Genomes and secondary metabolomes of Streptomyces spp. isolated from Leontopodium nivale ssp. alpinum.

Bacterial endophytes dwelling in medicinal plants represent an as yet underexplored source of bioactive natural products with the potential to be developed into drugs against various human diseases. For the first time, several Streptomyces spp. were isolated from the rare and endangered traditional medicinal plant Leontopodium nivale ssp. alpinum, also known as Edelweiss. In the search for novel natural products, nine endophytic Streptomyces spp. from Edelweiss were investigated via genome sequencing and analysis, followed by fermentation in different media and investigation of secondary metabolomes. A total of 214 secondary metabolite biosynthetic gene clusters (BGCs), of which 35 are presumably unique, were identified by the bioinformatics tool antiSMASH in the genomes of these isolates. LC-MS analyses of the secondary metabolomes of these isolates revealed their potential to produce both known and presumably novel secondary metabolites, whereby most of the identified molecules could be linked to their cognate BGCs. This work sets the stage for further investigation of endophytic streptomycetes from Edelweiss aimed at the discovery and characterization of novel bioactive natural products.

The Parkinson's drug entacapone disrupts gut microbiome homeostasis via iron sequestration.

Increasing evidence shows that many human-targeted drugs alter the gut microbiome, leading to implications for host health. However, much less is known about the mechanisms by which drugs target the microbiome and how drugs affect microbial function. Here we combined quantitative microbiome profiling, long-read metagenomics, stable isotope probing and single-cell chemical imaging to investigate the impact of two widely prescribed nervous system-targeted drugs on the gut microbiome. Ex vivo supplementation of physiologically relevant concentrations of entacapone or loxapine succinate to faecal samples significantly impacted the abundance of up to one third of the microbial species present. Importantly, we demonstrate that the impact of these drugs on microbial metabolism is much more pronounced than their impact on abundances, with low concentrations of drugs reducing the activity, but not the abundance of key microbiome members like Bacteroides, Ruminococcus or Clostridium species. We further demonstrate that entacapone impacts the microbiome due to its ability to complex and deplete available iron, and that microbial growth can be rescued by replenishing levels of microbiota-accessible iron. Remarkably, entacapone-induced iron starvation selected for iron-scavenging organisms carrying antimicrobial resistance and virulence genes. Collectively, our study unveils the impact of two under-investigated drugs on whole microbiomes and identifies metal sequestration as a mechanism of drug-induced microbiome disturbance.

Cultivation and genomic characterization of novel and ubiquitous marine nitrite-oxidizing bacteria from the Nitrospirales.

Nitrospirales, including the genus Nitrospira, are environmentally widespread chemolithoautotrophic nitrite-oxidizing bacteria. These mostly uncultured microorganisms gain energy through nitrite oxidation, fix CO2, and thus play vital roles in nitrogen and carbon cycling. Over the last decade, our understanding of their physiology has advanced through several new discoveries, such as alternative energy metabolisms and complete ammonia oxidizers (comammox Nitrospira). These findings mainly resulted from studies of terrestrial species, whereas less attention has been given to marine Nitrospirales. In this study, we cultured three new marine Nitrospirales enrichments and one isolate. Three of these four NOB represent new Nitrospira species while the fourth represents a novel genus. This fourth organism, tentatively named "Ca. Nitronereus thalassa", represents the first cultured member of a Nitrospirales lineage that encompasses both free-living and sponge-associated nitrite oxidizers, is highly abundant in the environment, and shows distinct habitat distribution patterns compared to the marine Nitrospira species. Partially explaining this, "Ca. Nitronereus thalassa" harbors a unique combination of genes involved in carbon fixation and respiration, suggesting differential adaptations to fluctuating oxygen concentrations. Furthermore, "Ca. Nitronereus thalassa" appears to have a more narrow substrate range compared to many other marine nitrite oxidizers, as it lacks the genomic potential to utilize formate, cyanate, and urea. Lastly, we show that the presumed marine Nitrospirales lineages are not restricted to oceanic and saline environments, as previously assumed.

Secondary Metabolite Production Potential in a Microbiome of the Freshwater Sponge Spongilla lacustris.

