Progress 03/01/12 to 02/28/17
Outputs
Target Audience:The target audience is primarily microbiome researchers and fungal biologists. Audience has been reached by presentations at venues such as the Fungal Genetics Conference and the Mycological Society of America annual meeting. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Over the lifetime of the project, two graduate students have worked on this project; each received an MS on the project and one received a PhD. The PhD work was completed, and the degree awarded, during this reporting period. How have the results been disseminated to communities of interest?During the reporting period, by two posters at the 2017 Fungal Genetics Conference in Pacific Grove, CA (~850 in attendence), one talk at the Mycological Society of America 2017 conference in Athens, GA (~40 in attendence for the talk), and a public PhD dissertation defense. Multiple conference presentations, two MS theses, one PhD dissertation, and six peer-reviewed presentations were produced during the five year project duration. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Theoverall goalof this project is to characterize the fungal component ("mycobiome") of the healthy human microbiome, and to examine ecological interactions involving gut fungi. The composition of the microbiome (especially the gastrointestinal microbiome) has been linked to numerous conditions in humans and animals (obesity/leanness, diabetes, and even brain function), and fungi are the most significant eukaryotes in the human microbiome in developed countries. Distinguishing resident fungi from those that do not colonize the gut or interact with other gut microbes, and determining which fungi are consistently resident in healthy humans, is necessary to determine their role in health and disease. Interactions involving fungi in the gut may shift the balance of gut microbes to a more or less healthy state. As part of this project'simpact, we have developed a comprehensive picture of the healthy human gut mycobiome. Notably, while >270 species of fungi have been reported from the human gut, only ~15 species are regularly detected, from multiple individuals or studies. These colonizers share the necessary trait of being able to grow at human body temperatures, and are primarily yeasts. Fungi are less stable members of the microbiome than bacteria; commonly, the same fungus may not be detected in two consecutive samples from the same individual, while resident bacteria can be routinely detected in samples from the same individual over a period of years. Fungal and bacterial species show little correlation in the gut, with the only significant correlations being between the most abundant fungi and bacteria. The foodborne yeastDebaryomyces hansenii(not a colonizer, as it does not grow at 37 C) shows a weak negative correlation with the resident, opportunistic pathogenicCandidayeasts. Current studies are examining the interactions betweenD. hanseniiandCandidain detail,in vitroandin vivo. Work during this reporting period was conducted by a PhD student, with the supervision and assistance of Dr. Hallen-Adams. Objective 1. To develop a profile of the normal fungal inhabitants of healthy humans. Done in previous years; published as Hallen-Adams et al. (2015) Fungi inhabiting the healthy human gastrointestinal tract: a diverse and dynamic community.Fungal Ecol15:9-17.; Suhr & Hallen-Adams (2015) The human gut mycobiome: pitfalls and potentials - a mycologist's perspective.Mycologia107:1057-1073; Suhr et al. (2016) Sequence-based methods for detecting and evaluating the human gut mycobiome.Lett Appl Microbiol62:209-215; and Hallen-Adams & Suhr (2016) Fungi in the healthy human gastrointestinal tract.Virulence8:352-358. Objective 2. To compare the fungal profile with extant bacterial community profiling of the same individuals at the same