Jack Saczawa1, Racheal Wilkinson1, Xiang Zhang PhD2, Senu Apewokin MD1 

1Department of Internal Medicine, University of Cincinnati College of Medicine 

2Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine 

Introduction: Micro-RNAs play roles in gene regulation for both prokaryotic and eukaryotic genes. Recent studies have shown that human intestinal epithelial cells secrete exosomes that contain micro-RNAs. These exosomes are stable in the physiological environment and can directly affect bacterial growth.  This lays the foundation for a deeper understanding into the relationship between a host and its microbiota. Utilization of fecal micro-RNAs can potentially serve as a tool to reason how and why gut dysbiosis can occur in humans how the host can control bacterial functions such as toxin production. CDI. a leading cause of morbidity and mortality, results from the production of exotoxins A and B. Factors that trigger the progression from asymptomatic Clostridioides difficile (CD) colonization to the symptomatic exotoxin production that hallmarks CDI has not been clearly elucidated. This project provides insight into this process by evaluating the regulatory role of micro-rna in the control of toxin production.  

Hypothesis: We hypothesize that host intestinal epithelial cell-derived micro-RNAs align to the CD genome or mRNA and can be extracted from human stool samples  

Methods: Human stool samples were collected at various time points during a chemotherapy regiment and stored in proper storage units. Stool samples were then tested for toxin B gene (tcdb) positivity using the Cephid GeneXpert PCR test and toxin production using EIA. Stool samples were then isolated following a modified miRvana micro-RNA isolation procedure. Isolated micro-RNA samples were analyzed using gel electrophoresis. Samples were sequenced using illumine sequencing and sequence analysis to the Clostridioides difficile genome was done using miRdeep.  

Results: Micro-RNA was successfully isolated from human stool samples and enriched in the 25 base pair range for nucleotide length. These isolated micro-RNAs were able to be mapped to Clostridioides difficile genome or mRNA. 

Conclusion: The extraction method was viable in isolating fecal micro-RNAs and showed that stored human stool can be successfully isolated for micro-RNAs. These isolated micro-RNAs align to the Clostridioides difficile genome or mRNA and could provide insight into the shift from asymptomatic to symptomatic Clostridioides difficile colonization. 

Acknowledgements: This study was supported in part by NIH grant T35DK060444.