Developing teeth as biomarker of dioxin exposure.
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Actinobacillus actinomycetemcomitans isolates from 356 individuals were screened for identification of serotype e in order to investigate its distribution in relation to periodontal status. From subjects with serotype e, 1-6 isolates per subject (n = 61) were genotyped using arbitrarily primed-polymerase chain reaction (AP-PCR) and apaH gene polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis to determine the genetic heterogeneity within the serotype. Furthermore, one serotype e strain per subject was tested for fermentation of 8 carbohydrates for biotyping. Among patients with adult periodontitis (n = 219), localized juvenile periodontitis (n = 55) and other forms of early-onset periodontitis (n = 18) serotypes b, a and c, respectively, were the most frequently detected serotypes. Non-periodontitis subjects (n = 64) were predominantly colonized with serotype c. Serotype e was found in 30 (14%) adult periodontitis patients, 2 (11%) early-onset periodontitis patients and in 5 (8%) non-periodontitis individuals, but in none of the 55 localized juvenile periodontitis patients. AP-PCR distinguished 3 and apaH gene PCR-RFLP analysis 2 genotypes among the 61 A. actinomycetemcomitans serotype e isolates, one genotype per subject. The AP-PCR genotypes 1 and 3 represented the apaH genotype 1 and the AP-PCR genotype 2 the apaH genotype 2. On the basis of variable fermentation of galactose and xylose, 3 biotypes among A. actinomycetemcomitans serotype e were established. Contrary to the absence of A. actinomycetemcomitans serotype e in localized juvenile periodontitis patients, its detection frequency was comparable among other forms of periodontitis and periodontal health. Clinical serotype e isolates form at least 2 genetic types and 3 biotypes.
BACKGROUND: The Gram-negative facultatively anaerobic coccobacillus Actinobacillus actinomycetemcomitans is the major pathogen in localized juvenile periodontitis (LJP) and some forms of adult periodontitis (AP). A. actinomycetemcomitans can be grouped into 5 serotypes (a through e) based on differences in the carbohydrate moiety of cell surface lipopolysaccharide. The A. actinomycetemcomitans population is genetically heterogeneous. Since the studies on A. actinomycetemcomitans colonization have mostly applied only culture techniques, the clonality of the follow-up isolates has not been established. Thus, it is possible that, although A. actinomycetemcomitans could be repeatedly isolated from an individual, the initial colonizing strain was replaced by another strain. The aim of the study was to determine whether oral A. actinomycetemcomitans strains change spontaneously over time or after periodontal treatment. METHODS: A total of 922 A. actinomycetemcomitans isolates were recovered from 115 subjects. From each subject A. actinomycetemcomitans isolates were obtained from 2 to 9 follow-up samples 0.5 to 11.5 years apart. After the first sampling occasion, 99 subjects were treated for either LJP or AP, whereas the 16 non-periodontitis subjects received no treatment. All A. actinomycetemcomitans isolates were serotyped and 235 isolates from 52 subjects genotyped with AP-PCR and/or with ribotyping. RESULTS: Isolates of only one serotype, or non-serotypeable isolates alone, were repeatedly found in 104 subjects; serotype a occurred in 25%, b in 33%, c in 23%, d in 5%, e in 7%, and non-serotypeable isolates in 8% of these subjects. Two serotypes (or serotypeable isolates together with non-serotypeable isolates) occurred simultaneously in 9 subjects and in each of these subjects at least one of the serotypes was detected at each sampling occasion. In one subject the initial serotype reappeared although a different serotype was once seen alone, whereas in another subject the initial serotype could not be recovered later. Identical genotypes of A. actinomycetemcomitans were repeatedly detected in each of 52 subjects with follow-up isolates of the same serotype. CONCLUSIONS: The results showed that spontaneous or treatment-induced change in the oral A. actinomycetemcomitans strain(s) is extremely rare and that colonization with the same strain(s) seems to be remarkably persistent.
