Genomic integration and expression of the Aggregatibacter actinomycetemcomitans catalase gene in Aggregatibacter aphrophilus.

OBJECTIVE: To test the hypothesis that virulence genes of Aggregatibacter actinomycetemcomitans can be expressed and confer fitness advantages in the closely related Aggregatibacter aphrophilus. DESIGN: Clinical isolates of A. aphrophilus were screened for natural competence with marked genomic DNA from A. actinomycetemcomitans and A. aphrophilus. The gene katA of A. actinomycetemcomitans D7S-1 and its flanking regions were constructed and inserted into a comparable locus in the genome of a naturally competent A. aphrophilus strain by a markerless protocol via natural transformation. Mutants of A. actinomycetemcomitans with or without katA were also constructed by a similar protocol. Discs soaked with either 0.03% hydrogen peroxide or broth culture of Streptococcus gordonii Challis were placed on the agar with cultures of A. actinomycetemcomitans or A. aphrophilus. The size of the growth inhibition zone associated with the disc was measured after 2-day culture. RESULTS: Five of the 13A. aphrophilus strains exhibited a transformation frequency of 10-6 or higher. The intra- and inter-species transformation frequencies were comparable. The inhibition zones for katA-negative strains of A. actinomycetemcomitans or A. aphrophilus were 3- to 7-fold larger than those associated with katA-positive strains (p<0.05). CONCLUSIONS: There was no apparent species barrier for the transfer and expression of A. actinomycetemcomitans katA in A. aphrophilus. The inserted A. actinomycetemcomitans-specific katA gene in A. aphrophilus strain NJ8700 conferred resistance to inhibition by hydrogen peroxide or S. gordonii. The potential to swap genes between these two closely related oral species may be an alternative approach for investigating the virulence determinants of A. actinomycetemcomitans.

Influence of cyclic heating on physical property and biocompatibility of α- and ß-form gutta-percha.

BACKGROUND/PURPOSE: Thermoplasticized techniques with high temperature and repetitive heating in root canal filling may cause degeneration of gutta-percha, producing cytotoxic byproducts and interfering sealing quality. This study was conducted to investigate the influence of cyclic heating on the physical property and biocompatibility of α- and ß-form gutta-perchas. METHODS: Both α- and ß-form gutta-perchas were submitted to two heating processes: continuous heating and cyclic heating. Continuous heating was carried out by heating the samples up to 300°C and 400°C. The samples were then analyzed by differential scanning calorimetry, differential thermal analysis (DTA), and thermogravimetry. For cyclic heating process, samples were heated from 30°C to 200°C for seven cycles and analyzed with DTA and thermogravimetry. For cell adhesion assay, samples were treated (30°C to 200°C, one and seven cycles), submitted to cell culture and examined by scanning electron microscope. RESULTS: Differential scanning calorimetry and DTA indicated that α-form gutta-percha presented a major endothermic peak at 50-57°C, while ß-form gutta-percha showed two major endothermic peaks at 46-50°C and 60-63°C. Total weight loss of ß-form gutta-percha was about 2-fold greater than that of α-form gutta-percha after continuous heating up to 300°C, or cyclic heating for seven times. Scanning electron microscopy showed no obvious difference of cell adhesion on α- and ß-form samples, even with seven cyclic heating or one heating cycle. However, the attachment of the cells to the culture plate (the control) is better than to the gutta-percha samples. CONCLUSION: The increase of heating cycles for α- and ß-form gutta-percha exerts no adverse influence on their biocompatibility. Because the physical property of ß-form gutta-percha becomes unstable when it is heated at over 300°C or subjected to cyclic heating, ß-form gutta-percha may not be recommended for use in thermoplasticized gutta-percha techniques.

A markerless protocol for genetic analysis of Aggregatibacter actinomycetemcomitans.

BACKGROUND/PURPOSE: The genomes of different Aggregatibacter actinomycetemcomitans (A actinomycetemcomitans) strains contain many strain-specific genes and genomic islands (defined as DNA found in some but not all strains) of unknown functions. Genetic analysis for the functions of these islands will be constrained by the limited availability of genetic markers and vectors for A actinomycetemcomitans. In this study, we tested a novel genetic approach of gene deletion and restoration in a naturally competent A actinomycetemcomitans strain D7S-1. METHODS: Specific genes' deletion mutants and mutants restored with the deleted genes were constructed by a markerless loxP/Cre system. In mutants with sequential deletion of multiple genes loxP with different spacer regions were used to avoid unwanted recombinations between loxP sites. RESULTS: Eight single-gene deletion mutants, four multiple-gene deletion mutants, and two mutants with restored genes were constructed. No unintended non-specific deletion mutants were generated by this protocol. The protocol did not negatively affect the growth and biofilm formation of A actinomycetemcomitans. CONCLUSION: The protocol described in this study is efficient and specific for genetic manipulation of A actinomycetemcomitans, and will be amenable for functional analysis of multiple genes in A actinomycetemcomitans.

Microstructural changes of enamel, dentin-enamel junction, and dentin induced by irradiating outer enamel surfaces with CO2 laser.

The microstructural changes of enamel, dentin-enamel junction (DEJ), and dentin after CO(2) laser irradiation were studied. Buccal enamel surfaces of human third molars about 2 mm from the DEJ were irradiated, and the laser tip was moved 5 mm in a mesiodistal direction with 5 s of irradiation time. The output powers were 2 to 10 W in a continuous mode, and the average doses were approximately from 250 to 1,250 J/cm(2). All specimens were examined by a scanning electron microscope, and heat-transfer simulation was also applied to calculate temperature distribution. Surface ablation of enamel and separation of enamel and dentin along the DEJ were observed when the laser power output exceeded 3 W. Heat-transfer simulation indicated that dentin was prone to store more thermal energy than enamel. In conclusion, operation parameters of the CO(2) laser should be carefully selected to avoid thermal damage to the DEJ.

Repair of perforating internal resorption with mineral trioxide aggregate: a case report.

A case of internal resorption with buccal perforation was found in a maxillary central incisor. Because of the extensive lesion and continuous exudation, surgical intervention was used. The apical third was obturated with gutta-percha, and the perforated lesion was repaired with mineral trioxide aggregate. The residual canal space was filled by thermoplasticized gutta-percha technique, and the coronal cavity was restored with composite resin. The symptoms and signs ceased, and the results were satisfactory at 1-year follow-up.