Smokeless Tobacco Harbors Bacteria Involved in Biofilm Formation as Well as Salt and Heavy Metal Tolerance Activity

In our previous culture-independent study on smokeless tobacco products, we have observed a strong positive correlation between several bacteria and genes involved in nitrate/nitrite reduction, biofilm formation, and pro-inflammation. Therefore, the present investigation was carried out to analyze the inhabitant bacterial population of the Indian ST products for assessing the health-associated risk attributes using culture-dependent approach. Traditional cultivation approaches recovered several bacterial isolates from commercial ST products on different culture media. A high colony formation unit (CFU) count was observed that ranged from 173 × 10⁴ to 630.4 × 10⁵ per gram of ST products. Of the 74 randomly selected and distinct bacterial isolates, 17 isolates showed a significantly enhanced growth (p-value < 0.05) in the presence of the aqueous tobacco extract. On biochemical characterization, these bacteria were identified as the member of Bacillus, Enterobacter, Micrococcus, Providencia, Serratia, Pantoea, Proteus, and Pseudomonas. Most of these bacteria also exhibited biofilm-forming activity, where eight bacterial isolates were identified for strong biofilm-forming action. 16S rRNA-based molecular characterization of these bacteria identified them as Bacillus subtilis, Bacillus paralicheniformis, Enterobacter sp., Serratia marcescens, Pantoea anthophila, and Enterobacter cloacae. Moreover, these bacteria also exhibited the potential to withstand high salt and heavy metal concentrations. The findings demonstrate that Indian ST products are heavily populated with wide bacterial species exhibiting potential in biofilm formation, heavy metal resistance, and salt tolerance.