Gut bacteria may interact with therapeutic drugs in ways that can result in both reduced microbial fitness or reduced drug efficacy. For example, drugs may be inactivated or otherwise chemically transformed by the bacterial metabolism, which in some cases may then produce a toxic metabolite, and they may be sequestered by bacteria in a species-specific manner, though the latter is less well understood.
In a research paper recently published in the journal Nature by Klünemann et al. (September 8th, 2021), the interaction of 15 diverse drugs with 25 common strains of gut bacteria is investigated, revealing 70 bacteria-drug interactions that include 29 entirely novel mechanisms not yet reported.
Drug-bacteria specific bioaccumulation
Bacterial species were selected to represent a diverse and healthy microbiota. Each was exposed in culture to drug concentrations of 50 µM, within the estimated range of concentrations found in the colon during ordinary administration. Surviving bacteria were separated from the culture medium, and each was tested by ultra-performance liquid chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry, allowing the level of bioaccumulation of each drug to be assessed in each species of bacteria.
Seventeen of the tested drugs were found to be depleted in culture medium and not in bacteria in most of the tested species, implying that accumulation had taken place within the cells without chemical transformation. For example, the antidepressant drug duloxetine and the antidiabetic drug rosiglitazone were each exclusively accumulated in several species. In contrast, asthma drug montelukast and chronic obstructive pulmonary disease drug roflumilast were found to be accumulated in some species and transformed in others.
Twelve other interactions, between eight species of bacteria and five drugs, were noted to represent biotransformation events, where the metabolism of the bacteria had modified the drug. Fusobacterium nucleatum was the only bacterial strain not to demonstrate any accumulation interactions with any of the drugs tested and only engaged in transformation.
Bioaccumulation alters bacterial metabolism
While the mechanism of biotransformation can usually be attributed to the activity of metabolic enzymes, the reason for drug bioaccumulation is less well understood. To explore this, the research group firstly focused on bacterial strains in which duloxetine accumulated and set out to identify the protein targets of the drug.
An alkynated form of a drug bearing desthiobiotin was manufactured to allow drug-protein conjugates to be extracted, and 55 target proteins were identified. These were mainly found to be involved in amino acid metabolism and nucleotide biosynthesis, frequently bearing structures intended for bonding with nucleotides.
Bacterial strains that demonstrated more significant accumulation were found to have a greater incidence of drug-protein interactions.
In comparative assays, lysed cells demonstrated higher numbers of drug-protein interactions than in cells with an intact envelope, as more proteins throughout the cell can be reached without gateway proteins blocking entry, and therefore the specificity observed in accumulation between bacterial species is likely due to differences in uptake and efflux rate and specificity between the cells.
As duloxetine was found to bind to metabolic enzymes, the metabolites produced by each strain were quantified by high-performance liquid chromatography-mass spectrometry, with some species such as Clostridium saccharolyticum undergoing a significant shift in their exo-metabolome that the group state is comparable to the difference between species.
Bioaccumulation alters the microbiome
Metabolic interactions between bacterium modulate the composition of the bacterial community. Thus a change in the metabolic character of one species may influence the frequency of other species in the neighboring population.
Communities containing five species of gut bacteria, one each of which bioaccumulated or was inhibited by duloxetine, respectively, were cultured and exposed to the drug.
The group found that the composition of the community changed significantly, with E. rectale, the species inhibited by duloxetine, becoming dominant, increasing in frequency 100-fold.
The altered secondary metabolite profile of the bioaccumulating species, S. salivarius, was thought to be promoting the growth of E. rectale.
Several metabolites were identified that were depleted during independent E. rectale growth and accumulated during S. salivarius growth, including linolenic acid and glycocholic acid, and nucleotide-related metabolites such as uridine-5′-diphosphate. Therefore, the altered metabolite profile of S. salivarius when exposed to duloxetine results in enhanced growth of E. rectale.
To examine the effect of altered bacterial metabolism, the group exposed roundworm (C. elegans) to duloxetine, which as a serotonin-norepinephrine reuptake inhibitor regulates muscular movement in these animals in a dose-dependent manner, allowing measurement of movement to be used to infer drug exposure.
A bioaccumulating species of E. coli in the hosts were found to cause a less dramatic drop in movement than exposure to the drug with a non-accumulating species, as sequestration of duloxetine with bacterial proteins had reduced the effective dose of the drug received.