. After describing the current use of carpet indoors, questions focus on five specific areas: 1) indoor chemistry, 2) indoor microbiology, 3) resuspension and exposure, 4) current practices and future needs, and 5) sustainability. Overall, it is clear that carpet can influence our exposures to particles and volatile compounds in the indoor environment by acting as a direct source, as a reservoir of environmental contaminants, and as a surface supporting chemical and biological transformations. However, the health implications of these processes are not well known, nor how cleaning practices could be optimized to minimize potential negative impacts. Current standards and recommendations focus largely on carpets as a primary source of chemicals and on limiting moisture that would support microbial growth. Future research should consider enhancing knowledge related to the impact of carpet in the indoor environment and how we might improve the design and maintenance of this common material to reduce our exposure to harmful contaminants while retaining the benefits to consumers.

Emission Factors of Microbial Volatile Organic Compounds from Environmental Bacteria and Fungi.

Knowledge of the factors controlling the diverse chemical emissions of common environmental bacteria and fungi is crucial because they are important signal molecules for these microbes that also could influence humans. We show here not only a high diversity of mVOCs but that their abundance can differ greatly in different environmental contexts. Microbial volatiles exhibit dynamic changes across microbial growth phases, resulting in variance of composition and emission rate of species-specific and generic mVOCs. In vitro experiments documented emissions of a wide range of mVOCs (>400 different chemicals) at high time resolution from diverse microbial species grown under different controlled conditions on nutrient media, or residential structural materials ( N = 54, Ncontrol = 23). Emissions of mVOCs varied not only between microbial taxa at a given condition but also as a function of life stage and substrate type. We quantify emission factors for total and specific mVOCs normalized for respiration rates to account for the microbial activity during their stationary phase. Our VOC measurements of different microbial taxa indicate that a variety of factors beyond temperature and water activity, such as substrate type, microbial symbiosis, growth phase, and lifecycle affect the magnitude and composition of mVOC emission.

Taxonomic annotation of public fungal ITS sequences from the built environment - a report from an April 10-11, 2017 workshop (Aberdeen, UK).

Recent DNA-based studies have shown that the built environment is surprisingly rich in fungi. These indoor fungi - whether transient visitors or more persistent residents - may hold clues to the rising levels of human allergies and other medical and building-related health problems observed globally. The taxonomic identity of these fungi is crucial in such pursuits. Molecular identification of the built mycobiome is no trivial undertaking, however, given the large number of unidentified, misidentified, and technically compromised fungal sequences in public sequence databases. In addition, the sequence metadata required to make informed taxonomic decisions - such as country and host/substrate of collection - are often lacking even from reference and ex-type sequences. Here we report on a taxonomic annotation workshop (April 10-11, 2017) organized at the James Hutton Institute/University of Aberdeen (UK) to facilitate reproducible studies of the built mycobiome. The 32 participants went through public fungal ITS barcode sequences related to the built mycobiome for taxonomic and nomenclatural correctness, technical quality, and metadata availability. A total of 19,508 changes - including 4,783 name changes, 14,121 metadata annotations, and the removal of 99 technically compromised sequences - were implemented in the UNITE database for molecular identification of fungi (https://unite.ut.ee/) and shared with a range of other databases and downstream resources. Among the genera that saw the largest number of changes were Penicillium, Talaromyces, Cladosporium, Acremonium, and Alternaria, all of them of significant importance in both culture-based and culture-independent surveys of the built environment.

Indoor microbiota in severely moisture damaged homes and the impact of interventions.

BACKGROUND: The limited understanding of microbial characteristics in moisture-damaged buildings impedes efforts to clarify which adverse health effects in the occupants are associated with the damage and to develop effective building intervention strategies. The objectives of this current study were (i) to characterize fungal and bacterial microbiota in house dust of severely moisture-damaged residences, (ii) to identify microbial taxa associated with moisture damage renovations, and (iii) to test whether the associations between the identified taxa and moisture damage are replicable in another cohort of homes. We applied bacterial 16S rRNA gene and fungal ITS amplicon sequencing complemented with quantitative PCR and chemical-analytical approaches to samples of house dust, and also performed traditional cultivation of bacteria and fungi from building material samples. RESULTS: Active microbial growth on building materials had significant though small influence on the house dust bacterial and fungal communities. Moisture damage interventions-including actual renovation of damaged homes and cases where families moved to another home-had only a subtle effect on bacterial community structure, seen as shifts in abundance weighted bacterial profiles after intervention. While bacterial and fungal species richness were reduced in homes that were renovated, they were not reduced for families that moved houses. Using different discriminant analysis tools, we were able identify taxa that were significantly reduced in relative abundance during renovation of moisture damage. For bacteria, the majority of candidates belonged to different families within the Actinomycetales order. Results for fungi were overall less consistent. A replication study in approximately 400 homes highlighted some of the identified taxa, confirming associations with observations of moisture damage and mold. CONCLUSIONS: The present study is one of the first studies to analyze changes in microbiota due to moisture damage interventions using high-throughput sequencing. Our results suggest that effects of moisture damage and moisture damage interventions may appear as changes in the abundance of individual, less common, and especially bacterial taxa, rather than in overall community structure.

