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Abstract Background Termitarium is an intricate biological home with exceptional physicochemical soil properties and numerous beneficial microbiomes. The termitarium microbiome plays a role in several bionetwork processes. It contributes significantly to sustainable agriculture, forestry, and horticulture. In contrast to the immense number of research works focusing on termitarium bacteria and archaea, the amount of study about termitarium fungal communities is limited. This is despite the fact that fungi significantly contribute to the cycling of energy and matter on Earth. Thus, this study investigates the metagenomic datasets from termitarium soil collected from agricultural soils, to obtain a depiction of their mycobiome of ecological importance. Results Our dataset showed that Ascomycota was the most rank phylum, while Basidiomycota was the next in rank. Specifically, we identified numerous fungi of commercially importance, for example, producers of cellulases and cellulosomes. This was strengthened with the presence of functional genes/enzymes capable of promoting plant growth traits and defence system. Analysis further shown that soil properties influenced the distribution of fungal communities. Magnesium, nitrogen, and potassium, for example, were observed to be among the best predictor of fungal distribution and explained 87.40% of the total variation. Conclusion This study epitomize further insights into the mycobiome of termitarium soils that are of ecological interest. Again, with some of our sequences assembled as uncategorized fungi, further studies are recommended for the evaluation of the metabolic potentials of these fungi as there is a possibility of the discovery of new fungi or genes of industrial or environmental applications.

Microbiota is a collection of microorganisms such as bacteria, fungi, viruses, and archaea, with bacteria being the most numerous in the human body. Meanwhile, the mycobiome consists of commensal fungi, which are a small part of the microbiota. Examples found in the human body, from the skin to the internal organs, play a role in the immune response, homeostasis, metabolism, and disease. The composition of mycobiome varies over time, and the human intestine is the most studied organ due to the diversity of microbiota. Furthermore, lung cancer is the leading cause of death in oncology patients worldwide. Various studies suggest that mycobiomes play a role in cancer associated with dysbiosis. Chronic inflammation, biofilm formation, and carcinogen products are parts of cancer pathogenesis. Metagenome analysis has provided information about the diversity of microbiota, specifically mycobiome in the human body. The common method for gene sequencing in the metagenome is next-generation sequencing (NGS). Analysis through metagenomes in chronic diseases such as cancer shows that mycobiomes play a role in the process of cancer formation. However, the causal relationship between mycobiomes and cancer remains unknown.

Microbial endophytes live asymptomatically within a host plant and contribute positively to its fitness. Endophytes associate ubiquitously with all plants examined so far and form an integral part of the host for processes such as nutrient uptake and stress response and traits like disease resistance. Well-characterized host–endophyte interaction models exist, but little is known about the microbiome of the monocot plant host, palm. In this study, we identified the endophytic fungal community associated with mule palm leaves with an objective to test their antagonist effects on lethal pathogens of palms. The leaflets of mule palms were sampled across five sites in Central and South Florida, and fungal endophytes were identified using culture-dependent (CD) and amplicon sequence-based culture-independent (CI) methods. Operational taxonomic units (OTUs) corresponding to phylum Ascomycota were identified by both approaches, whereas Basidiomycota were only detected using the CI method. Among the ascomycetes, a small (22%) overlap in the OTUs at the genus level was observed between the two methods, whereas the majority (64%) of OTUs were detected using the CI approach. In vitro cocultivation of endophytes with palm pathogens was observed to produce three distinct interaction phenotypes: inhibition zone, niche establishment, and overlapping growth. Eight endophytes capable of inhibiting fungal pathogen growth in vitro were also identified, suggesting a biocontrol potential for endophyte-derived secondary metabolites. A better understanding of the composition, assembly, and ecology of palm-associated microbial communities would eventually lead to healthy and resilient palms. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Wheat (Triticum spp.) is a staple food crop, providing a fifth of the world's protein and caloric needs. Our research examines the impact of multigeneration postflowering drought stress on the wheat seed endophytic fungal community. Understanding how wheat seed fungal communities respond to drought stress over several generations can improve our knowledge of legacy drought stress. In this article, we aim to identify seed-associated fungi that play critical roles within the wheat seed under drought stress conditions. We examined the endophytic seed fungal communities of three winter wheat cultivars, Catawba, Shirley, and USG 3640. Moderate drought was imposed on a subset of plants immediately after flowering, with plants relieved from drought stress after 1 week. Seeds harvested from generation 1 were planted for a second generation of drought experiments. When examining the postflowering drought impact on wheat physiology, drought-exposed plants consistently exhibited lower daily transpiration rates, chlorophyll-a values, and seed yield compared with control plants, indicating that drought implementation was successful. Internal transcribed spacer 1 metabarcoding revealed that wheat seed fungal community species richness decreased during postanthesis drought stress across both generations. We also observed that generation accounted for variation in fungal species richness and community structure, independent of drought treatment. The most abundant taxa recovered across all cultivars, treatments, and generations included Cladosporium, Penicillium, Alternaria, and Epicoccum. These results support our hypothesis that postanthesis drought shapes the wheat seed fungal community.

Abstract Background Lichens, traditionally considered as a simple partnership primarily between mycobiont and photobiont, are, in reality, complex holobionts comprised of a multitude of microorganisms. Lichen mycobiome represents fungal community residing within lichen thalli. While it is acknowledged that factors like the host lichen species and environmental conditions influence the structure of the lichen mycobiome, the existing research remains insufficient. To investigate which factor, host genus or location, has a greater impact on the lichen mycobiome, we conducted a comparative analysis of mycobiomes within Parmelia and Peltigera collected from both Turkey and South Korea, using high-throughput sequencing based on internal transcribed spacer region amplification. Results Overall, the lichen mycobiome was dominated by Capnodiales (Dothideomycetes), regardless of host or location. At the order level, the taxonomic composition was not significantly different according to lichen genus host or geographical distance. Hierarchical clustering of the top 100 abundant ASVs did not clearly indicate whether the lichen mycobiome was more influenced by host genus or location. Analyses of community similarity and partitioning variables revealed that the structure of the lichen mycobiome is more significantly influenced by location than by host genus. When analyzing the core mycobiome by host genus, the Peltigera mycobiome contained more ASV members than the Parmelia mycobiome. These two core mycobiomes also share common fungal strains, including basidiomycete yeast. Additionally, we used chi-squared tests to identify host genus-specialists and location-specialists. Conclusions By comparing lichen mycobiomes of the same genera across different countries, our study advances our comprehension of these microbial communities. Our study elucidates that, although host species play a contributory role, geographic distance exerts a more pronounced impact on the structure of lichen mycobiome. We have made foundational contributions to understanding the lichen mycobiome occupying ecologically crucial niches. We anticipate that broader global-scale investigations into the fungal community structures will provide more detailed insights into fungal residents within lichens.

ObjectivesAscites, often associated with critical pathologies such as liver cirrhosis or bowel perforation, can be complicated by fungal infection, increasing mortality especially in intensive care settings and demanding rapid diagnosis and adequate treatment. Traditional microbiological diagnostic methods have limited sensitivity in accurately identifying fungal pathogens in ascitic fluid. Alternative diagnostic methods may offer important insights to enable guiding of antifungal therapy and refining empirical treatment strategies. The objective of this study was to evaluate the potential of next-generation sequencing methods to identify specific fungal pathogens responsible for ascitic fluid infections.MethodsWe prospectively collected 50 ascitic fluid samples from ICU patients with suspected ascites infection. In addition to standard culture-based microbiological testing, an ascitic fluid aliquot underwent fungal DNA isolation and was analyzed by next-generation sequencing (NGS) methods for identification of fungal species.ResultsOf 50 ascitic samples collected, five samples showed growth of Candida spp. in culture. After DNA isolation and ITS2 PCR, detectable amplification was achieved in 10 samples. Sequencing of the 50 patients’ samples identified facultative pathogenic fungi in 19 patients. In 15 cases, culture alone would not have permitted the identification of all facultative pathogenic fungi. The identification of fungal DNA by sequencing was significantly associated with poor patient outcome and a number of clinical parameters.ConclusionsOur results show a higher sensitivity for NGS-based diagnostic methods in the identification of ascitic fluid fungal infections compared to culture-based diagnostics. This may be beneficial especially for patients in a critical care setting, who have an increased prevalence of comorbidities and high mortality. The implementation of such methods in standard diagnosis will require increased standardization of the workflows and interpretation of the sequencing results with respect to patients’ clinical picture.