The production of bacteriocin-like inhibitory substances, mutacins, by mutans streptococci varies among isolates. To find if the degree of mutacin activity of an isolate was related to its transmission between mother and her child, 19 mothers and their 18-month- to 3-year-old children were sampled for their oral mutans streptococci. In addition, the stability of mutacin activity was studied with isolates from the mothers and with isolates from five unrelated 5-year-old children in 5- to 7-year follow-up studies. A total of 145 oral mutans streptococcal isolates were serotyped by immunodiffusion, ribotyped, and mutacin typed by the stab culture technique. Mutacin was produced by 88% of the strains against more than 1 of the 14 indicator strains, representing mutans streptococci, Streptococcus sanguis, Streptococcus salivarius, Streptococcus oralis, Streptococcus gordonii, and Streptococcus pyogenes. Streptococcus mutans isolates showed more inhibitory activity than did Streptococcus sobrinus isolates. Identical ribotypes had similar mutacin activity profiles within a subject, initially and in the follow-up studies, in all but two cases. The mothers harbored a total of 37 different mutans streptococcal ribotypes. Six children were negative for mutans streptococci. Transmission was probable in 9 of 20 mother-child pairs on the basis of the presence of identical strains, as determined by ribotyping and bacteriocin (mutacin) typing. S. mutans strains shared between a mother and her child showed a broader spectrum of inhibitory activity than did nontransmitted strains. In conclusion, the mutacin activity of clinical isolates is reasonably stable, and this virulence factor seems to be of clinical importance in early colonization by S. mutans.
Simple sample-processing methods for PCR detection of Porphyromonas gingivalis, a major pathogen causing adult periodontitis, from saliva were studied. The ability to detect P. gingivalis from 118 salivary samples by PCR after boiling and Chelex 100 processing was compared with bacterial culture. P. gingivalis was detected three times more often by PCR than by culture. Chelex 100 processing of saliva proved to be effective in preventing PCR inhibition and was applied to determine the occurrence of P. gingivalis in saliva samples from 263 Finnish subjects between 5 and 80 years of age. The occurrence of P. gingivalis increased with age, and it was detected by PCR in the saliva of 5.0% of subjects between 5 and 10 years of age, 13.8% of subjects between 11 and 20 years of age, 13.4% of subjects between 21 and 30 years of age, and 63.3% of subjects between 31 and 80 years of age. The results indicate that P. gingivalis is a rare finding in saliva from periodontally healthy children and young adults but a frequent one in saliva from adult periodontitis patients.
Our previous studies have shown that Actinobacillus actinomycetemcomitans isolates of a given arbitrarily primed PCR (AP-PCR) genotype belong to the same serotype (of serotypes a through e). In the present study we investigated whether the AP-PCR genotypes of nonserotypeable A. actinomycetemcomitans isolates match those of the serotypeable isolates. The isolates were additionally characterized by restriction analysis of the apaH PCR amplification products. The material included 75 nonserotypeable and 18 serotypeable A. actinomycetemcomitans isolates from 34 epidemiologically unrelated subjects. The serotypeable isolates were obtained from subjects who also harbored nonserotypeable isolates. Eight AP-PCR genotypes were distinguished among the isolates; six genotypes matched those detected in our previous studies, whereas two genotypes were new. Intraindividually, the A. actinomycetemcomitans isolates produced identical AP-PCR banding patterns, regardless of whether they were serotypeable or nonserotypeable, in 22 of 23 subjects participating with multiple isolates. AP-PCR genotype 3, corresponding to serotype c, was by far the most common among the nonserotypeable isolates (62% of subjects). Results obtained with the apaH restriction analysis confirmed the results obtained with AP-PCR for 31 of the 34 subjects. The results suggest that nonserotypeable A. actinomycetemcomitans isolates originate from serotypeable isolates, especially from serotype c isolates, and the likelihood of the existence of additional serotypes is small.
