Early microbial succession in redeveloping dental biofilms in periodontal health and disease.

BACKGROUND AND OBJECTIVE: The development of dental biofilms after professional plaque removal is very rapid. However, it is not clear whether most bacterial species return at similar rates in periodontally healthy and periodontitis subjects or if there are differences in bacterial recolonization between supragingival and subgingival biofilms in periodontal health and disease. MATERIAL AND METHODS: Supragingival and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects immediately after professional cleaning. Samples were taken again from seven teeth in randomly selected quadrants after 1, 2, 4 and 7 d of no oral hygiene and analyzed using checkerboard DNA-DNA hybridization. The percentage of DNA probe counts were averaged within subjects at each time-point. Ecological succession was determined using a modified moving-window analysis. RESULTS: Succession in supragingival biofilms from subjects with periodontitis and from healthy individuals was similar. At 1 d, Streptococcus mitis and Neisseria mucosa showed increased proportions, followed by Capnocytophaga gingivalis, Eikenella corrodens, Veillonella parvula and Streptococcus oralis at 1-4 d. At 4-7 d, Campylobacter rectus, Campylobacter showae, Prevotella melaninogenica and Prevotella nigrescens became elevated. Subgingival plaque redevelopment was slower and very different from supragingival plaque redevelopment. Increased proportions were first observed for S. mitis, followed by V. parvula and C. gingivalis and, at 7 d, by Capnocytophaga sputigena and P. nigrescens. No significant increase in the proportions of periodontal pathogens was observed in any of the clinical groups or locations. CONCLUSION: There is a defined order in bacterial species succession in early supragingival and subgingival biofilm redevelopment after professional cleaning.

Subgingival microbiota in adult Down syndrome periodontitis.

BACKGROUND AND OBJECTIVE: The subgingival microbiota in Down syndrome and non-Down syndrome adults receiving periodic dental care was examined for 40 bacterial species using checkerboard DNA-DNA hybridization and the results were related to clinical periodontal attachment loss. MATERIAL AND METHODS: A total of 44 Down syndrome, 66 non-Down syndrome mentally retarded and 83 mentally normal adults were clinically evaluated. This involved, for each subject, the removal of subgingival specimens from three interproximal sites on different teeth; all subgingival samples per subject were then pooled and assessed for the presence and levels of 40 bacterial species using species-specific whole-genomic DNA probes and checkerboard DNA-DNA hybridization. Significant group differences in species proportions averaged across subjects were evaluated using the Kruskal-Wallis test, and associations between subgingival species and mean subject attachment loss within Down syndrome and non-Down syndrome subject groups were quantified using Pearson correlation and multiple linear regression analysis. RESULTS: Down syndrome subjects exhibited greater attachment loss than non-Down syndrome subjects (p=0.05). Most microbial species were present in Down syndrome subjects at levels similar to non-Down syndrome subjects, except for higher proportions of Selenomonas noxia, Propionibacterium acnes, Streptococcus gordonii, Streptococcus mitis and Streptococcus oralis in Down syndrome subjects compared with non-Down syndrome study subjects, higher proportions of Treponema socranskii in Down syndrome subjects compared with non-Down syndrome mentally retarded subjects, and higher proportions of Streptococcus constellatus in Down syndrome subjects compared with mentally normal subjects. Down syndrome adults classified with periodontitis revealed higher subgingival levels of T. socranskii than Down syndrome subjects with no periodontitis (p=0.02). Higher subgingival proportions of S. constellatus, Fusobacterium nucleatum ssp. nucleatum, S. noxia and Prevotella nigrescens showed significant positive correlations (r=0.35-0.42) and higher proportions of Actinomyces naeslundii II and Actinomyces odontolyticus showed negative correlations (r=-0.36 to -0.40), with increasing mean subject attachment loss in Down syndrome adults. CONCLUSION: Individuals with Down syndrome show higher levels of some subgingival bacterial species and specific associations between certain subgingival bacterial species and loss of periodontal attachment. These findings are consistent with the notion that certain subgingival bacteria may contribute to the increased level of periodontal disease seen in Down syndrome individuals and raise the question as to the reason for increased colonization in Down syndrome.

