Fungal Profiling and Ecology of the Healthy Human Gastrointestinal Tract

<p>NON-TECHNICAL SUMMARY: <br/>Recent research into microorganisms inhabiting the human body is revealing a lot of information about the role of microbes in human health, including beneficial roles for various organisms such as Lactobacillus and Bifidobacterium. Fungi are known to be present in the human gut, but have been little studied except in the context of disease (e.g. irritable bowel disease; yeast infections; following perturbation by antibiotics). This work examines the role of fungi in the healthy human gut, beginning with a tally of gut-associated fungi and their relative abundances. Preliminary data suggests that certain common gut fungi, like the Candida yeasts and their allies, are very common in the human gut, but that one species may replace another related species, a finding which, if confirmed, could have serious implications in treating and preventing
yeast infections (which occur when Candida species normally inhabiting the gut begin growing invasively and acting as pathogens). Additionally, a better understanding of human gut fungi could reveal some beneficial roles, such as nutrient release (as has been documented for fungi in animal models).
<p>APPROACH:<br/> Initial fungal profiling has already been performed on 45 healthy adults at 1-2 time points each, using fungal-specific PCR primers to amplify the ITS region of the nuclear ribosomal RNA operon and multiplexed 454 sequencing. Statistical and bioinformatic analyses of this data are ongoing. Expanded populations are being recruited for fecal sampling, with an emphasis on people following specialized dietary regimens (vegetarian, vegan, low-carb, gluten-free; to study effect of diet on fungal microbiome); on people from different geographic regions (to study effects of infant and childhood exposure to local microbiota); and infants (to study the developing/immature microbiome). This sampling includes culturing for further characterization of fungal species as well as DNA extraction for fungal-specific PCR and sequencing. Laboratory culture studies will
evaluate different pairs of yeast species isolated from human feces (Candida spp., Debaryomyces hansenii), and from foodstuffs (D. hansenii), and a probiotic yeast (Saccharomyces boulardii). Studies will involve solid and liquid media, co-inoculations, and inoculation and establishment of one fungus followed by inoculation of another. Aerobic and anaerobic (to simulation the human gut) conditions will be examined. Interactions of the different yeast species will also be examined in human cell culture (Caco 2), where the effect of innocuous species on Candida adherence will be examined. If warranted, probiotic studies will involve the consumption of foodstuffs (cheese and/or fermented meats) naturally colonized by D. hansenii (as confirmed by culture) by healthy humans whose gut microbiota have been shown to include Candida yeasts, and further fecal analysis for results. Results will be
presented at scientific meetings (e.g. American Society of Microbiology) and published in peer-reviewed literature. Results may dictate educational efforts to better inform the public about Candida yeasts (is it necessary to attempt to eradicate them from the body, or are they fulfilling some useful role in the 60-80% of humans where they occur asymptomatically), and possible new treatments for Candida infections (replacement of Candida by an ecologically-similar, non-pathogenic fungus).
<p>PROGRESS: 2012/10 TO 2013/09<br/>Target Audience: Three poster presentations (two by graduate students) were given to a scientific audience of ~1900, attending the Fungal Genetics Meeting (Pacific Grove, CA, March 2013) and the joint American Phytopathological Society-Mycological Society of America Meeting (Austin, TX, August 2013). Changes/Problems: We plan to add thein vivotesting of D. hansenii killer toxin in a mouse model of Candida infection.D. hanseniiis an organism used in food fermentations and with Qualified Presumption of Safety status, so we feel justified in proceeding directly to healthy human trials for theD. hanseniiprobiotic. No yeast killer toxin has been evaluated for its safety in humans (though killer toxins are widely used in wine production, to control unwanted fermentations), so a mouse study for safety and efficacy is necessary. I have an
IACUC protocol in preparation. What opportunities for training and professional development has the project provided? Two graduate students are working on various aspects of this project, one on characterizing the gut fungi and one on the yeast interactions. For this purpose, I have trained them in: DNA extraction and techniques (PCR, qPCR, gel electrophoresis, dideoxy sequencing and data analysis, 454 pyrosequencing data analysis, allele-specific primer extension); fungal culture techniques; and yeast killer toxin assays and purification. The graduate students and I have trained three undergraduates to contribute to the project through basic DNA and culture techniques. Techniques we have learned from this project have been adapted into three teaching laboratories (in two classes) serving 14 upperclassmen and 17 graduate students in the Department of Food Science and Technology at the
University of Nebraska-Lincoln. How have the results been disseminated to communities of interest? Three posters have been presented at scientific meetings in 2013. Two manuscripts are currently in preparation. What do you plan to do during the next reporting period to accomplish the goals? Complete and submit the manuscripts in preparation. Recruit 36 healthy adults - 18 male and 18 female - for a study to evaluate the effect of probiotic Debaryomyces hansenii on other gut fungi, notably Candida yeasts, and the persistance of D. hansenii in the human gut. Test the efficacy of purified D. hansenii killer toxin against Candida yeasts in a mouse model of gastrointestinal Candida colonization.
