We are far from understanding function of fungi in the human microbiome. Our understanding is at the 1st rung, the most primitive stage, where we continue to examine mammalian-microbial interactions through the biased lens of pathogenesis, as in ‘eeks! Microbes are bad, cause diseases‘. For the picture to become clearer, this bias needs to go the way of the dodo and that’ll take a few years yet.
Fungal communities of the human body: the Human Mycobiome
I’ve organized this answer into 4 sections:
I. To identify and enumerate the human mycobiome (human body fungal communities) is no easy task. Why?
II. Mycobiome-human relationship in health and disease.
III. Location-wise human mycobiome in health and disease (skin-scalp, gut-oral cavity, lung, plasma, vagina).
IV. Saccharaomyces boulardii, example of probiotic fungus.
I. To identify and enumerate the human mycobiome is no easy task. Why?
Need to identify to understand function. However, human microbiota fungi are so rare as to make identification a challenge.
- Traditionally, fungi were identified by culture. Human mycobiome inhabitants are likely novel and we don’t yet know how to grow them in culture, at least many of them.
- Taking a leaf out of bacterial studies, where efforts center on the bacterial 18S rRNA locus, now PCR (Polymerase Chain Reaction)-based methods focus on the fungal genetic locus encompassing 18S, 5.8S and 28S rDNA genes, and the internal transcribed spacer regions (ITS1 and ITS2) that encode nonfunctional RNA transcribed during rRNA synthesis.
- Which region of this fungal locus to amplify is still debated, and different studies make different choices. With as many as 6 choices, studies run the gamut in target choice. Problem? Yes, makes it difficult to generalize datasets across studies.
- Debate also rages on which is better choice, 18S rDNA or ITS, 28S rDNA having been side-lined recently.
As expected from a rapidly growing field, much chaos, redundancy and mis-interpretation reigns.
- While the Fungal Genome Initiative (FGI; ) aims to establish genomes throughout the fungal kingdom, such databases prioritize human disease-associated fungi with as yet little or no information on human mycobiome, i.e. commensal, inhabitants.
- Fungal databases are puny and growing slowly compared to those for bacteria. For example, a major database on fungal ITS regions maintained by Henrik Nillsson at the University of Gothenburg, Sweden, was last updated in 2012 .
- Genome sequences of over a hundred fungal species are publicly available (3) but few of them are human-associated.
- Among Next-Generation Sequencing (NGS) technologies, RNA deep sequencing or RNA-seq offers several advantages for human mycobiome analysis: not hybridization-based; provide insight into transcriptional mechanisms (boundaries, links between exons).
- RNA-seq studies that pyrosequence ( ) fungal ITS and rDNA genes to study the human mycobiome started appearing in the past 4 or 5 years.
- Different studies use hugely varying approaches, ranging from different DNA isolation kits, analysis of different genes, different qPCR primer pairs and reactions ranging from 25 to even 45 cycles. Let’s remember PCR is not only exquisitely sensitive but also exponential so a 20 cycle difference runs the gamut from entirely missing low abundance species to detecting many artifacts.
- Molecular techniques such as qPCR are also so sensitive that environmental contaminants turn up frequently in the datasets. Lacking prior knowledge of what to look for, it’s difficult to exclude them.
- Not only do such differences make meta-analyses near-impossible, they make it difficult to distinguish clinically relevant datasets from experimental artifacts, unwittingly generated by sub-optimal experimental design and decisions.
- Problem with molecular analyses in a field of sparse databases is that in each study, data that doesn’t align to database sequences is discarded. Is such discarded data truly irrelevant or is it the missed iceberg?
- Fungal nomenclature is a major problem. Synonyms and different names for different sexual stages of the same species abound. For example, a recent study re-annotated ‘set of marker reference sequences that represent each currently accepted order of Fungi‘ (4). Careful and methodical curation required? Certainly!
II. Mycobiome-human relationship in health and disease
Commensal and environmental fungi constantly interact with the human body. How do they cause disease? Typically, underlying body perturbations such as immuodeficiencies and dysregulated immune function promote opportunistic fungal growth.
- For e.g., environmental Aspergillus spores that normally get killed could instead develop hyphae and invade tissue.
- or commensal Candida could switch from yeast to biofilms, which in turn provide rich nutritious milieu for variety of pathogenic bacteria as well as an effective barrier against antibiotics.
