David Figdor - Academia.edu (original) (raw)

Papers by David Figdor

Research paper thumbnail of Cell surface structures of Actinomyces israelii

Australian Dental Journal

Actinomyces israelii is the most common cause of human actinomycosis, a chronic granulomatous inf... more Actinomyces israelii is the most common cause of human actinomycosis, a chronic granulomatous infection. Periapical actinomycosis involving A. israelii has been identified as an important cause of failure of conventional endodontic treatment. Structures on the bacterial cell surface have been implicated in the pathogenicity of Actinomyces. In this study the ultrastructure of A. israelii was investigated by electron microscopy. Negatively stained preparations revealed the presence of hairlike fimbriae protruding through a thick surface coat on some species, whilst thin sectioning disclosed a Gram-positive cell wall surrounded by a fuzzy outer coat. These structures may be important for the pathogenicity of A. israelii.

Research paper thumbnail of Endodontic treatment: what can and what can't be saved

Annals of the Royal Australasian College of Dental Surgeons, 1998

Although endodontic treatment is generally considered to have a high success rate, not all teeth ... more Although endodontic treatment is generally considered to have a high success rate, not all teeth provide an opportunity for predictable salvage by endodontic therapy and subsequent restoration. New studies are yielding valuable information about factors that affect the outcome of treatment, which enhances the ability of the clinician to make a more accurate preoperative assessment and improve the quality and predictability of treatment. Most teeth can be saved by endodontic treatment; however, some cases fail despite good treatment. How can we assess and predict the outcome of treatment? This paper reviews reasons for biological failure of endodontic treatment and describes clinical factors that influence whether you might treat, refer, or extract the tooth.

Research paper thumbnail of Apical dentin permeability and microleakage associated with root end resection and retrograde filling

Journal of endodontics, 1994

This study evaluated the apical leakage associated with various depths of retrograde fillings pla... more This study evaluated the apical leakage associated with various depths of retrograde fillings placed in root apices which had been resected at one of three different angles. Leakage was assessed with a hydraulic conductance apparatus. Teeth were divided into groups corresponding to the angle of apical resection (0, 30, and 45 degrees to the long axis of the root) and apical leakage was determined following incremental increases in the depth of the retrograde filling (Ketac Silver). Increasing the depth of the retrograde filling significantly decreased apical leakage; there was also a significant increase in leakage as the amount of bevel increased. Both the permeability of resected apical dentin and microleakage around the retrograde filling material had a significant influence on apical leakage.

Research paper thumbnail of Aspects of dentinal and pulpal pain. Pain of dentinal and pulpal origin--a review for the clinician

Annals of the Royal Australasian College of Dental Surgeons, 1994

Recent advances in understanding the mechanisms of pain arising from the dental pulp serve to ben... more Recent advances in understanding the mechanisms of pain arising from the dental pulp serve to benefit patients by improving the clinician's ability to diagnose and treat pain. There are two types of pain arising from the pulp which are mediated by entirely different nerve fibres, each with their own individual characteristics. One is a short, sharp fast pain which is induced by stimuli which cause a rapid fluid flow within the dentinal tubules. Such stimuli include cold, heat, air, drilling, and osmotic stimuli. Once the affected teeth are identified, they can often be treated by sealing the open, exposed dentine. The second type of pain is experienced as a slow, dull, aching, poorly localized pain which is mediated by pain fibres activated by stimuli which are noxious to the pulp, such as prolonged damaging heat and inflammatory mediators. Pain of this character can be difficult to diagnose and often indicates serious pulp damage necessitating removal of the offending pulp by e...

Research paper thumbnail of Endodontic cellulitis 'flare-up

Australian dental journal, 1995

Research paper thumbnail of Preservation of Bacterial DNA by Human Dentin

Journal of Endodontics, 2014

The capacity of dentin and collagen to bind DNA and protect against spontaneous and nuclease-indu... more 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. 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. 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). 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.

Research paper thumbnail of DNA binding to hydroxyapatite: a potential mechanism for preservation of microbial DNA

Journal of endodontics, 2013

Molecular methods are increasingly being deployed for analysis of the microbial flora in the root... more 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 centuries-old 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. 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 electrop...