Plant-associated mycobiomes can influence important host plant traits including those related to disease, nutrient acquisition, flowering phenology, stress tolerance, and productivity. Disentangling the complex multifaceted relationships between host plants and their associated mycobiomes is a critical first step toward generating improvements in environmental sustainability and/or plant productivity. Despite decades of work on these plant–fungal interactions, consequences of and mechanisms controlling these interactions are not well resolved, especially in the face of environmental stress such as drought. Moreover, plant differential genotypic responses under stress and associated mycobiome assembly dynamics are likely important in structuring communities but have been less well studied. We conducted a controlled drought stress experiment by manipulating water treatment in a diverse set of cultivated sunflower lines with different inherent drought resistance levels to evaluate how sunflower host genotypes and drought interacts to affect belowground fungal mycobiomes at the rhizospheric and endospheric levels. Our results demonstrated that fungal community structure was driven by watering treatment, plant genotype, treatment by genotype interactions, genotype by plant compartment interactions, and treatment by genotype by compartment interactions. Additionally, our analyses demonstrated the relative abundance of plant pathogens and arbuscular mycorrhizal fungi increased with host genetic variation, or heterozygosity, levels. Our study provides evidence for drought and genotypic drivers of belowground sunflower–fungi interactions and offers a framework for leveraging these interactions to further understand how mycobiome community structure can improve plant productivity under stress.

Abstract Background The mosquito microbiome is an important modulator of vector competence and vectoral capacity. Unlike the extensively studied bacterial microbiome, fungal communities in the mosquito microbiome (the mycobiome) remain largely unexplored. To work towards getting an improved understanding of the fungi associated with mosquitoes, we sequenced the mycobiome of three field-collected and laboratory-reared mosquito species (Aedes albopictus, Aedes aegypti, and Culex quinquefasciatus). Results Our analysis showed both environment and host species were contributing to the diversity of the fungal microbiome of mosquitoes. When comparing species, Ae. albopictus possessed a higher number of diverse fungal taxa than Cx. quinquefasciatus, while strikingly less than 1% of reads from Ae. aegypti samples were fungal. Fungal reads from Ae. aegypti were

The gut microbiome is a complex, unique entity implicated in the prevention, pathogenesis, and progression of common gastrointestinal diseases. While largely dominated by bacterial populations, advanced sequencing techniques have identified co-inhabiting fungal communities, collectively referred to as the mycobiome. Early studies identified that gut inflammation is associated with altered microbial composition, known as gut dysbiosis. Altered microbial profiles are implicated in various pathological diseases, such as inflammatory bowel disease (IBD), though their role as a cause or consequence of systemic inflammation remains the subject of ongoing research. Diet plays a crucial role in the prevention and management of various diseases and is considered to be an essential regulator of systemic inflammation. This review compiles current literature on the impact of dietary modulation on the mycobiome, showing that dietary changes can alter the fungal architecture of the gut. Further research is required to understand the impact of diet on gut fungi, including the metabolic pathways and enzymes involved in fungal fermentation. Additionally, investigating whether dietary modulation of the gut mycobiome could be utilized as a therapy in IBD is essential.

The gut fungi play important roles in human health and are involved in energy metabolism. This study aimed to examine gut mycobiome composition in obese subjects in two geographically different regions in China and to identify specific gut fungi associated with obesity. A total of 217 subjects from two regions with different urbanization levels [Hong Kong (HK): obese, n = 59; lean, n = 59; Kunming (KM): obese, n = 50; lean, n = 49. Mean body mass index (BMI) for obesity = 33.7] were recruited. We performed deep shotgun metagenomic sequencing on fecal samples to compare gut mycobiome composition and trophic functions in lean and obese subjects across these two regions. The gut mycobiome of obese subjects in both HK and KM were altered compared to those of lean subjects, characterized by a decrease in the relative abundance of Nakaseomyces, Schizosaccharomyces pombe, Candida dubliniensis and an increase in the abundance of Lanchanceathermotolerans, Saccharomyces paradox, Parastagonospora nodorum and Myceliophthorathermophila. Reduced fungal – bacterial and fungal – fungal correlations as well as increased negative fungal-bacterial correlations were observed in the gut of obese subjects. Furthermore, the anti-obesity effect of fungus S. pombe was further validated using a mouse model. Supplementing high-fat diet-induced obese mice with the fungus for 12 weeks led to a significant reduction in body weight gain (p

ABSTRACTThe fungal microbiota plays an important role in the pathogenesis of alcohol-associated liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD). In this study, we aimed to compare changes of the fecal fungal microbiota between patients with ALD and NAFLD and to elucidate patterns in different disease stages between the two conditions. We analyzed fungal internal transcribed spacer 2 (ITS2) sequencing using fecal samples from a cohort of 48 patients with ALD, 78 patients with NAFLD, and 34 controls. The fungal microbiota differed significantly between ALD and NAFLD. The genera Saccharomyces, Kluyveromyces, Scopulariopsis, and the species Candida albicans (C. albicans), Malassezia restricta (M. restricta), Scopulariopsis cordiae (S. cordiae) were significantly increased in patients with ALD, whereas the genera Kazachstania and Mucor were significantly increased in the NAFLD cohort. We identified the fungal signature consisting of Scopulariopsis, Kluyveromyces, M. restricta, and Mucor to have the highest discriminative ability to detect ALD vs NAFLD with an area under the curve (AUC) of 0.93. When stratifying the ALD and NAFLD cohorts by fibrosis severity, the fungal signature with the highest AUC of 0.92 to distinguish ALD F0-F1 vs NAFLD F0-F1 comprised Scopulariopsis, Kluyveromyces, Mucor, M. restricta, and Kazachstania. For more advanced fibrosis stages (F2-F4), the fungal signature composed of Scopulariopsis, Kluyveromyces, Mucor, and M. restricta achieved the highest AUC of 0.99 to differentiate ALD from NAFLD. This is the first study to identify a fungal signature to differentiate two metabolic fatty liver diseases from each other, specifically ALD from NAFLD. This might have clinical utility in unclear cases and might hence help shape treatment approaches. However, larger studies are required to validate this fungal signature in other populations of ALD and NAFLD.

The structure of the leaf microbiome can alter host fitness and change in response to abiotic and biotic factors, like seasonality, climate, and leaf age. However, relatively few studies consider the influence of host age on microbial communities at a time scale of a few days, a short time scale relevant to microbes. To understand how host age modulates changes in the fungal and bacterial leaf microbiome on a short time scale, we ran independent field and greenhouse-based studies and characterized phyllosphere communities using next-generation sequencing approaches. Our field study characterized changes in the fungal and bacterial phyllosphere by examining leaves of different relative ages across individuals, whereas the greenhouse study examined changes in the fungal microbiome by absolute leaf age across individuals. Together, these results indicate that fungal communities are susceptible to change as a leaf ages as evidenced by shifts in the diversity of fungal taxa both in the field and the greenhouse. Similarly, there were increases in the diversity of fungal taxa by leaf age in the greenhouse. In bacterial communities in the field, we observed changes in the diversity, composition, and relative abundance of common taxa. These findings build upon previous literature characterizing host-associated communities at longer time scales and provide a foundation for targeted work examining how specific microbial taxa might interact with each other, such as fine-scale interactions between pathogenic and non-pathogenic species.

Obesity, type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD) are three common metabolic diseases with high prevalence worldwide. Emerging evidence suggests that gut dysbiosis may influence the development of metabolic diseases, in which gut fungal microbiome (mycobiome) is actively involved. In this review, we summarize the studies exploring the composition changes of gut mycobiome in metabolic diseases and mechanisms by which fungi affect the development of metabolic diseases. The current mycobiome-based therapies, including probiotic fungi, fungal products, anti-fungal agents and fecal microbiota transplantation (FMT), and their implication in treating metabolic diseases are discussed. We highlight the unique role of gut mycobiome in metabolic diseases, providing perspectives for future research on gut mycobiome in metabolic diseases.