time points in order to determine whether there are clear correlations between the different microbial communities. Done in previous years; published as Hallen-Adams et al. (2015) Fungi inhabiting the healthy human gastrointestinal tract: a diverse and dynamic community.Fungal Ecol15:9-17. Objective 3. To test the hypothesis of competitive exclusion of gut yeasts by in vitro studies of such yeasts isolated from human feces. Activities completed/experiments conducted: The ability of the foodborne yeastDebaryomyces hanseniito kill or inhibit growth ofCandida albicans, a commensal human gut yeast and opportunistic human pathogen, was tested against multipleC.albicans strains isolated from feces of six persons over five time points.C. albicansisolates were identified to strain by multilocus sequence typing (MLST), a genetic method. Data collected: MLST types forC. albicansstrains, pairwise interactions betweenC. albicansandD. hansenii. Results: Seven MLST types (strains) ofC. albicanswere found in 55 isolates from 6 healthy human subjects, with three subjects harboring two strains and three harboring one strain. All strains ofC. albicansisolated from human feces were inhibited by eight of elevenD. hanseniistrains tested (sevenD. hanseniistrains isolated from feces of human volunteers who had eaten cheese naturally colonized withD. hansenii, and one strain isolated directly from cheese. Three of the sevenC. albicansstrains were consistently more sensitive to inhibition byD. hansenii. Key outcomes:Multiple strains ofDebaryomyces hanseniiyeasts naturally occurring in cheese kill or inhibitCandida albicansfrom the human gutin vitro. This killing activity is common inD. hanseniithat has passed through the human GI tract, suggesting thatD. hanseniimay inhibitC. albicansinside the gut. Objective 4. Sampling of additional human demographics as warranted by results of Objectives 1 and 2. Done in previous years (for vegetarians); published as Suhr et al. (2016) Sequence-based methods for detecting and evaluating the human gut mycobiome.Lett Appl Microbiol62:209-215. Objective 5. Probiotic studies as warranted by results of Objective 3. Activities completed/experiments conducted: Six healthy adult human volunteers consumed cheese naturally containing a "killer" strain ofDebaryomyces hansenii(a strain shown to kill or inhibitCandida albicansandCandida tropicalisin the lab) for five days, and provided fecal samples prior to consuming the cheese; daily during the five days of cheese consumption; and three, five, and seven days after cheese consumption had ceased. DNA was extracted from the fecal samples and fungal DNA was specifically amplified and sequenced. Data collected: Daily dietary information from participants was collected during the two-week course of the experiment. Fungal DNA sequence data was collected from the sample prior to cheese consumption (baseline), two samples during cheese consumption, and the sample one week after cheese consumption had ceased (washout). Data includes fungal species, and an estimate of species abundance (number of times a particular sequence is detected in a sample). Results:D. hanseniiabundance increased significantly from baseline during cheese consumption, and returned to baseline levels seven days after the last day of cheese consumption. OverallCandida, andC. albicans, abundance was lower during cheese consumption, but did not change significantly between baseline, consumption, and washout.CandidaandD. hanseniiwere weakly negatively correlated (Pearson's correlation coefficient r= -0.39). Key outcomes:Debaryomyces hanseniifrom consuming cheese is detectable in human fecal samples, and does not persist (colonize the gut) long-term once cheese consumption has ceased. A weak negative correlation betweenD. hanseniiandC. albicanspresence was detected in this small (six participant, 24 sample) study.?
