Betel quid (BQ) chewing, a prevalent practice affecting over 600 million people globally, is associated with systemic toxicity and neurological alterations. While dysbiosis of the gut microbiota is implicated in neuropsychiatric disorders via the gut-brain axis (GBA), its role in BQ chewers remains unexplored. This exploratory study aimed to investigate whether chronic BQ chewing is associated with gut dysbiosis and alterations in spontaneous brain activity. Fecal samples (n = 30 BQ chewers, n = 19 healthy controls) were subjected to whole metagenome shotgun sequencing (WMGS) to assess microbial composition and function. Amplitude of low-frequency fluctuations (ALFF) values, a resting-state functional magnetic resonance imaging metric reflecting regional spontaneous neural activity, were assessed in a subset of 29 BQ chewers and 21 healthy controls. Group differences in microbiota and ALFF were analyzed using the Wilcoxon rank-sum test and two-sample t-test (adjusted for age, sex, education, smoking and alchohol). Partial Spearman's correlation analysis was performed to link microbial taxa with ALFF alterations. Motivated by the presence of complex polysaccharides and polyphenols in BQ, carbohydrate-active enzyme (CAZyme) profiles were also assessed. Chronic BQ chewers exhibited significant gut microbiome alterations, characterized by reduced microbial diversity, enrichment of pro-inflammatory genera, and depletion of beneficial taxa. Analysis of carbohydrate-active enzymes further revealed altered metabolic potential in BQ chewers. Furthermore, reduced ALFF was observed in the limbic lobe of BQ chewers. At a nominal significance level, Streptococcus abundance correlated positively with limbic ALFF (partial ρ = 0.35, 95% CI [0.07, 0.58], raw p = 0.04), whereas Dorea formicigenerans exhibited a negative correlation (partial ρ = -0.36, 95% CI [- 0.55, - 0.08], raw p = 0.04). Chronic BQ chewing is associated with gut microbial dysbiosis and functional metabolic shifts. Exploratory analyses suggest that these microbial features may correlate with spontaneous neural activity in the limbic lobe, providing preliminary evidence for a potential involvement of the GBA in BQ‑associated neurological sequelae. These findings highlight the need for further investigation into microbiota‑targeted strategies in BQ chewers.
