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Division of Community Oral Health Science, Department of Health Promotion, Kyushu Dental College, 2-6-1 Manazuru, Kokurakitaku, Kitakyushu 803-8580, Japan
Correspondence
Shuji Awano
awa-shu{at}kyu-dent.ac.jp
Received 19 October 2007
Accepted 5 February 2008
Abbreviations: BOP, bleeding on probing; GC, gas chromatograph; PD, probing depth of more than 4mm; VSC, volatile sulfur compound; VSCT, VSCs above the organoleptic threshold level.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Furthermore, increased tongue biofilm as assessed by observable tongue coating contributes to oral malodour more than other related factors, such as increased periodontal pockets or poor oral hygiene (Miyazaki et al., 1995). Moreover, the tongue has been reported to be even more malodourous in subjects with periodontitis (Mantilla Gomez et al., 2001; Yaegaki & Sanada, 1992b). Recently, tongue microbiota has been the focus of oral malodour research, and several studies have reported a relationship between periodontal pathogens in the tongue biofilm coating and oral malodour (Tanaka et al., 2004; Washio et al., 2005). Washio et al. (2005) reported that periodontal disease-associated bacteria, such as Porphyromonas gingivalis and Prevotella intermedia, may not be associated with oral malodour in patients without periodontal disease or with low-to-moderate levels of oral malodour, whilst Tanaka et al. (2004) reported that periodontal pathogens on the tongue dorsa can contribute greatly to VSC production. Additionally, several studies have indicated a relationship between micro-organisms present on the tongue and those present in saliva; it has been suggested that a relationship exists between oral malodour and periodontal pathogens in saliva, in addition to those in the tongue coating (Awano et al., 2002; Kozlovsky et al., 1994). However, it remains unclear whether the diversity of periodontal pathogens in saliva is associated with the production of oral malodour as well as those in the tongue coating.
Generally, differences in the prevalence of periodontal pathogens in the tongue coating and saliva are thought to be related to periodontal health conditions (Kozlovsky et al., 1994; Mantilla Gomez et al., 2001; Umeda et al., 1998). Thus an improvement in periodontal health may be linked to reductions in periodontal pathogens in saliva and the tongue coating, with improvements in oral malodour. We sought to investigate whether the prevalence of periodontal pathogens in tongue coating and saliva was related to periodontal health conditions and oral malodour, and whether improved periodontal health resulted in changes in the prevalence of periodontopathogenic bacteria in saliva and the tongue coating, with a reduction in oral malodour.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Informed consent was obtained from each subject. The protocol was reviewed by the ethics committee of Kyushu Dental College.
Study design. Subjects were selected randomly from patients who complained of oral malodour. At baseline, they underwent a clinical examination, which included measurement of the concentrations of H2S and CH3SH in mouth air, periodontal probing depth of six sites per tooth, verification of bleeding on probing (BOP) and evaluation of the tongue coating. Furthermore, resting saliva and tongue coating were sampled to evaluate the prevalence of periodontal bacteria following the measurements of H2S and CH3SH. After clinical examination, they received oral hygiene instruction at baseline and underwent initial periodontal therapy, such as scaling and professional mechanical teeth cleaning, followed twice more at 2 and 4 weeks after baseline, whilst all subjects were prohibited from tongue cleaning during the 2 months until the second assessment. Two months from baseline, the parameters assessed at baseline were re-evaluated. All examinations and samplings were performed by a trained dentist.
Clinical examination. The H2S and CH3SH concentrations in mouth air were determined using a gas chromatograph (GC) (G2800; Yanaco) equipped with a flame photometric detector and a 3.4 mmx3 m glass column packed with 25 % 1,2,3-Tris(2-cyanoethoxy)propane in chromosorb W (AW-DMCS, 60/80 mesh), as described previously (Awano et al., 2002). Briefly, after closing the lips for 30 s, 10 ml mouth air was collected in the sample loop of the multi-port injector connecting to the GC equipment and immediately injected into the GC with the carrier gas (nitrogen gas) by switching the multi-port injector from the sample loading port to the sample injection port.
The number of sites with a periodontal probing depth of 4 mm or greater or with BOP was scored. Tongue coating scores were estimated by accumulated thickness and were defined as follows: 0, no coating apparent; 1, less than one-third of the area of tongue dorsum coated; 2, between one-third and two-thirds of the surface coated; 3, more than two-thirds of the surface coated (Miyazaki et al., 1995).
Sampling and preparation of saliva and tongue coating. Saliva samples were collected by spitting resting saliva into sterile plastic tubes for 5 min. After removing the saliva on the lingual surface using a three-way syringe, tongue coat samples from a 1 cm2 area of the thickest tongue biofilm on the posterior tongue dorsum were collected by scraping two or three times, using the cap of a sterile microcentrifuge tube. All samples were stored at –30 °C after collection.
