We surveyed the mucosa-associated mycobiota across the longitudinal axis of normal-appearing adult human colon using the ITS2 amplicon sequencing. Ascomycota and Basidiomycota were the two major phyla. Malassezia and Candida were the two major genera. M. restricta, C. albicans, P. paneum, and M. spp were the major species. We found that almost all individuals were colonized with Malassezia, with sporadic colonization with other fungal genera. Notably, although we observed significant inter-individual variability in fungal composition across individuals, the distribution of fungi longitudinally along the colon was not statistically heterogeneous within each individual. The prevalence of fungi with other co-occurring bacteria was observed only for Malasseziaceae and a few bacterial families including Barnesiellaceae, Rikenellaceae, Acidaminococcaceae, and Enterobacteriaceae.
Ascomycota, Basidiomycota, and unclassified fungi accounted for 97% of the fungi phyla. A previous study using the adjacent normal colonic mucosa of 27 patients with colorectal adenoma found Ascomycota was the dominant phylum (80.5%), followed by Glomeromycota (3.1%) and Basidiomycota (2.5%) [18]. The other study using the mucosal sample of 14 patients with UC and 14 healthy controls found Ascomycota and Basidiomycota were the two dominant phyla [19]. In these two deep sequencing studies conducted in China, the relative abundance of Ascomycota was much higher than Basidiomycota. However, in our study, these two common phyla had equal abundance as detected in the normal mucosa of polyp-free individuals.
Malassezia, which belongs to Basidiomycota, was the most common genus with M. restricta being the most common species in our study. Malassezia is abundant in human breast milk and colonizes the gut during the neonatal period [31] and can survive in the gut [32]. Malassezia has previously been recognized as the dominant fungal genus in the oral mycobiome [33]. Malassezia is also a skin commensal fungus frequently associated with mild skin infections such as tinea versicolor, and occasionally results in fungemia in immunosuppressed individuals [34, 35]. Using the fecal sample, Malassezia was identified as an opportunistic fungus in patients with colorectal tumor [36]. Malassezia restricta was shown to be a key player in the pathogenesis of Crohn’s disease in a mouse model because this fungus can elicit innate inflammatory responses through caspase recruitment domain family member 9 (CARD9) [37]. While our study identified that Malassezia was the most abundant fungal genus, another study reported that Candida was the most abundant genus using 54 mucosal samples taken from cecum or rectum of 28 healthy individuals, followed by Cyberlindnera, Fusarium, Galactomyces, and Malassezia. The differences in these two studies included geography (California vs. Texas), race/ethnicity (Asian vs. African-American predominantly), and the taxonomic assignment tool (ASV vs. OTU-based) used [37]. Our finding suggested that the role of Malassezia in colonic physiology and pathology deserves more investigations.
Candida was the second most abundant genus in our study with a mean relative abundance of 11.8%, which is similar to the abundance reported by Nash using 317 fecal samples in the Human Microbiome Project [22]. Fechney et al reported that Candida is the most abundant oral colonizer in 17 children with or without dental caries [33]. C. albicans, .and C tropicalis were the major Candida spp in our study. C. albicans is not only a commensal in healthy individuals but also a pathogen of the gastrointestinal (GI) tract, at mucosal surfaces, and the blood under opportunistic conditions including disease- and drug-related immunosuppression or injury. Candida has recently been implicated in the pathogenesis of IBD [20], Clostridium difficile colitis [38], and graft-versus-host disease [39]. C. tropicalis is a common opportunistic infectious agent, especially in patients hospitalized in intensive care units [40]. C. tropicalis has also been associated with Familial Crohn’s disease [41]. In our study, Candida was not present in the colon of all participants. However, its occurrence in 43% of our colonically normal participants coupled with its known potential pathogenic significance suggested its presence and role should be further investigated.
Penicillium, commonly found in soil and belongs to Ascomycetes phylum and Aspergillaceae family [42], also colonizes the colon. P. paneum was the only species identified in our samples. P. paneum, isolated from the baled grass silage, has been shown to produce mycotoxins such as Roquefortine C, marcfortine A, and andrastin A and diverse secondary metabolites [43]. However, the implication of P. paneum in human health is essentially unknown.
Yamadazyma mexicana, unclassified fungi, and unclassified members of Cladosporium were also detected in the colonic mucosa. Yamadazyma mexicana is one of the six yeast strains that can degrade environmental hydrocarbons [44] and its role in the gut is completely unknown. Cladosporium spp is a ubiquitous fungus, which can cause skin infections and has been linked to allergic rhinitis, with its cell wall being classified as a potential allergen [45]. Among the 20 most abundant species, 10 were unclassified using our sequencing and bioinformatics pipeline. Additional research will be necessary to characterize these unclassified fungi in order to understand their potential function and significance in colon health and disease pathogenesis.