This issue of the Biomedical Journal features a special section exploring mycobiota. Three articles examine the role of fungi in common metabolic disorders in, Clostridium difficile infection, and in immunocompromised patients. Additionally, the potential and challenges of the metaverse in healthcare are reviewed, alongside a holistic approach to improve patient outcomes in pancreatic cancer. In this issue also possible mechanism contributing to long COVID are discussed, as well as biomarkers that effectively predict sepsis outcomes, and key targets in osteosarcoma progression. Moreover, factors leading to peri-intubation cardiac arrest are analyzed, healthcare strategies from various regions are employed to predict cardiovascular events in Asian populations, two approaches to cavernous sinus dural arteriovenous fistula are compared, and a combination therapy against soft tissue sarcoma is presented.

ABSTRACT Bacterial communities are often concomitantly present with numerous microorganisms in the human body and other natural environments. Amplicon-based microbiome studies have generally paid skewed attention, that too at a rather shallow genus level resolution, to the highly abundant bacteriome, with interest now forking toward the other microorganisms, particularly fungi. Given the generally sparse abundance of other microbes in the total microbiome, simultaneous sequencing of amplicons targeting multiple microbial kingdoms could be possible even with full multiplexing. Guiding studies are currently needed for performing and monitoring multi-kingdom-amplicon sequencing and data capture at scale. Aiming to address these gaps, amplification of full-length bacterial 16S rRNA gene and entire fungal internal-transcribed spacer (ITS) region was performed for human saliva samples (n = 96, including negative and positive controls). Combined amplicon DNA libraries were prepared for nanopore sequencing using a major fraction of 16S molecules and a minor fraction of ITS amplicons. Sequencing was performed in a single run of an R10.4.1 flow cell employing the latest V14 chemistry. An approach for real-time monitoring of the species saturation using dynamic rarefaction was designed as a guiding determinant of optimal run time. Real-time saturation monitoring for both bacterial and fungal species enabled the completion of sequencing within 30 hours, utilizing less than 60% of the total nanopores. Approximately 5 million high quality (HQ) taxonomically assigned reads were generated (~4.2 million bacterial and 0.7 million fungal), providing a wider (beyond bacteriome) snapshot of human oral microbiota at species-level resolution. Among the more than 400 bacterial and 240 fungal species identified in the studied samples, the species of Streptococcus (e.g., Streptococcus mitis and Streptococcus oralis) and Candida (e.g., Candida albicans and Candida tropicalis) were observed to be the dominating microbes in the oral cavity, respectively. This conformed well with the previous reports of the human oral microbiota. EnsembleSeq provides a proof-of-concept toward the identification of both fungal and bacterial species simultaneously in a single fully multiplexed nanopore sequencing run in a time- and resource-effective manner. Details of this workflow, along with the associated codebase, are provided to enable large-scale application for a holistic species-level microbiome study.IMPORTANCEHuman microbiome is a sum total of a variety of microbial genomes (including bacteria, fungi, protists, viruses, etc.) present in and on the human body. Yet, a majority of amplicon-based microbiome studies have largely remained skewed toward bacteriome as an assumed proxy of the total microbiome, primarily at a shallow genus level. Cost, time, effort, data quality/management, and importantly lack of guiding studies often limit progress in the direction of moving beyond bacteriome. Here, EnsembleSeq presents a proof-of-concept toward concomitantly capturing multiple-kingdoms of microorganisms (bacteriome and mycobiome) in a fully multiplexed (96-sample) single run of long-read amplicon sequencing. In addition, the workflow captures dynamic tracking of species-level saturation in a time- and resource-effective manner.

Abstract Background Amplicon-based mycobiome analysis has the potential to identify all fungal species within a sample and hence could provide a valuable diagnostic assay for use in clinical mycology settings. In the last decade, the mycobiome has been increasingly characterised by targeting the internal transcribed spacer (ITS) regions. Although ITS targets give broad coverage and high sensitivity, they fail to provide accurate quantitation as the copy number of ITS regions in fungal genomes is highly variable even within species. To address these issues, this study aimed to develop a novel NGS fungal diagnostic assay using an alternative amplicon target. Methods Novel universal primers were designed to amplify a highly diverse single copy and uniformly sized DNA target (Tef1) to enable mycobiome analysis on the Illumina iSeq100 which is a low cost, small footprint and simple to use next-generation sequencing platform. To enable automated analysis and rapid results, a streamlined bioinformatics workflow and sequence database were also developed. Sequencing of mock fungal communities was performed to compare the Tef1 assay and established ITS1-based method. The assay was further evaluated using clinical respiratory samples and the feasibility of using internal spike-in quantitative controls was assessed. Results The Tef1 assay successfully identified and quantified Aspergillus, Penicillium, Candida, Cryptococcus, Rhizopus, Fusarium and Lomentospora species from mock communities. The Tef1 assay was also capable of differentiating closely related species such as A. fumigatus and A. fischeri. In addition, it outperformed ITS1 at identifying A. fumigatus and other filamentous pathogens in mixed fungal communities (in the presence or absence of background human DNA). The assay could detect as few as 2 haploid genome equivalents of A. fumigatus from clinical respiratory samples. Lastly, spike-in controls were demonstrated to enable semi-quantitation of A. fumigatus load in clinical respiratory samples using sequencing data. Conclusions This study has developed and tested a novel metabarcoding target and found the assay outperforms ITS1 at identifying clinically relevant filamentous fungi. The assay is a promising diagnostic candidate that could provide affordable NGS analysis to clinical mycology laboratories.

Abstract 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.

The effects that mycobiomes have on physiological traits in turfgrasses are poorly understood. Drought tolerance, an economically and ecologically important trait, can be influenced by symbiotic fungi. In this two-year study, we evaluated the mycobiome associated with tall fescue exposed to prolonged periods of drought stress in a rainout shelter. Twelve plants, comprising six sets of half-sibs (progenies having one parent in common), one exhibiting a drought-tolerant phenotype and the other a drought-susceptible phenotype, were selected for analysis each year. The mycobiomes associated with the shoot, root, and rhizosphere soil were evaluated for each tall fescue half-sib pair using both short-read Illumina MiSeq and long-read Oxford Nanopore Technology (ONT) MinION sequencing pipelines. Both platforms sequenced portions of the fungal nuclear ribosomal RNA genes. The Illumina MiSeq sequenced the internal transcribed spacer region (ITS, 600 bp), while the ONT MinION covered the small subunit, ITS, and partial large subunit (4,600 bp). Both sequencing pipelines revealed that the mycobiomes associated with the roots, shoots, and soil were significantly different. The ONT MinION pipeline identified more diverse fungal lineages and had higher taxonomic resolution than the Illumina pipeline. Our results also indicated that root pathogens may play a more important role than endophytic or mycorrhizal symbionts in tall fescue drought stress tolerance.