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Banjara N, Hallen-Adams HE (2017) Purification and characterization of killer toxin from Debaryomyces hansenii isolated from cheese. Abstract 638T in 29th Fungal Genetics Conference Asilomar 17 Abstract Book, p.273
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Hallen-Adams HE, Banjara N (2017) Debaryomyces hansenii may antagonize Candida yeasts in the human gut. Abstract 468F in 29th Fungal Genetics Conference Asilomar 17 Abstract Book, p.223
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Hallen-Adams HE, Banjara N (2017) Genetic diversity of Candida in the healthy human gut, and interactions with a foodborne yeast. Abstract MON 39 for 2017 MSA Conference
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2017
Citation:
Banjara N (2017) Debaryomyces hansenii and anti-Candida activity. A dissertation presented to the Faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Doctor of Philosophy. 135 pp.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Work during the October 1, 2015 - September 30, 2016 reporting period impacted scientists, and was disseminated at one scientific meeting of international scope (approximate attendence 500), and one invited presentation; and in two peer-reviewed publications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?I have supervised two graduate students and one undergraduate student on this project during this reporting period. One graduate student received her MS degree, and the other will recieve his PhD in spring 2017. Both students presented posters at the American Society for Microbiology Conference on Candida and Candidiasis in 2016, and have manuscripts and papers in various stages of preparation and publication. How have the results been disseminated to communities of interest?The resuts have been disseminated by twograduate students each presenting one poster at a meeting with international attendence (approximately 750 attendees), and an invited talk given by myself at the Kunming Institute of Botany (~ 30 attendees). Further, results have been disseminated by publications in peer-reviewed journals: International Journal of Food Microbiology, Virulence, article in review with Mycoses. What do you plan to do during the next reporting period to accomplish the goals?Goals 1 and 2, regarding characterizing the fungal flora of the healthy human gut and its relationship to the bacterial flora are complete. Continuing efforts focus on the evaluation of certain strains of the cheese yeast Debrayomyces hansenii as a potential probiotic organism, including evaluating sequencing data from a small, IRB-approved human feeding trial. Further work involves identifying and characterizingthe anti-Candida factor(s) produced by D. hansenii, by protein purification and mass spectrometry, and genetic comparisons of D. hansenii strains which do and do not kill Candida yeasts.
Impacts
What was accomplished under these goals?
Goal 1) We have developed a profile of the normal fungal inhabitants of the healthy human gut, and further analyzed and disseminated the conclusions we first reported last year, namely, that no more than 15 or 20 fungal species are capable of playing an ecological role in the human gut, although many other species may be detected because of their presence on food or in the environment. We have identified the resident gut fungi - those commonly detected in healthy humans and capable of colonizing the gut - as yeasts in the genus Candida and in the family Dipodascaceae. Candida yeasts are normal residents of healthy humans, but may act as pathogens if the gut is damaged or gut bacteria are perturbed, for example, by the use of antibiotics. Certain yeaststhat are used in making food (Saccharomyces cerevisieae, bakers' and brewers' yeast; Debaryomyces hansenii, common in cheese) do not colonize the gut long term, but are frequently introduced through the diet and appear to interact with other gut microbes, notably D. hansenii, which appears to lower the abundance of Candida yeasts. We are currently pursuing the potential of this yeast as a probiotic. Goal 2) We have examined the populations of fungi and bacteria in the human gut, and observed few meainingful correlations. We did not pursue these studies further in 2015-2016. Goal 3 and 4) We have obtained fungal DNA from 24 fecal samples from a feeding study in whichvolunteers ate cheese known to harbor Debaryomyces hansenii yeasts, and have submitted these samples fro DNA sequencing in order to determine the identities and relative abundances of D. hansenii and native gut fungi before, during and after the cheese consumption. Participants also kept a detailed food diary, from which we will be able to determine if and when other fungi were entering the diet. These data will be analyzed early 2017, and are expected to result in a student dissertation chapter, a publication, and possibly a grant proposal.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Banjara N, Nickerson KW, Suhr MJ, Hallen-Adams HE (2016) Killer toxin from several food-derived Debaryomyces hansenii strains effective against pathogenic Candida yeasts. Int J Food Microbiol 222:23-29.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Hallen-Adams HE, Suhr MJ (2016) Fungi in the healthy human gastrointestinal tract. Virulence, DOI: 10.1080/21505594.2016.1247140
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2016
Citation:
Crespo Ram�rez RA (2016) Studies of Debaryomyces hansenii killer toxin and its effect on pathogenic bloodstream Candida isolates. A thesis submitted to the Faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree Master of Science.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Crespo Ram�rez RA, Banjara N, Hallen-Adams H (2016) Genetic basis of Debaryomyces hansenii killer toxin and its effect on pathogenic Candida species. 13th ASM Conference on Candida and Candidiasis, April 13-17, 2016, Seattle, WA. Abstract # 109.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Banjara N, Hallen-Adams HE (2016) Killer toxin from Debaryomyces hansenii strains effective against pathogenic Candida yeasts. 13th ASM Conference on Candida and Candidiasis, April 13-17, 2016, Seattle, WA. Abstract # 106.
