JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2009, p. 235–238 0095-1137/09/$08.00⫹0 doi:10.1128/JCM.01824-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 1
Detection of Multiple Pathogenic Species in Saliva Is Associated with Periodontal Infection in Adults䌤 Susanna Paju,1* Pirkko J. Pussinen,1 Liisa Suominen-Taipale,2 Mari Hyvo ¨nen,3 4 3,5 Matti Knuuttila, and Eija Ko ¨no ¨nen Institute of Dentistry, University of Helsinki and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland1; Department of Health and Functional Capacity2 and Department of Bacterial and Inflammatory Diseases,3 National Public Health Institute (KTL), Helsinki, Finland; Institute of Dentistry, University of Oulu, Oulu, Finland4; and Institute of Dentistry, University of Turku, Turku, Finland5 Received 20 September 2008/Returned for modification 1 November 2008/Accepted 7 November 2008
We investigated whether certain bacterial species and their combinations in saliva can be used as markers for periodontitis. In 1,198 subjects, the detection of multiple species, rather than the presence of a certain pathogen, in saliva was associated with periodontitis as determined by the number of teeth with deepened periodontal pockets. Periodontitis, infection of the tooth-supporting tissues, results from the accumulation of pathogenic bacterial plaque at and below the gingival margin (12). The composition of the dental plaque community plays a central role in the etiology of periodontitis (7, 11, 15). The major periodontal pathogens are Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia (previously forsythensis), Campylobacter rectus, and Treponema denticola (2, 7, 22). In subgingival plaque, P. gingivalis, T. forsythia, and T. denticola have the strongest relation to periodontal tissue destruction (16). The presence of pathogens in subgingival sites of early and advanced periodontitis and in the healthy periodontium has been studied previously (1, 6, 16, 17, 22), while the natural carriage rates of periodontal pathogens in saliva are hardly known. Recently, we showed in a population-based study of Finnish adults that distinct periodontal bacteria have different carriage profiles depending on the age, educational level, and periodontal status of the subjects (9). The salivary carriage of periodontal pathogens proved to be common: out of the six examined periodontal pathogens, at least one was found in 88% of the subjects (9). Since major periodontal bacteria are commonly found in adults, a combination of pathogenic bacteria in saliva may represent a marker for disease. The objective of the present study was to investigate whether saliva, an easily and noninvasively collectable specimen material, can be used for diagnostic purposes of periodontitis. The study subjects belong to a national population-based “Health 2000 Health Examination Survey,” coordinated by the National Public Health Institute (KTL), Finland (http://www .ktl.fi/health2000/index.uk.html/). All protocols were approved by the institutional ethic committees. Methods and patient recruitment have been previously published (9). The present study includes data for 1,198 dentate subjects, belonging to the southern Finland sample (n ⫽ 2,616), from whom both clinical
TABLE 1. Basic characteristics of the study subjects (n ⫽ 1,198) No. of study subjects
% Study subjects (SE)
Sex Male Female Total
538 660 1,198
47 (1.4) 53 (1.4)
Age group (yr) 30–39 40–49 50–59 60–69 70⫹ Total
358 307 311 125 97 1,198
29 (1.2) 26 (1.2) 28 (1.3) 10 (0.9) 7 (0.7)
Level of education Basic Secondary Higher Total
280 374 539 1,193
23 (1.3) 32 (1.3) 45 (1.7)
Smoking history Never Formerly Occasionally Daily Total
586 244 72 291 1,193
48 (1.5) 21 (1.1) 6 (0.7) 25 (1.3)
No. of teeth 25–32 20–24 10–19 1–9 Total
868 167 96 67 1,198
72 (1.3) 14 (0.9) 8 (0.8) 6 (0.6)
No. of teeth with ⱖ4-mm pockets 0 1–3 4–6 ⱖ7 Total
222 330 232 414 1,198
18 (1.0) 28 (1.4) 19 (1.1) 35 (1.4)
No. of teeth with ⱖ6-mm pockets 0 1–3 4–6 ⱖ7 Total
910 195 41 52 1,198
76 (1.2) 16 (1.0) 4 (0.5) 4 (0.6)
Characteristics
* Corresponding author. Mailing address: Institute of Dentistry, PL 41, FI-00014 University of Helsinki, Helsinki, Finland. Phone: 358-919127270. Fax: 358-9-19125194. E-mail:
[email protected]. 䌤 Published ahead of print on 19 November 2008. 235
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TABLE 2. Distribution of subjects (n ⫽ 1,198) and the mean number of teeth with deepened pockets by the number of periodontal speciesc
No. of species
No. of subjects
Teeth with PPD of (mm)
