Many people dread going to the dentist for their dental check-ups because they know there might be a possibility of hearing the infamous line, "Everything looks great, but you do have one cavity." Maintaining perfect oral health is not as easy as just brushing one's teeth. The main objective is to remove dental plaque, a biofilm that builds up on teeth. The microorganisms that form this colorless biofilm are mainly Streptococcus mutans (S. mutans). Streptococcus mutans is a Gram-positive bacterium with a thick peptidoglycan layer in the cell wall. Streptococcus mutans derive from the genus Streptococcus that form spherical, round chains (9). They were first described by J.K. Clark in 1924 when he isolated the bacterium from a carious lesion (8). Streptococcus mutans are facultative anaerobic organisms and are mainly found in the human oral cavity. Because they build up dental plaque on teeth, Streptococcus mutans are the key contributors of tooth decay also called dental caries or cavities (7).
Dental caries disease is an ongoing universal problem. Many people all over the world are affected by tooth decay, especially those that do not receive proper dental care starting from childhood. In order to prevent tooth decay in teeth, researchers are conducting various types of research on Streptococcus mutans. Researchers tested different types of carbohydrates, in particular natural sweeteners, to determine if they could benefit in the prevention of Streptococcus mutans growth and adherence (3)(4). Also mouthwashes containing essential oils and alcohol-free chlorhexidine were researched to observe the effects they had on human plaque acidogenicity (2).
In a recent research study completed in Sweden, investigators hypothesized that mouth rinses containing essentials oils were as effective as mouth rinses containing alcohol-free chlorhexidine in the prevention of plaque acidogenicity caused by Streptococcus mutans (2). Twenty healthy volunteers with a mean age of 59 used three different mouthwashes along with their normal oral hygiene maintenance for 16 days. The first mouthwash contained a solution with essential oils (Listerine); the second mouthwash contained a solution with alcohol-free chlorhexidine (Paroex), and the third mouthwash contained a solution with water which was the negative control. The unsupervised mouth washings were started after each subject received an oral examination and plaque pH measurement using the microtouch method. The subjects rinsed once a day on day 0 and twice a day from day 1 to day 16. The manufacturer's recommended dosage was the amount used for the each mouthwash. Each subject attended six plaque pH measurement sessions. They were done at day 0 at baseline, and at the end of the three mouthwash periods at day 17. Using the microtouch method, researchers used a microelectrode to measure the pH at two sites of the maxillary anterior region. After measuring the baseline plaque pH, the subjects rinsed with 10 ml of a 10% sucrose solution for one minute. This allowed Streptococcus mutans to metabolize sucrose to lactic acid making the pH more acidic. The plaque pH was then measured at timed intervals leading up to 30 minutes (2).
The key findings in this study were that both essential oils and alcohol-free chlorhexidine reduced plaque acidogenicity after a sucrose challenge. They both showed a notably lower pH change compared with the control mouthwash solution containing water (2). The hypothesis was accepted. The presented study agreed with previous studies on chlorhexidine and the negative effect it imposed dental plaque caused by Streptococcus mutans and other bacteria. Previous research also stated that chlorhexidine reduced the acidogenicity of dental plaque (2). As for the use of essential oils in daily oral maintenance, no previous research has been done on plaque pH acidogenicity (2). But a prior study observed a reduction in the amount of Streptococcus mutans to the total Streptococci in dental plaque after an one hour rinse with a mouthwash containing essentials oils in a 12 day study (1). The study on plaque acidogenicity and how it reacts to essential oils and chlorhexidine for the most part, agrees with other researchers' findings on relevant experiments.
Another research conducted on Streptococcus mutans was to observe how the presence of Xylitol and Erythritol would decrease the adherence of Streptococcus mutans to tooth enamel (4). Xylitol and Erythritol are natural sugar alcohols that have fewer calories than regular sugar. The study took various types of Streptococcus mutans and other bacteria and added 4% of Xylitol and 4% of Erythritol to the medium on which the bacterium would be grown on. The control medium did not receive Xylitol or Erythritol. The purpose of this experiment was to see how Streptococcus mutans would adhere to a glass surface in the presence of Xylitol and Erythritol (4). The results strongly showed that both Xylitol and Erythritol reduced the adherence of all Streptococcus mutans in the experiment. Overall there was no relationship detected in the degree of decreases in glass adherence caused by the two sugars. For example, the Streptococcus mutans 117 was highly inhibited by Erythritol but was weakly inhibited by Xylitol in relation to glass adherence (4). But, for the most part it was clear to say that both Xylitol and Erythritol inhibited the adhesion of Streptococcus mutans to teeth because the glass had a similar surface. These findings did relate to previous research since 1975 on Xylitol. It was stated then that Xylitol inhibited growth of Streptococcus mutans (10). As for Erythritol, previous research found unusual inhibitory patterns where else in the presented study, the patterns were constant (11).
Since Xylitol has been proven to reduce the growth of Streptococcus mutans, many various types of experiments have been made on the bacterium using a particular carbohydrate (10). One significant study was carried out by Lee Young-Eun and his colleagues in Korea. His experiment studied the long-term effect of the morphology and virulence of Streptococcus mutans in the presence of Xylitol gum. The hypothesis stated that by inducing the use of Xylitol on the bacterium, it would differ the structural shape of the specimen and inhibit its pathogenic ability to cause dental plaque (3).
