Draft Genome Sequence of Andreprevotia sp. Strain IGB-42, a Chitinolytic Bacterium Isolated from a Soil Sample of an Anthill in Stuttgart, Germany.

Andreprevotia sp. strain IGB-42 is a chitin-degrading bacterium isolated from the soil of an anthill. The genome contains 4.7 Mb, a G+C content of 61.31%, 4,257 predicted open reading frames, and a set of industrially interesting chitinase genes.

Rapid Next-Generation Sequencing-Based Diagnostics of Bacteremia in Septic Patients.

The increasing incidence of bloodstream infections including sepsis is a major challenge in intensive care units worldwide. However, current diagnostics for pathogen identification mainly depend on culture- and molecular-based approaches, which are not satisfactory regarding specificity, sensitivity, and time to diagnosis. Herein, we established a complete diagnostic workflow for real-time high-throughput sequencing of cell-free DNA from plasma based on nanopore sequencing for the detection of the causative agents, which was applied to the analyses of eight samples from four septic patients and three healthy controls, and subsequently validated against standard next-generation sequencing results. By optimization of library preparation protocols for short fragments with low input amounts, a 3.5-fold increase in sequencing throughput could be achieved. With tailored bioinformatics workflows, all eight septic patient samples were found to be positive for relevant pathogens. When considering time to diagnosis, pathogens were identified within minutes after start of sequencing. Moreover, an extrapolation of real-time sequencing performance on a cohort of 239 septic patient samples revealed that more than 90% of pathogen hits would have also been detected using the optimized MinION workflow. Reliable identification of pathogens based on circulating cell-free DNA sequencing using optimized workflows and real-time nanopore-based sequencing can be accomplished within 5 to 6 hours following blood draw. Therefore, this approach might provide therapy-relevant results in a clinically critical timeframe.

Stable Redox-Cycling Nitroxide Tempol Has Antifungal and Immune-Modulatory Properties.

Invasive mycoses remain underdiagnosed and difficult to treat. Hospitalized individuals with compromised immunity increase in number and constitute the main risk group for severe fungal infections. Current antifungal therapy is hampered by slow and insensitive diagnostics and frequent toxic side effects of standard antifungal drugs. Identification of new antifungal compounds with high efficacy and low toxicity is therefore urgently required. We investigated the antifungal activity of tempol, a cell-permeable nitroxide. To narrow down possible mode of action we used RNA-seq technology and metabolomics to probe for pathways specifically disrupted in the human fungal pathogen Candida albicans due to tempol administration. We found genes upregulated which are involved in iron homeostasis, mitochondrial stress, steroid synthesis, and amino acid metabolism. In an ex vivo whole blood infection, tempol treatment reduced C. albicans colony forming units and at the same time increased the release of pro-inflammatory cytokines, such as interleukin 8 (IL-8, monocyte chemoattractant protein-1, and macrophage migration inhibitory factor). In a systemic mouse model, tempol was partially protective with a significant reduction of fungal burden in the kidneys of infected animals during infection onset. The results obtained propose tempol as a promising new antifungal compound and open new opportunities for the future development of novel therapies.

Enhanced Performance of Next-Generation Sequencing Diagnostics Compared With Standard of Care Microbiological Diagnostics in Patients Suffering From Septic Shock.

OBJECTIVES: Culture-based diagnostics represent the standard of care in septic patients, but are highly insensitive and in many cases unspecific. We recently demonstrated the general feasibility of next-generation sequencing-based diagnostics using free circulating nucleic acids (cell-free DNA) in plasma samples of septic patients. Within the presented investigation, higher performance of next-generation sequencing-based diagnostics was validated by comparison to matched blood cultures. DESIGN: A secondary analysis of a prospective, observational, single-center study. SETTING: Surgical ICU of a university hospital and research laboratory. PATIENTS: Fifty patients with septic shock, 20 uninfected patients with elective surgery as control cohort. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: From 256 plasma samples of 48 septic patients at up to seven consecutive time points within the 28-day observation period, cell-free DNA was isolated and analyzed by next-generation sequencing and relevance scoring. In parallel, results from culture-based diagnostics (e.g., blood culture) were obtained. Plausibility of blood culture and next-generation sequencing results as well as adequacy of antibiotic therapy was evaluated by an independent expert panel. In contrast to blood culture with a positivity rate of 33% at sepsis onset, the positivity rate for next-generation sequencing-based pathogen identification was 72%. Over the whole study period, blood culture positivity was 11%, and next-generation sequencing positivity was 71%. Ninety-six percent of positive next-generation sequencing results for acute sepsis time points were plausible and would have led to a change to a more adequate therapy in 53% of cases as assessed by the expert evaluation. CONCLUSIONS: Our results show that next-generation sequencing-based analyses of bloodstream infections provide a valuable diagnostic platform for the identification of clinically relevant pathogens with higher sensitivity and specificity than blood culture, indicating that patients might benefit from a more appropriate therapy based on next-generation sequencing-based diagnosis.

