(122f) Priming of Live Therapeutic Bacteria for Improved Survival in Gastric Fluid

There is increasing interest in the use of bacteria as live cell therapies, as they can produce and secrete biologics, sense and therapeutically respond to their environments, and modulate the composition of the human microbiome. However, the acidic conditions of the stomach significantly reduce the viability of live therapeutic bacteria (LTB) during gastric transit, presenting a delivery challenge for LTBs. It is known that pathogenic bacteria activate specific transcriptional programs during transit through the gastrointestinal tract (GIT) that improve their viability in the stomach. Therefore, we propose that LTBs can be primed to activate similar transcriptional programs in vitro to improve their resistance to gastric pH. To test this, we primed the candidate LTB E. coli Nissle 1917 (EcN) to acid conditions during overnight culture in acidic media (pH = 5.0). We show that overnight priming significantly improves the survival of EcN during a challenge in simulated gastric fluid (SGF, pH = 2.8) (p < 0.01). We found that the acid-resistance of EcN is dependent on its growth phase, with improvements in survival during the stationary phase relative to the exponential phase (p < 0.01). By screening EcN colonies that were viable after an SGF challenge (‘survivors’) and growing them in neutral media (pH = 7.0), we show that acid-resistance is transient, as the survivors have no improvement in viability compared to an SGF-naïve control during the in vitro challenge. This suggests that acidic priming activates transcriptional programs that improve survival, rather than triggering the formation of stable genetic mutants. The reversibility of EcN acid-resistance makes it an attractive delivery strategy, as it enables improvement in LTB survival during short-term gastric transit without permanently altering the EcN genome or phenotype. Collectively, this approach allows for the study of LTB adaptations to the GIT in a controlled, in vitro environment. By priming LTBs to intestinal challenges prior to oral administration, this delivery approach could provide a low-cost and rapid method for improving gastric survival and colonization of LTBs, enhancing their therapeutic potential.