Vertical stratification of the air microbiome in the lower troposphere.

The troposphere constitutes the final frontier of global ecosystem research due to technical challenges arising from its size, low biomass, and gaseous state. Using a vertical testing array comprising a meteorological tower and a research aircraft, we conducted synchronized measurements of meteorological parameters and airborne biomass (n = 480) in the vertical air column up to 3,500 m. The taxonomic analysis of metagenomic data revealed differing patterns of airborne microbial community composition with respect to time of day and height above ground. The temporal and spatial resolution of our study demonstrated that the diel cycle of airborne microorganisms is a ground-based phenomenon that is entirely absent at heights >1,000 m. In an integrated analysis combining meteorological and biological data, we demonstrate that atmospheric turbulence, identified by potential temperature and high-frequency three-component wind measurements, is the key driver of bioaerosol dynamics in the lower troposphere. Multivariate regression analysis shows that at least 50% of identified airborne microbial taxa (n = ∼10,000) are associated with either ground or height, allowing for an understanding of dispersal patterns of microbial taxa in the vertical air column. Due to the interconnectedness of atmospheric turbulence and temperature, the dynamics of microbial dispersal are likely to be impacted by rising global temperatures, thereby also affecting ecosystems on the planetary surface.

Microbial communities in the tropical air ecosystem follow a precise diel cycle.

The atmosphere is vastly underexplored as a habitable ecosystem for microbial organisms. In this study, we investigated 795 time-resolved metagenomes from tropical air, generating 2.27 terabases of data. Despite only 9 to 17% of the generated sequence data currently being assignable to taxa, the air harbored a microbial diversity that rivals the complexity of other planetary ecosystems. The airborne microbial organisms followed a clear diel cycle, possibly driven by environmental factors. Interday taxonomic diversity exceeded day-to-day and month-to-month variation. Environmental time series revealed the existence of a large core of microbial taxa that remained invariable over 13 mo, thereby underlining the long-term robustness of the airborne community structure. Unlike terrestrial or aquatic environments, where prokaryotes are prevalent, the tropical airborne biomass was dominated by DNA from eukaryotic phyla. Specific fungal and bacterial species were strongly correlated with temperature, humidity, and CO2 concentration, making them suitable biomarkers for studying the bioaerosol dynamics of the atmosphere.

Metagenomics Reveals a Core Macrolide Resistome Related to Microbiota in Chronic Respiratory Disease.

Rationale: Long-term antibiotic use for managing chronic respiratory disease is increasing; however, the role of the airway resistome and its relationship to host microbiomes remains unknown.Objectives: To evaluate airway resistomes and relate them to host and environmental microbiomes using ultradeep metagenomic shotgun sequencing.Methods: Airway specimens from 85 individuals with and without chronic respiratory disease (severe asthma, chronic obstructive pulmonary disease, and bronchiectasis) were subjected to metagenomic sequencing to an average depth exceeding 20 million reads. Respiratory and device-associated microbiomes were evaluated on the basis of taxonomical classification and functional annotation including the Comprehensive Antibiotic Resistance Database to determine airway resistomes. Co-occurrence networks of gene-microbe association were constructed to determine potential microbial sources of the airway resistome. Paired patient-inhaler metagenomes were compared (n = 31) to assess for the presence of airway-environment overlap in microbiomes and/or resistomes.Measurements and Main Results: Airway metagenomes exhibit taxonomic and metabolic diversity and distinct antimicrobial resistance patterns. A "core" airway resistome dominated by macrolide but with high prevalence of ß-lactam, fluoroquinolone, and tetracycline resistance genes exists and is independent of disease status or antibiotic exposure. Streptococcus and Actinomyces are key potential microbial reservoirs of macrolide resistance including the ermX, ermF, and msrD genes. Significant patient-inhaler overlap in airway microbiomes and their resistomes is identified where the latter may be a proxy for airway microbiome assessment in chronic respiratory disease.Conclusions: Metagenomic analysis of the airway reveals a core macrolide resistome harbored by the host microbiome.

Environmental fungal sensitisation associates with poorer clinical outcomes in COPD.

