Preservation of Fusobacterium nucleatum and Peptostreptococcus anaerobius DNA after loss of cell viability.

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.

A big role for the very small--understanding the endodontic microbial flora.

Apical periodontitis, an inflammatory process around the apex of a tooth root, is primarily a sequel to microbial infection of the pulp space. The microbial flora is composed of a restricted group of the total oral flora, selected by environmental pressures of anaerobiosis, nutrition and competition with other species and inhabits the root canal as a biofilm of coaggregated communities in an extracellular matrix. The untreated infected canal is generally composed of a polymicrobial mix with approximately equal proportions of Gram-positive and Gram-negative species, dominated by obligate anaerobes. The type of microbial flora in the root-filled tooth with persistent apical periodontitis has very different characteristics. These infections are characterized by one or just a few species, predominantly Gram-positive micro-organisms with an equal distribution of facultative and obligate anaerobes. Enterococcus faecalis has been a conspicuous finding in most studies. Because the primary aetiological problem is infection, endodontic treatment is directed at control and elimination of the root canal flora by working in a sterile way. Based on current knowledge, the best available method for obtaining clean, microbe-free root canals is by instrumentation with antimicrobial irrigation reinforced by an intracanal dressing with calcium hydroxide.

Starvation survival, growth and recovery of Enterococcus faecalis in human serum.

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.

Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide.

AIM: This study sought to clarify the mechanisms that enable E. faecalis to survive the high pH of calcium hydroxide. METHODOLOGY: E. faecalis strain JH2-2 was exposed to sublethal concentrations of calcium hydroxide, with and without various pretreatments. Blocking agents were added to determine the role of stress-induced protein synthesis and the cell wall-associated proton pump. RESULTS: E. faecalis was resistant to calcium hydroxide at a pH of 11.1, but not pH 11.5. Pre-treatment with calcium hydroxide pH 10.3 induced no tolerance to further exposure at pH 11.5. No difference in cell survival was observed when protein synthesis was blocked during stress induction, however, addition of a proton pump inhibitor resulted in a dramatic reduction of cell viability of E. faecalis in calcium hydroxide. CONCLUSIONS: Survival of E. faecalis in calcium hydroxide appears to be unrelated to stress induced protein synthesis, but a functioning proton pump is critical for survival of E. faecalis at high pH.

A new bacterial species associated with failed endodontic treatment: identification and description of Actinomyces radicidentis.

OBJECTIVE: This report describes 2 endodontic patients who had persistent signs and symptoms after conventional root canal treatment. The aim of this study was to determine what microorganisms were present in the root canals of the teeth with failed endodontic therapy. STUDY DESIGN: After removal of the root fillings, the canals were sampled by advanced microbiological techniques and the isolates were characterized by various tests. RESULTS: Bacteria, which grew in pure cultures, were isolated in each case. The bacteria were similar to each other and were classified as Actinomyces on the basis of phylogenic and phenotypic evidence. The bacteria were different from others within the genus, thus warranting designation as a new species, Actinomyces radicidentis. CONCLUSIONS: The 2 cases of endodontic failure were infected with A radicidentis, a new Actinomyces species. This bacterium joins a restricted group of other microorganisms that have been associated with failure of root canal treatment.

Molecular approaches to the differentiation of Actinomyces species.

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.

Persistent periapical radiolucencies of root-filled human teeth, failed endodontic treatments, and periapical scars.

OBJECTIVE: This report describes 6 cases that demonstrate persistent periapical radiolucent lesions after conventional root canal treatment. STUDY DESIGN: Six teeth that had conventional root canal treatment or re-treatment with nonresolving periapical radiolucencies underwent periapical surgery. Biopsies were obtained and analyzed descriptively by correlative light and transmission electron microscopy for general features and microbial findings. RESULTS: Three findings were identified: periapical lesions with persisting infection in the apical root canal system (2 cases); a cyst (1 case); and periapical healing by scar tissue formation (2 cases). CONCLUSIONS: These results confirm previous observations that associated factors in the failure of endodontic treatment include persistent intraradicular infection and periapical cysts. In addition, unresolved periapical radiolucencies may occasionally be due to healing by scar tissue, which may be mistaken as a sign of failed endodontic treatment.

Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment.

OBJECTIVE: The purposes of this study were to determine what microbial flora were present in teeth after failed root canal therapy and to establish the outcome of conservative re-treatment. STUDY DESIGN: Fifty-four root-filled teeth with persisting periapical lesions were selected for re-treatment. After removal of the root filling, canals were sampled by means of advanced microbiologic techniques. The teeth were then re-treated and followed for up to 5 years. RESULTS: The microbial flora was mainly single species of predominantly gram-positive organisms. The isolates most commonly recovered were bacteria of the species Enterococcus faecalis. The overall success rate of re-treatment was 74%. CONCLUSIONS: The microbial flora in canals after failed endodontic therapy differed markedly from the flora in untreated teeth. Infection at the time of root filling and size of the periapical lesion were factors that had a negative influence on the prognosis. Three of four endodontic failures were successfully managed by re-treatment.

Endodontic treatment: what can and what can't be saved.

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.

Cell surface structures of Actinomyces israelii.

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.

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

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 single-rooted 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 followed-up 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.

Susceptibility of Actinomyces israelii to antibiotics, sodium hypochlorite and calcium hydroxide.

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.

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

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 endodontic therapy.

Apical dentin permeability and microleakage associated with root end resection and retrograde filling.

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.

pH changes in root dentin over a 4-week period following root canal dressing with calcium hydroxide.

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.

Pathogenicity of Actinomyces israelii and Arachnia propionica: experimental infection in guinea pigs and phagocytosis and intracellular killing by human polymorphonuclear leukocytes in vitro.

Strains of Actinomyces 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 human 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 enmeshed 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.

The antimicrobial effect of calcium hydroxide as a short-term intracanal dressing.

The antibacterial effect of calcium hydroxide as a short-term intracanal dressing was clinically evaluated by applying the medicament for 10 minutes or 7 days in root canals of teeth with periapical lesions. The results showed that the 7-day dressing efficiently eliminated bacteria which survived biomechanical instrumentation of the canal, while the 10-minute application was ineffective.

Phagocytosis and virulence of different strains of Porphyromonas gingivalis.

In this study 17 strains of Porphyromonas gingivalis, both reference and clinical isolates, were investigated for their in vitro interaction with human polymorphonuclear leukocytes, hydrophobicity, density, and virulence in a mouse model. The results of the phagocytosis, hydrophobicity, and density experiments showed that P. gingivalis strains could be divided into two distinct groups. One group of strains were readily attached and phagocytosed when exposed to the leukocytes. These bacteria were hydrophobic and had a higher buoyant density than the other group, which were poorly phagocytosed, had a low buoyant density, and were hydrophilic. This latter group also exhibited an extracellular meshwork resembling a glycocalyx when examined by electron microscopy. There were also significant differences between strains in the mouse pathogenicity model. Two strains caused an invasive, spreading infection compared with the other 15 strains which produced small, localized abscesses. There was no clear correlation between the results of the phagocytosis assay and the virulence of the bacteria when injected subcutaneously in mice. Resistance to phagocytosis may be important for survival of these bacteria, but it does not in itself imply the ability to cause damage to the host.