Yeasts in apical periodontitis.

Microbiological reports of apical periodontitis have revealed that yeasts can be isolated from approximately 5-20% of infected root canals. They occur either in pure cultures or together with bacteria. Almost all isolated yeasts belong to the genus Candida, and the predominant species is C. albicans. Pheno- and genotypic profiles of C. albicans isolates show heterogeneity comparable with those of isolates from other oral sites. C. albicans expresses several virulence factors that are capable of infecting the dentin-pulp complex, including dentinal tubules. This causes, consequentially, an inflammatory response around the root apex, which suggests a pathogenic role for this organism in apical periodontitis. Yeasts are particularly associated with persistent root canal infections that do not respond favorably to conservative root canal therapy. This may be due to the resistance of all oral Candida species against a commonly used topical medicament, calcium hydroxide. However, other antimicrobial agents may offer alternative therapeutic approaches and improve the treatment of these persistent cases of apical periodontitis.

Phenotypes and randomly amplified polymorphic DNA profiles of Candida albicans isolates from root canal infections in a Finnish population.

A total of thirty-seven Candida albicans isolates from root canal infections in a Finnish population were subtyped using phenotypic and genotypic methods. A previously described biotyping method based on the presence of five different enzymes, assimilation of eleven different carbohydrates and boric acid sensitivity of the yeasts was used to determine the phenotype. Commercially available API ZYM and API 20 C test kits were used to determine the presence of enzymes and assimilation of carbohydrates. The sensitivity of the isolates to boric acid was tested by their ability to grow on yeast-nitrogen-agar with incorporated boric acid (1.8 mg. ml(-1)). Combination of the tests revealed a total of 14 different phenotypes. The majority of the isolates, 26 strains, were classifiable into three major phenotypes: 16 isolates (43.2%) belonged to phenotype A1R, six (16.2%) to A1S and four (10.8%) to B1S. The remaining 11 phenotypes represented only a single isolate each. The randomly amplified polymorphic DNA profiles were used to determine the genotypes. For this purpose two different primers, RSD6 and RSD12 were used to develop a combination randomly amplified polymorphic DNA profile for each isolate. Altogether 31 genotypes were noted among the 37 isolates, of which only three pairs of isolates presented with congruent phenotypic and genotypic profiles. The heterogeneity of both the phenotypic and randomly amplified polymorphic DNA profiles of C. albicans isolates from root canal infections is akin to previous reports from other oral and non-oral sources in different geographic locales.

In vitro yeast infection of human dentin.

An in vitro model was developed for investigation of Candida albicans penetration into human dentinal tubules. The model consisted of a dentin disc mounted between two cuvettes that each had a circular opening facing the disc. The cuvettes were filled with Tryptic-Soy-Broth, and the pulpal side cuvette was inoculated with C. albicans and incubated at 37 degrees C in air until growth occurred in the uninoculated cuvette or up to 30 days. The system was also used with Enterococcus faecalis. Completely glue-covered dentin specimens served as negative controls. Brown & Brenn-stained histological preparations of the specimens were examined with light microscopy. The time needed before growth occurred in the uninoculated cuvette showed great variation with C. albicans, whereas E. faecalis penetrated within 1 to 5 days of incubation. Slight penetration both by hyphae and yeast cells was observed in specimens inoculated with C. albicans, whereas specimens inoculated with E. faecalis showed deep and effective penetration. This study demonstrates the penetration of dentin as a possible pathway of infection by C. albicans. However, dentin penetration by C. albicans was slow and limited in comparison with E. faecalis.

In vitro susceptibility of Candida albicans isolates from apical and marginal periodontitis to common antifungal agents.

The susceptibility of a total of 70 Candida albicans strains to five common antifungal agents was determined. Thirty-five of the strains were isolated from persistent cases of apical periodontitis and 35 from cases of marginal periodontitis. The susceptibility of the strains to amphotericin B, 5-fluorocytosine and three azoles: fluconazole, miconazole and clotrimazole, was tested. The antifungal agents and yeast inoculums were prepared according to the NCCLS (National Committee for Clinical Laboratory Standards) recommendations. The yeasts were incubated with ten different concentrations of antifungal agents at 35 degrees C for 48 h. Yeast growth was measured spectrophotometrically. All strains from both isolation sources were susceptible to low concentrations of amphotericin B and 5-fluorocytosine, whereas the susceptibility to the three azoles varied, and three of the strains showed azole cross-resistance. These findings are in agreement with recent reports of increased azole resistance in Candida species in general and suggest the possibility that the oral cavity may act as a reservoir of resistant yeast isolates in systemic infections.