INTRODUCTION: Allergic sensitisation to fungi such as Aspergillus are associated to poor clinical outcomes in asthma, bronchiectasis and cystic fibrosis; however, clinical relevance in COPD remains unclear. METHODS: Patients with stable COPD (n=446) and nondiseased controls (n=51) were prospectively recruited across three countries (Singapore, Malaysia and Hong Kong) and screened against a comprehensive allergen panel including house dust mites, pollens, cockroach and fungi. For the first time, using a metagenomics approach, we assessed outdoor and indoor environmental allergen exposure in COPD. We identified key fungi in outdoor air and developed specific-IgE assays against the top culturable fungi, linking sensitisation responses to COPD outcomes. Indoor air and surface allergens were prospectively evaluated by metagenomics in the homes of 11 COPD patients and linked to clinical outcome. RESULTS: High frequencies of sensitisation to a broad range of allergens occur in COPD. Fungal sensitisation associates with frequent exacerbations, and unsupervised clustering reveals a "highly sensitised fungal predominant" subgroup demonstrating significant symptomatology, frequent exacerbations and poor lung function. Outdoor and indoor environments serve as important reservoirs of fungal allergen exposure in COPD and promote a sensitisation response to outdoor air fungi. Indoor (home) environments with high fungal allergens associate with greater COPD symptoms and poorer lung function, illustrating the importance of environmental exposures on clinical outcomes in COPD. CONCLUSION: Fungal sensitisation is prevalent in COPD and associates with frequent exacerbations representing a potential treatable trait. Outdoor and indoor (home) environments represent a key source of fungal allergen exposure, amenable to intervention, in "sensitised" COPD.

The Mycobiome in Health and Disease: Emerging Concepts, Methodologies and Challenges.

Fungal disease is an increasingly recognised global clinical challenge associated with high mortality. Early diagnosis of fungal infection remains problematic due to the poor sensitivity and specificity of current diagnostic modalities. Advances in sequencing technologies hold promise in addressing these shortcomings and for improved fungal detection and identification. To translate such emerging approaches into mainstream clinical care will require refinement of current sequencing and analytical platforms, ensuring standardisation and consistency through robust clinical benchmarking and its validation across a range of patient populations. In this state-of-the-art review, we discuss current diagnostic and therapeutic challenges associated with fungal disease and provide key examples where the application of sequencing technologies has potential diagnostic application in assessing the human 'mycobiome'. We assess how ready access to fungal sequencing may be exploited in broadening our insight into host-fungal interaction, providing scope for clinical diagnostics and the translation of emerging mycobiome research into clinical practice.

Draft genome sequence of Penicillium citrinum B9, a plant growth-promoting symbiont from barley rhizosphere.

Penicillium citrinum strain B9 is a plant growth-promoting fungus isolated from Barley (Hordeum vulgare) rhizosphere. We report the first draft genome of P. citrinum B9 assembled using single-molecule real-time sequencing and Illumina reads. The assembled genome spans 31.3 Mb comprising nine contigs and 10,106 protein-encoding genes.

Draft genome sequence of Xylaria bambusicola isolate GMP-LS, the root and basal stem rot pathogen of sugarcane in Indonesia.

The first draft genome of X. bambusicola GMP-LS, the causal pathogen of the Root, and Basal Stem Rot disease in Sugarcane is presented based on single-molecule real-time PacBio sequencing. Xylaria genome (72.43 Mb) is predicted to encode 13,430 proteins and will contribute to molecular understanding of fungal pathogenesis.

Structure vs. chemistry: Alternate mechanisms for controlling leaf microbiomes.