Microbiologic profile of endodontic infections from HIV- and HIV+ patients using multiple-displacement amplification and checkerboard DNA-DNA hybridization.

OBJECTIVE: To compare the microbiota of endodontic infections in necrotic pulp from HIV-negative and HIV-positive subjects. MATERIALS AND METHODS: Root canal samples from necrotic pulp were collected from 40 HIV- and 20 HIV+ subjects. Pulps were amplified using multiple displacement amplification (MDA). Then, checkerboard DNA-DNA hybridization was employed to assess the levels of 107 microbial taxa. The percentage of DNA probe count and the percentage of teeth colonized by each test species were investigated. Significant differences between groups regarding proportions of taxa and prevalence of the test species were sought using the Mann-Whitney test and the Chi-square analysis, respectively. RESULTS: The most prevalent taxa detected were Dialister pneumosintes, Stenotrophomonas maltophilia, Streptococcus sobrinus, Corynebacterium diphteriae, and Helicobacter pylori among HIV- subjects and D. pneumosintes, Prevotella tannerae, Porphyromonas gingivalis, Parvimonas micra, Prevotella nigrescens, and Corynebacterium diphtheriae among HIV+ individuals. D. pneumosintes, C. diphtheria, and C. albicans were the most abundant species in the HIV- group, whereas the predominant taxa in HIV+ samples were P. tannerae, D. pneumosintes and Olsenella uli. P. tannerae, O. uli, Veilonella dispar, Bacteroides fragilis, and Actinomyces meyeri were significantly more abundant in HIV+ samples. CONCLUSIONS: There were significant differences in the prevalence and proportions of specific microbial taxa between HIV- and HIV+ individuals. The root canal microbiota may represent a reservoir of important oral and medical pathogens, mainly in HIV+ individuals.

Microbiota of deciduous endodontic infections analysed by MDA and Checkerboard DNA-DNA hybridization.

AIMS: To evaluate the microbiota of endodontic infections in deciduous teeth by Checkerboard DNA-DNA hybridization after uniform amplification of DNA in samples by multiple displacement amplification (MDA). METHODOLOGY: Forty samples from the root canal system of deciduous teeth exhibiting pulp necrosis with or without radiographically detectable periradicular/interradicular bone resorption were collected and 32 were analysed, with three individuals contributing two samples; these were MDA-amplified and analysed by Checkerboard DNA-DNA hybridization for levels of 83 bacterial taxa. Two outcome measures were used: the percentage of teeth colonized by each species and the mean proportion of each bacterial taxon present across all samples. RESULTS: The mean amount of DNA in the samples prior to amplification was 5.2 (±4.7) ng and 6.1 (±2.3) µg after MDA. The mean number of species detected per sample was 19 (±4) (range: 3-66) to the nearest whole number. The most prevalent taxa were Prevotella intermedia (96.9%), Neisseria mucosa (65.6%), Prevotella nigrescens (56.2%) and Tannerella forsythia (56.2%). Aggregatibacter (Haemophilus) aphrophilus and Helicobacter pylori were not detected. P. intermedia (10%), Prevotella tannerae (7%) and Prevotella nigrescens (4.3%) presented the highest mean proportions of the target species averaged across the positive samples. CONCLUSION: Root canals of infected deciduous teeth had a diverse bacterial population. Prevotella sp. were commonly found with P. intermedia, Prevotella tannerae and Prevotella nigrescens amongst the most prominent species detected.

Salivary cytokine levels in subjects with chronic periodontitis and in periodontally healthy individuals: a cross-sectional study.