Obstructed defecation-an enteric neuropathy? An exploratory study of patient samples.

PURPOSE: Although various strategies exist for chronic constipation therapy, the pathogenesis of chronic constipation is still not completely understood. The aim of this exploratory experimental study is to elucidate alterations of the autonomous enteric nervous system at the molecular level in patients with obstructed defecation, who represent one of the most predominant groups of constipated patients. METHODS: Full-thickness rectal wall samples of patients with obstructed defecation were analyzed and compared with controls. Differential gene expression analyses by RNA-Seq transcriptome profiling were performed and gene expression profiles were assigned to gene ontology pathways by application of different biological libraries. RESULTS: Analysis of the transcriptome showed that genes associated with the enteric nervous system functions were significantly downregulated in patients with obstructed defecation. These affected functions included developmental processes and synaptic transmission. CONCLUSIONS: Our results therefore indicate that obstructed defecation may represent an enteric neuropathy, comparable to Hirschsprung disease and slow-transit constipation.

Genetic repertoires of anaerobic microbiomes driving generation of biogas.

BACKGROUND: Biogas production is an attractive technology for a sustainable generation of renewable energy. Although the microbial community is fundamental for such production, the process control is still limited to technological and chemical parameters. Currently, most of the efforts on microbial management system (MiMaS) are focused on process-specific marker species and community dynamics, but a practical implementation is in its infancy. The high number of unknown and uncharacterized microorganisms in general is one of the reasons hindering further advancements. RESULTS: A Biogas Metagenomics Hybrid Assembly (BioMETHA) database, derived from microbiomes of biogas plants, was generated using a dedicated assembly strategy for different metagenomic datasets. Long reads from nanopore sequencing (MinION) were combined with short, more accurate second-generation sequencing reads (Illumina). The hybrid assembly resulted in 231 genomic bins each representing a taxonomic unit with an average completeness of 47%. Functional annotation identified 13,190 non-redundant genes covering roughly 207 k coding sequences. Mapping rates of metagenomics DNA derived from diverse biogas plants and laboratory reactors increased up to 73%. In addition, an EC (enzyme commission) reference sequence collection (ERSC) was generated whose genes are crucial for biogas-related processes, consisting of 235 unique EC numbers organized in 52 metabolic modules. Mapping rates of metatranscriptomic data to this ERSC revealed coverages of up to 93%. Process parameters and imbalances of laboratory reactors could be reconstructed by evaluating abundance of biogas-specific metabolic modules using metatranscriptomic data derived from various fermenter systems. CONCLUSION: This newly established metagenomic hybrid assembly in combination with an EC reference sequence collection might help to shed light on the microbial dark matter of biogas plants by contributing to the development of a reference for biogas plant microbiome-specific gene sequences. Considering a biogas microbiome as a complex meta-organism expressing a meta-transcriptome, the approach established here could lay the foundation for a function-based microbial management system.

A novel cytochrome P450 mono-oxygenase from Streptomyces platensis resembles activities of human drug metabolizing P450s.

Cytochrome P450 mono-oxygenases (P450) are versatile enzymes which play essential roles in C-source assimilation, secondary metabolism, and in degradations of endo- and exogenous xenobiotics. In humans, several P450 isoforms constitute the largest part of phase I metabolizing enzymes and catalyze oxidation reactions which convert lipophilic xenobiotics, including drugs, to more water soluble species. Recombinant human P450s and microorganisms are applied in the pharmaceutical industry for the synthesis of drug metabolites for pharmacokinetics and toxicity studies. Compared to the membrane-bound eukaryotic P450s, prokaryotic ones exhibit some advantageous features, such as high stability and generally easier heterologous expression. Here, we describe a novel P450 from Streptomyces platensis DSM 40041 classified as CYP107L that efficiently converts several commercial drugs of various size and properties. This P450 was identified by screening of actinobacterial strains for amodiaquine and ritonavir metabolizing activities, followed by genome sequencing and expression of the annotated S. platensis P450s in Escherichia coli. Performance of CYP107L in biotransformations of amodiaquine, ritonavir, amitriptyline, and thioridazine resembles activities of the main human metabolizing P450s, namely CYPs 3A4, 2C8, 2C19, and 2D6. For application in the pharmaceutical industry, an E. coli whole-cell biocatalyst expressing CYP107L was developed and evaluated for preparative amodiaquine metabolite production.

