Identification of Bacteroides forsythus in subgingival dental plaque with the aid of a rapid PCR method.

Bacteroides forysthus has been shown to be prevalent among patients with periodontitis. Conventional microbiological methods used to identify this bacterium, however, are laborious and time-consuming and are therefore not well-suited for screening purposes. We have developed a polymerase chain-reaction (PCR) method which is rapid, specific, and simple to perform and does not require other sample pre-treatment except a brief centrifugation. This method was applied to the detection of B. forsythus in subgingival plaque of 58 periodontitis patients. When compared with the results of conventional culturing, the PCR method always confirmed the culture-positive results, while none of the PCR negative samples was shown to be culture-positive. The PCR method appeared to give more than double the number of samples positive for B. forsythus than culturing (89.7% vs. 37.9%). The analysis requires less than 4 hrs to perform, and is specific only to B. forsythus and sensitive enough to detect fewer than 5 bacteria.

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.

Value of some laboratory and clinical measurements in the treatment plan for advanced periodontitis.

In our previous study, we reported that only 13 of 46 adult patients with advanced periodontitis responded well to initial non-surgical periodontal therapy. In the present follow-up study, the remaining 33 patients were randomly treated further using either modified Widman flap surgery or systemic metronidazole. The patients responding unsatisfactorily to this 2nd treatment phase, received supplementary systemic chemotherapy or surgery, respectively. By using this study design, we determined which baseline clinical variables and/or laboratory findings predicted the treatment outcome in these study patients. Clinical variables included the assessment of bleeding, suppuration, probing pocket depth, furcation lesions, relative attachment level and radiographic infrabony defects. Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were cultured from subgingival plaque samples. The specific IgG and IgA antibody levels against 5 serotypes of A. actinomycetemcomitans were determined in serum and saliva. Elastase-like, trypsin-like and general protease activities were assessed from saliva. The bivariate statistical analyses showed that the most pronounced difference between the patients responding well to initial non-surgical therapy (group MC, n = 13), to either supplementary surgery or chemotherapy (group FT1, n = 11), or those responding to the complex therapy (group FT2, n = 17), was the prior extent of periodontal destruction expressed as the proportion of > or = 6 mm deep periodontal pockets. When multiple linear regression was used to investigate the influence of clinical and laboratory findings on the variation of treatment response between the 3 groups, the most significant explanatory factor was the simultaneous presence of subgingival A. actinomycetemcomitans and multiple deep periodontal pockets. None of the immunological or biochemical variables used had any further influence in the model. Pretreatment microbiological examination, especially for the detection of A. actinomycetemcomitans, seems to be a valuable laboratory screening method for identifying complex treatment need in adult patients with advanced periodontitis. However, the evaluation of the extent and pattern of periodontal breakdown remains crucial for choosing the treatment strategy including surgery and/or chemotherapy in A. actinomycetemcomitans-infected adult periodontitis patients.

Distribution and genetic analysis of oral Prevotella intermedia and Prevotella nigrescens.

A total of 344 Prevotella intermedia and nigrescens group isolates from 59 subjects were identified by hybridization with nonradioactively labeled species-specific oligonucleotide probes. Identification of 20 P. intermedia and 46 P. nigrescens isolates was confirmed by analyzing the electrophoretic mobilities of malate and glutamate dehydrogenase enzymes. A total of 111 isolates (32%) were identified as P. intermedia and 233 isolates (68%) as P. nigrescens. Identification performed with oligonucleotide probes and with malate and glutamate dehydrogenase electrophoresis matched perfectly. The distribution of oral P. intermedia and P. nigrescens in various periodontal status groups was investigated in periodontally healthy or diseased individuals. To reveal intra- and interindividual genetic diversity and possible intrafamilial transmission, P. intermedia and P. nigrescens isolates from 16 to 59 subjects, representing 8 married couples, were ribotyped. The stability of colonization was examined in 12 of the 59 subjects, of whom 6 received periodontal treatment and 6 were untreated. All children and periodontally healthy adults and most subjects with initial periodontitis (13/21) harbored only P. nigrescens. Of the 20 subjects with advanced periodontitis, 7 harbored both P. intermedia and P. nigrescens, 7 only P. intermedia and 6 only P. nigrescens. One or two ribotypes of P. intermedia and/or P. nigrescens were found intraindividually. The spouses in 5 of the 8 married couples shared an identical ribotype of P. intermedia or P. nigrescens, whereas ribotypes from unrelated subjects were mostly unique. Colonization was stable, since the same ribotypes were found 1-6 months apart in both periodontally treated and untreated subjects. In conclusion, the study indicates that P. intermedia and P. nigrescens may occur simultaneously in the oral cavity, the colonization is stable and P. intermedia is associated with periodontal diseases. Ribotyping revealed considerable genetic heterogeneity in unrelated subjects, whereas isolates obtained from spouses could represent the same ribotype, which suggests transmission of these species.

