The gut microbiome is a diverse ecosystem, dominated by bacteria; however, fungi, phages/viruses, archaea, and protozoa are also important members of the gut microbiota. Exploration of taxonomic compositions beyond bacteria as well as an understanding of the interaction between the bacteriome with the other members is limited using 16S rDNA sequencing. Here, we developed a pipeline enabling the simultaneous interrogation of the gut microbiome (bacteriome, mycobiome, archaeome, eukaryome, DNA virome) and of antibiotic resistance genes based on optimized long-read shotgun metagenomics protocols and custom bioinformatics. Using our pipeline we investigated the longitudinal composition of the gut microbiome in an exploratory clinical study in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT; n = 31). Pre-transplantation microbiomes exhibited a 3-cluster structure, characterized by Bacteroides spp. /Phocaeicola spp., mixed composition and Enterococcus abundances. We revealed substantial inter-individual and temporal variabilities of microbial domain compositions, human DNA, and antibiotic resistance genes during the course of alloHSCT. Interestingly, viruses and fungi accounted for substantial proportions of microbiome content in individual samples. In the course of HSCT, bacterial strains were stable or newly acquired. Our results demonstrate the disruptive potential of alloHSCTon the gut microbiome and pave the way for future comprehensive microbiome studies based on long-read metagenomics.

A new prenylated indole diketopiperazine alkaloid, rubrumline P (1), was isolated along with six more analogues and characterized from the fermentation culture of a marine sediment-derived fungus, Aspergillus chevalieri, collected at a depth of 15 m near the lighthouse in Dahab, Red Sea, Egypt. In the current study, a bioassay-guided fractionation allowed for the identification of an active fraction displaying significant cytotoxic activity against the human pancreatic adenocarcinoma cell line PANC-1 from the EtOAc extract of the investigated fungus compared to the standard paclitaxel. The structures of the isolated compounds from the active fraction were established using 1D/2D NMR spectroscopy and mass spectrometry, together with comparisons with the literature. The absolute configuration of the obtained indole diketopiperazines was established based on single-crystal X-ray diffraction analyses of rubrumline I (2) and comparisons of optical rotations and NMR data, as well as on biogenetic considerations. Genome sequencing indicated the formation of prenyltransferases, which was subsequently confirmed by the isolation of mono-, di-, tri-, and tetraprenylated compounds. Compounds rubrumline P (1) and neoechinulin D (4) confirmed preferential cytotoxic activity against PANC-1 cancer cells with IC₅₀ values of 25.8 and 23.4 µM, respectively. Although the underlying mechanism-of-action remains elusive in this study, cell cycle analysis indicated a slight increase in the sub-G1 peak after treatment with compounds 1 and 4.

(1) Background: Microbial keratitis is a serious eye infection that carries a significant risk of vision loss. Acanthamoeba spp. are known to cause keratitis and their bacterial endosymbionts can increase virulence and/or treatment resistance and thus significantly worsen the course of the disease. (2) Methods and Results: In a suspected case of Acanthamoeba keratitis, in addition to Acanthamoeba spp., an endosymbiont of acanthamoebae belonging to the taxonomic order of Holosporales was detected by chance in a bacterial 16S rDNA-based pan-PCR and subsequently classified as Candidatus Paracaedibacter symbiosus through an analysis of an enlarged 16S rDNA region. We used Oxford Nanopore Technology to evaluate the usefulness of whole-genome sequencing (WGS) as a one-step diagnostics method. Here, Acanthamoeba castellanii and the endosymbiont Candidatus Paracaedibacter symbiosus could be directly detected at the species level. No other microbes were identified in the specimen. (3) Conclusions: We recommend the introduction of WGS as a diagnostic approach for keratitis to replace the need for multiple species-specific qPCRs in future routine diagnostics and to enable an all-encompassing characterisation of the polymicrobial community in one step.