III. Human mycobiome health-versus-disease comparison by location
Human Skin Mycobiome
Malassezia, a common human skin mycobiome inhabitant
- Lipid-dependent fungus Malassezia are the most abundant fungi living on human skin (7, 8)
- Lack fatty acid synthase, and express lipases and hydrolases, helping them adapt to human skin (3).
- Almost all epidermal skin cells express aryl-hydrocarbon receptor (AhR) (9) and Malassezia synthesizes powerful AhR ligands, indirubin and indolo [3,2-b] carbazole (ICZ) (10). Thus, Malassezia influences skin metabolism and function by exploiting the AhR-AhR ligand pathway.
- Requiring lipids for its growth, Malassezia preferentially colonizes face, scalp and upper trunk rather than limbs , i.e. the sebaceous gland-rich areas of skin.
- Malassezia is dimorphic, i.e. has yeast and mycelial (hyphal) phases.
- Its lipid dependence makes it challenging to isolate and grow in culture. For example, Sabouraud’s dextrose agar is a commonly used fungal culture medium. Yet it does not support growth of many Malassezia species (11). Rather all Malassezia yeasts identified thus far grow in the nutritionally complex (containing Ox bile and Tween 60, a fatty acid) and unconventional Leeming and Notham agar (LNA). Even so, such approaches can miss M. restricta. Slower-growing than others in such cultures, it’s easily overgrown by related species.
- Mis-identification is another common problem with purely phenotypic approaches.
- Careful characterization of microbial species requires isolation in culture, freeze-down and subculture of frozen aliquots. Even this routine task is difficult in the case of Malassezia which, poorly viable in culture, only maintains viability when stored at -80oC, and not at 4 to 12oC, the norm for yeasts (12).
- Culture-independent, molecular approaches are thus more suitable. Tape or swab is used to take the skin sample, fungal DNA extracted and subjected to PCR. DNA extraction method, and sensitivity and specificity of the particular PCR method used greatly influence the outcome. The specific approach taken to accurately identify Malassezia needs to keep in mind that human skin is after all host to multitude of bacteria as well as other fungi such as Candida.
Malassezia in human skin
- Presumed to colonize immediately post-birth (13).
- Found in skin swabs from 78 out of 245 British neonates (<28 days of age) on LNA culture, with 41 out of 42 still positive at follow-up (14).
- M. furfur and globosa found in 60.5% and 7.2%, respectively, cultures of 195 Iranian neonates (15). Melassezia species colonizing human skin are thus neither random nor inter-changeable.
- M. dermatis found in skin of 19 healthy Koreans aged 17 to 55 years by both culture-dependent and -independent (ITS-1 and 26 rDNA) (16). Geographic difference?
Why do we have Malassezia in our skin? Are its lipid dependence and its human skin colonization pattern related to age-related human sebaceous gland activity? Data suggest so.
- Minimal in children, sebaceous gland activity increases during the teens in response to androgens, and then stabilizes from late teens until old age (17).
- Sebum, product of sebaceous glands, consists of ceramides, fatty acids, cholesterol, squalene (cholesterol precursor and also popular vaccine adjuvant but that’s another story!), triglycerides and wax esters. Sebum fatty acid composition changes markedly with puberty (18).
- Healthy human skin from 245 Canadians ranked by age (0–3, 4–14, 15–25, 26–40, 41–60, and >60-years old), swabbed on LNA cultures, showed marked increase in Malassezia-positive cultures starting from age 15, with no noticeable difference in positivity between genders (19).
- DNA analysis of 770 healthy Japanese aged 0 to 82 years also showed marked increase in early teens but had major gender differences, being much more abundant in males. M. restricta predominated in males of all ages, while doing so only in females >23 years, with M. globosa dominating at 1o to 18 years (20).
- Similar dominance of M. restricta followed by M. globosa was also found by 5.8S/ ITS2 rDNA analysis in a small Brazilian study of scalp and forehead skin from healthy and seborrheic dermatitis subjects (21).
- Sampling trunk, arms, plantar heel, toenail and toeweb fungal communities using ITS 1 and 18S rDNA gene in 10 healthy adults, Findley et al found Malassezia dominance at all sites with much greater species diversity in the foot sites (22).
- Clearly Malassezia colonization of human skin closely mirrors sebaceous gland distribution and activity and sebum fatty acid composition.
Malassezia-human skin interactions (adapted from 23) range all the way from
- Healthy commensalism
- Mild, non-clinical altered skin melanocyte pigmentation and plaques that mildly alter epidermal barrier function (Pityriasis Versicolor)
- Inflammation without adaptive immune function involvement (Seborrheic dermatitis; SD and dandruff)
- Inflammation with adaptive immune function involvement (Atopic dermatitis; AD)
- Hair follicle invasion and inflammation (Malassezia folliculitis).
