This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Paju, S.
Right arrow Articles by Könönen, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Paju, S.
Right arrow Articles by Könönen, E.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, January 2009, p. 235-238, Vol. 47, No. 1
0095-1137/09/$08.00+0     doi:10.1128/JCM.01824-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Susanna Paju,1* Pirkko J. Pussinen,1 Liisa Suominen-Taipale,2 Mari Hyvönen,3 Matti Knuuttila,4 and Eija Könönen3,5

Institute of Dentistry, University of Helsinki and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland,1 Department of Health and Functional Capacity,2 Department of Bacterial and Inflammatory Diseases, National Public Health Institute (KTL), Helsinki, Finland,3 Institute of Dentistry, University of Oulu, Oulu, Finland,4 Institute of Dentistry, University of Turku, Turku, Finland5

Received 20 September 2008/ Returned for modification 1 November 2008/ Accepted 7 November 2008


arrow
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.


arrow
TEXT
 
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.


View this table:
[in this window]
[in a new window]

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


View this table:
[in this window]
[in a new window]

  		TABLE 2. Distribution of subjects (n = 1,198) and the mean number of teeth with deepened pockets by the number of periodontal speciesc


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		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).


View this table:
[in this window]
[in a new window]

  		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)


Figure 2
View larger version (7K):
[in this window]
[in a new window]

  		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.


arrow
ACKNOWLEDGMENTS
 
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.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Dentistry, PL 41, FI-00014 University of Helsinki, Helsinki, Finland. Phone: 358-9-19127270. Fax: 358-9-19125194. E-mail: Susanna.Paju{at}helsinki.fi Back