Abstract Background The accuracy of internal-transcribed-spacer (ITS) and shotgun metagenomics has not been robustly evaluated, and the effect of diet on the composition and function of the bacterial and fungal gut microbiome in a longitudinal setting has been poorly investigated. Here we compared two approaches to study the fungal community (ITS and shotgun metagenomics), proposed an enrichment protocol to perform a reliable mycobiome analysis using a comprehensive in-house fungal database, and correlated dietary data with both bacterial and fungal communities. Results We found that shotgun DNA sequencing after a new enrichment protocol combined with the most comprehensive and novel fungal databases provided a cost-effective approach to perform gut mycobiome profiling at the species level and to integrate bacterial and fungal community analyses in fecal samples. The mycobiome was significantly more variable than the bacterial community at the compositional and functional levels. Notably, we showed that microbial diversity, composition, and functions were associated with habitual diet composition instead of driven by global dietary changes. Our study indicates a potential competitive inter-kingdom interaction between bacteria and fungi for food foraging. Conclusion Together, our present work proposes an efficient workflow to study the human gut microbiome integrating robustly fungal, bacterial, and dietary data. These findings will further advance our knowledge of the interaction between gut bacteria and fungi and pave the way for future investigations in human mycobiome. Video Abstract

Inflammatory bowel diseases (IBD) have a complex, poorly understood pathogenesis and lack long-lasting effective treatments. Recent research suggests that intestinal fungal dysbiosis may play a role in IBD development. This study investigates the effects of the glucocorticoid-induced leucine zipper protein (GILZp)”, known for its protective role in gut mucosa, and a yeast extract (Py) with prebiotic properties, either alone or combined, in DSS-induced colitis. Both treatments alleviated symptoms via overlapping or distinct mechanisms. In particular, they reduced the transcription levels of pro-inflammatory cytokines IL-1β and TNF-α, as well as the expression of the tight junction protein Claudin-2. Additionally, GILZp increased MUC2 transcription, while Py reduced IL-12p40 and IL-6 levels. Notably, both treatments were effective in restoring the intestinal burden of clinically important Candida and related species. Intestinal mycobiome analysis revealed that they were able to reduce colitis-associated fungal dysbiosis, and this effect was mainly the result of a decreased abundance of the Meyerozima genus, which was dominant in colitic mice. Overall, our results suggest that combined treatment regimens with GILZp and Py could represent a new strategy for the treatment of IBD by targeting multiple mechanisms, including the fungal dysbiosis.

ABSTRACTParasiticide fungi are considered an accurate, sustainable, and safe solution for the biocontrol of animal gastrointestinal (GI) parasites. This research provides an initial characterization of the virulence of the native parasiticide fungus Mucor circinelloides (FMV-FR1) and an assessment of its impact on birds’ gut microbes. The genome of this fungus was sequenced to identify the genes coding for virulence factors. Also, this fungus was checked for the phenotypic expression of proteinase, lecithinase, DNase, gelatinase, hemolysin, and biofilm production. Finally, an in vivo trial was developed based on feeding M. circinelloides spores to laying hens and peacocks three times a week. Bird feces were collected for 3 months, with total genomic DNA being extracted and subjected to long-read 16S and 25S-28S sequencing. Genes coding for an iron permease (FTR1), iron receptors (FOB1 and FOB2), ADP-ribosylation factors (ARFs) (ARF2 and ARF6), and a GTPase (CDC42) were identified in this M. circinelloides genome. Also, this fungus was positive only for lecithinase activity. The field trial revealed a fecal microbiome dominated by Firmicutes and Proteobacteria in laying hens, and Firmicutes and Bacteroidetes in peacocks, whereas the fecal mycobiome of both bird species was mainly composed of Ascomycetes and Basidiomycetes fungi. Bacterial and fungal alpha-diversities did not differ between sampling time points after M. circinelloides administrations (P = 0.62 and P = 0.15, respectively). Although findings from this research suggest the lack of virulence of this M. circinelloides parasiticide isolate, more complementary in vitro and in vivo research is needed to conclude about the safety of its administration to birds, aiming at controlling their GI parasites.IMPORTANCEA previous study revealed that the native Mucor circinelloides isolate (FMV-FR1) can develop parasiticide activity toward coccidia oocysts, one of the most pathogenic GI parasites in birds. However, ensuring its safety for birds is of utmost importance, namely by studying its virulence profile and potential effect on commensal gut microbes. This initial study revealed that although this M. circinelloides isolate had genes coding for four types of virulence factors—iron permease, iron receptors, ADP-ribosylation factors, and GTPase—and only expressed phenotypically the enzyme lecithinase, the administration of its spores to laying hens and peacocks did not interfere with the abundances and diversities of their gut commensal bacteria and fungi. Although overall results suggest the lack of virulence of this M. circinelloides isolate, more complementary research is needed to conclude about the safety of its administration to birds in the scope of parasite biocontrol programs.

Abstract The human microbiome exhibits a profound connection with the cancer development, progression, and therapeutic response, with particular emphasis on its components of the mycobiome, which are still in the early stages of research. In this review, we comprehensively summarize cancer‐related symbiotic and pathogenic fungal genera. The intricate mechanisms through which fungi impact cancer as an integral member of both gut and tissue‐resident microbiomes are further discussed. In addition, we shed light on the pivotal physiological roles of various nutrients, including cholesterol, carbohydrates, proteins and minerals, in facilitating the growth, reproduction, and invasive pathogenesis of the fungi. While our exploration of the interplay between nutrients and cancer, mediated by the mycobiome, is ongoing, the current findings have yet to yield conclusive results. Thus, delving into the relationship between nutrients and fungal pathogenesis in cancer development and progression would provide valuable insights into anticancer therapy and foster precision nutrition and individualized treatments that target fungi from bench to bedside.

Clostridioides difficile infection (CDI) is responsible for around 300,000 hospitalizations yearly in the United States, with the associated monetary cost being billions of dollars. Gut microbiome dysbiosis is known to be important to CDI. To the best of our knowledge, metatranscriptomics (MT) has only been used to characterize gut microbiome composition and function in one prior study involving CDI patients. Therefore, we utilized MT to investigate differences in active community diversity and composition between CDI+ (n = 20) and CDI− (n = 19) samples with respect to microbial taxa and expressed genes. No significant (Kruskal-Wallis, p > 0.05) differences were detected for richness or evenness based on CDI status. However, clustering based on CDI status was significant for both active microbial taxa and expressed genes datasets (PERMANOVA, p ≤ 0.05). Furthermore, differential feature analysis revealed greater expression of the opportunistic pathogens Enterocloster bolteae and Ruminococcus gnavus in CDI+ compared to CDI− samples. When only fungal sequences were considered, the family Saccharomycetaceae expressed more genes in CDI−, while 31 other fungal taxa were identified as significantly (Kruskal-Wallis p ≤ 0.05, log(LDA) ≥ 2) associated with CDI+. We also detected a variety of genes and pathways that differed significantly (Kruskal-Wallis p ≤ 0.05, log(LDA) ≥ 2) based on CDI status. Notably, differential genes associated with biofilm formation were expressed by C. difficile. This provides evidence of another possible contributor to C. difficile’s resistance to antibiotics and frequent recurrence in vivo. Furthermore, the greater number of CDI+ associated fungal taxa constitute additional evidence that the mycobiome is important to CDI pathogenesis. Future work will focus on establishing if C. difficile is actively producing biofilms during infection and if any specific fungal taxa are particularly influential in CDI.

The role of the oral microbiota in the overall health and in systemic diseases has gained more importance in the recent years, mainly due to the systemic effects that are mediated by the chronic inflammation caused by oral diseases, such as periodontitis, through the microbial communities of the mouth. The chronic infection by the human immunodeficiency virus (HIV) interacts at the tissue level (e.g. gut, genital tract, brain) to create reservoirs; the modulation of the gut microbiota by HIV infection is a good example of these interactions. The purpose of the present review is to assess the state of knowledge on the oral microbiota (microbiome, mycobiome and virome) of HIV-infected patients in comparison to that of HIV-negative individuals and to discuss the reciprocal influence of HIV infection and oral microbiota in patients with periodontitis on the potential establishment of a viral gingival reservoir. The influence of different clinical and biological parameters are reviewed including age, immune and viral status, potent antiretroviral therapies, smoking, infection of the airway and viral coinfections, all factors that can modulate the oral microbiota during HIV infection. The analysis of the literature proposed in this review indicates that the comparisons of the available studies are difficult due to their great heterogeneity. However, some important findings emerge: (i) the oral microbiota is less influenced than that of the gut during HIV infection, although some recurrent changes in the microbiome are identified in many studies; (ii) severe immunosuppression is correlated with altered microbiota and potent antiretroviral therapies correct partially these modifications; (iii) periodontitis constitutes a major factor of dysbiosis, which is exacerbated in HIV-infected patients; its pathogenesis can be described as a reciprocal reinforcement of the two conditions, where the local dysbiosis present in the periodontal pocket leads to inflammation, bacterial translocation and destruction of the supporting tissues, which in turn enhances an inflammatory environment that perpetuates the periodontitis cycle. With the objective of curing viral reservoirs of HIV-infected patients in the future years, it appears important to develop further researches aimed at defining whether the inflamed gingiva can serve of viral reservoir in HIV-infected patients with periodontitis.