BACKGROUND AND OBJECTIVE: Saliva has been proposed as a noninvasive diagnostic fluid that could be used in the diagnosis of oral and systemic diseases. The levels of salivary biomarkers, such as cytokines, could potentially be used as a surrogate to distinguish periodontally healthy individuals from subjects with periodontitis. Therefore, the goal of the present investigation was to determine if the levels of 10 different cytokines in saliva differed between a group of periodontally healthy individuals and a group of subjects with periodontitis. Correlations between the concentrations of these 10 cytokines and clinical parameters of periodontal disease were also examined. MATERIAL AND METHODS: In this cross-sectional study, 74 subjects with chronic periodontitis and 44 periodontally healthy individuals were periodontally examined and had the levels of granulocyte-macrophage colony-stimulating factor, interleukin-1beta, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-10, interferon-gamma and tumor necrosis factor-alpha measured in whole saliva using a multiplexed bead immunoassay (Luminex). Significance of statistical differences in the levels of salivary cytokines between groups was determined using nonparametric analysis of covariance, adjusting for age and smoking status. The Spearman rank correlation coefficient was used to explore associations between the mean levels of salivary cytokines and mean clinical parameters. RESULTS: There were no statistically significant differences between groups for any of the cytokines. There were weak, statistically significant positive associations between salivary interleukin-8 and pocket depth (r(s) = 0.2, p < 0.05) and bleeding on probing (r(s) = 0.2, p < 0.05), and weak negative correlations between salivary interleukin-10 and attachment level (r(s) = -0.2, p < 0.05) and bleeding on probing (r(s) = -0.3, p < 0.001). CONCLUSION: Mean salivary levels of granulocyte-macrophage colony-stimulating factor, interleukin-1beta, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-10, interferon-gamma and tumor necrosis factor-alpha could not discriminate between periodontal health and disease.

Factors affecting human supragingival biofilm composition. II. Tooth position.

BACKGROUND AND OBJECTIVE: Little is known regarding the factors that affect the microbial composition of supragingival biofilms. This study was designed to test the hypothesis that tooth location affects the microbial composition of supragingival plaque beyond the effect due to plaque mass as reflected by total DNA probe count. MATERIAL AND METHODS: Supragingival plaque samples were taken from the mesiobuccal aspect of each tooth in 187 subjects (n = 4745 samples). All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. Significance of differences in mean species counts and proportions were determined among tooth surfaces and six tooth type categories: molars, bicuspids, incisors/canines in the mandible and maxilla separately using the Kruskal-Wallis test. Stepwise multiple linear regression was employed to examine the relationship between species proportions and total DNA probe count, tooth location, periodontal and smoking status, age and sex. RESULTS: All species differed significantly among tooth types and among the six tooth categories. Higher plaque levels were seen on molars and lower incisors. Some differences observed between tooth types could be partly explained by the level of plaque. Teeth with high plaque mass exhibited high levels of Capnocytophaga gingivalis, Actinomyces naeslundii genospecies 2, Campylobacter rectus and Campylobacter showae. However, certain species, such as Veillonella parvula and Streptococcus sanguinis, differed significantly at different tooth locations despite similarities in plaque mass. Twenty of the test species exhibited a significant association with tooth location after adjusting for total DNA probe count and subject level factors. CONCLUSION: While plaque mass was associated with differences in proportions of many species in supragingival biofilms, tooth location also was strongly associated with species proportions in both univariate and multivariate analyses.

Factors affecting human supragingival biofilm composition. I. Plaque mass.