Research paper thumbnail of Bacterial DNA Persists for Extended Periods after Cell Death

Journal of Endodontics, 2007

The fate of DNA from bacteria that infect the root canal but cannot survive is currently unknown,... more The fate of DNA from bacteria that infect the root canal but cannot survive is currently unknown, yet such information is essential in establishing the validity of polymerase chain reaction (PCR)-based identification methods for root canal samples. This in vitro study tested the hypothesis that PCR-detectable DNA from dead bacteria might persist after cell death and investigated the efficiency of sodium hypochlorite (NaOCl) as a field decontamination agent. Using heat-killed Enterococcus faecalis, the persistence of DNA encoding the 16S rRNA gene was monitored by PCR. While most probable number analysis showed an approximate 1000-fold decay in amplifiable template, E. faecalis DNA was still PCR-detectable 1 year after cell death. NaOCl (1%) eliminated amplifiable DNA within 60 seconds of exposure. Our findings also disclosed a previously overlooked problem of concentration-dependent inhibition of the PCR reaction by thiosulfate-inactivated NaOCl. These results highlight the challenges of reliably identifying the authentic living root canal flora with PCR techniques.

Research paper thumbnail of Apical periodontitis: A very prevalent problem

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 2002

Research paper thumbnail of Persistent periapical radiolucencies of root-filled human teeth, failed endodontic treatments, and periapical scars

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 1999

Research paper thumbnail of Starvation response and growth in serum of Fusobacterium nucleatum, Peptostreptococcus anaerobius, Prevotella intermedia, and Pseudoramibacter alactolyticus

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 2009

The microbiota inhabiting the untreated root canal differ markedly from those found in post-treat... more 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 starvationsurvival 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 Ն10% 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.

Research paper thumbnail of Starvation survival and recovery in serum of Candida albicans compared with Enterococcus faecalis

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 2010

Objective. Candida albicans has been a common isolate in posttreatment disease, usually as a mono... more Objective. Candida albicans has been a common isolate in posttreatment disease, usually as a monoinfection of the root filled canal. A factor likely to contribute to its pathogenic potential in posttreatment infection is an ability to endure starvation and use serum as a nutritional source. This study evaluated the starvation-survival behavior, growth, and recovery in human serum of C. albicans and compared it with Enterococcus faecalis. Study design. Varying cell densities of C. albicans and E. faecalis were suspended in 5% human serum or water for 4-6 months. Starvation recovery was assessed by addition of 50% serum to starved cells. Cell survival was monitored by periodic removal of aliquots and viable counts. Results. Initial cell density was important for starvation survival. Candida albicans and E. faecalis survived starvation in water for 6 months when the starting cell density was Ͼ10 5 and Ն10 8 colony-forming units (cfu)/mL, respectively. Both species thrived in 5% serum from low initial densities (Ͼ10 2 and Ͼ10 4 cfu/mL for C. albicans and E. faecalis, respectively), and starvation-state cells recovered on addition of 50% serum. Conclusion. Candida albicans is well suited for survival in nutrient-limited conditions and can use serum as a source of nutrition and for recovery from starvation. These findings parallel the behavior of E. faecalis, which possesses a similar capacity for starvation survival and growth in serum, traits that are of likely importance for their participation in posttreatment infection.

Research paper thumbnail of Molecular approaches to the differentiation of Actinomyces species

Oral Microbiology and Immunology, 1999

The definition of the genus Actinomyces relies heavily on traditional methods of taxonomy. This s... more The definition of the genus Actinomyces relies heavily on traditional methods of taxonomy. This study sought to develop molecular tools for the identification of strains of Actinomyces israelii and Actinomyces gerencseriae. Oligonucleotide probes were designed and one of these successfully differentiated. A. gerencseriae from ten strains of A. israelii and three other Actinomyces species by DNA:DNA hybridization. However, probes based on known 16S rRNA sequences failed to hybridize to all the strains previously identified as A. israelii. Using the PCR technique, a region encoding a portion of the 16S rRNA was amplified from genomic DNA. The results showed that A. israelii can be divided into three different groups based on comparison of the amplified DNA sequences. This information should allow the development of probes that are specific for these newly identified groups of strains within the species A. israelii.

