Chlorine kinetic isotope effects on the haloalkane dehalogenase reaction.

We have found chlorine kinetic isotope effects on the dehalogenation catalyzed by haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 to be 1.0045 +/- 0.0004 for 1,2-dichloroethane and 1.0066 +/- 0.0004 for 1-chlorobutane. The latter isotope effect approaches the intrinsic chlorine kinetic isotope effect for the dehalogenation step. The intrinsic isotope effect has been modeled using semiempirical and DFT theory levels using the ONIOM QM/QM scheme. Our results indicate that the dehalogenation step is reversible; the overall irreversibility of the enzyme-catalyzed reaction is brought about by a step following the dehalogenation.

Systemic diseases caused by oral infection.

Recently, it has been recognized that oral infection, especially periodontitis, may affect the course and pathogenesis of a number of systemic diseases, such as cardiovascular disease, bacterial pneumonia, diabetes mellitus, and low birth weight. The purpose of this review is to evaluate the current status of oral infections, especially periodontitis, as a causal factor for systemic diseases. Three mechanisms or pathways linking oral infections to secondary systemic effects have been proposed: (i) metastatic spread of infection from the oral cavity as a result of transient bacteremia, (ii) metastatic injury from the effects of circulating oral microbial toxins, and (iii) metastatic inflammation caused by immunological injury induced by oral microorganisms. Periodontitis as a major oral infection may affect the host's susceptibility to systemic disease in three ways: by shared risk factors; subgingival biofilms acting as reservoirs of gram-negative bacteria; and the periodontium acting as a reservoir of inflammatory mediators. Proposed evidence and mechanisms of the above odontogenic systemic diseases are given.

Extraradicular infection: a methodological study.

Whether bacteria live or die in periapical lesions of endodontic origin is debated. Sampling of periapical bacteria is difficult due to possible contamination from the indigenous microflora. The aim of this study was to examine whether bacteria were present in periapical lesions of asymptomatic teeth before sampling or were transferred there during sampling. Thirty patients with root-filled teeth and periapical radiolucencies were divided into two groups, each containing 15 patients. In Group 1, a marginal incision was made to explore the periapical lesion. In Group 2, a submarginal incision was made. Before incision, the gingiva and mucosa were washed with 0.2% chlorhexidine gluconate. Bacterial samples were taken from the mucosa before reflecting the flap, and from the alveolar bone and the periapical lesion immediately after. All samples were cultured anaerobically on all-purpose and selective media. In Group 1, 12 of the 15 patients (80%) yielded bacteria from their mucosal samples despite the chlorhexidine wash. Bacterial growth was observed in all samples (100%) from the alveolar bone while the periapical lesions gave bacterial growth in 11 of 15 cases (73%). In Group 2, bacteria were cultured from the mucosa in 11 of 15 (73%) patients. Three samples (20%) from the alveolar bone and 10 from the periapical lesions (67%) gave positive growth. The predominant cultivable bacteria were anaerobic. Phenotypic profiling, performed with the data-based API bioMérieux system, indicated that the sampling technique used prevented mucosal bacteria from reaching the exposed bone and the periapical lesions. Profiling also indicated that following marginal incision, bacteria from the periodontal pocket might have reached the underlying tissues by surgeon-released bacteremia or direct translocation. Most organisms detected in the periapical lesions were clearly different from the bacteria present at neighboring sites and appeared to have been there before sampling.

Assessment of periradicular microbiota by DNA-DNA hybridization.

In the present study the "checkerboard" DNA-DNA hybridization technique was used to identify bacteria in periapical endodontic lesions of asymptomatic teeth. Thirty-four patients with root-filled teeth and apical periodontitis were divided into two groups, each containing 17 patients. In Group 1, a marginal incision was performed during surgery to expose the lesion, and in Group 2, a submarginal incision was applied. The gingiva and mucosa were swabbed with an 0.2% chlorhexidine gluconate solution prior to surgery. Bacterial DNA was identified in all samples from the two groups using 40 different whole genomic probes. The mean number (+/- SD) of species detected was 33.7 +/- 3.3 in Group 1 and 21.3 +/- 6.3 in Group 2 (P < 0.001). The majority of the probe-detected bacteria were present in more lesions from Group 1 than from Group 2. The differences were most notable for Campylobacter gracilis, Porphyromonas endodontalis, Propionibacterium acnes, Capnocytophaga gingivalis, Fusobacterium nucleatum ssp. nucleatum, Fusobacterium nucleatum ssp. polymorphum, Prevotella intermedia, Treponema denticola, Streptococcus constellatus and Actinomyces naeslundii I. Bacterial species such as Actinobacillus actinomycetemcomitans and Bacteroides forsythus were detected in more than 60% of the lesions from both groups. Also, P. endodontalis was abundant in periapical tissue. The data supported the idea that following a marginal incision, bacteria from the periodontal pocket might reach the underlying tissues by surgeon-released bacteremia. The study provided solid evidence that bacteria invade the periapical tissue of asymptomatic teeth with apical periodontitis. The detection of much more bacteria with the "checkerboard" DNA-DNA hybridization method than has previously been recovered by anaerobic culture indicated that the endodontic (and periodontal) microfloras should be redefined using molecular methods.

