Abstract
The human gut is host to a diverse range of fungal species, collectively referred to as the gut “mycobiome”. The gut mycobiome is emerging as an area of considerable research interest due to the potential roles of these fungi in human health and disease. However, there is no consensus as to what the best or most suitable methodologies available are with respect to characterizing the human gut mycobiome. The aim of this study is to provide a comparative analysis of several previously published mycobiome-specific culture-dependent and -independent methodologies, including choice of culture media, incubation conditions (aerobic versus anaerobic), DNA extraction method, primer set and freezing of fecal samples to assess their relative merits and suitability for gut mycobiome analysis. There was no significant effect of media type or aeration on culture-dependent results. However, freezing was found to have a significant effect on fungal viability, with significantly lower fungal numbers recovered from frozen samples. DNA extraction method had a significant effect on DNA yield and quality. However, freezing and extraction method did not have any impact on either α or β diversity. There was also considerable variation in the ability of different fungal-specific primer sets to generate PCR products for subsequent sequence analysis. Through this investigation two DNA extraction methods and one primer set was identified which facilitated the analysis of the mycobiome for all samples in this study. Ultimately, a diverse range of fungal species were recovered using both approaches, with Candida and Saccharomyces identified as the most common fungal species recovered using culture-dependent and culture-independent methods, respectively. As has been apparent from ecological surveys of the bacterial fraction of the gut microbiota, the use of different methodologies can also impact on our understanding of gut mycobiome composition and therefore requires careful consideration. Future research into the gut mycobiome needs to adopt a common strategy to minimize potentially confounding effects of methodological choice and to facilitate comparative analysis of datasets.
Highlights
Our knowledge of the role that the gut microbiota plays in human health and disease has been greatly facilitated by advances in generation sequencing (NGS) technologies (van Dijk et al, 2014)
We found no significant effect of aeration on fungal counts irrespective of the media type tested (p > 0.05 repeated measures ANOVA), see Figure 2B
Freezing of samples significantly reduced the recovery of culturable fungi from samples with one freeze thaw cycle resulting in approximately a 10-fold reduction in fungal numbers post freezing compared to fresh samples (p < 0.0001, repeated measures ANOVA, adjusted p < 0.01 Holm–Sidak’s multiple comparisons test) (Figure 2C)
Summary
Our knowledge of the role that the gut microbiota plays in human health and disease has been greatly facilitated by advances in generation sequencing (NGS) technologies (van Dijk et al, 2014). NGS allows for the detailed investigation of microbial diversity and abundance, enabling researchers to identify specific microbes and microbial populations that are associated with the Methodologies to Study the Gut Mycobiome health or disease status of the host (Iliev et al, 2012; Mar Rodríguez et al, 2015). NGS can be used by researchers to investigate the influence of host genetics, host environment, and specific intervention strategies (e.g., diet, antibiotic administration, probiotics) on gut microbiota composition (Hoffmann et al, 2013). The relative impact of other factors (e.g., antibiotic intervention and host genetics) on both the composition and functionality of the gut mycobiome, and/or how they affect the ability of specific fungi to transition from commensalism to pathogenicity (Underhill and Iliev, 2014) remains largely unknown. Greater research into the fungal fraction of the gut microbiome is required to better understand its potential role in human health and disease
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.