Comprehensive comparative analysis of the intestinal mycobiomes of pediatric patients with Crohn's disease using next-generation sequencing (NGS)
Agnieszka Krawczyk1*, Dominika Salamon1, Agnieszka Sroka-Oleksiak1, Barbara Zapala2, Tomasz Gosiewski1
1 Department of Molecular Medical Microbiology, Jagiellonian University Collegium Medicum, Poland.
2 Department of Clinical Biochemistry, Jagiellonian University Collegium Medicum, Poland
*E-mail: agnieszka.krawczyk@doctoral.uj.edu.pl
INTRODUCTION: Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract characterized by periods of exacerbation, in which an increase in disease symptoms is observed, interspersed with episodes of remission, in which the discomfort is alleviated or completely resolved. The pathogenesis of the disease remains unknown, but a growing body of data suggests that in addition to genetic and immunological factors, the gut microbiome plays a key role in the development of inflammation in the course of CD. Previous research has focused primarily on the role of bacteria in the etiology of the disease, while the contribution of fungi, which are also part of the human gut microbiota, has rarely been considered.
OBJECTIVE: The aim of this study was to comprehensively analyze the composition of the mycobiome, using next-generation sequencing (NGS), among pediatric patients with Crohn's disease compared to healthy subjects, and to correlate the results with biochemical and clinical parameters of the patients.
MATERIAL AND METHODS: The study material consisted of stool samples collected from children:
I) with CDL-C in the period of disease exacerbation (n=66)
II) with CDL-C in remission (n=39)
III) healthy, constituting the control group (n=40)
Fungal DNA was isolated from fecal samples using enzymatic and mechanical lysis to increase the efficiency of the isolation process. Then, each sample was amplified during a PCR reaction, with primers targeting the ITS-1 region to create genomic libraries. NGS sequencing was performed using the MiSeq sequencing protocol and platform (Illumina).
RESULTS: The use of NGS sequencing provided a complete picture of the composition of the intestinal mycobiota from the phylum (L2) to species (L7) level. Differences in mycobiotic profiles were observed between the group of patients with CDC and the healthy population.
CONCLUSION: Further studies are needed to clarify whether the observed changes may play a role in disease induction in individuals genetically predisposed to chL-C, or whether they are merely a secondary effect of the disease.