We have previously shown that dioxins at prevailing levels in mothers' milk may cause mineralization defects in the developing teeth of their children. Developmental dental defects have also been reported in rhesus macaques and rats experimentally exposed to dioxin. The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a potent modulator of epithelial cell growth and differentiation. To clarify whether epidermal growth factor receptor (EGFR), implicated in the mediation of the developmental toxicity of TCDD, is involved in dental toxicity, we cultured embryonic molar teeth from EGFR-deficient mice with TCDD, epidermal growth factor (EGF), and both agents in combination. In teeth of the normal embryos, TCDD caused depolarization of odontoblasts and ameloblasts. Consequently, the dentin matrix failed to undergo mineralization, the enamel matrix was not deposited, and cuspal morphology was disrupted. In teeth of the null mutant embryos, only the cuspal contour was mildly modified. EGF alone retarded the molar tooth development of normal embryos, but not that of EGFR-deficient embryos. When coadministered with TCDD, EGF for the most part prevented the adverse effects of TCDD on teeth of the normal embryos. These results show that the interference of TCDD with mouse molar tooth development in vitro involves EGFR signaling. Thus, EGFR may also play a role in the developmental defects that dioxins cause in human teeth. Because EGFR is widely expressed in developing organs, EGFR signaling may even be of general relevance in the mediation of the developmental toxicity of TCDD.
Forty-four clinical isolates of mutans streptococci were examined by a semiquantitative cross-dot assay for in vitro production of glucosyltransferases GTF-I, GTF-SI and GTF-S of Streptococcus mutans, and GTF-I of Strep. sobrinus, using monospecific antibodies. The isolates were obtained from 12 1.5- to 3-year old children, six caries-active and six caries-free, and from their mothers. The isolates were selected originally from 243 isolates and they represented 35 genetically distinct types as analysed by serotyping and ribotyping. 27 isolates were of serotype c, nine of serotype e and eight of serotype g. Mother child pairs shared nine ribotypes, suggesting vertical transmission. The results showed that, when cultured in Todd-Hewitt broth supplemented with 1% glucose, all Strep. mutans isolates produced GTF-I and GTF-S and all except two produced GTF-SI of Strep. mutans. All Strep. sobrinus isolates produced GTF-I of Strep. sobrinus. The Strep. mutans GTF-I, GTF-SI and GTF-S production of isolates exhibiting a different ribotype showed variability. The variability of GTF-SI and GTF-S production was less pronounced for serotype e isolates. The GTF-I production by Strep. sobrinus isolates did not vary. Transmitted strains produced the same levels of GTFs as strains that were distinct (not transmitted). Strep. mutans isolates of caries-active children produced the same levels of GTF-I and GTF-S, but tended to produce lower levels of GTF-SI than isolates of caries-free children. In conclusion, the results suggested that Strep. mutans isolates exhibiting a different ribotype often had differences in production of GTFs. However, no clear superiority of the high-producer over the low-producer strains was found in regard to their colonization or caries promotion in young children.
A total of 4-22 isolates of oral yeasts per subjects from 48 yeast-positive Finnish and American subjects (25 females and 23 males) were phenotyped and genotyped to determine the frequency of simultaneous oral carriage of multiple yeast taxa. An oral sample from either periodontal pockets, oral mucosa or saliva was obtained. All subjects yielded Candida albicans and 3 subjects an additional yeast species (Candida krusei, Candida glabrata or Saccharomyces cerevisiae). The API 20C Aux kit distinguished 9 different carbohydrate assimilation profiles among the C. albicans isolates. Thirty-eight of 46 C. albicans biotype I isolates were categorized in a single numerical profile. PCR analysis, using a random primer OPA-03 and a repetitive primer (GACA)4, detected 2 major genotypic groups among the C. albicans isolates; 44 subjects showing isolates with a "typical" PCR-profile and 4 subjects isolates with an "atypical" PCR-profile. The "atypical" PCR-profile was similar to that of Candida dubliniensis. All C. albicans isolates assimilated xylose, except 5, including the 4 with an "atypical" PCR-profile. No difference was found in distribution of oral yeast species, and of C. albicans phenotypes and genotypes between Finnish and American subjects. The present PCR method may offer a rapid and easy means of distinguishing oral Candida species.
