Gut microbiome profiling in systemic sclerosis: a metagenomic approach.

OBJECTIVES: The early gastrointestinal (GI) manifestation of systemic sclerosis (SSc) suggests a possible GI microbiota engagement in the pathophysiology and/or progression of SSc. Previous studies have revealed dysbiosis among Caucasian SSc patients. This study extends these findings to Asian SSc patients. METHODS: Adult SSc patients, stratified according to 1) on immunosuppressive (On-IS) drugs or 2) no immunosuppressive drugs (No-IS), and age-and-sex-matched healthy controls (HC) were recruited. Metagenomic sequencing of stool DNA was compared between SSc patients and HC, and between SSc (On-IS) and (No-IS) patients. Alpha and beta-diversity, taxonomic and functional profiling were evaluated. RESULTS: Twenty-three female SSc patients (12 On-IS; 11 No-IS; 5 diffuse and 18 limited SSc subtype) and 19 female HC, with median age of 54 years and 56 years, respectively, were recruited. Median SSc disease duration was 3.3 years. Alpha diversity was significantly higher in SSc versus HC (p=0.014) and in SSc (No-IS) versus HC (p=0.006). There was no significant difference in beta diversity between SSc and HC (p=0.307). At the phyla level, there were significantly increased abundance of Firmicutes and Actinobacteria in SSc versus HC, and reduced abundance of Bacteroidetes (all p<0.001). At the species level, there were significantly increased abundance of several Lactobacillus, Bifidobacterium, and Coprococcus species in SSc, and increased abundance of Odoribacter, Bacteroides and Prevotella species in HC. KEGG pathway analysis demonstrated distinct differences between SSc versus HC, and between SSc (No-IS) and SSc (On-IS). CONCLUSIONS: Using metagenomic sequencing, our study further underlines distinct alterations in microbiota profiling among Asian SSc patients.

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.

A synbiotic intervention modulates meta-omics signatures of gut redox potential and acidity in elective caesarean born infants.

BACKGROUND: The compromised gut microbiome that results from C-section birth has been hypothesized as a risk factor for the development of non-communicable diseases (NCD). In a double-blind randomized controlled study, 153 infants born by elective C-section received an infant formula supplemented with either synbiotic, prebiotics, or unsupplemented from birth until 4 months old. Vaginally born infants were included as a reference group. Stool samples were collected from day 3 till week 22. Multi-omics were deployed to investigate the impact of mode of delivery and nutrition on the development of the infant gut microbiome, and uncover putative biological mechanisms underlying the role of a compromised microbiome as a risk factor for NCD. RESULTS: As early as day 3, infants born vaginally presented a hypoxic and acidic gut environment characterized by an enrichment of strict anaerobes (Bifidobacteriaceae). Infants born by C-section presented the hallmark of a compromised microbiome driven by an enrichment of Enterobacteriaceae. This was associated with meta-omics signatures characteristic of a microbiome adapted to a more oxygen-rich gut environment, enriched with genes associated with reactive oxygen species metabolism and lipopolysaccharide biosynthesis, and depleted in genes involved in the metabolism of milk carbohydrates. The synbiotic formula modulated expression of microbial genes involved in (oligo)saccharide metabolism, which emulates the eco-physiological gut environment observed in vaginally born infants. The resulting hypoxic and acidic milieu prevented the establishment of a compromised microbiome. CONCLUSIONS: This study deciphers the putative functional hallmarks of a compromised microbiome acquired during C-section birth, and the impact of nutrition that may counteract disturbed microbiome development. TRIAL REGISTRATION: The study was registered in the Dutch Trial Register (Number: 2838 ) on 4th April 2011.

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.

The genomic characterisation and comparison of Bacillus cereus strains isolated from indoor air.

