The reestablishment of microbial communities after surface cleaning in schools.

AIMS: The goal of this study was to quantify the indoor microbiome dynamics of bacterial and fungal communities on school desk surfaces during a cleaning intervention. METHODS AND RESULTS: Quantitative PCR and DNA sequenced-based approaches were employed to describe microbial community dynamics on ten desk surfaces, spread across three schools, located in the Northeast region of the United States. Six samples were taken from each desk, one precleaning, and five postcleaning at 30 min, 1, 3, 7 and 21 days. Cleaning of the desks physically removed c. 50% of bacteria, fungi, and human cells and a full recovery of the surface microbial concentrations occurred within 2-5 days. This recovery period is much shorter than the schools' once per semester cleaning schedule. The dominant source of bacteria and fungi on desks at all time points came from the human microbiome (skin, oral cavity, and gut). More than 50% fungi on desks were members of genera that contain known allergens. CONCLUSIONS: Microbial communities on these school desks are primarily generated and maintained from the deposition of human-associated bacteria and fungi. Current school surface cleaning protocols and cycles may be ineffective at reducing student exposure to fungal allergens and microbes of human origin. SIGNIFICANCE AND IMPACT OF STUDY: Multiple students often share desks in schools. Results on the removal and reestablishment of microbial communities on these surfaces are critical for setting cleaning schedules and practices that effectively interrupt exposure to surface-associated pathogens and allergens.

Building and environmental factors that influence bacterial and fungal loading on air conditioning cooling coils.

We investigated bacterial and fungal concentrations on cooling coils of commercial AC units and quantified associations between microbial loads and AC unit or building operational parameters. A field campaign was conducted to sample 25 AC units in the humid, subtropical climate of Southern CT, USA and 15 AC units in the hot-summer Mediterranean climate of Sacramento, CA, USA. Median concentrations (with interquartile range) of bacteria and fungi on the cooling coils were 1.2 × 107 (5.1 × 106 -3.9 × 107 ) cells/m2 and 7.6 × 105 (5.6 × 104 -4.4 × 106 ) spore equivalents (SE)/m2 , respectively. Concentrations varied among units with median unit concentrations ranging three orders of magnitude for bacteria and seven orders of magnitude for fungi. Controlled comparisons and multivariable regressions indicate that dominant factors associated with AC coil loading include the nominal efficiency of upstream filters (P = .008 for bacteria and P < .001 for fungi) and coil moisture, which was reflected in fungal loading differences between top and bottom halves of the AC coils in Southern CT (P = .05) and the dew points of the two climates considered (P = .04). Environmental and building characteristics explained 42% (P < .001) of bacterial concentration variability and 66% (P < .001) of fungal concentration variability among samples.

Gene expression of indoor fungal communities under damp building conditions: Implications for human health.

Dampness and visible mold growth in homes are associated with negative human health outcomes, but causal relationships between fungal exposure and health are not well established. The purpose of this study was to determine whether dampness in buildings impacts fungal community gene expression and how, in turn, gene expression may modulate human health impacts. A metatranscriptomic study was performed on house dust fungal communities to investigate the expression of genes and metabolic processes in chamber experiments at water activity levels of 0.5, 0.85, and 1.0. Fungi at water activities as low as 0.5 were metabolically active, focusing their transcriptional resources on primary processes essential for cell maintenance. Metabolic complexity increased with water activity where communities at 1.0 displayed more diverse secondary metabolic processes. Greater gene expression at increasing water activity has important implications for human health: Fungal communities at 1.0 aw upregulated a greater number of allergen-, mycotoxin-, and pathogenicity-encoding genes versus communities at 0.85 and 0.5 aw . In damp buildings, fungi may display increases in secondary metabolic processes with the potential for greater per-cell production of allergens, toxins, and pathogenicity. Assessments in wet versus dry buildings that do not account for this elevated health impact may not accurately reflect exposure.

Fungal and bacterial growth in floor dust at elevated relative humidity levels.

