“Reverse” DMA hybridization method for the rapid identification of subgingival microorganisms

A “reverse” hybridization method is described, in which whole chromosomal DNA was extracted from 10-20 colonies of “unknown” strains in pure culture and labelled with digoxigenin by a random primer technique. DNA probes were prepared from a total of 23 strains and hybridized with targets containing 100 ng purified, denatured DNA from 38 reference strains fixed to nitrocellulose. 21/23 digoxigenin-labelled DNA probes successfully detected all members of the homologous species present on filters. Probes to Fusobacterium nucleatum strains 364 and MG detected 3/4 and 1/4 members of this species, respectively; 13/23 probes were 100% specific, but cross reactions between 10 probes and DNA targets from closely related, heterologous species occurred in 15/834 possible instances. False-positive reactions that occurred between closely related species were, however, easily distinguished and did not prevent the accurate identification of probe strains. Digoxigenin-labelled probes were capable of detecting 100 pg of homologous DNA. The reverse hybridization procedure allows identification or grouping of a large number of isolates within 3 days and provides a more economical means of characterizing subgingival isolates than predominant cultivable techniques and conventional phenotypic testing. This method could be adapted for the direct identification of microorganisms in subgingival plaque samples     

Non-isotopic DNA probes for the identification of subgingival microorganisms

The purpose of the present investigation was to examine the potential of non-isotopic DNA probes to identify pure cultures in predominant cultivable microbiota studies. Non-isotopic DNA probes to 7 subgingival species were prepared by 2 methods. In the first, biotin-labelled probes were prepared by nick translation. In the second, single-stranded DNA was covalently linked to horseradish peroxidase via polyethyleneimine. The relative sensitivities and specificities of these probes were tested against pure cultures of a range of subgingival species. Aliquots of broth cultures were standardized by optical densities, placed on nitrocellulose or Whatman 541 filters and then treated to lyse the cells, denature and fix DNA to the filter. Using a streptavidin-alkaline phosphatase detection system, 10(4)-10(5) cells were detected by homologous biotin-labelled probes. Horseradish peroxidase-labelled probes were approximately one order of magnitude less sensitive. Non-specific reactions with unrelated species, displayed by both biotin- and horseradish peroxidase-labelled probes, were eliminated by treatment of filters with proteinase K and organic solvents. Cross-reactions between closely related species could be discriminated by comparing reaction intensities of the test strains with probes to the each of the cross-reacting species involved. Thus, nonisotopic DNA probes could be used for the rapid identification of subgingival isolates. The technique could also be used for the recognition and grouping of strains of unknown species. A probe made to the strain of an unknown species may be used to rapidly screen hundred of unknown isolates for related strains.     

Rapid method for the purification of DNA from subgingival microorganisms

A method is described which facilitates the rapid purification of high molecular weight chromosomal DNA from gram positive and gram negative bacteria grown on solid media. A total of 32 reference strains and fresh isolates were examined in this study. The purification procedure involved lysis of cells with SDS in the presence of proteinase K, followed by removal of cellular polysaccharides and proteins with hexadecyltrimethyl ammonium bromide (CTAB) and phenol:chloroform:isoamyl alcohol. Preparations were incubated with RNase and, after removal of the enzyme, DNA was precipitated with ethanol. Several hundred micrograms of DNA could be prepared within 5 h from cells grown on 1-2 agar plates. None of the final preparations contained RNA; protein was detected in 12/32 preparations. The resultant DNA proved suitable for restriction enzyme digestion and biotin-labelling by a random primer technique. DNA probes constructed from these preparations were capable of detecting 100 pg of homologous target DNA fixed to nitrocellulose. Cross reactions between closely related species displayed weaker signal intensities than, and, thus, were easily distinguished from, true positive reactions between homologous species. DNA obtained by this procedure may also be suitable for DNA-DNA homology studies, recombinant DNA experiments and molecular fingerprinting.     