BACKGROUND AND OBJECTIVE: Little is known about the factors that affect the microbial composition of supragingival biofilms. This study was designed to examine the relationship between total DNA probe counts of supragingival biofilm samples, clinical parameters and supragingival biofilm composition. MATERIAL AND METHODS: Supragingival plaque samples were taken from 187 systemically healthy adult subjects (n = 4745 samples). All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. The relationship between total DNA probe counts and microbial composition was examined by subsetting the data into 10 groups based on 10 percentile increments of the total DNA probe counts. Differences among groups in terms of species counts and proportions were sought, as well as relationships of total plaque DNA probe count and clinical parameters. RESULTS: There was a wide distribution in mean total DNA probe counts among the 187 subjects. With increasing total plaque levels there was a change in the proportions of individual species and microbial complexes. 'Small plaques' were characterized by high proportions of species in the yellow, orange, purple and 'other' complexes; plaques of moderate mass were characterized by high proportions of Actinomyces and purple complex species, while 'large plaques' exhibited increased proportions of green and orange complex species. Measures of gingival inflammation, pocket depth and recession were significantly positively associated with total DNA probe counts. Increased plaque numbers were related to increased pocket depth irrespective of presence or absence of gingival inflammation. CONCLUSION: The proportions of individual species and microbial complexes in supragingival biofilms are influenced by the total numbers of organisms in the biofilm.

Microbial complexes in supragingival plaque.

BACKGROUND/AIMS: To examine microbial communities in supragingival biofilm samples. METHODS: Supragingival plaque samples were taken from 187 subjects at baseline (n = 4745). Fifty-five subjects provided supragingival plaque samples at 1-7 days after professional tooth cleaning (n = 1456); 93 subjects provided 8044 samples between 3 and 24 months post-therapy. All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. Microbial associations among species were sought using cluster analysis and community ordination techniques for the three groups separately. RESULTS: Six complexes were formed for the baseline samples. Similar complexes were formed for the samples taken 3-24 months post-therapy. However, distinct changes were observed in microbial communities in samples taken during the 7 days of plaque redevelopment. The complexes related to clinical parameters of periodontal disease. CONCLUSION: There were specific microbial complexes in supragingival plaque that were similar to those found in subgingival plaque samples with a few minor differences. The relation of previously unclustered taxa to the complexes was also described.

Disease progression in periodontally healthy and maintenance subjects.

BACKGROUND: The aim of this study was to determine whether the rate of attachment loss in periodontally healthy subjects in a prevention regimen would differ from the rate of disease progression in periodontitis subjects enrolled in a maintenance program. METHODS: Fifty-five periodontally healthy subjects and 57 periodontitis subjects were clinically and microbiologically monitored at baseline and at 1, 2, and 3 years. Clinical parameters measured at six sites per tooth included bleeding on probing, visible plaque, probing depth, and attachment level. Subgingival plaque samples were taken from the mesio-buccal aspect of every tooth and were analyzed for the levels of 40 bacterial species using checkerboard DNA-DNA hybridization. The significance of differences over time in the clinical parameters was determined using repeated-measures analysis of variance, whereas the significance of differences between groups was determined using the unpaired t test. The Mann-Whitney test was used for microbial analyses, and P values were adjusted for multiple comparisons. RESULTS: Mean clinical parameters improved for both groups over time. By the end of the study, 4% of the sites in maintenance subjects lost > or =2 mm of attachment, whereas in the prophylaxis subjects only 1% of the sites lost > or =2 mm of attachment. Maintenance subjects lost attachment primarily at shallow buccal and lingual sites. The maintenance subjects harbored significantly higher levels of most test species throughout the study. The maintenance program did not reduce the levels of red complex species to those typical of healthy subjects. CONCLUSIONS: Treated periodontitis subjects under maintenance displayed more rapid attachment loss than periodontally healthy subjects in a preventive regimen. The greater propensity to disease progression may be related to an elevated exposure to periodontal pathogens.

Initial subgingival colonization of 'pristine' pockets.

