PCR detection of 5 putative periodontal pathogens in dental plaque samples from children 2 to 12 years of age

BACKGROUND, AIMS: The purpose of this study was to detect the presence of Prevotella intermedia, P. nigrescens, Bacteroides forsythus, Treponema denticola, and Campylobacter rectus in plaque samples from 119 children, collected from their toothbrushes using a polymerase chain reaction (PCR). METHOD: The subjects were 24, 83, and 12 children with healthy gingiva, gingivitis, and periodontitis, respectively, ranging in age from 2-12 years old. Plaque samples were collected from all erupted teeth sites using a sterile toothbrush. The mean concentration of DNA recovered from the plaque samples was approximately 640 microg/ml, which was deemed sufficient for performing a PCR-based survey. RESULTS: The prevalence by PCR in healthy, gingivitis, and periodontitis subjects was 0.0%, 6.0% and 25.0% for P. intermedia, 45.8%, 79.5% and 50.0% for P. nigrescens, 33.3%, 63.9% and 58.3% for B. forsythus, 0.0%, 18.1% and 16.7% for T. denticola, and 100% in duplicate for C. rectus, respectively. CONCLUSION: Our survey indicated that P. intermedia and T. denticola were more associated with periodontal diseases, B. forsythus and P. nigrescens had a moderate prevalence in all clinical groups, while C. rectus were the most commonly detected species in the oral cavities of children suggesting establishment in their early years.     

Characterisation of black-pigmented anaerobes isolated from diseased and healthy periodontal sites

Prevotella intermedia has recently been re-defined and a new species, Prevotella nigrescens has been proposed. However, there is little data available on the incidence of these new species in periodontal health or disease. Black-pigmented anaerobes isolated from diseased and healthy subgingival sites were identified by serotyping, SDS-PAGE and physiological tests. In adult periodontitis subjects, 64% of active sites, 35.7% of inactive sites and 38.5% of healthy sites yielded black-pigmented anaerobes. Of these, Porphyromonas gingivalis was found in 11% of active and 5% of healthy sites in diseased patients, Prevotella intermedia in 15.5% of active and 20.5% of healthy sites, Prevotella nigrescens in 37.7% of active and 11.5% of healthy sites and Prevotella denticola in 3% of active and 1% of healthy sites. In healthy subjects, 50% of sites yielded black-pigmented anaerobes. P. gingivalis was not found in healthy subjects but P. intermedia was found in 18% and P. nigrescens in 31% of sites. SDS-PAGE proved to be a useful method for routinely differentiating P. intermedia and P. nigrescens and two sub-types of the latter species were detected on the basis of band pattern. Only one P. nigrescens sub-type was found in any given individual and one type, typified by ATCC 25261, was more commonly found in deep pockets. However, overall both P. nigrescens and P. intermedia as species were just as frequently found at healthy sites as diseased sites. Thus, these species, in contrast to P. gingivalis , appear to be common commensals but they may act as opportunistic pathogens.     

Similarity of salivary and subgingival Prevotella intermedia and Prevotella nigrescens isolates by arbitrarily primed polymerase chain reaction

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.     

The distribution of periodontopathic bacteria among Japanese children and their parents