Microbiome dynamics and adaptation of expression signatures during methane production failure and process recovery.

This study aimed to uncover microbial dynamics and transcriptional adaptations during mesophilic AD of maize silage and slurry. While one digester performed under optimal conditions, the investigations also evaluated the microbiome during a temperature drop mediated process failure accompanied by acidification and how it contributed to a process recovery. Composition and pathway activities were analyzed by whole genome shotgun (WGS) and metatranscriptome sequencing, respectively. A biodiversity of 112 species was observed with noticeable shifts over process time. Although four distinct groups of microbes could be identified with a correlating versatility according to substrate and to process disturbance, also tremendous effects on gene expression were monitored especially of the archaeal methane metabolism. Particularly, the expression of acetogenotrophic methanogenesis related genes was identified to be relevant for process regeneration.

Antifungal defense of probiotic Lactobacillus rhamnosus GG is mediated by blocking adhesion and nutrient depletion.

Candida albicans is an inhabitant of mucosal surfaces in healthy individuals but also the most common cause of fungal nosocomial blood stream infections, associated with high morbidity and mortality. As such life-threatening infections often disseminate from superficial mucosal infections we aimed to study the use of probiotic Lactobacillus rhamnosus GG (LGG) in prevention of mucosal C. albicans infections. Here, we demonstrate that LGG protects oral epithelial tissue from damage caused by C. albicans in our in vitro model of oral candidiasis. Furthermore, we provide insights into the mechanisms behind this protection and dissect direct and indirect effects of LGG on C. albicans pathogenicity. C. albicans viability was not affected by LGG. Instead, transcriptional profiling using RNA-Seq indicated dramatic metabolic reprogramming of C. albicans. Additionally, LGG had a significant impact on major virulence attributes, including adhesion, invasion, and hyphal extension, whose reduction, consequently, prevented epithelial damage. This was accompanied by glucose depletion and repression of ergosterol synthesis, caused by LGG, but also due to blocked adhesion sites. Therefore, LGG protects oral epithelia against C. albicans infection by preventing fungal adhesion, invasion and damage, driven, at least in parts, by metabolic reprogramming due to nutrient limitation caused by LGG.

Dual transcriptome of the immediate neutrophil and Candida albicans interplay.

BACKGROUND: Neutrophils are traditionally considered transcriptionally inactive. Compared to other immune cells, little is known about their transcriptional profile during interaction with pathogens. METHODS: We analyzed the meta-transcriptome of the neutrophil-Candida albicans interplay and the transcriptome of C. albicans challenged with neutrophil extracellular traps (NETs) by RNA-Seq, considering yeast and hypha individually in each approach. RESULTS: The neutrophil response to C. albicans yeast and hyphae was dominated by a morphotype-independent core response. However, 11 % of all differentially expressed genes were regulated in a specific manner when neutrophils encountered the hyphal form of C. albicans. While involving genes for transcriptional regulators, receptors, and cytokines, the neutrophil core response lacked typical antimicrobial effectors genes. Genes of the NOD-like receptor pathway, including NLRP3, were enriched. Neutrophil- and NET-provoked responses in C. albicans differed. At the same time, the Candida transcriptome upon neutrophil encounter and upon NET challenge included genes from various metabolic processes and indicate a mutual role of the regulators Tup1p, Efg1p, Hap43p, and Cap1p. Upon challenge with neutrophils and NETs, the overall Candida response was partially morphotype-specific. Yet again, actual oppositional regulation in yeasts and hyphae was only detected for the arginine metabolism in neutrophil-infecting C. albicans. CONCLUSIONS:  Taken together, our study provides a comprehensive and quantitative transcript profile of the neutrophil-C. albicans interaction. By considering the two major appearances of both, neutrophils and C. albicans, our study reveals yet undescribed insights into this medically relevant encounter. Hence, our findings will facilitate future research and potentially inspire novel therapy developments.