Oral colonization by more than one clonal type of mutans streptococcus in children with nursing-bottle dental caries.

By ribotyping the genetic diversity of mutans streptococci in six 1.5-3-yr-old children with nursing-bottle caries and in six caries-free, age-matched children and in their mothers was examined. The proportion of mutans streptococci in the dental plaque of the children and their levels in the saliva of the mothers were also examined. For ribotyping, chromosomal DNA of isolates obtained from the plaque of the children (3-12 isolates per child) and from the saliva of the mothers (4-13 isolates per mother) was digested with restriction endonuclease HindIII. The DNA fragments were hybridized to the plasmid pKK3535 which contains the rRNA operon of the Escherichia coli chromosome. The results showed that children with nursing-bottle caries exposed to frequent consumption of sucrose had a high proportion of mutans streptococci in plaque and four of them were colonized with more than one ribotype, whereas caries-free children had a low proportion of mutans streptococci in plaque and only one of them harboured more than one ribotype. Mothers of children with nursing bottle caries had similar levels and numbers of ribotypes of mutans streptococci in saliva as the mothers of the caries-free children. In both child groups, mothers were probably the main source of infection with mutans streptococci. Thus, children with nursing-bottle caries were not only heavily infected with mutans streptococci but also often colonized with more than one clonal type. In the child's acquisition of such clones, frequent sugar consumption may have an important role.

Simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by a rapid PCR method.

The identification of periodontal pathogens by conventional methods is time-consuming and difficult. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.) and Porphyromonas gingivalis (P.g.) was developed for rapid and easy determination of these risk-indicator bacteria in human periodontal disease. The PCR primers were designed to hybridize to various regions of 16S rRNA genes, and a hot-start technique was used to obtain maximum sensitivity and specificity. This method can detect both of these bacteria in subgingival plaque samples at concentrations as low as 5 to 50 cells per sample. The sensitivity, however, was even 10 times better when the bacteria were analyzed in a water suspension. Since the only step between sample collection and the actual analysis is a brief centrifugation of the patient sample, the detection can be readily carried out in four hours. The performance of the method was studied with 36 patient samples. The results showed that the PCR method detected A.a. (44% vs. 25%, respectively) and P.g. (56% vs. 42%, respectively) more often than the conventional culture in plaque samples. Thus, our multiplex PCR method is rapid and more effective than conventional protocols in detecting these periodontal pathogens.

Salivary levels of suspected periodontal pathogens in relation to periodontal status and treatment.

The primary ecological niche for suspected periodontal pathogens seems to be the subgingival area, even though periodontal pathogens are also frequently recovered from saliva. The interrelationship of different periodontal conditions and the salivary levels of suspected periodontal pathogens is not known. In the present study, salivary levels of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, and Peptostreptococcus micros were determined by bacterial culture and related to clinical periodontal status in 40 subjects with either advanced, moderate, or initial/no periodontitis. Culture-positive subjects harbored the 5 bacterial species in mean numbers ranging from 2 x 10(5) to 6 x 10(7) colony-forming units (CFU)/mL saliva. A. actinomycetemcomitans was found in none and P. gingivalis in one of the subjects with initial periodontitis, whereas both species were found in 33% and 44%, respectively, of the subjects with moderate periodontitis and in 60% and 40%, respectively, of the subjects with advanced periodontitis. The mean numbers of CFU/mL of P. intermedia, C. rectus and P. micros were significantly higher in subjects with advanced periodontitis than in subjects with initial/no periodontitis. Ten patients with advanced periodontitis were treated mechanically and with adjunctive systemic metronidazole, and were re-examined 1 and 6 months after treatment. Periodontal treatment eradicated or significantly reduced the levels of salivary periodontal pathogens for half a year, whereas in untreated subjects, the levels and the detection frequencies generally remained fairly stable. In conclusion, the results showed that the salivary levels of periodontal pathogens reflect the periodontal status of the patient.

Periodontal findings in spouses. A clinical, radiographic and microbiological study.