Genomic DNA from the saliva sample (100 µl) and the tongue coat sample was isolated using an Easy DNA kit (Invitrogen) according to the manufacturer's instructions, and was used as template for real-time PCR.
Real-time PCR.
The species-specific primers and TaqMan probes for five periodontopathogenic bacteria (Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, Prevotella intermedia and Prevotella nigrescens) are shown in Table 1
. Initially, conventional PCR was used to confirm the presence of each of these bacteria in the saliva and tongue coating of each subject, and real-time PCR was then performed using the specimens in which their presence had been confirmed. Amplification and detection were performed using a LightCycler Sequence Detection System (Roche Diagnostics). Each PCR was performed as described previously (Kato et al., 2005). For relative quantification, the copy number of each target bacterial gene was normalized to the copy number of the 16S rRNA gene using a simplified comparative threshold cycle method (Yoshida et al., 2003).
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1.5 ng per 10 ml air or CH3SH 0.4 ng per 10 ml air) as follows: –PD/–VSCT group, patients without PD and VSCT; –PD/+VSCT group, patients without PD and with VSCT; +PD/–VSCT group, patients with PD and without VSCT; and +PD/+VSCT group, patients with PD and VSCT. Comparisons among the four groups were assessed by analysis of variance and a post-hoc test, and those between baseline data and follow-up data were performed using a paired Student's t-test. Comparisons of the proportions of the five periodontopathogenic bacteria compared with total bacterial cells in saliva and tongue coating were evaluated using the Kruskal–Wallis test and the Mann–Whitney U-test among the four groups, and Wilcoxon's rank-sum test between baseline and follow-up data. Correlations between the proportion of each periodontopathogenic bacterium and total bacterial cells in saliva and tongue were analysed using Spearman's rank correlation coefficient. All analyses were performed using SPSS software (version 11; SPSS Japan).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. In the baseline data, the mean ages in the +PD/–VSCT and +PD/+VSCT groups, which were composed of patients with PD, were significantly higher than those in the two groups without PD. A recent study reported that probing depth increased in prevalence with increasing age, and levelled off at around 50 years of age and above (Susin et al., 2005). This study also suggested that the number of subjects with PD tended to increase with increasing age. The number of teeth in patients in the +PD/–VSCT group was significantly lower than in the –PD/–VSCT group [Fisher's least significant difference (LSD) test, P <0.05]. Furthermore, no significant difference in numbers of PD was observed between the +PD/–VSCT and +PD/+VSCT groups at baseline, whilst the number of BOP in the +PD/+VSCT group was significantly greater than those in the other groups (Fisher's LSD test, P <0.05). Thus periodontal health status in patients with PD and VSCT at baseline got worse, with increases in the number of sites with BOP, compared with patients with PD alone (without VSCT). Furthermore, the mean scores of tongue coating were significantly greater in the +PD/+VSCT group than in the other groups. Thus increased tongue coating may be related to deterioration of periodontal health with an increase in both PD and BOP, and an increase in tongue biofilm coating may be associated with oral malodour, as in previous reports (Bosy et al., 1994; Miyazaki et al., 1995; Yaegaki & Sanada, 1992a).
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In the follow-up data, after treatment, the numbers of PD in the +PD/–VSCT and +PD/+VSCT groups, BOP in the –PD/–VSCT and +PD/+VSCT groups, the mean score of tongue coating in the +PD/+VSCT group and the concentration of H2S in the –PD/+VSCT and +PD/+VSCT groups and CH3SH in the +PD/+VSCT group were all significantly reduced compared with those at baseline (paired t-test, P<0.05). Moreover, no significant difference was detected in any parameter among the four groups in the follow-up data. Thus it was evident that the scaling and professional mechanical teeth cleaning that all subjects underwent resulted in improvements in periodontal health and oral malodour.
Comparison of five periodontopathogenic bacteria in saliva between baseline and follow-up
The proportions of the five periodontopathogenic bacteria compared with total bacterial cells in saliva at baseline and follow-up are shown in Table 3. In the baseline data, the mean percentage (±SD) of the five periodontopathogenic bacteria in the saliva of all subjects was approximately 3.2 % (±5.5). The proportions of Porphyromonas gingivalis and Treponema denticola and the total proportion of the five bacteria in the +PD/–VSCT group were significantly higher than in the two groups without PD (Mann–Whitney U-test, P <0.05). Furthermore, the proportion of Treponema denticola in the +PD/+VSCT group was significantly higher than in the two groups without PD, and the proportions of Tannerella forsythensis and all five bacteria in the +PD/+VSCT group were significantly higher than in the –PD/–VSCT group (Mann–Whitney U-test, P <0.05). Thus the present findings showed that patients with PD had higher proportions of periodontopathogenic bacteria in saliva compared with those without PD. In particular, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythensis, referred to as the ‘red complex’ and regarded as the main pathogens in periodontitis, had a higher prevalence in patient groups with PD than in those without PD. We reported previously that the presence of Tannerella forsythensis in subjects with periodontitis was strongly correlated with the concentration of VSCs in mouth air (Awano et al., 2002). This study also found that the higher prevalence of Tannerella forsythensis in patients with PD was associated with enhanced oral malodour. Tannerella forsythensis has been identified along with Porphyromonas gingivalis in subgingival plaque and saliva samples from subjects with severe periodontal disease (Holt & Ebersole, 2005; Yang et al., 2004). Thus the increase in the proportion of Tannerella forsythensis may be related to the severity of periodontal disease, which may increase the VSC level in mouth air.
