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. 1996 Feb;34(2):376–384. doi: 10.1128/jcm.34.2.376-384.1996

Rapid characterization of periodontal bacterial isolates by using fluorogenic substrate tests.

M F Maiden 1, A Tanner 1, P J Macuch 1
PMCID: PMC228801  PMID: 8789019

Abstract

Eighty-nine species of subgingival bacteria, represented by 121 reference strains and 892 patient isolates, including gram-negative, gram-positive, aerobic, facultatively anaerobic, microaerophilic, and anaerobic species, were characterized with a panel of fluorogenic, 4-methylumbelliferyl-linked substrate tests. Identifications of all patient isolates were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins relative to reference strains. Characteristic profiles of positive fluorogenic reactions differentiated most of the species, including five Porphyromonas species, six pigmenting and five nonpigmenting Prevotella species, Bacteroides forsythus, three Capnocytophaga species, six Actinomyces species, four Propionibacterium species, and eight Streptococcus species. Two mannoside isomers differentiated Actinomyces israelii and Actinomyces gerencseriae. In addition to Porphyromonas gingivalis, B. forsythus, and Capnocytophaga species, Fusobacterium alocis, Actinomyces odontolyticus, Actinomyces meyeri, and Bifidobacterium dentium were all positive for so-called trypsin-like activity. Fusobacterium nucleatum, Eikenella corrodens, Actinobacillus actinomycetemcomitans, and Campylobacter species were nonreactive with the carbohydrate-based substrates tested. Fluorogenic substrate tests provided a sensitive and simple method for biochemical characterization that could presumptively identify to species level most subgingival isolates within 4 h. The method was ideal for rapidly obtaining presumptive identifications of isolates prior to confirming identifications by definitive methods, such as SDS-PAGE.