Microbes and associated soluble and volatile chemicals on periodically wet household surfaces.

BACKGROUND: Microorganisms influence the chemical milieu of their environment, and chemical metabolites can affect ecological processes. In built environments, where people spend the majority of their time, very little is known about how surface-borne microorganisms influence the chemistry of the indoor spaces. Here, we applied multidisciplinary approaches to investigate aspects of chemical microbiology in a house. METHODS: We characterized the microbial and chemical composition of two common and frequently wet surfaces in a residential setting: kitchen sink and bathroom shower. Microbial communities were studied using culture-dependent and independent techniques, including targeting RNA for amplicon sequencing. Volatile and soluble chemicals from paired samples were analyzed using state-of-the-art techniques to explore the links between the observed microbiota and chemical exudates. RESULTS: Microbial analysis revealed a rich biological presence on the surfaces exposed in kitchen sinks and bathroom shower stalls. Microbial composition, matched for DNA and RNA targets, varied by surface type and sampling period. Bacteria were found to have an average of 25× more gene copies than fungi. Biomass estimates based on qPCR were well correlated with measured total volatile organic compound (VOC) emissions. Abundant VOCs included products associated with fatty acid production. Molecular networking revealed a diversity of surface-borne compounds that likely originate from microbes and from household products. CONCLUSIONS: Microbes played a role in structuring the chemical profiles on and emitted from kitchen sinks and shower stalls. Microbial VOCs (mVOCs) were predominately associated with the processing of fatty acids. The mVOC composition may be more stable than that of microbial communities, which can show temporal and spatial variation in their responses to changing environmental conditions. The mVOC output from microbial metabolism on kitchen sinks and bathroom showers should be apparent through careful measurement, even against a broader background of VOCs in homes, some of which may originate from microbes in other locations within the home. A deeper understanding of the chemical interactions between microbes on household surfaces will require experimentation under relevant environmental conditions, with a finer temporal resolution, to build on the observational study results presented here.

Schrödinger's microbes: Tools for distinguishing the living from the dead in microbial ecosystems.

While often obvious for macroscopic organisms, determining whether a microbe is dead or alive is fraught with complications. Fields such as microbial ecology, environmental health, and medical microbiology each determine how best to assess which members of the microbial community are alive, according to their respective scientific and/or regulatory needs. Many of these fields have gone from studying communities on a bulk level to the fine-scale resolution of microbial populations within consortia. For example, advances in nucleic acid sequencing technologies and downstream bioinformatic analyses have allowed for high-resolution insight into microbial community composition and metabolic potential, yet we know very little about whether such community DNA sequences represent viable microorganisms. In this review, we describe a number of techniques, from microscopy- to molecular-based, that have been used to test for viability (live/dead determination) and/or activity in various contexts, including newer techniques that are compatible with or complementary to downstream nucleic acid sequencing. We describe the compatibility of these viability assessments with high-throughput quantification techniques, including flow cytometry and quantitative PCR (qPCR). Although bacterial viability-linked community characterizations are now feasible in many environments and thus are the focus of this critical review, further methods development is needed for complex environmental samples and to more fully capture the diversity of microbes (e.g., eukaryotic microbes and viruses) and metabolic states (e.g., spores) of microbes in natural environments.

Draft genome sequences of eight bacteria isolated from the indoor environment: Staphylococcus capitis strain H36, S. capitis strain H65, S. cohnii strain H62, S. hominis strain H69, Microbacterium sp. strain H83, Mycobacterium iranicum strain H39, Plantibacter sp. strain H53, and Pseudomonas oryzihabitans strain H72.