Research paper thumbnail of Pathogenicity of Actinomyces israelii and Arachnid propionica: experimental infection in guinea pigs and phagocytosis and intracellular killing by human polymorphonuclear leukocytes in vitro

Oral Microbiology and Immunology, 1992

Strains of Actitiomyces israelii and Arachnia propionica, isolated from clinical cases of failed ... more Strains of Actitiomyces israelii and Arachnia propionica, isolated from clinical cases of failed endodontic therapy, were examined for: (i) their ability to survive and establish themselves in the soft connective tissue that grew into subcutaneously implanted tissue cages in guinea pigs; (ii) cell-surface hydrophobicity; and (iii) phagocytosis and killing by human polymorphonuclear leukocytes (PMNs) under aerobic and anaerobic conditions. Bacteria were inoculated into the tissue cages in guinea pigs and the cage contents were retrieved after 1, 7, 14 and 21 d for culturing and light and electron microscopy. Both bacterial species showed substantial decline in the number of bacteria by day 7 after the inoculation. Thereafter, the A. israelii strain recovered and, by day 21, had started to increase in number. Light and electron microscopy revealed the formation of typical actinomycotic colonies. A. propionica, on the other hand, continued to decline in number during the entire period of experimental infection and did not form colonies. Both strains were hydrophobic, readily phagocytosed and were efficiently killed by hutnan PMNs under aerobic and anaerobic conditions in vitro. These results suggest that the pathogenicity of A. israelii is due to its ability to establish characteristic cohesive colonies consisting of branching filamentous organisms that are entneshed in an extracellular matrix. It seems that the organisms existing in such colonies can collectively evade destruction and elimination by host phagocytic cells, whereas in vitro suspensions of the bacteria are easily phagocytosed and efficiently killed by PMNs. With respect to A. propionica, further investigations are necessary to understand its pathogenicity.

Research paper thumbnail of Starvation survival, growth and recovery of Enterococcus faecalis in human serum

Oral Microbiology and Immunology, 2003

The ability of Enterococcus faecalis to survive starvation for long periods in the obturated root... more The ability of Enterococcus faecalis to survive starvation for long periods in the obturated root canal is likely to be an important factor in the pathogenesis and maintenance of a persistent infection after endodontic treatment. The response of E. faecalis to starvation survival in water and glucose-, phosphate- or amino acid-limited chemically defined medium was studied, along with the capacity for growth and recovery of starved cells of E. faecalis in pooled human serum. After an initial rapid fall in cell numbers, a small remaining population of E. faecalis was able to survive in water for over 4 months and in nutrient-limited media for extended periods. A high cell density at the onset of starvation was critical for the ability of E. faecalis to endure prolonged nutrient limitation. Upon starvation, a static population of starved cells developed and were apparently in a minimal metabolic state, since blocking cell wall synthesis with penicillin G or inhibiting DNA synthesis with norfloxacin during starvation resulted in limited change in the rate of loss of viable cells. In 50% serum, E. faecalis grew, then stabilized at a relatively constant population of 106 colony-forming units/ml for 4 months, irrespective of the initial cell density. In summary, E. faecalis is capable of withstanding prolonged periods of starvation in a minimal metabolic state provided that there is a high cell density at the onset of starvation. Starved cells were capable of recovery upon addition of human serum.

Research paper thumbnail of pH changes in root dentin over a 4-week period following root canal dressing with calcium hydroxide

Journal of Endodontics, 1993

Root canals in extracted human teeth were cleaned and shaped and subsequently dressed with a calc... more Root canals in extracted human teeth were cleaned and shaped and subsequently dressed with a calcium hydroxide root canal dressing. pH Changes in the root dentin were measured over a 4-wk period with microelectrodes in small cavities at apical and cervical levels in inner and outer dentin. The pH increased within hours in the inner dentin, peaking at pH 10.8 cervically and 9.7 apically. However, 1 to 7 days elapsed before the pH began to rise in the outer root dentin, reaching peak levels of pH 9.3 cervically and 9.0 apically after 2 to 3 wk. The results show that hydroxyl ions derived from a calcium hydroxide dressing do diffuse through root dentin. They diffuse faster and reach higher levels cervically than apically. Surface pH measurements showed that hydroxyl ions do not diffuse in more than a minor way through the intact root surface.