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Selected References

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  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. Bedi G. S., Williams T. Purification and characterization of a collagen-degrading protease from Porphyromonas gingivalis. J Biol Chem. 1994 Jan 7;269(1):599–606. [PubMed] [Google Scholar]
  3. Beighton D., Hardie J. M., Whiley R. A. A scheme for the identification of viridans streptococci. J Med Microbiol. 1991 Dec;35(6):367–372. doi: 10.1099/00222615-35-6-367. [DOI] [PubMed] [Google Scholar]
  4. Bruun B., Ying Y., Kirkegaard E., Frederiksen W. Phenotypic differentiation of Cardiobacterium hominis, Kingella indologenes and CDC group EF-4. Eur J Clin Microbiol. 1984 Jun;3(3):230–235. doi: 10.1007/BF02014891. [DOI] [PubMed] [Google Scholar]
  5. Collins M. D., Love D. N., Karjalainen J., Kanervo A., Forsblom B., Willems A., Stubbs S., Sarkiala E., Bailey G. D., Wigney D. I. Phylogenetic analysis of members of the genus Porphyromonas and description of Porphyromonas cangingivalis sp. nov. and Porphyromonas cansulci sp. nov. Int J Syst Bacteriol. 1994 Oct;44(4):674–679. doi: 10.1099/00207713-44-4-674. [DOI] [PubMed] [Google Scholar]
  6. Dellinger C. A., Moore L. V. Use of the RapID-ANA System to screen for enzyme activities that differ among species of bile-inhibited Bacteroides. J Clin Microbiol. 1986 Feb;23(2):289–293. doi: 10.1128/jcm.23.2.289-293.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Elliott J. A., Facklam R. R. Identification of Leuconostoc spp. by analysis of soluble whole-cell protein patterns. J Clin Microbiol. 1993 May;31(5):1030–1033. doi: 10.1128/jcm.31.5.1030-1033.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Heukeshoven J., Dernick R. Increased sensitivity for Coomassie staining of sodium dodecyl sulfate-polyacrylamide gels using PhastSystem Development Unit. Electrophoresis. 1988 Jan;9(1):60–61. doi: 10.1002/elps.1150090112. [DOI] [PubMed] [Google Scholar]
  9. Hinnebusch C. J., Nikolai D. M., Bruckner D. A. Comparison of API Rapid Strep, Baxter MicroScan Rapid Pos ID Panel, BBL Minitek Differential Identification System, IDS RapID STR System, and Vitek GPI to conventional biochemical tests for identification of viridans streptococci. Am J Clin Pathol. 1991 Oct;96(4):459–463. doi: 10.1093/ajcp/96.4.459. [DOI] [PubMed] [Google Scholar]
  10. Johnson J. L., Moore L. V., Kaneko B., Moore W. E. Actinomyces georgiae sp. nov., Actinomyces gerencseriae sp. nov., designation of two genospecies of Actinomyces naeslundii, and inclusion of A. naeslundii serotypes II and III and Actinomyces viscosus serotype II in A. naeslundii genospecies 2. Int J Syst Bacteriol. 1990 Jul;40(3):273–286. doi: 10.1099/00207713-40-3-273. [DOI] [PubMed] [Google Scholar]
  11. Khwaja K. J., Parish P., Aldred M. J., Wade W. G. Protein profiles of Capnocytophaga species. J Appl Bacteriol. 1990 Apr;68(4):385–390. doi: 10.1111/j.1365-2672.1990.tb02888.x. [DOI] [PubMed] [Google Scholar]
  12. Kilian M. Rapid identification of Actinomycetaceae and related bacteria. J Clin Microbiol. 1978 Aug;8(2):127–133. doi: 10.1128/jcm.8.2.127-133.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kinder S. A., Kornman K. S., Holt S. C. Characterization of selected gram-negative oral microorganisms by SDS-PAGE. Oral Microbiol Immunol. 1989 Mar;4(1):52–56. doi: 10.1111/j.1399-302x.1989.tb00407.x. [DOI] [PubMed] [Google Scholar]
  14. Lala A., Amano A., Sojar H. T., Radel S. J., De Nardin E. Porphyromonas gingivalis trypsin-like protease: a possible natural ligand for the neutrophil formyl peptide receptor. Biochem Biophys Res Commun. 1994 Mar 30;199(3):1489–1496. doi: 10.1006/bbrc.1994.1399. [DOI] [PubMed] [Google Scholar]
  15. Laughon B. E., Syed S. A., Loesche W. J. API ZYM system for identification of Bacteroides spp., Capnocytophaga spp., and spirochetes of oral origin. J Clin Microbiol. 1982 Jan;15(1):97–102. doi: 10.1128/jcm.15.1.97-102.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Love D. N., Bailey G. D., Collings S., Briscoe D. A. Description of Porphyromonas circumdentaria sp. nov. and reassignment of Bacteroides salivosus (Love, Johnson, Jones, and Calverley 1987) as Porphyromonas (Shah and Collins 1988) salivosa comb. nov. Int J Syst Bacteriol. 1992 Jul;42(3):434–438. doi: 10.1099/00207713-42-3-434. [DOI] [PubMed] [Google Scholar]
