Title: Nicotine Increases Streptococcus mutans Biofilm and Arginine Negates the Effect
Dawn Wagenknecht (Presenter)
Indiana University School of Dentistry
Elizabeth A.S. Moser, Indiana University School of Medicine & Richard M Fairbanks School of Public Health
Richard Gregory, Indiana University
Objectives: S. mutans is detected in diseased cardiovascular tissues and strains expressing collagen binding proteins Cbm and Cnm invade endothelial cells in vitro. Nicotine (NIC) increases S. mutans biofilm and arginine (ARG) changes the biofilm by altering structure and adhesion of the extracellular polysaccharides (EPS). We investigated the effects of NIC and ARG on biofilm formation by S. mutans serotypes c and k strains (n=10, 10) genotyped for collagen binding protein genes (cbm, cnm).
Methods: S. mutans grown in tryptic soy broth with 1% sucrose (TSBS) was seeded into 96-well plates with and without NIC (4 mg/ml) and/or ARG (10 mg/ml) and incubated 24 hrs at 37oC in 5% CO2. Biofilm mass (crystal violet staining) and metabolic activity (XTT assay) were measured. S. mutans (Syto-9) and EPS (dextran-Alexa Fluor 568) mass were quantitated by confocal laser scanning microscopy (CLSM). Two-way ANOVA with interaction and random effect for multiple measurements was used for statistical analyses. Biofilm and metabolic data required a rank-transformation prior to analysis.
Results: NIC, ARG and NIC+ARG had significant effects on biofilm mass and metabolic activity (p<0.0001). Genotype, but not serotype, had an effect on biofilm mass (p=0.002 & 0.245, respectively). Both genotype and serotype affected metabolic activity (p<0.0001). Biofilm mass and metabolism were greatest in cbm-/cnm+ strains. In CLSM NIC increased bacterial but not EPS mass (p=0.007 & 0.085). Genotype affected EPS production (p<0.0001); cbm-/cnm+ strains produced more EPS with and without nicotine than cbm+/cnm+ (p=0.008) and cbm+/cnm- strains (p<0.0001). ARG alone did not have an effect on bacteria or EPS mass, however, the effect of NIC on EPS was significantly suppressed in the presence of ARG (p=0.006).
Conclusions: Nicotine significantly increased S. mutans but not EPS mass in biofilm. The cnm+ strains produced more EPS than cnm- strains. Addition of ARG to toothpaste for smokers may negate NIC-enhanced biofilm production.