Baseline measurements

At baseline, there were no significant differences for WTCI, amounts of collected tongue coating and dental plaque samples, and amounts of total bacteria and F. nucleatum in whole collected samples as well as those in 1-mg samples between subjects with and without tongue cleaning (Table1). Furthermore, there were no significant differences for those parameters between the first and second baseline measurements after the 3-week washout period (data not shown). These results showed that the oral inhabitants returned to baseline levels in regard to amounts and bacterial load during the washout period, indicating that 3 weeks was sufficient for this crossover study.

Table 1 Baseline measurements for tongue coating and dental plaque samples Full size table

The volume of tongue coating was greater than dental plaque, while the amounts of both total bacteria and F. nucleatum in the 1-mg samples were greater in those from dental plaque. These findings showed that the density of total bacteria and F. nucleatum was higher in dental plaque than tongue coating at the baseline. In addition, F. nucleatum was detected in all samples of tongue coating and dental plaque collected at the baseline.

Change in amounts of total bacteria in tongue coating and dental plaque following tongue cleaning

In subjects who performed tongue cleaning, the average amount of total bacteria in whole collected tongue coating samples was lower on day 3 (4.11 ± 1.13 pg, average ± SD) than at the baseline (4.76 ± 1.18 pg). Intra-group comparisons using a paired t test showed a p-value for the difference between day 3 and baseline of less than 0.01, which indicated a statistically significant difference in multiple comparisons of the 3 examination days after Bonferroni adjustment. A lower level of total bacteria was also observed on day 10 (4.14 ± 1.30 pg), though the difference as compared to the baseline was not significant (Figure1A). In contrast, in subjects who did not perform tongue cleaning, the total bacterial amounts were not significantly different between the examination days (Figure1B).

Figure 1 Change in amount of total bacteria in tongue coating following tongue cleaning. Error bars indicate 95% confidential intervals. Values shown in closed circles are averages of the amount of total bacteria expressed as logarithm values of the genome weight of 16S rRNA universal in whole collected tongue coating samples (A) following tongue cleaning and (B) without tongue cleaning. *Statistically significant, multiple paired t test with Bonferroni adjustment. Full size image

As for genome weight, the difference between baseline and day 3 in the group with tongue cleaning was 4.55 pg (actual value). Our preliminary examination showed that with E. coli at a genome weight of 1 ng corresponded to 3.6 × 104 CFU, thus a weight of 4.55 pg was approximately equivalent to 1.9 log CFU of E. coli.

Quirynen et al.[16] reported that the reduction of bacterial load on the tongue dorsum after 6 months of daily tongue cleaning was less than 0.4 log CFU, which was not significant as compared to the baseline value. They also suggested that difficulty in reducing the bacterial load on the tongue is due to the surface characteristics of the tongue dorsum where innumerable depressions exist, as that structure provides ideal niches for bacterial adhesion and growth, and shelter from cleaning actions. However, Bordas et al.[17] reported significant changes in bacterial load on the tongue dorsum following 3 days of tongue scraping, with the reduction ranging from 1.11-1.96 log CFU. Our finding seems to be in agreement with the latter, though they used a cultivation method. On the other hand, when considering differences in sampling volume and frequency of tongue cleaning, the bacterial reduction by single tongue cleaning was greater and continued for a longer period than found in that previous study. Real-time PCR is able to quantify the total bacterial amount including non-cultivable bacteria with high sensitivity, whereas as much as 50% or more of the microbiota in oral biofilm have yet be successfully cultured[32, 33]. Therefore, the differences between the present and previous studies may be mainly derived from different bacterial detection methods utilized.

Subsequently, for inter-group comparisons, the rates of total bacterial amounts on days 3 and 10 against the baseline were compared between subjects with and without tongue cleaning using a Wilcoxon test. There was a tendency that subjects had more for a greater reduction in bacterial load against the baseline in subjects who cleaned their tongue, though the difference was not significant (p = 0.106). Furthermore, there was no difference between the groups on day 10 (p = 0.478). Thus, the previous and present results show that the effect of tongue cleaning on reduction of bacterial amount is not remarkable, and it remains unclear whether tongue cleaning has a practical effect to reduce bacterial load in the whole oral cavity.