Research paper thumbnail of Preservation of Fusobacterium nucleatum and Peptostreptococcus anaerobius DNA after loss of cell viability

International Endodontic Journal, 2014

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

Research paper thumbnail of Susceptibility of Actinomyces israelii to antibiotics, sodium hypochlorite and calcium hydroxide

International Endodontic Journal, 1996

Actinomyces israelii has been repeatedly implicated as a cause of failure of endodontic therapy. ... more Actinomyces israelii has been repeatedly implicated as a cause of failure of endodontic therapy. This study investigated the antimicrobial effect of antibiotics as well as intracanal medicaments, sodium hypochlorite solution and calcium hydroxide, on this important pathogen. Growth of A. israelii was inhibited by low concentrations of antibiotics, yet high concentrations were not bactericidal for A. israelii over 1 week. When A. israelii was exposed for 2-6 weeks at concentrations equivalent to clinical serum levels, the antibiotics were lethal. The results reveal a species-specific antibiotic tolerance for A. israelii. Both sodium hypochlorite solution and calcium hydroxide were found to be highly effective in killing A. israelii.

Research paper thumbnail of Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis

International Endodontic Journal, 1997

This study investigated the role of infection on the prognosis of endodontic therapy by following... more This study investigated the role of infection on the prognosis of endodontic therapy by following-up teeth that had had their canals cleaned and obturated during a single appointment. The root canals of 55 singlerooted teeth with apical periodontitis were thoroughly instrumented and irrigated with sodium hypochlorite solution. Using advanced anaerobic bacteriological techniques, post-instrumentation samples were taken and the teeth were then root-filled during the same appointment. All teeth were initially infected; after instrumentation low numbers of bacteria were detected in 22 of 55 root canals. Periapical healing was followedup for 5 years. Complete periapical healing occurred in 94% of cases that yielded a negative culture. Where the samples were positive prior to root filling, the success rate of treatment was just 68% -a statistically significant difference. Further investigation of three failures revealed the presence of Actinomyces species in each case; no other specific bacteria were implicated in failure cases. These findings emphasize the importance of completely eliminating bacteria from the root canal system before obturation. This objective cannot be reliably achieved in a one-visit treatment because it is not possible to eradicate all infection from the root canal without the support of an inter-appointment antimicrobial dressing.

Research paper thumbnail of The antimicrobial effect of calcium hydroxide as a short-term intracanal dressing

International Endodontic Journal, 1991

andtDepartment o.f E nd o d ont o I ogy, Llniaer si t.y of C onne c t icut, F armingt 0n, C onne ... more andtDepartment o.f E nd o d ont o I ogy, Llniaer si t.y of C onne c t icut, F armingt 0n, C onne c t icut, (J S A Surnrnary.

Research paper thumbnail of Cell surface structures of Actinomyces israelii

Australian Dental Journal

Actinomyces israelii is the most common cause of human actinomycosis, a chronic granulomatous inf... more Actinomyces israelii is the most common cause of human actinomycosis, a chronic granulomatous infection. Periapical actinomycosis involving A. israelii has been identified as an important cause of failure of conventional endodontic treatment. Structures on the bacterial cell surface have been implicated in the pathogenicity of Actinomyces. In this study the ultrastructure of A. israelii was investigated by electron microscopy. Negatively stained preparations revealed the presence of hairlike fimbriae protruding through a thick surface coat on some species, whilst thin sectioning disclosed a Gram-positive cell wall surrounded by a fuzzy outer coat. These structures may be important for the pathogenicity of A. israelii.

Research paper thumbnail of Endodontic treatment: what can and what can't be saved

Annals of the Royal Australasian College of Dental Surgeons, 1998

Although endodontic treatment is generally considered to have a high success rate, not all teeth ... more Although endodontic treatment is generally considered to have a high success rate, not all teeth provide an opportunity for predictable salvage by endodontic therapy and subsequent restoration. New studies are yielding valuable information about factors that affect the outcome of treatment, which enhances the ability of the clinician to make a more accurate preoperative assessment and improve the quality and predictability of treatment. Most teeth can be saved by endodontic treatment; however, some cases fail despite good treatment. How can we assess and predict the outcome of treatment? This paper reviews reasons for biological failure of endodontic treatment and describes clinical factors that influence whether you might treat, refer, or extract the tooth.

Research paper thumbnail of Apical dentin permeability and microleakage associated with root end resection and retrograde filling

Journal of endodontics, 1994

This study evaluated the apical leakage associated with various depths of retrograde fillings pla... more This study evaluated the apical leakage associated with various depths of retrograde fillings placed in root apices which had been resected at one of three different angles. Leakage was assessed with a hydraulic conductance apparatus. Teeth were divided into groups corresponding to the angle of apical resection (0, 30, and 45 degrees to the long axis of the root) and apical leakage was determined following incremental increases in the depth of the retrograde filling (Ketac Silver). Increasing the depth of the retrograde filling significantly decreased apical leakage; there was also a significant increase in leakage as the amount of bevel increased. Both the permeability of resected apical dentin and microleakage around the retrograde filling material had a significant influence on apical leakage.

