Laboratory Maintenance of Fusobacteria

Yiping W. Han1

1 Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 13A.1
DOI:  10.1002/9780471729259.mc13a01s00
Online Posting Date:  January, 2006
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This unit describes routine laboratory handling of fusobacteria. Different media that can be used to grow or enrich Fusobacterium nucleatum and other species of this genus are described. The growth and stock conditions as well as the susceptibility of F. nucleatum to oxygen in a pure culture are also discussed.

Keywords: fusobacteria; anaerobe; periodontal disease; preterm birth; adherence; coaggregation; dental plaque; oxygen sensitivity

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growth of Pure Fusobacterial Cultures
  • Basic Protocol 2: Enrichment of Fusobacteria from a Mixed Population
  • Basic Protocol 3: Storage of Fusobacteria
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Growth of Pure Fusobacterial Cultures

  • Columbia blood agar plates (see recipe)
  • Columbia broth (see recipe)
  • Fusobacterium cultures (clinical isolates or ATCC strains)
  • Anaerobic incubator: 37°C anaerobic chamber, desiccant, palladium catalyst pellets, nitrogen gas tank, gas mix (90% N 2, 5% CO 2, and 5% H 2) tank—e.g., Coy or Forma or
  • Anaerobic jar system (e.g., 2.5‐liter Pack‐Rectangular Jar, AnaeroPack System; Mitsubishi Gas Chemical Co.; http://www.mgc‐
  • Sachets for anaerobic jar system (Mitsubishi Gas Chemical Co.)
  • Anaerobic indicator strips (Becton Dickinson)

Basic Protocol 2: Enrichment of Fusobacteria from a Mixed Population

  • Reduced transport fluid (RTF; see recipe)
  • FEA or CVE plates (see reciperecipes)
  • Dental plaque (provided by a dental care professional)
  • Anaerobic incubator: 37°C anaerobic chamber, desiccant, palladium catalyst pellets, nitrogen gas tank, gas mix (90% N 2, 5% CO 2, and 5% H 2) tank—e.g., Coy or Forma or
  • Anaerobic jar system (e.g., 2.5‐liter Pack‐Rectangular Jar, AnaeroPack System; Mitsubishi Gas Chemical Co.; http://www.mgc‐
  • Sachets for anaerobic jar system (Mitsubishi Gas Chemical Co.)
  • Anaerobic indicator strips (Becton Dickinson)

Basic Protocol 3: Storage of Fusobacteria

  • Fusobacterium cultures
  • Columbia broth (see recipe)
  • Glycerol (autoclaved)
  • Anaerobic chamber or anaerobic jar (see protocol 1)
  • 1.5‐ml microcentrifuge tubes, sterile
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Literature Cited