Clinical, radiographic and microbiological examination of periodontal conditions was carried out in 2 groups of married couples to assess similarities between husband and wife. The diseased probands (n = 10) exhibited advanced periodontitis and the healthy ones (n = 10) were periodontally normal. The clinical examination comprised the assessment of plaque, probing pocket depths, gingival bleeding on probing, suppuration, supragingival and subgingival calculus. The extent and type of alveolar bone loss was determined from panoramic radiographs. Bacterial samples were taken from the 6 deepest and most inflamed periodontal pockets and from stimulated saliva. The samples were cultured for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus and Peptostreptococcus micros. The mean detection frequency of moderately deep pockets (4-5 mm) and deep pockets (> or = 6 mm) was significantly higher in the diseased probands than in their spouses. The mean detection frequency of moderately deep pockets was significantly higher in the spouses of the diseased probands than in the spouses of the healthy ones. Deep pockets were found in 6 spouses of the diseased probands, whereas only in 2 spouses of the healthy ones. Both diseased proband and his/her spouse harbored A. actinomycetemcomitans, P. gingivalis, P. intermedia, C. rectus and P. micros in 4, 6, 9, 9 and 4 couples, respectively. Both healthy proband and his/her spouse harbored the pathogens in 0, 1, 9, 5 and 3 couples, respectively. P. gingivalis was found in 7 spouses of the diseased probands, but only in 2 spouses of the healthy ones.(ABSTRACT TRUNCATED AT 250 WORDS)

The demonstration by ribotyping of the stability of oral Streptococcus mutans infection over 5 to 7 years in children.

The distribution of serotypes and ribotypes of mutans streptococcal isolates obtained from seven unrelated children at 5 and at 10 or 12 yr of age was investigated. For ribotyping, chromosomal DNA from 5 to 13 isolates per subject was digested with restriction endonucleases EcoRI and HindIII. The DNA fragments were electrophoretically separated, blotted on to nylon membrane and hybridized to the plasmid pKK3535, which contains the rRNA operon of the Escherichia coli chromosome. The ribotypes were unique for each child. In five children only one ribotype and serotype (c, e or f) was found. In one child two serotypes (c and f) were found at baseline and only one (serotype c) in the follow-up sample. In one child the same serotype was not found in the baseline (serotype e) and in the follow-up (serotype c) samples. Every child except one had a ribotype that was identical to one found 5-7 yr later. The results suggest that, at the age of 5 yr, infection by Streptococcus mutans has already stabilized and the colonizing strain remains permanent.

Transmission of oral bacterial species between spouses.

The transmission of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and mutans streptococci was studied between 4 married couples who suffered from advanced periodontitis. Of the 20 couples investigated, the 4 in which both spouses harbored A. actinomycetemcomitans and P. gingivalis were chosen for the transmission study. Three of these couples also harbored mutans streptococci. A. actinomycetemcomitans isolates (8-24 per subject) and mutans streptococcal isolates (5-23 per subject) were serotyped by immunodiffusion technique. For ribotyping, chromosomal DNA from A. actinomycetemcomitans isolates (4-5 per subject) and mutans streptococcal isolates (4-11 per subject) was digested with restriction endonucleases ClaI or BglI and HindIII or SmaI, respectively. P. gingivalis isolates (2-15 per subject) were ribotyped by using ClaI, BglI and SmaI. The blotted restriction fragments were hybridized to the plasmid pKK3535, which contains the rRNA operon of the E. coli chromosome. The spouses in 2 couples shared the same sero- and ribotypes of A. actinomycetemcomitans and S. mutans. P. gingivalis ribotypes were identical in 2 couples. The result suggests transmission of oral bacteria between spouses.

One-year trial of the effect of rinsing with an amine fluoride-stannous-fluoride-containing mouthwash on gingival index scores and salivary microbial counts in lymphoma patients receiving cytostatic drugs.

This double-blind study was undertaken to evaluate the effect of twice-daily use of a mouthwash containing 0.025% of fluoride as amine fluoride-stannous fluoride (AmF + SnF) or 0.05% of fluoride as sodium fluoride (NaF) on visible plaque index (VPI) and gingival bleeding index (GBI) and on some salivary micro-organisms in patients suffering from non-Hodgkin or Hodgkin lymphomas. 79 patients were allocated at random to the two mouthwash groups. Mouthwashing began at the start of cancer chemotherapy. Results relating to 45 patients who completed a 1-year rinsing protocol showed a significant decrease in VPI and GBI in the AmF + SnF group. An increase was found in the NaF group. Mean values for stimulated salivary secretion rates and buffering capacities mostly did not differ significantly from baseline values during the study. In both groups, mutans streptococci counts decreased significantly after the study began and remained low in the AmF + SnF group. No corresponding effect was seen in relation to lactobacilli and yeast counts. In the NaF group, lactobacilli counts increased significantly over a year. Significantly more patients reported adverse or unpleasant effects in the AmF + SnF group (52%) than in the NaF group (6%), although both solutions had the same colour and taste. However, all patients continued with rinsing.