Mean no. of teeth with deepened pockets (SE)b
RR (95% CI)
Unadjusted
Adjusteda
0 1 2 3 4–6 Total avg
123 316 375 229 155
ⱖ4 ⱖ4 ⱖ4 ⱖ4 ⱖ4
2.6 (0.4) 4.1 (0.3) 6.0 (0.3) 7.7 (0.4) 10.2 (0.6) 6.0 (0.2)
Ref. 1.5 (1.1–2.1) 2.3 (1.8–3.0) 2.9 (2.2–3.9) 3.9 (2.9–5.1)
Ref. 1.4 (1.0–1.8) 1.9 (1.5–2.5) 2.2 (1.7–2.9) 2.6 (2.0–3.5)
0 1 2 3 4–6 Total avg
123 316 375 229 155
ⱖ6 ⱖ6 ⱖ6 ⱖ6 ⱖ6
0.1 (0.1) 0.3 (0.1) 0.8 (0.1) 1.4 (0.2) 2.2 (0.4) 0.9 (0.1)
Ref. 2.6 (1.0–6.3) 6.9 (2.9–16.1) 12.1 (5.2–27.8) 18.2 (7.7–43.2)
Ref. 2.2 (0.9–5.3) 4.8 (2.1–10.6) 6.5 (2.9–14.6) 8.7 (3.8–19.9)
a Adjusted by age, gender, level of education, smoking history, and number of teeth of the subjects. b Results of the Kruskal-Wallis analysis of variance were P values of ⬍0.001 for diffrences of means for teeth with ⱖ4-mm pockets and teeth with ⱖ6-mm pockets for different numbers of pathogens. c The relation of the number of pathogens to teeth with periodontal pockets was explained by means of Poisson regression models: unadjusted and adjusted RR with 95% CI. Ref., no studied species were present.
data on oral health examination and microbiological data on salivary bacteria were available (9). The number of teeth (all teeth and tooth remnants) and the number of periodontally diseased teeth (excluding third molars) determined by having probing pocket depths (PPDs) of ⱖ4 mm and ⱖ6 mm were recorded by a specially trained dentist. Smoking history and level of education were gathered by interviews (9). Bacterial DNA from saliva samples was extracted (9), and PCR detection for six periodontal pathogens, A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia, C. rectus, and T. denticola, was performed using speciesspecific primers (3, 13, 20) as previously described. Due to the skewed distribution of the outcome variables, a nonparametric test (Kruskal-Wallis analysis of variance) was used to analyze differences of the means in different numbers of pathogens. Relative risk (RR) and 95% confidence interval (95% CI) were estimated using Poisson regression models. The outcome variables included the number of teeth with PPDs of ⱖ4 mm and ⱖ6 mm. The number of species, presence of pathogens, and their various combinations were used as independent variables in addition to the age, sex, smoking history, and level of education of the subjects. The SUDAAN statistical package was used in the analyses to take into account twostage cluster sampling. Table 1 shows the characteristics of 1,198 study subjects. The number of the six studied periodontal species in saliva was associated with the number of teeth with PPDs of both ⱖ4 mm and ⱖ6 mm (P ⬍ 0.001) (Table 2). Among women who had never smoked, the higher number of pathogens was associated with the higher number of teeth with PPDs of ⱖ4 mm (data not shown). No such clear observations were made with men and women who smoked daily. Figure 1 shows percentages of subjects with certain bacterial species or different bacterial combinations. The associations between the presence of cer-
FIG. 1. Percentage of subjects with periodontal pathogens or different bacterial combinations in saliva in the study population (n ⫽ 1,198). Studied bacterial species are Aggregatibacter actinomycetemcomitans (Aa), Campylobacter rectus (Cr), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Treponema denticola (Td), and Tannerella forsythia (Tf).
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TABLE 3. The relation of occurrence of pathogenic species with teeth with periodontal pockets explained by means of Poisson regression models in study subjects (n ⫽ 1,198) RR (95% CI) for teeth with PPD of: Pathogenic species alone or in any combination
Aggregatibacter actinomycetemcomitans Campylobacter rectus Porphyromonas gingivalis Prevotella intermedia Treponema denticola Tannerella forsythia a b
ⱖ4 mm
ⱖ6 mm
Unadjusted
Adjusteda
Adjustedb
Unadjusted
Adjusteda
Adjustedb
1.3 (1.1–1.5) 1.2 (1.1–1.4) 1.6 (1.4–1.8) 1.8 (1.6–2.0) 1.9 (1.7–2.1) 1.1 (0.9–1.2)
0.9 (0.8–1.1) 0.8 (0.7–1.0) 1.2 (1.0–1.3) 1.2 (1.0–1.4) 1.3 (1.2–1.5) 0.8 (0.7–0.9)
1.0 (0.9–1.2) 0.9 (0.7–1.0) 1.2 (1.0–1.3) 1.1 (1.0–1.3) 1.2 (1.0–1.4) 0.8 (0.7–0.9)
1.5 (1.1–2.1) 1.7 (1.2–2.3) 3.1 (2.2–4.3) 3.4 (2.5–4.7) 3.5 (2.6–4.9) 0.8 (0.5–1.1)
0.8 (0.5–1.1) 0.8 (0.6–1.1) 2.0 (1.4–2.8) 1.5 (1.0–2.2) 1.7 (1.2–2.6) 0.4 (0.3–0.6)
1.0 (0.7–1.5) 0.8 (0.6–1.2) 1.7 (1.2–2.4) 1.4 (1.0–1.9) 1.4 (0.9–2.1) 0.5 (0.3–0.6)
Adjusted by number of bacterial species. Adjusted by age, sex, level of education, number of teeth, and smoking habits of the subjects and by number of bacterial species.
