‘Bugs on drugs’: implications for gut health

Intense research is ongoing to dissect the reciprocal interactions between microbiota and drugs. New work finds that a drug to dampen host inflammation can also have off-target effects on the microbiota at transcriptional, metabolic and compositional levels, with resultant expanded benefits to the host.

Refers to Nayak, R. R. et al. Methotrexate impacts conserved pathways in diverse human gut bacteria leading to decreased host immune activation. Cell Host Microbe https://doi.org/10.1016/j.chom.2020.12.008 (2021).

Beginning in 1947, Sidney Farber extended the lives of children with leukaemia by up to 9 months using the antimetabolite methotrexate. Since the 1980s, low methotrexate doses have been used to treat rheumatoid arthritis. Methotrexate inhibits dihydrofolate reductase (DHFR), an evolutionarily conserved enzyme that converts folic acid into tetrahydrofolate — a cofactor necessary for producing purines, pyrimidines, and amino acids. Methotrexate also dampens inflammation by increasing extracellular adenosine levels. New work from Nayak et al. extends these mechanisms to the gut microbiota — in vitro and in vivo approaches reveal that methotrexate alters gut microbiota at compositional and functional levels¹. As methotrexate is an arche typal immunomodulatory drug used to treat inflammatory disorders associated with intes tinal dysbiosis, including Crohn’s disease², this work establishes the need to assess how other commonly used pharmaceuticals functionally impact intestinal microbiota.

Bacteria and their products have been used as drugs for >100 years. Streptococcus erysipelas and Serratia marascens toxins were used to treat sarcoma as early as 1891, and Mycobacterium bovis is still used for superficial bladder cancer. More recently, faecal microbial transplantation and defined resident bacterial subsets can treat recurrent Clostridiodes difficile and ulcerative colitis³.

How bacteria alter drugs is also a focus of intense study (Fig. 1). Using their vast capacity for enzymatic reactions, the gut microbiota can alter metabolism of drugs in unique ways⁴. Bacteria alter drug metabolism via direct biotransformation, thereby activating or inactivating drugs, generating toxic inter mediates or altering bioavailability. A classic example is colonic bacterial azoreductase, which releases 5aminosalycilate from the prodrug sulfasalazine. Another is the competitive binding of microbial p-cresol to the hepatic enzyme sulfotransferase (SULT1A1), reducing its ability to metabolize paracetamol, consequently diverting it to cytochrome P450 enzymes that metabolize it into the highly reactive N-acetylpbenzoquinone imine, a toxic metabolite responsible for paracetamol hepatotoxicity. Furthermore, the gut bacterial enzyme ß-glucuronidase converts the detoxified metabolite of irinotecan, SN38G, into the active, toxic SN38 metabolite that inflicts dose-limiting gastrointestinal toxicity, which can be prevented using targeted, selective nonlethal ß-glucuronidase inhibitors; this process improves the efficacy and tolerability of irinotecan⁵. Moreover, bacteria can indirectly influence therapeutic responses: for example, microbiota composition influences variable responses to immune checkpoint inhibitors⁶.

Fig. 1 |. overview of drug–microbiota interactions.

Microbiota alter the metabolism of a number of pharmaceuticals⁴. Moreover, bacteria and their products are themselves used as drugs⁴. Nayak et al.¹ expand our understanding of drug–microbiota interactions, demonstrating that methotrexate changes the growth, transcription, and metabolic activity of gut microbiota, which can collectively influence host immunity. FMT, faecal microbiota transplantation. https://doi.org/10.1016/j.chom.2020.12.008

Beyond intensive research revealing that human-targeted drugs from diverse classes (such as antidiabetic agents, proton pump inhibitors and NSAIDs) alter composition of the gut microbiota, the latest research reveals that many of these drugs also influence bacterial viability. Maier et al. used high throughput screens to systematically test 1,197 FDA-approved compounds against 40 diverse gut bacterial isolates, finding that 24% of these compounds inhibited growth of at least one strain⁷. Notably, methotrexate at concentrations found within the intestinal lumen substantially reduced Escherichia coli viability, reinforcing a prior report of methotrexate inhibiting growth of various clinical and laboratory E. coli strains⁸.

