Optimization of DNA extraction from human urinary samples for mycobiome community profiling.

IntroductionRecent data suggest the urinary tract hosts a microbial community of varying composition, even in the absence of infection. Culture-independent methodologies, such as next-generation sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, identifying both common commensals and fastidious organisms. A fundamental challenge has been the isolation of DNA representative of the entire resident microbial community, including fungi.Materials and methodsWe evaluated multiple modifications of commonly-used DNA extraction procedures using standardized male and female urine samples, comparing resulting overall, fungal and bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. This optimized protocol was tested against commercially available methodologies to compare overall and microbial DNA yields, community representation and diversity by next-generation sequencing (NGS).ResultsOverall and fungal-specific DNA yields from standardized urine samples demonstrated that microbial abundances differed significantly among the eight methods used. Methodologies that included multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase digestion, particularly in combination with small volume processing and pooling steps, provided more comprehensive representation of the range of bacterial and fungal species. Concentration of larger volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA levels and providing greater microbial representation and diversity.ConclusionsAlterations in the methodology of urine storage, preparation, and DNA processing improve microbial community profiling using culture-independent sequencing methods. Our optimized protocol for DNA extraction from urine samples provided improved fungal community representation. Use of this technique resulted in equivalent representation of the bacterial populations as well, making this a useful technique for the concurrent evaluation of bacterial and fungal populations by NGS

Alterations of the gut mycobiome in patients with MS

BACKGROUND: The mycobiome is the fungal component of the gut microbiome and is implicated in several autoimmune diseases. However, its role in MS has not been studied. METHODS: In this case-control observational study, we performed ITS sequencing and characterised the gut mycobiome in people with MS (pwMS) and healthy controls at baseline and after six months. FINDINGS: The mycobiome had significantly higher alpha diversity and inter-subject variation in pwMS than controls. Saccharomyces and Aspergillus were over-represented in pwMS. Saccharomyces was positively correlated with circulating basophils and negatively correlated with regulatory B cells, while Aspergillus was positively correlated with activated CD16 INTERPRETATION: There is an alteration of the gut mycobiome in pwMS, compared to healthy controls. Further study is required to assess any causal association of the mycobiome with MS and its direct or indirect interactions with bacteria and autoimmunity. FUNDING: This work was supported by the Washington University in St. Louis Institute of Clinical and Translational Sciences, funded, in part, by Grant Number # UL1 TR000448 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award (Zhou Y, Piccio, L, Lovett-Racke A and Tarr PI); R01 NS102633-04 (Zhou Y, Piccio L); the Leon and Harriet Felman Fund for Human MS Research (Piccio L and Cross AH). Cantoni C. was supported by the National MS Society Career Transition Fellowship (TA-1805-31003) and by donations from Whitelaw Terry, Jr. / Valerie Terry Fund. Ghezzi L. was supported by the Italian Multiple Sclerosis Society research fellowship (FISM 2018/B/1) and the National Multiple Sclerosis Society Post-Doctoral Fellowship (FG- 1907-34474). Anne Cross was supported by The Manny & Rosalyn Rosenthal-Dr. John L. Trotter MS Center Chair in Neuroimmunology of the Barnes-Jewish Hospital Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Gut Microbial Metabolism and Nonalcoholic Fatty Liver Disease.

The gut microbiome, the multispecies community of microbes that exists in the gastrointestinal tract, encodes several orders of magnitude more functional genes than the human genome. It also plays a pivotal role in human health, in part due to metabolism of environmental, dietary, and host-derived substrates, which produce bioactive metabolites. Perturbations to the composition and associated metabolic output of the gut microbiome have been associated with a number of chronic liver diseases, including nonalcoholic fatty liver disease (NAFLD). Here, we review the rapidly evolving suite of next-generation techniques used for studying gut microbiome composition, functional gene content, and bioactive products and discuss relationships with the pathogenesis of NAFLD

The Fungal Frontier: A Comparative Analysis of Methods Used in the Study of the Human Gut Mycobiome

