Multiple sclerosis (MS) is a chronic inflammatory demyelinating disorder of the central nervous system influenced by genetic and environmental factors. The gut microbiota has emerged as a key environmental component potentially playing a role in MS development, immune dysregulation and disease progression.

To characterise gut microbiota composition and enterotypes among patients with multiple sclerosis compared with healthy controls in Ukraine, and to assess associations with disease-modifying therapy (DMT) status.

A single-center cross-sectional study included 33 participants (28 with MS and 5 healthy controls). Stool samples, clinical data, and neurological examinations were collected during November 2024 to February 2025. Microbiome profiling was performed using 16S rRNA gene sequencing on the Illumina MiSeq platform. Statistical analyses evaluated differences in microbial composition, diversity, and enterotype distribution between MS patients and controls, considering treatment status.

The MS and control groups were demographically comparable (median age 33 years; IQR 31-37). Enterotype distribution demonstrated a moderate-to-strong association with both MS diagnosis and disease-modifying therapy (DMT) use (Cramér’s V = 0.41). Comparative analysis of bacterial phyla revealed that MS patients exhibited decreased relative abundance of Firmicutes (d = 0.44) and Bacteroidetes (d = 0.27), and increased abundance of Proteobacteria (d = -0.36), all representing moderate effect sizes. Significant differences were observed for Firmicutes (H = 12.26, p = 0.016) and Proteobacteria (H = 10.18, p = 0.037) among control, untreated, and DMT-treated groups, indicating treatment-associated shifts in microbial composition. Other phyla, including Actinobacteria, Verrucomicrobia, Tenericutes, Fusobacteria, and Euryarchaeota showed small, non-significant variations between groups.

Gut microbiota composition in Ukrainian MS patients differs from that of healthy individuals, with enterotype patterns potentially influenced by DMT use. The increase in proinflammatory Proteobacteria supports the role of gut dysbiosis in MS pathogenesis. Further genus- and species-level studies are warranted to clarify microbiome-disease interactions and identify potential therapeutic targets.