In order to carry out this study, Young-Eun directed participants for a women's oral health prevention program in Korea. Twenty women between the ages of 24 and 35 were evaluated for one year. These women were randomly divided into two groups. One group consumed two pellets of Xylitol chewing gum three times a day. While the second group, which was the control group, also used the chewing gum, but sparingly. The Xylitol chewing gum was composed mainly of Xylitol making up 77% of the gum's ingredients. The two groups were given toothbrushing instructions and encouraged to maintain their usual diets. Saliva samples were obtained from both groups at the same location and time at baseline, 6 months, and 12 months.
Each saliva sample was then smeared onto Mitis Salivarius agar containing 0.2 U/ml of Bacitracin and 15% sucrose. The agar plates were incubated at 37°C for 48 hours. The number of colonies forming Streptococcus mutans was counted. One colony was then taken and smeared onto a new Mitis Salivarius agar plate to obtain a single colony. After the second colony growth occurred, colonies containing Streptococcus mutans were inoculated into Brain Heart Infusion Medium for 48 hours resulting in the complete isolation of Streptococcus mutans (3).
Various tests had to be completed in order to observe the morphological changes and quantity of the bacterium and the concentration of glucosyltransferase B (gtfB) gene expression in each group. Morphological changes were observed under Field Emission Electron Microscope procedures. Bacterium counts in each group were conducted with the Friedman test along with the Bonferroni-corrected Wilcoxon signed-rank test used for post examination. A third test called the Mann-Whitney test was made to compare the means of the Streptococcus mutans counts and the gtfB gene expression values between the two groups. Also real-time reverse transcription polymerase chain reaction was conducted (3). During the Field Emission Scanning Electron Microscope procedure, the isolated Streptococcus mutans colonies were washed in 10 mM phosphate-buffered saline, and fixed in 2ml of 3.7% formaldehyde at room temperature for 1 day (3). Since the electron microscope is a vacuum all the water on the specimens must be removed (12). The specimens were dried out through ethanol rinses. In order for the specimens to be vitally dry, liquid CO2 was induced. Finally, the Streptococcus mutans specimens were coated with gold-palladium. At the end of this step they could be examined under the Field Emission Scanning Electron Microscope where their shapes could be seen at x200 and x10,000 magnification for more detail (3).
Results from the images received by the electron microscope mostly complied with the hypothesis. Streptococcus mutans experienced morphological changes from both the control and the Xylitol group. Even though the isolated colonies from the Xylitol group had a typical morphology, they were smaller and smoother compared to the control group (Figs. 1, 2). Also the adherence ability of Streptococcus mutans to the Mitis Salivarius agar in the Xylitol group was lower than the control. This evidence goes along with the results from the Xylitol and Erythritol adherence study. The decreased adherence of the specimen was the result of lower production of sticky substances on the surface on colonies in the Xylitol group (Fig. 2). These sticky substances were assumed to be glucan polysaccharides (3).
Along with the morphological changes, colony counts of the bacterium decreased over the 12 month period in the Xylitol group. Also, the gtfB gene expression drastically reduced at the 6 and 12 month saliva collections. There was no significant change of the gtfB gene in the control group of women. The gtfB gene regulates the production of insoluble glucan on the surface of bacterial cells. Young-Eun determined that the decrease of sticky substances may have occurred in part of the changes of the gtfB gene expression (3). The results of the experiment concluded that long-term chewing of Xylitol gum can diminish the growth and size of Streptococcus mutans. This could lead to the decreased accumulation of dental plaque on teeth preventing the formation of caries.
Figure 1.
Morphological changes of S. mutans specimen (control group) under field emission scanning electron microscope on MSB agar. Note the size similarity from baseline to 12 months (A-C) and the presence of sticky substances (D-F).
Baseline; A and D
6 months; B and E
12 months; C and F
x200 magnification; A-C
x10,000 magnification (surface); D-F
(3).
Figure 2.
Morphological changes of S. mutans specimen (Xylitol group) under field emission scanning electron microscope on MSB agar. Note the decrease in size from baseline to 12 months (A-C) and the disintegration of sticky substances (D-F).
Baseline; A and D
6 months; B and E
12 months; C and F
x200 magnification; A-C
x10,000 magnification (surface); D-F
(3).
Ultimately, research in the field of Streptococcus mutans is in an overall positive direction. Researchers are developing different strategies in the fight against these microorganisms. Examples include chewing Xylitol containing gum, and rinsing the mouth with chlorhexidine and essential oils. Although some scientists have suggested a vaccine for Streptococcus mutans it has had no progress on humans till this day (7). Even thought researchers have come to conclusions during their research, several limitations prevented the generalization of the results. For example, in the experiment on the pH acidogenicity, because the study was done on subjects with similar baseline pH levels, it is unknown how subjects with much higher pH would react to this study (2). To generalize the preventive effect of essential oils and chlorhexidine on all individuals is therefore not possible. The study would have to be done for a longer period of time and on subjects with variable pH levels. Another example is in the study of the Xylitol chewing gum, the experiment was evaluated on twenty women only (3). The sample size of the participants was fairly small so further studies with larger subjects are crucial to gain reliable results on the morphological changes in Streptococcus mutans in the presence of Xylitol. Also this particular study was carried out on women only making the results questionable to the male population. Scientists need to discover the reason behind the colonization of Streptococcus mutans in the oral cavity and find a method to stop colonization permanently. Further tests must be done in order to find suitable solutions to the problems caused by this bacterium. One possible study that can be conducted is the use of laser treatment in the deterioration of Streptococcus mutans and observing if there is any positive result. So the next time you are thinking about skipping out on flossing your teeth after brushing, think about how many Streptococcus mutans are still hiding in between them.