Xylitol and the bactericidal effect of chlorhexidine and fluoride on Streptococcus mutans and Streptococcus sanguis.

The present study was made to investigate the effect of xylitol on the bactericidal and bacteriostatic action of chlorohexidine diacetate (CHX) and sodium fluoride (F) in ATCC strains of Streptococcus mutans and S. sanguis. Standardized bacterial cell suspensions were used in tests for bactericidal effect and for inhibition of growth and sucrose fermentation. The results showed no interference of xylitol with the antibacterial effect of CHX and F combinations. Xylitol did not show any additive effect either but appeared inert in the combinations used.

Occurrence of Porphyromonas gingivalis with Prevotella intermedia in periodontal samples.

To further examine the previously suggested inverse relationship between Porphyromonas gingivalis and Prevotella intermedia in periodontal disease, 1016 samples taken from single or multiple (pooled) subgingival sites were cultured anaerobically and examined for the simultaneous occurrence of the microorganisms. P. gingivalis was isolated from 297 (29%) and Pr. intermedia from 501 (49%) samples. P. gingivalis was found as frequently with (14%) as without (15%) Pr. intermedia. The type of sampling had no effect on the occurrence of P. gingivalis with Pr. intermedia. However, female subjects harboured them in combination more frequently than male subjects. The mean proportions of P. gingivalis in the cultivable flora appeared to be lower when found with than without Pr. intermedia. Whether the detection of the combination, or P. gingivalis alone, has clinical relevance needs further clarification.

Caries and saliva in 12-18-year-old diabetics and controls.

Eighty-five 12-18-yr-old adolescents suffering from insulin-dependent diabetes mellitus (IDDM) and their healthy age- and sex-matched controls were investigated with respect to dental caries, salivary flow rate, pH and buffering capacity of saliva, counts for lactobacilli and mutans streptococci, and salivary glucose content. The diabetics had their disease well controlled according to the HbA1 levels. The results showed no statistically significant difference between diabetics and controls in DMF and DMFS indexes and the number of initial caries lesions. Mean number of initial caries lesions was 3.2 in diabetics, 2.3 in controls. Mean stimulated salivary flow rate was 1.2 ml/min in the patients, 1.4 ml/min in the controls. The pH and buffering capacity values were 7.3 and 4.8 in the patients, 7.4 and 5.1 in the controls, respectively. High counts of mutans streptococci (> 10(6) CFU/ml) and lactobacilli (> 10(5) CFU/ml) were observed more often, but not significantly so, among the patients than in the controls. The mean concentration of glucose in saliva was 10.3 micrograms/ml in the patients, 9.7 1 microgram/ml in the controls. Thus, if the patients' IDDM is well controlled, their salivary and caries data does not differ from that of healthy controls.

Effect of extraction of partly erupted third molars on subgingival microorganisms.

This study was made to investigate the effect of extraction of third molars on subgingival microbes in 39 generally and gingivally healthy men with an average age of 20.2 years (SD 0.9). Microbial samples were taken from the pericoronal space of symptom-free partly erupted lower third molars and from the adjacent gingival pockets of the second molars. The samples were cultivated anaerobically. All partly erupted third molars were extracted from 20 subjects. A control group of 19 subjects was left untreated. Microbe sampling was repeated 2 and 5 months postoperatively with highly significant results. It was shown that at baseline the number of black-pigmented gram-negative bacteria and Fusobacterium species was more frequent in third molar than in second molar sites. The total bacterial count decreased significantly at the second molar sites after extraction of the third molars when compared with the control group. Before the extractions, black-pigmented gram-negative bacteria were detected in 45% of the test subjects and Actinobacillus actinomycetemcomitans in 20%. The respective postoperative figures were 30% for black-pigmented gram-negative bacteria and 10% for Actinobacillus actinomycetemcomitans. Capnocytophaga species were not affected by the extractions. The findings suggest that erupting third molars may harbor harmful bacteria that can be reduced by eradicating the foci.

Effect of an experimental antiplaque preparation on salivary microbial counts in military academy cadets refraining from mechanical cleaning of the teeth.

Dental emergencies in the army are mostly due to infections of the teeth, caused by dental plaque micro-organisms. Because practicing normal oral hygiene is restricted during terrain maneuvers, for example, we developed a new antiseptic tablet preparation which can be used to make an antiplaque solution or even chewed and thus mixed with saliva to rinse the mouth. The preparation contains chlorhexidine, fluoride, and xylitol (XYLIHEX) and can be added to the soldier's kit. The efficiency of the preparation on selected oral micro-organisms was tested against mouthwash solutions containing plain chlorhexidine (CHX) and sodium fluoride (F) in 45 military cadets who volunteered to participate in this double-blind cross-over study. The results showed a significant decrease in salivary mutants streptococci after rinsing periods with XYLIHEX and CHX when compared with F (p less than 0.001). Lactobacilli and yeast counts were not affected. The new preparation appeared promising as a new means for improving soldiers' oral hygiene.