Under sustained, elevated building moisture conditions, bacterial and fungal growth occurs. The goal of this study was to characterize microbial growth in floor dust at variable equilibrium relative humidity (ERH) levels. Floor dust from one home was embedded in coupons cut from a worn medium-pile nylon carpet and incubated at 50%, 80%, 85%, 90%, 95%, and 100% ERH levels. Quantitative PCR and DNA sequencing of ribosomal DNA for bacteria and fungi were used to quantify growth and community shifts. Over a 1-wk period, fungal growth occurred above 80% ERH. Growth rates at 85% and 100% ERH were 1.1 × 104 and 1.5 × 105 spore equivalents d-1 mg dust-1 , respectively. Bacterial growth occurred only at 100% ERH after 1 wk (9.0 × 104 genomes d-1 mg dust-1 ). Growth resulted in significant changes in fungal (P<.00001) and bacterial community structure (P<.00001) at varying ERH levels. Comparisons between fungal taxa incubated at different ERH levels revealed more than 100 fungal and bacterial species that were attributable to elevated ERH. Resuspension modeling indicated that more than 50% of airborne microbes could originate from the resuspension of fungi grown at ERH levels of 85% and above.

Influence of housing characteristics on bacterial and fungal communities in homes of asthmatic children.

Variations in home characteristics, such as moisture and occupancy, affect indoor microbial ecology as well as human exposure to microorganisms. Our objective was to determine how indoor bacterial and fungal community structure and diversity are associated with the broader home environment and its occupants. Next-generation DNA sequencing was used to describe fungal and bacterial communities in house dust sampled from 198 homes of asthmatic children in southern New England. Housing characteristics included number of people/children, level of urbanization, single/multifamily home, reported mold, reported water leaks, air conditioning (AC) use, and presence of pets. Both fungal and bacterial community structures were non-random and demonstrated species segregation (C-score, P < 0.00001). Increased microbial richness was associated with the presence of pets, water leaks, longer AC use, suburban (vs. urban) homes, and dust composition measures (P < 0.05). The most significant differences in community composition were observed for AC use and occupancy (people, children, and pets) characteristics. Occupant density measures were associated with beneficial bacterial taxa, including Lactobacillus johnsonii as measured by qPCR. A more complete knowledge of indoor microbial communities is useful for linking housing characteristics to human health outcomes. Microbial assemblies in house dust result, in part, from the building's physical and occupant characteristics.

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.

Next-generation DNA sequencing reveals that low fungal diversity in house dust is associated with childhood asthma development.

UNLABELLED: Dampness and visible mold in homes are associated with asthma development, but causal mechanisms remain unclear. The goal of this research was to explore associations among measured dampness, fungal exposure, and childhood asthma development without the bias of culture-based microbial analysis. In the low-income, Latino CHAMACOS birth cohort, house dust was collected at age 12 months, and asthma status was determined at age 7 years.The current analysis included 13 asthma cases and 28 controls. Next-generation DNA sequencing methods quantified fungal taxa and diversity. Lower fungal diversity (number of fungal operational taxonomic units) was significantly associated with increased risk of asthma development: unadjusted odds ratio(OR) 4.80 (95% confidence interval (CI) 1.04­22.1). Control for potential confounders strengthened this relationship. Decreased diversity within the genus Cryptococcus was significantly associated with increased asthma risk (OR 21.0, 95% CI 2.16­204). No fungal taxon (species, genus, class) was significantly positively associated with asthma development, and one was significantly negatively associated. Elevated moisture was associated with increased fungal diversity, and moisture/mold indicators were associated with four fungal taxa. Next-generation DNA sequencing provided comprehensive estimates of fungal identity and diversity, demonstrating significant associations between low fungal diversity and childhood asthma development in this community. PRACTICAL IMPLICATIONS: Early life exposure to low fungal diversity in house dust was associated with increased risk for later asthma developmen tin this low-income, immigrant community. No individual fungal taxon (species, genus, or class) was associated with asthma development, although exposure to low diversity within the genus Cryptococcus was associated with asthma development. Future asthma development studies should incorporate fungal diversity measurements, in addition to measuring individual fungal taxa. These results represent a step toward identifying the aspect(s) of indoor microbial populations that are associated with asthma development and suggest that understanding the factors that control diversity in the indoor environment may lead to public health recommendations for asthma prevention in the future.

