Tutorial: Microbiome studies in drug metabolism

Abstract

The human gastrointestinal tract is home to a dense population of microorganisms whose metabolism impacts human health and physiology. The gut microbiome encodes millions of genes, the products of which endow our bodies with unique biochemical activities. In the context of drug metabolism, microbial biochemistry in the gut influences humans in two major ways: (1) by producing small molecules that modulate expression and activity of human phase I and II pathways; and (2) by directly modifying drugs administered to humans to yield active, inactive, or toxic metabolites. Although the capacity of the microbiome to modulate drug metabolism has long been known, recent studies have explored these interactions on a much broader scale and have revealed an unprecedented scope of microbial drug metabolism. The implication of this work is that we might be able to predict the capacity of an individual's microbiome to metabolize drugs and use this information to avoid toxicity and inform proper dosing. Here, we provide a tutorial of how to study the microbiome in the context of drug metabolism, focusing on in vitro, rodent, and human studies. We then highlight some limitations and opportunities for the field.

FIGURE 1

Dominant bacterial genera (or families) across the human gastrointestinal tract. Major bacterial genera identified through 16S rRNA gene surveys and metagenomic studies discussed in the text are highlighted here.

FIGURE 2

Factors affecting the gut microbiota. Multiple factors influence the composition and structure of the gut microbiota. A subset highlighted in the text are diagrammed here. GI, gastrointestinal; HMO, human milk oligosaccharide; NSAID, nonsteroidal anti‐inflammatory drug.

FIGURE 3

Representative metabolic activities of the gut microbiota. (a) Arabinoxylan metabolism by gut Bacteroidota. Arabinoxylan consists of a beta‐1,4‐linked xylose backbone decorated with 4‐O‐methylglucuronic acid, acetate, and arabinose linkages. Bacteria encode multiple different enzyme activities required to hydrolyze these individual bonds which include endo‐xylanases, β‐xylosidases, α‐glucuronidases, acetyl‐xylan esterases, and arabinofuranosidases. (b) Bile acid 7‐α‐dehydroxylation. Several strains dehydroxylate primary bile acids (e.g., cholic acid), converting them to secondary bile acids (e.g., deoxycholic acid). (c) Digoxin is reduced by cardiac glycoside reductases forming dihydrodigoxin. (d) Bacterial glucuronidases convert the inactive metabolite of irinotecan (SN38G) to the active metabolite (SN38).

FIGURE 4

In vitro cultures of gut microbiota strains or stool to identify microbiota‐drug interactions. (a) Anaerobic gut microbial strains are arrayed into 96‐well plates and cultured in the presence of drugs. After growth, drugs are extracted from culture supernatants and analyzed by LC–MS. Extracted ion chromatograms for drugs present in supernatants of each strain can reveal drug‐microbe interactions. (b) Stool samples from human donors are inoculated into different culture media. After growth, 16S rRNA gene profiling of cultures can be compared to the original stool donor to identify media that best recapitulate the donor community composition. After identifying optimal media, stool is cultured in the presence of drugs and drug remaining in the supernatant after growth is analyzed by LC–MS. Extracted ion chromatograms for drugs present in supernatants of different stool donors can reveal individualized drug‐microbiota interactions. LC–MS, liquid‐chromatography mass spectrometry.

FIGURE 5

Loss of function and gain of function experiments to identify genes responsible for microbiota‐drug interactions. (a) Loss‐of‐function studies. A transposon library for a bacterium of interest is generated and arrayed into individual wells of microtiter plates. The mutants are then cultured with drugs and levels of drugs in the supernatant are analyzed by LC–MS. Extracted ion chromatograms reveal mutants which do not metabolize drugs as compared to the wild‐type (WT) strain. Sequencing of the transposon insertion site of such mutants can reveal genes responsible for drug metabolism. (b) Gain‐of‐function studies. Genomic DNA isolated from an individual bacterial strain or stool metagenomic DNA is sheared, then fragments are cloned into a plasmid vector and transformed into heterologous hosts (such as Escherichia coli) to make a clone library. Individual clones or pools of clones are cultured with drugs and levels of drugs in the culture supernatants are analyzed by LC–MS. Extracted ion chromatograms reveal clones which deplete drugs in the supernatants as compared to empty vector controls. Sequencing of the DNA within the plasmid can reveal genes responsible for drug metabolism. LC–MS, liquid‐chromatography mass spectrometry.