On the other hand, the average amounts of total bacteria in the whole collected dental plaque samples were significantly lower at 3 and 10 days after removal as compared to the baseline value, as shown by a paired t test with Bonfferoni adjustment (Figure2A). This was also true in subjects without tongue cleaning (Figure2B). Furthermore, a Wilcoxon test performed similarly to analyze tongue coating revealed there was no significant difference between the groups on either day (p = 0.280 on day 3, p = 0.380 on day 10). Together, these results suggest that tongue cleaning does not contribute to inhibition of dental plaque formation.

Figure 2 Changes in amount of total bacteria in dental plaque after removal. Values shown in closed circles are averages of the amount of total bacteria expressed as logarithm values of the genome weight of 16S rRNA universal in whole collected dental plaque samples (A) following tongue cleaning and (B) without tongue cleaning. Error bars and asterisks are the same as in Figure1. Full size image

Change in WTCI score after tongue cleaning

In contrast to the changing profile of total bacterial amount in tongue coating, WTCI score did not show a significant difference among the examination days in both groups (Figure3). These findings agree with a study by Chérel, et al., who reported that average tongue coating scores returned to baseline levels 2 days after tongue cleaning[34]. Other reports have also noted disagreement between change in bacterial load on the tongue and tongue coating score after tongue cleaning[15–17]. Thus, components other than microorganisms in tongue coating are generally evaluated with an ocular inspection method. On the other hand, slight reductions in WTCI as compared to the baseline even in subjects without tongue cleaning on days 3 and 10 were noted, while a reduction in amount of total bacteria in tongue coating samples from subjects without tongue cleaning was also observed (Figure1B). Those findings may have been related to naturally occurring inter-day changes.

Figure 3 Changes in WTCI following tongue cleaning. Values shown in open circles are averages in subjects without tongue cleaning and those in closed circles are averages in subjects with tongue cleaning. There were no significant differences between the subject groups or examination days. Full size image

Amounts of F. nucleatum and total bacteria in tongue coating and dental plaque

To assess etiological shift, we examined the changes in amounts of F. nucleatum in tongue coating and dental plaque samples. Three days after tongue cleaning, the average amount of F. nucleatum in tongue coating was significantly reduced as compared with the baseline (2.19 ± 1.18 to 1.75 ± 1.29 log pg; p = 0.006). When tongue cleaning was not performed, there was no significant difference between day 3 and the baseline (1.94 ± 1.27 vs. 2.02 ± 1.27 log pg; p = 0.726). In addition, there was no difference between with and without tongue cleaning on day 10.

In dental plaque, the average amount of F. nucleatum on day 3 was reduced after tongue cleaning but not significant (2.07 ± 1.00 to 1.88 ± 0.87 log pg; p = 0.145). A reduction of F. nucleatum on day 3 as compared to the baseline was also observed in subjects without tongue cleaning (1.98 ± 1.23 to 1.59 ± 1.00 log pg; p = 0.019), though the difference was not significant in multiple comparisons using Bonferroni adjustment. Since the profiles of change after removal were similar to total bacteria, we analyzed the relationship between amount of total bacteria and that of F. nucleatum in the samples using Pearson’s correlation analysis, and found a significant correlation coefficient. The relationship level was constantly high in both tongue coating and dental plaque with all sampling conditions used in the present protocol (Figures4 and5). These results showed that F. nucleatum occupied a certain proportion of total bacteria in both tongue coating and dental plaque during both development and under stable conditions. Furthermore, in the present study, F. nucleatum was detected in all tongue coating and dental plaque samples from periodontally healthy individuals, in whom the detection rates of periodontopathic bacteria such as red complex spices are often reported to be extremely low[3–6, 32]. Another report noted that colonization of F. nucleatum induced red complex species habitation by binding both early and late colonizers in dental plaque[24]. Furthermore, we previously reported a strong correlation between dental plaque and tongue coating in regard to colonization of red complex spices[6]. Therefore, it is possible that an increase in the amount of F. nucleatum in tongue coating as well as dental plaque indicates an environment that is acceptable for virulent bacteria, consequently increasing the risk for periodontitis. However, we did not determine the presence of the red complex species, which is a limitation of this study.

