The human mycobiome in health and disease

Cui et al. Genome Medicine 2013, 5:63 http://genomemedicine.com/content/5/7/63 REVIEW The human mycobiome in health and disease Lijia Cui1, Alison Morris2 and Elodie Ghedin1,3* Abstract The mycobiome, referring primarily to the fungal biota in an environment, is an important component of the human microbiome. Despite its importance, it has remained understudied. New culture-independent approaches to determine microbial diversity, such as next-generation sequencing methods, are greatly broadening our view of fungal importance. An integrative analysis of current studies shows that different body sites harbor specific fungal populations, and that diverse mycobiome patterns are associated with various diseases. By interfacing with other biomes, as well as with the host, the mycobiome probably contributes to the progression of fungus-associated diseases and plays an important role in health and disease. Introduction Studies of fungi have been ongoing for more than 150 years, beginning with the first mycological paper published in 1852 [1,2]. It is only in the past 10 years, however, that the microbiome as a novel microbiological concept, in which all microbes (bacteria, archaea, viruses and fungi) in an environment are taken into account, has drawn closer attention [3]. In particular, two large government-backed projects launched in the past five years - the Human Microbiome Project (HMP) in the United States and Metagenomics of the Human Intestinal Tract (MetaHit) in Europe - have provided progress in this new field of research [4-6]. These projects have defined what constitutes the normal bacterial microbiome of various human body sites, such as the oral cavity, skin, vagina, and gut. Exciting as the results have been, the characterization of fungal diversity in these body sites is still lacking. In fact, the word ‘microbiome’ has implied reference to only commensal and pathogenic *Correspondence: 1 Center for Vaccine Research, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA Full list of author information is available at the end of the article © 2010 BioMed Central Ltd © 2013 BioMed Central Ltd bacteria [7]. This connotation changed in 2010, when the term ‘mycobiome’ (a combination of the words ‘mycology’ and ‘microbiome’) was first used to refer to the fungal microbiome [8]. Still, in a recent search of PubMed (6 July 2013), the term ‘mycobiome’ appeared in only 10 publications, and relevant studies - with or without using this specific word - numbered fewer than 40. Clearly, this field is still in its infancy. There are several reasons to include the mycobiome in assessments of the biota of specific environments. First, the incidence of fungal infection has increased greatly in the past two decades, primarily in opportunistic infections of immunosuppressed populations, such as in those who have AIDS or have undergone organ transplantation or cancer chemotherapy [9]. Second, several diseases that were formerly considered to have no association with fungi, such as hepatitis B [10], cystic fibrosis [11,12] and inflammatory bowel disease (IBD) [13,14], are now found to be associated with particular mycobiomes. Finally, the interaction between different biomes [15], and between the host and the mycobiome [16], are critical in disease progression. In this review, we summarize the methodology used to study the mycobiome, its composition and role in health and disease, and the outlook for this field. Tools used to explore the fungal biome The most basic fungal culture technique, dating back to the 1920s, involved growing fungal mats in flasks of sterile liquid media. The experimental results were evaluated by looking at the growth structures and assessing their composition within the media [17]. Fungal culture methodology has improved over the decades, but even now, organisms that occur in low abundance and those that require microbe-microbe interactions to grow cannot be cultivated optimally [18]. Like unculturable bacteria in the microbiome, unculturable fungi comprise the largest part of the human mycobiome. In a study of the bacterial and fungal microbiome of patients with cystic fibrosis, more than 60% of the species or genera were not detected by culture [19]. A study focusing on the mycobiome of the oral cavity reported that 11 of the 85 fungal genera identified could not be cultured [6]. In the gastrointestinal mycobiome, culture-independent methods identified 37 Cui et al. Genome Medicine 2013, 5:63 http://genomemedicine.com/content/5/7/63 different fungal groups compared to only 5 species found by culture-dependent analyses [10]. The limitations of culture-dependent methods for mycobiome studies have led to the introduction, over the past 20 years, of culture-independent approaches. Methods for classifying fungi that do not rely on microbial culture include restriction fragment length polymorphism (RFLP) analysis, oligonucleotide fingerprinting of rRNA genes (OFRG), denaturing gradient gel electrophoresis (DGGE), and in-situ hybridization (Table 1). These tech niques are useful for comparing fungal diversities between different groups, but they lack the specificity necessary to identify the different fungal species in a large-scale study. Direct sequencing of fungal genes has proven to be the most efficient method for classifying the mycobiome. Furthermore, with the development of next-generation sequencing (NGS) platforms, direct sequencing has become more cost-effective than was the case when only classical Sanger sequencing was available. Selecting target genomic regions to serve as proxy for the fulllength genome is a popular approach for studies of fungal diversity, as it is in the determination of bacterial diversity in microbiome studies. The fungal ribosomal RNA gene cluster (rRNA) is the region most commonly selected as proxy, with sequencing efforts primarily targeting the 18S small subunit rDNA (SSU), 28S large subunit rDNA (LSU) or the internal transcribed spacer (ITS) [20]. Although 28S has gradually been eliminated as a target sequence because of its lack of discrimination power for many species, the debate over whether the 18S or the ITS sequences are most useful is still fierce. Compared with 18S, ITS is more diverse and enables greater genus-level phylogenetic placement. However, in our own experience, the higher conservation of the 18S facilitates the amplifi cation of rDNA from various fungi, and also enables the detection of non-fungal eukaryotes, such as the parasitic protozoa Leishmania and Toxoplasma [21,22]. Which region within the ITS is selected as a target also remains a matter of choice. Several studies amplify the ITS1 or ITS2 regions, whereas others favor amplifying the entire length of ITS1-5.8S-ITS2 (Table 2; Figure 1). This selection should be made carefully because some primers, such as the ITS1 and ITS1-F primers, are biased toward amplification of Basidiomycetes, whereas others, such as the ITS2 an (...truncated)