(P22-030-23) Establishing Growth Curve Assays for Bacterial Glucosinolate Metabolism: A Study Protocol

Objectives: Inflammatory bowel diseases (IBD) cause dysfunction of the gastrointestinal (GI) tract and can result in hospitalization, suffering and disruption to overall health. Recent work has demonstrated the anti-inflammatory capacity of a broccoli sprout-diet in artificially-induced GI inflammation in pathogen free C57BL/6 mice. Microbiota samples obtained from the GI tract of these mice will be used to study the presence and activity of broccoli glucosinolate hydrolysis to create microbial-sourced bioactives, to further understand the relationship between broccoli-diets and inflammation reduction. It is imperative to validate or replicate qPCR protocols which have been established for glucosinolate metabolism in Bacteroides thetaiotaomicron (B. theta), in other bacterial species. Additionally, this project will focus on developing new growth curve assays for glucosinolate metabolism, as these methods are lacking in published literature.

Methods: Pathogen free C57BL/6 mice were given dextran sodium sulfate (DSS) into their drinking water to create a disease profile similar in development and morphology to human ulcerative colitis (UC), a type of IBD. DSS and a steamed broccoli sprout diet were administered. Samples were taken from the digesta, jejunum, cecum, and colon of mice fed broccoli diets. About 806 bacterial isolates will be grown up/cultured anaerobically on minimal/selective media containing glucosinolate-related compounds (glucoraphanin, sinigrin) to determine hydrolysis activity via spectroscopy to measure optical density of growth in competent isolates. Successful isolates will be further analyzed with LC/MS to confirm production of bioactive products, and with qPCR using the B. theta positive control genome to help identify gene targets (α-1,6-mannanase, glycosyl hydrolase, nicotinamide-dependent oxidoreductase, and transcriptional regulator protein) for glucosinolate conversion in isolates.

Results: N/A

Conclusions: In the initial study from which these samples are sourced, mice fed a broccoli diet had less inflammation than those fed a control diet and DSS, and had higher bacterial diversity in their gut. We expect that bacteria isolated from the GI of broccoli-fed mice will contain more glucosinolate-metabolizing genes.

Funding Sources: NIH (Li and Ishaq) and USDA (Li)