OBJECTIVE AND BACKGROUND: It is not well known how periodontopathic bacteria colonize in the oral cavity during childhood. The purpose of this study was to investigate the distribution of periodontopathic bacteria in oral cavities of children and their parents and the relationship between the bacterial findings and clinical parameters. METHODS: Fifty-six children (mean age: 8.3 +/- 3.5, range: 1-15 years), including 15 with deciduous dentition, 26 with mixed dentition and 15 with permanent dentition, and their parents participated in this study. Whole saliva and dental plaque of the children and whole saliva of their parents were collected for detection of seven species of periodontopathic bacteria (Actinobacillus actinomycetemcomitans, Tannerella forsythensis (Bacteroides forsythus), Campylobacter rectus, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Treponema denticola) using the polymerase chain reaction method. Clinical parameters including simplified Oral Hygiene Index and Papillary-Marginal-Attachment Index were recorded for the children and their accompanied parents. RESULTS: The detection frequencies of T. forsythensis, C. rectus, P. nigrescens, T. denticola, A. actinomycetemcomitans and P. gingivalis in the oral cavities of children were 42.9%, 94.6%, 42.9%, 48.2%, 1.8% and 8.9%, respectively. T. forsythensis, P. gingivalis and T. denticola were detected more frequently in the saliva of parents (54.8%, 54.8%, 88.1%, respectively) than in the saliva of children (25.5%, 7.3%, 41.8%, respectively). Different detection frequencies of P. nigrescens were found among the oral cavities of children with deciduous, mixed and permanent dentitions. In mixed dentition, females harbored T. forsythensis more frequently than males did. Children who harbored T. forsythensis, P. intermedia, P. nigrescens and T. denticola showed high scores for oral debris measurement by simplified Oral Hygiene Index. T. forsythensis, P. intermedia and P. nigrescens were detected more frequently in children whose parents were positive for these pathogens than in children whose parents were negative. CONCLUSIONS: High plaque retention seems to promote the colonization of periodontal pathogens in the oral cavities of children. T. forsythensis, P. intermedia and P. nigrescens were detected more frequently in the oral cavities of children whose parents already harbored these bacteria. Familial transmission of these bacteria is suggested.     

Subgingival microbial profile of Papillon-Lefévre patients assessed by DNA-probes

Abstract. The prevalence of 18 selected bacterial species was assessed by means of "checkerboard" DNA-DNA hybridisation in a group of 12 Saudi-Arabian adolescents with Papillon-Lefévre syndrome. A total of 36 tooth sites were investigated. The patients exhibited severe periodontal disease with deep pockets. All 12 patients harboured the putative bacterial pathogens P. intermedia, F. nucleatum, P. micros and S. intermedius while T. denticola, B. forsythus, P. nigrescens, E. corrodens, S. noxia and C. rectus were recovered from 11 patients. P. gingivalis was recovered from 9 patients and 18 sites while corresponding figures for A. actinomycetemcomitans were 8 and 19, respectively A number of the investigated species (B. forsythus, T. denticola, P. intermedia, C. rectus) reached high levels (≥10⁶ cells) in more than 1/2 of the patients. On the other hand, bacteria such as A. actinomycetemcomitans and P. gingiyalis were infrequently encountered at high levels in these subgingival samples. In conclusion, the analysis failed to demonstrate a PLS-specific profile of the subgingival infection, since the bacterial composition of the sampled sites closely resembled that characterising deep pockets in adult periodontitis patients.     

Hydrolytic and depolymerising enzyme activity of Prevotella intermedia and Prevotella nigrescens

OBJECTIVE: Prevotella intermedia has been reported to be associated with periodontal disease whilst P. nigrescens has predominantly been isolated from more specific conditions and healthy sites. The aim of the present study was to compare the enzyme activity of these species.
MATERIALS AND METHODS Nine strains of P. intermedio and 12 strains of P. nigrescens were studied. Lipolytic. saccharolytic, nucleolytic and proteolytic activity was determined by traditional microbiological and chromo-genic substrate methods.
RESULTS: All strains hydrolysed gelatine, casein. DNA and RNA. Lipase activity was produced by all strains except P. nigrescens ATCC 33563^T. Lipolytic activity of P. nigrescens strains decreased as the environmental glucose concentration was increased. Only two strains, both P. intermedia , hydrolysed benzyl-arg-p-nitroanilide. All strains hydrolysed alkaline pnitrophenolphosphate (except P. intermedia DAL 100). produced glycylprolyl dipeptidase activity and demonstrated elastase-like activity. All but three strains (2 P. intermedia and I P. nigrescens) hydrolysed suc-ala-ala-pro-phe-p-nitroanilide. Overall, no qualitatively analysed enzyme activity was exclusive to all strains of either species. Quantitatively analysed activity exhibited a high degree of variability both within and between species.
CONCLUSIONS: P. intermedia and P. nigrescens degrade natural and synthetic substrates, but intra- and interspec-ies activity is variable.     