- Much less is known about Malassezia’s role in psoriasis (20).
– Geographic differences in Malassezia distribution: Rare elsewhere, M. dermatis and M. japonica are more frequently found in East Asia, (20, 24, 25), and in India (26).
Malassezia and Psoriasis
- Psoriasis is a chronic skin inflammation marked by hyperproliferation and hyperkeratinization of the epidermis. Malassezia‘s role in this disease is as yet undetermined. Data are all over the place.
- While an Indian study that examined ITS 2 in addition to 28S rDNA gene found no difference in Malassezia prevalence in skin from psoriatic and healthy subjects (n=50 each) (26), a Japanese analysis of 28S rDNA gene sequences from skin samples from healthy (n=12) and psoriasis (n=12) subjects found psoriatic skin contained more diverse fungi compared to healthy skin though Malassezia was less abundant (27).
- Itraconazole, ketoconazole, and posaconazole are the most effective drugs for treating Malassezia infections (28, 29).
Malassezia and Dandruff
- In a French study of 49 volunteers examining fungal ITS 1-5.8S-ITS2 and part of the 28S rDNA genes, Malassezia restricta was found more frequently associated with dandruff (30).
- Similar dominance of M. restricta (and globosa) in dandruff scalps also seen in comparison of 62 and 57 dandruff and healthy scalp in Japanese volunteers (31).
Human Gut Mycobiome
Human Gut Mycobiome is influenced by diet
- Yeasts in human stool were first reported in 1917 (32) so human gut-fungal association is not a novel finding.
- Geotrichum candidum and Saccharomyces cerevisiae were found in gut mycobiome of people who ate cheese and drank sake among French and Japanese, respectively (33, 34).
- Reduced gut fungal diversity in US urban/suburban residents (Boulder, CO and Philadelphia, PA) eating typical western diets compared to rural Malawi residents eating diets ‘rich in maize, legumes and other plants‘ (35) revives the old question of the hygiene hypothesis, namely, whether loss of our co-evolved microbial diversity triggered the greater autoimmune prevalence in Western populations. Differences between these 2 populations include diet, hygiene and contact with animals to mention just a few of the more obvious ones.
- ITS 1 pyrosequencing found fungal genome signals in every one of 96 stool samples from healthy American volunteers (36). Proportionally Saccharomyces (89%), Candida (57%) and Cladosporium (42%). Candida correlated positively with carbohydrates and negatively with total saturated fatty acids, while Aspergillus correlated negatively with SCFA (Short-chain fatty acids). Saccharomyces showed no particular dietary trend.
- The are an indigenous Amerindian tribe living in French Guiana and Brazil in South America. Fungal ITS1-ITS4 and NL1-NL4 PCR and pyrosequencing of stool samples from 151 healthy volunteers on two different occasions, 2006 and 2010, showed not Candida albicans but Candida krusei and Saccharomyces cerevisiae were the most abundant gut fungal species. In other words a very different gut mycobiome in an isolated rural human population from the one observed in humans living in industrialized environments.
Human Gut Mycobiome in health and disease
- A 2005 stool culture study of 80 pediatric bone marrow transplant or cancer patients and 61 healthy controls on Sabouraud’s Dextrose agar found Candida albicans in 41.2% and 40.5%, respectively, and non-albicans Candida in 50% and 40.5%, respectively (38). In other words, similar Candida proportion in stool.
- Anti-Saccharomyces cerevisiae antibodies are found more frequently in Crohn’s disease (CD) patients compared to Ulcerative colits patients and healthy controls (39, 40). No consensus yet on what this signifies.
- 18S rDNA pyrosequencing of distal colon (rectal/sigmoid) biopsies from 25 children with IBD (Inflammatory Bowel Disease) compared to 12 age-matched controls, and 2 adults each either normal or with Ulcerative Colitis (UC) showed that Ascomycota and Basidiomycota were the dominant phyla. Fungal DNA was only found in few, not all, subjects (41). Antibiotics or immunosuppression weren’t responsible for these differences since these newly diagnosed IBD patients hadn’t been administered them yet. Why such poor recovery? Could it be site (colon biopsy versus stool) or choice of fungal gene (18S rDNA versus ITS 1 in other studies)?
- A Chinese ileal biopsy and stool sample study of 19 CD patients and 7 healthy volunteers (42) found CD patients had increased Candida prevalence and different gut mucosa- and stool-associated fungi species compared to controls. Red: CD; Green: Controls.
