title: Biofilm formation enhances the oxygen tolerance and invasiveness of Fusobacterium nucleatum in an oral mucosa culture model.
abstract: BACKGROUND
  The present study evaluates the survival capability of Fusobacterium nucleatum strains in an aerobic environment and compares the invasive capability of F. nucleatum in biofilm and planktonic forms in an organotypic cell culture (OCC) model.
  
  
  METHODS
  Biofilms of F. nucleatum American Type Culture Collection (ATCC) 25586 or Anaerobe Helsinki Negative (AHN) 9508 were produced by culturing on semipermeable membranes on brucella agar plates. The oxygen tolerance of the F. nucleatum strains was examined by incubating 3-day-old anaerobically grown biofilms in an aerobic environment (CO(2) [5% in air] incubator) for an additional 48 hours. The OCC model was constructed by seeding keratinocytes on a fibroblast-containing collagen gel. In invasion assays, a 3-day-old anaerobically grown biofilm (and planktonic bacteria in solution as the control) was placed upside down on the top of OCC and incubated under 5% CO(2) for 24 hours. Invasion of the bacteria and morphologic changes in OCC were assessed using hematoxylin and eosin, Ki-67, and periodic acid-Schiff stainings.
  
  
  RESULTS
  In biofilms, both F. nucleatum strains continuously increased their cell numbers in an aerobic environment for 48 hours. After incubating the bacterial biofilm in contact with the OCC model, F. nucleatum AHN 9508 was able to pass through the epithelial/basement membrane barrier and invade the collagen matrix. The invasiveness of biofilm F. nucleatum ATCC 25586 was limited to the epithelium. Cytotoxic effects and invasiveness of F. nucleatum on the OCC were much stronger when the bacteria were in biofilms than in the planktonic form.
  
  
  CONCLUSIONS
  Biofilm formation regulates the survival and invasiveness of F. nucleatum in an aerobic environment.
authors.affiliation: Institute of Dentistry and Department of Oral and Maxillofacial Surgery, University of Helsinki, Helsinki University Central Hospital, Helsinki, Finland. ulvi.gursoy@utu.fi
authors.forename: Ulvi Kahraman
authors.initials: UK
authors.lastname: Gursoy
authors.forename: Marja
authors.initials: M
authors.lastname: Pöllänen
authors.forename: Eija
authors.initials: E
authors.lastname: Könönen
authors.forename: Veli-Jukka
authors.initials: VJ
authors.lastname: Uitto
mesh_terms: D000332:Aerobiosis / Q000502:physiology; D001422:Bacterial Adhesion / Q000502:physiology; D018407:Bacterial Physiological Phenomena; D018441:Biofilms / Q000254:growth & development*; D049109:Cell Proliferation; D002470:Cell Survival; D003094:Collagen; D015169:Colony Count, Microbial; D004396:Coloring Agents; D004847:Epithelial Cells / Q000382:microbiology; D005347:Fibroblasts / Q000382:microbiology; D016967:Fusobacterium nucleatum / Q000502:physiology*; D006801:Humans; D015603:Keratinocytes / Q000382:microbiology; D019394:Ki-67 Antigen / Q000032:analysis; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D050296:Microbial Viability; D009061:Mouth Mucosa / Q000166:cytology / Q000382:microbiology*; D010100:Oxygen; D046509:Tissue Culture Techniques
chemical_list: D004396:Coloring Agents; D019394:Ki-67 Antigen; D003094:Collagen; D007770:L-Lactate Dehydrogenase; D010100:Oxygen