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  • 1.
    Brundin, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Stability of bacterial DNA in relation to microbial detection in teeth2013Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    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.

  • 2.
    Brundin, Malin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Figdor, David
    Johansson, Anders
    Sjögren, Ulf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Preservation of bacterial DNA by binding to dentinManuskript (preprint) (Övrigt vetenskapligt)
  • 3.
    Brundin, Malin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Figdor, David
    Department of Microbiology, Monash University, Melbourne, Australia.
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Sjögren, Ulf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Preservation of bacterial dna by human dentin2014Ingår i: Journal of Endodontics, ISSN 0099-2399, E-ISSN 1878-3554, Vol. 40, nr 2, s. 241-245Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    INTRODUCTION: The capacity of dentin and collagen to bind DNA and protect against spontaneous and nuclease-induced degradation was evaluated individually and by the incubation of DNA with nuclease-producing bacteria in a mixed culture.

    METHODS: Extracted Fusobacterium nucleatum DNA was incubated with dentin shavings or collagen for 90 minutes. The DNA-bound substrates were incubated in different media (water, sera, and DNase I) for up to 3 months. Amplifiable DNA was released from dentin using EDTA,or from collagen using proteinase K, and evaluated by polymerase chain reaction (PCR). The stability of dentin-bound DNA was also assessed in a mixed culture (Parvimonas micra and Pseudoramibacter alactolyticus) containing a DNase-producing species, Prevotella intermedia. Samples were analyzed for amplifiable DNA.

    RESULTS: In water, dentin-bound DNA was recoverable by PCR at 3 months compared with no detectable DNA after 4 weeks in controls (no dentin). DNA bound to collagen was detectable by PCR after 3 months of incubation in water. In 10% human sera, amplifiable DNA was detectable at 3 months when dentin bound and in controls (no dentin). In mixed bacterial culture, dentin-bound DNA was recoverable throughout the experimental period (3 months), compared with no recoverable F. nucleatum DNA within 24 hours in controls (no dentin).

    CONCLUSIONS: There is a strong binding affinity between DNA and dentin, and between DNA and serum proteins or collagen. These substrates preserve DNA against natural decomposition and protect DNA from nuclease activity, factors that may confound molecular analysis of the endodontic microbiota yet favor paleomicrobiological studies of ancient DNA.

  • 4.
    Brundin, Malin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Endodonti.
    Figdor, David
    Roth, Chrissie
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Endodonti.
    Davies, John K
    Sundqvist, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Endodonti.
    Sjögren, Ulf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Endodonti.
    Persistence of dead-cell bacterial DNA in ex vivo root canals and influence of nucleases on DNA decay in vitro2010Ingår i: Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics, ISSN 1079-2104, E-ISSN 1528-395X, Vol. 110, nr 6, s. 789-794Artikel i tidskrift (Refereegranskat)
    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.

  • 5.
    Brundin, Malin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Endodonti.
    Figdor, David
    Sundqvist, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Endodonti.
    Sjögren, Ulf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Endodonti.
    DNA Binding to hydroxyapatite: a potential mechanism for preservation of microbial DNA2013Ingår i: Journal of Endodontics, ISSN 0099-2399, E-ISSN 1878-3554, Vol. 39, nr 2, s. 211-216Artikel i tidskrift (Refereegranskat)
    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.

  • 6.
    Brundin, Malin
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Figdor, David
    Department of Microbiology, Monash University, Melbourne, Victoria, Australia.
    Sundqvist, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Sjögren, Ulf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Preservation of Fusobacterium nucleatum and Peptostreptococcus anaerobius DNA after loss of cell viability2015Ingår i: International Endodontic Journal, ISSN 0143-2885, E-ISSN 1365-2591, Vol. 48, nr 1, s. 37-45Artikel i tidskrift (Övrigt vetenskapligt)
    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.

  • 7.
    Brundin, Malin
    et al.
    Umeå universitet, Medicinsk fakultet, Odontologi. Umeå universitet, Medicinsk fakultet, Odontologi, Endodonti.
    Figdor, David
    Sundqvist, Göran
    Sjögren, Ulf
    Umeå universitet, Medicinsk fakultet, Odontologi. Umeå universitet, Medicinsk fakultet, Odontologi, Endodonti.
    Starvation response and growth in serum of Fusobacterium nucleatum, Peptostreptococcus anaerobius, Prevotella intermedia, and Pseudoramibacter alactolyticus.2009Ingår i: Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics, ISSN 1079-2104, E-ISSN 1528-395X, Vol. 108, nr 1, s. 129-34Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The microbiota inhabiting the untreated root canal differ markedly from those found in post-treatment disease, yet there is limited information on the microbial characteristics distinguishing the different infections. We hypothesized that starvation survival is a key microbial property in species selection. This study analyzed starvation-survival behavior over 60 days of species representative of the untreated root canal infection: Fusobacterium nucleatum, Peptostreptococcus anaerobius, Prevotella intermedia and Pseudoramibacter alactolyticus. All species did not survive 1 day in water. In 1% serum, the 4 species could not survive beyond 2-3 weeks. They required a high initial cell density and >or=10% serum to survive the observation period. The results highlight a poor starvation-survival capacity of these 4 species compared with species prevalent in post-treatment infection, which are well equipped to endure starvation and survive in low numbers on minimal serum. These findings point to starvation-survival capacity as a selection factor for microbial participation in post-treatment disease.

