Characteristics of trypsin-like activity in subgingival plaque samples

Previous studies have demonstrated that the hydrolysis of the trypsin substrate N-benzoyl-DL-arginine-2-naphthylamide (BANA), by subgingival plaque obtained from a single site, correlates best with the numbers and proportions of spirochetes in plaque samples and may serve as an indicator of clinical disease. In this investigation, we determined whether the association between BANA hydrolysis and spirochetes could be obtained in pooled subgingival plaque samples. Concomitantly, the characteristics of this reaction in terms of substrate type and concentration, microbial numbers needed to give a positive reaction as assessed by microscopic counts, rapidity of hydrolysis, and the effect of pH and various additives on the plaque BANA hydrolytic activity have been studied in pooled plaque samples from patients who were periodontally healthy or diseased. In addition, it was determined whether BANA hydrolytic activity found in subgingival plaque reflected contributions from saliva and supragingival plaque. Results indicated that the assay can best be performed with 0.67 mmol/L BANA at pH 7.0. EDTA and CaCl2 gave a slight inhibition and DTT a slight enhancement of the BANA reaction by the pooled plaque suspensions. The majority of the reactions (85%) developed their full color after overnight incubation. BANA hydrolysis was not found in saliva and occurred with much greater frequency in subgingival plaque as opposed to supragingival plaque. Analysis of the data indicated that BANA hydrolysis by pooled subgingival plaque samples is a suitable test for the detection of spirochetes when two or three spirochetes per high microscopic field are present in the sample.     

Gingivitis in young adults with Actinobacillus actinomycetemcomitans

High intraoral load of A. actinomycetemcomitans in subjects with no or minimal periodontal disease may induce subtle changes in clinical periodontal conditions. The aim of the present study was to compare, at a site level, clinical conditions in two groups of young adults with plaque-induced gingivitis. In one group, more than 20% subgingival sites harboured cultivable A. actinomycetemcomitans (n=9), whereas in the other group, the organism was present in 20% or fewer subgingival plaque samples (n=8). Whereas no overt differences in clinical conditions could be ascertained, on average, the association between the presence of subgingival plaque and bleeding upon probing was considerably stronger (Mantel-Haenszel's common odds ratio RMH and 95% confidence interval 3.903, 2.951-5.165, P<0.001) in subjects with only a few subgingival sites harbouring A. actinomycetemcomitans as compared to subjects with a widespread intraoral distribution of the organism (R(MH)=1.637, 1.226-2.184, P<0.001). Since the proportion of sites not bleeding upon probing in the presence of supragingival plaque was slightly elevated in these subjects, the present findings may suggest a suppressed inflammatory reaction on supragingival plaque in the presence of a pronounced intraoral load of A. actinomycetemcomitans.     

The effect of supragingival plaque removal on anaerobic bacteria deep periodontal pockets

A study was made to determine if the numbers of subgingival anaerobes in deep periodontal pockets can be controlled by removal of only supragingival plaque. The study was based on the premises that the subgingival flora is dependent on the supragingival plaque for its source of organisms as well as for its perpetuation. Daily professional removal of only supragingival plaque produced a statistically significant reduction per sample in subgingival facultative and obligatory anaerobes.     

The microbiological profiles of saliva, supragingival and subgingival plaque and dental caries in adults with and without type 2 diabetes mellitus

INTRODUCTION: The relationships between suspected bacteria in saliva, yeasts in oral rinse, and supragingival and subgingival plaque versus root surface and coronal caries in adults with type 2 diabetes mellitus and a non-diabetic group were explored. METHODS: One-hundred and five patients with type 2 diabetes and 103 non-diabetic subjects were recruited; their periodontal status, plaque index and magnitude of root surface and coronal caries were assessed. Saliva and an oral rinse were cultured for mutans streptococci, lactobacilli and yeasts. Toothbrush samples of supragingival plaque and curette samples of subgingival plaque were assessed for 17 bacterial species using the checkerboard DNA-DNA hybridization method. RESULTS: Type 2 diabetes patients had significantly more severe periodontitis, a higher plaque index and a higher prevalence and magnitude of root surface caries than non-diabetic subjects. Significantly more diabetic subjects had higher levels of Treponema denticola, Prevotella nigrescens, Streptococcus sanguinis, Streptococcus oralis and Streptococcus intermedius in their supragingival plaque than non-diabetic subjects. No significant difference was found for the organisms in saliva, oral rinse and subgingival plaque between the two groups. After adjustment for diabetic status, root surface caries was associated with an increased count of mutans streptococci, lactobacilli and yeasts in saliva and of Streptococcus mutans in supragingival plaque samples. Coronal caries was only associated with lactobacilli and yeasts in saliva. CONCLUSION: The number of cariogenic organisms in saliva and oral rinse estimated by culture demonstrated a stronger association with both root surface and coronal caries compared to those 17 species assessed with the checkerboard method in supragingival and subgingival plaque.     