{triangledown} Published ahead of print on 19 November 2008. Back


arrow
REFERENCES
 
    1
  1. Aas, J. A., B. J. Paster, L. N. Stokes, I. Olsen, and F. E. Dewhirst. 2005. Defining the normal bacterial flora of the oral cavity. J. Clin. Microbiol. 43:5721-5732.[Abstract/Free Full Text]
    2. 2
  2. American Academy of Periodontology. 1996. Consensus report. Periodontal diseases: pathogenesis and microbial factors. Ann. Periodontol. 1:926-932.[Medline]
    3. 3
  3. Ashimoto, A., C. Chen, I. Bakker, and J. Slots. 1996. Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol. Immunol. 11:266-273.[Medline]
    4. 4
  4. Boutaga, K., P. H. Savelkoul, E. G. Winkel, and A. J. van Winkelhoff. 2007. Comparison of subgingival bacterial sampling with oral lavage for detection and quantification of periodontal pathogens by real-time polymerase chain reaction. J. Periodontol. 78:79-86.[CrossRef][Medline]
    5. 5
  5. Cortelli, S. C., M. Feres, A. A. Rodrigues, D. R. Aquino, J. A. Shibli, and J. R. Cortelli. 2005. Detection of Actinobacillus actinomycetemcomitans in unstimulated saliva of patients with chronic periodontitis. J. Periodontol. 76:204-209.[CrossRef][Medline]
    6. 6
  6. Haffajee, A. D., A. Bogren, H. Hasturk, M. Feres, N. J. Lopez, and S. S. Socransky. 2004. Subgingival microbiota of chronic periodontitis subjects from different geographic locations. J. Clin. Periodontol. 31:996-1002.[CrossRef][Medline]
    7. 7
  7. Haffajee, A. D., and S. S. Socransky. 1994. Microbial etiological agents of destructive periodontal diseases. Periodontol. 2000 5:78-111.[CrossRef]
    8. 8
  8. Könönen, E., H. Jousimies-Somer, and S. Asikainen. 1994. The most frequently isolated gram-negative anaerobes in saliva and subgingival samples taken from young women. Oral Microbiol. Immunol. 9:126-128.[Medline]
    9. 9
  9. Könönen, E., S. Paju, P. J. Pussinen, M. Hyvönen, P. Di Tella, L. Suominen-Taipale, and M. Knuuttila. 2007. Population-based study of salivary carriage of periodontal pathogens in adults. J. Clin. Microbiol. 45:2446-2451.[Abstract/Free Full Text]
    10. 10
  10. Mager, D. L., A. D. Haffajee, and S. S. Socransky. 2003. Effects of periodontitis and smoking on the microbiota of oral mucous membranes and saliva in systemically healthy subjects. J. Clin. Periodontol. 30:1031-1037.[CrossRef][Medline]
    11. 11
  11. Marsh, P. D. 2003. Are dental diseases examples of ecological catastrophes? Microbiology 149:279-294.[Abstract/Free Full Text]
    12. 12
  12. Pihlstrom, B. L., B. S. Michalowicz, and N. W. Johnson. 2005. Periodontal diseases. Lancet 366:1809-1820.[CrossRef][Medline]
    13. 13
  13. Premaraj, T., N. Kato, K. Fukui, H. Kato, and K. Watanabe. 1999. Use of PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques for differentiation of Prevotella intermedia sensu stricto and Prevotella nigrescens. J. Clin. Microbiol. 37:1057-1061.[Abstract/Free Full Text]
    14. 14
  14. Ready, D., F. D'Aiuto, D. A. Spratt, J. Suvan, M. S. Tonetti, and M. Wilson. 2008. Disease severity associated with the presence in subgingival plaque of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Tannerella forsythia, singularly or in combination, as detected by nested multiplex PCR. J. Clin. Microbiol. 46:3380-3383.[Abstract/Free Full Text]
    15. 15
  15. Socransky, S. S., and A. D. Haffajee. 2005. Periodontal microbial ecology. Periodontol. 2000 38:135-187.[CrossRef]
    16. 16
  16. Socransky, S. S., A. D. Haffajee, M. A. Cugini, C. Smith, and R. L. Kent, Jr. 1998. Microbial complexes in subgingival plaque. J. Clin. Periodontol. 25:134-144.[CrossRef][Medline]
    17. 17
  17. Tanner, A. C., R. Kent, Jr., E. Kanasi, S. C. Lu, B. J. Paster, S. T. Sonis, L. A. Murray, and T. E. Van Dyke. 2007. Clinical characteristics and microbiota of progressing slight chronic periodontitis in adults. J. Clin. Periodontol. 34:917-930.[CrossRef][Medline]
    18. 18
  18. Teles, F. R., A. D. Haffajee, and S. S. Socransky. 2008. The reproducibility of curet sampling of subgingival biofilms. J. Periodontol. 79:705-713.[CrossRef][Medline]
    19. 19
  19. Timmerman, M. F., G. A. Van der Weijden, S. Armand, F. Abbas, E. G. Winkel, A. J. van Winkelhoff, and U. Van der Velden. 1998. Untreated periodontal disease in Indonesian adolescents. Clinical and microbiological baseline data. J. Clin. Periodontol. 25:215-224.[CrossRef][Medline]
    20. 20
  20. Tran, S. D., and J. D. Rudney. 1999. Improved multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis. J. Clin. Microbiol. 37:3504-3508.[Abstract/Free Full Text]
    21. 21
  21. Umeda, M., A. Contreras, C. Chen, I. Bakker, and J. Slots. 1998. The utility of whole saliva to detect the oral presence of periodontopathic bacteria. J. Periodontol. 69:828-833.[Medline]
    22. 22
  22. van Winkelhoff, A. J., B. G. Loos, W. A. van der Reijden, and U. van der Velden. 2002. Porphyromonas gingivalis, Bacteroides forsythus and other putative periodontal pathogens in subjects with and without periodontal destruction. J. Clin. Periodontol. 29:1023-1028.[CrossRef][Medline]

Journal of Clinical Microbiology, January 2009, p. 235-238, Vol. 47, No. 1
0095-1137/09/$08.00+0     doi:10.1128/JCM.01824-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hyvarinen, K., Laitinen, S., Paju, S., Hakala, A., Suominen-Taipale, L., Skurnik, M., Kononen, E., Pussinen, P. J. (2009). Detection and quantification of five major periodontal pathogens by single copy gene-based real-time PCR. Innate Immunity 15: 195-204 [Abstract]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Paju, S.
Right arrow Articles by Könönen, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Paju, S.
Right arrow Articles by Könönen, E.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»