This research was conducted with the financial support of Science Foundation Ireland (SFI) under Grant Number SFI/12/RC/2273. PS is funded by a Royal Society-Science Foundation Ireland University Research Fellowship.peer reviewedThe human gut is host to a diverse range of fungal species, collectively referred to as the gut “mycobiome”. The gut mycobiome is emerging as an area of considerable research interest due to the potential roles of these fungi in human health and disease. However, there is no consensus as to what the best or most suitable methodologies available are with respect to characterizing the human gut mycobiome. The aim of this study is to provide a comparative analysis of several previously published mycobiome-specific culture-dependent and -independent methodologies, including choice of culture media, incubation conditions (aerobic versus anaerobic), DNA extraction method, primer set and freezing of fecal samples to assess their relative merits and suitability for gut mycobiome analysis. There was no significant effect of media type or aeration on culture-dependent results. However, freezing was found to have a significant effect on fungal viability, with significantly lower fungal numbers recovered from frozen samples. DNA extraction method had a significant effect on DNA yield and quality. However, freezing and extraction method did not have any impact on either α or β diversity. There was also considerable variation in the ability of different fungal-specific primer sets to generate PCR products for subsequent sequence analysis. Through this investigation two DNA extraction methods and one primer set was identified which facilitated the analysis of the mycobiome for all samples in this study. Ultimately, a diverse range of fungal species were recovered using both approaches, with Candida and Saccharomyces identified as the most common fungal species recovered using culture-dependent and culture-independent methods, respectively. As has been apparent from ecological surveys of the bacterial fraction of the gut microbiota, the use of different methodologies can also impact on our understanding of gut mycobiome composition and therefore requires careful consideration. Future research into the gut mycobiome needs to adopt a common strategy to minimize potentially confounding effects of methodological choice and to facilitate comparative analysis of datasets.Science Foundation Irelan

Alterations of the Gut Mycobiome in Patients with MS - a Bioinformatic Approach

The mycobiome is the fungal component of the gut microbiome and is implicated in several autoimmune diseases. However, its role in multiple sclerosis (MS) has not been studied. We performed descriptive and formal statistical tests using the R language to characterize the gut mycobiome in people with MS (pwMS) and healthy controls. We found that the microbiome composition of multiple sclerosis patients is different from healthy people. The mycobiome had significantly higher alpha diversity and inter-subject variation in pwMS than controls. Additionally, Saccharomyces and Aspergillus were over-represented in pwMS. Different mycobiome profiles, defined as mycotypes, were associated with different bacterial abundances. Computer-based analysis of vast sequencing data will continue improving our understanding of the complicated microbiome community and their interactions with the host

Cytokine tuning of intestinal epithelial function

The intestine serves as both our largest single barrier to the external environment and the host of more immune cells than any other location in our bodies. Separating these potential combatants is a single layer of dynamic epithelium composed of heterogeneous epithelial subtypes, each uniquely adapted to carry out a subset of the intestine’s diverse functions. In addition to its obvious role in digestion, the intestinal epithelium is responsible for a wide array of critical tasks, including maintaining barrier integrity, preventing invasion by microbial commensals and pathogens, and modulating the intestinal immune system. Communication between these epithelial cells and resident immune cells is crucial for maintaining homeostasis and coordinating appropriate responses to disease and can occur through cell-to-cell contact or by the release or recognition of soluble mediators. The objective of this review is to highlight recent literature illuminating how cytokines and chemokines, both those made by and acting on the intestinal epithelium, orchestrate many of the diverse functions of the intestinal epithelium and its interactions with immune cells in health and disease. Areas of focus include cytokine control of intestinal epithelial proliferation, cell death, and barrier permeability. In addition, the modulation of epithelial-derived cytokines and chemokines by factors such as interactions with stromal and immune cells, pathogen and commensal exposure, and diet will be discussed

Humans as holobionts: systems-level approaches for disease prevention and therapy