BACKGROUND: Bacillus cereus is ubiquitous in nature, found in environments such as soil, plants, air, and part of the insect and human gut microbiome. The ability to produce endospores and biofilms contribute to their pathogenicity, classified in two types of food poisoning: diarrheal and emetic syndromes. Here we report gap-free, whole-genome sequences of two B. cereus strains isolated from air samples and analyse their emetic and diarrheal potential. RESULTS: Genome assemblies of the B. cereus strains consist of one chromosome and seven plasmids each. The genome size of strain SGAir0260 is 6.30-Mb with 6590 predicted coding sequences (CDS) and strain SGAir0263 is 6.47-Mb with 6811 predicted CDS. Macrosynteny analysis showed 99% collinearity between the strains isolated from air and 90.2% with the reference genome. Comparative genomics with 57 complete B. cereus genomes suggests these strains from air are closely associated with strains isolated from foodborne illnesses outbreaks. Due to virulence potential of B. cereus and its reported involvement in nosocomial infections, antibiotic resistance analyses were performed and confirmed resistance to ampicillin and fosfomycin, with susceptibility to ciprofloxacin, tetracycline and vancomycin in both strains. CONCLUSION: Phylogenetic analysis combined with detection of haemolytic (hblA, hblC, and hblD) and non-haemolytic (nheA, nheB, and nheC) enterotoxin genes in both air-isolated strains point to the diarrheic potential of the air isolates, though not emetic. Characterization of these airborne strains and investigation of their potential disease-causing genes could facilitate identification of environmental sources of contamination leading to foodborne illnesses and nosocomial infections transported by air.

Taxonomic composition and seasonal dynamics of the air microbiome in West Siberia.

Here, we describe taxonomical composition, as well as seasonal and diel dynamics of airborne microbial communities in West Siberia. A total of 78 airborne biomass samples from 39 time intervals were analysed, within a temperature range of 48 °C (26 °C to - 22 °C). We observed a 5-170-fold decrease in DNA yield extracted from the airborne biomass in winter compared to summer, nevertheless, yielding sufficient material for metagenomic analysis. The airborne microbial communities included Actinobacteria and Proteobacteria, Ascomycota and Basidiomycota fungi as major components, as well as some Streptophyta plants. In summer, bacterial and fungal plant pathogens, and wood-rotting saprophytes were predominant. In winter, Ascomycota moulds and cold-related or stress environment bacterial species were enriched, while the fraction of wood-rotting and mushroom-forming Basidiomycota fungi was largely reduced. As recently reported for the tropical climate, the airborne microbial communities performed a diel cycle in summer, however, in winter diel dynamics were not observed.

A compromised developmental trajectory of the infant gut microbiome and metabolome in atopic eczema.

Evidence is accumulating that the establishment of the gut microbiome in early life influences the development of atopic eczema. In this longitudinal study, we used integrated multi-omics analyses to infer functional mechanisms by which the microbiome modulates atopic eczema risk. We measured the functionality of the gut microbiome and metabolome of 63 infants between ages 3 weeks and 12 months with well-defined eczema cases and controls in a sub-cohort from the Growing Up in Singapore Toward healthy Outcomes (GUSTO) mother-offspring cohort. At 3 weeks, the microbiome and metabolome of allergen-sensitized atopic eczema infants were characterized by an enrichment of Escherichia coli and Klebsiella pneumoniae, associated with increased stool D-glucose concentration and increased gene expression of associated virulence factors. A delayed colonization by beneficial Bacteroides fragilis and subsequent delayed accumulation of butyrate and propionate producers after 3 months was also observed. Here, we describe an aberrant developmental trajectory of the gut microbiome and stool metabolome in allergen sensitized atopic eczema infants. The infographic describes an impaired developmental trajectory of the gut microbiome and metabolome in allergen-sensitized atopic eczema (AE) infants and infer its contribution in modulating allergy risk in the Singaporean mother-offspring GUSTO cohort. The key microbial signature of AE is characterized by (1) an enrichment of Escherichia coli and Klebsiella pneumoniae which are associated with accumulation of pre-glycolysis intermediates (D-glucose) via the trehalose metabolic pathway, increased gene expression of associated virulence factors (invasin, adhesin, flagellin and lipopolysaccharides) by utilizing ATP from oxidative phosphorylation and delayed production of butyrate and propionate, (2) depletion of Bacteroides fragilis which resulted in lower expression of immunostimulatory bacterial cell envelope structure and folate (vitamin B9) biosynthesis pathway, and (3) accompanied depletion of bacterial groups with the ability to derive butyrate and propionate through direct or indirect pathways which collectively resulted in reduced glycolysis, butyrate and propionate biosynthesis.