- Type:
Other
Status:
Other
Year Published:
2016
Citation:
Hallen-Adams H (2016) The significance of fungi in human gut microbiome studies. Invited talk at Kunming Institute of Botany, Kunming, Yunnan, China, June 8, 2016.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Suhr MJ, Gomes-Neto JC, Banjara N, Florescu DF, Mercer DF, Iwen PC, Hallen-Adams HE. Epidemiological investigation of Candida species causing bloodstream infection in pediatric small bowel transplant patients. Submitted to Mycoses.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Work during the October 1, 2014 - September 30, 2015 reporting period impacted scientists, and was disseminated at two scientific meetings of international scope (combined attendence of approximately 2500), and in threepeer-reviewed publications. Changes/Problems:In a minor change to goal 3 (to test the hypothesis of competitive exclusion of gut yeasts by in vitro studies of such yeasts isolated from human feces), we are usingDebaryomyces hanseniiyeasts isolated from food (cheese and fermented sausages - instead of from human feces) in competition assays againstCandidayeasts. AsD. hanseniiis a recognized food additive and regarded as safe, and as we hope to conduct feeding trials in humans to determin howD. hanseniiinfluences gut populations ofCandida, we believe using strains directly isolated from food is safer and more straightforward than usingD. hanseniiisolated from feces. What opportunities for training and professional development has the project provided?I have supervised three graduate students and one undergraduate on this project during the reporting period. One graduate student received her MS degree, and is now pursuing a PhD; another will receive her MS in spring of 2016. The graduate students all attended the Fungal Genetics Conference in 2015 and presented posters on their work, and two have had papers both published and accepted in 2015. How have the results been disseminated to communities of interest?Results have been disseminatedby three posters (presented by graduate students) at the Fungal Genetics Conference (an international meeting held March 2015, with 950 in attendence) and one talk (presented by myself) at the Botany 2015 Conference (an international meeting held in July 2015, with ~1500 in attendence). Further, results have been disseminated by publications in peer-reviewed journals: Fungal Ecology, Current Microbiology, Mycologia, Letters in Applied Microbiology. What do you plan to do during the next reporting period to accomplish the goals?Goals 1 and 2 (to develop a profile of the normal fungal inhabitants of the healthy human gut;to compare the fungal profile with the bacterial community profiling of the sameindividuals) are complete. For goal 3 (to test the hypothesis of competitive exclusion of gut yeasts by in vitro studies of such yeasts isolated from human feces), we will continue to evaluate new strains ofD. hanseniifor activity against Candidayeastsas we find them; however, we have a working collection of 23 strains that can killCandidaunder varying conditions, and will focus on these as we proceed with goal 4 (probiotic studies as warranted by results of [goal] 3). For goal 4, we are currently analyzing data from a small human trial; if results are encourgaing (Candidaprevelance decreases when cheese with "killer"D. hansenii is consumed), we will seek funding for larger, more comprehensive trials, including pureD. hanseniiin probiotic capsules.
Impacts
What was accomplished under these goals?