Literature Cited
   Andersen, R.N., Ganeshkumar, N., and Kolenbrander, P.E. 1998. Helicobacter pylori adheres selectively to Fusobacterium spp. Oral Microbiol. Immunol. 13:51‐54.
   Babu, J.P., Dean, J.W., and Pabst, M.J. 1995. Attachment of Fusobacterium nucleatum to fibronectin immobilized on gingival epithelial cells or glass coverslips. J. Periodontol. 66:285‐290.
   Bennett, K.W. and Eley, A. 1993. Fusobacteria: New taxonomy and related diseases. J. Med. Microbiol. 39:246‐254.
   Bradshaw, D.J., Marsh, P.D., Watson, G.K., and Allison, C. 1998. Role of Fusobacterium nucleatum and coaggregation in anaerobe survival in planktonic and biofilm oral microbial communities during aeration. Infect. Immun. 66:4729‐4732.
   Darveau, R.P., Belton, C.M., Reife, R.A., and Lamont, R.J. 1998. Local chemokine paralysis: A novel pathogenic mechanism for Porphyromonas gingivalis. Infect. Immun. 66:1660‐1665.
   Dzink, J.L., Sheenan, M.T., and Socransky, S.S. 1990. Proposal of three subspecies of Fusobacterium nucleatum Knorr 1922: Fusobacterium nucleatum subsp. nucleatum subsp. nov., comb. nov.; Fusobacterium nucleatum subsp. polymorphum subsp. nov., nom. rev., comb. nov.; and Fusobacterium nucleatum subsp. vincentii subsp. nov., nom. rev., comb. nov. Int. J. Syst. Bacteriol. 40:74‐78.
   Garrity, G.M. (ed.) 2001. Bergey's Manual of Systematic Bacteriology, Vol. 1, 2nd ed. Springer‐Verlag, New York.
   George, K.S. and Falkler, W.A. Jr. 1992. Coaggregation studies of the Eubacterium species. Oral Microbiol. Immunol. 7:285‐290.
   Grimaudo, N.J. and Nesbitt, W.E. 1997. Coaggregation of Candida albicans with oral Fusobacterium species. Oral Microbiol. Immunol. 12:168‐173.
   Guo, M., Han, Y.W., Sharma, A., and De Nardin, E. 2000. Identification and characterization of human immunoglobulin G Fc receptors of Fusobacterium nucleatum. Oral Microbiol. Immunol. 15:119‐123.
   Han, Y.W., Redline, R.W., Li, M., Yin, L., Hill, G.B., and McCormick, T.S. 2004. Fusobacterium nucleatum induces premature and term stillbirths in pregnant mice: Implication of oral bacteria in preterm birth. Infect. Immun. 72:2272‐2279.
   Han, Y.W., Shi, W., Huang, G.T., Kinder Haake, S., Park, N.H., Kuramitsu, H., and Genco, R.J. 2000. Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells. Infect. Immun. 68:3140‐3146.
   Hill, G.B. 1993. Investigating the source of amniotic fluid isolates of fusobacteria. Clin. Infect. Dis. 16:S423‐424.
   Hill, G.B. 1998. Preterm birth: Associations with genital and possibly oral microflora. Ann Periodontol. 3:222‐232.
   Jabra‐Rizk, M.A., Falkler, W.A., Jr., Merz, W.G., Kelley, J.I., Baqui, A.A., and Meiller, T.F. 1999. Coaggregation of Candida dubliniensis with Fusobacterium nucleatum. J. Clin. Microbiol. 37:1464‐1468.
   Jewett, A., Hume, W.R., Le, H., Huynh, T.N., Han, Y.W., Cheng, G., and Shi, W. 2000. Induction of apoptotic cell death in peripheral blood mononuclear and polymorphonuclear cells by an oral bacterium, Fusobacterium nucleatum. Infect. Immun. 68:1893‐1898.
   Kapatral, V., Anderson, I., Ivanova, N., Reznik, G., Los, T., Lykidis, A., Bhattacharyya, A., Bartman, A., Gardner, W., Grechkin, G., Zhu, L., Vasieva, O., Chu, L., Kogan, Y., Chaga, O., Goltsman, E., Bernal, A., Larsen, N., D'Souza, M., Walunas, T., Pusch, G., Haselkorn, R., Fonstein, M., Kyrpides, N., and Overbeek, R. 2002. Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586. J. Bacteriol. 184:2005‐2018.
   Kapatral, V., Ivanova, N., Anderson, I., Reznik, G., Bhattacharyya, A., Gardner, W.L., Mikhailova, N., Lapidus, A., Larsen, N., D'Souza, M., Walunas, T., Haselkorn, R., Overbeek, R., and Kyrpides, N. 2003. Genome analysis of F. nucleatum sub spp vincentii and its comparison with the genome of F. nucleatum ATCC 25586. Genome Res. 13:1180‐1189.
   Kaufman, J. and DiRienzo, J.M. 1989. Isolation of a corncob (coaggregation) receptor polypeptide from Fusobacterium nucleatum. Infect. Immun. 57:331‐337.
   Kinder, S.A. and Holt, S.C. 1993. Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22. J. Bacteriol. 175:840‐850.
   Kolenbrander, P.E. and Andersen, R.N. 1989. Inhibition of coaggregation between Fusobacterium nucleatum and Porphyromonas (Bacteroides) gingivalis by lactose and related sugars. Infect. Immun. 57:3204‐3209.
   Kolenbrander, P.E., Andersen, R.N., and Moore, L.V. 1989. Coaggregation of Fusobacterium nucleatum, Selenomonas flueggei, Selenomonas infelix, Selenomonas noxia, and Selenomonas sputigena with strains from 11 genera of oral bacteria. Infect. Immun. 57:3194‐3203.