The treatment of periodontitis/peri-implantitis involves the reduction/eradication of periopathogens. After therapy, beneficial and pathogenic species recolonize the subgingival area. The dynamics of recolonization and especially the role of the supragingival environment in this process are still not well-understood. This prospective, split-mouth study followed the early colonization of 'pristine' pockets created during implant surgery (16 partially edentulous patients), to record the time needed before a complex subgingival flora could be established with the supragingival area as the single source. Four subgingival plaque samples were taken from shallow and medium pockets around implants (test), and neighboring teeth (undisturbed microbiota as reference) 1, 2, and 4 wks after abutment connection. Checkerboard DNA-DNA hybridization and culture data revealed a complex microbiota (including several pathogenic species) in the pristine pockets within a wk, with a minimal increase in counts up to 4 wks. Analysis of these data demonstrated that, even with the supragingival environment as the single source for colonizing bacteria, a complex subgingival microbiota can develop within 1 wk.

"Checkerboard" DNA-DNA hybridization.

A method is introduced for hybridizing large numbers of DNA samples against large numbers of DNA probes on a single support membrane. Denatured DNA from up to 43 samples was fixed in separate lanes on a single membrane mounted in a Miniblotter 45. The membrane was then rotated 90 degrees in the same device, which enabled simultaneous hybridization with 43 different DNA probes. Hybridizations were also performed on lysates of bacterial cells blotted to membranes. A MiniSlot device allowed lysates loaded in parallel channels to be aspirated through the membrane, depositing horizontal lanes on the membrane surface. Hybridizations were performed in vertical lanes with either digoxigenin-labeled whole genomic probes or 16S rRNA-based oligonucleotide probes directly conjugated to alkaline phosphatase. The method permits the simultaneous determination of the presence of multiple bacterial species in single or multiple dental plaque samples, thus suggesting its usefulness for a range of clinical or environmental samples.

Limit of resolution of flow cytometry for the detection of selected bacterial species.

The enumeration of bacteria in dental plaque samples is a vital but time-consuming procedure that uses standard cultural methods. Flow cytometry has proven to be a useful tool for the analysis of eukaryotic cells. In the present investigation, the utility of this technology for the enumeration of bacteria in mixtures was explored. Rabbit antisera were produced against the putative periodontal pathogens A. actinomycetemcomitans, B. intermedius, B. gingivalis, E. corrodens, W. recta, B. forsythus, as well as the frequently isolated supragingival species S. sanguis. Cross-reactive antibodies were removed by absorption, and the specificity of each antiserum was confirmed by being tested against a panel of 235 oral microbial strains (79 genera; 94 species) by means of ELISA. Conditions were established for the indirect immunofluorescent labeling of cells without agglutination with use of a goat anti-rabbit Ig-FITC second antibody. When an internal bead standard was used, it was found that unstained bacteria were enumerated by light-scattering parameters with poor efficiency (less than 3%). However, cells exposed to FITC either in the presence of specific or non-specific first antibody were enumerated with high efficiency (102.6 +/- 29.3%), indicating that a small amount of non-specific binding of fluorochrome facilitates bacterial detection. Clear discrimination between specifically- and non-specifically-stained bacteria was achieved with all six rabbit antisera. Mixtures of known composition were made (1) with pure cultures or (2) with a known species and supragingival plaque devoid of that species by culture. The results from both approaches with various species combinations revealed that the limit of resolution for accurate quantitation of a selected species was approximately 5%, although specific organisms could be detected qualitatively when present at approximately 1%.

Effect of therapy on periodontal infections.

Periodontal disease progression requires the simultaneous presence of high numbers of pathogens, low numbers of compatible or beneficial species, a conductive local environment, and a susceptible host. Effective therapy acts by altering one or more of these factors. Data from an ongoing study were used to examine the biological basis of treatment success or failure. Seventeen subjects showing disease progression were treated by Widman flap surgery at deep sites, scaling at shallow sites, and 1 of 4 randomly-assigned, systemically-administered adjunctive agents including amoxicillin/clavulanate potassium (Au) (n = 3), ibuprofen (n = 3), tetracycline (n = 9), or a placebo (n = 2). Clinical measurements and microbiological samples (enumerated using DNA probes) taken from the mesial aspect of each tooth pre-treatment and 12 months post-treatment were compared and 418 pre- and 418 post-therapy plaque samples were enumerated. Overall, the 4 treatments resulted in pocket depth reduction and "gain" in attachment. After therapy, the percentage of sites colonized by Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, and Bacteroides forsythus was decreased and counts > 10(6) were less frequent. Large attachment level gains were accompanied by major decreases in these species and were more frequent in subjects receiving antibiotics. A small number of sites in each treatment group became deeper and/or lost attachment. More than half of these sites were detected in 2 subjects who were older (65 vs. 44), had higher serum antibody to Actinobacillus actinomycetemcomitans serotype a (506 vs. 125 ELISA units), A. actinomycetemcomitans serotype b (518 vs. 130), and Campylobacter rectus (39 vs. 18).(ABSTRACT TRUNCATED AT 250 WORDS)

