Detection of Multiple Pathogenic Species in Saliva Is Associated with Periodontal Infection in Adults

Abstract

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 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 ≥4 mm and ≥6 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 species-specific 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 ≥4 mm and ≥6 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 two-stage 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 ≥4 mm and ≥6 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 ≥4 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 certain 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 ≥6 mm (Table 3). The association of specific bacterial combinations (of two or three pathogens) with PPDs of ≥4 mm (Fig. 2A) was similar to the results with PPDs of ≥6 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.

TABLE 1.

Basic characteristics of the study subjects (n = 1,198)

Characteristics	No. of study subjects	% Study subjects (SE)
Sex
Male	538	47 (1.4)
Female	660	53 (1.4)
Total	1,198
Age group (yr)
30-39	358	29 (1.2)
40-49	307	26 (1.2)
50-59	311	28 (1.3)
60-69	125	10 (0.9)
70+	97	7 (0.7)
Total	1,198
Level of education
Basic	280	23 (1.3)
Secondary	374	32 (1.3)
Higher	539	45 (1.7)
Total	1,193
Smoking history
Never	586	48 (1.5)
Formerly	244	21 (1.1)
Occasionally	72	6 (0.7)
Daily	291	25 (1.3)
Total	1,193
No. of teeth
25-32	868	72 (1.3)
20-24	167	14 (0.9)
10-19	96	8 (0.8)
1-9	67	6 (0.6)
Total	1,198
No. of teeth with ≥4-mm pockets
0	222	18 (1.0)
1-3	330	28 (1.4)
4-6	232	19 (1.1)
≥7	414	35 (1.4)
Total	1,198
No. of teeth with ≥6-mm pockets
0	910	76 (1.2)
1-3	195	16 (1.0)
4-6	41	4 (0.5)
≥7	52	4 (0.6)
Total	1,198

TABLE 2.

Distribution of subjects (n = 1,198) and the mean number of teeth with deepened pockets by the number of periodontal species^c

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	123	≥4	2.6 (0.4)	Ref.	Ref.
1	316	≥4	4.1 (0.3)	1.5 (1.1-2.1)	1.4 (1.0-1.8)
2	375	≥4	6.0 (0.3)	2.3 (1.8-3.0)	1.9 (1.5-2.5)
3	229	≥4	7.7 (0.4)	2.9 (2.2-3.9)	2.2 (1.7-2.9)
4-6	155	≥4	10.2 (0.6)	3.9 (2.9-5.1)	2.6 (2.0-3.5)
Total avg			6.0 (0.2)
0	123	≥6	0.1 (0.1)	Ref.	Ref.
1	316	≥6	0.3 (0.1)	2.6 (1.0-6.3)	2.2 (0.9-5.3)
2	375	≥6	0.8 (0.1)	6.9 (2.9-16.1)	4.8 (2.1-10.6)
3	229	≥6	1.4 (0.2)	12.1 (5.2-27.8)	6.5 (2.9-14.6)
4-6	155	≥6	2.2 (0.4)	18.2 (7.7-43.2)	8.7 (3.8-19.9)
Total avg			0.9 (0.1)

^aAdjusted by age, gender, level of education, smoking history, and number of teeth of the subjects.

^bResults of the Kruskal-Wallis analysis of variance were P values of <0.001 for diffrences of means for teeth with ≥4-mm pockets and teeth with ≥6-mm pockets for different numbers of pathogens.

^cThe 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.

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).

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)

Pathogenic species alone or in any combination	RR (95% CI) for teeth with PPD of:
	≥4 mm			≥6 mm
	Unadjusted	Adjusteda	Adjustedb	Unadjusted	Adjusteda	Adjustedb
Aggregatibacter actinomycetemcomitans	1.3 (1.1-1.5)	0.9 (0.8-1.1)	1.0 (0.9-1.2)	1.5 (1.1-2.1)	0.8 (0.5-1.1)	1.0 (0.7-1.5)
Campylobacter rectus	1.2 (1.1-1.4)	0.8 (0.7-1.0)	0.9 (0.7-1.0)	1.7 (1.2-2.3)	0.8 (0.6-1.1)	0.8 (0.6-1.2)
Porphyromonas gingivalis	1.6 (1.4-1.8)	1.2 (1.0-1.3)	1.2 (1.0-1.3)	3.1 (2.2-4.3)	2.0 (1.4-2.8)	1.7 (1.2-2.4)
Prevotella intermedia	1.8 (1.6-2.0)	1.2 (1.0-1.4)	1.1 (1.0-1.3)	3.4 (2.5-4.7)	1.5 (1.0-2.2)	1.4 (1.0-1.9)
Treponema denticola	1.9 (1.7-2.1)	1.3 (1.2-1.5)	1.2 (1.0-1.4)	3.5 (2.6-4.9)	1.7 (1.2-2.6)	1.4 (0.9-2.1)
Tannerella forsythia	1.1 (0.9-1.2)	0.8 (0.7-0.9)	0.8 (0.7-0.9)	0.8 (0.5-1.1)	0.4 (0.3-0.6)	0.5 (0.3-0.6)

^aAdjusted by number of bacterial species.

^bAdjusted by age, sex, level of education, number of teeth, and smoking habits of the subjects and by number of bacterial species.

FIG. 2.

RR with 95% CI for the presence of teeth with PPDs of ≥4 mm (A) and ≥6 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.

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 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.