   Kolenbrander, P.E., Parrish, K.D., Andersen, R.N., and Greenberg, E.P. 1995. Intergeneric coaggregation of oral Treponema spp. with Fusobacterium spp. and intrageneric coaggregation among Fusobacterium spp. Infect. Immun. 63:4584‐4588.
   Krisanaprakornkit, S., Kimball, J.R., Weinberg, A., Darveau, R.P., Bainbridge, B.W., and Dale, B.A. 2000. Inducible expression of human beta‐defensin 2 by Fusobacterium nucleatum in oral epithelial cells: Multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier. Infect. Immun. 68:2907‐2915.
   Lawson, P.A., Gharbia, S.E., Shah, H.N., Clark, D.R., and Collins, M.D. 1991. Intrageneric relationships of members of the genus Fusobacterium as determined by reverse transcriptase sequencing of small‐subunit rRNA. Int. J. Syst. Bacteriol. 41:347‐354.
   Loesche, W.J., Hockett, R.N., and Syed, S.A. 1972. The predominant cultivable flora of tooth surface plaque removed from institutionalized subjects. Arch. Oral Biol. 17:1311‐1325.
   Mangan, D.F., Novak, M.J., Vora, S.A., Mourad, J., and Kriger, P.S. 1989. Lectinlike interactions of Fusobacterium nucleatum with human neutrophils. Infect. Immun. 57:3601‐3611.
   Metzger, Z., Featherstone, L.G., Ambrose, W.W., Trope, M., and Arnold, R.R. 2001. Kinetics of coaggregation of Porphyromonas gingivalis with Fusobacterium nucleatum using an automated microtiter plate assay. Oral Microbiol. Immunol. 16:163‐169.
   Moore, W.E. and Moore, L.V. 1994. The bacteria of periodontal diseases. Periodontol 2000 5:66‐77.
   Morgenstein, A.A., Citron, D.M., and Finegold, S.M. 1981. New medium selective for Fusobacterium species and differential for Fusobacterium necrophorum. J. Clin. Microbiol. 13:666‐669.
   Murray, P.A., Kern, D.G., and Winkler, J.R. 1988. Identification of a galactose‐binding lectin on Fusobacterium nucleatum FN‐2. Infect. Immun. 56:1314‐1319.
   Ozaki, M., Miyake, Y., Shirakawa, M., Takemoto, T., Okamoto, H., and Suginaka, H. 1990. Binding specificity of Fusobacterium nucleatum to human erythrocytes, polymorphonuclear leukocytes, fibroblasts, and HeLa cells. J. Periodont. Res. 25:129‐134.
   Rosen, G., Nisimov, I., Helcer, M., and Sela, M.N. 2003. Actinobacillus actinomycetemcomitans serotype b lipopolysaccharide mediates coaggregation with Fusobacterium nucleatum. Infect. Immun. 71:3652‐3656.
   Shaniztki, B., Ganeshkumar, N., and Weiss, E.I. 1998. Characterization of a novel N‐acetylneuraminic acid‐specific Fusobacterium nucleatum PK1594 adhesin. Oral Microbiol. Immunol. 13:47‐50.
   Shaniztki, B., Hurwitz, D., Smorodinsky, N., Ganeshkumar, N., and Weiss, E.I. 1997. Identification of a Fusobacterium nucleatum PK1594 galactose‐binding adhesin which mediates coaggregation with periopathogenic bacteria and hemagglutination. Infect. Immun. 65:5231‐5237.
   Shenker, B.J. and Datar, S. 1995. Fusobacterium nucleatum inhibits human T‐cell activation by arresting cells in the mid‐G1 phase of the cell cycle. Infect. Immun. 63:4830‐4836.
   Takemoto, T., Hino, T., Yoshida, M., Nakanishi, K., Shirakawa, M., and Okamoto, H. 1995. Characteristics of multimodal co‐aggregation between Fusobacterium nucleatum and streptococci. J. Periodont. Res. 30:252‐257.
   Tuttle, R.S. and Mangan, D.F. 1990. Interaction of Fusobacterium nucleatum 191 with human peripheral blood lymphocytes. J. Periodont. Res. 25:64‐371.
   Tuttle, R.S., Strubel, N.A., Mourad, J., and Mangan, D.F. 1992. A non‐lectin‐like mechanism by which Fusobacterium nucleatum 10953 adheres to and activates human lymphocytes. Oral Microbiol. Immunol. 7:78‐83.
   Walker, C.B., Ratliff, D., Muller, D., Mandell, R., and Socransky, S.S. 1979. Medium for selective isolation of Fusobacterium nucleatum from human periodontal pockets. J. Clin. Microbiol. 10:844‐849.
   Weiss, E.I., Shaniztki, B., Dotan, M., Ganeshkumar, N., Kolenbrander, P.E., and Metzger, Z. 2000. Attachment of Fusobacterium nucleatum PK1594 to mammalian cells and its coaggregation with periodontopathogenic bacteria are mediated by the same galactose‐binding adhesin. Oral Microbiol. Immunol. 15:371‐377.
   Winkler, J.R., John, S.R., Kramer, R.H., Hoover, C.I., and Murray, P. A. 1987. Attachment of oral bacteria to a basement‐membrane‐like matrix and to purified matrix proteins. Infect. Immun. 55:2721‐2726.
   Xie, H., Gibbons, R.J., and Hay, D.I. 1991. Adhesive properties of strains of Fusobacterium nucleatum of the subspecies nucleatum, vincentii and polymorphum. Oral Microbiol. Immunol. 6:257‐263.
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