Human Oral Mycobiome
- In a study of 20 healthy volunteers, ITS 1 pyrosequencing found Candida (75%), Cladosporium (65%), Aureobasidium and Saccharomycetales (50% each), Aspergillus (35%), Fusarium (30%) and Cryptococcus (20%) but no Malassezia (43).
- But a more recent ITS 1 pyrosequencing found Malassezia in saliva of 6 out of 6 healthy adult volunteers, identifying it for the 1st time as a commensal fungal inhabitant of the human oral cavity (44). This raises the question how previous studies missed such a basic finding? Malassezia culture is difficult, requiring specialized culture media, nomenclature is muddled, fungal databases are incomplete and confusing, Malassezia is dimorphic, all possible reasons.
Summary of Human Gut-Oral Mycobiome studies
- The table to the right is my original summation of the differences between the major human gut and oral mycobiome studies published thus far.
- Differences include different diseases, tissue samples and methods, and absence of controls.
- Boy, are the methods different!
- Any generalizable observations? Yes, fungal diversity increases in GI tract-associated diseases such as CD, HBV (Hepatitis B).
Human Lung Mycobiome changes with lung disease: cause or effect? Not clear
– Mouthwash/gargle (oropharyngeal wash, OW) and BAL (bronchoalveolar lavage) ITS PCR and pyrosequencing comparison of lung transplant patients and controls indicated fungi had colonized deeper lung tissues in lung transplant patients.
- Same group showed clinically relevant fungi like Candida and Aspergillus were enriched in BAL of HIV-infected and lung transplant patients, and more frequently present, i.e. more samples positive compared to healthy controls (47).
- Comparison of mouthwash, induced sputum and BAL (bronchoalveolar lavage) of HIV-infected, COPD (Chronic Obstructive Pulmonary Disease) and normal people (48) using 18S rDNA and ITS PCR and pyrosequencing. Showed two things, one, the three sites had overlapping as well as distinct fungi with Candida dominating in mouthwash and sputum, two, Pneumocystis jirovecii was enriched in HIV-infected and COPD samples.
- Common theme emerging as in human lung mycobiome changes with diseases as it does in gut. Cause-effect distinction not yet clear.
Human Lung Mycobiome Summary
Human Plasma and Vagina Mycobiome
- Reports of mycobiome in human plasma, milk, vagina are currently restricted to only one or few peer-reviewed studies.
- One human plasma study found a surprise. Ascomycota, in particular the order Hypocreales, was dominant fungal signature in 5 of 6 subjects (50). Could source be gut since Ascomycota are prevalent there?
- A large study of 494 pre-menopausal Estonian women examined fungal mycobiome using ITS1 and 2 pyrosequencing in vaginal fornix and cervix brush samples. They found great diversity consisting of 196 OTUs including 16 for Candida alone (51). As with other mycobiome studies, results were plagued with the issue of air-borne contamination.
IV. Saccharaomyces boulardii, example of probiotic fungus
- Effective against diarrhea in human clinical trial (52) in a study of 35 children each given either S. boulardii (250mg orally twice a day) or not. Children given S. boulardii recovered faster from both diarrhea (3.4 versus 5.5 days) and vomiting (2.5 versus 3.3 days) (statistically significant).
- Could such differences be biologically relevant? Absolutely and this table explains why.
- Other S. boulardii studies: 11 randomised clinical trials for acute infectious diarrhea (AID), 9 for antibiotic-associated diarrhea (ADD), 4 each for Helicobater pylori infection, and Crohn’s disease (CD), 1 for Ulcerative colitis (UC), 5 newborn studies, and 3 for IBS (Irritable Bowel Syndrome).
- S. boulardii is very effective in disease treatment either alone (for AID and ADD) or as an adjunct (for H. pylori, CD, UC).
- Extensive and larger trials of S. boulardii for CD, UC and IBS are very much warranted.
- A good place to learn more about S. boulardii, especially its history is here:
How does S.boulardii work against so many diseases? Many hypotheses (adapted from 53):
- Secretes polyamines
- Restores normal levels of colonic Short-chain fatty acids (SCFA)
- Stabilizes gut epithelium barrier function
- Restores fluid transport pathways
- Induces enhanced gut mucosal secretory IgA production
- Neutralizes bacterial toxins, specifically those of Clostridium difficile
- Its metabolic functions, such as polyamines, accelerate re-establishment of normal gut microbiota
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