In vitro examination of the effect of honey on the survival of the foodborne pathogen enterotoxigenic E. coli in a small intestine microbial mock community

Authors: TRAXLER, STE - University Of California, Davis; Storms, David; FASO, ALYCE - University Of California, Davis; Lemay, Danielle; Kable, Mary

Submitted to: Current Developments in Nutrition
Publication Type: Abstract Only
Publication Acceptance Date: 4/3/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary: Objectives: Previously, our lab has shown that in an in vitro gastric digestion with honey, the foodborne pathogen enterotoxigenic E. coli (ETEC) displays a significant decrease in survival as compared to a matched simple sugar control or media alone. Here, we investigate whether this effect persists in an in vitro small intestine mock community fermentation. Methods: An in vitro gastric digestion of both sterile filtered honey and a simple sugar control (42% fructose, 35% glucose, 23% water) was performed in a simulated gastric electrolyte solution containing pepsin and hydrochloric acid (HCl) and duplicate digests were spiked with 8x10^5 CFU/mL ETEC. Resulting digesta was added at 1% w/v to small intestine microbial mock community fermentations, containing 1.0x10^5 cells each of 8 common small intestinal commensal bacteria, and fermented anaerobically for 6 hours at 37' and an initial pH of 5.8. Samples were collected for plating on selective MacConkey agar to assess total E. coli survival; optical density and pH were measured to determine total bacterial growth and estimate the relative production of acids in the culture media, respectively. Colony isolates from ETEC survival plates were cultured in brain heart infusion (BHI) broth and processed for plasmid DNA. PCR was performed with primers for either the housekeeping gene arcA or the virulence factor Sta2. Results: Mock community-only cultures showed an initial trend for increased optical density (total growth) in the sugar control condition; at 6 hours this trend reversed and sterile filtered honey saw a significant increase compared to control. Fermentations spiked with pathogenic ETEC saw no significant differences in total growth between conditions. Colony counts of total E. coli growth showed a trend for increased survival in sterile honey, and this trend was repeated for ETEC survival, though it did not reach statistical significance in either case. Conclusion: Our experiments suggest honey may promote more robust growth of small intestine microbial communities, compared to a sugar control, but this growth is not protective against ETEC within the context of an in vitro small intestine environment. Research is ongoing to investigate changes in community composition and expression of toxins.

Technical Abstract: Objectives: Previously, our lab has shown that in an in vitro gastric digestion with honey, the foodborne pathogen enterotoxigenic E. coli (ETEC) displays a significant decrease in survival as compared to a matched simple sugar control or media alone. Here, we investigate whether this effect persists in an in vitro small intestine mock community fermentation. Methods: An in vitro gastric digestion of both sterile filtered honey and a simple sugar control (42% fructose, 35% glucose, 23% water) was performed in a simulated gastric electrolyte solution containing pepsin and hydrochloric acid (HCl) and duplicate digests were spiked with 8x10^5 CFU/mL ETEC. Resulting digesta was added at 1% w/v to small intestine microbial mock community fermentations, containing 1.0x10^5 cells each of 8 common small intestinal commensal bacteria, and fermented anaerobically for 6 hours at 37' and an initial pH of 5.8. Samples were collected for plating on selective MacConkey agar to assess total E. coli survival; optical density and pH were measured to determine total bacterial growth and estimate the relative production of acids in the culture media, respectively. Colony isolates from ETEC survival plates were cultured in brain heart infusion (BHI) broth and processed for plasmid DNA. PCR was performed with primers for either the housekeeping gene arcA or the virulence factor Sta2. Results: Mock community-only cultures showed an initial trend for increased optical density (total growth) in the sugar control condition; at 6 hours this trend reversed and sterile filtered honey saw a significant increase compared to control. Fermentations spiked with pathogenic ETEC saw no significant differences in total growth between conditions. Colony counts of total E. coli growth showed a trend for increased survival in sterile honey, and this trend was repeated for ETEC survival, though it did not reach statistical significance in either case. Conclusion: Our experiments suggest honey may promote more robust growth of small intestine microbial communities, compared to a sugar control, but this growth is not protective against ETEC within the context of an in vitro small intestine environment. Research is ongoing to investigate changes in community composition and expression of toxins.