Location: Immunity and Disease Prevention Research
Project Number: 2032-51530-026-009-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Jul 1, 2019
End Date: Jun 30, 2024
Objective:
Objective 1: Determine whether honey can promote a healthy microbial composition in the small intestine that is resistant to colonization by food-borne pathogens.
Objective 2: Analyze the effect of honey fermentation products on gut barrier function and the inflammatory state of intestinal epithelial cells in a polarized intestinal epithelial cell culture model.
Approach:
Approach 1:
- In vitro gastric digestion: Honey or a fructose/glucose control will be mixed with simulated gastric juice and incubated at 37 °C for 3 hours. The digesta will be used to inoculate small intestine microbial fermentations.
- Small intestine microbial fermentations: Bacterial communities will be obtained from six individuals with an ileostomy. Fermentations will be performed in a minimal growth medium with 0.5% honey, fructose/glucose, or basal media alone.
- Pathogen growth and survival: C. jejuni (ATCC 33560) or ETEC cells will be inoculated into half of the cultured communities. All fermentations will proceed at 37°C for 72 hours. The growth and survival of C. jejuni or ETEC will be measured at 12 hour intervals by plating dilutions of the bacterial mixture on Campy CVA or MacConkey agar. Assessment of changes in the microbial community: An aliquot will be taken from the fermentation at time 0 and at 12 hour intervals until 72 hours. The 16S rRNA V4 region will be amplified and sequenced from bacterial DNA and the sequences will be analyzed using QIIME 2.
Approach 2:
- Treatment of Intestinal Caco-2 cells: Caco-2 cell monolayers in transwells will be incubated with or without 250 uL fermentation supernatant (apical) and in the absence or presence of LPS (apical) and TNF-alpha (basolateral).
- Assessment of barrier function and inflammation: Tight junction integrity will be assessed by measuring the transepithelial electrical resistance (TEER) of the monolayer. Pro-inflammatory cytokine IL-8 will be measured by ELISA.