ABSTRACTReptiles and amphibians (herptiles) are some of the most endangered and threatened species on the planet and numerous conservation strategies are being implemented with the goal of ensuring species recovery. Little is known, however, about the gut microbiome of wild herptiles and how it relates to the health of these populations. Here, we report results from the gut microbiome characterization of both a broad survey of herptiles, and the correlation between the fungus Basidiobolus, and the bacterial community supported by a deeper, more intensive sampling of Plethodon glutinosus, known as slimy salamanders. We demonstrate that bacterial communities sampled from frogs, lizards, and salamanders are structured by the host taxonomy and that Basidiobolus is a common and natural component of these wild gut microbiomes. Intensive sampling of multiple hosts across the ecoregions of Tennessee revealed that geography and host:geography interactions are strong predictors of distinct Basidiobolus operational taxonomic units present within a given host. Co-occurrence analyses of Basidiobolus and bacterial community diversity support a correlation and interaction between Basidiobolus and bacteria, suggesting that Basidiobolus may play a role in structuring the bacterial community. We further the hypothesis that this interaction is advanced by unique specialized metabolism originating from horizontal gene transfer from bacteria to Basidiobolus and demonstrate that Basidiobolus is capable of producing a diversity of specialized metabolites including small cyclic peptides.IMPORTANCEThis work significantly advances our understanding of biodiversity and microbial interactions in herptile microbiomes, the role that fungi play as a structural and functional members of herptile gut microbiomes, and the chemical functions that structure microbiome phenotypes. We also provide an important observational system of how the gut microbiome represents a unique environment that selects for novel metabolic functions through horizontal gene transfer between fungi and bacteria. Such studies are needed to better understand the complexity of gut microbiomes in nature and will inform conservation strategies for threatened species of herpetofauna.

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer, with a high mortality rate. There is growing evidence supporting a link between oral cancer and the microbiome. The microbiome can impact various aspects of cancer, such as pathogenesis, diagnosis, treatment, and prognosis. While there is existing information on bacteria and its connection to oral cancer, the fungi residing in the oral cavity represent a significant component of the microbiome that remains in its early stages of exploration and understanding. Fungi comprise a minuscule part of the human microbiome called the mycobiome. Mycobiome is ubiquitous in the human body but a weakened immune system offers a leeway space for fungi to showcase its virulence. The role of mycobiome as a colonizer, facilitator, or driver of carcinogenesis is still ambiguous. Reactivating the mycobiome that undergoes collateral damage associated with cancer treatment can be watershed event in cancer research. The coordinated, virulent, non-virulent behavior of the fungi once they reach a critical density must be hacked, considering its diagnostic, prognostic and therapeutic implications in cancer. This review highlights the diversity of the mycobiome and its potential role in oral cancer.

Plants recruit diverse microbial communities from the soil to their roots. Intermicrobial interactions and connectivity in the root microbiome play essential roles in plant health by promoting resistance to soilborne pathogens. However, the understanding of these interactions under field conditions is still scarce. Using a strawberry crop model, we characterized the prokaryotic and the fungal communities in the rhizosphere and the roots of three strawberry cultivars grown under field conditions and displaying varying degrees of resistance to the soilborne fungal pathogen Macrophomina phaseolina. We tested the hypothesis that resistant cultivars assemble distinct bacterial and fungal communities that foster microbial connectivity and mediate disease resistance. Our results show that the soilborne pathogen M. phaseolina does not alter the root microbiome of the three strawberry cultivars. Microbiome comparative analysis indicated that the highly susceptible cultivar Sweet Ann assembled a distinct rhizosphere and root microbiome, whereas the microbiome of the strawberry cultivars Marquis and Manresa were more similar and enriched with potential beneficial microbes. Cooccurrence network analysis revealed that the fungal pathogen M. phaseolina was more peripheral in the microbial network of Sweet Ann compared with Manresa and Marquis. Collectively, these results stress the role of the plant microbiome in mediating resistance against soilborne pathogens and further suggest the role of plant genetic traits in the assembly of beneficial microbiome members. Our study reinforces the eminent role of the plant microbiome as trait selection in breeding programs and the need for further understanding of the genetic and biological mechanisms that mediate microbiome assembly. Uncovering these mechanisms will be key for the future success of plant breeding programs in their fight against soilborne pathogens. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Invasive plants, which cause substantial economic and ecological impacts, acquire both pathogens and beneficial microbes in their introduced ranges. Communities of fungal endophytes are known to mediate impacts of pathogens on plant fitness but few studies have examined the temporal dynamics of fungal communities on invasive plants. The annual grass Microstegium vimineum, an invader of forests and riparian areas throughout the eastern United States, experiences annual epidemics of disease caused by Bipolaris pathogens. Our objective was to characterize the dynamics of foliar fungal communities on M. vimineum over a growing season during a foliar disease epidemic. First, we asked how the fungal community in the phyllosphere changed over 2 months that corresponded with increasing disease severity. Second, we experimentally suppressed disease with fungicide in half of the plots and asked how the treatment affected fungal community diversity and composition. We found increasingly diverse foliar fungal communities and substantial changes in community composition between timepoints using high-throughput amplicon sequencing of the internal transcribed spacer 2 region. Monthly fungicide application caused shifts in fungal community composition relative to control samples. Fungicide application increased diversity at the late-season timepoint, suggesting that it suppressed dominant fungicide sensitive taxa and allowed other fungal taxa to flourish. These results raise new questions regarding the roles of putative endophytes found in the phyllosphere given the limited number of pathogens known to cause disease on M. vimineum in its invasive range.

The human gastrointestinal ecosystem, or microbiome (comprising the total bacterial genome in an environment), plays a crucial role in influencing host physiology, immune function, metabolism, and the gut–brain axis. While bacteria, fungi, viruses, and archaea are all present in the gastrointestinal ecosystem, research on the human microbiome has predominantly focused on the bacterial component. The colonization of the human intestine by microbes during the first two years of life significantly impacts subsequent composition and diversity, influencing immune system development and long-term health. Early-life exposure to pathogens is crucial for establishing immunological memory and acquired immunity. Factors such as maternal health habits, delivery mode, and breastfeeding duration contribute to gut dysbiosis. Despite fungi’s critical role in health, particularly for vulnerable newborns, research on the gut mycobiome in infants and children remains limited. Understanding early-life factors shaping the gut mycobiome and its interactions with other microbial communities is a significant research challenge. This review explores potential factors influencing the gut mycobiome, microbial kingdom interactions, and their connections to health outcomes from childhood to adulthood. We identify gaps in current knowledge and propose future research directions in this complex field.

The human lung microbiome remains largely underexplored, despite its potential implications in the pharmacokinetics of inhaled drugs and its involvement in lung diseases. Interactions within these bacterial communities and with the host are complex processes which often involve microbial small molecules. In this study, we employed a computational approach to describe the metabolic potential of the human lung microbiome. By utilizing antiSMASH and BiG-SCAPE software, we identified 1831 biosynthetic gene clusters for the production of specialized metabolites in a carefully compiled genome database of lung-associated bacteria and fungi. It was shown that RiPPs represent the largest class of natural products within the bacteriome, while NRPs constitute the largest class of natural products in the lung mycobiome. All predicted BGCs were further categorized into 767 gene cluster families, and a subsequent network analysis highlighted that these families are widely distributed and contain many uncharacterized members. Moreover, in-depth annotation allowed the assignment of certain gene clusters to putative lung-specific functions within the microbiome, such as osmoadaptation or surfactant synthesis. This study establishes the lung microbiome as a prolific source for secondary metabolites and lays the groundwork for detailed investigation of this unique environment.

