Evaluating the effects of a bioengineered live biotherapeutic on a colitis-associated dysbiotic microbiome and horizontal gene transfer

The current treatments for inflammatory bowel disease (IBD), an incurable group of diseases characterized by relapsing intestinal inflammation, harbour significant side effects. Disease-associated changes, like a loss of symbiosis to the gut microbiome, the collection of intestinal microorganisms, is a well-established hallmark of IBD. Attributed to microbiome changes, microbial therapeutics like probiotics have long been proposed as an IBD therapy. However, their efficacy in IBD clinical trials is inconsistent, possibly due to lack of effective persistence, especially in a chronically inflamed gut. The Gibson lab bioengineered Escherichia coli Nissle 1917 (EcN::ttr) to leverage inflammatory byproducts for sustained IBD gut persistence. While EcN::ttr was previously shown to be protective in pre-clinical colitis models, its impact on the dysbiotic gut microbiome during colitis was examined herein, including if the engineered genes were transferred to other gut bacteria via horizontal gene transfer (HGT). To approach this, we administered EcN::ttr to Muc2-/- mice with chronic spontaneous colitis and examined colon tissue samples via shotgun metagenomics sequencing. EcN::ttr intervention made specific, nuanced ecosystem and predicted functional changes including enriching beneficial bacteria, reducing samples with detected levels of a pathogen, and enriching pathways/genes that may promote host colitis recovery and microbial adaptation to inflammation. In another EcN::ttr treated-Muc2⁻⧸⁻ mice cohort, we sought evidence of HGT events at 8 months of age. We rationalized that the colitic environment of 32 weeks post-EcN::ttr inoculation would provide the stressor for enhanced selection pressure for HGT. We collected stool and grew bacterial colonies under various conditions and tested in a tetrathionate colorimetric assay. We screened over 1000 colonies for tetrathionate reduction, of which 64% were tetrathionate positive. From these, 100% were confirmed to be EcN::ttr via PCR. We conclude that if HGT occurs, it is at a much lower frequency that we report here. Overall, this research illuminates how EcN::ttr can mitigate colitis through gut microbiome modulations, informing its suitability and safety for IBD patients. Given the lack of frequent HGT events, revealing the inherent stability of the EcN chromosomal genetic modification, this data supports EcN::ttr safe use in human and veterinary applications for treatment of intestinal inflammation-associated diseases.