Characterization of Prevotella intermedia and Prevotella nigrescens by enzyme production, restriction endonuclease and ribosomal RNA gene restriction analyses

Restriction endonuclease analysis, rRNA gene restriction analysis (ribotyping), multilocus enzyme electrophoresis and lipase production were investigated for their potential to differentiate isolates belonging to the closely-related species Prevotella intermedia and Prevotella nigrescens . Of 122 strains identified originally as P. intermedia , 52 were assigned to P. intermedia and 68 to P. nigrescens using multilocus enzyme electrophoresis. All 39 P. intermedia and 52 out of 53 P. nigrescens tested produced lipase. Restriction endonuclease analysis identified clonal variants, but did not facilitate the differentiation of strains into species. Tag I ribotyping of 99 strains revealed that all P. intermedia demonstrated a species-specific fragment of 0.40 kbp, which was always associated with a second fragment of 0.57 kbp, and all P. nigrescens tested shared a species-specific fragment of 2.21 kbp. Two strains atypical by multilocus enzyme electrophoresis had none of the above species-specific fragments. Thus, lipase production and restriction endonuclease analysis did not distinguish between P. intermedia and P. nigrescens , but Tag I ribotyping did and also allowed the characterization of individual strains.     

Quantitative detection of periodontal pathogens using real-time polymerase chain reaction with TaqMan probes

Quantitative analysis, with identification of periodontopathic bacteria, is important for the diagnosis, therapeutic evaluation and risk assessment of periodontal disease. We developed a highly sensitive and specific method using real-time polymerase chain reaction (PCR) to detect and quantify six periodontal bacteria: Porphyromonas gingivalis, Tannerella forsythia, Actinobacillus actinomycetemcomitans, Treponema denticola, Prevotella intermedia, and Prevotella nigrescens. Species-specific TaqMan probe/primer sets were designed according to 16S ribosomal RNA gene sequences. Plaque and tongue debris specimens were collected from 10 patients with advanced periodontitis and 10 periodontal healthy individuals and analyzed. All species, except for P. nigrescens, were detected in samples from diseased sites in significantly greater numbers than in those from healthy sites, whereas greater numbers of P. nigrescens were found in the controls. These results suggest that the present real-time PCR method with the designed probe/primer sets enabled sensitive detection of the six periodontal bacteria, and may also assist future microbial studies of periodontal diseases.     

Relationship between herpesviruses and adult periodontitis and periodontopathic bacteria.

BACKGROUND: Various mammalian viruses and specific bacteria seem to play important roles in the pathogenesis of human periodontitis. This study examined the relationship between subgingival herpesviruses and periodontal disease and potential periodontopathic bacteria in 140 adults exhibiting either periodontitis or gingivitis. METHODS: A nested-polymerase chain reaction (PCR) method determined the presence of Epstein-Barr virus type 1 and type 2 (EBV-1, EBV-2), human cytomegalovirus (HCMV), and herpes simplex virus (HSV) and a 16S rRNA PCR detection method identified Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Bacteroides forsythus, Prevotella intermedia, Prevotella nigrescens, and Treponema denticola. RESULTS: Using a logistic analysis, EBV-1 showed significant positive association with P. gingivalis (odds ratio [OR] 3.37), and with coinfections of P. gingivalis and P. intermedia (OR 4.03); P. gingivalis and B. forsythus (OR 3.84); P. gingivalis and T. denticola (OR 4.17); P. gingivalis, B. forsythus, and T. denticola (OR 4.06); and P. gingivalis, P. nigrescens, and T. denticola (OR 3.29). EBV-1 also showed positive association with severe periodontitis (OR 5.09), with increasing age (OR 1.03), and with periodontal probing depth at the sample sites (OR 1.77). HCMV was positively associated with coinfections of P. gingivalis and P. nigrescens (OR 3.23); P. gingivalis, B. forsythus, and P. nigrescens (OR 3.23); and P. gingivalis, P. nigrescens, and T. denticola (OR 2.59); with severe periodontitis (OR 4.65); and with age (OR 1.03). Patients with mixed viral infections revealed significant associations with P. gingivalis (OR 2.27), and with coinfections of P. gingivalis and B. forsythus (OR 2.06); P. gingivalis and P. nigrescens (OR 2.91); P. gingivalis, B. forsythus, and P. nigrescens (OR 2.91); and P. gingivalis, P. nigrescens, and T. denticola (OR 2.70) with the clinical diagnosis of slight (OR 3.73), moderate (OR 3.82), or severe periodontitis (OR 4.36), and with probing depth at the sample sites (OR 1.39). HSV and EBV-2 showed no significant associations with any of the variables tested. CONCLUSIONS: The results indicate that subgingival EBV-1, HCMV, and viral coinfections are associated with the subgingival presence of some periodontal pathogens and periodontitis. Herpesviruses may exert periodontopathic potential by decreasing the host resistance against subgingival colonization and multiplication of periodontal pathogens.     