Comparison of two methods for the small-scale extraction of DNA from subgingival microorganisms

Two methods were compared for the extraction of DNA from small numbers of bacterial cells. The first method involved lysis of cells with SDS in the presence of proteinase K, treatment with hexadecyltrimethyl ammonium bromide (CTAB) and precipitation of DNA with isopropanol. In the second method, DNA was extracted by treatment of the cells with guanidine hydrochloride (GHCl) and precipitated with ethanol. Thirty strains of representative gram positive and gram negative species were included in the study. Preparations derived from confluent growth on one-quarter of the surface of agar plates and from 10(8) cells were subjected to each extraction procedure and analyzed for their content of DNA, RNA and protein. The suitabilities of the resultant DNA for restriction enzyme digestion and biotin-labelling by a random primer technique were also assessed. In general, the CTAB method yielded greater amounts of DNA than the GHCl procedure. RNA was present in most preparations of both types, but in amounts detectable only by agarose gel electrophoresis. The latter technique also revealed that DNA was not excessively sheared by either procedure. Protein was detected in some CTAB and GHCl preparations, but was not consistently associated with one or the other method. DNA obtained by both methods could be digested by the restriction enzyme EcoR I. In addition, biotin-labelled DNA probes prepared from CTAB and GHCl preparations were capable of hybridizing with homologous target DNA fixed to nitrocellulose. Since the CTAB method was consistently successful in recovering DNA from preparations containing 10(8) cells, it may be more suitable for the direct treatment of single colonies taken from primary isolation plates or plaque samples.     

Comparison between polymerase chain reaction-based and checkerboard DNA hybridization techniques for microbial assessment of subgingival plaque samples

Aim: To compare polymerase chain reaction (PCR) with subsequent reverse hybridization (micro-IDent test) and checkerboard DNA–DNA hybridization for the identification of 13 bacterial species in subgingival plaque samples.
Material and Methods: Subgingival plaque samples were taken using paper points and curettes from two sites each with pocket depth <4, 4–6 and >6 mm at baseline and 3 months in 25 periodontitis subjects and two sites in 25 periodontally healthy subjects. Samples were analysed for their content of 13 bacterial species using both assays. Similarities for each species between techniques were determined using regression analysis. Differences between health and periodontitis were determined using the Mann–Whitney test.
Results: Three hundred and fifty samples were evaluated using both techniques. Regression analysis indicated that 10/13 test species showed significant positive correlations between the counts determined by checkerboard analysis and levels determined by the PCR-based test after adjusting for 13 comparisons. The highest rank correlations of 0.58, 0.49 and 0.46 were seen for Treponema denticola, Fusobacterium nucleatum and Eubacterium nodatum , respectively ( p <0.0001). Both tests could distinguish samples from healthy and periodontitis subjects.
Conclusion: Detection patterns of 10/13 test species in subgingival plaque samples from periodontitis and healthy subjects were similar using the two molecular techniques.     

Comparison of the subgingival microbiota of periodontally healthy and diseased adults in Northern Cameroon

Abstract Our study is the 1st report on subgingival microbiota in adult Cameronians. The aim was to investigate, using the checkerboard DNA-DNA hybridization technique, the prevalence of 18 oral species in subgingival plaque samples obtained from sex- and age-matched Cameronian adults with and without periodontal destruction. We also compared cultivation and the Affirm^TM DP test with the checkerboard technique in their capability to detect some selected species among the 18, 21 adult periodontitis patients and 21 periodontally healthy subjects were examined and the results were compared statistically. Each periodontitis patient had at least 4 pockets of ≥ 6 mm depth, while the healthy subjects had no sites more than 3 mm deep. Results of the checkerboard analysis showed that significantly (p<0.05) more periodontitis patients tested positive for most of the 18 bacterial species. The Gram-positive species Actinomyces naeslundii, Streptococcus mitis and Streptococcus sanguis, known as microbiota of healthy sites, were detected significantly more frequently in the healthy group. Cultivation demonstrated P. gingivalis, B. forsythus, A. actinomycetemcomitans, P. intermedia and F. nucleatum in significantly lower %s of patients as compared to the checkerboard technique. Furthermore, the Affirm^TM DP test detected P. gingivalis and B. forsythus in significantly fewer patients than did the checkerboard technique. A. actinomycetemcomitans was detected in 52.3% of the patients by the latter technique while the Affirm^TM DP test failed to detect the bacterium in any of the samples. Overall, the results of the present study confirm the importance of the screening method and indicate that the prevalences of the investigated putative periodontal pathogens and beneficial species in the healthy and diseased adult Cameronians were similar to those reported for adults in the West and in some developing countries.     