The microbes that live in the gut, also known as the gut microbiota, play an important role in the well-being of the host. In the last years, the development of metagenomics and metabolomics have helped to better understand the vital role of the gut microbiome in human health and disease, however, the mechanisms and its implications are still not fully clarified. Therefore, more research and improved pipelines, protocols, and tools are needed to further investigate and understand the connection of the gut microbiome with host health. This dissertation aimed to implement and develop bioinformatic and statistical analyses to improve our understanding of the gut microbiome's role in non-alcoholic fatty liver disease (NAFLD) pathogenesis and management. In addition, during my Ph.D., I also investigated novel microbiome-based therapeutic strategies. In the different projects that form this dissertation, it is shown that the human body together with its microbiome forms a unity of life or holobiont indispensable for the well-functioning of the organism. The different bioinformatic analyses performed highlight the important role of the gut microbiome in human health and disease, especially giving new insights in relation to NAFLD pathogenesis and management. In addition, different microbiome-based strategies are explored showing the high potential of the gut microbiome in the development of new therapies. Therefore, the use of microbiome-related information for patient therapeutics needs to be further explored and applied to improve and develop new and more personalized treatments

Probiotics, Prebiotics and the Nervous System

Treballs Finals de Grau de Farmàcia, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, 2018. Tutora: Magdalena Rafecas.[eng] The relationship between the gut microbiota and the central nervous system (the microbiota-gut-brain axis) is an area of increasing interest and research. Studies based on germ-free models have provided a big amount of evidence of the connection between the gut and the brain. This research has done a great step forward in recent years, due to the application of metagenomics and bioinformatics. We now know that the human gut microbiome can be classified into three enterotypes, characterized by the variation in three genera: Bacteroides, Bacteroidetes, and Prevotella. Type of birth, formula feeding, and antibiotic intake are among the main factors that impact on infant microbiome assembly. Moreover, the composition of the gut microbiota is strongly associated with diet. A review of the bibliographical evidence connecting the alterations in the microbiome and some central nervous system disorders (as Alzheimer disease, Parkinson disease or autism spectrum disorder) shows us that the levels of Prevotella and the Firmicutes/Bacteroidetes ratio are altered in these pathologies. Accumulating data reveals that the microbiota-gut-brain axis can be modulated by the administration of probiotics and prebiotics. Moreover, some traditional fermented food has been seen to have probiotic properties and high-fiber containing diets have been associated with a lower Firmicutes/Bacteroidetes ratio and higher levels of Prevotella.[cat] La relació entre la microbiota intestinal i el sistema nerviós central (l'eix microbiota-intestí-cervell) és una àrea d'interès i investigació creixents. Els estudis basats en models lliures de gèrmens han proporcionat gran quantitat d’evidències de la connexió entre l'intestí i el cervell. Aquesta investigació ha fet un gran avenç en els últims anys gràcies a la metagenòmica i la bioinformàtica. Ara sabem que el microbioma intestinal humà es pot classificar en tres enterotips, caracteritzats per la variació en tres gèneres: Bacteroides, Bacteroidetes i Prevotella. Els tipus de naixement, el tipus d’alletament i la ingesta d'antibiòtics són els principals factors que afecten el desenvolupament del microbioma infantil. A més, la composició de la microbiota intestinal està fortament relacionada amb la dieta. Una revisió de les evidències bibliogràfiques que connecten les alteracions en el microbioma i alguns trastorns del sistema nerviós central (com la malaltia d'Alzheimer, la malaltia de Parkinson o el trastorn de l'espectre autista) ens mostra que els nivells de Prevotella i la relació Firmicutes/Bacteroidetes estan alterats en aquestes patologies. Cada cop hi ha més dades que revelen que l'eix microbiota-intestí-cervell pot ser modulat per l'administració de probiòtics i prebiòtics. D'altra banda, s'ha observat que alguns aliments fermentats tradicionals tenen propietats probiòtiques i que les dietes amb alt contingut de fibra s’associen a nivells inferiors de la raó Firmicutes /Bacteroidetes i superiors de Prevotella