tain pathogens, alone or in any combination, and the number of teeth with deepened pockets are shown in Table 3. After adjustment, the carriage of P. gingivalis, despite the presence of other species, was significantly associated with the presence of PPDs of ⱖ6 mm (Table 3). The association of specific bacterial combinations (of two or three pathogens) with PPDs of ⱖ4 mm (Fig. 2A) was similar to the results with PPDs of ⱖ6 mm (Fig. 2B). Several combinations of four, five, and six pathogens were significantly associated with the occurrence of deepened pockets but were left out because the number of bacterial species, rather than the presence of certain species, proved to be important. For the first time, in the present sample of 1,198 dentate Finnish adults, we report that the salivary carriage of multiple periodontal bacterial species is associated with periodontitis at the population level. Saliva is a representative diagnostic specimen for an overall view of the oral microbiota, since bacteria from various sites and surfaces of the oral cavity are found in saliva and mouth rinses (4, 8, 10, 19, 21). For example, A. actinomycetemcomitans has been detected in unstimulated saliva with no statistical difference to pooled subgingival samples (5). Subgingival curette sampling is a reproducible and reliable method for studying proportions of bacteria in periodontal biofilms (18). However, this technique requires a person with periodontal education/experience for selecting subgingival sites representative of periodontal status, whereas saliva can easily and less time-consumingly be collected at a dental hygienist’s or nurse’s appointment. As an easy and noninvasive specimen, saliva offers an excellent sample material for large
population-based studies of periodontal health or carriage of periodontal pathogens. The detection of multiple pathogenic species in saliva, rather than the presence of any single pathogen in saliva, was associated with periodontitis in our study. Although no specific combination was significantly more disease linked than others, A. actinomycetemcomitans, P. gingivalis, T. forsythia, and T. denticola, species that have been previously shown to have the strongest relationship to periodontal breakdown (2, 16), were also major players in the present bacterial combinations associated with the disease. Certain combinations in saliva were associated with the number of teeth with deepened pockets but not as strongly as has been reported for subgingival samples (3, 6, 16, 19, 22). This may be partially explained by the geographic location; subgingival microbial profiles have been found to differ in subjects from Europe and North and South America (6). In a recent study using a multiplex PCR method for detecting the subgingival presence of A. actinomycetemcomitans, P. gingivalis, and T. forsythia in periodontitis patients, subjects with a single pathogen had more severe disease than subjects with two or three pathogens, which suggests that both positive and negative bacterial interactions are important in periodontal biofilms (14). We aimed to find out if saliva can be used for diagnostic purposes of periodontitis. Salivary sampling and PCR technique allow rapid identification of periodontal bacteria. In our study population, however, there were many distinctions between subjects of different ages, sexes, and behavioral habits such as smoking, and no specific disease marker could be
FIG. 2. RR with 95% CI for the presence of teeth with PPDs of ⱖ4 mm (A) and ⱖ6 mm (B) in combinations of two or three pathogens in saliva (n ⫽ 1,198). Adjustments were made for the age, sex, education, number of teeth, and smoking habits of subjects. Studied bacterial species are as described in the legend to Fig. 1.
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established. The associations found in our cross-sectional study between the number of species and periodontal pockets were strong, however, suggesting possible predictive markers for periodontitis and encouraging further longitudinal studies. The present study in an adult population indicated that, rather than the presence of certain periodontal pathogens or specific combinations, the number of pathogenic species in saliva associates with clinical signs of periodontitis. We thank the Health 2000 organization. Tiina Karvonen and Pirjo Nurmi are acknowledged for technical assistance. Bacterial work has received financial support from the Academy of Finland (grant 78443 to E.K., grant 209152 to S.P., and grants 211129 and 118391 to P.J.P.). The oral health examination was partly supported by the Finnish Dental Society Apollonia and the Finnish Dental Association.
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