Immune-Response Patterns and Next Generation Sequencing Diagnostics for the Detection of Mycoses in Patients with Septic Shock-Results of a Combined Clinical and Experimental Investigation.

Fungi are of increasing importance in sepsis. However, culture-based diagnostic procedures are associated with relevant weaknesses. Therefore, culture- and next-generation sequencing (NGS)-based fungal findings as well as corresponding plasma levels of ß-d-glucan, interferon gamma (INF-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, -4, -6, -10, -17A, and mid-regional proadrenomedullin (MR-proADM) were evaluated in 50 septic patients at six consecutive time points within 28 days after sepsis onset. Furthermore, immune-response patterns during infections with Candida spp. were studied in a reconstituted human epithelium model. In total, 22% (n = 11) of patients suffered from a fungal infection. An NGS-based diagnostic approach appeared to be suitable for the identification of fungal pathogens in patients suffering from fungemia as well as in patients with negative blood cultures. Moreover, MR-proADM and IL-17A in plasma proved suitable for the identification of patients with a fungal infection. Using RNA-seq., adrenomedullin (ADM) was shown to be a target gene which is upregulated early after an epithelial infection with Candida spp. In summary, an NGS-based diagnostic approach was able to close the diagnostic gap of routinely used culture-based diagnostic procedures, which can be further facilitated by plasmatic measurements of MR-proADM and IL-17A. In addition, ADM was identified as an early target gene in response to epithelial infections with Candida spp.

Draft Genome Sequences of Three Actinobacteria Strains Presenting New Candidate Organisms with High Potentials for Specific P450 Cytochromes.

The three Actinobacteria strains Streptomyces platensis DSM 40041, Pseudonocardia autotrophica DSM 535, and Streptomyces fradiae DSM 40063 were described to selectively oxyfunctionalize several drugs. Here, we present their draft genomes to unravel their gene sets encoding promising cytochrome P450 monooxygenases associated with the generation of drug metabolites.

Central Role for Dermal Fibroblasts in Skin Model Protection against Candida albicans.

The fungal pathogen Candida albicans colonizes basically all human epithelial surfaces, including the skin. Under certain conditions, such as immunosuppression, invasion of the epithelia occurs. Not much is known about defense mechanisms against C. albicans in subepithelial layers such as the dermis. Using immune cell-supplemented 3D skin models we defined a new role for fibroblasts in the dermis and identified a minimal set of cell types for skin protection against C. albicans invasion. Dual RNA sequencing of individual host cell populations and C. albicans revealed that dermal invasion is directly impeded by dermal fibroblasts. They are able to integrate signals from the pathogen and CD4+ T cells and shift toward an antimicrobial phenotype with broad specificity that is dependent on Toll-like receptor 2 and interleukin 1ß. These results highlight a central function of dermal fibroblasts for skin protection, opening new possibilities for treatment of infectious diseases.

Draft Genome Sequence of Pseudonocardia autotrophica Strain DSM 43083, an Efficient Producer of Peroxidases for Lignin Modification.

Pseudonocardia autotrophica strain DSM 43083 is a filamentous actinobacterium and was described to degrade or modify lignin. Here, we present its draft genome sequence, with a size of 5.8 Mb, to unravel the gene set coding for promising monooxygenases, dioxygenases, and DyP-type peroxidases associated with aromatic metabolism and lignin modification.

The Molecular Blueprint of a Fungus by Next-Generation Sequencing (NGS).

Sequencing the whole genome of an organism is invaluable for its comprehensive molecular characterization and has been drastically facilitated by the advent of high-throughput sequencing techniques. Especially in clinical microbiology the impact of sequenced strains increases as resistance and virulence markers can easily be detected. Here, we describe a combined approach for sequencing a fungal genome and transcriptome from initial nucleic acid isolation through the generation of ready-to-load DNA libraries for the Illumina platform and the final step of genome assembly with subsequent gene annotation.

Next-generation sequencing diagnostics of bacteremia in septic patients.

