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Stability of bacterial DNA in relation to microbial detection in teeth
Umeå University, Faculty of Medicine, Department of Odontology.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The fate of DNA from dead cells is an important issue when interpreting results from root canal infections analysed by the PCR technique. DNA from dead bacterial cells is known to be detectable long time after cell death and its stability is dependent on many different factors. This work investigated factors found in the root canal that could affect the recovery of microbial DNA. In an ex vivo experiment, DNA from non-viable gram-positive Enterococcus faecalis was inoculated in instrumented root canals and recovery of DNA was assessed by PCR over a two-year period. DNA was still recoverable two years after cell death in 21/25 teeth. The fate of DNA from the gram-negative bacteria Fusobacterium nucleatum and the gram-positive Peptostreptococcus anaerobius was assessed in vitro. DNA from dead F. nucleatum and P. anaerobius could be detected by PCR six months post cell death even though it was clear that the DNA was released from the cells due to lost of cell wall integrity during the experimental period. The decomposition rate of extracellular DNA was compared to cell-bound and it was evident that DNA still located inside the bacterium was much less prone to decay than extracellular DNA.

Free (extracellular) DNA is very prone to decay in a naked form. Binding to minerals is known to protect DNA from degradation. The fate of extracellular DNA was assessed after binding to ceramic hydroxyapatite and dentine. The data showed that free DNA, bound to these materials, was protected from spontaneous decay and from enzymatic decomposition by nucleases.

The main conclusions from this thesis were: i) DNA from dead bacteria can be detected by PCR years after cell death ex vivo and in vitro. ii) Cell-bound DNA is less prone to decomposition than extracellular DNA. iii) DNA is released from the bacterium some time after cell death. iv) Extracellular DNA bound to hydroxyapatite or dentine is protected from spontaneous decomposition and enzymatic degradation.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2013. , 35 p.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 127
Keyword [en]
Cell-bound DNA, cell-death, dentine, DNA binding affinity, DNA decomposition, DNA preservation, extracellular DNA, hydroxyapatite, PCR, polymerase chain reaction
National Category
Dentistry
Research subject
Odontology
Identifiers
URN: urn:nbn:se:umu:diva-82735ISBN: 978-91-7459-743-1 (print)OAI: oai:DiVA.org:umu-82735DiVA: diva2:662554
Public defence
2013-12-05, Sal B, tandläkarhögskolan 9 tr, Norrlands universitetssjukhus, Umeå, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2013-11-08 Created: 2013-11-07 Last updated: 2013-11-08Bibliographically approved
List of papers
1. Persistence of dead-cell bacterial DNA in ex vivo root canals and influence of nucleases on DNA decay in vitro
Open this publication in new window or tab >>Persistence of dead-cell bacterial DNA in ex vivo root canals and influence of nucleases on DNA decay in vitro
Show others...
2010 (English)In: Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics, ISSN 1079-2104, E-ISSN 1528-395X, Vol. 110, no 6, 789-794 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
polymerase-chain-reaction; human dental-pulp; endodontic infections; porphyromonas-gingivalis; identification; serum; microorganisms; microflora; assay; pcr
National Category
Dentistry
Identifiers
urn:nbn:se:umu:diva-40692 (URN)10.1016/j.tripleo.2010.07.010 (DOI)000284625500029 ()21112536 (PubMedID)
Available from: 2011-03-05 Created: 2011-03-05 Last updated: 2017-12-11Bibliographically approved
2. Preservation of Fusobacterium nucleatum and Peptostreptococcus anaerobius DNA after loss of cell viability
Open this publication in new window or tab >>Preservation of Fusobacterium nucleatum and Peptostreptococcus anaerobius DNA after loss of cell viability
2015 (English)In: International Endodontic Journal, ISSN 0143-2885, E-ISSN 1365-2591, Vol. 48, no 1, 37-45 p.Article in journal (Other academic) Published
Abstract [en]

Aim: To investigate whether DNA from two obligate anaerobes, Fusobacterium nucleatum and Peptostreptococcus anaerobius, is recoverable after loss of cell viability induced by air exposure. Methodology: 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. Conclusion: 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.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2015
Keyword
DNA post-cell death, Fusobacterium nucleatum, Peptostreptococcus anaerobius, PMA-PCR, quantitative PCR, scanning electron microscopy
National Category
Dentistry
Identifiers
urn:nbn:se:umu:diva-82734 (URN)10.1111/iej.12273 (DOI)000346034300005 ()24611977 (PubMedID)
Available from: 2013-11-07 Created: 2013-11-07 Last updated: 2017-12-06Bibliographically approved
3. DNA Binding to hydroxyapatite: a potential mechanism for preservation of microbial DNA
Open this publication in new window or tab >>DNA Binding to hydroxyapatite: a potential mechanism for preservation of microbial DNA
2013 (English)In: Journal of Endodontics, ISSN 0099-2399, E-ISSN 1878-3554, Vol. 39, no 2, 211-216 p.Article in journal (Refereed) Published
Abstract [en]

Introduction: Molecular methods are increasingly being deployed for analysis of the microbial flora in the root canal. Such methods are based on the assumption that recovered DNA is associated with the active endodontic infection, yet paleomicrobiology research is based on the recovery of ancient DNA from centuriesold tooth and bone samples, which points to considerable longevity of the DNA molecule in these tissues. The main component of dentin and bone is the mineral hydroxyapatite. This study assessed DNA binding to hydroxyapatite and whether thiS binding affinity stabilizes the DNA molecule in various media.

Methods: DNA was extracted from Fusobacterium nucleatum and added to ceramic hydroxyapatite for 90 minutes. The DNA-bound hydroxyapatite was incubated in different media (ie, water, sera, and DNase I) for up to 3 months. At predetermined intervals, the recovery of detectable DNA was assessed by releasing the DNA from the hydroxyapatite using EDTA and evaluating the presence of DNA by gel electrophoresis and polymerase chain reaction (PCR) amplification.

Results: When incubated with hydroxyapatite, nonamplified DNA was detectable after 3 months in water, sera, and DNase I. In contrast, DNA incubated in the same media (without hydroxyapatite) decomposed to levels below the detection level of PCR within 3 weeks, with the exception of DNA in sera in which PCR revealed a weak positive amplification product.

Conclusions: These results confirm a specific binding affinity of hydroxyapatite for DNA. Hydroxyapatite-bound DNA is more resistant to decay and less susceptible to degradation by serum and nucleases, which may account for the long-term persistence of DNA in bone and tooth.

Place, publisher, year, edition, pages
Elsevier, 2013
Keyword
Ancient DNA, DNA binding affinity, DNA decomposition, DNA preservation, hydroxyapatite, polymerase chain reaction
National Category
Dentistry
Identifiers
urn:nbn:se:umu:diva-67045 (URN)10.1016/j.joen.2012.09.013 (DOI)000314482500013 ()
Available from: 2013-03-24 Created: 2013-03-12 Last updated: 2017-12-06Bibliographically approved
4. Preservation of bacterial DNA by binding to dentin
Open this publication in new window or tab >>Preservation of bacterial DNA by binding to dentin
(English)Manuscript (preprint) (Other academic)
National Category
Dentistry
Identifiers
urn:nbn:se:umu:diva-82733 (URN)
Available from: 2013-11-07 Created: 2013-11-07 Last updated: 2013-11-08Bibliographically approved

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