Fungal Dysbiosis in Mucosa-associated Microbiota of Crohn's Disease Patients

Abstract

Background and aims: Gut microbiota is involved in many physiological functions and its imbalance is associated with several diseases, particularly with inflammatory bowel diseases. Mucosa-associated microbiota could have a key role in induction of host immunity and in inflammatory process. Although the role of fungi has been suggested in inflammatory disease pathogenesis, the fungal microbiota has not yet been deeply explored. Here we analysed the bacterial and fungal composition of the mucosa-associated microbiota of Crohn's disease patients and healthy subjects.

Methods: Our prospective, observational study evaluated bacterial and fungal composition of mucosa-associated microbiota of 23 Crohn's disease patients [16 in flare, 7 in remission] and 10 healthy subjects, using 16S [MiSeq] and ITS2 [pyrosequencing] sequencing, respectively. Global fungal load was assessed by real time quantitative polymerase chain reaction.

Results: Bacterial microbiota in Crohn's disease patients was characterised by a restriction in biodiversity. with an increase of Proteobacteria and Fusobacteria. Global fungus load was significantly increased in Crohn's disease flare compared with healthy subjects [p < 0.05]. In both groups, the colonic mucosa-associated fungal microbiota was dominated by Basidiomycota and Ascomycota phyla. Cystofilobasidiaceae family and Candida glabrata species were overrepresented in Crohn's disease. Saccharomyces cerevisiae and Filobasidium uniguttulatum species were associated with non-inflamed mucosa, whereas Xylariales order was associated with inflamed mucosa.

Conclusions: Our study confirms the alteration of the bacterial microbiota and is the first demonstration of the existence of an altered fungal microbiota in Crohn's disease patients, suggesting that fungi may play a role in pathogenesis.

Keywords: Inflammatory bowel disease; fungal microbiota; mucosa-associated microbiota.

Figure 1.

Bacterial microbiota biodiversity and composition. [A] Operational taxonomic units [OTUs] number describing the diversity of the bacterial microbiota in the different groups studied [t test, ** = p < 0.01; *** = p < 0.001]. [B] Beta diversity. Principal coordinate analysis of weighted Unifrac distance with each sample coloured by the disease phenotype. PC1, PC2, and PC3 represent the top three principal coordinates that captured most of the diversity, with the fraction of diversity captured by that coordinate shown percent. [C] Global composition of bacteria at phylum level. Healthy subjects [HS] and patient sub-groups are labelled on the x-axis and expressed as relative OTUs abundance per each group.

Figure 2.

Bacterial genus differentially abundant in Crohn’s disease [CD] and healthy subject [HS] mucosa. HS [n = 10] versus CD [n = 23] patients. The histogram shows the Linear Discriminant Analysis [LDA] score computed for genus differentially abundant between the CD and HS mucosa and identified using linear discriminant analysis effect size [LEfSe].

Figure 3.

Fungal microbiota quantification and biodiversity. [A] Real-time quantitative polymerase chain reaction [qPCR] results of fungi normalised to total bacterial 16S rRNA gene copies. Global fungus level was significantly increased in Crohn’s disease [CD] flare, both in inflamed [n = 16] and non-inflamed [n = 16] mucosa compared with healthy subjects [HS] [n = 10] [t test, * = p < 0.05]. [B] Operational taxonomic units [OTUs] number describing the biodiversity of fungal microbiota in the different groups studied. [C] Beta diversity. Principal coordinate analysis of the BrayCurtis distance [for internal transcribed spacer 2 ITS2] with each sample coloured by the disease phenotype. PC1, PC2, and PC3 represent the top three principal coordinates that captured most of the diversity, with the fraction of diversity captured by that coordinate shown percent.

Figure 4.

Global composition of fungi. [A] Fungal composition at phylum level. [B] Fungal composition at genus level. Healthy subjects [HS] and patients sub-groups are labelled on the x-axis and expressed as relative perational taxonomic units [OTUs] abundance per each group.

Figure 5.

Fungal taxa differentially abundant in Crohn’s disease [CD] and healthy subject [HS] mucosa. HS [n = 10] versus CD [n = 23] patients. The histogram shows the Linear Discriminant Analysis [LDA] score computed for taxa differentially abundant between the CD and HS mucosa and identified using linear discriminant analysis effect size [LEfSe].

Figure 6.

Fungal taxa differentially abundant in Crohn’s disease [CD] flare, remission, inflamed, and non-inflamed mucosa: CD remission [n = 7], CD flare [n = 16]: inflamed mucosa [n = 16], and non-inflamed [n = 16]. The histogram shows the Linear Discriminant Analysis [LDA] score computed for taxa differentially abundant between the different groups: [A] CD flare inflamed mucosa [n = 16] versus CD in remission [n = 7]. [B] CD flare inflamed mucosa [n = 16] versus CD flare non-inflamed mucosa [n = 16].