Inactivation of local root canal medicaments by dentine: an in vitro study.

AIMS: The aim of the study was to investigate the inactivation by dentine of the antibacterial activity of various commonly used local root canal medicaments. METHODOLOGY: The medicaments tested were saturated calcium hydroxide solution, 1% sodium hypochlorite, 0.5% and 0.05% chlorhexidine acetate, and 2/4% and 0.2/0.4% iodine potassium iodide. Dentine was sterilized by autoclaving and crushed into powder with a particle size of 0.2-20 microns. Aliquots of dentine suspension were incubated with the medicaments in sealed test tubes at 37 degrees C for 24 h or 1 h before adding the bacteria. In some experiments bacteria were added simultaneously with dentine powder and the medicament. Enterococcus faecalis A197A was used as a test organism. Samples for bacterial culturing were taken from the suspensions at 5 min, 1 h and 24 h after adding the bacteria. RESULTS: Dentine powder had an inhibitory effect on all medicaments tested. The effect was dependent on the concentration of the medicament as well as on the length of the time the medicament was preincubated with dentine powder before adding the bacteria. The effect of calcium hydroxide on E. faecalis was totally abolished by the presence of dentine powder. Similarly, 0.2/0.4% iodine potassium iodide lost its effect after preincubation for 1 h with dentine before adding the bacteria. The effect of 0.05% chlorhexidine and 1% sodium hypochlorite on E. faecalis was reduced but not totally eliminated by the presence of dentine. No inhibition could be measured when full strength solutions of chlorhexidine and iodine potassium iodide were used in killing E. faecalis. CONCLUSIONS: The dentine powder model appears to be an efficient tool for the study of interactions between local endodontic medicaments, dentine, and microbes.

Radiation sensitivity of Bacillus cereus with and without a crystalline surface protein layer.

The radiation sensitivity of four strains of Bacillus cereus was investigated with attention to bacterial surface structure. All four strains were sensitive to radiation with gamma rays (D(10)=0.4 kGy). No crystalline surface protein layer could be detected on the cell surface. When cultured on solid media, an S-layer covered the cells of the two strains, and they were 2.6 times as resistant to radiation as the two reference strains without an S-layer. In SDS-PAGE, a major 97-kDa band from the resistant strains from plate cultures was replaced by a ca. 85-kDa protein band in samples from broth cultures. Electron microscopy, SDS-PAGE, Western blot and fluorescent antibody staining indicated that the higher resistance to radiation of the clinical strains from plate cultures was associated with the presence of the S-layer on the cell surface.

Susceptibility of oral Candida species to calcium hydroxide in vitro.

AIM: The susceptibility of common oral Candida species to saturated aqueous calcium hydroxide solution was studied. METHODOLOGY: The yeast species tested were C. albicans (16 strains). C. glabrata (three strains), C. guilliermondii (three strains), C. krusei (two strains), and C. tropicalis (two strains). At least one reference strain of each species was used; the others were clinical isolates either from persistent apical periodontitis or from marginal periodontitis. The susceptibility of Enterococcus faecalis (ATCC 29212) was studied for comparative purposes. Standardized inocula of the strains were incubated in aqueous calcium hydroxide solution, pH 12.4, for time-periods ranging from 5 min to 6 h. Volumes of 0.1 mL of the test suspension were cultured directly on Brucella blood agar and incubated in air at 37C. The plates were inspected for growth at 24 and 48 h and the colonies were counted. The time required to reduce the number of colony-forming units to less than 0.1% of the initial number was determined for each strain. RESULTS: The sensitivity of the C. albicans strains was generally low, with 16 h of incubation required to kill 99.9% of the colony-forming units. No differences in susceptibility between C. albicans strains isolated from root-canal infections and from periodontitis were found. Both strains of C. tropicalis were killed between 3 and 6 h of incubation, whilst strains of C. guilliermondii were killed after only 1020 min of incubation. All strains of C. glabrata survived 20 min, but not 1 h, of incubation, whilst 13 h were required to kill C. krusei. Compared with E. faecalis, all Candida spp. showed either equally high or higher resistance to aqueous calcium hydroxide. CONCLUSIONS: This study indicates that Candida spp. are resistant to calcium hydroxide in vitro, which may explain the isolation of yeasts from cases of persistent apical periodontitis.

In vitro susceptibility of Candida albicans to four disinfectants and their combinations.