The human microbiome, i.e., microorganisms that live in the human body and especially in the gut, is an area of intense research. Gut microbial composition has been shown to exert a profound influence on human health, including obesity, and diabetes and other autoimmune diseases. Fungi in the human gut includeCandidayeasts, which are usually harmless but may cause severe disease in some circumstances; many other fungi have been reported in the gut but their role in health or disease, if any, has not been known. Our research has identified a core of no more than 15 species of fungi that persist in the human gut and may impact human health (compared with several hundred bacterial species). We have also demonstrated that a yeast used to make cheese,Debaryomyces hansenii, can kill disease-causingCandidaspecies in the laboratory, and thatD. hanseniiin the healthy gut correlates with low quantities ofCandida.D. hanseniicould provide a safe, inexpensive means of reducing or eliminatingCandidain the gut when a person is at risk of infection (for example, when they are taking antibiotics or will be undergoing surgery that affects the GI tract, or involves implantating a catheter). Goal 1) To develop a profile of the normal fungal inhabitants of the healthy human gut. We have done this,byusing next-generation DNA sequencing techniques to identify the fungi in fecal samples from 59healthy adult volunteers (30female, 29 male; 16 vegetarian, 43 on a conventional Western diet); furthermore, we have conducted a comprehensive literature review of papers published on the topic of gut fungi since 1917. From this we determined that no more than 15 species are likely to be members of the gut community (many other species are reported, but these are unable to grow or persist in the gut, and probably are either eaten or inhaled, and "passing through"); and that gut fungi may interact and influence the ability of other fungi to inhabit the same gut. We found no effect from whether a volunteer was male or female. Vegetarians possessed the same kinds of fungi as people on a conventional diet, but in different proportions. (Data published in Fungal Ecology, 2015;Mycologia, 2015;and forthcoming in Letters in Applied Microbiology, 2016.)Goal 2) To compare the fungal profile with the bacterial community profiling of the sameindividuals. We found a few statistically-significant correlations between certain fungi and bacteria (Fungal Ecology, 2015); none of obvious utility. The yeastDebaryomyces hanseniiis negatively correlated withCandidayeasts, suggesting competition. Goal 3) To test the hypothesis of competitive exclusion of gut yeasts by in vitro studies of such yeasts isolated from human feces. We have chosen to use food isolates ofD. hansenii(naturally found in cheese) against human isolates ofC. albicansandC. tropicalis. Of 42D. hanseniistrains, isolated from 22 cheeses, 23 were able to killC. albicansand/orC. tropicalis in the lab; furthermore, cheese containing such "killer" strains could also killCandidafor several days when incubated at human body temperature (manuscript in review). Goal 4) Probiotic studies as warranted by results of objective 3. We have conducted a pilot study in which6volunteers ate 2 ounces of cheese daily for five days, where the cheese had been pre-determined to harborD. hanseniicapable of killingCandida. Volunteers provided fecal samples before, during and after the five days of cheese consumption, and we are in the process of determining the relative abundances ofD. hanseniiandCandidain thesesamples. Impacts: Our work has increased knowledge of fungi in the human gut, by providing the first analyses of gut fungi that include interpretation of the role of the fungi in the gut. We are the first researchers to recognize that most fungi detected in fecal samples are not gut organisms (i.e., they are not capable of growing at human body temperature or gut pH, and their detection can be readily explained by ingestion, inhalation or other environmental exposure). As this knowledge spreads, we hope that it will prevent waste of time and resources, as researchers begin devoting study to the few important fungi, and cease cataloging every organism.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Hallen-Adams HE, Kachman SD, Kim J, Legge RM, Martinez I (2015) Fungi inhabiting the healthy human gastrointestinal tract: a diverse and dynamic community. Fungal Ecology 15:9-17.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Suhr MJ, Hallen-Adams HE (2015) The human gut mycobiome: pitfalls and potentials - a mycologist's perspective. Mycologia 107; DOI: 10.3852/ 15-147
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Banjara N, Suhr MJ, Hallen-Adams HE (2015) Diversity of yeast and mold species from a variety of cheese types. Current Microbiology 70:792-800.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Suhr MJ, Banjara N, Hallen-Adams HE. Sequence-based methods for detecting and evaluating the human gut mycobime. Letters in Applied Microbiology, accepted
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2015
