Culture-dependent microbial profiling of Indian smokeless tobacco products

In India, wide range of SLT products are available using variable production processes and additives. Present study characterize cultivable microbial diversity among eighteen commercially available Indian SLT products. Through conventional culture-based approaches 1776 cultivable aerobic, anaerobic, fungal, and nitrate/nitrite-reducing colonies were reported, out of which thirty-five bacterial isolates were further characterized as nitrate-reducing using biochemical identification assays. Antibiotic susceptibility testing was done to evaluate any potential health risks associated. Pan Masala samples exhibited the highest microbial diversity, followed by chewing tobacco leaves, whereas Khaini predominantly supported anaerobic populations. Zarda samples demonstrated comparatively low microbial loads, potentially due to higher chemical additives. The heterogeneous product group displayed variable fungal and nitrate-reducing communities but lacked detectable anaerobic populations. Among the nitrate-reducing isolates, none demonstrated pathogenic characteristics or multidrug resistance. Five strains exhibited enhanced nitrite-reduction capacity, indicating potential to reduce nitrite pool generation and hence possibly impacting the nitrosation processes linked to TSNA formation. Biochemical characterization suggested all nitrite-reducing isolates belongs to Enterobactereaceae group though, further characterization is required. Present study is first to systematically demonstrate the presence of metabolically active Nitrate/Nitrite-reducers in Indian SLT products. Collectively, the findings highlight pronounced microbiological variability across Indian SLT products and confirmed presence of nitrate and nitrite-reducing microbes. This study provides foundational evidence linking variable product characteristics with microbial ecology in SLTs.