Potential subjects from Seattle, WA, USA (n = 422), were interviewed for conditions that would preclude participation. Individuals were excluded if they had taken antibiotics during the preceding 4 wks or anticipated doing so during the study. Subjects with a history of GI problems or phenylalanine intolerance were excluded. Screening plaque samples were taken for mutans streptococci enumeration from potential subjects who met the inclusion criteria (n = 405). Plaque was placed into 1 mL of sterile pre-reduced saline, and subjects with ≥ 104 CFU mutans streptococci/mL in their sample were invited to participate (n = 189). From this group, 132 (70%) subjects completed all baseline procedures and were enrolled.
The 132 participants had a mean age of 35 yrs (range, 18-73), and 68% were women. Participants were 70%, 19%, and 8% Caucasian, Asian, and African American, respectively. Less than 2% were either Hispanic, Native American, or of mixed race/ethnicity. The University of Washington Institutional Review Board approved this study, and informed consent was obtained from the participants.
This prospective controlled, double-blind clinical trial had a four-group design. Each group chewed 3 gum pellets, 4 times/day. Subjects were randomly assigned to groups by a blocked randomization procedure and were blinded to group assignment, as were investigators. Block randomization ensured similar proportions of participants in each group.
The 3 active groups received a mixture of control gum and/or xylitol gum, resulting in a total daily xylitol dose of 3.44 g (G2), 6.88 g (G3), or 10.32 g (G4). The controls received 9.83 g of sorbitol and 0.702 g of maltitol per day, but no xylitol. Plaque and unstimulated saliva samples were taken at baseline, 5 wks, and 6 mos of exposure. The study was carried out at the University of Washington Regional Clinical Dental Research Center.
Each xylitol pellet contained 0.858 g xylitol plus gum base, peppermint, menthol, gum Arabic, glycerol, soy lecithin, and glazing agents. Each pellet of the control gum contained the same non-active ingredients plus 0.819 g sorbitol, 0.0585 g maltitol, and 0.0015 g Acesulfame K. All gum pellets were formulated (Fennobon-Oy, Karkkila, Finland) to be similar in size, consistency, color, and sweetness.
We determined that the sample size provided sufficient power to test the primary hypothesis that mutans streptococci in plaque and saliva will decrease in response to increasing doses of xylitol. For dose-effect analysis, log10 transformation of mutans streptococci counts and linear regression model were used. A sample size of n = 26 subjects per group provided 81% power (two-sided, α = 0.05), where a difference in total mutans streptococci counts between the lowest and highest groups was assumed to be log0.75 = 5.6-fold. Assuming 10-20% attrition, 33 subjects per group were necessary.
Participants were coached on development of a daily routine for gum chewing and asked to chew the gum for ≥ 5 min. Daily gum packets were distributed on a weekly basis for the first 3 wks, then bi-weekly to promote and monitor compliance.
Unstimulated Saliva and Plaque Sampling
Plaque samples were first collected from the cervical third of the buccal surfaces of all teeth, with 1 sterile Kerr applicator used per arch, and placed in 5-mL tubes containing glass beads and 1 mL of pre-reduced saline. Then, subjects were instructed to let saliva collect without swallowing for at least 1 min, and then to expectorate into the collection tube. This process was repeated as necessary until the minimum 1 mL of saliva was collected. The tubes were immediately carried to the laboratory, where they were stored at room temperature until processed, generally within 3 hrs. However, samples after 3:30 p.m. were not processed until the following morning (15-17 hrs) after delivery. Staff was trained to conduct plaque and saliva samplings. No differences in total counts were observed between samples processed the same day and those processed the following morning.
Culture of Mutans Streptococci
Samples were taken from study participants at baseline, 5 wks, and 6 mos after enrollment. Plaque was prepared in pre-reduced saline, and 10-fold dilutions of plaque and saliva were prepared separately as described above. For each sample, Brucella agar (Difco Laboratories, Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with 0.05 μg/mL vitamin K, 5 μg/mL hemin, and 5% sheep blood (BA) was used for the enumeration of total cultivable facultative/aerobic bacteria, and a modified Mitis-salivarius agar (Difco Laboratories) supplemented with 500 μg/mL kanamycin, 1% potassium tellurite solution, and 50 U/mL bacitracin (MSKB) was used for the enumeration of mutans streptococci. The MSKB medium was more specific than MSB for mutans streptococci isolation (Kimmel and Tinanoff, 1991
; Roberts et al., 2002
), but did not distinguish between S. mutans
and S. sobrinus
. Thus, mutans streptococci counts from MSKB plates in this report included both of these species. Only freshly prepared plates were used.
The plaque samples were vortexed to break up the plaque. Then, plaque and saliva samples were diluted. The 10-0
dilutions were plated on BA and MSKB media and incubated in 5% CO2
at 36.5°C for 2-3 days or 7 days, respectively, prior to enumeration. In addition, dilutions (10-0
) of plaque and saliva samples were plated onto MSKB plates supplemented with 5%, 10%, or 15% (w/v) xylitol and incubated for 7 days as above (Roberts et al., 2002
). This provided the number of mutans streptococci able to grow at various xylitol concentrations.
Verification of Mutans Streptococci Counts
To verify that only mutans streptococci colonies were counted, we randomly selected 1400 colonies from the MSKB media and hybridized them with DNA probes (SSP001 and SM002/SM010) specific for the rRNA variable region of S. mutans
and S. sobrinus
(Roberts et al., 2002
). Greater than 90% were positive, indicating that they were either S. mutans
or S. sobrinus
Using the SPSS (v.11.5), and SAS (v.8e) software packages, we determined the significance levels between means at baseline vs. follow-ups and evaluated linear regression models for dose-response. Where appropriate, descriptive statistics, t test, and analysis of variance (ANOVA) were used to determine differences among the groups at baseline and between baseline and levels at 5 wks and 6 mos.