  17. Maiden M. F., Tanner A. Identification of oral yeasts by polyacrylamide gel electrophoresis. Oral Microbiol Immunol. 1991 Jun;6(3):187–190. doi: 10.1111/j.1399-302x.1991.tb00475.x. [DOI] [PubMed] [Google Scholar]
  18. Maiden M. F., Tanner A., Moore W. E. Identification of Selenomonas species by whole-genomic DNA probes, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, biochemical tests and cellular fatty acid analysis. Oral Microbiol Immunol. 1992 Feb;7(1):7–13. doi: 10.1111/j.1399-302x.1992.tb00012.x. [DOI] [PubMed] [Google Scholar]
  19. Manafi M., Kneifel W., Bascomb S. Fluorogenic and chromogenic substrates used in bacterial diagnostics. Microbiol Rev. 1991 Sep;55(3):335–348. doi: 10.1128/mr.55.3.335-348.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Manafi M., Kneifel W. Rapid methods for differentiating gram-positive from gram-negative aerobic and facultative anaerobic bacteria. J Appl Bacteriol. 1990 Dec;69(6):822–827. doi: 10.1111/j.1365-2672.1990.tb01579.x. [DOI] [PubMed] [Google Scholar]
  21. Manafi M., Rotter M. L. A new plate medium for rapid presumptive identification and differentiation of Enterobacteriaceae. Int J Food Microbiol. 1991 Nov;14(2):127–134. doi: 10.1016/0168-1605(91)90099-b. [DOI] [PubMed] [Google Scholar]
  22. Millership S. E., Want S. V. Whole-cell protein electrophoresis for typing Mycobacterium tuberculosis. J Clin Microbiol. 1992 Nov;30(11):2784–2787. doi: 10.1128/jcm.30.11.2784-2787.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moncla B. J., Braham P. Detection of sialidase (neuraminidase) activity in Actinomyces species by using 2'-(4-methylumbelliferyl)alpha-D-N-acetylneuraminic acid in a filter paper spot test. J Clin Microbiol. 1989 Jan;27(1):182–184. doi: 10.1128/jcm.27.1.182-184.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moncla B. J., Braham P., Hillier S. L. Sialidase (neuraminidase) activity among gram-negative anaerobic and capnophilic bacteria. J Clin Microbiol. 1990 Mar;28(3):422–425. doi: 10.1128/jcm.28.3.422-425.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moncla B. J., Braham P., Rabe L. K., Hillier S. L. Rapid presumptive identification of black-pigmented gram-negative anaerobic bacteria by using 4-methylumbelliferone derivatives. J Clin Microbiol. 1991 Sep;29(9):1955–1958. doi: 10.1128/jcm.29.9.1955-1958.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moore L. V., Johnson J. L., Moore W. E. Descriptions of Prevotella tannerae sp. nov. and Prevotella enoeca sp. nov. from the human gingival crevice and emendation of the description of Prevotella zoogleoformans. Int J Syst Bacteriol. 1994 Oct;44(4):599–602. doi: 10.1099/00207713-44-4-599. [DOI] [PubMed] [Google Scholar]
  27. Moore L. V., Moore W. E. Oribaculum catoniae gen. nov., sp. nov.; Catonella morbi gen. nov., sp. nov.; Hallella seregens gen. nov., sp. nov.; Johnsonella ignava gen. nov., sp. nov.; and Dialister pneumosintes gen. nov., comb. nov., nom. rev., Anaerobic gram-negative bacilli from the human gingival crevice. Int J Syst Bacteriol. 1994 Apr;44(2):187–192. doi: 10.1099/00207713-44-2-187. [DOI] [PubMed] [Google Scholar]
  28. Moore W. E., Hash D. E., Holdeman L. V., Cato E. P. Polyacrylamide slab gel electrophoresis of soluble proteins for studies of bacterial floras. Appl Environ Microbiol. 1980 Apr;39(4):900–907. doi: 10.1128/aem.39.4.900-907.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Paster B. J., Dewhirst F. E., Olsen I., Fraser G. J. Phylogeny of Bacteroides, Prevotella, and Porphyromonas spp. and related bacteria. J Bacteriol. 1994 Feb;176(3):725–732. doi: 10.1128/jb.176.3.725-732.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pederson E. D., Miller J. W., Matheson S., Simonson L. G., Chadwick D. E., Covill P. J., Turner D. W., Lamberts B. L., Morton H. E. Trypsin-like activity levels of Treponema denticola and Porphyromonas gingivalis in adults with periodontitis. J Clin Periodontol. 1994 Sep;21(8):519–525. doi: 10.1111/j.1600-051x.1994.tb01167.x. [DOI] [PubMed] [Google Scholar]