Influence of coronal restorations on the periapical health of endodontically treated teeth.

The purpose of the study was to evaluate a possible relationship between the quality of the coronal restoration, the root canal obturation and the periapical status of endodontically treated teeth. Full mouth series of radiographs from randomly selected patient charts at the Dental Faculty, University of Oslo were examined. A total of 1001 endodontically treated teeth restored with a permanent restoration were evaluated independently by two examiners. According to a predetermined set of radiographic criteria, the technical quality of the root filling of each tooth was scored as either good (GE) or poor (PE), and the technical quality of the coronal restoration was scored as good (GR) or poor (PR). The root and the surrounding structures were then evaluated and according to the periradicular findings, the treatment was categorized as success or failure. The success rate for all endodontically treated teeth was 67.4% (n = 1001). Teeth with root canal posts had a success rate of 70.7% (n = 527) and teeth without posts had a success rate of 63.6% (n = 472). The two groups with technically good endodontics had the highest success rates. In combination with technically good restorations the success rate was 81% (GE + GR, 81%) and combined with technically poor restorations the success rate was 71% (GE + PR, 71%). The two groups with technically poor endodontics combined with either good restorations or poor restorations had significantly lower success rates (PE + GR, 56% and PE + PR, 57%). The technical quality of the endodontic treatment as judged radiographically was significantly more important than the technical quality of the coronal restoration when the periapical status of endodontically treated teeth was evaluated.

Brain abscesses caused by oral infection.

Brain abscesses are rare but can be life-threatening infections. Recent progress in microbiological classification and identification has indicated that they are sometimes caused by oral infection and dental treatment. It has been postulated that oral microorganisms may enter the cranium by several pathways: 1) by direct extension, 2) by hematogenous spread, 3) by local lymphatics, and 4) indirectly, by extraoral odontogenic infection. In the direct extension, oral infections spread along the fascial planes. Hematogenous spreading occurs along the facial, angular, ophthalmic, or other veins which lack valves, through the cavernous sinus and into the cranium. Another hematogenous pathway is through the general circulation. Oral bacteria may cause systemic infections, e.g., endocarditis, and then indirectly initiate brain abscess. Microbiota, complications, and the prevention and management of odontogenic brain abscesses are also discussed in this review.

Anaerobic bacteremia and fungemia in patients undergoing endodontic therapy: an overview.

Oral focal infection, a concept neglected for several decades, is a subject of controversy. Recent progress in classification and identification of oral microorganisms has renewed interest in focal infection. The aim of this study was to use phenotypic and genetic methods to trace microorganisms released into the bloodstream during and after endodontic treatment back to their presumed source--the root canal. Microbiological samples were taken from the root canals of 26 patients with asymptomatic apical periodontitis of single-rooted teeth. The blood of the patients was drawn during and 10 minutes after endodontic therapy. Microorganisms in blood were collected after anaerobic lysis filtration and cultured anaerobically on blood agar plates. The phenotypic methods used for characterization and tracing of microorganisms in blood and root canals were: biochemical and antimicrobial susceptibility test, SDS-PAGE of whole-cell soluble proteins, and gas chromatography of cellular fatty acids. Phenotypic data were verified by DNA restriction patterns and corresponding ribotypes of the root canal and blood isolates by using a computer-assisted system fro gel analysis. All root canals contained anaerobic bacteria. The frequency of bacteremia varied from 31% to 54%. The microorganisms from the root canal and blood presented identical phenotype and genetic characteristics within the patients examined. These characteristics differed between patients. The present study demonstrated that endodontic treatment can be the cause of anaerobic bacteremia and fungemia. The phenotypic and genetic methods used appeared valuable for tracing microorganisms in the blood back to their origin.