Detection of Prevotella intermedia in subgingival plaque of adult periodontitis patients by polymerase chain reaction

A PCR assay was developed that could specifically amplify DNA from the periodontal pathogen Prevotella intermedia. A pair of primers was selected from regions of the 16S rRNA gene of P. intermedia that were both divergent in sequence at their 3' ends with respect to the corresponding regions of the 16S rRNA gene of P. nigreseens , its most closely related species, and used in the PCR assay. Positivity was indicated by amplification of an 855 bp product. Using purified genomic DNA from these 2 species, assay conditions were determined under which only P. intermedia DNA and not P. nigrescens DNA was amplifiable. Absolute specificity of the assay was confirmed by the fact that no amplification products were obtained when using DNA from several other important periodontal organisms. The optimized PCR assay was used to identify P. intermedia in subgingival plaque samples of patients with adult periodontitis. Confirmation of amplification of P. intermedia DNA was achieved by digestion of PCR products with the restriction endonuclease R sal, which gives different restriction patterns for P. intermedia and P. nigrescens. Of the 97 samples analysed, 38 (39%) were positive for P. intermedia. The results obtained confirm P. intermedia as a possible aetiological agent of adult periodontitis. Additionally, PCR primers targeting the corresponding region of the 16S rRNA gene of P. nigrescens were shown to be specific for the organism when used in a PCR assay, although P. nigrescens was not detectable in any of the subgingival plaques analysed.     

The Prevotella intermedia group organisms in young children and their mothers as related to maternal periodontal status

Currently, the Prevotella intermedia group includes three biochemically and phylogenetically related species: Prevotella intermedia, Prevotella nigrescens, and the newly described Prevotella pallens. The two first-named species are mentioned with varying emphasis in connection with periodontal diseases, while such a connection of P. pallens is not known. Mothers serve as a plausible source of bacteria to their children, and conceivably, a mother with periodontitis as a recurrent reservoir of periodontally infecting organisms. In the present study, 23 mothers and their young children were examined for the presence of the P. intermedia group organisms in relation to maternal periodontal status (I: periodontal health, II: initial periodontitis, and III: advanced periodontitis). Species differentiation was based on established biochemical methods, electrophoretic mobility patterns, SDS-PAGE, and DNA hybridization. P. intermedia was not recovered from children but nearly exclusively from mothers in group III, thus confirming its association with periodontitis. P. nigrescens and P. pallens were frequently found in mothers and children. To determine bacterial transmission between a mother and her child, 72 isolates from 13 mother-child pairs were analyzed by arbitrarily primed PCR (AP-PCR). Similar AP-PCR types of P. nigrescens and/or P. pallens were recovered from 3/4 pairs in group I, 2/5 pairs in group II, and none in group III. Our results indicate that different species within the P. intermedia group have a different colonization pattern in childhood and that the periodontal status reflects qualitatively their presence in maternal saliva. Intra-familial transmission of P. nigrescens and P. pallens can occur in early childhood, however similar AP-PCR types were most obvious within periodontally healthy mother-child pairs.     