Use of randomly cloned DMA fragments for the identification of oral spirochetes

DNA probes were produced for the detection and identification of 4 cultivable species of oral spirochetes, Treponema denticola, Treponema socranskii, Treponema vincentii and Treponema pectinovorum. To obtain probe sequences, chromosomal DNA, isolated from representative strains within each species, was cloned in Escherichia coli K-12. Cloned DNA fragments were screened for the ability to hybridize to DNA only from homologous strains. Several such fragments were identified and shown to be specific when tested against a series of DNAs from gram-negative and gram-positive oral bacteria. The selected probe sequences were semi-conserved within strains of T. denticola and T. socranskii such that restriction fragment length polymorphism (RFLP) was observed. In the case of T. socranskii, RFLP was useful in distinguishing between the 3 known subspecies. Chromosomal DNA fragments from 2 strains of T. vincentii failed to cross-hybridize, under stringent conditions, to genomic DNA from each of these strains. The hybridization probes were suitable for the identification of clinical isolates of T. denticola and could be used to detect the presence of individual Treponema species in mixed cultures. On this basis, the probes were used successfully to detect T. denticola in uncultured plaque samples.     

The use of DNA probes to examine the distribution of subgingival species in subjects with different levels of periodontal destruction

The present investigation examined the distribution of 14 subgingival species at a total of 2299 sites in 90 subjects with different levels of periodontal destruction. Subgingival plaque samples taken from the mesial aspect of each tooth were anaerobically dispersed, diluted and plated on non selective media. After anaerobic incubation, colonies were lifted to nylon filters and specific species detected using digoxygenin-labeled whole chromosomal DNA probes. The mean total viable count for all sites in all subjects was 8.3 x 10(6). The probes accounted for an average of 27.8% of the total viable count. The % of subjects in which each species was detected was as follows; V. parvula, 98; B. intermedius I, 98; S. sanguis II, 96; B. intermedius II, 95; C. ochracea, 94; B. gingivalis, 91; S. sanguis I, 85; W. recta, 83; F. nucleatum ss. vincentii, 82; S. intermedius, 80; B. forsythus, 76; P. micros, 74; A. actinomycetemcomitans serotype a, 62 and A. actinomycetemcomitans serotype b, 52. The % of sites colonized by each of the 14 test species varied considerably within different subjects. The median number of sites colonized by different species ranged from 3.6% for A. actinomycetemcomitans serotype b to 43.5% for V. parvula. In half the subjects, the mean % of the total viable counts for each of the test species was less than 4%. When subjects were divided on the basis of % of sites at baseline with greater than 3 mm attachment loss, the 14 probes accounted for 29.9% of the microbiota in the localized disease group and 25% in the widespread disease group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a non-radioactive DMA probe for detecting Porphyromonas gingivalis in subgingival specimens

This study compared the ability of a nonradioactive digoxigenin-labeled DNA probe and anaerobic culture to identify subgingival Porphyromonas gingivalis. Total cellular DNA from P. gingivalis ATCC 33277T was labeled using the Genius kit from Boehringer Mannheim Biochemicals. Anaerobic culture was performed using VMGA III transport medium and enriched brucella blood agar. The DNA probe could detect as little as 1000 P. gingivalis cells added to supragingival plaque. Also, the probe could detect P. gingivalis when it was present in proportions too low to be visualized on overgrown bacterial plates. The probe showed no visible reaction with strains of various oral species or with thousands of non-P. gingivalis colonies from plaque samples. VMGA III could maintain the viability of P. gingivalis for up to 6 days, as evidenced by DNA probing of colony blot of subgingival cultures. A total cellular DNA probe for detecting P. gingivalis seems to offer a simple and reliable method of detecting the organism in subgingival specimens.     

Microbial composition of supra- and subgingival plaque in subjects with adult periodontitis