Research paper thumbnail of Aspects of dentinal and pulpal pain. Pain of dentinal and pulpal origin--a review for the clinician

Annals of the Royal Australasian College of Dental Surgeons, 1994

Recent advances in understanding the mechanisms of pain arising from the dental pulp serve to ben... more Recent advances in understanding the mechanisms of pain arising from the dental pulp serve to benefit patients by improving the clinician's ability to diagnose and treat pain. There are two types of pain arising from the pulp which are mediated by entirely different nerve fibres, each with their own individual characteristics. One is a short, sharp fast pain which is induced by stimuli which cause a rapid fluid flow within the dentinal tubules. Such stimuli include cold, heat, air, drilling, and osmotic stimuli. Once the affected teeth are identified, they can often be treated by sealing the open, exposed dentine. The second type of pain is experienced as a slow, dull, aching, poorly localized pain which is mediated by pain fibres activated by stimuli which are noxious to the pulp, such as prolonged damaging heat and inflammatory mediators. Pain of this character can be difficult to diagnose and often indicates serious pulp damage necessitating removal of the offending pulp by e...

Research paper thumbnail of Endodontic cellulitis 'flare-up

Australian dental journal, 1995

Research paper thumbnail of Preservation of Bacterial DNA by Human Dentin

Journal of Endodontics, 2014

The capacity of dentin and collagen to bind DNA and protect against spontaneous and nuclease-indu... more 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. 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. 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). 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.

Research paper thumbnail of DNA binding to hydroxyapatite: a potential mechanism for preservation of microbial DNA

Journal of endodontics, 2013

Molecular methods are increasingly being deployed for analysis of the microbial flora in the root... more 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 centuries-old 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. 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 electrop...

Research paper thumbnail of Bacterial DNA Persists for Extended Periods after Cell Death

Journal of Endodontics, 2007

The fate of DNA from bacteria that infect the root canal but cannot survive is currently unknown,... more The fate of DNA from bacteria that infect the root canal but cannot survive is currently unknown, yet such information is essential in establishing the validity of polymerase chain reaction (PCR)-based identification methods for root canal samples. This in vitro study tested the hypothesis that PCR-detectable DNA from dead bacteria might persist after cell death and investigated the efficiency of sodium hypochlorite (NaOCl) as a field decontamination agent. Using heat-killed Enterococcus faecalis, the persistence of DNA encoding the 16S rRNA gene was monitored by PCR. While most probable number analysis showed an approximate 1000-fold decay in amplifiable template, E. faecalis DNA was still PCR-detectable 1 year after cell death. NaOCl (1%) eliminated amplifiable DNA within 60 seconds of exposure. Our findings also disclosed a previously overlooked problem of concentration-dependent inhibition of the PCR reaction by thiosulfate-inactivated NaOCl. These results highlight the challenges of reliably identifying the authentic living root canal flora with PCR techniques.

Research paper thumbnail of Apical periodontitis: A very prevalent problem

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 2002

Research paper thumbnail of Persistent periapical radiolucencies of root-filled human teeth, failed endodontic treatments, and periapical scars

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 1999

Research paper thumbnail of Starvation response and growth in serum of Fusobacterium nucleatum, Peptostreptococcus anaerobius, Prevotella intermedia, and Pseudoramibacter alactolyticus

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 2009

The microbiota inhabiting the untreated root canal differ markedly from those found in post-treat... more 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 starvationsurvival 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 Ն10% 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.

Research paper thumbnail of Starvation survival and recovery in serum of Candida albicans compared with Enterococcus faecalis