BACKGROUND: Bloodstream infections remain one of the major challenges in intensive care units, leading to sepsis or even septic shock in many cases. Due to the lack of timely diagnostic approaches with sufficient sensitivity, mortality rates of sepsis are still unacceptably high. However a prompt diagnosis of the causative microorganism is critical to significantly improve outcome of bloodstream infections. Although various targeted molecular tests for blood samples are available, time-consuming blood culture-based approaches still represent the standard of care for the identification of bacteria. METHODS: Here we describe the establishment of a complete diagnostic workflow for the identification of infectious microorganisms from seven septic patients based on unbiased sequence analyses of free circulating DNA from plasma by next-generation sequencing. RESULTS: We found significant levels of DNA fragments derived from pathogenic bacteria in samples from septic patients. Quantitative evaluation of normalized read counts and introduction of a sepsis indicating quantifier (SIQ) score allowed for an unambiguous identification of Gram-positive as well as Gram-negative bacteria that exactly matched with blood cultures from corresponding patient samples. In addition, we also identified species from samples where blood cultures were negative. Reads of non-human origin also comprised fragments derived from antimicrobial resistance genes, showing that, in principle, prediction of specific types of resistance might be possible. CONCLUSIONS: The complete workflow from sample preparation to species identification report could be accomplished in roughly 30 h, thus making this approach a promising diagnostic platform for critically ill patients suffering from bloodstream infections.

Transcriptomics and physiological analyses reveal co-ordinated alteration of metabolic pathways in Jatropha curcas drought tolerance.

Jatropha curcas, a multipurpose plant attracting a great deal of attention due to its high oil content and quality for biofuel, is recognized as a drought-tolerant species. However, this drought tolerance is still poorly characterized. This study aims to contribute to uncover the molecular background of this tolerance, using a combined approach of transcriptional profiling and morphophysiological characterization during a period of water-withholding (49 d) followed by rewatering (7 d). Morphophysiological measurements showed that J. curcas plants present different adaptation strategies to withstand moderate and severe drought. Therefore, RNA sequencing was performed for samples collected under moderate and severe stress followed by rewatering, for both roots and leaves. Jatropha curcas transcriptomic analysis revealed shoot- and root-specific adaptations across all investigated conditions, except under severe stress, when the dramatic transcriptomic reorganization at the root and shoot level surpassed organ specificity. These changes in gene expression were clearly shown by the down-regulation of genes involved in growth and water uptake, and up-regulation of genes related to osmotic adjustments and cellular homeostasis. However, organ-specific gene variations were also detected, such as strong up-regulation of abscisic acid synthesis in roots under moderate stress and of chlorophyll metabolism in leaves under severe stress. Functional validation further corroborated the differential expression of genes coding for enzymes involved in chlorophyll metabolism, which correlates with the metabolite content of this pathway.

Draft Genome Sequence of Amantichitinum ursilacus IGB-41, a New Chitin-Degrading Bacterium.

Amantichitinum ursilacus IGB-41 is a new species of chitin-degrading bacterium isolated from soil, which secretes potential industrial enzymes. The genome of A. ursilacus was sequenced, and the gene set encoding chitinases was identified. Here, we present the draft genome of 4.9 Mb, comprising 38 contigs, and the corresponding annotation.

The transcriptomic profile of Pseudozyma aphidis during production of mannosylerythritol lipids.

The basidiomycetous fungus Pseudozyma aphidis is able to convert vegetable oils to abundant amounts of the biosurfactant mannosylerythritol lipid (MEL) with a unique product pattern of MEL-A, MEL-B, MEL-C, and MEL-D. To investigate the metabolism of MEL production, we analyzed the transcriptome of P. aphidis DSM 70725 under MEL-inducing and non-inducing conditions using deep sequencing. Following manual curation of the previously described in silico gene models based on RNA-Seq data, we were able to generate an experimentally verified gene annotation containing 6347 genes. Using this database, our expression analysis revealed that only four of the five cluster genes required for MEL synthesis were clearly induced by the presence of soybean oil. The acetyltransferase encoding gene PaGMAT1 was expressed on a much lower level, which may explain the secretion of MEL with different degrees of acetylation in P. aphidis. In parallel to MEL synthesis, microscopic observations showed morphological changes accompanied by expression of genes responsible for cell development, indicative of a coregulation between MEL synthesis and cell morphology. In addition a set of transcription factors was identified which may be responsible for regulation of MEL synthesis and cell development. The upregulation of genes required for nitrogen metabolism and other assimilation processes indicate additional metabolic pathways required under the MEL-inducing conditions used. We also searched for a conserved gene cluster for cellobiose lipids (CL) but only found seven genes with limited homology distributed over the genome. However, we detected characteristic TLC spots in fermentations using P. aphidis DSM 70725, indicative of CL secretion.