Irradiation of human dental tissues with CO2-, Nd:YAG-, and CO2-Nd:YAG combination laser.

Extracted third molars were used to study the effect of Nd:YAG laser irradiation combined with CO2 laser beam on dental hard tissues. The specimens were studied with SEM after lasing and the size of the impact areas and beam penetration into enamel and dentin were planimetrically analyzed. High-energy CO2 laser (e.g. 10 s irradiation with 10 W output energy) penetrated all enamel and dentin. The simultaneous addition of Nd:YAG irradiation to the CO2 beam was found to increase the effect of CO2 laser, while Nd:YAG irradiation alone, used with equivalent energy densities, did not cause any effect on enamel surface. Thus, Nd:YAG laser was found to potentiate statistically significantly the effect of CO2 irradiation, but the morphologic alterations on dental hard tissues, such as crater formation at the beam focus site, appeared to be due to CO2 irradiation alone.

Effect of antiseptic mouthwashes on some clinical and microbiological findings in the mouths of lymphoma patients receiving cytostatic drugs.

51 patients suffering from Hodgkin's disease or non-Hodgkin's lymphoma participated in this double-blind, cross-over study in which 2 antiseptic mouthwashes were tested for their effects on various periodontal index scores and salivary microbial counts. All patients were receiving combination cytostatic treatment based on methotrexate and doxorubicin. The patients (49 +/- 14 years old, 28 men, 23 women) were allotted at random to 2 groups. One rinsed with a 0.12% chlorhexidine gluconate (CHX) solution, the other with a 0.025% amine-stannous fluoride (AmF + SnF) solution 2x daily for 2 weeks. Both groups then continued rinsing with a 0.05% sodium fluoride (F) solution for 2 weeks, before switching over to AmF + SnF or CHX, respectively. All solutions had been prepared in such a way that they had the same colour and taste. Visible plaque index and gingival bleeding index scores were significantly reduced after periods of rinsing with CHX solution (P less than 0.001) and AmF + SnF solution (P less than 0.05). Microbiological cultivations of saliva specimens revealed significant reductions in mutans streptococci immediately after commencing rinsing, while lactobacilli and yeast counts were not affected.

Antineoplastic agents inhibit the growth of Streptococcus mutans and Streptococcus sanguis in vitro.

The effect of methotrexate (MTX) and doxorubicin on the growth, metabolism and ultrastructure of Streptococcus mutans and Streptococcus sanguis was studied in vitro. Both anticancer drugs exerted an inhibitory effect on the oral streptococci. MTX was more inhibitory than doxorubicin. The minimum inhibitory concentrations (MICs) of MTX to S. mutans were 0.25-2.5 micrograms/ml and that of doxorubicin 0.2 mg/ml. The MICs of MTX and doxorubicin to S. sanguis were 0.025 micrograms/ml and 2.0-0.02 mg/ml, respectively. When saliva samples of patients with malignant tumors receiving various doses of MTX were analyzed, MTX was found to be secreted into the oral cavity at concentrations ranging from 0.014 to 4.486 micrograms/ml. The saliva of these patients was also found to inhibit the growth of S. mutans, and the inhibition zones were in accordance with the MIC values observed. The results suggest that anticancer therapy must be taken into account when the salivary microbiological findings of cancer patients are interpreted.

Application of a new mechanical properties microprobe to study hardness of eroded bovine enamel in vitro.

A mechanical properties microprobe technique was used to study bovine tooth specimens that had been immersed in 50 ml of either cola beverage or sports drink for 1 min, 5 min, and 15 min. A Vickers diamond was used to analyze the test and control surfaces at 7V input. This corresponds to a 100 g load of a conventional indentation microhardness apparatus which was used for comparison. The results showed good agreement between the conventional and the microprobe hardness analyses. Enamel softening was found to be significant after 5 min immersion in both acidic drinks, while 1 min immersion did not cause softening. This shows that shorter immersion times than previously used can be applied in dental erosion studies in vitro. Compared with the conventional indentation microhardness measurements, however, the microprobe technique did not offer any advantage, such as assessment of the microelastic properties of enamel, probably because the biologic tissue did not allow reliable data collection during the loading and unloading phases of the instrument.