Micro-eukaryotic diversity of the human distal gut microbiota: qualitative assessment using culture-dependent and -independent analysis of faeces

Abstract

Molecular ecological surveys of the human gut microbiota to date have focused on the prokaryotic fraction of the community and have revealed a remarkable degree of bacterial diversity and functionality. However, there is a dearth of information on the eukaryotic composition of the microbiota, and no culture-independent sequence-based surveys of human faeces are available. Culture-independent analyses based on DNA extraction and polymerase chain reaction targeting both the total eukaryotic 18S rRNA genes and fungal internal transcribed regions (ITS), together with culture-dependent analyses of fungi, were performed on a group of healthy volunteers. Temporal analysis was also included wherever possible. Collectively, the data presented in this study indicate that eukaryotic diversity of the human gut is low, largely temporally stable and predominated by different subtypes of Blastocystis. Specific analyses of the fungal populations indicate that a disparity exists between the cultivable fraction, which is dominated by Candida sp, and cultureindependent analysis, where sequences identical to members of the genera Gloeotinia/Paecilomyces and Galactomyces were most frequently retrieved from both fungal ITS profiles and subsequent clone libraries. Collectively, these results highlight the presence of unprecedented intestinal eukaryotic inhabitants whose functional roles are as yet unknown in healthy individuals. Furthermore, differences between results obtained from traditionally employed culture-based methods and those obtained from culture-independent techniques highlight similar anomalies to that encountered when first analysing the bacterial diversity of the human faecal microbiota using culture-independent surveys.

The Gut Microbiota: Ecology and Function

“…The cultivable fraction of the fungal microbiota in human feces is dominated by Candida spp. strains (Scanlan and Marchesi, 2008), whereas culture-independent analysis revealed that members of the genera Gloeotinia/Paecilomyces and Galactomyces were the most abundant (Scanlan and Marchesi, 2008). However, the diversity and abundance of fungi in the GI tract is quite low relative to that seen with the bacteria.…”

“…One study identified a wide variety of fungi in mouse feces, but it was not determined whether they were colonizing commensals or simply transient organisms attached to dietary particles (Scupham et al, 2006). Repeated sampling of the human gut indicated that some fungi were temporally stable and actually colonized the intestine (Scanlan and Marchesi, 2008). Similar to bacteria, there is a great disparity between what can be cultivated and what can be detected using DNA-based methods.…”

In situ proteo-metabolomics reveals metabolite secretion by the acid mine drainage bio-indicator, Euglena mutabilis

“…All extracted genomic DNA samples were stored at À20 1C until further processing. 18S rRNA genes of the eukaryotic microbial community were amplified using primers Euk1A and Euk516r (Scanlan and Marchesi, 2008). Three independent amplifications were performed as follows: denaturation at 94 1C for 1 min, annealing at 64 1C for 1 min, amplification at 72 1C for 1 min and repeated 35 times.…”

The human gut mycobiome: pitfalls and potentials--a mycologists perspective

“…The protist Blastocystis and fungi (as a group) both have been reported as the dominant eukaryotes in the gut (Rajilic-Stojanovic et al 2007, Nam et al 2008, Scanlan and Marchesi 2008. The term mycobiome was coined in 2010 to differentiate the fungal biota from the bacterial biota, in that the word "microbiota" is frequently treated as synonymous with bacteria (Ghannoum et al 2010).…”