Abstract The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among DoD organizations and to facilitate resource, material and information sharing amongst consortium members, which includes collaborators in academia and industry. The 6th Annual TSMC Symposium was a hybrid meeting held in Fairlee, Vermont on 27–28 September 2022 with presentations and discussions centered on microbiome-related topics within seven broad thematic areas: (1) Human Microbiomes: Stress Response; (2) Microbiome Analysis & Surveillance; (3) Human Microbiomes Enablers & Engineering; (4) Human Microbiomes: Countermeasures; (5) Human Microbiomes Discovery - Earth & Space; (6) Environmental Micro & Myco-biome; and (7) Environmental Microbiome Analysis & Engineering. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted innovative research being done in academia and industry that can be leveraged by the DoD, and fostered collaborative opportunities. This report summarizes the activities and outcomes from the 6th annual TSMC symposium.

There is growing evidence of the role of fungal microbiota in the pathogenesis of inflammatory bowel disease (IBD). Fungi can exert direct pro-inflammatory effects or modify the bacterial composition via interkingdom interactions. Although several studies have demonstrated alterations in the fecal fungal microbiota composition in IBD, there is a wide variation in the mycobiome in different populations, with no definite pattern that can define the mycobiome in IBD having yet been identified. Recent work has suggested that characterizing the fecal fungal composition may influence therapeutic decisions and help to predict outcomes in a subset of IBD patients. In this study, we review the current literature on the emerging role of the fecal mycobiome as a potential tool for precision medicine in IBD.

Objective: This study analyzes the mycobiome in wild and captive Sumatran orangutans. Materials and Methods: Nine orangutan feces samples from the wild and nine from captivity were divided into three repeats from 11- to 15-year-olds in good health. The Illumina platform for analysis of ITS bioinformatics was used according to the Qiime2 and CCMetagen approaches. Results: Wild Sumatran orangutans include 53% Ascomycota, 38% uncultured fungi, and 4% Basidiomycota. Orangutans in captivity are 57% Ascomycota, 26% uncultured fungi, and 2% Basidiomycota. Based on genus level, uncultured Neurospora (31%), Penicillium (10%), Aspergillus (3%), Fusarium (3%), Candida (2%), Cutaneotrichosporon (2%), and Limonomyces (2%) are found in wild orangutans. The most prevalent genus among captivity orangutans is Aspergillus (32%), followed by fungal sp. (11%), Lasiodiplodia (18%), Devriesia (2%), and Sordariomycetes (2%). According to the Chao1 diversity index and Shannon and Simpson, there was no significant difference between wild and captive Sumatran orangutans. Conclusion: Neurospora is unique to wild Sumatran orangutans, although Aspergillus predom¬inates in captive orangutans. We hypothesize that the gut mycobiome of wild orangutans will resemble that of orangutans in captivity. The excellent range of food sources in the forest does not result in the prevalence of fungi in the typical gut microbiome. [J Adv Vet Anim Res 2023; 10(2.000): 228-236]

Seeds acquire fungal endophytes either from the environment or from their progenitors. These transmission modes are central in shaping the microbiome because they affect species composition and balance. We studied fungal endophyte communities (FECs) in three plant species: bread wheat (Triticum aestivum), wild emmer wheat (Triticum turgidum dicoccoides), and wild barley (Hordeum spontaneum). We conducted two experiments to test seed-to-seed transmission: (i) we compared FECs in stems and seeds collected from agricultural and natural habitats and (ii) we grew plants under greenhouse conditions to isolate the effect of vertical transmission on the plant FECs. The analysis of seed communities revealed differences in FEC composition and diversity among plant species; however, Alternaria infectoria dominated FECs in all plant species. In field-collected plants, the number of taxa in the seeds was less than half the number in stems, and close to 90% of the seed taxa were found in stems. The FECs from stems and seeds of greenhouse-grown plants were more diverse than the FECs of original seeds; they lacked a single dominant taxon, and the FECs in the new seeds had a similar richness and diversity to stem FECs, with only 40% overlap. The controlled-environment experiment confirmed vertical transmission of certain species but also showed that external infection of the seeds is the main source for specific taxa. Our results show that many taxa can reach the seeds internally, albeit in different abundance, that both infection sources affect seed FEC composition, and that external conditions affect the balance between FECs within the plant.

ABSTRACT Plants harbor complex and highly diverse fungal endophyte communities (FECs), making it difficult to evaluate the functional role of individual taxa, subsets of the community, or the FEC as a whole. To reduce the complexity of this system, we aimed to produce fungi-null wheat (Triticum aestivum) plants. To this end, we treated seeds with heat and fungicides and generated plants from rescued embryos and callus tissue. A culture-based approach and reverse transcription PCR analysis were negative, indicating that all treatments produced plants apparently free of fungi. However, the analysis of DNA using digital droplet PCR and next-generation sequencing revealed that tissues from all treatments retained low levels but diversity-rich FECs. While the FECs varied in composition across treatments and tissues, they all included core taxa of the mycobiome. The reduced fungal biomass, along with the changes in FEC composition, negatively affected plant development, supporting a FEC contribution to proper plant development and fitness. Our discovery that a large part of the FEC cannot be separated from plants and can be transmitted through seeds and tissue culture calls for reevaluation of particular microbiome paradigms, such as core taxa concepts, transmission modes, and functional species.IMPORTANCEThe native microbiome in a given plant must be considered when evaluating the effect of a single taxon or synthetic community. The pre-existing microbiome can interact with artificially added microbial cargo, which affects the final outcome. Such issues can be at least partially solved by the use of endophyte-free plants, which provide a clean background that should be useful in determining the effect of a single taxon, taxa combinations, or the entire microbiome on plant performance. Previous reports regarded plants as endophyte-free or axenic by the lack of fungal growth on culture media or the generation of plants from tissue cultures. We showed here that while fungi could not be isolated from fungicide-treated or tissue culture-regenerated plants, nevertheless, all plants contained rich fungal endophyte communities; namely, it was impossible to create fungi-free wheat plants. Our results call for rethinking fundamental microbiome-related concepts, such as core taxa, transmission mode, and functional species.

Large intestine-dwelling helminths affect microbiome composition. In sub-Saharan Africa, where helminth infections are endemic, the use of chemotherapeutic drugs is the primary strategy for controlling soil-transmitted helminthiases (STHs). However, the emergence of anthelmintic resistance necessitates the urgent exploration of alternative and complementary treatments to achieve the World Health Organization’s goal of eliminating STHs. One promising avenue involves the manipulation of gut microbiota in at-risk populations. This study aimed to enhance the understanding of the interplay between Trichuris trichiura and the gut microbiome. In this study, we used the Mini-FLOTAC technique for parasitological analyses and a shotgun metagenomic sequencing approach to investigate the effect of T. trichiura on the gut microbiome by comparing infected and non-infected women of reproductive age (WRA) from Pemba. Structural and functional analyses of the gut microbiome revealed that T. trichiura infection shaped the host gut microbiome in WRA. Some taxa vary according to infection status. Prevotella genus was more abundant in healthy participants, whereas species such as Weissella cibaria, Leuconostoc citreum (new emergent probiotics), and Leuconostoc lactis (starter) decreased in infected individuals, suggesting the use of potential probiotic treatments to mitigate dysbiosis induced by STHs. Furthermore, the overall number of common fungi, irrespective of species, was significantly higher in the mycobiome of Trichuris infected participants. Functional analysis revealed significant differences in metabolic pathways (p < 0.05), with cholesterol metabolism and pathogenic infections being more abundant in the infected samples than in the non-infected samples. In conclusion, this study sheds light on the intricate interactions between helminth infections and the gut microbiome in the WRA, particularly in STH-endemic regions. The identified associations between specific gut microbial changes and T. trichiura infection may pave the way for innovative complementary treatments to effectively combat STHs.