Ribotyping of bacteria from root canal and blood of patients receiving endodontic therapy.

DNA restriction patterns and corresponding ribotypes of 26 bacterial isolates (Propionibacterium acnes, Peptostreptococcus prevotii, Fusobacterium nucleatum, Prevotella intermedia, Prevotella nigrescens, Actinomyces israelii, Streptococcus intermedius, and Streptococcus sanguis) recovered from patients with infected root canals and their peripheral blood, collected during and after endodontic therapy, were examined. Eleven additional reference strains including type strains were also examined. Purified DNA was digested with BglI,EcoRI, and HindIII. Hybridization was carried out with a digoxigenin-labeled cDNA probe obtained by reverse transcription of Escherichia coli 16S + 23S rRNA. Ribotypes of the bacteria recovered from root canal and blood showed identical characteristics within the patients, and differed between the patients. The results were confirmed when the similarity coefficient (S(AB)) of the ribotypes from the isolates were assessed by the Dendron computer-assisted system. These results suggested that the bacteria isolated from the blood originated from the root canal.

Distinction of Prevotella intermedia and Prevotella nigrescens from endodontic bacteremia through their fatty acid contents.

Prevotella nigrescens has recently been recognized as a new species distinct from Prevotella intermedia. The distinction is based largely on DNA-DNA hybridization, electrophoretic migration of malate and glutamate dehydrogenase, and peptidase and lipase activities of type strains. Gas chromatography of cellular fatty acids can be a useful adjunct for characterization and identification of bacterial species. In the present study, cellular fatty acid profiles were determined for seven strains of P. intermedia and six strains of P. nigrescens. Six of these 13 strains were isolated from the root canal and blood of three patients during endodontic therapy of teeth with Asymptomatic apical periodontitis. The bacteria were cultivated anaerobically in 10 mL prereduced anaerobically sterilized peptone-yeast extract-glucose broth for 24 h. Dried cells of each isolate were methanolysed and their fatty acid contents determined by the Microbial Identification System software package by MIDI. The data were treated by principal component analysis, which distinguished P. nigrescensfromP. intermedia. Cellular fatty acid profiles of these strains of the species in blood matched the profiles of their respective root canal isolates, as demonstrated by Euclidean Distance Square assessment. This suggested that the organisms in the root canal had spread to the bloodstream during endodontic treatment.

Observation of Saccharomyces cerevisiae in blood of patient undergoing root canal treatment.

In this study, an unusual observation of Saccharomyces cerevisiae isolated from an infected root canal and from the blood of a patient undergoing endodontic therapy of a tooth with asymptomatic apical periodontitis is reported. Phenotypic (biochemical tests, antifungal susceptibility tests and SDS-PAGE of cellular proteins) and genetic (ribotyping) methods were used to characterize the strains. By using these methods it was found that the blood and root canal isolates were identical but differed from S. cerevisiae strains of other sources. It was therefore more than likely that the root canal was the source of the blood isolate and that it had been transferred unintentionally to the bloodstream during root canal treatment.

Electrophoresis of whole-cell soluble proteins of microorganisms isolated from bacteremias in endodontic therapy.

We have previously demonstrated that anaerobic bacteria are the microorganisms most frequently isolated from blood following endodontic therapy of teeth with apical periodontitis. Phenotypic characterisation of the isolates suggested that the bacteria in the blood originated from the root canal. The present experiment using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in an effort to verify these findings, and to further study the microorganisms involved in endodontic bacteremias. Soluble cellular proteins were extracted from 11 reference strains and 26 bacterial isolates recovered from the root canal and blood. These included Propionibacterium acnes, Peptostreptococcus prevotii, Fusobacterium nucleatum, Prevotella intermedia. Actinomyces israelii, Streptococcus intermedius, Streptococcus sanguis. The electrophoretic patterns mostly confirmed the identity of the isolates as determined by the biochemical and antimicrobial resistance tests. Furthermore, with this typing method the species Prevotella intermedia and Prevotella nigrescens could be differentiated. These species had been recovered from both root canal and blood. Also, differences between subspecies of Fusobacterium nucleatum became evident with SDS-PAGE, and the results indicated that the organism recovered from the root canal and blood was Fusobacterium nucleatum subsp. vincentii. The electrophoretic patterns of the different organisms isolated from the root canal and the blood were similar, providing further evidence that the bacteria found in the blood originated from the root canal.