Background, aims: The purpose of the present study was to compare and relate the microbial composition of supra and subgingival plaque in 23 adult periodontitis subjects (mean age 51±14 years). Methods: A total of 1,170 samples of supra and subgingival plaque were collected from the mesial aspect of every tooth (up to 28 supra and 28 subgingival samples) from each subject and evaluated for the presence and levels of 40 bacterial taxa using whole genomic DNA probes and checkerboard DNA‐DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. The counts (levels), % DNA probe count (proportion) and % of sites colonized (prevalence) of each species in supra and separately in subgingival plaque were computed for each subject. Significance of differences between supra and subgingival plaque for each species was sought using the Wilcoxon signed ranks test and adjusted for multiple comparisons. Results: All 40 taxa were detected in both supra and subgingival plaque. Actinomyces species were the most prevalent taxa in both habitats. 75 to 100% of supra and 62 to 100% of subgingival sites were colonized by at least one of the 5 Actinomyces species. Supragingival samples exhibited significantly higher counts of Actinomyces naeslundii genospecies 1, Actinomyces israelii, Actinomyces odontolyticus, Neisseria mucosa, Streptococcus gordonii, Capnocytophaga ochracea and Capnocytophaga sputigena when compared with mean counts in subgingival samples taken from the same tooth surfaces. Subgingival plaque samples presented significantly higher counts of Prevotella nigrescens, Prevotella intermedia, Bacteroides forsythus and Porphyromonas gingivalis. Subgingival samples exhibited a significantly higher proportion of “red” and “orange complex” species, while supragingival plaque exhibited higher proportions of “green” and “purple” complex species as well as Actinomyces species. Suspected periodontal pathogens could be detected in supragingival plaque from sites where subgingival samples were negative for the same species. Conclusions: The data indicate that supragingival plaque can harbor putative periodontal pathogens, suggesting a possible rôle of this environment as a reservoir of such species for the spread or reinfection of subgingival sites.     

Comparison of the microbiota of supra- and subgingival plaque in health and periodontitis

BACKGROUND, AIMS: The purpose of the present investigation was to compare the microbial composition of supra and subgingival plaque in 22 periodontally healthy (mean age 32+/-16 years) and 23 adult periodontitis subjects (mean age 51+/-14 years). METHODS: A total of 2358 supra and separately subgingival plaque samples were collected from the mesial aspect of all teeth excluding 3rd molars in each subject. Samples were examined for the presence and levels of 40 bacterial taxa using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. Mean counts (x10(5), % DNA probe count and % sites colonized for each species were determined separately for supra and subgingival samples in each subject and then averaged across subjects in the 2 clinical groups. Significance of differences between healthy and periodontitis subjects was determined using the Mann-Whitney test and adjusted for multiple comparisons. RESULTS: Mean total DNA probe counts (x10(5), +/-SEM) for healthy and periodontitis subjects in supragingival plaque were 72.1+/-11 and 132+/-17.5, respectively (p<0.01), and in subgingival plaque 22.1+/-6.6 and 100.3+/-18.4, (p<0.001). Porphyromonas gingivalis, Bacteroides forsythus and Treponema denticola could be detected in supragingival plaque samples of both healthy and periodontitis subjects. Actinomyces species were the dominant taxa in both supra- and subgingival plaque from healthy and periodontitis subjects. 4 Actinomyces species accounted for 63.2%, of supragingival and 47.2% of subgingival plaque in healthy subjects and 48.% and 37.8% in periodontitis subjects respectively. Increased proportions of P. gingivalis, B. forsythus, and species of Prevotella, Fusobacterium, Campylobacter and Treponema were detected subgingivally in the periodontitis subjects. P. gingivalis, B. forsythus and T. denticola were significantly more prevalent in both supra- and subgingival plaque samples from periodontitis subjects. CONCLUSIONS: The main differences between supra and subgingival plaque as well as between health and disease were in the proportions and to some extent levels of Actinomyces, "orange" and "red" complex species.     

A new method for assessing the % of spirochaetes in subgingival plaque

A new method for counting the numbers of spirochaetes in subgingival plaque is described. The technique involves the combined use of a negative stained preparation, dark field microscopy, and an image analysing system. Advantages of the method include: smears need not be made until 2 days after sampling, and can be examined microscopically for up to 1 year after preparation; reproducibility of counts are more than 90% and the data can be computerised. The new method should prove useful in the routine assessment of subgingival plaque from patients with various forms of periodontitis, and in epidemiological surveys.     