β-lactamase-producing strains in the species Prevotella intermedia and Prevotella nigrescens

A total of 96 strains were collected that included laboratory strains and clinical isolates classified Prevotella intermedia sensu lato and the type strains of the species P. intermedia sensu stricto and Prevotella nigrescens. Susceptibility to amoxicillin and amoxicillin-clavulanic acid was determined by the Etest. PCRDNA probe assays were used to speciate each strain as P. intermedia sensu stricto or P. nigrescens. By Etest, 71 strains (74%) were susceptible to both amoxicillin and amoxicillin-clavulanic acid with minimum inhibitory concentrations in the 0.016–0.064 μg/ml range. In contrast, amoxicillin minimum inhibitory concentrations of 25 strains (26%) were in the range of 1.5–96 μg/ml with concomitant amoxicillin-clavulanic acid minimum inhibitory concentrations in the low range 0.016–0.38 μg/ml, indicating a production of β-lactamase as confirmed by nitrocefin tests. Of these β-lactamase-producing strains, 20% (5/25) were identified as P. intermedia sensu stricto by the PCR-DNA probe assay and 72% (18/25) as P. nigrescens. Our results provide support for the major role of P. nigrescens in the failure of therapy using β-lactam antibiotics.     

Occurrence of periodontal bacteria in healthy children: a 2-year longitudinal study

OBJECTIVES: To examine the occurrence of specific periodontal bacteria in children and adolescents. METHODS: Ten putative periodontal bacteria were longitudinally examined in plaque and saliva samples from 119 periodontally healthy children (2-15 years old) using a polymerase chain reaction method. RESULTS: Capnocytophaga ochracea, C. sputigena, and Actinobacillus actinomycetemcomitans were frequently found in saliva, and tended to persist in saliva for the remainder of the study, whereas Porphyromonas gingivalis, Treponema denticola, and Prevotella intermedia were rarely detected. P. nigrescens was more frequently detected in plaque and its prevalence increased with age. Eikenella corrodens and Campylobacter rectus were sometimes detected in both plaque and saliva, while Tannerella forsythensis was occasionally detected in saliva. CONCLUSION: A. actinomycetemcomitans, C. ochracea, C. sputigena, P. nigrescens, C. rectus, and E. corrodens are common members of the oral microbial flora of healthy children, whereas P. gingivalis, P. intermedia, and T. denticola appear to be transient organisms.     

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polymerase chain reaction for differentiation of Prevotella intermedia and Prevotella nigrescens.

Isolates previously thought to be Prevotella intermedia have been shown to be a closely related species now known as Prevotella nigrescens. The purpose of this study was to determine the efficacy of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and polymerase chain reaction (PCR) to differentiate endodontic isolates of P. nigrescens from P. intermedia. Fifty-six strains of black-pigmented bacteria isolated from endodontic infections and conventionally identified as P. intermedia were used in this study. Using SDS-PAGE, novel polypeptide bands were used to differentiate P. nigrescens from P. intermedia. PCR was accomplished with specific primers for the 16S ribosomal RNA gene of both strains. Of 56 endodontic isolates, 41 (73%) strains were identified by SDS-PAGE as P. nigrescens and 15 (27%) strains as P. intermedia. Of the 41 strains of P. nigrescens identified by SDS-PAGE, PCR identified 37 strains as P. nigrescens. Restriction endonuclease digestion of amplified 16S ribosomal RNA genes indicated that the remaining four strains originally identified by SDS-PAGE as P. nigrescens were actually strains of Prevotella distinct from P. nigrescens and P. intermedia. Of 15 strains of P. intermedia identified by SDS-PAGE, PCR identified 14 strains as P. intermedia; but, one strain was identified as P. nigrescens. The results indicated that PCR was a more precise method than SDS-PAGE to differentiate P. intermedia from P. nigrescens. This study confirms that P. nigrescens is more commonly isolated in pure culture from endodontic infections than P. intermedia.     

Does the frequency of Prevotella intermedia increase during pregnancy?