Background/Objectives: Patient-specific diagnostic and therapeutic approaches are important in the care of people with chronic wounds. The heterogeneity of underlying disease profiles and the diversity of the wound micro-environment make generalized approaches difficult. While high-throughput molecular diagnostic methods are increasingly widespread and available, the analysis of objective biomolecular disease patterns has not found its way into everyday wound management. The aim of this study is to evaluate the use of wound swab samples for the analysis of biomarkers and disease patterns in people with chronic wounds. Methods: A sample cohort from the multicenter “Wound-BIOME” project was analyzed. The project aims to comprehensively investigate the local micro-environment of chronic wounds of various entities, healing tendencies and regeneration stages at the biomolecular level. A sample collection and handling protocol suitable for everyday use was tested and evaluated regarding feasibility for multiplex immunoassay, proteomics, small RNA sequencing (miRNA) and metagenome analyses (microbiomics). Results: It could be shown that standard wound swabs are well-suited for the analysis of the complex wound micro-environment using various high-throughput methods. Despite the sample heterogeneity, the quality was adequate to analyze biomolecular patterns. Conclusions: Initial analyses of protein signatures, microbial wound communities and miRNA patterns show promising results for future individualized diagnostics and targeted interventions.

Fungal respiratory tract colonization is a common finding in patients with hematologic neoplasms due to immunosuppression inherent in the diseases and exacerbated by therapy. This greatly increases the risk of fungal infections of the lungs, which is associated with significant mortality. Therefore, reliable diagnostic methods with rapidly available results are needed to administer adequate antifungal therapy. We have established an improved method for fungal DNA extraction and amplification that allows simultaneous detection of fungal families based on a set of multiplexed real-time PCR reactions (fuPCR). We analyzed respiratory rinses and blood of 94 patients with hematological systemic diseases by fuPCR and compared it with the results of culture and serological diagnostic methods. 40 healthy subjects served as controls. Regarding Candida species, the highest prevalence resulted from microbiological culture of respiratory rinses and from detection of antibodies in blood serum in patients (61 and 47%, respectively) and in the control group (29 and 51%, respectively). Detection of other pathogenic yeasts, such as Cryptococcus and Trichosporon, and molds, such as Fusarium, was only possible in patients by fuPCR from both respiratory rinses and whole blood and serum. These fungal species were found statistically significantly more frequent in respiratory rinses collected from patients after myeloablative therapy for stem cell transplantation compared to samples collected before treatment (P < 0.05i). The results show that fuPCR is a valuable complement to culturing and its inclusion in routine mycological diagnostics might be helpful for early detection of pathophysiologically relevant respiratory colonization for patients with hematologic neoplasms. We validated a set of PCR reactions (fuPCR) for use in routine diagnostic. In contrast to culture and serological methods, only by fuPCR pathogenic yeasts (Cryptococcus and Trichosporon) and molds (Aspergillus and Fusarium) were detected in respiratory rinses and blood of hematological patients.

Rare invasive fungal infections are increasingly emerging in hosts with predisposing factors such as immunodeficiency. Their timely diagnosis remains difficult, as their clinical picture may initially mimic infections with more common fungal species and species identification may be difficult with routine methods or may require time-consuming subcultures. This often results in ineffective drug administration and fatal outcomes. We report on a patient in their early twenties with mixed cellularity classical Hodgkin lymphoma with a disseminated Trichosporon asahii (T. asahii) infection. Even though pathogen detection and identification was possible via the standard procedure consisting of culture followed by matrix-assisted laser desorption ionisation–time of flight (MALDI-TOF) mass spectrometry, the patient passed away in the course of multi organ failure. Herein, we report on a retrospectively applied experimental diagnostic fungal PCR-analysis used on an EDTA blood sample and consisting of two pan-fungal reactions and seven branch-specific reactions. Regarding invasive T. asahii infection, this PCR array could considerably shorten time to diagnosis and switch to a targeted therapy with triazoles.