The bacterial etiology of destructive periodontal disease: current concepts.

The interpretation of diagnostic tests for the detection of subgingival bacterial species is dependent on knowledge of the microbial etiology of destructive periodontal diseases. Specific etiologic agents of these diseases have been sought for over 100 years; however, the complexity of the microbiota, an incomplete understanding of the biology of periodontal diseases, and technical problems have handicapped this search. Nonetheless, a number of possible pathogens have been suggested on the basis of their association with disease, animal pathogenicity, and virulence factors. The immunological response of the host to a species and the relation of successful therapy to the elimination of the species have also been used to support or refute suspected periodontal pathogens. Current data suggest that pathogens are necessary but not sufficient for disease activity to occur. Factors which influence activity include susceptibility of the individual host and the presence of interacting bacterial species which facilitate or impede disease progression. Recent studies have attempted to distinguish virulent and avirulent clonal types of suspected pathogenic species and seek transmission of genetic elements needed for pathogenic species to cause disease. Finally, the local environment of the periodontal pocket may be important in the regulation of expression of virulence factors by pathogenic species. Thus, in order that disease result from a pathogen, 1) it must be a virulent clonal type; 2) it must possess the chromosomal and extra-chromosomal genetic factors to initiate disease; 3) the host must be susceptible to this pathogen; 4) the pathogen must be in numbers sufficient to exceed the threshold for that host; 5) it must be located at the right place; 6) other bacterial species must foster, or at least not inhibit, the process; and 7) the local environment must be one which is conducive to the expression of the species' virulence properties.

Microbiological and clinical effects of surgery plus doxycycline on juvenile periodontitis.

It was previously determined that surgery plus the antibiotic doxycycline were effective in eliminating or suppressing Actinobacillus actinomycetemcomitans (Aa), an organism strongly associated with disease in localized juvenile periodontitis (LJP). Eight patients with LJP participated in this surgical study. Patients were reexamined three and 12 months following therapy. The results of this study strongly suggest that surgery plus doxycycline effectively eliminate Aa from periodontal pockets. This elimination results in clinical improvement and attachment gain at three and 12 months following surgery. Further destruction was seen in individuals who continued to harbor high levels of Aa.

A selective medium for Actinobacillus actinomycetemcomitans and the incidence of the organism in juvenile periodontitis.

A selective medium, malachite green bacitracin agar, was developed for the isolation of Actinobacillus actinomycetemcomitans from subgingival plaque of periodontally diseased patients. The medium consisted of Trypticase soy agar 40 gm/liter, bacitracin 128 micrograms/ml, malachite green 8 micrograms/ml and 5% defibrinated sheep blood. The medium, when incubated in an atmosphere of air plus 10% CO2 for 5 days, permitted greater than 80% recovery of pure cultures of A. actinomycetemcomitans when compared with a nonselective medium. The most frequent contaminant in plaque samples from different clinical conditions was Haemophilus aphrophilus. Decomposition of H2O2 was useful in differentiating these two species. Clinical studies employing the malachite green bacitracin medium revealed a significant association between the presence of the organism, A. actinomycetemcomitans and juvenile periodontitis.

