Standardized multi-omics of Earth's microbiomes reveals microbial and metabolite diversity.

Despite advances in sequencing, lack of standardization makes comparisons across studies challenging and hampers insights into the structure and function of microbial communities across multiple habitats on a planetary scale. Here we present a multi-omics analysis of a diverse set of 880 microbial community samples collected for the Earth Microbiome Project. We include amplicon (16S, 18S, ITS) and shotgun metagenomic sequence data, and untargeted metabolomics data (liquid chromatography-tandem mass spectrometry and gas chromatography mass spectrometry). We used standardized protocols and analytical methods to characterize microbial communities, focusing on relationships and co-occurrences of microbially related metabolites and microbial taxa across environments, thus allowing us to explore diversity at extraordinary scale. In addition to a reference database for metagenomic and metabolomic data, we provide a framework for incorporating additional studies, enabling the expansion of existing knowledge in the form of an evolving community resource. We demonstrate the utility of this database by testing the hypothesis that every microbe and metabolite is everywhere but the environment selects. Our results show that metabolite diversity exhibits turnover and nestedness related to both microbial communities and the environment, whereas the relative abundances of microbially related metabolites vary and co-occur with specific microbial consortia in a habitat-specific manner. We additionally show the power of certain chemistry, in particular terpenoids, in distinguishing Earth's environments (for example, terrestrial plant surfaces and soils, freshwater and marine animal stool), as well as that of certain microbes including Conexibacter woesei (terrestrial soils), Haloquadratum walsbyi (marine deposits) and Pantoea dispersa (terrestrial plant detritus). This Resource provides insight into the taxa and metabolites within microbial communities from diverse habitats across Earth, informing both microbial and chemical ecology, and provides a foundation and methods for multi-omics microbiome studies of hosts and the environment.

Asthma and Wheeze Severity and the Oropharyngeal Microbiota in Children and Adolescents.

Rationale: There is a major unmet need for improving the care of children and adolescents with severe asthma and wheeze. Identifying factors contributing to disease severity may lead to improved diagnostics, biomarkers, or therapies. The airway microbiota may be such a key factor. Objectives: To compare the oropharyngeal airway microbiota of children and adolescents with severe and mild/moderate asthma/wheeze. Methods: Oropharyngeal swab samples from school-age and preschool children in the European U-BIOPRED (Unbiased BIOmarkers in the PREDiction of respiratory disease outcomes) multicenter study of severe asthma, all receiving severity-appropriate treatment, were examined using 16S ribosomal RNA gene sequencing. Bacterial taxa were defined as amplicon sequence variants. Results: We analyzed 241 samples from four cohorts: A) 86 school-age children with severe asthma; B) 39 school-age children with mild/moderate asthma; C) 65 preschool children with severe wheeze; and D) 51 preschool children with mild/moderate wheeze. The most common bacteria were Streptococcus (mean relative abundance, 33.5%), Veillonella (10.3%), Haemophilus (7.0%), Prevotella (5.9%), and Rothia (5.5%). Age group (school-age vs. preschool) was associated with the microbiota in ß-diversity analysis (F = 3.32, P = 0.011) and in a differential abundance analysis (28 significant amplicon sequence variants). Among all children, we found no significant difference in the microbiota between children with severe and mild/moderate asthma/wheeze in univariable ß-diversity analysis (F = 1.99, P = 0.08, N = 241), but a significant difference in a multivariable model (F = 2.66, P = 0.035), including the number of exacerbations in the previous year. Age was also significant when expressed as a microbial maturity score (Spearman Rho, 0.39; P = 4.6 × 10-10); however, this score was not associated with asthma/wheeze severity. Conclusions: There was a modest difference in the oropharyngeal airway microbiota between children with severe and mild/moderate asthma/wheeze across all children but not in individual age groups, and a strong association between the microbiota and age. This suggests the oropharyngeal airway microbiota as an interesting entity in studying asthma severity, but probably without the strength to serve as a biomarker for targeted intervention.

Chemically informed analyses of metabolomics mass spectrometry data with Qemistree.

Untargeted mass spectrometry is employed to detect small molecules in complex biospecimens, generating data that are difficult to interpret. We developed Qemistree, a data exploration strategy based on the hierarchical organization of molecular fingerprints predicted from fragmentation spectra. Qemistree allows mass spectrometry data to be represented in the context of sample metadata and chemical ontologies. By expressing molecular relationships as a tree, we can apply ecological tools that are designed to analyze and visualize the relatedness of DNA sequences to metabolomics data. Here we demonstrate the use of tree-guided data exploration tools to compare metabolomics samples across different experimental conditions such as chromatographic shifts. Additionally, we leverage a tree representation to visualize chemical diversity in a heterogeneous collection of samples. The Qemistree software pipeline is freely available to the microbiome and metabolomics communities in the form of a QIIME2 plugin, and a global natural products social molecular networking workflow.

Community-intrinsic properties enhance keratin degradation from bacterial consortia.