As in-feed antibiotics are phased out of swine production, producers are seeking alternatives to facilitate improvements in growth typically seen from this previously common feed additive. Kazachstania slooffiae is a prominent commensal fungus in the swine gut that peaks in relative abundance shortly after weaning and has beneficial interactions with other bacteriome members important for piglet health. In this study, piglets were supplemented with K. slooffiae to characterize responses in piglet health as well as fungal and bacterial components of the microbiome both spatially (along the entire gastrointestinal tract and feces) and temporally (before, during, and after weaning). Litters were assigned to one of four treatments: no K. slooffiae (CONT); one dose of K. slooffiae 7 days before weaning (day 14; PRE); one dose of K. slooffiae at weaning (day 21; POST); or one dose of K. slooffiae 7 days before weaning and one dose at weaning (PREPOST). The bacteriome and mycobiome were analyzed from fecal samples collected from all piglets at day 14, day 21, and day 49, and from organ samples along the gastrointestinal (GI) tract at day 21 and day 49. Blood samples were taken at day 14 and day 49 for cytokine analysis, and fecal samples were assayed for antimicrobial resistance. While some regional shifts were seen in response to K. slooffiae administration in the mycobiome of the GI tract, no remarkable changes in weight gain or health of the animals were observed, and changes were more likely due to sow and the environment. Ultimately, the combined microbiome changed most considerably following the transition from suckling to nursery diets. This work describes the mycobiome along the piglet GI tract through the weaning transition for the first time. Based on these findings, K. slooffiae administered at this concentration may not be an effective tool to hasten colonization of K. slooffiae in the piglet GI tract around the weaning transition nor support piglet growth, microbial gut health, or immunity. However, diet and environment greatly influence microbial community development.

IntroductionAlterations of the gut microbiome are involved in the pathogenesis of Crohn’s disease (CD). The role of fungi in this context is unclear. This study aimed to determine postoperative changes in the bacterial and fungal gut communities of CD patients undergoing intestinal resection, and to evaluate interactions between the bacteriome and mycobiome and their impact on the patients’ outcome.MethodsWe report a subgroup analysis of a prospective cohort study, focusing on 10 CD patients whose fecal samples were collected for bacterial 16S rRNA and fungal ITS2 genes next-generation sequencing the day before surgery and on the 5th or 6th postoperative day.ResultsNo significant differences in bacterial and fungal diversity were observed between preoperative and postoperative stool samples. By in-depth analysis, significant postoperative abundance changes of bacteria and fungi and 17 interkingdom correlations were detected. Network analysis identified 13 microbial clusters in the perioperative gut communities, revealing symbiotic and competitive interactions. Relevant factors were gender, age, BMI, lifestyle habits (smoking, alcohol consumption) and surgical technique. Postoperative abundance changes and identified clusters were associated with clinical outcomes (length of hospital stay, complications) and levels of inflammatory markers.ConclusionsOur findings highlight the importance of dissecting the interactions of gut bacterial and fungal communities in CD patients and their potential influence on postoperative and disease outcomes.

The gut mycobiome plays an important role in the health and disease of the human gut, but its exact function is still under investigation. While there is a wealth of information available on the bacterial community of the human gut microbiome, research on the fungal community is still relatively limited. In particular, technical methodologies for mycobiome analysis, especially the DNA extraction method for human faecal samples, varied in different studies. In the current study, two commercial kits commonly used in DNA extraction, the QIAamp® Fast DNA Stool Mini Kit and DNeasy PowerSoil Pro Kit, and one manual method, the International Human Microbiome Standards Protocol Q, were compared. Furthermore, the effectiveness of two different bead-beating machines, the Mini-Beadbeater-16 and FastPrep-24TM 5G, was compared in parallel. A mock fungal community with a known composition of fungal strains was also generated and included to compare different DNA extraction methods. Our results suggested that the method using the DNeasy PowerSoil Pro Kit and Mini-Beadbeater-16 provides the best results to extract DNA from human faecal samples. Based on our data, we propose a standard operating procedure for DNA extraction from human faecal samples for mycobiome analysis.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is the most common chronic liver disease worldwide. Circadian disruptors, such as chronic jet lag (CJ), may be new risk factors for MAFLD development. However, the roles of CJ on MAFLD are insufficiently understood, with mechanisms remaining elusive. Studies suggest a link between gut microbiome dysbiosis and MAFLD, but most of the studies are mainly focused on gut bacteria, ignoring other components of gut microbes, such as gut fungi (mycobiome), and few studies have addressed the rhythm of the gut fungi. This study explored the effects of CJ on MAFLD and its related microbiotic and mycobiotic mechanisms in mice fed a high fat and high fructose diet (HFHFD). Forty-eight C57BL6J male mice were divided into four groups: mice on a normal diet exposed to a normal circadian cycle (ND-NC), mice on a normal diet subjected to CJ (ND-CJ), mice on a HFHFD exposed to a normal circadian cycle (HFHFD-NC), and mice on a HFHFD subjected to CJ (HFHFD-CJ). After 16 weeks, the composition and rhythm of microbiota and mycobiome in colon contents were compared among groups. The results showed that CJ exacerbated hepatic steatohepatitis in the HFHFD-fed mice. Compared with HFHFD-NC mice, HFHFD-CJ mice had increases in Aspergillus, Blumeria and lower abundances of Akkermansia, Lactococcus, Prevotella, Clostridium, Bifidobacterium, Wickerhamomyces, and Saccharomycopsis genera. The fungi-bacterial interaction network became more complex after HFHFD and/or CJ interventions. The study revealed that CJ altered the composition and structure of the gut bacteria and fungi, disrupted the rhythmic oscillation of the gut microbiota and mycobiome, affected interactions among the gut microbiome, and promoted the progression of MAFLD in HFHFD mice.

ABSTRACTIn recent years, it has become clear that gut microbiota plays a major role in the human body, both in health and disease. Because of that, the gut microbiome and its impact on human well-being are getting wider and wider attention. Studies focused on the liver are not an exception. However, the majority of the analyses are concentrated on the bacterial part of the gut microbiota, while the fungi living in the human intestines are often omitted or underappreciated. This review is focused on the gut mycobiome as an important factor that should be taken into consideration regarding liver homeostasis and its perturbations. We have collected the findings in this field and we discuss their importance. We aim to emphasize the fungal compositional changes related to liver diseases and, by that, provide novel insights into the directions of liver research and gut microbiota as a therapeutic target for liver diseases.

Abstract Background Eukaryotes such as fungi and protists frequently accompany bacteria and archaea in microbial communities. Unfortunately, their presence is difficult to study with “shotgun” metagenomic sequencing since prokaryotic signals dominate in most environments. Recent methods for eukaryotic detection use eukaryote-specific marker genes, but they do not incorporate strategies to handle the presence of eukaryotes that are not represented in the reference marker gene set, and they are not compatible with web-based tools for downstream analysis. Results Here, we present CORRAL (for Clustering Of Related Reference ALignments), a tool for the identification of eukaryotes in shotgun metagenomic data based on alignments to eukaryote-specific marker genes and Markov clustering. Using a combination of simulated datasets, mock community standards, and large publicly available human microbiome studies, we demonstrate that our method is not only sensitive and accurate but is also capable of inferring the presence of eukaryotes not included in the marker gene reference, such as novel strains. Finally, we deploy CORRAL on our MicrobiomeDB.org resource, producing an atlas of eukaryotes present in various environments of the human body and linking their presence to study covariates. Conclusions CORRAL allows eukaryotic detection to be automated and carried out at scale. Implementation of CORRAL in MicrobiomeDB.org creates a running atlas of microbial eukaryotes in metagenomic studies. Since our approach is independent of the reference used, it may be applicable to other contexts where shotgun metagenomic reads are matched against redundant but non-exhaustive databases, such as the identification of bacterial virulence genes or taxonomic classification of viral reads. Video Abstract

Abstract As the Human Microbiome Project (HMP) progresses, the relationship between microbes and human health has been receiving increasing attention. A growing number of reports support the correlation between cancer and microbes. However, most studies have focused on bacteria, rather than fungal communities. In this study, we studied the alteration in lung mycobiome in patients with non-small-cell lung cancer (NSCLC) using metagenomic sequencing and qPCR. The higher fungal diversity and more complex network were observed in the patients with NSCLC. In addition, Alternaria arborescens was found as the most relevant fungus to NSCLC, and the enrichment of it in cancerous tissue was also detected. This study proposes that the changes in fungal communities may be closely related to lung cancer, and provides insights into further exploration the relationship between lung cancer and fungi.