Suspected periodontal pathogens can be transmitted between spouses. The treatment response may be unsuccessful in periodontitis patients, if the spouse harbors these bacteria. The aim of the present 6-month follow-up study was to clarify whether the microbiological treatment outcome of periodontitis patients is related to the detection of suspected periodontal pathogens in the saliva of the spouse. 10 patients with advanced periodontitis and their spouses were included in the study. The patients received mechanical periodontal treatment and 500 mg metronidazole systemically 2x a day for 7 days. The presence of visible plaque, gingival bleeding after probing, suppuration, supragingival and subgingival calculus and pocket depths were assessed at baseline and 1 and 6 months after treatment. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia/Prevotella nigrescens, Campylobacter rectus and Peptostreptococcus micros were cultured from pooled subgingival samples from the patients and from salivary samples from the spouses at corresponding occasions. Periodontal conditions in the patients improved after treatment as determined by the significantly lower values of clinical variables 1 and 6 months after treatment compared to those at baseline. However, the re-emergence of periodontal bacteria after treatment of the patients was not related to the concurrent detection of the respective bacteria in the saliva of the spouses. In this study design, it seemed that the salivary bacterial load in the spouse was of minor importance for the microbiological treatment outcome of the patient.
Recent findings suggest that two major periodontal pathogenes, Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, are transmitted among family members. The authors discuss the evidence of person-to-person transmission of periodontal bacteria, the significance of saliva as a vehicle of transmission and the methods of verifying clonal similarity of bacterial strains obtained from family members. The authors also discuss the prophylactic and therapeutic implications of the person-to-person spread of periodontal bacteria.
Radiographic periodontal status and microbiological findings of periodontal pockets in subjects with Cohen syndrome are presented in this report. This hereditary disorder causes mental retardation, and neutropenia is one feature of the syndrome. Fifteen patients with Cohen syndrome and 15 controls matched for age and sex and, as far as possible, according to the degree of mental retardation were examined. Alveolar bone loss was evaluated from the panoramic radiographs. Two subgingival samples were obtained from the most affected anterior and posterior periodontal sites in each dentate subject and examined for the occurrence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Peptostreptococcus micros, Bacteroides forsythus, and Campylobacter rectus. Subjects with Cohen syndrome had alveolar bone loss more frequently and the bone loss was more extensive (Mann-Whitney U-test: P < 0.05) than in the controls. They also harbored one or several of the putative periodontal pathogens (Mann-Whitney U-test: P < 0.001) more often than the controls. We conclude that subjects with Cohen syndrome have increased susceptibility to early periodontal breakdown which is likely to be associated with neutropenia.
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.
The distribution and the genetic similarity of Prevotella intermedia and Prevotella nigrescens in saliva and in subgingival samples recovered from the same subject were studied in 16 subjects with different periodontal status. The isolates (4 salivary and 4 subgingival P. intermedia/nigrescens group isolates per subject) were identified to species level by hybridization with species-specific oligonucleotide probes, and the clonal analysis was performed using arbitrarily primed polymerase chain reaction (AP-PCR) (all isolates) and ribotyping (isolates from 5 subjects). In addition, the applicability of AP-PCR in differentiating between P. intermedia and P. nigrescens species was tested using 18 P. intermedia and 20 P. nigrescens isolates from 34 subjects. P. intermedia was detected in 7 and P. nigrescens in 14 of the 16 subjects. In all subjects the same species was found both in saliva and in subgingival plaque. In 15 of the 16 subjects, similar AP-PCR types of P. intermedia and/or P. nigrescens between salivary and subgingival samples were found. The salivary and subgingival isolates that were similar by AP-PCR were indistinguishable also by ribotyping. The AP-PCR analysis revealed a P. intermedia or P. nigrescens species-specific AP-PCR product in most isolates. This study indicates that both P. intermedia and P. nigrescens were found both in salivary and in subgingival samples, and both sampling sites within the same individual were usually colonized with identical AP-PCR types of the species. Thus, in addition to a subgingival sample a salivary sample seems to be suitable for detection and clonal analysis of these species. The AP-PCR method proved to be a simple method applicable for differentiation and clonal analysis of P. intermedia and P. 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.