Evaluation of the relationship between smoking during pregnancy and subgingival microbiota

BACKGROUND: Numerous studies have shown that smoking negatively affects periodontal health. Hormonal changes, which occur during pregnancy have also been reported to have adverse effects on the periodontal tissues or indirectly through alterations in the subgingival bacterial flora. At present, no knowledge exists concerning possible effects of smoking on the composition of subgingival plaque in pregnancy. The purpose of the present study was to evaluate the effects of smoking during pregnancy on the subgingival plaque bacteria most commonly associated with periodontal disease. METHODS: A total number of 181 women were examined within 72 h post-partum. Smoking status was recorded by means of a self-reported questionnaire and the study population was divided into three groups; non-smokers, light smokers, and heavy smokers. In each woman, two subgingival plaque samples were obtained from mesio- or disto-buccal aspect of randomly selected one molar and one incisor tooth by sterile paperpoints. Clinical periodontal recordings comprising presence of dental plaque, bleeding on probing (BOP), and probing pocket depth (PPD) were performed at six sites per each tooth at all teeth. Plaque samples were analysed by checkerboard DNA-DNA hybridization with respect to 12 bacterial species. In all analyses, the individual subject was the computational unit. Thus, mean values for all clinical parameters were calculated and bacterial scores from each individual sample were averaged. Statistical methods included chi2 test, Kruskal-Wallis test and Mann-Whitney U-test. RESULTS: Mean ages were similar in the study groups. Plaque, BOP and PPD recordings were lower in the heavy-smoker group, but the differences were not statistically significant (p>0.05). The detection rates and bacterial loads of the specific subgingival bacteria exhibited no significant differences between the groups. No correlation could be found between smoking status and detection rates and bacterial loads of various bacterial species. CONCLUSION: The present findings suggest that smoking during pregnancy does not have a significant effect on the composition of subgingival plaque bacteria.     

Changes in the subgingival biofilm composition after coronally positioned flap

Objectives

This study evaluated the effects of coronally positioned flap (CPF) on the subgingival biofilm composition.

Material and Methods

Twenty-two subjects with gingival recessions were treated with CPF. Clinical parameters were assessed before and at 6 months after surgery. Subgingival biofilms were analyzed by checkerboard DNADNA hybridization technique for 40 bacterial species.

Results

Recession height, clinical attachment level and bleeding on probing improved significantly (p<0.05) at 6 months post- CPF. The proportions of 10 periodontal pathogens and the proportions of red and orange complexes decreased at 6 months.

Conclusion

In conclusion, CPF can induce beneficial effects on the composition of the subgingival microbiota after 6 months.     

The effect of repeated professional supragingival plaque removal on the composition of the supra- and subgingival microbiota

BACKGROUND, AIMS: The purpose of the present investigation was to determine the effect of weekly professionally administered supragingival plaque removal on the composition of the supra and subgingival microbiota. METHODS: 18 adult subjects with periodontitis who had been treated and were in a maintenance phase of therapy were clinically and microbiologically monitored at baseline, 3, 6 and 12 months. After the baseline visit, the subjects received scaling and root planing followed by professional supragingival plaque removal every week for 3 months. Clinical measures of plaque accumulation, bleeding on probing (BOP), gingival redness, suppuration, pocket depth and attachment level were made at 6 sites per tooth at each visit. Separate supra (N = 1804) and subgingival (N = 1804) plaque samples were taken from the mesial aspect of all teeth excluding third molars in each subject at each time point and evaluated for their content of 40 bacterial taxa using checkerboard DNA-DNA hybridization. Significance of changes in mean counts, prevalence and proportions of bacterial species over time in both supra and subgingival samples were determined using the Quade test and adjusted for multiple comparisons. RESULTS: Mean % of sites exhibiting plaque, gingival redness and BOP were significantly reduced during the course of the study. Significant decreases in mean counts were observed in both supra and subgingival samples. Mean total DNA probe counts (x10(5), +/-SEM) at baseline, 3, 6 and 12 months were: 133+/-19, 95+/-25, 66+/-6, 41+/-6 (p<0.001) for supragingival samples and 105+/-22, 40+/-10, 19+/-4, 13+/-3 (p<0.001) for subgingival samples. Mean counts of 22 of 40 and 34 of 40 species tested were significantly reduced in the supra and subgingival samples respectively over the monitoring period. For example, mean counts of Porphyromonas gingivalis x10(5) at baseline, 3, 6 and 12 months in the subgingival plaque samples were 2.0+/-0.4, 0.5+/-0.2, 0.6+/-0.3, 0.3+/-0.1 (p<0.001); Bacteroides forsythus 2.0+/-0.6, 0.4+/-0.1, 0.4+/-0.2, 0.1+/-0.2 (p<0.001); Treponema denticola 3.4+/-1.1, 0.8+/-0.3, 0.4+/-0.2, 0.3+/-0.3 (p<0.01). Similar reductions were seen in supragingival plaque samples. While counts were markedly reduced by professional plaque removal, the proportion and prevalence of the 40 test species were marginally affected. CONCLUSIONS: Weekly professional supragingival plaque removal profoundly diminished counts of both supra- and subgingival species creating a microbial profile comparable to that observed in periodontal health. This profile was maintained at the final monitoring visit, 9 months after completion of therapy.     