Longitudinal assessment of antibiotic resistance gene profiles in gut microbiomes of infants at risk of eczema.

BACKGROUND: While there is increasing knowledge about the gut microbiome, the factors influencing and the significance of the gut resistome are still not well understood. Infant gut commensals risk transferring multidrug-resistant antibiotic resistance genes (ARGs) to pathogenic bacteria. The rapid spread of multidrug-resistant pathogenic bacteria is a worldwide public health concern. Better understanding of the naïve infant gut resistome may build the evidence base for antimicrobial stewardship in both humans and in the food industry. Given the high carriage rate of extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Asia, we aimed to evaluate community prevalence, dynamics, and longitudinal changes in antibiotic resistance gene (ARG) profiles and prevalence of ESBL-producing E. coli and K. pneumoniae in the intestinal microbiome of infants participating in the Growing Up in Singapore Towards Healthy Outcomes (GUSTO) study, a longitudinal cohort study of pregnant women and their infants. METHODS: We analysed ARGs in the first year of life among 75 infants at risk of eczema who had stool samples collected at multiple timepoints using metagenomics. RESULTS: The mean number of ARGs per infant increased with age. The most common ARGs identified confer resistance to aminoglycoside, beta-lactam, macrolide and tetracycline antibiotics; all infants harboured these antibiotic resistance genes at some point in the first year of life. Few ARGs persisted throughout the first year of life. Beta-lactam resistant Escherichia coli and Klebsiella pneumoniae were detected in 4 (5.3%) and 32 (42.7%) of subjects respectively. CONCLUSION: In this longitudinal cohort study of infants living in a region with high endemic antibacterial resistance, we demonstrate that majority of the infants harboured several antibiotic resistance genes in their gut and showed that the infant gut resistome is diverse and dynamic over the first year of life.

Complete Genome Sequence of Penicillium oxalicum Strain SGAir0226 Isolated from Outdoor Tropical Air in Singapore.

Penicillium oxalicum strain SGAir0226 was isolated from a tropical air sample collected in Singapore. The complete genome was assembled from long reads obtained from single-molecule real-time sequencing and was further polished and error corrected using short read sequencing data. The assembly comprises 20 contigs with a total length of 30.7 Mb. The genome was predicted to contain 8310 protein-coding genes, 237 tRNAs and 83 rRNAs.

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.

Complete genome of Enterobacter sichuanensis strain SGAir0282 isolated from air in Singapore.

BACKGROUND: Enterobacter cloacae complex (ECC) bacteria, such as E. cloacae, E. sichuanensis, E. kobei, and E. roggenkampii, have been emerging as nosocomial pathogens. Many strains isolated from medical clinics were found to be resistant to antibiotics, and in the worst cases, acquired multidrug resistance. We present the whole genome sequence of SGAir0282, isolated from the outdoor air in Singapore, and its relevance to other ECC bacteria by in silico genomic analysis. RESULTS: Complete genome assembly of E. sichuanensis strain SGAir0282 was generated using PacBio RSII and Illumina MiSeq platforms, and the datasets were used for de novo assembly using Hierarchical Genome Assembly Process (HGAP) and error corrected with Pilon. The genome assembly consisted of a single contig of 4.71 Mb and with a G+C content of 55.5%. No plasmid was detected in the assembly. The genome contained 4371 coding genes, 83 tRNA and 25 rRNA genes, as predicted by NCBI's Prokaryotic Genome Annotation Pipeline (PGAP). Among the genes, the antibiotic resistance related genes were included: Streptothricin acetdyltransferase (SatA), fosfomycin resistance protein (FosA) and metal-dependent hydrolases of the beta-lactamase superfamily I (BLI). CONCLUSION: Based on whole genome alignment and phylogenetic analysis, the strain SGAir0282 was identified to be Enterobacter sichuanensis. The strain possesses gene clusters for virulence, disease and defence, that can also be found in other multidrug resistant ECC type strains.

