Persistence of dead-cell bacterial DNA in ex vivo root canals and influence of nucleases on DNA decay in vitro.
OBJECTIVE
The fate of DNA from bacteria that do not survive in the root canal is uncertain, yet DNA longevity may confound recovery of authentic etiologic participants in the disease process. This study assessed the recovery of PCR-detectable DNA in ex vivo human root canals and some environmental factors on the decay of microbial DNA.


METHODS
Heat-killed Enterococcus faecalis cells were inoculated into instrumented human root canals ex vivo, and samples were taken at intervals over 2 years and analyzed by polymerase chain reaction. In an in vitro assay, heat-killed E. faecalis cells and extracted E. faecalis DNA were inoculated into various media, DNase, and culture of a DNase-producing species, Prevotella intermedia. Recovery of DNA was assessed by gel electrophoresis.


RESULTS
In ex vivo human teeth, amplifiable DNA was recovered after 1 and 2 years (in 14/15 and 21/25 teeth, respectively). In vitro experiments showed that extracted DNA incubated in different media (water, 10%-50% sera, and DNase) progressively decomposed to levels below the detection limit. In corresponding assays, cell-bound DNA was more resistant to decay.


CONCLUSIONS
Amplifiable DNA is preserved after cell death, but the critical determinant is the form of DNA. Free DNA undergoes spontaneous and enzymatic decomposition, whereas cell-bound E. faecalis DNA persists for long periods.
Department of Odontology/Endodontics, Faculty of Medicine, Umeå University, Umeå, Sweden. malin.brundin@odont.umu.se
Malin
M
Brundin
David
D
Figdor
Chrissie
C
Roth
John K
JK
Davies
Göran
G
Sundqvist
Ulf
U
Sjögren
D002473:Cell Wall / Q000648:ultrastructure; D003470:Culture Media; D004249:DNA Damage; D004269:DNA, Bacterial / Q000032:analysis* / Q000187:drug effects; D003786:Dental Pulp Cavity / Q000382:microbiology*; D003850:Deoxyribonuclease I / Q000494:pharmacology*; D004586:Electrophoresis; D013293:Enterococcus faecalis / Q000201:enzymology / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D050296:Microbial Viability; D016133:Polymerase Chain Reaction; D018713:Prevotella intermedia / Q000201:enzymology; D012329:RNA, Bacterial / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012387:Root Canal Filling Materials / Q000627:therapeutic use; D018915:Root Canal Preparation / Q000379:methods; D013997:Time Factors; D015035:Zinc Oxide-Eugenol Cement / Q000627:therapeutic use
D003470:Culture Media; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012387:Root Canal Filling Materials; D015035:Zinc Oxide-Eugenol Cement; D003850:Deoxyribonuclease I