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology, 2010

Objective. Candida albicans has been a common isolate in posttreatment disease, usually as a mono... more Objective. Candida albicans has been a common isolate in posttreatment disease, usually as a monoinfection of the root filled canal. A factor likely to contribute to its pathogenic potential in posttreatment infection is an ability to endure starvation and use serum as a nutritional source. This study evaluated the starvation-survival behavior, growth, and recovery in human serum of C. albicans and compared it with Enterococcus faecalis. Study design. Varying cell densities of C. albicans and E. faecalis were suspended in 5% human serum or water for 4-6 months. Starvation recovery was assessed by addition of 50% serum to starved cells. Cell survival was monitored by periodic removal of aliquots and viable counts. Results. Initial cell density was important for starvation survival. Candida albicans and E. faecalis survived starvation in water for 6 months when the starting cell density was Ͼ10 5 and Ն10 8 colony-forming units (cfu)/mL, respectively. Both species thrived in 5% serum from low initial densities (Ͼ10 2 and Ͼ10 4 cfu/mL for C. albicans and E. faecalis, respectively), and starvation-state cells recovered on addition of 50% serum. Conclusion. Candida albicans is well suited for survival in nutrient-limited conditions and can use serum as a source of nutrition and for recovery from starvation. These findings parallel the behavior of E. faecalis, which possesses a similar capacity for starvation survival and growth in serum, traits that are of likely importance for their participation in posttreatment infection.

Research paper thumbnail of Molecular approaches to the differentiation of Actinomyces species

Oral Microbiology and Immunology, 1999

The definition of the genus Actinomyces relies heavily on traditional methods of taxonomy. This s... more The definition of the genus Actinomyces relies heavily on traditional methods of taxonomy. This study sought to develop molecular tools for the identification of strains of Actinomyces israelii and Actinomyces gerencseriae. Oligonucleotide probes were designed and one of these successfully differentiated. A. gerencseriae from ten strains of A. israelii and three other Actinomyces species by DNA:DNA hybridization. However, probes based on known 16S rRNA sequences failed to hybridize to all the strains previously identified as A. israelii. Using the PCR technique, a region encoding a portion of the 16S rRNA was amplified from genomic DNA. The results showed that A. israelii can be divided into three different groups based on comparison of the amplified DNA sequences. This information should allow the development of probes that are specific for these newly identified groups of strains within the species A. israelii.

Research paper thumbnail of Pathogenicity of Actinomyces israelii and Arachnid propionica: experimental infection in guinea pigs and phagocytosis and intracellular killing by human polymorphonuclear leukocytes in vitro

Oral Microbiology and Immunology, 1992

Strains of Actitiomyces israelii and Arachnia propionica, isolated from clinical cases of failed ... more Strains of Actitiomyces israelii and Arachnia propionica, isolated from clinical cases of failed endodontic therapy, were examined for: (i) their ability to survive and establish themselves in the soft connective tissue that grew into subcutaneously implanted tissue cages in guinea pigs; (ii) cell-surface hydrophobicity; and (iii) phagocytosis and killing by human polymorphonuclear leukocytes (PMNs) under aerobic and anaerobic conditions. Bacteria were inoculated into the tissue cages in guinea pigs and the cage contents were retrieved after 1, 7, 14 and 21 d for culturing and light and electron microscopy. Both bacterial species showed substantial decline in the number of bacteria by day 7 after the inoculation. Thereafter, the A. israelii strain recovered and, by day 21, had started to increase in number. Light and electron microscopy revealed the formation of typical actinomycotic colonies. A. propionica, on the other hand, continued to decline in number during the entire period of experimental infection and did not form colonies. Both strains were hydrophobic, readily phagocytosed and were efficiently killed by hutnan PMNs under aerobic and anaerobic conditions in vitro. These results suggest that the pathogenicity of A. israelii is due to its ability to establish characteristic cohesive colonies consisting of branching filamentous organisms that are entneshed in an extracellular matrix. It seems that the organisms existing in such colonies can collectively evade destruction and elimination by host phagocytic cells, whereas in vitro suspensions of the bacteria are easily phagocytosed and efficiently killed by PMNs. With respect to A. propionica, further investigations are necessary to understand its pathogenicity.

Research paper thumbnail of Starvation survival, growth and recovery of Enterococcus faecalis in human serum