Use of checkerboard DNA-DNA hybridization to study complex microbial ecosystems

It has been difficult to conduct large scale studies of microbiologically complex ecosystems using conventional microbiological techniques. Molecular identification techniques in new probe-target formats, such as checkerboard DNA-DNA hybridization, permit enumeration of large numbers of species in very large numbers of samples. Digoxigenin-labeled whole genomic probes to 40 common subgingival species were tested in a checkerboard hydridization format. Chemifluorescent signals resulting from the hybridization reactions were quantified using a Fluorimager and used to evaluate sensitivity and specificity of the probes. Sensitivity of the DNA probes was adjusted to detect 10(4) cells. In all, 93.5% of potential cross-reactions to 80 cultivable species exhibited signals <5% of that detected for the homologous probe signal. Competitive hybridization and probes prepared by subtraction hybridization and polymerase chain reaction were effective in minimizing cross-reactions for closely related taxa. To demonstrate utility, the technique was used to evaluate 8887 subgingival plaque samples from 79 periodontally healthy and 272 chronic periodontitis subjects and 8126 samples from 166 subjects taken prior to and after periodontal therapy. Significant differences were detected for many taxa for mean counts, proportion of total sample, and percentage of sites colonized between samples from periodontally healthy and periodontitis subjects. Further, significant reductions were observed post therapy for many subgingival species including periodontal pathogens. DNA probes used in the checkerboard DNA-DNA format provide a useful tool for the enumeration of bacterial species in microbiologically complex systems.     

A rapid DNA probe test compared to culture methods for identification of subgingival plaque bacteria

There has been a significant amount of interest in developing a more rapid and cost-effective test to identify bacterial pathogens in plaque. DNA probe technology may meet both these objectives, it is more rapid and cost-effective when compared to culture methods. The purpose of this study was to compare an automated DNA probe test with classical culture methods for identifying Bacteroides forsythus and Porphyromonas gingivalis in subgingival plaque of patients with adult periodontitis. Subgingival plaque samples were collected from sites with moderate to severe periodontitis and divided into two aliquots for analysis by either DNA probe or culture methods. When the DNA probe method was compared with the culture method (gold standard), the sensitivity and specificity for B. forsythus were 92.0% (SE = 3.4%) and 50.5% (SE = 7.8%), respectively; for P. gingivalis they were 52.2% (SE = 8.7%) and 74.7% (SE = 5.9%), respectively. Detection of B. forsythus and P. gingivalis by DNA probe correlated with probing depth (P = 0.01 for B. forsythus and P = 0.03 for P. gingivalis). It was concluded the DNA probe test was comparable to culture methods in detecting B. forsythus. In addition, when compared to the culture method, a better correlation was obtained with DNA probe detection of B. forsythus or P. gingivalis and clinical parameters.     