Whole-Genome Sequencing of Aspergillus terreus Species Complex.

Aspergillus terreus species complex is an opportunistic fungal pathogen increasingly implicated in invasive infection, as well as chronic respiratory disease. Currently, an understanding of A. terreus pathogenicity is impeded by a limited number of whole-genome sequences of this fungal pathogen. We here describe a high-quality whole-genome assembly of European A. terreus clinical isolate M6925, derived by single-molecule real-time sequencing with short-read polishing.

Whole-Genome Sequence of Bacillus megaterium Strain SGAir0080, Isolated from an Indoor Air Sample.

Bacillus megaterium strain SGAir0080 was isolated from a tropical air sample in Singapore. Its genome was assembled using single-molecule real-time (SMRT) sequencing and MiSeq reads. It has one chromosome of 5.06 Mbp and seven plasmids (average length, 62.8 kbp). It possesses 5,339 protein-coding genes, 130 tRNAs, and 35 rRNAs.

Neurogenesis and prolongevity signaling in young germ-free mice transplanted with the gut microbiota of old mice.

The gut microbiota evolves as the host ages, yet the effects of these microbial changes on host physiology and energy homeostasis are poorly understood. To investigate these potential effects, we transplanted the gut microbiota of old or young mice into young germ-free recipient mice. Both groups showed similar weight gain and skeletal muscle mass, but germ-free mice receiving a gut microbiota transplant from old donor mice unexpectedly showed increased neurogenesis in the hippocampus of the brain and increased intestinal growth. Metagenomic analysis revealed age-sensitive enrichment in butyrate-producing microbes in young germ-free mice transplanted with the gut microbiota of old donor mice. The higher concentration of gut microbiota-derived butyrate in these young transplanted mice was associated with an increase in the pleiotropic and prolongevity hormone fibroblast growth factor 21 (FGF21). An increase in FGF21 correlated with increased AMPK and SIRT-1 activation and reduced mTOR signaling. Young germ-free mice treated with exogenous sodium butyrate recapitulated the prolongevity phenotype observed in young germ-free mice receiving a gut microbiota transplant from old donor mice. These results suggest that gut microbiota transplants from aged hosts conferred beneficial effects in responsive young recipients.

Complete Genome Sequence of Curtobacterium sp. Strain SGAir0471, Isolated from Singapore Air Samples.

Curtobacterium sp. strain SGAir0471 was isolated from tropical air samples collected in Singapore. The genome was assembled using PacBio RS II long reads and Illumina MiSeq short paired-end reads. The complete genome measures 3.53 Mb and consists of 3,151 protein-coding genes, 49 tRNAs, and 12 rRNAs.

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.

Complete Genome Sequence of Rhodococcus sp. Strain SGAir0479, Isolated from Indoor Air Collected in Singapore.

The complete genome sequence of Rhodococcus sp. strain SGAir0479 is presented here. This organism was isolated from an air sample collected in an indoor location in Singapore. The consensus assembly generated one chromosome of 4.86 Mb (G+C content of 69.8%) and one plasmid of 104,493 bp.

Complete Genome Sequence of Micrococcus luteus Strain SGAir0127, Isolated from Indoor Air Samples from Singapore.

Micrococcus luteus strain SGAir0127 was isolated from indoor air samples collected in Singapore. The assembly, based on single-molecule real-time sequencing reads, resulted in two contigs, one chromosomal contig with a length of 2.57 Mbp and one nonchromosomal contig of 8.68 kbp. The genome has a total of 2,564 genes.

Complete Genome Sequence of Lysinibacillus sp. Strain SGAir0095, Isolated from Tropical Air Samples Collected in Singapore.

Lysinibacillus sp. strain SGAir0095 was isolated from tropical air samples collected in Singapore, and its complete genome was sequenced with a hybrid strategy using single-molecule real-time sequencing and short reads. The genome consists of one chromosome of 4.14 Mbp and encompasses 3,885 protein-coding genes, 39 rRNAs, and 101 tRNAs.