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

UNLABELLED: 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 × 10(6) genome copies, and 7.3 × 10(6) 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.

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

UNLABELLED: 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.

Viral metagenome analysis to guide human pathogen monitoring in environmental samples.

AIMS: The aim of this study was to develop and demonstrate an approach for describing the diversity of human pathogenic viruses in an environmentally isolated viral metagenome. METHODS AND RESULTS: In silico bioinformatic experiments were used to select an optimum annotation strategy for discovering human viruses in virome data sets and applied to annotate a class B biosolid virome. Results from the in silico study indicated that <1% errors in virus identification could be achieved when nucleotide-based search programs (BLASTn or tBLASTx), viral genome only databases and sequence reads >200 nt were considered. Within the 51,925 annotated sequences, 94 DNA and 19 RNA sequences were identified as human viruses. Virus diversity included environmentally transmitted agents such as parechovirus, coronavirus, adenovirus and aichi virus, as well as viruses associated with chronic human infections such as human herpes and hepatitis C viruses. CONCLUSIONS: This study provided a bioinformatic approach for identifying pathogens in a virome data set and demonstrated the human virus diversity in a relevant environmental sample. SIGNIFICANCE AND IMPACT OF THE STUDY: As the costs of next-generation sequencing decrease, the pathogen diversity described by virus metagenomes will provide an unbiased guide for subsequent cell culture and quantitative pathogen analyses and ensures that highly enriched and relevant pathogens are not neglected in exposure and risk assessments.

Photoreactivation in airborne Mycobacterium parafortuitum.

Photoreactivation was observed in airborne Mycobacterium parafortuitum exposed concurrently to UV radiation (254 nm) and visible light. Photoreactivation rates of airborne cells increased with increasing relative humidity (RH) and decreased with increasing UV dose. Under a constant UV dose with visible light absent, the UV inactivation rate of airborne M. parafortuitum cells decreased by a factor of 4 as RH increased from 40 to 95%; however, under identical conditions with visible light present, the UV inactivation rate of airborne cells decreased only by a factor of 2. When irradiated in the absence of visible light, cellular cyclobutane thymine dimer content of UV-irradiated airborne M. parafortuitum and Serratia marcescens increased in response to RH increases. Results suggest that, unlike in waterborne bacteria, cyclobutane thymine dimers are not the most significant form of UV-induced DNA damage incurred by airborne bacteria and that the distribution of DNA photoproducts incorporated into UV-irradiated airborne cells is a function of RH.

Development and application of small-subunit rRNA probes for assessment of selected Thiobacillus species and members of the genus Acidiphilium.

Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using (32)P radiolabels, probe specificity was characterized by hybridization dissociation temperature (T(d)) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined T(d)s. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris.

Neurotensin-induced hypothermia prevents hippocampal neuronal damage and increased locomotor activity in ischemic gerbils.

Hypothermia induced by surface cooling has shown to protect vulnerable regions of the brain during an ischemic insult. This study evaluated the neuroprotective efficacy of neurotensin, a potent hypothermic agent, using a 5-min carotid occlusion procedure in the gerbil. In Experiment 1, the dose-response and time course of neurotensin-induced hypothermia were evaluated (n = 5/dose). Central infusion of 10, 20, and 30 micrograms neurotensin were found to significantly decrease core body temperature of conscious gerbils within 30 min of administration. In Experiment 2, gerbils pretreated with 30 micrograms neurotensin were permitted to become hypothermic or were maintained at 37 degrees-38 degrees C (rectal) during ischemic insult. Other gerbils were pretreated with peptide vehicle prior to ischemic insult (at 37 degrees -38 degrees C) or underwent a sham procedure (n = 6/condition). At 24 h after surgery, gerbils were tested for increased locomotor activity in an open-field apparatus. Gerbils pretreated with peptide vehicle or neurotensin and maintained at 37 degrees-38 degrees C during ischemia had significantly higher activity levels compared to the other treated groups. In contrast, gerbils made hypothermic with neurotensin exhibited activity levels similar to sham gerbils. Histological assessment revealed that neurotensin-induced hypothermia protected the CA1 region from ischemic damage.