The fecal mycobiome has been examined using culture-based and non-culture-based methods. The following core fungal genera were proposed: Candida (especially C. albicans), Saccharomyces, Penicillium, Cryptococcus, Malassezia (especially M. restricta), Cladosporium, Galactomyces, Aspergillus, Debaryomyces, and Trichosporon [46]. However, Nash et al were unable to find the latter four genera using NGS in 317 stool samples [22]. Similarly, we did not identify Cryptococcus and Galactomyces in our mucosal samples. Saccharomyces was only detected in the cecum of one individual with a relative abundance of 0.18%. Saccharomyces cerevisiae was not detected in our samples. Because Saccharomyces is a yeast that is most abundant in foods, our observation supported the notion that foodborne Saccharomyces cerevisiae is likely not a mucosa-associated fungus and is only transiently present in the human GI tract [47]. A Saccharomyces-free diet has been shown to eliminate its presence in the stool [15]. In our study, participants had a full colonic preparation as well as fasted for 12 hours before the procedure. The absence of food-related fungi may be the indicator of proper colonic preparation. On the other hand, the colonization of fungi in the colon mucosa should be further investigated. Overall, the structure of the fungi in the colonic mucosa differed from that of feces shown by other studies.
Our findings did not concur with a previous research using the mucosal sample. Li et al used the 18S rRNA region to survey the mycobiome in normal terminal ileum tissues (non-colonic) samples of seven healthy volunteers [20]. They found Saccharomyces cerevisiae, Saccharomyces castellii, Candida albicans, Candida tropicalis, Gibberella moniliformis, and Sclerotinia sclerotiorum to be the most abundant species. The difference between our study and Li’s study included anatomic locations surveyed, target regions sequenced, and the genome assemblies for fungal identification. ITS2 has improved resolution of the mycobiome membership compared to metagenomics and 18S rRNA gene sequencing [48]. More research is needed to characterize the fungi entity in humans using the standardized approach.
In an earlier study, we found the bacterial distribution was largely homogeneous along the colon axis [6]. In the present study, we found that the distribution of the common fungal genus, Malassezia, was homogeneous along the colon. Most of other fungal species were only sporadically detected in the colon. Whether the rare or sporadic microorganism has any role to play in patchy colonic lesions should be elucidated further.
We found the bacterial-bacterial interaction in colonic mucosa of these normal individuals detected by SparCC. However, because fungi were sporadic, we only observed the interaction between the most common fungi, Malasseziaceae family, with Acidaminococcaceae and Barnesiellaceae, and possibly with Enterobacteriaceae. We also observed co-abundance of Aspergillaceae and unclassified member of Lactobacillales. Taken together, our data suggested that in the normal colon, there was no extensive interaction between fungi and bacteria because of the rarity of fungi.
Our study had several strengths. It was among the first to characterize fungi in the normal colon in humans. Although we provided a snapshot of the fungal profile, the survey of the mucosal mycobiota likely reflects the long-term colonic indigenous mycobiota that are less likely to be disturbed by bowel preparation. In addition, we used the identical procedure to collect biopsies where the contamination was minimized in a clinical setting. Lastly, we used the ASVs for taxonomic assignment, which is thought to be more precise than the OTU-based assignment [49].
Our study also had several limitations. First, the ITS2 ribosomal domain for fungal identification is different from that of ITS1. The taxonomic characteristics may be biased towards Ascomycota and Basidiomycota [50]. Second, the generalizability of this study was limited as it included predominantly overweight and obese middle-aged men using the VA healthcare system. Third, our sample size was limited in testing the distribution of less abundant fungi across the colon segment. Lastly, the research provided a snap-shot of the fungal distribution in the gut after bowel preparation. Additional study is needed to describe the trajectory of fungal colonization over time and with aging.
In summary, using the ITS2 targeted-amplicon sequencing and the present fungal genome assemblies, we found fungi that are commonly present in indoor and outdoor environments also inhabit the lower GI tract in humans. Our study findings argue against the absence of fungi in “healthy” adult colons. Nevertheless, Malassezia was the only prevalent fungi and others were sporadic. The potential co-occurring and co-exclusive correlation between Malasseziaceae and bacteria and its physiological implication deserve further research. The characterization of the fungal origin, richness, composition, and structure in the human colon is essential for informing future research examining the role of fungi in both maintenances of colonic health and the pathogenesis of colonic diseases. Appropriate use of antifungal therapy will be dependent on understanding the role of fungi in health and disease.