To clarify the source of re-emerging periodontal pathogens after treatment, we compared the ribotypes of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia/Prevotella nigrescens group and Campylobacter rectus before and after treatment in 7 periodontitis patients and in 6 of the spouses. The patients harbored A. actinomycetemcomitans, P. gingivalis, P. intermedia/P. nigrescens group or C. rectus in their subgingival or salivary samples before treatment. The respective bacterial species were not detected 1 month after treatment, but reappeared by 6 months later. When available, 4 random colonies of each of the 4 species were isolated from both subgingival and salivary samples at each sampling occasion, the isolates were subcultured, identified and typed applying pheno- and genotypic intraspecies characterization methods. Altogether 90 strains of A. actinomycetemcomitans, P. gingivalis, P. intermedia/P. nigrescens group and C. rectus were available from 2, 3, 2 and 4 patients, respectively. The pre- and post-treatment ribotypes of A. actinomycetemcomitans-, P. gingivalis- and P. intermedia/P. nigrescens group-isolates were identical in all respective patients. The pre- and post-treatment ribotypes of C. rectus were identical in 1 of 4 patients, whereas 2 patients harbored a previously not detected post-treatment ribotype and 1 patient harbored the initial and a previously not detected post-treatment ribotype. To study the possibility that periodontitis patients may acquire strains from the spouse after treatment, isolates of A. actinomycetemcomitans, P. gingivalis, P. intermedia/P. nigrescens group and C. rectus (n = 95) from the patients' spouses were ribotyped and compared with those of the patients. The patient exhibited his own post-treatment ribotypes, different from those of the spouse, of A. actinomycetemcomitans and P. gingivalis in 1 couple and of P. intermedia/P. nigrescens group and C. rectus in 1 couple. In the 2 patients who harbored a previously not detected post-treatment ribotype of C. rectus, one patient shared the new ribotype with the spouse, whereas the other one did not. Although an exogenous source cannot be fully ruled out, the patient's own oral flora seems to be the main source of re-emerging periodontal pathogens after treatment.
The susceptibilities of 379 clinical mutans streptococcal isolates to chlorhexidine (CHX) were tested by agar dilution according to the standards of the National Committee for Clinical Laboratory Standards. Isolates were obtained from saliva samples of 34 young mothers who had high or moderate salivary levels of mutans streptococci at baseline. Samples were collected on three occasions, before childbirth, when each child was 6 months old, and 1 year later. Of these isolates, 50% were inhibited at 1 microgram of CHX per ml, 90% were inhibited at 2.0 micrograms/ml, and all were inhibited at 4.0 micrograms/ml. The MICs for Streptococcus mutans isolates (serotypes c, e, and f) were lower than those for Streptococcus sobrinus isolates (serotypes d and g). In some subjects, the MICs for isolates of the same serotype were different. This phenomenon was studied by ribotyping isolates (n = 45) from selected subjects (n = 7). It was found that if there were intraindividual differences in the MICs for isolates of the same serotype, then the ribotypes of these isolates were different. In order to decrease the mutans streptococcal infection risk for children, 24 mothers (test group) brushed their teeth periodically with a gel that contained 0.3% CHX digluconate and 0.2% NaF, pH 5.8, between the second and third sampling occasions. The gel was used twice a day for the first 10 days of each month. Development of resistant strains during CHX-NaF gel use was not detected. The serotype distribution of isolates from the test group after 1 year of periodic CHX-NaF gel use did not differ from that at baseline. Periodic CHX-NaF gel brushing did not lead to lower salivary mutans streptococcal counts.
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.