The analysis of phyllosphere microbiomes traditionally relied on DNA extracted from whole leaves. To investigate the microbial communities on the adaxial (upper) and abaxial (lower) leaf surfaces, swabs were collected from both surfaces of two garden plants, Rhapis excelsa and Cordyline fruticosa. Samples were collected at noon and midnight and at five different locations to investigate if the phyllosphere microbial communities change with time and location. The abaxial surface of Rhapis excelsa and Cordyline fruticosa had fewer bacteria in contrast to its adaxial counterpart. This observation was consistent between noon and midnight and across five different locations. Our co-occurrence network analysis further showed that bacteria were found almost exclusively on the adaxial surface while only a small group of leaf blotch fungi thrived on the abaxial surface. There are higher densities of stomata on the abaxial surface and these openings are vulnerable ports of entry into the plant host. While one might argue about the settling of dust particles and microorganisms on the adaxial surface, we detected differences in reactive chemical activities and microstructures between the adaxial and abaxial surfaces. Our results further suggest that both plant species deploy different defence strategies to deter invading pathogens on the abaxial surface. We hypothesize that chemical and mechanical defence strategies evolved independently for harnessing and controlling phyllosphere microbiomes. Our findings have also advanced our understanding that the abaxial leaf surface is distinct from the adaxial surface and that the reduced microbial diversity is likely a consequence of plant-microbe interactions.

Short-range contributions of local sources to ambient air.

Recent developments in aerobiology have enabled the investigation of airborne biomass with high temporal and taxonomic resolution. In this study, we assess the contributions of local sources to ambient air within a 160,000 m2 tropical avian park (AP). We sequenced and analyzed 120 air samples from seven locations situated 160 to 400 m apart, representing distinct microhabitats. Each microhabitat contained a characteristic air microbiome, defined by the abundance and richness of its airborne microbial community members, supported by both, PCoA and Random Forest analysis. Each outdoor microhabitat contained 1% to 18.6% location-specific taxa, while a core microbiome of 27.1% of the total taxa was shared. To identify and assess local sources, we compared the AP dataset with a DVE reference dataset from a location 2 km away, collected during a year-round sampling campaign. Intersection of data from the two sites demonstrated 61.6% of airborne species originated from local sources of the AP, 34.5% from ambient air background, and only 3.9% of species were specific to the DVE reference site. In-depth taxonomic analysis demonstrated association of bacteria-dominated air microbiomes with indoor spaces, while fungi-dominated airborne microbial biomass was predominant in outdoor settings with ample vegetation. The approach presented here demonstrates an ability to identify local source contributions against an ambient air background, despite the prevailing mixing of air masses caused by atmospheric turbulences.

Experimental parameters defining ultra-low biomass bioaerosol analysis.

Investigation of the microbial ecology of terrestrial, aquatic and atmospheric ecosystems requires specific sampling and analytical technologies, owing to vastly different biomass densities typically encountered. In particular, the ultra-low biomass nature of air presents an inherent analytical challenge that is confounded by temporal fluctuations in community structure. Our ultra-low biomass pipeline advances the field of bioaerosol research by significantly reducing sampling times from days/weeks/months to minutes/hours, while maintaining the ability to perform species-level identification through direct metagenomic sequencing. The study further addresses all experimental factors contributing to analysis outcome, such as amassment, storage and extraction, as well as factors that impact on nucleic acid analysis. Quantity and quality of nucleic acid extracts from each optimisation step are evaluated using fluorometry, qPCR and sequencing. Both metagenomics and marker gene amplification-based (16S and ITS) sequencing are assessed with regard to their taxonomic resolution and inter-comparability. The pipeline is robust across a wide range of climatic settings, ranging from arctic to desert to tropical environments. Ultimately, the pipeline can be adapted to environmental settings, such as dust and surfaces, which also require ultra-low biomass analytics.

Complete Genome Sequence of Agrococcus sp. Strain SGAir0287, Isolated from Tropical Air Collected in Singapore.

Agrococcus sp. strain SGAir0287 was isolated from tropical air samples collected in Singapore. Assembled using single-molecule real-time (SMRT) sequencing and MiSeq reads, the genome consists of one circular chromosome of 3,084,767 bp. The entire genome has 2,870 protein-coding genes, 45 tRNAs, and 3 rRNAs.

Complete Genome Sequence of Bacillus altitudinis Type Strain SGAir0031 Isolated from Tropical Air Collected in Singapore.

Bacillus altitudinis strain SGAir0031 (Firmicutes) was isolated from tropical air samples collected in Singapore. Its genome was assembled using short reads and single-molecule real-time sequencing, comprising one chromosome with 3.81 Mb and one plasmid with 32 kb. The genome consists of 3,820 protein-coding genes, 81 tRNAs, and 24 rRNAs.