We report here the draft genome sequences of eight bacterial strains of the genera Staphylococcus, Microbacterium, Mycobacterium, Plantibacter, and Pseudomonas. These isolates were obtained from aerosol sampling of bathrooms of five residences in the San Francisco Bay area. Taxonomic classifications as well as the genome sequence and gene annotation of the isolates are described. As part of the "Built Environment Reference Genome" project, these isolates and associated genome data provide valuable resources for studying the microbiology of the built environment.

Contribution of Vegetation to the Microbial Composition of Nearby Outdoor Air.

UNLABELLED: Given that epiphytic microbes are often found in large population sizes on plants, we tested the hypothesis that plants are quantitatively important local sources of airborne microorganisms. The abundance of microbial communities, determined by quantifying bacterial 16S RNA genes and the fungal internal transcribed spacer (ITS) region, in air collected directly above vegetation was 2- to 10-fold higher than that in air collected simultaneously in an adjacent nonvegetated area 50 m upwind. Nonmetric multidimensional scaling revealed that the composition of airborne bacteria in upwind air samples grouped separately from that of downwind air samples, while communities on plants and downwind air could not be distinguished. In contrast, fungal taxa in air samples were more similar to each other than to the fungal epiphytes. A source-tracking algorithm revealed that up to 50% of airborne bacteria in downwind air samples were presumably of local plant origin. The difference in the proportional abundances of a given operational taxonomic unit (OTU) between downwind and upwind air when regressed against the proportional representation of this OTU on the plant yielded a positive slope for both bacteria and fungi, indicating that those taxa that were most abundant on plants proportionally contributed more to downwind air. Epiphytic fungi were less of a determinant of the microbiological distinctiveness of downwind air and upwind air than epiphytic bacteria. Emigration of epiphytic bacteria and, to a lesser extent, fungi, from plants can thus influence the microbial composition of nearby air, a finding that has important implications for surrounding ecosystems, including the built environment into which outdoor air can penetrate. IMPORTANCE: This paper addresses the poorly understood role of bacterial and fungal epiphytes, the inhabitants of the aboveground plant parts, in the composition of airborne microbes in outdoor air. It is widely held that epiphytes contribute to atmospheric microbial assemblages, but much of what we know is limited to qualitative assessments. Elucidating the sources of microbes in outdoor air can inform basic biological processes seen in airborne communities (e.g., dispersal and biogeographical patterns). Furthermore, given the considerable contribution of outdoor air to microbial communities found within indoor environments, the understanding of plants as sources of airborne microbes in outdoor air might contribute to our understanding of indoor air quality. With an experimental design developed to minimize the likelihood of other-than-local plant sources contributing to the composition of airborne microbes, we provide direct evidence that plants are quantitatively important local sources of airborne microorganisms, with implications for the surrounding ecosystems.

Microbiota of the indoor environment: a meta-analysis.

BACKGROUND: As modern humans, we spend the majority of our time in indoor environments. Consequently, environmental exposure to microorganisms has important implications for human health, and a better understanding of the ecological drivers and processes that impact indoor microbial assemblages will be key for expanding our knowledge of the built environment. In the present investigation, we combined recent studies examining the microbiota of the built environment in order to identify unifying community patterns and the relative importance of indoor environmental factors. Ultimately, the present meta-analysis focused on studies of bacteria and archaea due to the limited number of high-throughput fungal studies from the indoor environment. We combined 16S ribosomal RNA (rRNA) gene datasets from 16 surveys of indoor environments conducted worldwide, additionally including 7 other studies representing putative environmental sources of microbial taxa (outdoor air, soil, and the human body). RESULTS: Combined analysis of subsets of studies that shared specific experimental protocols or indoor habitats revealed community patterns indicative of consistent source environments and environmental filtering. Additionally, we were able to identify several consistent sources for indoor microorganisms, particularly outdoor air and skin, mirroring what has been shown in individual studies. Technical variation across studies had a strong effect on comparisons of microbial community assemblages, with differences in experimental protocols limiting our ability to extensively explore the importance of, for example, sampling locality, building function and use, or environmental substrate in structuring indoor microbial communities. CONCLUSIONS: We present a snapshot of an important scientific field in its early stages, where studies have tended to focus on heavy sampling in a few geographic areas. From the practical perspective, this endeavor reinforces the importance of negative "kit" controls in microbiome studies. From the perspective of understanding mechanistic processes in the built environment, this meta-analysis confirms that broad factors, such as geography and building type, structure indoor microbes. However, this exercise suggests that individual studies with common sampling techniques may be more appropriate to explore the relative importance of subtle indoor environmental factors on the indoor microbiome.