Metagenomic Characterization of Microbial Communities In Situ Within the Deeper Layers of the Ileum in Crohnâs DiseaseSummary

Background & Aims: Microbial dysbiosis and aberrant hostâmicrobe interactions in the gut are believed to contribute to the development and progression of Crohnâs disease (CD). Microbiome studies in CD typically have focused on microbiota in feces or superficial mucosal layers of the colon because accessing DNA from deeper layers of the bowel is challenging. In this study, we analyzed the deep tissue microbiome in patients who underwent surgical resection of the small intestine. Methods: Paraffin blocks were obtained from 12 CD patients undergoing ileocecal resection, and healthy ileum samples (inflammatory bowel diseaseâfree controls) were obtained from 12 patients undergoing surgery for right-sided colon cancer. Diseased and healthy-appearing ileum was identified using microscopy, and paraffin blocks were macrodissected using a core needle to specifically isolate DNA. Illumina Whole Genome Sequencing was used for microbial sequence identification and subsequent taxonomic classification using the PathSeq tool. Results: We observed significant differences between the microbiome of CD samples vs inflammatory bowel diseaseâfree controls, including depletion of Bacteroidetes and Clostridia. Notably, microbial composition at the phyla level did not differ markedly between healthy and diseased areas of CD patients. However, we observed enrichment of potentially pathogenic organisms at the species level. Conclusions: Our study showed dysbiosis within deeper layers of the ileum of CD patients, specifically enrichment of enterotoxigenic Staphylococcus aureus and an environmental Mycobacterium species not described previously. Future studies with larger cohort sizes are warranted to confirm these findings. Studies would benefit from effective microbial DNA extraction methods from paraffin sections and host nucleic acid depletion approaches to increase microbial read coverage. Keywords: Crohn's Disease, Deeper Mucosal Layers, Microbial Dysbiosis