Although organic matter may accumulate sometimes (e.g. lignocellulose in peat bog), most natural biodegradation processes are completed until full mineralization. Such transformations are often achieved by the concerted action of communities of interacting microbes, involving different species each performing specific tasks. These interactions can give rise to novel "community-intrinsic" properties, through e.g. activation of so-called "silent genetic pathways" or synergistic interplay between microbial activities and functions. Here we studied the microbial community-based degradation of keratin, a recalcitrant biological material, by four soil isolates, which have previously been shown to display synergistic interactions during biofilm formation; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus. We observed enhanced keratin weight loss in cultures with X. retroflexus, both in dual and four-species co-cultures, as compared to expected keratin degradation by X. retroflexus alone. Additional community intrinsic properties included accelerated keratin degradation rates and increased biofilm formation on keratin particles. Comparison of secretome profiles of X. retroflexus mono-cultures to co-cultures revealed that certain proteases (e.g. serine protease S08) were significantly more abundant in mono-cultures, whereas co-cultures had an increased abundance of proteins related to maintaining the redox environment, e.g. glutathione peroxidase. Hence, one of the mechanisms related to the community intrinsic properties, leading to enhanced degradation from co-cultures, might be related to a switch from sulfitolytic to proteolytic functions between mono- and co-cultures, respectively.

Bacterial community analysis for investigating bacterial transfer from tonsils to the pig carcass.

Tonsils in the oral cavity are an important source of contamination during pig slaughter, but have not received as much attention as faecal contamination. In the present study, ten pigs were sampled from tonsils, faeces and three different areas on each carcass. The samples were analysed by both culturing of Escherichia coli and Yersinia enterocolitica and by 16S rRNA gene sequencing to characterize the bacterial communities. Comparing culture data from deep tonsil tissue and tonsil surface showed similar numbers of E. coli but significantly higher numbers of Y. enterocolitica in the deep tissue samples. Microbiota analysis showed similar bacterial communities in the two sample types at phylum level, while comparison at genus level showed significant differences between the relative abundance of several genera in the two sample types. The finding of a significantly higher relative abundance of Yersinia in tonsil tissue compared to tonsil surface supported the culture analysis. The microbiota analysis also investigated characteristics of the bacterial community that could discriminate bacterial transfer from tonsils and faeces to the carcass during slaughter. The microbiota analyses demonstrated that Fusobacteria and Proteobacteria are the most abundant phyla in tonsils, while Firmicutes showed the highest relative abundance in faeces. The dominating phylum on carcasses was Proteobacteria. Besides Proteobacteria, the swabbing area on the forepart of the carcass, showed a higher relative abundance of Firmicutes and Fusobacteria compared to swabbing areas on the rear part and mid-section of the carcass. Principal coordinate analysis showed clear clustering of samples based on sample source (tonsils, faeces and carcass). Carcass swab samples from the forepart tended to cluster closer to the tonsil samples compared to carcass swab samples from the rear part and mid-section. Identification of the genera Fusobacterium, Moraxella, Actinobacillus and non-E. coli genera of the family Enterobacteriaceae in carcass swabs could indicate tonsil contamination, while faecal contamination would more likely include higher prevalence of bacteria belonging to the class of Clostridia. The present study supports that it is possible to identify bacterial groups that are indicative for either tonsil or faecal carcass contamination. The level and composition of Enterobacteriaceae on the carcasses did, however, indicate that other sources of meat contamination than tonsils and faeces may be important, such as the process environment.

Publisher Correction: Maturation of the gut microbiome and risk of asthma in childhood.

The originally published version of this Article contained an incorrect version of Figure 3 that was introduced following peer review and inadvertently not corrected during the production process. Both versions contain the same set of abundance data, but the incorrect version has the children's asthma status erroneously disconnected from the abundance data, thereby producing the non-representative p values and graphic presentations. These errors have now been rectified, with the correct version of Figure 3 replaced in both the PDF and HTML versions of the Article.

Maturation of the gut microbiome and risk of asthma in childhood.

The composition of the human gut microbiome matures within the first years of life. It has been hypothesized that microbial compositions in this period can cause immune dysregulations and potentially cause asthma. Here we show, by associating gut microbial composition from 16S rRNA gene amplicon sequencing during the first year of life with subsequent risk of asthma in 690 participants, that 1-year-old children with an immature microbial composition have an increased risk of asthma at age 5 years. This association is only apparent among children born to asthmatic mothers, suggesting that lacking microbial stimulation during the first year of life can trigger their inherited asthma risk. Conversely, adequate maturation of the gut microbiome in this period may protect these pre-disposed children.

Genetic barcoding of dark-spored myxomycetes (Amoebozoa)-Identification, evaluation and application of a sequence similarity threshold for species differentiation in NGS studies.

Unicellular, eukaryotic organisms (protists) play a key role in soil food webs as major predators of microorganisms. However, due to the polyphyletic nature of protists, no single universal barcode can be established for this group, and the structure of many protistean communities remains unresolved. Plasmodial slime moulds (Myxogastria or Myxomycetes) stand out among protists by their formation of fruit bodies, which allow for a morphological species concept. By Sanger sequencing of a large collection of morphospecies, this study presents the largest database to date of dark-spored myxomycetes and evaluate a partial 18S SSU gene marker for species annotation. We identify and discuss the use of an intraspecific sequence similarity threshold of 99.1% for species differentiation (OTU picking) in environmental PCR studies (ePCR) and estimate a hidden diversity of putative species, exceeding those of described morphospecies by 99%. When applying the identified threshold to an ePCR data set (including sequences from both NGS and cloning), we find 64 OTUs of which 21.9% had a direct match (>99.1% similarity) to the database and the remaining had on average 90.2 ± 0.8% similarity to their best match, thus thought to represent undiscovered diversity of dark-spored myxomycetes.