3-hydroxy fatty acids in a lipopolysaccharide-like material from Treponema denticola strain FM.

Treponemes are associated with major oral diseases such as apical and marginal periodontitis. Lipopolysaccharide (LPS) is regarded as an important virulence factor in these diseases. It is unclear whether LPS is present in oral treponemes. Therefore, the present study was undertaken to examine if a common oral treponeme-Treponema denticola-possesses LPS. A modified Westphal method (phenol water-ethanol-hexane extraction) was used to extract LPS-like material from T. denticola, reference strain FM. It was cultivated in prereduced anaerobically sterilized pectin medium. Gas chromatography and gas chromatography-mass spectrometry of the extract detected three hydroxy fatty acids: C3-OH-i-13:0, C3-OH-i-15:0, and C3-OH-16:0 which constituted 12% of its fatty acid content. These acids, commonly regarded as markers of LPS, suggested the presence of LPS in T. denticola.

Characterization of new periodontal and endodontic isolates of spirochetes.

Gas chromatographic analysis of cellular fatty acids (CFA), biochemical reactions, electrophoresis of soluble cellular proteins, as well as immunodiffusion were used to discriminate between 16 fresh spirochete isolates from periodontal and endodontic infections. CFA patterns were compared by the Hewlett Packard MIDI library to all available reference strains, and results of the biochemical tests were compared to VPI's records for treponemal strains. The electrophoretograms of soluble proteins of the fresh isolates were compared to those of previously well described strains of Treponema denticola, T. pectinovorum, T. vincentii, T. socranskii subsp. socranskii, T. socranskii subsp. paredis, T. socranskii subsp. buccale, and T. socranskii 04. Immunodiffusion was carried out by using adsorbed polyclonal rabbit antibodies to representative strains of the species mentioned. These methods separated most clinical strains from approved species strains, suggesting that new species had been isolated.

A new method for routine isolation of oral treponemes using U-tubes and pectin medium.

Determination of the composition of the oral microflora has traditionally been based on cultivation. Treponemes are prevalent in many oral infections but, unfortunately, are not regularly cultured. In this study a new method was established for routine isolation of oral treponemes from clinical samples. Bacterial samples from 47 periodontal pockets and 4 endodontic infections were incubated anaerobically under nitrogen atmosphere at 37 degrees C in U-tubes containing pectin medium. In the U-tube a 'bacterial sample side' and a 'sterile medium side' were established on separate sides of a membrane filter and an agar plug. Using this method we were able to isolate viable treponemes from all bacterial samples. This was in contrast to previously established methods such as the agar dilution technique, the technique involving the membrane filter placed on the surface of solid agar media and the well in agar plate technique. We believe that the 'U-tube method' is a valuable supplement to previously described techniques in routine isolation of treponemes from clinical samples.

Population genetic analysis of oral treponemes by multilocus enzyme electrophoresis.

Seventeen treponemes recently isolated from necrotic pulps, periodontal and periapical infections and 17 previously well characterized oral treponemal strains were analyzed by multilocus enzyme electrophoresis. Ten genetic loci were characterized on the basis of the electrophoretic mobilities of their enzymatic products. All loci were polymorphic. The average number of alleles per locus was 7.8. The genetic diversity among the electrophoretic types at each locus ranged from 0.624 to 0.836 with a mean genetic diversity per locus of 0.751. The 34 strains represented 34 electrophoretic types, constituting 6 main divisions (I-VI) separated at genetic distances greater than 0.75. Several of the previously characterized treponemes revealed multiple bands of enzyme activity at several loci, indicating that they were not pure. The characterized strains usually clustered within established species, whereas fresh clinical isolates overlapped species borders. There was a large genetic difference between some reference and clinical strains, indicating that the latter may contain undescribed species. Treponema socranskii and Treponema denticola strains clustered in distinct divisions (IV and V, respectively), with the exception of T. denticola strain FDC 51B2 and T. socranskii subsp. paredis strain VPI D46CPE1, both previously well described. This indicated that the taxonomic assignment of these 2 strains should be reconsidered.

Bacteremia in conjunction with endodontic therapy.