Clinical and microbiological features of refractory periodontitis subjects

Abstract. The purpose of this investigation was lo compare the clinical parameters and the site prevalence and levels of 40 subgingival species in successfully treated and refractory periodontitis subjects. 94 subjects received scaling and root planing and if needed, periodontal surgery and systemically administered tetracycline. 28 refractory subjects showed mean full mouth attachment loss and/or >3 sites showing attachment loss >2.5 mm within 1 year post-therapy. 66 successfully treated subjects showed mean attachment level gain and no sites with attachment loss >2.5 mm. Baseline subgingival plaque samples were taken from the mesial aspect of each tooth and the presence and levels of 40 subgingival taxa were determined using whole genomic DNA probes and checkerboard DNA-DNA hybridization. The mean levels and % of sites colonized by each species (prevalence) was computed for each subject and differences between groups sought using the Mann-Whitney test. Most of the 40 species tested, including Actinobacillus actinomycetemcomitans. Porphyromonas gingivalis, Treponema denticola and Bacteroides forsythus, were equally or less prevalent in the refraclory group. Prevotella nigrescens was significantly more prevalent in successfully treated subjects, while refractory subjects harbored a larger proportion of Streptococcus species, particularly Streptococcus constellatus. The odds of a subject being refractory was 8.6 (p<0.001) if S. constellatus constituted ≤3.5% of the total DNA probe count. Since few microbiological differences existed between treatment outcome groups using DNA probes to known species, the predominant cultivable microbiota of 33 subgingival samples from 14 refractory subjects was examined, 85% of the 1649 isolates were identified using probes to 69 recognized subgingival species. The remaining unidentified strains were classified by analyzing 16S rRNA gene sequences. Many sequenced isolates were of taxa not considered a common part of the oral microbiota such as Acinetobacter baumanni, Gemella haemolysans, Enterococcus faecalts, Staphyhcoccus warneri, Pseudomonas aeruginosa and novel species in the genera Bartonella, Ralstonia, Neisseria, Eubacterium, Rothia, Gordona, Gemella, Corynebacterium, Leptotrichia, and Actinomyces. Refractory subjects constituted a heterogeneous group based on their subgingival microbiota. As a group, they did not harbor more of the “classic” periodontopathogens, although elevated proportions of S. constellatus were found.     

Use of checkerboard DNA–DNA hybridization to study complex microbial ecosystems

It has been difficult to conduct large scale studies of microbiologically complex ecosystems using conventional microbiological techniques. Molecular identification techniques in new probe‐target formats, such as checkerboard DNA–DNA hybridization, permit enumeration of large numbers of species in very large numbers of samples. Digoxigenin‐labeled whole genomic probes to 40 common subgingival species were tested in a checkerboard hydridization format. Chemifluorescent signals resulting from the hybridization reactions were quantified using a Fluorimager and used to evaluate sensitivity and specificity of the probes. Sensitivity of the DNA probes was adjusted to detect 10⁴ cells. In all, 93.5% of potential cross‐reactions to 80 cultivable species exhibited signals <5% of that detected for the homologous probe signal. Competitive hybridization and probes prepared by subtraction hybridization and polymerase chain reaction were effective in minimizing cross‐reactions for closely related taxa. To demonstrate utility, the technique was used to evaluate 8887 subgingival plaque samples from 79 periodontally healthy and 272 chronic periodontitis subjects and 8126 samples from 166 subjects taken prior to and after periodontal therapy. Significant differences were detected for many taxa for mean counts, proportion of total sample, and percentage of sites colonized between samples from periodontally healthy and periodontitis subjects. Further, significant reductions were observed post therapy for many subgingival species including periodontal pathogens. DNA probes used in the checkerboard DNA–DNA format provide a useful tool for the enumeration of bacterial species in microbiologically complex systems.     

Proportional distribution of the red complex and its individual pathogens after sample storage using the checkerboard DNA-DNA hybridization technique

BACKGROUND: Information on the impact of sample storage prior to analysis by DNA methods is limited. AIMS: To investigate the effect of microbial sample storage on bacterial detection and proportional distribution of the red complex and its individual pathogens. MATERIAL AND METHODS: Subgingival plaque samples were analysed by (1) immediate processing, (2) after storage at +4 degrees C for 6 weeks, (3) after storage at -20 degrees C for 6 months or (4) after storage at -20 degrees C for 12 months using the checkerboard DNA-DNA hybridization. RESULTS: Proportional distribution of the red complex did not differ between the first three protocols. However, the total bacterial DNA for pathogens studied decreased significantly in protocols 3 and 4. Relative amounts of Tannerella forsythensis, Porphyromonas gingivalis and Treponema denticola remained stable in the second protocols and changed in an unpredictable way if stored for 6 or 12 months. CONCLUSIONS: Results from samples stored for maximum 6 months at -20 degrees C with high proportional amounts of the red complex and T. denticola may be used as an indicator of persistence. All bacterial samples for DNA extraction should be processed following a standardized storage protocol (i.e. samples stored at +4 degrees C for maximum 6 weeks) in order to get comparable qualitative and quantitative results for total DNA, bacterial complexes and individual pathogens. Most representative results are yielded if processing and hybridization could be performed immediately after sampling.