Introduction:  The former Bacteroides intermedius , currently including Prevotella intermedia and Prevotella nigrescens , has been associated with hormone-induced pregnancy gingivitis. The aim of the present longitudinal study was to determine whether only P. intermedia or P. nigrescens , or both species, are involved in the demonstrated microbial shift during pregnancy.
Methods:  Subgingival plaque and saliva samples, collected from 30 healthy pregnant women and 24 healthy non-pregnant women as their controls, were examined for the presence of pigmented gram-negative anaerobes. Altogether 2628 isolates were preliminarily identified as P. intermedia sensu lato , based on phenotypic testing. Their further identification was performed by using a 16S ribosomal DNA-based polymerase chain reaction (PCR).
Results:  A mean of 8.3 P. intermedia sensu lato isolates from each subject/sampling was examined. During the second trimester, the mean number of P. intermedia sensu lato in plaque increased along with increasing signs of pregnancy gingivitis, and then both decreased. After delivery, gingival inflammation still decreased while the number of P. intermedia sensu lato transiently increased both in plaque and saliva. In the present study, the vast majority of isolates (95.3%) proved to be P. nigrescens and 2.5% were P. intermedia . The remaining 2.2% of the isolates could not be identified with PCR as P. intermedia or P. nigrescens . The corresponding percentages in the control population were 94.2%, 5.5%, and 0.3%.
Conclusion:  In the oral cavity of relatively young women without periodontitis, P. nigrescens , unlike P. intermedia , is a frequent finding. Conceivably, pregnant women harbor increasing numbers of P. nigrescens associated with pregnancy gingivitis.     

Microbial analysis of canals of root-filled teeth with periapical lesions using polymerase chain reaction

The objective of the present study was to investigate the presence of nine bacterial species in root-filled teeth associated with periapical lesions using a polymerase chain reaction analysis and to correlate these species with clinical features of the cases. DNA was extracted from 45 canal samples of root-filled teeth with periapical lesions. A PCR assay using species-specific primers of 16S rDNA and the downstream intergenic spacer region was used for microbial detection. Enterococcus faecalis was the most prevalent species, detected in 77.8% of the study teeth, followed by Peptostreptococcus micros, detected in 51.1%. Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Prevotella nigrescens were detected in 35.6%, 22.2%, 11.1%, and 11.1% of the sampled teeth, respectively. Moreover, PCR detected Filifactor alocis in 26.7%, Treponema denticola in 24.4%, and Tannerella forsythia in 4.4% of the samples. T. denticola and P. micros were statistically associated with tenderness to percussion (p < 0.05). P. nigrescens was associated with the presence of spontaneous pain and abscess (p < 0.05). P. endodontalis and P. nigrescens were associated with purulent exudates (p < 0.05). Synergistic relationship was also observed between some species. The results of this study indicated that E. faecalis was the most frequently identified test species by PCR in teeth with failing endodontic treatment.     

β-lactamase-producing strains in the species Prevotella intermedia and Prevotella nigrescens

A total of 96 strains were collected that included laboratory strains and clinical isolates classified Prevotella intermedia sensu lato and the type strains of the species P. intermedia sensu stricto and Prevotella nigrescens. Susceptibility to amoxicillin and amoxicillin-clavulanic acid was determined by the Etest. PCRDNA probe assays were used to speciate each strain as P. intermedia sensu stricto or P. nigrescens. By Etest, 71 strains (74%) were susceptible to both amoxicillin and amoxicillin-clavulanic acid with minimum inhibitory concentrations in the 0.016–0.064 μg/ml range. In contrast, amoxicillin minimum inhibitory concentrations of 25 strains (26%) were in the range of 1.5–96 μg/ml with concomitant amoxicillin-clavulanic acid minimum inhibitory concentrations in the low range 0.016–0.38 μg/ml, indicating a production of β-lactamase as confirmed by nitrocefin tests. Of these β-lactamase-producing strains, 20% (5/25) were identified as P. intermedia sensu stricto by the PCR-DNA probe assay and 72% (18/25) as P. nigrescens. Our results provide support for the major role of P. nigrescens in the failure of therapy using β-lactam antibiotics.     