Figure 4 Relationship between amounts of total bacteria and F. nucleatum in tongue coating. Scatter plots of the amounts of total bacteria (X-axes) and F. nucleatum (Y-axes) in whole collected tongue coating samples expressed as logarithm values of the genome weight (A) in case with tongue cleaning and (B) without tongue cleaning. Closed circles, open circles, and triangles show values obtained at baseline, and days 3 and 10, respectively. Solid, dotted, and broken lines indicate approximate straight lines for baseline, and days 3 and 10, respectively. Correlation coefficients in subjects with tongue cleaning were 0.746, 0.837, and 0.928 at baseline, and on days 3 and 10, respectively, while those in subjects without tongue cleaning were 0.884, 0.844, and 0.896, respectively. Full size image

Figure 5 Relationship between amounts of total bacteria and F. nucleatum in dental plaque. Scatter plots of the amounts of total bacteria (X-axes) and F. nucleatum (Y-axes) in whole collected dental plaque samples expressed as logarithm values of the genome weight (A) in case with tongue cleaning and (B) without tongue cleaning. Symbols are the same as in Figure4. The correlation coefficients in subjects with tongue cleaning were 0.570, 0.849, and 0.870 at baseline, and on days 3 and 10, respectively, while those in subjects without tongue cleaning were 0.952, 0.868, and 0.745, respectively. Full size image

Overall relationships of volume and bacterial load in tongue coating and dental plaque

To review the contradictions and relationships among assessment results in the present and previous studies, we performed a principle component analysis. We applied wet weight, and amounts of total bacteria (pg/mg) and of F. nucleatum (pg/mg) in both tongue coating and dental plaque samples along with WTCI scores to the analysis. Table2 summarizes the factor loadings for the measurements after Varimax rotation. The first component was strongly associated with the amounts of total bacteria and F. nucleatum in dental plaque, and moderately with wet weight of dental plaque. In contrast, the second component was exclusively related to amounts of total bacteria and F. nucleatum in tongue coating. The wet weight of tongue coating and WTCI formed another group related to the third component. These results indicated that the variations in bacterial amounts in tongue coating and dental plaque samples were largely independent of each other. Furthermore, WTCI scores were closely associated with the wet weight of tongue coating. Lundgren, et al. also found a high correlation between wet weight of tongue scrapings and WTCI[35]. On the other hand, in our study, measurements that assessed the volume of oral specimens showed a weak to moderate association with bacterial amounts among the overall variation of measurements. These results may explain the disagreement of changes after tongue cleaning between bacterial amounts and WTCI noted in our study.

Table 2 Component matrix after Varimax rotation following principal analysis for overall samples (n = 180) Full size table

Our present findings provide additional evidence to elucidate the effects of tongue cleaning, though there are some limitations. First, precise quantification using real-time PCR showed that mechanical tongue cleaning has a longer effect over time to reduce bacterial load than found in previous studies that used cultivation methods. However, it remains unclear whether the small scale reduction in bacteria observed in this study contributes to overall oral health. Second, tongue cleaning did not contribute to inhibit dental plaque formation, since the bacterial amounts in the 2 aggregates had quite different variations in an oral cavity. Finally, the volumes in tongue coating and dental plaque do not accurately represent the bacterial load in sites of attachment. In addition, the amount of F. nucleatum in tongue coating and dental plaque increases along with bacterial growth, which suggests an increment of virulent species in the tongue coating. These findings led us to conclude that tongue cleaning and tooth brushing should both be performed in order to reduce the amount of bacteria on the tongue and tooth surfaces, and improve the periodontal etiology.