Nayak et al. built upon these observations by reporting the functional consequences of methotrexate treatment on gut bacteria¹. Germfree mice colonized with stools from a healthy human donor were treated with methotrexate doses that reflected doses used for clinical rheumatoid arthritis and cancer treatments. Longitudinal 16s RNA sequencing of stool and caecal contents revealed that methotrexate persistently altered gut microbiota within 1 day: methotrexate decreased the abundance of Bacteroidetes at both doses, whereas the higher cancer dose altered the abundance of multiple phyla including Actinobacteria, Firmicutes, and Proteobacteria. In vitro, pharmacological methotrexate concentrations inhibited Bacteroidetes growth and shifted the Bacteroides thetaiotaomicron transcriptome, altering the expression of 83% of all genes. Clostridia species had variable transcriptional responses, ranging from 21 to 468 genes. Among the differentially altered pathways were purine and pyrimidine metabolism, presumably through DHFR inhibition by methotrexate.

Using stool samples collected from treatment naive patients with rheumatoid arthritis, clinically relevant methotrexate concentrations inhibited ex vivo bacterial growth, selectively reducing Bacteroidetes abundance. Stools collected from 23 patients with rheumatoid arthritis at baseline and one month after initiating methotrexate treatment did not differ in gut microbiota community richness, structure, or bacterial abundance. By contrast, >6,000 bacterial gene families were differentially abundant following treatment, many of which were pathways involved in pyrimidine and protein synthesis. Interestingly, patients who responded to methotrexate were characterized by reduced abundance of Bacteroidetes, relative to methotrexate non-responders.

Although mouse models of rheumatoid arthritis might have been more relevant to the study setting, the authors utilized a dextran sodium sulfate (DSS)induced coli tis mouse model to test the functional effect of methotrexate altered gut microbiota on mucosal and peripheral T cell responses during inflammation¹. Germfree mice were colonized with pretreatment or posttreatment stools from three methotrexate-responsive patients with rheumatoid arthritis with the greatest Bacteroidetes reduction, and were then treated with DSS to elicit colitis, or with water as a control. Resultant colitis symptoms were uniform in all groups, and DSS-challenged mice colonized with post methotrexate stool had several reduced effector immunocyte populations (for example, activated T cells and IFNγ+ T cells) relative to other groups. Moreover, the intestinal mucosa had reduced numbers of activated myeloid cells, T cells and T helper 17 cells.

In humans and mouse models, methotrexate consistently reduced levels of Bacteroidetes, which comprise the most abundant phylum of gut microbiota, are known to pro mote homeostasis, and influence host immunity and metabolism⁹. The well-characterized Bacteroides fragilis has opposing roles in mucosal homeostasis to Bacteroidetes phyla in general: certain strains express B. fragilis toxin (BFT), which disrupts the intestinal epithelial barrier and promotes intestinal inflammation. Another Bacteroidetes phylum representative, the opportunistic pathogen Bacteroides thetaiotaomicron, induces experimental colitis in certain susceptible mouse models¹⁰. Methotrexate inhibited the growth of both B. fragilis and B. thetaiotaomicron in vitro, and reduced their abundance when mice received methotrexate by oral and intra peritoneal administration in the new study¹. Although this study did not resolve bacterial strains, the consistent decreased abundance of Bacteroidetes by methotrexate suggests a loss of inflammatory Bacteroides strains to account for reduced intestinal immune activation.

This new study by Nayak et al.¹ is important for numerous reasons. First, it reinforces the interspecies conservation of specific bio chemical pathways. Second, it underscores the importance of off target effects of human drugs on bacterial enzymes with orthologous structures, which necessitates elucidating the molecular and structural basis for bacterial enzymatic function. Finally, this work emphasizes that drugs can have additional desirable therapeutic repercussions beyond on target effects on the host — reducing the inflammatory drive of bacteria provides an additional mechanism by which methotrexate treatment reduces inflammation in the host. Future work will be needed to determine whether these effects are enduring. Thus, Nayak et al.¹ present a primer to utilize integrative, multidisciplinary methods to delve deeper into drug–microbiota interactions, their applicability for developing predictive biomarkers of drug response, and their therapeutic potential.