Sequence-based methods for detecting and evaluating the human gut mycobiome

Abstract

We surveyed the fungal microbiota in 16 faecal samples from healthy humans with a vegetarian diet. Fungi were identified using molecular cloning, 454 pyrosequencing and a Luminex analyte‐specific reagent (ASR) assay, all targeting the ITS region of the rRNA genes. Fungi were detected in each faecal sample and at least 46 distinct fungal operational taxonomic units (OTUs) were detected, from two phyla — Ascomycota and Basidiomycota. Fusarium was the most abundant genus, followed by Malassezia, Penicillium, Aspergillus and Candida. Commonly detected fungi such as Aspergillus and Penicillium, as well as known dietary fungi Agaricus bisporus and Ophiocordyceps sinensis, are presumed to be transient, allochthonous members due to their abundance in the environment or dietary associations. No single method identified the full diversity of fungi in all samples; pyrosequencing detected more distinct OTUs than the other methods, but failed to detect OTUs in some samples that were detected by cloning and/or ASR assays. ASRs were limited by the commercially available assays, but the potential to design new, optimized assays, coupled with speed and cost, makes the ASR method worthy of further study. Significance and Impact of the Study Fungi play a role in human gut ecology and health. The field lags immensely behind bacterial gut microbiota research, and studies continue to identify new fungi in faecal samples from healthy humans. However, many of these ‘new’ species are incapable of growth in the human GI tract, let alone making a meaningful contribution to the gut microbial community. Fungi actually inhabiting and impacting the gut likely constitute a small set of species, and an optimized, targeted, probe‐based assay may prove to be the most sensible way of quantifying their abundances.

Fungi in the healthy human gastrointestinal tract

“…51 Finally, Suhr and colleagues examined 16 samples from 15 vegetarians, while Hallen-Adams et al used the same methodology in the same laboratory to isolate and sequence fungal DNA from 69 samples from 45 people on a conventional Western diet. 22, 23 The distribution of fungi differed considerably between the 2 groups ( Table 2). Plant pathogenic Fusarium was detected in all but 2 samples from vegetarians (14 samples; 88%), while it was only detected in 2 samples from participants on conventional diets (3%).…”

“…Malassezia has also been reported in significant abundance in fecal samples and may play a role in the gut. 29,30,23 To determine whether any Malassezia is indigenous to the gut would require more invasive sampling techniques than the standard fecal collection, to eliminate the possibility of inoculation of the feces by skin flora.…”

“…commune, foodborne species incapable of growing at 37 C. 39,40 Finally, there is the long tail of rarely-detected fungi which make up most of the species richness in gut mycobiota studies but whose presence in human fecal samples is strictly incidental and cannot be assumed to influence the gut ecology. These include edible mushrooms, 23 plant pathogens (widely reported; see, e.g. refs.…”

“…The studies from our own lab identified 97 distinct fungal taxa in 85 samples from 60 subjects. 22,23 Forty-eight taxa were limited to a single sample, while 14 occurred in 10 samples or more. The most commonly reported genera and species of gut fungi are given in Table 1.…”

Primary sclerosing cholangitis: 50 years of a gut–liver relationship and still no love?

“…DNA sequencing now allows for relatively cheap characterisation of the bacterial composition of any given sample. The same technology allows for complete characterisation of the genetic variability of the bacterial content (metagenome sequencing),14 as well as similar assessments of gut-resident viruses (the gut virome) and fungi (the gut mycobiome) 15 16. The gut microbiota in metabolic and inflammatory diseases show significant deviations from the healthy state, typically with reduced diversity of the overall bacterial flora (loss of microbial diversity) and altered abundance of particular bacteria.…”