Citation:
Suhr MJ (2015) Characterization and investigation of fungi inhabiting the gastrointestinal tract of healthy and diseased humans. A thesis presented to the Faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Master of Science.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Banjara N, Hallen-Adams H (2015) Debaryomyces hansenii killer toxin against Candida species. Fungal Genetics Reports 61(Suppl): Abstract #27.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Crespo Ramirez RA, Hallen-Adams H (2015) Genetic basis of Debaryomyces hansenii killer toxin, and its effect on Candida albicans and Candida tropicalis. Fungal Genetics Reports 61(Suppl): Abstract #199.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Suhr MJ, Florescu DF, Mercer DF, Hallen-Adams HE. Ecology of candidemia in pediatric small bowel transplant recipients. Fungal Genetics Reports 61(Suppl): Abstract #449.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Hallen-Adams HE (2015) The significance of fungi in human gut microbiome studies. Botany 2015, Edmonton, AB July 25-29, 2015.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Three poster presentations (two by graduate students) were given to a scientific audience of ~400 attending the American Society of Microbiology 12th Candida and Candidiasis Meeting (New Orleans, LA, March 2014); and an oral presentation was made to a scientific audience of ~75 attending the Mycological Society of America Annual Meeting (East Lansing, MI, June 2014). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Three graduate students are working on various aspects of this project, one on fungal identification methodology (sequencing- and probe-based techniques), one on the killer yeast ecology and proteins, and a third on the killer yeast genomics. One student has completed his MS in 2014 and is currently working on his PhD; one manuscript (tangential to this Hatch project, describing the isolation of yeasts and mold from cheese) has been submitted; a second manuscript, on the killer toxin nature and activity, is in preparation. One graduate student has had extensive training on the Luminex MagPix methodology for detection and identification of species in mixed-matrix samples (such as DNA isolated from huamn feces), inclduing correspondence with Luminex scientists and attending a Luminex training session in Austin, TX; this methodology has been included in her submitted manuscript on vegetarian gut fungi. Information on this and other advances in sequencing and identification methods has been incorporated into FDST 458/858 (Advanced Food Analysis) lectures, given to 18 undergraduates and 8 graduate students in 2014. How have the results been disseminated to communities of interest? Three posters and a talk were presented at scientific meetings in 2014. One manuscript has been accepted, and another has been submitted, to journals with international distribution. What do you plan to do during the next reporting period to accomplish the goals? Complete characterization of killer toxin and evaluation in a mouse model of Candida infection. Continue to submit publications as warranted. Human probiotic trials (objective 4) are on hold while I search for a manufacturer who can prepare set quantities of viable, encapsulated, food-grade yeast (my initial probiotic manufacturer has changed their emphasis to exclusively bacteria). Some probiotic trials will be conducted in mouse in the interim, for which the same rigorous standards as those for human probiotics do not apply.
Impacts
What was accomplished under these goals?
The human microbiome is an area of increasingly active research, and studies in the past several years are beginning to reveal the importance of human gut microbes in preventing or promoting obesity, autoimmune disorders and mental health. While bacteria have been well-studied, little is known about other gut microbe, of which the fungi are the most dominant. My lab is at the forefront in conducting larger scale studies of gut fungi in healthy humans. Findings to date include: · evidence of ecological competition between Candida and related species, yeasts found in the gastrointestinal tract of most healthy humans, but which are capable of causing yeast infections or more serious disease. This suggests the possibility of displacing more pathogenic species such as C. albicans with safer, food preparation species. · A core fungal microbiome consisting of two groups of yeasts, Candida and Geotrichum, occurring in >50% of samples from healthy humans, while a wide range of “rare” fungi detected from the gut come from from environmental sources (ingestion or inhalation) and are likely to play no role in gut ecology or function. This is counter to findings from the bacterial microbiome, in which organisms found in the gut are presumed to have direct effects on the host and/or each other. These findings have implications for human health and the relations of people and microbes to the environment and food systems. Food production yeasts are found in both of the core gut fungal groups; especially