Gut mycobiota and adenomas

Abstract

Fungal microbiota (mycobiota) is potentially involved in the intestinal illness. The characterization of the mycobiota in patients with adenomas is essential for understanding the etiology of the pre-cancerous lesion since the mycobiota is potentially associated with the presents of adenomas. The recovery of the mycobiome may also help to identify the potential biomarkers which may closely relate to different stages of adenoma.

Keywords:

• gut microbiota
• intestinal adenoma
• mycobiota

This article refers to:

Abbreviations

CD	=	Crohn’s Disease
CRC	=	Colorectal Cancer
DGGE	=	Denaturing Gradient Gel Electrophoresis
GI	=	Gastrointestinal
IBD	=	Inflammatory Bowel Disease.

Colorectal cancer (CRC) caused the third of cancer-related deaths world-wide. More than 80% of CRC are sporadic CRC cases which were induced by, the precancerous lesion, colorectal adenomas.¹ So far, the etiology of adenomas is still unclear. Based on the current study, several risk factors closely related with it and gut microbiota is the most significant ones may associate with the formation and progression of intestinal adenoma and CRC. Despite extensive literature on the human gut microbiota, little is known regarding the gut fungal microbiota, mycobiota, especially the intestinal mucosal-associated mycobiota and the dynamic changes during gut disease development. Because the dysbiosis of intestinal mucosal-associated fungal microbiota may be associated with the etiology of IBD⁵ and CD,⁶ changes in the gut fungal microbiota may be involved in the development of adenoma. In a study published on January 23th, 2015 in the journal of Scientific Reports, we characterized the intestinal fungal microbiota in patients with adenoma using high through-put sequencing platform.⁴ 27 paired tissue of adenoma and adjacent biopsies collected from adenomas patients were involved in this study. The results showed that 5 fungi phyla were characterized and 3 of which were predominant in all biopsies samples including Ascomycota, Glomeromycota and Basidiomycota. The same tendency was also been found in the study which focused on inflammatory bowel disease (IBD) patients using denaturing gradient gel electrophoresis (DGGE).⁵ However, fewer fungi phyla were found compared with the high-through put sequencing platform. More importantly, in the 60 fungi genus characterized, 2 opportunistic fungi genera, Phoma and Candida, were abundantly (45%) present in all 54 biopsy samples. Phoma was previously been observed accounting for 2.8% of the fungi in oral rinse samples from healthy subjects, was firstly been found predominantly in intestinal biopsy samples in subjects with adenomas. At species level, Candida tropicalis, perhaps associated with fungal infection in severe ulcerative colitis, was also found to be present in all 54 biopsy samples at relatively high abundance. Taken together, our results indicated that the pathogenetic fungi dominantly present in intestinal mycobiota may be common among patients with adenomas. And it is also likely to be involved in the development of intestinal adenoma.

In order to find out whether the individual OTUs may involve in the etiology of adenomas, we further explored the global structure of mycobiota at OTU level in biopsy samples of subjects with adenomas. Several helpful results were obtained. Firstly, more OTUs (232) were characterized in adenomas patients compared with IBD patients and healthy subjects (43).⁵ And a decreased diversity of adenomas was noticed compared with control biopsy samples in our study. However, at global level, there was no significant difference between adenomas and adjacent biopsy samples, even though several OTUs showed a significant difference at a phylum or order level. Moreover, colorectal adenomas are classified into advanced or non-advanced stages² according to the American Society for Gastrointestinal Endoscopy (ASGE) guideline. Advanced adenomas can further develop into carcinoma.³ Further analysis revealed that there was a discrepancy between advanced and non-advanced adenomas biopsy samples in PCA analysis. Two OTUs were significantly enriched in advanced biopsy samples compared with non-advanced adenomas tissue. And the other 2 OTUs, which can be assigned to Fusarium and Trichoderma genus, were significantly enriched in advanced subjects when comparing the adjacent biopsy samples. Since Fusarium⁷ and Trichoderma⁸ genus can cause infections in human, therefore their colonization might be associated with different stages of adenoma progression. The clinical data, especially adenoma size and disease stage, showed a close relationship with the OTU Shannon-Wiener diversity in our study.