Abstract Background The biodiversity of the mycobiota of soft cheese rinds such as Brie or Camembert has been extensively studied, but scant information is available on the fungi colonizing the rinds of cheese produced in the Southern Switzerland Alps. This study aimed at exploring the fungal communities present on rinds of cheese matured in five cellars in Southern Switzerland and to evaluate their composition with regards to temperature, relative humidity, type of cheese, as well as microenvironmental and geographic factors. We used macro- and microscopical morphology, matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry, and sequencing to characterize the fungal communities of the cheeses, and compared them with metabarcoding targeting the ITS region. Results Isolation by serial dilution yielded 201 isolates (39 yeasts and 162 filamentous fungi) belonging to 9 fungal species. Mucor and Penicillium were dominant, with Mucor racemosus, M. lanceolatus, P. biforme, and P. chrysogenum/rubens being the most frequent species. All but two yeast isolates were identified as Debaryomyces hansenii. Metabarcoding detected 80 fungal species. Culture work and metabarcoding produced comparable results in terms of similarity of the fungal cheese rind communities in the five cellars. Conclusions Our study has shown that the mycobiota on the rinds of the cheeses studied is a comparatively species-poor community influenced by temperature, relative humidity, type of cheese, and manufacturing steps, as well as microenvironmental and possibly geographic factors.

Abstract Background Suboptimal maternal oral health during pregnancy is potentially associated with adverse birth outcomes and increased dental caries risks in children. This study aimed to assess the oral microbiome and immune response following an innovative clinical regimen, Prenatal Total Oral Rehabilitation (PTOR), that fully restores women’s oral health to a “disease-free status” before delivery. Methods This prospective cohort study assessed 15 pregnant women at baseline and 3 follow-up visits (1 week, 2 weeks, and 2 months) after receiving PTOR. The salivary and supragingival plaque microbiomes were analyzed using metagenomic sequencing. Multiplexed Luminex cytokine assays were performed to examine immune response following PTOR. The association between salivary immune markers and oral microbiome was further examined. Results PTOR was associated with a reduction of periodontal pathogens in plaque, for instance, a lower relative abundance of Tannerella forsythia and Treponema denticola at 2 weeks compared to the baseline (p

Multiple sclerosis (MS) arises from a complex interplay between host genetic factors and environmental components, with the gut microbiota emerging as a key area of investigation. In the current study, we used ion torrent sequencing to delve into the bacteriome (bacterial microbiota) and mycobiome (fungal microbiota) of people with MS (pwMS), and compared them to healthy controls (HC). Through principal coordinate, diversity, and abundance analyses, as well as clustering and cross-kingdom microbial correlation assessments, we uncovered significant differences in the microbial profiles between pwMS and HC. Elevated levels of the fungus Torulaspora and the bacterial family Enterobacteriaceae were observed in pwMS, whereas beneficial bacterial taxa, such as Prevotelladaceae and Dialister, were reduced. Notably, clustering analysis revealed overlapping patterns in the bacteriome and mycobiome data for 74% of the participants, with weakened cross-kingdom interactions evident in the altered microbiota of pwMS. Our findings highlight the dysbiosis of both bacterial and fungal microbiota in MS, characterized by shifts in biodiversity and composition. Furthermore, the distinct disease-associated pattern of fungi–bacteria interactions suggests that fungi, in addition to bacteria, contribute to the pathogenesis of MS. Overall, our study sheds light on the intricate microbial dynamics underlying MS, paving the way for further investigation into the potential therapeutic targeting of the gut microbiota in MS management.

The checklist serves as an informative method for evaluating the diversity, geography, and ecology of established and reproducing macrofungi. Additionally, considering macrofungi as bioindicator species, their census should be incorporated into efforts to monitor the state of health of ecosystems and directly applied to conservation policies. Between 2019 and 2023, a census of macrofungal species was conducted in Taburno-Camposauro Regional Park (Campania, Italy) across nine distinct habitats. A total of 453 fungal taxa were identified, including several new records for the Campania region. The fungal diversity exhibited significant variations based on the dominant plant species in each habitat. Fagacean tree species and Carpinus spp. shared similar fungal communities. Equally, coniferous tree species displayed a comparable fungal composition. In Abies alba and mixed broad-leaved forests, low levels of ectomycorrhizal taxa were observed alongside a concurrent increase in saprotrophs, indicating a disturbed habitat and a reduction in the Gadgil effect. Notably, lower fungal diversity was documented in the grassland habitat, suggesting the potential implications of wildlife imbalance and excessive grazing. The provided checklist constitutes a valuable resource for local management authorities, providing insights to formulate specific management policies.

Candida (C.) infections represent a serious health risk for people affected by inflammatory bowel disease. An important fungal virulence factor is the capacity of the fungus to form biofilms on the colonized surface of the host. This research study aimed to determine the effect of a C. tropicalis and C. albicans co-infection on dextran sodium sulfate (DSS)-induced colitis in mice. The colitis severity was evaluated using histology and a colonoscopy. The mice were mono-inoculated with C. albicans or C. tropicalis or co-challenged with both species. The mice were administered 3% DSS to induce acute colitis. The biofilm activity was assessed using (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] 2H-tetrazoliumhydroxide (XTT) and dry-weight assays. The abundance of C. albicans in the colon tissues was assessed by immunohistochemistry. The co-challenged mice showed a decreased colitis severity compared to the mono-inoculated mice. The dry-weight assay demonstrated a marked decrease in C. albicans biofilm production in a C. albicans culture incubated with C. tropicalis supernatant. Immunohistochemical staining showed that C. albicans was more abundant in the mucosa of C. albicans mono-inoculated mice compared to the co-inoculated group. These data indicate an antagonistic microbial interaction between the two Candida species, where C. tropicalis may produce molecules capable of limiting the ability of C. albicans to adhere to the host intestinal surface, leading to a reduction in biofilm formation.

The severe and chronic inflammatory bowel diseases (IBD), Crohn disease and ulcerative colitis, are characterized by persistent inflammation and gut damage. There is an increasing recognition that the gut microbiota plays a pivotal role in IBD development and progression. However, studies of the complete microbiota composition (bacteria, fungi, viruses) from precise locations within the gut remain limited. In particular, studies have focused primarily on the bacteriome, with available methods limiting evaluation of the mycobiome (fungi) and virome (virus). Furthermore, while the different segments of the small and large intestine display different functions (e.g., digestion, absorption, fermentation) and varying microenvironment features (e.g., pH, metabolites), little is known about the biogeography of the microbiota in different segments of the intestinal tract or how this differs in IBD. Here, we highlight evidence of the differing microbiota communities of the intestinal sub-organs in healthy and IBD, along with method summaries to improve future studies.

BackgroundNearly everything on Earth harbors a microbiome. A microbiome is a community of microbes (bacteria, fungi, and viruses) with potential to form complex networks that involve mutualistic and antagonistic interactions. Resident microbiota on/in an organism are determined by the external environment, both biotic and abiotic, and the intrinsic adaptability of each organism. Although the maize microbiome has been characterized, community changes that result from the application of fungal biocontrol strains, such as non-aflatoxigenic Aspergillus flavus, have not.MethodsWe silk channel inoculated field-grown maize separately with a non-aflatoxigenic biocontrol strain (K49), a highly toxigenic strain (Tox4), and a combination of both A. flavus strains. Two maize inbreds were treated, A. flavus-susceptible B73 and A. flavus-resistant CML322. We then assessed the impacts of A. flavus introduction on the epibiota and endobiota of their maize kernels.ResultsWe found that the native microbial communities were significantly affected, irrespective of genotype or sampled tissue. Overall, bacteriomes exhibited greater diversity of genera than mycobiomes. The abundance of certain genera was unchanged by treatment, including genera of bacteria (e.g., Enterobacter, Pantoea) and fungi (e.g., Sarocladium, Meyerozyma) that are known to be beneficial, antagonistic, or both on plant growth and health.ConclusionBeneficial microbes like Sarocladium that responded well to A. flavus biocontrol strains are expected to enhance biocontrol efficacy, while also displacing/antagonizing harmful microbes.