Indiana University-Purdue University Indianapolis (IUPUI) / Tooth decay is a complex dieto-bacterial disease with an association of social, behavioral and biological factors. Streptococcus mutans plays a major role in tooth decay. This endogenous oral microorganism adheres to tooth surfaces and grows and develops into micro-communities that mature and form dental biofilm. Development of cariogenic biofilm is one of the major factors associated with the tooth decay process. The use of tobacco is considered a great risk factor for oral diseases. Several studies demonstrated the association of tooth decay and the use of tobacco as effects of first-hand or second- hand smoking. Nicotine has been reported to increase the biofilm growth and metabolism of S. mutans in a dose-dependent manner up to 16 mg/ml of nicotine. However, its effects on biofilm formation of S. mutans strains isolated from smokers are not known and should be investigated. Therefore, we proposed the use of an in-vitro model to better understand the effects of nicotine on biofilm formation of strains of S. mutans isolates from smokers versus non-smoking subjects.
Objectives: To investigate the effects of nicotine on biofilm formation of S. mutans isolates from oral washes of smoker and non-smoker human subjects.
Materials and Methods: This study was conducted using three S. mutans isolates collected from oral washes of 10 smoking subjects and 10 non-smoking subjects. The oral wash samples were stored at -80oC before S. mutans isolation. S. mutans isolates were obtained by plating on Mitis Salivarius Sucrose Bacitracin plates and species identity confirmed by carbohydrate fermentation assays. Nicotine from Sigma-Aldrich (St. Louis, MO, USA) was used. Biofilm was formed by overnight culturing of each S. mutans strain (10 μl) in 190 μl of tryptic soy broth (TSB) supplemented with 1.0-percent sucrose (TSBS) containing 0 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 2.0 mg/ml, 4.0 mg/ml, 8.0 mg/ml, 16.0 mg/ml, and 32.0 mg/ml of nicotine for 24 hours in 5.0-percent CO2 at 37oC in sterile (8 x 12) 96-well microtiter plates (Fisher Scientific, Newark, DE, USA). The absorbance values of biofilm were measured at 490 nm in a microplate spectrophotometer (SpectraMax 190; Molecular Devices, SunnyVale, CA, USA) after crystal violet staining.
Null Hypotheses: 1) Nicotine will not increase biofilm formation in both smoker and non-smoker S. mutans isolates. 2) An increase in nicotine concentrations will not increase biofilm formation in both smoker and non-smoker S. mutans isolates in a dose-dependent manner. 3) Nicotine will not produce significant differences in biofilm formation between smoker and non-smoker S. mutans isolates.
Alternative Hypotheses: 1) Nicotine increases the growth of biofilm formation in both smoker and non-smoker S. mutans isolates. 2) An increase in nicotine concentrations increase biofilm formation of both smoker and non-smoker S. mutans isolates in a dose-dependent manner. 3) However, nicotine increases biofilm formation of smoker S. mutans strains more than non-smoker S. mutans isolates. The rationale for this hypothesis is that our preliminary data indicated that S. mutans can become resistant to increased nicotine concentrations and that this resistance appears to be stable and may allow the smoker isolates to be able to respond more vigorously to higher nicotine concentrations than the non-smoker isolates.
Results: There was a significant effect (p < 0.05) of both nicotine concentrations and smoking on the growth of biofilm, planktonic cells, and total absorbance, for all strains of S. mutans (p < 0.0001). Isolates from smokers had significantly more biofilm at 0 mg/ml to 16 mg/ml of nicotine compared with those from non-smokers (p-value < 0.0001).
Conclusion: S. mutans smoker isolates are more affected by high nicotine concentrations than non-smoker isolates.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/13992 |
| Date | January 2017 |
| Creators | El-ezmerli, Nasreen Farouk |
| Contributors | Gregory, Richard L., Hara, Anderson T., Cook, Norman Blaine, Windsor, Jack L. |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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