exciting is the possibility of food production and foodborne yeasts reducing the chance of Candida infection. To this end, we have assembled a collection of strains of the cheese manufacturing yeast Debaryomyces hansenii, and identified several that can kill Candida in the lab. Work is underway to identify the protein used to kill Candida, and to test both the purified protein and the live yeast against Candida in a mouse model. Objective 1: To develop a profile of the normal fungal inhabitants of the healthy human gut. A manuscript providing the most thorough characterization of gut fungi in healthy humans to date (45 subjects, 69 samples) has been accepted by Fungal Ecology, a journal with international distribution; this will be available in hard copy in early 2015, and is anticipated to be available online by the end of 2014. A further manuscript, addressing gut fungi in 16 healthy vegetarians and comparing available methodology for characterizing gut fungi, has been submitted to PLoS ONE. New findings in these papers include the small number of common gut fungi, which could be said to be adapted to the human gut, and the relative variability of fungal gut communities, both between people and within a single person over time. On publication, this information will be available to a wide range of gut microbial researchers and will be incorporated into a holistic picture of human gut microbes. Objective 2: To compare the fungal profile with bacterial community profiling of the same individuals. An accepted manuscript on the gut fungi of 45 healthy humans (see above) also examines the correlation of fungi and bacteria. Little direct correlation (presence/absence of one organism predicts presence/absence of another) was observed, although abundance of particular fungi or bacteria may both be influenced by some additional factor (i.e. obesity). Objective 3: To test the hypothesis of competitive exclusion of gut yeasts by in vitro studies of such yeasts isolated from human feces. Anti-Candida killer toxin(s) have been isolated from several strains of the yeast Debaryomyces hansenii and tested as crude extracts; efforts to purify the toxin for mouse studies are underway. Ten D. hansenii isolates - seven killer and three non-killer - we collected have been subject to genome sequencing; comparisons between these sequences is underway to detect putative killer toxin gene(s), whose function will then be empirically confirmed in the lab. Protein electrophoresis and sequencing is being used in tandem to identify and characterize these killer toxins. Objective 4: Probiotic studies as warranted by results of objective 3. The yeast strain most effective at killing Candida albicans is being prepared for probiotic (and purified killer toxin) studies in a mouse model.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Hallen-Adams HE, Kachman SD, Kim J, Legge RM, Martinez I (2015) Fungi inhabiting the healthy human gastrointestinal tract: a diverse and dynamic community. Fungal Ecol in press
|
Progress 10/01/12 to 09/30/13
Outputs
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.
Impacts
What was accomplished under these goals?
Data collection for objectives 1 and 2 is complete; we have built a profile of the gut fungi of healthy adults on a conevntional Western diet, and compared it with the gut bacterial populations. Additionally, we have assembled a gut fungal profile of healthy vegetarian adults, and observe consistant differences between vegetarians and persons on a conventional Western diet - notably, all vegetarians sampled harbor Malassezia fungi (fungi with very particular lipid requirements from the host), while only approximately 15% of conventional diet participant harbored Malassezia. For objective 3, we assembled a collection of 40+ Debaryomyces hansenii yeast strains, isolated from locally-purchased cheese. We have conducted extensive in vitro testing, and have established that 80% of these D. hansenii strains produce a compound ("killer toxin") capable of killing or inhibiting growth of the opportunistic pathogenic yeasts, Candida albicans and C. tropicalis, in culture.
Publications
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Progress 10/01/11 to 09/30/12
Outputs
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.
Impacts
Data on the fungal component of the gut microflora of healthy humans suggests competitive interactions between a foodborne yeast and opportunistic pathogenic yeasts in the genus Candida. An NIH grant proposal has been submitted to study these interactions in detail, both in the laboratory and in feeding studies in which healthy humans are provided with known quantities of the foodborne yeast and its impact on natural Candida populations in the gut are monitored. This may inform development of an anti-Candida probiotic to be taken at times of elevated risk of Candida infection (when on long-term antibiotics, or prior to surgery that would disrupt gut barriers, for example).
Publications
- No publications reported this period
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