AIM: The aim of this study was to evaluate the susceptibility of seven strains of Candida albicans to four disinfectants: iodine potassium iodide, chlorhexidine acetate, sodium hypochlorite and calcium hydroxide. In addition, all possible pairs of the disinfectants were tested in order to compare the effect of the combination and its components. METHODOLOGY: Filter paper discs were immersed in standardized yeast suspensions and then transferred to disinfectant solutions of different concentrations and incubated at 37 degrees C for 30 s, 5 min, 1 h and 24 h. After incubation the filter paper discs were transferred to vials with PBS and glass beads that were then vigorously shaken for dispersal of the yeast cells. PBS with resuspended yeasts was serially diluted 10-fold. Droplets of 25 microL from each dilution were inoculated on TSB agar plates and incubated in air at 37 degrees C for 24 h. The number of colony-forming units was then calculated from appropriate dilutions. RESULTS: C. albicans cells were highly resistant to calcium hydroxide. Sodium hypochlorite (5% and 0.5%) and iodine (2%) potassium iodide (4%) killed all yeast cells within 30 s, whilst chlorhexidine acetate (0.5%) showed complete killing after 5 min. Combinations of disinfectants were equally or less effective than the more effective component. All C. albicans strains tested showed similar susceptibility to the medicaments tested. CONCLUSIONS: This study indicates that sodium hypochlorite, iodine potassium iodide and chlorhexidine acetate are more effective than calcium hydroxide against C. albicans in vitro. However, combining calcium hydroxide with sodium hypochlorite or chlorhexidine may provide a wide-spectrum antimicrobial preparation with a long-lasting effect.

Fungi in therapy-resistant apical periodontitis.

The occurrence of yeasts in 967 microbiological endodontic samples taken from root canals in persistent endodontic infections was studied. The sampling was done by general practitioners in various parts of Finland from root canal infections which did not respond favourably to standard conservative therapy. The samples were cultivated aerobically on a non-selective enriched horse blood agar medium, on TSBV agar medium in 5% CO2 and anaerobically on horse blood agar medium. Micro-organisms were found in 692 of the samples while 275 showed no growth. Forty-eight fungi were isolated from 47 samples which is 7% of the culture-positive samples. Twenty yeast strains were identified further by their colony morphology, growth and cellular characteristics and patterns of carbohydrate assimilation. All isolates except one belonged to the genus Candida. Candida albicans was the most common species. C. glabrata was found together with C. albicans in one sample. C. guilliermondii, C. inconspicua and Geotrichum candidum were each isolated once. Yeasts were found in pure culture in six samples and together with bacteria in 41 samples. In all the samples except two, the accompanying facultative bacteria were Gram positive. The most frequent of them were alpha- and non-haemolytic Streptococcus species which were found in 31 samples. Anaerobic bacteria were isolated together with yeasts from 12 root canals. They included both Gram positive species such as Peptostreptococcus micros and Gram negative species such as Fusobacterium nucleatum. The regular isolation of yeasts, also in pure culture, indicates that yeasts may have an important role in cases of apical periodontitis persisting after conventional treatment.

Microbiological findings and clinical treatment procedures in endodontic cases selected for microbiological investigation.

The relationship between bacteriological findings and clinical treatment procedures was investigated in root canal treatment cases that were selected for bacteriological investigation by general dental practitioners in Finland. The cultures were sent to the Oral Microbiological Service Laboratory at the Institute of Dentistry in Helsinki. Two groups of teeth were selected based on the type of infection present in the root canal system. The 'enteric bacteria' group consisted of 40 sequential cases where Enterococcus faecalis and/or other facultative enteric bacteria or Pseudomonas sp. were found in the samples in pure culture (35%) or together with other types of bacteria. The group 'non-enteric bacteria' consisted of 40 sequential cases where only non-enteric bacteria were found. The dentists who had sent the bacteriological samples received a questionnaire where they were asked about the treatment protocol and procedures. A total of 70 out of 80 questionnaires were returned. If the root canals had been unsealed at some point during the treatment, enteric bacteria were found more frequently than in canals with an adequate seal between the appointments. Of cases with enteric bacteria 55% had been open during the treatment, while in the group where only non-enteric bacteria were found 30% had been open. Enteric bacteria were also more frequently isolated in cases with a high number of appointments before sampling. In the enteric bacteria group 35% of the samples were taken at the 10th visit or later, while the corresponding percentage in the non-enteric group was 3%. In addition, the number of retreatment cases was significantly higher, 12 out of 34, in the enteric bacteria group than in non-enteric bacteria group, which was five out of 36. Other clinical parameters showed no differences between the two groups. The results emphasize the importance of controlled asepsis throughout the root canal treatment.