Oral Microbiology and Immunology, 2003

The ability of Enterococcus faecalis to survive starvation for long periods in the obturated root... more The ability of Enterococcus faecalis to survive starvation for long periods in the obturated root canal is likely to be an important factor in the pathogenesis and maintenance of a persistent infection after endodontic treatment. The response of E. faecalis to starvation survival in water and glucose-, phosphate- or amino acid-limited chemically defined medium was studied, along with the capacity for growth and recovery of starved cells of E. faecalis in pooled human serum. After an initial rapid fall in cell numbers, a small remaining population of E. faecalis was able to survive in water for over 4 months and in nutrient-limited media for extended periods. A high cell density at the onset of starvation was critical for the ability of E. faecalis to endure prolonged nutrient limitation. Upon starvation, a static population of starved cells developed and were apparently in a minimal metabolic state, since blocking cell wall synthesis with penicillin G or inhibiting DNA synthesis with norfloxacin during starvation resulted in limited change in the rate of loss of viable cells. In 50% serum, E. faecalis grew, then stabilized at a relatively constant population of 106 colony-forming units/ml for 4 months, irrespective of the initial cell density. In summary, E. faecalis is capable of withstanding prolonged periods of starvation in a minimal metabolic state provided that there is a high cell density at the onset of starvation. Starved cells were capable of recovery upon addition of human serum.

Research paper thumbnail of pH changes in root dentin over a 4-week period following root canal dressing with calcium hydroxide

Journal of Endodontics, 1993

Root canals in extracted human teeth were cleaned and shaped and subsequently dressed with a calc... more Root canals in extracted human teeth were cleaned and shaped and subsequently dressed with a calcium hydroxide root canal dressing. pH Changes in the root dentin were measured over a 4-wk period with microelectrodes in small cavities at apical and cervical levels in inner and outer dentin. The pH increased within hours in the inner dentin, peaking at pH 10.8 cervically and 9.7 apically. However, 1 to 7 days elapsed before the pH began to rise in the outer root dentin, reaching peak levels of pH 9.3 cervically and 9.0 apically after 2 to 3 wk. The results show that hydroxyl ions derived from a calcium hydroxide dressing do diffuse through root dentin. They diffuse faster and reach higher levels cervically than apically. Surface pH measurements showed that hydroxyl ions do not diffuse in more than a minor way through the intact root surface.

Research paper thumbnail of Preservation of Fusobacterium nucleatum and Peptostreptococcus anaerobius DNA after loss of cell viability

International Endodontic Journal, 2014

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

Research paper thumbnail of Susceptibility of Actinomyces israelii to antibiotics, sodium hypochlorite and calcium hydroxide

International Endodontic Journal, 1996

Actinomyces israelii has been repeatedly implicated as a cause of failure of endodontic therapy. ... more Actinomyces israelii has been repeatedly implicated as a cause of failure of endodontic therapy. This study investigated the antimicrobial effect of antibiotics as well as intracanal medicaments, sodium hypochlorite solution and calcium hydroxide, on this important pathogen. Growth of A. israelii was inhibited by low concentrations of antibiotics, yet high concentrations were not bactericidal for A. israelii over 1 week. When A. israelii was exposed for 2-6 weeks at concentrations equivalent to clinical serum levels, the antibiotics were lethal. The results reveal a species-specific antibiotic tolerance for A. israelii. Both sodium hypochlorite solution and calcium hydroxide were found to be highly effective in killing A. israelii.

Research paper thumbnail of Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis

International Endodontic Journal, 1997

This study investigated the role of infection on the prognosis of endodontic therapy by following... more This study investigated the role of infection on the prognosis of endodontic therapy by following-up teeth that had had their canals cleaned and obturated during a single appointment. The root canals of 55 singlerooted teeth with apical periodontitis were thoroughly instrumented and irrigated with sodium hypochlorite solution. Using advanced anaerobic bacteriological techniques, post-instrumentation samples were taken and the teeth were then root-filled during the same appointment. All teeth were initially infected; after instrumentation low numbers of bacteria were detected in 22 of 55 root canals. Periapical healing was followedup for 5 years. Complete periapical healing occurred in 94% of cases that yielded a negative culture. Where the samples were positive prior to root filling, the success rate of treatment was just 68% -a statistically significant difference. Further investigation of three failures revealed the presence of Actinomyces species in each case; no other specific bacteria were implicated in failure cases. These findings emphasize the importance of completely eliminating bacteria from the root canal system before obturation. This objective cannot be reliably achieved in a one-visit treatment because it is not possible to eradicate all infection from the root canal without the support of an inter-appointment antimicrobial dressing.

Research paper thumbnail of The antimicrobial effect of calcium hydroxide as a short-term intracanal dressing

International Endodontic Journal, 1991

andtDepartment o.f E nd o d ont o I ogy, Llniaer si t.y of C onne c t icut, F armingt 0n, C onne ... more andtDepartment o.f E nd o d ont o I ogy, Llniaer si t.y of C onne c t icut, F armingt 0n, C onne c t icut, (J S A Surnrnary.