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Page 8 of 23 Poznyak et al. Microbiome Res Rep. 2026;5:3

has been associated with a reduction in certain gut bacteria linked to RA, such as Actinobacteria (including
Collinsella species) and some Firmicutes. Conversely, Bacteroides species, which earlier studies suggested
might decrease in RA patients, were found to increase in healthy individuals following minocycline
treatment [97,98] .

In a comprehensive analysis of fecal, dental, and salivary samples from individuals with and without RA,
researchers found distinct microbiomes in RA patients compared to healthy participants, consistent with
previous findings. However, this difference diminished after three months of methotrexate treatment [99-101] .
Interestingly, a predictive model utilizing gene expression data from the microbiome organized into
metagenomic linkage groups proved effective in distinguishing RA patients who responded positively to
DMARD therapy from those who did not. A focused study involving 42 RA patients and 10 healthy controls,
using 16S rRNA sequencing to analyze fecal microbiomes, indicated that methotrexate treatment reduced the
presence of Enterobacteriales [102,103] . This effect may result from methotrexate’s interaction with the bacterial
enzyme dihydrofolate reductase, which is crucial for bacterial survival, suggesting that methotrexate can
unintentionally alter bacterial populations. The relationship between methotrexate and the gut microbiota is
becoming increasingly prominent, as emerging evidence suggests that gut microbiota may play a significant
role in methotrexate metabolism and pharmacokinetics, thereby influencing RA treatment efficacy [104-106] .

Sulfasalazine exhibits both antimicrobial and anti-inflammatory properties, but its effectiveness relies on
activation by the gut microbiota. In its inactive form, sulfasalazine is converted into the active compound
5-aminosalicylic acid by the bacterial enzyme azuroreductase, produced by microbes in the distal gut [107,108] .
Nevertheless, research on the impact of sulfasalazine on gut microbiota in RA remains limited. Before the
advent of next-generation sequencing, one study observed significant reductions in fecal levels of Clostridium
perfringens and Escherichia coli (E. coli) during sulfasalazine treatment. This reduction in E. coli was
confirmed by subsequent research, which also noted a concurrent decrease in Bacteroides spp. and an
increase in Bacillus spp. [109,110] . Furthermore, treatment with hydroxychloroquine, an anti-rheumatic
medication, was associated with increased bacterial richness and diversity in the gut, as shown in a study
utilizing 16S rRNA sequencing. This treatment appeared to enhance levels of Faecalibacterium spp., known
for producing butyrate, a SCFA that helps reduce inflammation and regulate the intestinal barrier [111,112] .

While tumor necrosis factor (TNF) inhibitors are a cornerstone of RA treatment, research on their effects on
the microbiome is lacking. For instance, treatment with etanercept in RA patients resulted in an increase in
Cyanobacteria and Nostocophycideae, alongside a decrease in Clostridiaceae and Deltaproteobacteria within
their fecal microbiomes [113,114] . Conversely, in CIA mice, etanercept treatment was associated with decreased
gut microbial richness and diversity, characterized by an increase in Escherichia and Shigella spp., and a
decrease in Lactobacillaceae, Clostridium cluster XIVa, and Tannerella spp. Further research is warranted to
explore the effects of cytokine inhibitors on the gut microbiota in RA. However, it is essential to approach
the application of findings from other conditions, such as inflammatory bowel disease (IBD), to RA with
caution. Despite some shared dysbiosis across various immune-mediated inflammatory diseases, unique
microbiome alterations have been observed in each condition [115-117] . This suggests that extrapolating
microbiome findings from other immune-mediated inflammatory diseases to RA may be misleading.
Disease-specific microbial alterations should be carefully characterized in RA, ideally in longitudinal studies
and across diverse patient populations, to ensure that interventions target mechanisms truly relevant to RA
pathogenesis rather than generic dysbiosis patterns observed in other conditions .
[118]

Dietary alternatives
The notion that current treatments for RA might also influence gut bacteria suggests that manipulating the
gut microbiome could have therapeutic benefits for RA patients. However, findings from a French cohort