Marine and freshwater sponges harbor diverse communities of bacteria with vast potential to produce secondary metabolites that may play an important role in protecting the host from predators and infections. In this work, we initially used cultivation and metagenomics to investigate the microbial community of the freshwater sponge Spongilla lacustris collected in an Austrian lake. Representatives of 41 bacterial genera were isolated from the sponge sample and classified according to their 16S rRNA gene sequences. The genomes of 33 representative isolates and the 20 recovered metagenome-assembled genomes (MAGs) contained in total 306 secondary metabolite biosynthesis gene clusters (BGCs). Comparative 16S rRNA gene and genome analyses showed very little taxon overlap between the recovered isolates and the sponge community as revealed by cultivation-independent methods. Both culture-independent and -dependent analyses suggested high biosynthetic potential of the S. lacustris microbiome, which was confirmed experimentally even at the subspecies level for two Streptomyces isolates. To our knowledge, this is the most thorough description of the secondary metabolite production potential of a freshwater sponge microbiome to date. IMPORTANCE A large body of research is dedicated to marine sponges, filter-feeding animals harboring rich bacterial microbiomes believed to play an important role in protecting the host from predators and infections. Freshwater sponges have received so far much less attention with respect to their microbiomes, members of which may produce bioactive secondary metabolites with potential to be developed into drugs to treat a variety of diseases. In this work, we investigated the potential of bacteria associated with the freshwater sponge Spongilla lacustris to biosynthesize diverse secondary metabolites. Using culture-dependent and -independent methods, we discovered over 300 biosynthetic gene clusters in sponge-associated bacteria and proved production of several compounds by selected isolates using genome mining. Our results illustrate the importance of a complex approach when dealing with microbiomes of multicellular organisms that may contain producers of medically important secondary metabolites.

Disturbances in microbial skin recolonization and cutaneous immune response following allogeneic stem cell transfer.

The composition of the gut microbiome influences the clinical course after allogeneic hematopoietic stem cell transplantation (HSCT), but little is known about the relevance of skin microorganisms. In a single-center, observational study, we recruited a cohort of 50 patients before undergoing conditioning treatment and took both stool and skin samples up to one year after HSCT. We could confirm intestinal dysbiosis following HSCT and report that the skin microbiome is likewise perturbed in HSCT-recipients. Overall bacterial colonization of the skin was decreased after conditioning. Particularly patients that developed acute skin graft-versus-host disease (aGVHD) presented with an overabundance of Staphylococcus spp. In addition, a loss in alpha diversity was indicative of aGVHD development already before disease onset and correlated with disease severity. Further, co-localization of CD45+ leukocytes and staphylococci was observed in the skin of aGVHD patients even before disease development and paralleled with upregulated genes required for antigen-presentation in mononuclear phagocytes. Overall, our data reveal disturbances of the skin microbiome as well as cutaneous immune response in HSCT recipients with changes associated with cutaneous aGVHD.

Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing.

Long-read Oxford Nanopore sequencing has democratized microbial genome sequencing and enables the recovery of highly contiguous microbial genomes from isolates or metagenomes. However, to obtain near-finished genomes it has been necessary to include short-read polishing to correct insertions and deletions derived from homopolymer regions. Here, we show that Oxford Nanopore R10.4 can be used to generate near-finished microbial genomes from isolates or metagenomes without short-read or reference polishing.

Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing.

Microorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

Characterizing the growing microorganisms at species level in 46 anaerobic digesters at Danish wastewater treatment plants: A six-year survey on microbial community structure and key drivers.

Anaerobic digestion (AD) is a key technology at many wastewater treatment plants (WWTPs) for converting primary and surplus activated sludge to methane-rich biogas. However, the limited number of surveys and the lack of comprehensive datasets have hindered a deeper understanding of the characteristics and associations between key variables and the microbial community composition. Here, we present a six-year survey of 46 anaerobic digesters, located at 22 WWTPs in Denmark, which is the first and largest known study of the microbial ecology of AD at WWTPs at a regional scale. For three types of AD (mesophilic, mesophilic with thermal hydrolysis pretreatment, and thermophilic), we present the typical value range of 12 key parameters including operational variables and performance parameters. High-resolution bacterial and archaeal community analyses were carried out at species level using amplicon sequencing of >1,000 samples and the new ecosystem-specific MiDAS 3 reference database. We detected 42 phyla, 1,600 genera, and 3,584 species in the bacterial community, where 70% of the genera and 93% of the species represented environmental taxa that were only classified based on MiDAS 3 de novo placeholder taxonomy. More than 40% of the bacterial species were found not to grow in the mesophilic and thermophilic digesters and were only present due to immigration with the feed sludge. Ammonium concentration was the main driver shaping the bacterial community while temperature and pH were main drivers for the archaea in the three types of ADs. Sub-setting for the growing microbes improved significantly the correlation analyses and revealed the main drivers for the presence of specific species. Within mesophilic digesters, feed sludge composition and other key parameters (organic loading rate, biogas yield, and ammonium concentration) correlated with specific growing species. This survey provides a comprehensive insight into community structure at species level, providing a foundation for future studies of the ecological significance/characteristics and function of the many novel or poorly described taxa.

High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing.

High-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies (ONT) or Pacific Biosciences circular consensus sequencing, yielding high-accuracy single-molecule consensus sequences of large genomic regions. We applied our approach to sequence ribosomal RNA operon amplicons (~4,500 bp) and genomic sequences (>10,000 bp) of reference microbial communities in which we observed a chimera rate <0.02%. To reach a mean UMI consensus error rate <0.01%, a UMI read coverage of 15× (ONT R10.3), 25× (ONT R9.4.1) and 3× (Pacific Biosciences circular consensus sequencing) is needed, which provides a mean error rate of 0.0042%, 0.0041% and 0.0007%, respectively.