Abstract

Smoking perturbs the oral environment leading to microbial dysbiosis that may contribute to adverse health outcomes. While smoking-related shifts in the oral microbiome have been increasingly studied, data from South Asian urban centers remain limited. In this study, we characterized the oral microbiome of Kathmandu residents to investigate differences between smokers and non-smokers. Using 16S rRNA sequencing targeting the V3–V4 regions, we profiled microbial composition at both the phylum and genus levels. Across all samples, Proteobacteria (~52%) and Firmicutes (~26%) were the dominant phyla, while Streptococcus (~15%) and Pseudomonas (~11%) were the most abundant genera. Microbial diversity at both alpha and beta levels did not differ significantly between smokers and non-smokers. However, subgroup analysis among smokers identified 64 differentially abundant genera distinguishing two smoker groups. One subgroup (Smoker-I) was enriched with several Gram-negative genera, including Aeromonas, Klebsiella, Enterobacter, Yersinia, Salmonella, Serratia, Vibrio, and Campylobacter, as well as rare genera like Nocardia, Gordonia, and Rhodococcus. These distinct microbial patterns suggest that varying levels of tobacco exposure selectively reshape oral microbiome composition and suggest that not all smokers are microbiologically alike. The emergence of distinct microbial signatures in smoker subpopulations points to potential differences in behavior, exposure, or health status that warrant further investigation. Incorporating microbiome stratification may offer new insights into differential health risks among smokers.