Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1995 Dec;33(12):3102–3105. doi: 10.1128/jcm.33.12.3102-3105.1995

Identification of Actinobacillus actinomycetemcomitans in subgingival plaque by PCR.

T F Flemmig 1, S Rüdiger 1, U Hofmann 1, H Schmidt 1, B Plaschke 1, A Strätz 1, B Klaiber 1, H Karch 1
PMCID: PMC228652  PMID: 8586681

Abstract

The purpose of this study was to assess the sensitivity and specificity of the PCR in detecting Actinobacillus actinomycetemcomitans. The PCR's detection capability was compared with those of three other methods: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conventional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was amplified by PCR with primers TT-15 and TT-16. For CH, the PCR product was labeled with digoxigenin and used as a hybridization probe. Nucleotide sequence analysis of the PCR product of A. actinomycetemcomitans 1D4 and 1664 and three clinical isolates revealed complete homology among the tested strains, with only one base substitution (at position 1344) in comparison with the published sequence. With artificially infected subgingival plaque, the detection limit of PCR for A. actinomycetemcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR (CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of subgingival plaque samples from 35 patients with periodontal disease and 10 periodontally healthy subjects revealed that CE-PCR and CH had the highest overall rate of A. actinomycetemcomitans detection (both 58%), followed by PCR and culture (both 42%). With CH as the "gold standard", the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respectively. The CE-PCR provided acceptable positive and negative predictive values (> or = 70%) when the prevalence of A. actinomycetemcomitans varied between 30 and 70%. PCR alone provided comparable predictive values over a narrower range of prevalence rates (30 to 50%), while culture did not afford acceptable predictive values at any prevalence rate. PCR and CE-PCR were found to be superior to culture with presumptive biochemical identification and should be the preferred methods for the detection of A. actinomycetemcomitans in subgingival plaque.

