e Streptococcus gordonii is a commensal species of human oral flora. It initiates dental biofilm formation and provides binding sites for later colonizers to attach to and generate mature biofilm. Smoking is the second highest risk factor for periodontal disease, and cigarette smoke extract has been reported to facilitate Porphyromonas gingivalis-S. gordonii dual-species biofilm formation. Our hypothesis is that nicotine, one of the most important and active components of tobacco, stimulates S. gordonii multiplication and aggregation. In the present study, S. gordonii planktonic cell growth (kinetic absorbance and CFU), biofilm formation (crystal violet stain and confocal laser scanning microscopy [CLSM]), aggregation with/without sucrose, and 11 genes that encode binding proteins or regulators of gene expression were investigated. Results demonstrated planktonic cell growth was stimulated by 1 to 4 mg/ml nicotine treatment. Biofilm formation was increased at 0.5 to 4 mg/ml nicotine. CLSM indicated bacterial cell mass was increased by 2 and 4 mg/ml nicotine, but biofilm extracellular polysaccharide was not significantly affected by nicotine. Cell aggregation was upregulated by 4, 8, and 16 mg/ml nicotine with sucrose and by 16 mg/ml nicotine without sucrose. Quantitative reverse transcriptase PCR indicated S. gordonii abpA, scaA, ccpA, and srtA were upregulated in planktonic cells by 2 mg/ml nicotine. In conclusion, nicotine stimulates S. gordonii planktonic cell growth, biofilm formation, aggregation, and gene expression of binding proteins. Those effects may promote later pathogen attachment to tooth surfaces, the accumulation of tooth calculus, and the development of periodontal disease in cigarette smokers.

Several epidemiology studies have reported a positive relationship between smoking and dental caries. Nicotine, an alkaloid component of tobacco, has been demonstrated to stimulate biofilm formation and metabolic activity of Streptococcus mutans, one of the most important pathogens of dental caries. The first aim of the present study was to explore the possible mechanisms leading to increased biofilm by nicotine treatment from three aspects, extracellular polysaccharides (EPS) synthesis, glucosyltransferase (Gtf) synthesis and glucan-binding protein (Gbp) synthesis at the mRNA and protein levels. The second aim was to investigate how nicotine affects S. mutans virulence, particular in lactate dehydrogenase (LDH) activity. Confocal laser scanning microscopy results demonstrated that both biofilm bacterial cell numbers and EPS were increased by nicotine. Gtf and GbpA protein expression of S. mutans planktonic cells were upregulated while GbpB protein expression of biofilm cells were downregulated by nicotine. The mRNA expression trends of those genes were mostly consistent with results on protein level but not statistically significant, and gtfD and gbpD of biofilm cells were inhibited. Nicotine was not directly involved in S. mutans LDH activity. However, since it increases the total number of bacterial cells in biofilm, the overall LDH activity of S. mutans biofilm is increased. In conclusion, nicotine stimulates S. mutans planktonic cell Gtf and Gbp expression. This leads to more planktonic cells attaching to the dental biofilm. Increased cell numbers within biofilm results in higher overall LDH activity. This contributes to caries development in smokers.

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