Creating a fluorogenic assay for characterization and functional profiling of the gut microbiota

Purpose: The purpose of this study is making the ability to biochemically generate a “fingerprint” to understand the functional properties of the gut microbiota. Also known as gut microflora, this ecosystem has a huge influence on a person’s ability to fight infection to how drugs are metabolized in the body. Furthermore, research has shown the impact (but not necessarily the specific primary hydrolytic and reductive reactions) of the microbial community. Research has also noted that gut microbiota can be changed by xenobiotics just as much as has the impact on them.

Methods: Bacterial have enzymes for xenobiotic metabolization/transformations and are responsible for biological functions. Enzymes often can be analyzed easily and quickly with fluorogenic reactions. The energy from the reactions are easily recorded and to prepare. For this particular study, coumarin based substrates are paired with the bacterial sample into individual wells on a 96 well plate. The substrates are aligned with control wells, tested in triplicates, and in increasing concentration. Control wells allows to observe the stability of the coumarin substrate in the environment without the sample. As for the increasing concentration of coumarin substrates, this eventually creates the opportunity to observe dose dependence of drugs and human gut microflora. Each of the microbial samples should then have a combination of reactions that would classify other microbial samples that are similar to itself. The complex microbial samples are fecal matter from wild type mice and a mouse model. The mouse model is a knockout MyD88, which is an critical innate immunity gene. Without MyD88, these mice were found to have metabolic issues.

Results: There were prominent patterns of enzymatic turnover rate between each of the individual bacterial strains. One of the differences between the individual bacterial strains observed was dose dependence. Another was that the lactobacilli had the most galactosidase activity when compared to Enterococcus and Streptococcus, which had more glucosidase turnover. The complex samples had enzymatic activity that could distinguish wild type versus diabetic mice. There were three particular enzymes differed between the two complex samples: glucuronidase, N-acetyl-B-d-glucosaminidase, and esterase. Glucuronidase is important to hormonal reabsorption from outside sources and MyD88 -/- showed less of this activity, which possibly contribute to why the unstable metabolic state can have hormonal issues. However, the MyD88 -/- had higher enzymatic turnover rate for other type of sugar processing enzymes.

Conclusions: Creating a fluorogenic panel provides the opportunity for clinics to diagnose microbial related diseases or use in adjunct to other diagnoses tests. Furthermore, the fluorescent panel would help to refine dosing for certain patients on different medications depending on their microbial composition, figure out what combination of drugs can help a patient achieve a more successful regimen to treat their disease state. There is also the potential to gain a better understanding of drug metabolism to support drug development. Ultimately, paving a way to more personalized medicine.