Caracterização do microbioma fecal de pacientes com retocolite ulcerativa através de metabarcoding

Abstract

The human gastrointestinal tract is composed of millions of microorganisms that form the intestinal microbiota (IM), which act in the modulation of metabolism and immunity aiming at homeostasis. The quantitative and/or qualitative imbalance of IM (dysbiosis) has been associated with the development and maintenance of inflammatory bowel diseases (IBDs), such as ulcerative colitis (UC), which has a complex pathogenesis and affects the protective layer of the colonic mucosa.Therefore, the objective was to identify the composition of the intestinal microbiome of patients with UC using high-throughput DNA sequencing (metabarcoding), comparing it with the IM of healthy individuals.A case-control study was carried out with volunteer patients diagnosed with UC (test group) at the Professor Alberto Antunes University Hospital (HUPAA), with application of a structured questionnaire and and obtaining information based on medical records. Individuals without IBD registered as healthy donors in a fecal material repository went through the same steps as the patients to compose the control group. Both patients and healthy donors were referred for laboratory tests and individually collected stool samples with a sterile kit. An aliquot was sent to the Probiome® company for DNA extraction and 16S rDNA sequencing, through the metabarcoding technique by the Illumina® platform, while another part was processed in the laboratory (weighing, filtration and dilution) for storage at - 80°C in a repository. Bioinformatics tools were used to organize the sequenced reads using QIIME2 (Quantitative Insights Into Microbial Ecology, version 1.9.1), and the online software MicrobiomeAnalyst was used to generate operational taxonomic units (OTU) with phylogenetic classification from phylum to genus. Eighteen patients with UC answered a structured questionnaire and different clinical situations were detected: distal colitis (44.44%), pancolitis (50%) and 5.56% of patients with both extensions of the disease. Seven patients (38.88%) had comorbidities in addition to UR, such as diabetes (11.11%) and hypertension (16.67%), controlled by specific medication. Regarding laboratory tests, it is noteworthy that 13 patients (72.2%) had high fecal calprotectin (FC) and 8 (44.44%) were reactive to the C-reactive protein test. After the taxonomic indications of OTUs, members of the phyla Bacteroidetes and Firmicutes were found to be predominant in 13 (72.22%) and 16 (88.89%) patients, respectively, accompanied by an increased relative abundance of Proteobacteria 72.22% (13). The Bacteroidaceae, Ruminococcaceae, Lachnospiraceae and Prevotellaceae families showed important variations in patients (10% to 47%, 1% to 28% and 1% to 52%, respectively), with the presence of Escherichia-Shigella in 5 (27.78% ). Among the patients, two had discrepancies in their microbiomes, such as the absence of Prevotellaceae in one and another with about 70% of the microbiome without identification. Depending on the patient's clinical condition (measuring by the inflammatory marker CF), we found Bacteroides spp. and Faecalibacterium spp. in high relative abundance in cases of UC remission, while patients with active UC had an abundance of the Prevotellaceae family. Rosebury spp. And Faecalibacterium spp. were identified in abundance in healthy individuals (control), in addition to noting the absence of Escherichia-Shigella in this group. Important differences in relation to enterobacteria (Phylum Proteobacteria) were observed when comparing the microbiomes of patients with UC with a control group, with an abundance of these in patients and absence in the healthy microbiome. Characteristic dysbiosis patterns were identified in the RCU microbiome with possible microbial markers, and the identification of high concentrations of beneficial microorganisms in the control group indicated potential for use in faecal microbiota transplantation to benefit patients with UC.