Some evidence suggests a role for an imbalance between GI commensal fungal and bacterial microbiota or the invasion of host niches by pathogenic fungi or fungal metabolites in inflammatory bowel disease (IBD),⁵ peptic ulcers,⁹ irritable bowel syndrome (IBS),¹⁰ antibiotic associated diarrhea (AAD)¹¹ and chemotherapy-induced enteric disorders.¹² Our results further revealed that there was also a change in the fungal microbiota structure in adenoma patients at different stages of adenoma and that the opportunistic pathogenetic fungi predominant in mucosa biopsy samples. Moreover, we characterized the microbiota of the mucosal biopsies and try to find out whether there is a correlation between fungi and bacteria in adenomas patients based on the core OTUs analysis (see Fig. 1). The results showed that 4 fungal OTUs, including Phoma, Acaulospora, Candida, Cladosporium and 2 bacteria genus Tatumella and Escherichia/Shigella involved in this relationship. Phoma and Acaulospora both negatively correlated with Tatumella. Cladosporium is positively correlated with Candida while negatively correlated with Escherichia/Shigella. As the opportunist pathogenic fungi Phoma and Candida were dominant in intestinal mucosa of adenomas patients⁴ and have been reported to be involved in human infections.^13,14 While no reports show that Acaulospora, a plant fungal pathogen, is involved in human infections. For the bacteria genus Tatumella, which covered 5 species, is negatively correlated with Phoma and Acaulospora. The typical species T. ptyseos is a rare opportunistic pathogen and can caused infection usually in patients with low immunity.¹⁵ So perhaps the fungi pathogens Phoma also involved in the formation and progression of adenomas but expel the potential opportunistic pathogen such as Tatumella. For the 2 pathogenic fungi genera Candida and Cladosporium were positively correlated with each other and this synergism relationship further inhibit the pathogenic bacteria genus Escherichia/Shigella by Cladosporium. Furthermore, there were 27 bacteria genera positively correlated with each other and they are strongly negatively associated with Bacteroides and Lachnospiracea_incertae_sedis. Based on the current study, Bacteroides are suspected to be associated with colon polyps¹⁶ and colorectal cancer.¹⁷ While Lachnospiracea_incertae_sedis is butyric acid producer which might provide relatively high concentrated products compared with physiological concentration in the microenvironment to help the formation of polys and the survival of Bacteroides.

Figure 1. The Correlation between bacteria and fungi in tissue biopsy in patients with adenomas based on the core OTUs analysis. The core OTUs represent in more than 20% of tissue biopsies and supported by more than 10 reads. OTUs nodes are marked with the genus names (some with more precise species names), and edges with spearman ranked correlation coefficient with cutoff ± 0.5 are indicated. Blue Squares indicated bacteria and orange squares are indicated fungi. Each node represents a taxon color-coded for co-abundance group and each line highlights a significant correlation between genera. The thickness of the lines is proportional to correlation strength.

However, up to date, the critical role of mycobiota in human healthy and disease was neglected. Few studies focused on human related fungi and the interactions between fungal-fungal, fungal-bacterial and fungal-host immunity system. Also the metabolites of fungi, such as mycotoxins, may involve in the development or progression of gastrointestinal illness also needs to be investigated. The mycobiota structure of fecal samples and mucosal biopsies is still unclear in healthy subjects and patients with gastrointestinal disease. The biomarkers closely related with the presence of gastrointestinal illness such as IBD, IBS, UC, Crohn’s disease and CRC et al also need to be discovered. More importantly the causal relationship between specific fungi or community structure and digestive disease need to be further investigated using animal models. Currently the high-through put sequencing platform is the most powerful tool to characterize the mycobiota and microbiota associated with human. The understanding of the diversity and abundance of the fungi in healthy subjects is a crucial step since it plays the fundamental role in the determination of the mycobiota structure in healthy conditions. Moreover, a large number of healthy biopsy samples should be collected in order to better define healthy mycobiota structure due to the variations in healthy individuals. Furthermore, the specific fungi and bacteria of intestinal biopsy samples should be investigated in the development of adenomas and whether the fungal microbiota is an independent etiologic factor of colorectal adenomas.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

The study was supported by grants from the China Ministry of Science and Technology Project 973 (numbers 2015CB554204).