Full Text

The Full Text of this article is available as a PDF (194.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aicoforado G. A., McKay T. L., Slots J. Rapid method for detection of lactose fermenting oral microorganisms. Oral Microbiol Immunol. 1987 Mar;2(1):35–38. doi: 10.1111/j.1399-302x.1987.tb00267.x. [DOI] [PubMed] [Google Scholar]
  2. Bonta Y., Zambon J. J., Genco R. J., Neiders M. E. Rapid identification of periodontal pathogens in subgingival plaque: comparison of indirect immunofluorescence microscopy with bacterial culture for detection of Actinobacillus actinomycetemcomitans. J Dent Res. 1985 May;64(5):793–798. doi: 10.1177/00220345850640050201. [DOI] [PubMed] [Google Scholar]
  3. Bragd L., Dahlén G., Wikström M., Slots J. The capability of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedius to indicate progressive periodontitis; a retrospective study. J Clin Periodontol. 1987 Feb;14(2):95–99. doi: 10.1111/j.1600-051x.1987.tb00949.x. [DOI] [PubMed] [Google Scholar]
  4. Genco R. J., Zambon J. J., Christersson L. A. The origin of periodontal infections. Adv Dent Res. 1988 Nov;2(2):245–259. doi: 10.1177/08959374880020020901. [DOI] [PubMed] [Google Scholar]
  5. Goncharoff P., Figurski D. H., Stevens R. H., Fine D. H. Identification of Actinobacillus actinomycetemcomitans: polymerase chain reaction amplification of lktA-specific sequences. Oral Microbiol Immunol. 1993 Apr;8(2):105–110. doi: 10.1111/j.1399-302x.1993.tb00554.x. [DOI] [PubMed] [Google Scholar]
  6. Gunaratnam M., Smith G. L., Socransky S. S., Smith C. M., Haffajee A. D. Enumeration of subgingival species on primary isolation plates using colony lifts. Oral Microbiol Immunol. 1992 Feb;7(1):14–18. doi: 10.1111/j.1399-302x.1992.tb00013.x. [DOI] [PubMed] [Google Scholar]
  7. Haffajee A. D., Socransky S. S. Effect of sampling strategy on the false-negative rate for detection of selected subgingival species. Oral Microbiol Immunol. 1992 Feb;7(1):57–59. doi: 10.1111/j.1399-302x.1992.tb00022.x. [DOI] [PubMed] [Google Scholar]
  8. Haubek D., Poulsen K., Asikainen S., Kilian M. Evidence for absence in northern Europe of especially virulent clonal types of Actinobacillus actinomycetemcomitans. J Clin Microbiol. 1995 Feb;33(2):395–401. doi: 10.1128/jcm.33.2.395-401.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kolodrubetz D., Dailey T., Ebersole J., Kraig E. Cloning and expression of the leukotoxin gene from Actinobacillus actinomycetemcomitans. Infect Immun. 1989 May;57(5):1465–1469. doi: 10.1128/iai.57.5.1465-1469.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kraig E., Dailey T., Kolodrubetz D. Nucleotide sequence of the leukotoxin gene from Actinobacillus actinomycetemcomitans: homology to the alpha-hemolysin/leukotoxin gene family. Infect Immun. 1990 Apr;58(4):920–929. doi: 10.1128/iai.58.4.920-929.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kwok S., Higuchi R. Avoiding false positives with PCR. Nature. 1989 May 18;339(6221):237–238. doi: 10.1038/339237a0. [DOI] [PubMed] [Google Scholar]
  12. Moore W. E., Moore L. V. The bacteria of periodontal diseases. Periodontol 2000. 1994 Jun;5:66–77. doi: 10.1111/j.1600-0757.1994.tb00019.x. [DOI] [PubMed] [Google Scholar]
  13. Moseley S. L., Echeverria P., Seriwatana J., Tirapat C., Chaicumpa W., Sakuldaipeara T., Falkow S. Identification of enterotoxigenic Escherichia coli by colony hybridization using three enterotoxin gene probes. J Infect Dis. 1982 Jun;145(6):863–869. doi: 10.1093/infdis/145.6.863. [DOI] [PubMed] [Google Scholar]
  14. Nakagawa S., Machida Y., Nakagawa T., Fujii H., Yamada S., Takazoe I., Okuda K. Infection by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, and antibody responses at different ages in humans. J Periodontal Res. 1994 Jan;29(1):9–16. doi: 10.1111/j.1600-0765.1994.tb01085.x. [DOI] [PubMed] [Google Scholar]
  15. Noordhoek G. T., Kolk A. H., Bjune G., Catty D., Dale J. W., Fine P. E., Godfrey-Faussett P., Cho S. N., Shinnick T., Svenson S. B. Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories. J Clin Microbiol. 1994 Feb;32(2):277–284. doi: 10.1128/jcm.32.2.277-284.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pavicić M. J., van Winkelhoff A. J., Douqué N. H., Steures R. W., de Graaff J. Microbiological and clinical effects of metronidazole and amoxicillin in Actinobacillus actinomycetemcomitans-associated periodontitis. A 2-year evaluation. J Clin Periodontol. 1994 Feb;21(2):107–112. doi: 10.1111/j.1600-051x.1994.tb00287.x. [DOI] [PubMed] [Google Scholar]
  17. Savitt E. D., Strzempko M. N., Vaccaro K. K., Peros W. J., French C. K. Comparison of cultural methods and DNA probe analyses for the detection of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius in subgingival plaque samples. J Periodontol. 1988 Jul;59(7):431–438. doi: 10.1902/jop.1988.59.7.431. [DOI] [PubMed] [Google Scholar]
  18. Slots J., Feik D., Rams T. E. Actinobacillus actinomycetemcomitans and Bacteroides intermedius in human periodontitis: age relationship and mutual association. J Clin Periodontol. 1990 Oct;17(9):659–662. [PubMed] [Google Scholar]
  19. Slots J. Rapid identification of important periodontal microorganisms by cultivation. Oral Microbiol Immunol. 1986 Nov;1(1):48–57. doi: 10.1111/j.1399-302x.1986.tb00318.x. [DOI] [PubMed] [Google Scholar]
  20. Slots J. Salient Biochemical Characters of Actinobacillus actinomycetemcomitans. Arch Microbiol. 1982 Feb;131(1):60–67. doi: 10.1007/BF00451500. [DOI] [PubMed] [Google Scholar]
  21. Slots J. Selective medium for isolation of Actinobacillus actinomycetemcomitans. J Clin Microbiol. 1982 Apr;15(4):606–609. doi: 10.1128/jcm.15.4.606-609.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Syed S. A., Loesche W. J. Survival of human dental plaque flora in various transport media. Appl Microbiol. 1972 Oct;24(4):638–644. doi: 10.1128/am.24.4.638-644.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tanner A. C., Goodson J. M. Sampling of microorganisms associated with periodontal disease. Oral Microbiol Immunol. 1986 Nov;1(1):15–22. doi: 10.1111/j.1399-302x.1986.tb00310.x. [DOI] [PubMed] [Google Scholar]
  24. Tønjum T., Haas R. Identification of Actinobacillus actinomycetemcomitans by leukotoxin gene-specific hybridization and polymerase chain reaction assays. J Clin Microbiol. 1993 Jul;31(7):1856–1859. doi: 10.1128/jcm.31.7.1856-1859.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wolff L. F., Anderson L., Sandberg G. P., Reither L., Binsfeld C. A., Corinaldesi G., Shelburne C. E. Bacterial concentration fluorescence immunoassay (BCFIA) for the detection of periodontopathogens in plaque. J Periodontol. 1992 Dec;63(12 Suppl):1093–1101. doi: 10.1902/jop.1992.63.12s.1093. [DOI] [PubMed] [Google Scholar]
  26. Zambon J. J. Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontol. 1985 Jan;12(1):1–20. doi: 10.1111/j.1600-051x.1985.tb01348.x. [DOI] [PubMed] [Google Scholar]
  27. van Winkelhoff A. J., de Graaff J. Microbiology in the management of destructive periodontal disease. J Clin Periodontol. 1991 Jul;18(6):406–410. doi: 10.1111/j.1600-051x.1991.tb02308.x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES