Detection frequency of periodontitis-associated bacteria by polymerase chain reaction in subgingival and supragingival plaque of periodontitis and healthy subjects

The aim of this study was to compare the detection frequencies of 25 bacterial species in subgingival and supragingival plaque of 18 untreated periodontitis subjects and 12 periodontally healthy subjects. Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by polymerase chain reaction of the 16S rRNA genes. Fourteen bacteria showed no relationship with periodontitis, and 11 of these 14 species were frequently detected (≥50%) in subgingival plaque in both periodontitis and healthy subjects. Nine bacteria such as Eubacterium saphenum, Prevotella intermedia, and Treponema denticola seemed to be related to periodontitis; their detection frequencies in subgingival plaque samples were higher in periodontitis than in healthy subjects, but these differences were not statistically significant by multiple comparisons (0.002≤ P< 0.05). Two species ( Mogibacterium timidum and Porphyromonas gingivalis ) were detected significantly more frequently in subgingival plaque of periodontitis subjects than of healthy subjects ( P< 0.002), with P. gingivalis being detected only in periodontitis subjects, suggesting that these two species are closely related to periodontitis. There were no significant differences in the detection frequencies of the 25 bacteria between subgingival and supragingival plaque, suggesting that the bacterial flora of supragingival plaque reflects that of subgingival plaque.     

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.     

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. nigrescens, 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 Rsal, 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.     

Comparative analysis of putative periodontopathic bacteria by multiplex polymerase chain reaction

Background and Objective:  The polymerase chain reaction (PCR) has been applied for the rapid and specific detection of periodontopathic bacteria in subgingival plaque and is potentially of clinical benefit in the diagnosis and treatment of periodontitis subjects. However, several technical points need to be modified before the conventional PCR detection system can be used by clinicians.
Material and Methods:  To develop a PCR-based technique more applicable for clinical use than conventional PCR, we established a multiplex PCR for five putative periodontopathic ( Treponema denticola , Porphyromonas gingivalis , Aggregatibacter actinomycetemcomitans , Prevotella intermedia and Tannerella forsythia ) and two nonperiodontopathic ( Streptococcus sanguinis and Streptococcus salivarius ) species of bacteria using whole-plaque suspension as templates, and detected bacteria in subgingival plaque taken from 85 subjects at the supportive periodontal therapy stage after active periodontal treatments.
Results:  Among putative periodontopathic bacteria, the detection frequency of T. denticola and P. gingivalis was elevated in parallel with higher probing pocket depth and clinical attachment loss, and had 4.2–14.1 times increasing odds of the clinical parameters tested. Detection of any of the five species of putative periodontopathic bacteria markedly increased the odds ratio of a higher probing pocket depth, clinical attachment loss and bleeding on probing.
Conclusion:  The multiplex PCR system developed in this study enabled the detection of all the bacteria under investigation in one reaction tube in a less time- and labor-intensive manner than conventional PCR. These results support the potential clinical use of multiplex PCR for detecting periodontopathic bacteria and for evaluating therapeutic strategies and predicting the prognosis for each subject.     

The effect of supragingival plaque control on the composition of the subgingival microflora in human periodontitis

The aim of this study was to evaluate the effect of supragingival plaque control on the composition of the subgingival microflora. 8 subjects with moderate to severe periodontitis were chosen for the study. Sites with periodontal destruction (GI greater than 2; probing depth greater than 6.5 mm; vertical alveolar bone loss on radiographs) were submitted to professional plaque control 3 X a week for 3 weeks. Contralateral sites received no prophylaxis and served as controls. Patients maintained usual oral hygiene during the observation period: it consisted exclusively of tooth brushing once or twice a day with no use of interdental cleaning aids. Clinical examination and bacterial sampling were performed every week. At the end of the study, PlI scores for the experimental sites showed a marked diminution compared with the control sites. No variations were observed in GI or probing depth in test or control sites during the study. The composition of subgingival plaque in both groups showed no significant variations during that period.     

The effect of supragingival plaque control on the composition of the subgingival flora in periodontal pockets

The effect of mechanical supragingival plaque control on the composition of the subgingival microflora in untreated 4-6 mm deep periodontal pockets was investigated. 13 subjects with chronic periodontitis were recruited for the study. Periodontally-diseased sites were subjected to professional plaque control 3 x weekly for a period of 3 weeks. Contralateral sites received no prophylaxis and served as controls. No instructions in oral hygiene procedures were given to the patients who maintained their habitual oral hygiene regime during the observation period. Clinical examination and darkfield microscopic analysis of bacterial samples were performed every week. The PlI scores for the experimental sites were reduced markedly, while those for the control sites remained stable throughout the observation period. No changes in the other clinical parameters were detected during the study. The composition of the subgingival microflora at the control sites did not change during the experimental period. In contrast, at the test sites, the proportion of spirochetes+motile rods decreased continuously. This decrease reached statistical significance at the end of the experiment. The results indicate that at periodontally diseased sites with an established subgingival ecosystem, supragingival plaque removal may influence the composition of the subgingival microflora.     

Absence of Helicobacter pylori in subgingival samples determined by polymerase chain reaction

The polymerase chain reaction was used for the detection of Helicobacter pylori from subgingival plaque in 336 periodontitis patients. A pair of primers derived from the H. pylori urease gene A served to amplify a targeted 411-bp fragment of genomic DNA. This technique permitted the detection of as few as 60 H. pylori cells. Paper point samples from 3 deep periodontal pockets per patient were immersed in 1 ml of phosphate-buffered saline or distilled water, DNA was solubilized by detergent/protease method, 3.7 μl or 37 μl of lysate supernatant was used as template, and the amplification product was analyzed in 1% agarose gel containing ethidium bromide. Each experiment included purified DNA and cell lysate of H. pylori as positive controls. The presence of bacteria in the sample was verified by a primer pair common to prokaryote 16S rRNA. The present study did not reveal the specific polymerase chain reaction amplification product characteristic of H. pylori. We conclude that periodontal pockets do not constitute a natural reservoir for H. pylori.     

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.     

Detection rates of presumptive periodontal pathogens in subgingival plaque samples of untreated periodontitis using either four or six pooled samples

Aim: A comparison of the detection frequency and number of periodontal pathogens in patients with aggressive or generalized, severe chronic periodontitis using a gene‐probe analysis. Methods: In 16 aggressive and 34 generalized, severe chronic periodontitis patients, plaque was sampled from the deepest pockets per quadrant (MT4) and per sextant (MT6). After sampling two paper points simultaneously, one paper point from each pocket was pooled with three paper points of the other pockets (MT4). The remaining four paper points were pooled with two paper points from the deepest pockets from the two remaining sextants (MT6). Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola were detected by 16S rRNA gene probes. Results: Log‐transformed counts for Aggregatibacter actinomycetemcomitans were statistically significantly higher with MT6 (aggressive: 3.21 ± 2.94; generalized, severe chronic: 2.22 ± 2.70) than MT4 (aggressive: 2.04 ± 2.74; generalized, severe chronic: 1.50 ± 2.37) (P < 0.05). The detection frequency and mean counts were high for Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola (>95%/>6.0). Conclusion: Aggregatibacter actinomycetemcomitans was detected in higher numbers for MT6 than MT4. For both MT4 and MT6, Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola were detected in >95% of all patients and with mean log‐transformed numbers >6.0.     

Capnocytophaga granulosa and Capnocytophaga haemolytica: novel species in subgingival plaque

BACKGROUND: The oral cavity accommodates one of the most diverse microfloras in the human body. Knowledge of this microflora, and of the periodontal microflora in particular, proves crucial towards an understanding of the bacterial-host interactions which lead to the development of infectious inflammatory periodontal diseases. Capnocytophaga species have been implicated as putative periodontal pathogens. To date, only 3 members of this genus (C. gingivalis, C. ochracea and C. sputigena) have been isolated from subgingival plaque. AIM: This communication reports the isolation of 2 recently-speciated strains, namely C. granulosa and C. haemolytica, from subgingival plaque collected from adult periodontitis patients. MATERIAL AND METHODS: Subgingival plaque was collected from 29 patients with chronic adult periodontitis. Plaque samples were inoculated onto fastidious anaerobe agar and incubated anaerobically for 5 days. Routine identification of clinical isolates was performed by 16S rRNA PCR-RFLP analysis, using Cfo I as restriction enzyme and corroborated by 16S rRNA gene sequencing. RESULTS: 16 of 29 patients (55%) tested positive for either C. granulosa and or C. haemolytica. A total of 70 isolates (63 C. granulosa and 7 C. haemolytica) were cultivated from subgingival plaque. 15 (51%) patients tested positive for C. granulosa, and 3 (10%) patients tested positive for C. haemolytica. CONCLUSION: This is the 1st report which recounts the presence of C. granulosa and C. haemolytica in subgingival plaque. Further research is required to establish the relative proportions of these species subgingivally in health and disease.     

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.     

Profiling of subgingival plaque biofilm microflora from periodontally healthy subjects and from subjects with periodontitis using quantitative real‐time PCR

Abiko Y, Sato T, Mayanagi G, Takahashi N. Profiling of subgingival plaque biofilm microflora from periodontally healthy subjects and from subjects with periodontitis using quantitative real‐time PCR. J Periodont Res 2010; 45: 389–395. © 2010 John Wiley & Sons A/S Background and Objective: Qualitative and quantitative changes of the subgingival plaque biofilm microflora in periodontal pockets are thought to be associated with the development and progression of periodontitis. The aims of the present study were to quantify the proportions of nine periodontitis‐associated bacterial species and four Streptococcus species in subgingival plaque, and to evaluate their relationship with periodontitis quantitatively. Material and Methods: Subgingival plaque samples were obtained from 12 periodontally healthy subjects and from 28 patients with periodontitis. The amounts of total and target bacteria were measured by quantitative real‐time PCR using universal and species‐specific primers, respectively. Results: The proportion of total obligate anaerobes was found to be higher in subjects with periodontitis than in periodontally healthy subjects (p < 0.05). Among obligate anaerobes, Tannerella forsythia (2.04 ± 5.27%, p < 0.05), Porphyromonas gingivalis (0.54 ± 1.41%) and Eubacterium saphenum (0.30 ± 0.96%) were detected at high proportions in subjects with periodontitis, but not in periodontally healthy subjects. By contrast, the proportion of total streptococci was lower in subjects with periodontitis (p < 0.05). Specifically, the proportion of T. forsythia, P. gingivalis or E. saphenum increased (≥ 2.78%) and the proportion of Streptococcus species decreased to virtually undetectable levels, in subjects with periodontitis. Conclusion: Obligate anaerobes, including T. forthysia, P. gingivalis and E. saphenum, were identified predominantly in microflora from subjects with periodontitis, whereas Streptococcus species were identified predominantly in microflora from periodontally healthy subjects, suggesting a change in the subgingival environment that resulted in conditions more suitable for the survival of obligate anaerobes. The proportion of these obligate anaerobes in the subgingival plaque of subjects with periodontitis appears to be associated with the status of human periodontitis.     

Detection of Eikenella corrodens and Actinobacillus actinomycetemcomitans by use of the polymerase chain reaction (PCR) in vitro and in subgingival plaque

Abstract The purpose of the present investigation was to identify 2 putative penodontal pathogens: Eikenella corrodens and Actinobacillus actinoinycetemcoiniiuns by polymerase chain reaction (PCR) in vilro and in subgingival plaque. On the basis of published sequences coding for 16S rRNA two primer pairs were designed which amplify a 410 bp sequence from E. corrodens DNA and a 547 bp fragment from A. actinomycetemcomitans DNA. respectively. As few as 50 cells could be detected from pure bacterial cultures. Each of the two primer pairs was found to be specific in that it did not give any amplification product neither with cell lysates from the respective alternative bacterium nor with lysates obtained from other putative periodontal pathogens and other bacteria. The PCR method developed turned out to be a simple, rapid and reliable diagnostic tool for the detection of the target microorganisms in clinical samples.     

Detection of periodontal bacteria in atheromatous plaque by nested polymerase chain reaction

Background: In recent years, increasing evidence regarding the potential association between periodontal diseases and cardiovascular diseases has been identified. The available evidence underlines the importance of detecting periodontal pathogens on atheromatous plaque as the first step in demonstrating the causal relationship between the two conditions. The main aim of this investigation is to detect periodontitis‐associated bacteria from carotid artery atheromatous plaque from patients who received an endarterectomy using strict sample procurement and laboratory procedures. Methods: Atheromatous plaque from endarterectomies from carotid arteries were scraped and homogenized, and bacterial DNA was extracted. To obtain a representative concentration of amplicons, two amplifications of the bacterial 16S ribosomal‐RNA gene were carried out for each sample with universal eubacteria primers by a polymerase chain reaction (PCR). A nested PCR with specific primers for the target bacteria was performed next. Statistical tests included the χ² test. Results: Forty‐two atheromatous plaque were analyzed. All of them were positive for ≥1 target bacterial species. The bacterial species most commonly found was Porphyromonas gingivalis (78.57%; 33 of 42), followed by Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) (66.67%; 28 of 42), Tannerella forsythia (previously T. forsythensis) (61.90%; 26 of 42), Eikenella corrodens (54.76%; 23 of 42), Fusobacterium nucleatum (50.00%; 21 of 42), and Campylobacter rectus (9.52%; four of 42). The simultaneous presence of various bacterial species within the same specimen was a common observation. Conclusion: Within the limitations of this study, the presence of DNA from periodontitis‐associated bacteria in carotid artery atheromatous plaque retrieved by endarterectomy is confirmed.     

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.     

慢性牙周炎龈下菌斑中五种牙周可疑致病微生物的分布

目的研究五种牙周可疑致病微生物在慢性牙周炎患者龈下菌斑的分布。方法选择27例慢性牙周炎患者,每位患者选取牙周袋最深的两个位点作为观察位点,采集龈下微生物样本,采用多重聚合酶链反应和反杂交的方法对伴放线菌嗜血菌、牙龈卟啉单胞菌、福赛斯坦纳菌、中间普雷沃菌和齿垢密螺旋体五种微生物进行半定量检测。结果在所检测的54个位点中,牙龈卟啉单胞菌、中间普雷沃菌、福赛斯坦纳菌和齿垢密螺旋体均有较高的检出率,分别为98.15%、92.59%、100%和98.15%;伴放线菌嗜血菌检出率较低,为20.37%。牙龈卟啉单胞菌和福赛斯坦纳菌的检出量明显高于其他三种微生物,其差异有统计学意义（P<0.05）。结论慢性牙周炎患者多存在牙龈卟啉单胞菌、福赛斯坦纳菌、中间普雷沃菌和齿垢密螺旋体的同时感染,且牙龈卟啉单胞菌和福赛斯坦纳菌的感染量较高。     

The effect of supragingival plaque control on the subgingival microbiota in subjects with periodontal disease

The present investigation was performed to study the effect on the subgingival microbiota, of a plaque control program which included meticulous oral hygiene instruction, supragingival scaling and professional monitoring during a 2 year period. 300 subjects were examined for periodontal disease and monitored for 2 years without treatment. After the 2 year examination, 80 subjects were invited to participate in a treatment program intended to improve the standard of their self-performed plaque control. 40 of the invitees had a gingivitis and only minor attachment loss, while 40 subjects had moderate signs of periodontitis. 62 subjects volunteered for this treatment. 23 of the volunteers (Group AB) had several sites with deep pockets (> 4 mm). 39 of the volunteers had gingivitis but shallow pockets only (Group C). Group AB contributed 31 shallow pocket sites (A-sites) and 40 deep pocket sites (B-sites), while Group C contributed 63 shallow sites (C-sites). After the clinical examination, samples of the subgingival microbiota were harvested from the 134 A, B and C sites. The 62 subjects were enrolled in a supervised oral hygiene program. Supragingival scaling was carried out. Oral hygiene instruction was provided and repeated on an individual need basis so that all subjects reached and maintained a supragingival plaque score which was < 20%. 24 months after the year 2 examination, the 62 subjects were examined again using both clinical and microbiological examination procedures. The findings demonstrated that carefully performed supragingival plaque control changed the quantity and the composition of the supragingival microbiota.(ABSTRACT TRUNCATED AT 250 WORDS)