<p>PROGRESS: 2011/10/01 TO 2012/09/30<br/>OUTPUTS: Fungal DNA has been selectively PCR amplified and sequenced from 45 healthy humans on conventional Western diets: from two time points for 24 individuals and from one time point for 21 individuals. Multiple unsuccessful efforts were made to amplify fungal DNA from the second time point for the 21 individuals; these DNA samples have successfully yielded bacterial DNA amplification and sequence, and we thus conclude fungal DNA to be below the level of detection in these samples. A manuscript discussing results is in preparation and will be submitted by the end of 2012. DNA has been extracted from fecal samples from 20 healthy humans on non-conventional diets (vegetarian, vegan, gluten-free, lactose-free) and 2 infants. Selective PCR amplification of fungal DNA from these samples is ongoing and will be followed by DNA
pyrosequencing in late 2012 and early 2013. Preliminary data on dominant fungi has been obtained by selective culturing and/or quantitative PCR, and results will be presented at the Fungal Genetics Meeting in Pacific Grove, CA, in March of 2013. PARTICIPANTS: The PI, Heather Hallen-Adams, is directing the project, and has participated in PCR, DNA sequencing, DNA sequence analysis, recruitment for the non-conventional diet study, data interpretation and manuscript preparation. Graduate student Mallory Suhr is participating in DNA extraction and PCR of the participants with non-conventional diets. She will be doing the DNA sequence analysis and data interpretation for this portion of the study, following relevant training. This is part of her graduate training. Graduate student Nabaraj Banjara is participating int he study of the potential of the foodborne yeast as a probiotic. He has
isolated numerous yeast strains and species from locally-obtained foods, and will conduct the competitive trials between foodborne yeasts and Candida yeasts in laboratory culture. He will participate in DNA extraction, PCR amplification and sequence analysis of any feeding studies. This is part of his graduate training. Undergraduate (now graduated) Geraldine Spinner performed the preliminary PCR amplifications on the DNA from fecal samples from people on conventional diets. This constituted part of her undergraduate and occupational training in molecular biology. TARGET AUDIENCES: Target audiences for project data include physicians and microbiologists. Potential beneficiaries in future years include any person harboring Candida yeasts as a component of their gut microbiota (approximately 60% of humans worldwide) and facing increased risk of Candida yeast infections due to various
factors including surgical intervention, immunosuppression and antibiotic use. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
<p>PROGRESS: 2011/10/01 TO 2012/09/30<br/>OUTPUTS: Fungal DNA has been selectively PCR amplified and sequenced from 45 healthy humans on conventional Western diets: from two time points for 24 individuals and from one time point for 21 individuals. Multiple unsuccessful efforts were made to amplify fungal DNA from the second time point for the 21 individuals; these DNA samples have successfully yielded bacterial DNA amplification and sequence, and we thus conclude fungal DNA to be below the level of detection in these samples. A manuscript discussing results is in preparation and will be submitted by the end of 2012. DNA has been extracted from fecal samples from 20 healthy humans on non-conventional diets (vegetarian, vegan, gluten-free, lactose-free) and 2 infants. Selective PCR amplification of fungal DNA from these samples is ongoing and will be followed by DNA
pyrosequencing in late 2012 and early 2013. Preliminary data on dominant fungi has been obtained by selective culturing and/or quantitative PCR, and results will be presented at the Fungal Genetics Meeting in Pacific Grove, CA, in March of 2013. PARTICIPANTS: The PI, Heather Hallen-Adams, is directing the project, and has participated in PCR, DNA sequencing, DNA sequence analysis, recruitment for the non-conventional diet study, data interpretation and manuscript preparation. Graduate student Mallory Suhr is participating in DNA extraction and PCR of the participants with non-conventional diets. She will be doing the DNA sequence analysis and data interpretation for this portion of the study, following relevant training. This is part of her graduate training. Graduate student Nabaraj Banjara is participating int he study of the potential of the foodborne yeast as a probiotic. He has
isolated numerous yeast strains and species from locally-obtained foods, and will conduct the competitive trials between foodborne yeasts and Candida yeasts in laboratory culture. He will participate in DNA extraction, PCR amplification and sequence analysis of any feeding studies. This is part of his graduate training. Undergraduate (now graduated) Geraldine Spinner performed the preliminary PCR amplifications on the DNA from fecal samples from people on conventional diets. This constituted part of her undergraduate and occupational training in molecular biology. TARGET AUDIENCES: Target audiences for project data include physicians and microbiologists. Potential beneficiaries in future years include any person harboring Candida yeasts as a component of their gut microbiota (approximately 60% of humans worldwide) and facing increased risk of Candida yeast infections due to various
factors including surgical intervention, immunosuppression and antibiotic use. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.