  • 8.
    Claesson, Rolf
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Sjögren, Ulf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Esberg, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Brundin, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Granlund, Margareta
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Actinomyces radicidentis and Actinomyces haliotis, coccoid Actinomyces species isolated from the human oral cavity2017Ingår i: Anaerobe, ISSN 1075-9964, E-ISSN 1095-8274, Vol. 48, s. 19-26Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There are few reports on the bacterial species Actinomyces radicidentis in the literature. In this study, putative A. radicidentis isolates were collected from 16 root canal samples from 601 examined patients. The isolates were examined by biochemical tests, 16S rRNA gene sequencing, Arbitrarily-primed (AP-) PCR, antibiotic susceptibility testing, and MALDI-TOF analyses. In parallel, two A. radicidentis reference strains and two putative A. radicidentis isolates from United Kingdom were tested. Sixteen of the 18 isolates were confirmed as A. radicidentis. The remaining two isolates, both of which were isolated from root canals (one from Sweden and the other from the UK), but were identified as Actinomyces haliotis by sequencing ∼ 1300 base pairs of the 16S rRNA-gene. This isolates had a divergent, but between them similar, AP-PCR pattern, and a common distribution of sequence signatures in the 16S rRNA gene, but were not identified by MALDI-TOF. A. haliotis is a close relative to A. radicidentis, hitherto only been described from a sea-snail. The identity of A. haliotis was confirmed by a phylogenetic tree based on 16S rRNA gene sequences with species specific sequences included, and by additional biochemical tests. The examined bacteria exhibited similar antibiotic susceptibility patterns when tested for 10 separate antibiotic classes with E-tests (bioMérieux). The MIC90 for β-lactams (benzylpenicillin and cefuroxime) and vancomycin was 0.5 mg/L, for colistin and ciprofloxacin 8 mg/mL and for the other antibiotic classes ≤ 25 mg/mL The isolation of A. haliotis from infected dental root canals cast doubt on the accepted opinion that all Actinomyces infections have an endogenous source.

  • 9. Figdor, David
    et al.
    Brundin, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Contamination Controls for Analysis of Root Canal Samples by Molecular Methods: An Overlooked and Unsolved Problem2016Ingår i: Journal of Endodontics, ISSN 0099-2399, E-ISSN 1878-3554, Vol. 42, nr 7, s. 1003-1008Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Introduction: It has been almost 20 years since molecular methods were first described for the analysis of root canal microbial flora. Contamination control samples are essential to establish DNA decontamination before taking root canal samples, and this review assessed those studies. Methods: Using PubMed, a search was conducted for studies using molecular microbial analysis for the investigation of endodontic samples. Studies were grouped according to the cleaning protocol, acquisition methods, and processing of control samples taken to check for contamination. Results: Of 136 studies applying molecular analysis to root canal samples, 21 studies performed surface cleaning and checking nucleotide decontamination with contamination control samples processed by polymerase chain reaction. Only 1 study described disinfection, sampling from the access cavity,, and processing by polymerase chain reaction and reported the result; that study reported that all samples contained contaminating bacterial DNA. Conclusions: Cleaning, disinfection, and checking for contamination are basic scientific prerequisites for this type of investigation; yet, this review identifies it as an overlooked issue. On the basis of this review, we call for improved scientific practice in this field.

  • 10. Nair, PN
    et al.
    Brundin, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Sundqvist, Göran
    Sjögren, Ulf
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi.
    Building biofilms in vital host tissues: a survival strategy of Actinomyces radicidentis.2008Ingår i: Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics, Vol. 106, nr 4, s. 595-603Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To investigate the ability of Actinomyces radicidentis to survive and establish in soft connective tissue that grew into subcutaneously implanted tissue cages in Sprague-Dawley rats. STUDY DESIGN: Known concentrations of A. radicidentis suspension, grown on blood agar and broth cultures, were inoculated into tissue cages in rats. The cage contents were retrieved after 7, 14, and 28 days for culturing and correlative light and transmission electron microscopy. RESULTS: Cell suspensions harvested from both types of cultures showed substantial decline in numbers in tissue cages during the observation period. However, correlative light and transmission electron microscopy revealed numerous aggregates of coccoid bacteria already by 7 days of observation compared with the formation of well established colonies with characteristic actinomycotic features by 14 days after inoculation. CONCLUSIONS: These results suggest that the pathogenicity of A. radicidentis is due to its ability to form large aggregates of cells held together by embedding themselves in an extracellular matrix in vital host tissues. Thus, A. radicidentis, like other pathogenic Actinomyces, existing in the protected biofilm-environment can collectively evade destruction and elimination by host defenses, including phagocytosis.

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