Tissue levels of bone resorptive cytokines in periodontal disease.

The levels of 3 bone resorptive cytokines, interleukin 1 alpha (IL-1 alpha), IL-1 beta, and tumor necrosis factor alpha (TNF alpha), were assessed in tissues from sites of periodontal disease. As determined by ELISA of tissue extracts, IL-1 beta and TNF alpha were detected in all diseased sites, whereas IL-1 alpha was present in 8/22 sites, IL-1 beta was present in highest concentration (mean +/- SEM: 11,695 +/- 2,888 pg/ml; 672 pM), followed by TNF alpha (434 +/- 135 pg/ml; 26 pM), and IL-1 alpha (342 +/- 160 pg/ml; 20 pM). The levels of all 3 mediators were significantly lower in clinically healthy tissues. There was a highly significant correlation between levels of IL-1 beta and TNF alpha (rs = 0.61, P less than 0.001), suggesting coordinated expression of these 2 mediators. The numbers of cells containing each mediator was also determined by indirect immunofluorescence on frozen tissue sections. Consistent with findings from tissue extracts, IL-1 beta-containing cells were present in approximately 5-fold higher numbers than TNF alpha-containing cells, and 40-fold higher numbers than IL-1-alpha-containing cells. Taken together with previous findings, these results indicate that IL-1 beta is likely to be an important mediator in the pathogenesis of periodontal disease.

A method for the geometric and densitometric standardization of intraoral radiographs.

The interpretation of dental radiographs for the diagnosis of periodontal disease conditions poses several difficulties. These include the inability to adequately reproduce the projection geometry and optical density of the exposures. In order to improve the ability to extract accurate quantitative information from a radiographic survey of periodontal status, a method was developed which provided for consistent reproduction of both geometric and densitometric exposure parameters. This technique employed vertical bitewing projections in holders customized to individual segments of the dentition. A copper stepwedge was designed to provide densitometric standardization, and wire markers were included to permit measurement of angular variation. In a series of 53 paired radiographs, measurement of alveolar crest heights was found to be reproducible within approximately 0.1 mm. This method provided a full mouth radiographic survey using seven films, each complete with internal standards suitable for computer-based image processing.

Tetracycline: levels achievable in gingival crevice fluid and in vitro effect on subgingival organisms. Part I. Concentrations in crevicular fluid after repeated doses.

The concentration of tetracycline in gingival crevice fluid and blood was determined using a sensitive bioassay after oral administration of repeated doses of tetracycline. Crevicular fluid was sampled by an intracrevicular technique from four gingival sites in each individual and blood was obtained by finger puncture. Four volunteers received doses of 250 mg of tetracycline-HCl either every 6 hours or every 12 hours and were sampled at hours 0 to 15, 21 to 36, 48 to 60 and 96 to 102. Volunteers given 250 mg every 6 hours had average crevicular fluid concentrations between 4 to 8 micrograms/ml and blood concentrations between 2 to 2.5 micrograms/ml after 48 hours. The levels in crevicular fluid and blood of volunteers who received 250 mg every 12 hours were 2 to 4 micrograms/ml and 0.3 to 1.4 micrograms/ml respectively after 48 hours. The results demonstrated that after repeated doses of tetracycline the crevicular fluid levels were typically 2 to 4 times the blood levels.

Tetracycline: levels of achievable in gingival crevice fluid and in vitro effect on subgingival organisms. Part II. Susceptibilities of periodontal bacteria.

The sensitivity to tetracycline of 345 bacterial isolates from periodontal lesions was determined. Most species of bacteria, including those thought to be involved in the initiation and progress of destructive periodontal disease, were inhibited in vitro by tetracycline concentrations of 4 to 8 micrograms/ml. This concentration is equivalent to crevicular fluid levels of tetracycline at dosages of 1 gm/day. These data indicate that tetracycline is inhibitory at levels achieved in crevicular fluid for bacteria currently implicated in destructive periodontal disease.