The Treatment-naïve Mycobiome in Newonset Inflammatory Bowel Disease

Denne Ph.d.-afhandling undersøger forholdet mellem tarmens mycobiome og patogenesen af kronisk tarmbetændelse. Afhandlingen omfatter et systematisk review om tarmens mycobiome i kronisk tarmbetændelse, en protokolartikel for NORDTREAT kohorten af ny-diagnosticerede patienter med kronisk tarmbetændelse og en omfattende analyse af mycobiomet hos behandlingsnaive, nydiagnosticerede patienter med kronisk tarmbetændelse samt symptomatiske kontroller.Baggrund Genetiske risikofaktorer for kronisk tarmbetændelse er blevet forbundet med en øget risiko for svampeinfektioner og signalering af medfødte svampe immunreceptorer. De genetiske risikofaktorer kan potentielt spille en rolle i de serologiske responser mod svampe der ligeledes er knyttet til risikoen for at udvikle kroniske tarmbetændelser, disse er kendt som anti-Saccharomyces cerevisiae antistoffer (ASCA). Ud over B-celle-responset er specifikke cytotoksiske T-celle-responser mod tarmsvampe også forbundet med kronisk tarmbetændelse, hvilket yderligere forstærker forbindelsen mellem immunsystemet, svampe og kronisk tarmbetændelse.Formål Det systematiske review havde til formål at kortlægge den aktuelle viden om mycobiomet ved kronisk tarmbetændelse gennem en systematisk litteraturgennemgang. Dernæst designede og gennemførte vi en kohorte undersøgelse, blandt andet, for at forbedre vores forståelse af svampes rolle i patogenesen af kronisk tarmbetændelse. Endelig analyserede vi mycobiomet ved colitis ulcerosa, Crohns sygdom og symptomatiske kontroller med fokus på relativ forekomst, diversitetsmål og serologisk ASCA-status. Undersøgelsen benyttede NORDTREAT-kohorten samt den populations baserede norske IBSEN III kohorte af ny-diagnosticerede patienter med kronisk tarmbetændelse.Resultater Det systematiske review analyserede 27 studier, der fremhævede forskelle i beta-diversitet ved kronisk tarmbetændelse sammenlignet med kontroller og inkonsekvente fund vedrørende ændringer i forekomsten af specifikke svampe blev også konstateret. Candida og Malassezia blev ofte bemærket, men resultaterne varierede på grund af effekter af behandlingen mod kronisk tarmbetændelse og metodologiske forskelle. Mycobiome analysen omfattede 964 afføringsprøver og 150 tyktarms biopsier fra behandlingsnaive patienter med kronisk tarmbetændelse og symptomatiske kontroller. Genus Saccharomyces dominerede både afføringsprøver og biopsier, og i alle diagnostiske grupper. Der blev ikke påvist signifikante forskelle i alfa-diversitet (forskelligheden indenfor den enkelte prøve) eller beta-diversitet (forskellighed på tværs af grupper) blandt patienter med colitis ulcerosa, Crohns sygdom og kontroller, bortset fra det slimhindeassocierede mycobiome (fra biopsier) i IBSEN III-kohorten, hvor der blev identificeret signifikante ændringer i beta-diversitet mellem de diagnostiske grupper. Undersøgelsen af det slimhindeassocierede mycobiome i NORDTREAT-kohorten bekræftede ikke dette fund. Som forventet korrelerede positive ASCA antistoffer med Crohns sygdom, men ikke med mycobiome diversitet.Konklusioner Tarmens mycobiome viste ikke konsekvente ændringer hos nydiagnosticerede, behandlingsnaive patienter med kronisk tarmbetændelse sammenlignet med kontroller, hvilket tyder på, at tidligere fund kan afspejle effekter af medicinsk behandling mod kronisk tarmbetændelse frem for sygdomsdrevne ændringer i tarmens mycobiome. Immunresponser rettet mod svampe ved kronisk tarmbetændelse kan muligvis skyldes unormale immunreaktioner på tarmens svampe, snarere end at de er drevet af ændringer i forekomsten af svampe i tarmen.Fremtidige perspektiver Fremtidige studier bør fokusere på det slimhindeassocierede mycobiome. Teknologiske fremskridt inden for genetik og andre omics-teknologier kan bidrage til en højere detaljeringsgrad og bedre funktionel forståelse af svampe i tarmen, både i raske og syge. Den potentielle indflydelse af mycobiomet på behandlingsrespons og sygdomsprogression i relation til kronisk tarmbetændelse kan i fremtiden yderligere afdækkes gennem longitudinelle analyser.This PhD thesis investigates the relationship between the gut mycobiome andthe pathogenesis of inflammatory bowel disease (IBD).The research includes a systematic review of the mycobiome in IBD, a protocolarticle for the NORDTREAT Inception Cohort Study, and an extensive analysisof the mycobiome of treatment-naïve, newly diagnosed patients with IBD, andsymptomatic controls.BackgroundGenetic risk factors for IBD