title: Preservation of Fusobacterium nucleatum and Peptostreptococcus anaerobius DNA after loss of cell viability.
abstract: OBJECTIVE
  To investigate whether DNA from two obligate anaerobes, Fusobacterium nucleatum and Peptostreptococcus anaerobius, is recoverable after loss of cell viability induced by air exposure.
  
  
  METHODS
  Harvested cultures of F. nucleatum and P. anaerobius were killed by exposure to air and stored in phosphate-buffered saline. Dead cells were incubated aerobically for up to 6 months. Every month, the presence of detectable DNA in the cell pellet and supernatant was assessed by conventional and quantitative PCR. Cell staining techniques were used to characterize the cell wall permeability of air-killed cells. Scanning electron microscopy was used to examine viable, freshly killed and stored cells.
  
  
  RESULTS
  With conventional PCR, amplifiable DNA was detectable over 6 months in all samples. Quantitative PCR showed a progressive fall in DNA concentration in nonviable cell pellets and a concomitant rise in DNA concentration in the supernatant. DNA staining showed that some air-killed cells retained an intact cell wall. After storage, SEM of both air-killed species revealed shrivelling of the cells, but some cells of P. anaerobius retained their initial form.
  
  
  CONCLUSIONS
  Amplifiable DNA from F. nucleatum and P. anaerobius was detectable 6 months after loss of viability. Air-killed anaerobes initially retained their cell form, but cells gradually shriveled over time. The morphological changes were more pronounced with the gram-negative F. nucleatum than the gram-positive P. anaerobius. Over 6 months, there was a gradual increase in cell wall permeability with progressive leakage of DNA. Bacterial DNA was recoverable long after loss of cell viability.
authors.affiliation: Department of Odontology/Endodontics, Faculty of Medicine, Umeå University, Umeå, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Brundin
authors.forename: D
authors.initials: D
authors.lastname: Figdor
authors.forename: G
authors.initials: G
authors.lastname: Sundqvist
authors.forename: U
authors.initials: U
authors.lastname: Sjögren
mesh_terms: D000388:Air; D001386:Azides / Q000494:pharmacology; D004269:DNA, Bacterial / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D016967:Fusobacterium nucleatum / Q000302:isolation & purification*; D006801:Humans; D008855:Microscopy, Electron, Scanning; D009336:Necrosis; D010462:Peptostreptococcus / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011419:Propidium / Q000031:analogs & derivatives / Q000494:pharmacology; D013194:Staining and Labeling
chemical_list: D001386:Azides; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium
keywords: DNA post-cell death; Fusobacterium nucleatum; PMA-PCR; Peptostreptococcus anaerobius; quantitative PCR; scanning electron microscopy