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). Furthermore, in the follow-up data, Porphyromonas gingivalis and Treponema denticola were significantly higher in the +PD/–VSCT group than in the two groups without PD as well as at baseline, and the total proportion of the five bacteria at follow-up was significantly higher in the groups with PD than in the groups without PD (Mann–Whitney U-test, P <0.05). Moreover, the proportion of Tannerella forsythensis in the +PD/+VSCT group differed significantly from that in the –PD/–VSCT group (P <0.05). These results showed that the initial periodontal therapy led to a significant reduction in the proportion of periodontal pathogens such as Porphyromonas gingivalis, Treponema denticola and Tannerella forsythensis in saliva, in addition to the improvement in periodontal health and oral malodour. However, the tendency for patients with PD at baseline to have a higher proportion of periodontopathogenic bacteria compared with patients without PD was maintained.
Comparison of the five periodontopathogenic bacteria in tongue coating at baseline and follow-up
The proportions of the five periodontopathogenic bacteria compared with total bacterial cells in the tongue coating at baseline and follow-up are shown in Table 4. The mean percentage of the total five periodontopathogenic bacteria in the tongue coating of all patients was approximately one-sixth lower than in saliva. In baseline data, the proportion of Treponema denticola in the +PD/+VSCT group was significantly higher than that in the –PD/–VSCT group (Mann–Whitney U-test, P <0.05). Furthermore, in follow-up data, the +PD/+VSCT group had a significantly higher proportion of Treponema denticola compared with the –PD/–VSCT and –PD/+VSCT groups (P <0.05), and a significantly higher proportion of Prevotella nigrescens than the other groups. However, no significant difference was observed in the proportion of each bacterium in all patients or among the four groups between the baseline and follow-up data. These results showed that no relationship existed between the proportion of periodontal pathogens in the tongue coating and differences in PD and VSCT. The improvement in periodontal health and oral malodour resulting from the periodontal therapy seemed not to be reflected in the prevalence of periodontal pathogens in the tongue coating.
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In another recent report, the numbers of total colonies and black/grey colonies sampled from the tongue coating in the odour group were significantly higher than those in the no/low odour group (Washio et al., 2005). Moreover, an increase in the number of H2S-producing bacteria, such as Veillonella, Actinomyces and Prevotella species, in the tongue biofilm was associated with oral malodour, even though the bacterial composition of the tongue biofilm was similar in the odour and the no/low odour groups (Washio et al., 2005). In this study, although the amounts of the five target bacteria in the tongue coating were not evaluated, it was evident that increased tongue coating was associated with VSC levels. Thus the difference in the amount of total bacteria in the tongue coating rather than the composition of microflora may influence the production of VSCs more strongly.
This study did not focus on the microflora in subgingival plaque as the diversity of the microbiota is influenced by differences in the sampling sites in addition to differences among subjects (Paster et al., 2001). The bacterial population in subgingival plaque is associated with the local periodontal health status of the sampling site (Haffajee et al., 1998); however, the bacterial population in subgingival plaque sampled from a local site may not reflect a comprehensive periodontal evaluation of a subject. Furthermore, it has been reported that it is useful to observe the prevalence of periodontal pathogens in saliva and tongue coating in addition to in subgingival plaque (Tanner et al., 2006; Umeda et al., 1998). Therefore, it may be more reasonable to use saliva and tongue coating than plaque sampled from the local site to comprehensively investigate the relationship between periodontal health status and the prevalence of periodontal pathogens in the whole mouth.
Conclusions
In this study, we found that the prevalence of the periodontal pathogens Porphyromonas gingivalis, Treponema denticola and Tannerella forsythensis in saliva was related to periodontal health status and VSC levels in mouth air, but no relationship was seen between the prevalence of these periodontal pathogens in the tongue coating and the status of periodontal health or VSC levels in mouth air. Moreover, an improvement in periodontal health resulted in lower prevalences of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythensis in saliva, with reduced VSC levels in mouth air. Thus our findings suggest that the prevalence of these periodontal pathogens in saliva is linked to periodontal health status and influences VSC levels in mouth air.
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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