have been associated with an increased susceptibility to fungal infections, downstream signalling of innate antifungal immune receptors, and potentially play a role in serologic antifungal responses linked toIBD. The most notable serological response associated with IBD involves antifungal anti-Saccharomyces cerevisiae antibodies (ASCA).In addition to the B-cell response, specific cytotoxic T-cell responses against gutfungi are also associated with IBD, further reinforcing the connection betweenthe immune system, fungi, and IBD.ObjectivesThe review aimed to map the current knowledge of the gut mycobiome in IBDdisease through a systematic literature review.Next, we designed and conducted a cohort study to, amongst other objectives,enhance our understanding of the role of fungi in the pathogenesis of IBD.Finally, we analysed the mycobiome in ulcerative colitis, Crohn’s disease, andsymptomatic controls, focusing on relative abundance, diversity measures, andserological ASCA status. The study utilised the newly conducted NORDTREATcohort and the population-based Norwegian IBSEN III inception cohort. ResultsThe systematic review analysed 27 studies highlighting beta diversity differences in IBD versus controls, with inconsistent findings on specific fungal taxa.Candida and Malassezia were frequently noted, but the results varied due totreatment effects and methodological differences.Mycobiome analysis included 964 stool samples and 150 colonic biopsies fromtreatment-naïve patients with IBD and symptomatic controls. The genus Sac-charomyces dominated all sample subtypes and diagnostic groups. No significant alpha diversity (within-sample dissimilarity) or beta diversity (dissimilarityacross groups) differences among patients with ulcerative colitis, Crohn’s disease, and controls were established, except for the mucosa-associated mycobiome (of biopsies) in the IBSEN III cohort, where significant changes in beta diversity were identified between diagnostic groups. The mucosa-associated mycobiome in the NORDTREAT cohort did not validate this finding.As expected, ASCA conentrations correlated with Crohn’s disease but not withmycobiome diversity.ConclusionsThe gut mycobiome did not show consistent alterations in new-onset, treatmentnaïve patients with IBD compared to controls, suggesting that previous findingsmay reflect treatment effects rather than disease-driven changes in the gut mycobiome.Antifungal immune responses in IBD may stem from abnormal immune reactions to commensal fungi rather than shifts in fungal abundance.Future PerspectivesFuture studies should focus on the mucosa-associated mycobiome.Technological advances in the field of metagenomics and other omics layersmay contribute to a higher resolution and better functional understanding offungi present in the gut, in health and disease. The potential impact of the mycobiome on treatment response and disease progression of IBD can be furtherunravelled through longitudinal analyses

The Transformative Possibilities of the Microbiota and Mycobiota for Health, Disease, Aging, and Technological Innovation

The gut microbiota is extremely important for the health of the host across its lifespan. Recent studies have elucidated connections between the gut microbiota and neurological disease and disorders such as depression, anxiety, Alzheimer\u27s disease (AD), autism, and a host of other brain illnesses. Dysbiosis of the normal gut flora can have negative consequences for humans, especially throughout key periods during our lifespan as the gut microbes change with age in both phenotype and number of bacterial species. Neurologic diseases, mental disorders, and euthymic states are influenced by alterations in the metabolites produced by gut microbial milieu. We introduce a new concept, namely, the mycobiota and microbiota-gut-brain neuroendocrine axis and discuss co-metabolism with emphasis on means to influence or correct disruptions to normal gut flora throughout the lifespan from early development to old age. These changes involve inflammation and involve the permeability of barriers, such as the intestine blood barrier, the blood-brain barrier, and others. The mycobiota and microbiota-gut-brain axis offer new research horizons and represents a great potential target for new therapeutics, including approaches based around inflammatory disruptive process, genetically engineered drug delivery systems, diseased cell culling kill switches , phage-like therapies, medicinal chemistry, or microbial parabiosis to name a few