  31. Pot B., Hertel C., Ludwig W., Descheemaeker P., Kersters K., Schleifer K. H. Identification and classification of Lactobacillus acidophilus, L. gasseri and L. johnsonii strains by SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization. J Gen Microbiol. 1993 Mar;139(3):513–517. doi: 10.1099/00221287-139-3-513. [DOI] [PubMed] [Google Scholar]
  32. Slayne M. A., Aldred M. J., Wade W. G. A rapid, semi-automated SDS-PAGE identification system for oral anaerobic bacteria. J Appl Bacteriol. 1990 Apr;68(4):391–395. doi: 10.1111/j.1365-2672.1990.tb02889.x. [DOI] [PubMed] [Google Scholar]
  33. Slots J. Detection of colonies of Bacteroides gingivalis by a rapid fluorescence assay for trypsin-like activity. Oral Microbiol Immunol. 1987 Sep;2(3):139–141. doi: 10.1111/j.1399-302x.1987.tb00279.x. [DOI] [PubMed] [Google Scholar]
  34. Slots J. Enzymatic characterization of some oral and nonoral gram-negative bacteria with the API ZYM system. J Clin Microbiol. 1981 Sep;14(3):288–294. doi: 10.1128/jcm.14.3.288-294.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tanner A. C. Characterization of Wolinella spp., Campylobacter concisus, Bacteroides gracilis, and Eikenella corrodens by polyacrylamide gel electrophoresis. J Clin Microbiol. 1986 Oct;24(4):562–565. doi: 10.1128/jcm.24.4.562-565.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tanner A. C., Strzempko M. N., Belsky C. A., McKinley G. A. API ZYM and API An-Ident reactions of fastidious oral gram-negative species. J Clin Microbiol. 1985 Sep;22(3):333–335. doi: 10.1128/jcm.22.3.333-335.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tanner A., Maiden M. F., Paster B. J., Dewhirst F. E. The impact of 16S ribosomal RNA-based phylogeny on the taxonomy of oral bacteria. Periodontol 2000. 1994 Jun;5:26–51. doi: 10.1111/j.1600-0757.1994.tb00017.x. [DOI] [PubMed] [Google Scholar]
  38. Vandamme P., Falsen E., Pot B., Hoste B., Kersters K., De Ley J. Identification of EF group 22 campylobacters from gastroenteritis cases as Campylobacter concisus. J Clin Microbiol. 1989 Aug;27(8):1775–1781. doi: 10.1128/jcm.27.8.1775-1781.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Whiley R. A., Fraser H. Y., Douglas C. W., Hardie J. M., Williams A. M., Collins M. D. Streptococcus parasanguis sp. nov., an atypical viridans Streptococcus from human clinical specimens. FEMS Microbiol Lett. 1990 Mar 1;56(1-2):115–121. doi: 10.1111/j.1574-6968.1990.tb04133.x. [DOI] [PubMed] [Google Scholar]
  40. Whiley R. A., Fraser H., Hardie J. M., Beighton D. Phenotypic differentiation of Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus strains within the "Streptococcus milleri group". J Clin Microbiol. 1990 Jul;28(7):1497–1501. doi: 10.1128/jcm.28.7.1497-1501.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Whiley R. A., Hardie J. M. DNA-DNA hybridization studies and phenotypic characteristics of strains within the 'Streptococcus milleri group'. J Gen Microbiol. 1989 Oct;135(10):2623–2633. doi: 10.1099/00221287-135-10-2623. [DOI] [PubMed] [Google Scholar]
  42. Willems A., Collins M. D. Phylogenetic placement of Dialister pneumosintes (formerly Bacteroides pneumosintes) within the Sporomusa subbranch of the Clostridium subphylum of the gram-positive bacteria. Int J Syst Bacteriol. 1995 Apr;45(2):403–405. doi: 10.1099/00207713-45-2-403. [DOI] [PubMed] [Google Scholar]
  43. Willems A., Collins M. D. Reclassification of Oribaculum catoniae (Moore and Moore 1994) as Porphyromonas catoniae comb. nov. and emendation of the genus Porphyromonas. Int J Syst Bacteriol. 1995 Jul;45(3):578–581. doi: 10.1099/00207713-45-3-578. [DOI] [PubMed] [Google Scholar]
  44. van Winkelhoff A. J., Clement M., de Graaff J. Rapid characterization of oral and nonoral pigmented Bacteroides species with the ATB Anaerobes ID system. J Clin Microbiol. 1988 May;26(5):1063–1065. doi: 10.1128/jcm.26.5.1063-1065.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. van Winkelhoff A. J., van Steenbergen T. J., Kippuw N., de Graaff J. Enzymatic characterization of oral and non-oral black-pigmented Bacteroides species. Antonie Van Leeuwenhoek. 1986;52(2):163–171. doi: 10.1007/BF00429320. [DOI] [PubMed] [Google Scholar]

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