This study characterizes oral microorganisms believed to have spread from the root canal into the blood stream during and after endodontic therapy of teeth with Asymptomatic apical periodontitis. Microbiological samples were taken under aseptic conditions from the root canal of 26 single-rooted teeth in 26 patients. In the endodontic treatment of 13 of the patients (Group 1), the first 3 reamers, sizes 15, 20 and 25, were used to a level 2 mm beyond the apical foramen. In the other 13 patients (Group 2), the instrumentation ended inside the root canal 1 mm short of the apical foramen. Blood samples were taken from the patients during the instrumentation and 10 min after the treatment was completed. Anaerobic microorganisms were isolated from all root canals. In 7 patients of Group 1, Propionibacterium acnes, Peptostreptococcus prevotii, Fusobacterium nucleatum, Prevotella intermedia and Saccharomyces cerevisiae were recovered from the blood. In 4 patients of Group 2, P. intermedia, Actinomyces israelii, Streptococcus intermedius and Streptococcus sanguis were isolated from the blood. Biochemical tests and antibiograms revealed that the isolates from the root canal and blood had identical profiles within the patients, strongly suggesting that the microorganisms isolated from the blood had the root canal as their source.

Reliability of laser Doppler flowmetry in testing vitality of human teeth.

The aim of this investigation was to study the reliability of the laser Doppler flowmetry (LDF) output signals when testing from 4 different positions on the buccal surface of human teeth. Recordings were made from 10 intact anterior teeth in 10 patients, mean age 27.4 years. Five types of probes were used. Each probe had 3 fibers arranged in a triangle, one lighted fiber and 2 receiving ones. The separation of the fibers was 1500, 1000, 800, 500 and 250 microns. The diameter of the fibers was 200 microns in all probes except the one with the smallest separation of fibers where it was 125 microns. A rubber base splint was used to position the probe on the buccal surface of the teeth tested. The first recording was made with the ingoing light in a gingival position. Then 3 additional recordings were made turning the probe 90 degrees each time consequently changing the position of the ingoing light accordingly. Thus, gingival, mesial, incisal and distal recordings were obtained. The output signals were fed into a lap-top computer upon which all calculations were done. The output signals from the incisalmost position of the teeth were significantly lower than the output signals from the other 3 positions with all probes except one. The turning of the probes into the 4 different positions affected the output signals from the probe with the smallest separation of fibers significantly more than the output signals from the other probes.

Systemic diseases caused by oral microorganisms.

Human endodontic and periodontal infections are associated with complex microfloras in which approximately 150, (in apical periodontitis) and 350 (in marginal periodontitis) bacterial species have been encountered. These infections are predominantly anaerobic, with gram-negative rods being the most common isolates. The anatomic closeness of this microflora to the bloodstream can facilitate bacteremia and systemic spread of bacterial by-products and immunocomplexes. A variety of clinical procedures such as tooth extraction, periodontal and endodontic treatment, may cause translocation of microorganisms from the oral cavity to the bloodstream. The microorganisms that gain entrance to the blood circulate throughout the body, but are usually eliminated by the host (reticuloendothelial system) within minutes. However, in patients with ineffective heart valves or vascular diseases, bacteremia can be a potential danger, leading most commonly to infective endocarditis and myocardial or cerebral infarction. Other forms of systemic diseases such as brain abscesses, hematological infections and implant infections have also been related to oral microorganisms.

Efficacy of laser Doppler flowmetry in determining pulp vitality of human teeth.

The aim of this study was to examine if laser Doppler flowmetry (LDF) could aid in distinguishing teeth with necrotic pulps from vital teeth and if so, which configuration of 5 experimental probes would give the best results. Each probe had 3 fibers arranged in a triangle. One fiber carried the laser light to the pulp tissue and 2 fibers carried the back-scattered light to the detector giving the output signal. The distance between the 3 fibers in the triangular arrangement in each probe was 250, 500, 800, 1000, and 1500 microns. A special rubber-base splint was used to hold the probe in place on the buccal surface of the tested teeth. Eleven anterior teeth with clinically diagnosed necrotic pulp were measured with the LDF method and the results compared to contralateral teeth with vital pulp. For the sake of comparison, 10 pairs of anterior teeth with vital pulps in 10 patients were tested as well. The LDF signals were fed into an analog printer and a lap-top computer where all calculations were done. With the LDF method, the output signals from the teeth with necrotic pulps were significantly lower with all 5 probes than the output signals from the contralateral vital teeth. On average, the signal was 42.7% lower from the teeth with necrotic pulps than from the vital teeth. Four of 11 teeth with necrotic pulp gave a positive response to an electrical pulp tester (EPT). The results suggested that the probe with the smallest separation of fibers was the most sensitive in distinguishing necrotic pulps from vital ones.