Presence and antibiotic resistance of Porphyromonas gingivalis,Prevotella intermedia,and Prevotella nigrescens in children

BACKGROUND/AIMS: Only limited information exists about the prevalence in children of pathogens associated with periodontitis. The aim of the present study was to determine by culture whether 8-11-year-old children carry Porphyromonas gingivalis, Prevotella intermedia, and/or P. nigrescens in samples from the gingiva and/or the buccal mucosa taken before, and after caries treatment and oral hygiene instruction. A second aim was to assess the proportion of subjects who had gram-negative anaerobes carrying the tet(Q) and erm(F) genes, suggesting antibiotic resistance to tetracycline or erythromycin. METHOD: A total of 150 children provided gingival and buccal swab bacterial samples that were cultured for P. gingivalis, P. intermedia, and P. nigrescens. The species was verified using DNA-DNA hybridization with species-specific probes made from the variable region of the 16S rRNA sequences. Antibiotic-resistant genes, tet(Q) and erm(F), were identified using specific DNA-DNA hybridization with specific DNA probes. RESULTS: A total of 116 isolates of black-pigmented bacteria were cultured from 47 (31%) of 150 children. Five isolates were identified as P. gingivalis, 29 as P. intermedia, 33 as P. nigrescens, and 49 as other species. In general, the bacteria were not culturable at more than one time period. We found that 55% of these 47 children harbored black pigmented bacteria that carried either one or both of the two antibiotic-resistant genes studied (tet(Q), and erm(F)). CONCLUSION: The present study demonstrated that children not exposed to regular dental treatment carry bacteria outside the gingival sulcus that have been associated with periodontitis, and that standard treatment procedures may not clear the presence of the putative pathogens. In addition, antibiotic-resistant genes are common in identifiable gram-negative anaerobes, including putative pathogens.     

Colonization pattern of periodontal bacteria in Japanese children and their mothers

Background and Objective:  The purpose of this study was to determine the time of infection by anaerobic gram-negative rods associated with periodontal disease, and to clarify their transmission from mother to child.
Material and Methods:  Seventy-eight Japanese children (including 10 siblings), aged from 3 to 9 years, and 68 mothers, were enrolled in this study. Colonization by 11 periodontal bacterial species was determined using polymerase chain reaction amplification of samples of subgingival plaque obtained from the children and their mothers.
Results:  The detection rates of Porphyromonas gingivalis , Tannerella forsythensis and Treponema denticola increased in children after the age of 6 years. We found a high consistency in colonization by P. gingivalis , T. denticola , Prevotella intermedia and Prevotella nigrescens in 9 of the 10 siblings. The average number of bacterial species in plaque samples harboring Fusobacterium nucleatum and/or Fusobacterium periodonticum was significantly greater than in those without, in both children and mothers. Kappa statistical analysis revealed that the detection of Capnocytophaga gingivalis , Capnocytophaga ochracea , Campylobacter rectus and T. denticola in children was consistent with that in the mother.
Conclusion:  Periodontal bacterial colonization in Japanese children increased with age and was associated with F. nucleatum and/or periodonticum , and the bacterial flora in children was similar to that in their mothers.     

Clustering of subgingival microbial species in adolescents with periodontitis

It has become increasingly recognized that groups of microorganisms interact within the subgingival plaque of adult subjects with periodontitis. It is much less clear, however, whether the consortia of microorganisms associated with periodontitis are different in early and more advanced cases of periodontitis. To investigate this point further, subgingival plaque was collected from six sites in 87 adolescents with periodontitis and 73 controls and the samples were analyzed for the detection of 18 microbial species using the DNA-DNA hybridization technique. Actinomyces oris accounted for the highest proportion of the flora and was more predominant among controls. Prevotella nigrescens, Prevotella intermedia, Porphyromonas gingivalis, and Tannerella forsythia were present at higher levels among the subjects with periodontitis. Factor analyses identified one factor characterized by highly positive loadings for T. forsythia, Campylobacter rectus, P. gingivalis, P. intermedia, P. nigrescens, Parvimonas micra, and Treponema denticola, and another factor characterized by highly positive loadings of A. oris, Capnocytophaga ochracea, Eikenella corrodens, Streptococcus intermedius, Selenomonas noxia, Streptococcus oralis, Streptococcus sanguinis, and Veillonella parvula. Aggregatibacter actinomycetemcomitans and Streptococcus mutans did not load on any of the two factors, while Fusobacterium nucleatum loaded on both. These findings confirm the occurrence of clustering of subgingival bacteria according to case status also among young individuals.