Comparison of the distribution of Actinomyces in dental plaque on inserted enamel and natural tooth surfaces in periodontal health and disease

The distribution of Actinomyces naeslundii, Actinomyces viscosus and Actinomyces odontolyticus in healthy and diseased adult populations was studied in 3 different ways. First, supragingival plaque formation at 2 through 72 h was examined in 12 periodontally healthy adults using a removable pre-measured surface of enamel bonded to molars and premolars. Second, a cross-sectional examination of the composition of both supragingival and subgingival plaque of unknown age was conducted in 205 adults exhibiting periodontal health to moderate disease. Third, the effects of oral hygiene instruction and root planing on the subgingival micro-flora of a subset of 19 subjects with moderate periodontitis were examined. The evaluation of 12 adults revealed that the predominant species in early plaque formation (2, 4 and 8 h) was A. odontolyticus, A. viscosus and A. naeslundii were present in developing plaques in almost all subjects in 2-h plaque, but absent in half the subjects when 4-, 8- or 24-h plaque was examined. These two species significantly increased in numbers per mm² enamel surface area in 48- and 72-h plaques. A. odontolyticus was not related to clinical signs of periodontal disease in 205 adults, and its subgingival proportions in plaque did not change following periodontal treatment of 19 individuals. A. naeslundii was found in significantly higher numbers in supragingival than subgingival plaques in the 205 adults examined. The mean proportion of A. naeslundii significantly decreased as the magnitude of probing depth and attachment loss increased. The proportions of A. naeslundii and A. viscosus were found to be significantly increased in subgingival plaques following periodontal treatment.     

Actinobacillus actinomycetemcomitans recovery from extracrevicular locations of the mouth

Associations between recovery of Actinobacillus actinomycetemcomitans from samples of subgingival plaque, and samples of buccal mucosa, tongue and unstimulated saliva were studied in 107 subjects. Ten subjects had gingivitis, 18 localized juvenile periodontitis, 45 rapidly progressive periodontitis and 32 adult periodontitis. Two children suffered from prepubertal periodontitis. Heterogeneity tests for associations in different study populations yielded nonsignificant results. Mantel-Haenszel's common odds ratios were 52.9, 37.2 and 19.8 for respective associations between pooled subgingival samples, and cheek, saliva and tongue samples. Significant McNemar's chi-square of 5.88, 11.25 and 16.96 for respective associations pointed to secondary occurrence of A. actinomycetemcomitans in extra-crevicular samples. Multiple linear regression yielded a significant influence of the number of deep periodontal pockets of 7 mm or more and a negative influence of the diagnosis "adult periodontitis" on the log-transformed number of colony-forming units of A. actinomycetemcomitans in samples from cheek mucosa in patients infected with the organism. Extracrevicular occurrence of A. actinomycetemcomitans seems to reflect total subgingival numbers of the organism. Especially sampling check mucosa appears to be a promising tool in the diagnosis of a periodontal infection with A. actinomycetemcomitans .     

The predominant cultivable dental plaque flora of beagle dogs with periodontitis

Abstract The predominant cultivable dental plaque flora was studied in 10 adult female beagle dogs with advanced periodontitis. Supragingival and subgingival plaque from a maxillary third premolar (P³) was removed and cultured anaerobically on various growth media and all colonies were subcultured and partially characterized. Histopathological specimens of the plaque sampling sites showed significant loss of connective tissue attachment. Spirochetes were found in ail samples. Anaerobic gram-negative organisms were predominant in both types of plaque accounting for about 55 % of the cultivable organisms in the supragingival plaque and almost 75% in the subgingival plaque. Bacteroides asaccharolyticus was the most prominant organism in the supragingival plaque, whereas Fusobacterium nucleatum predominated in the subgingival flora. Streptococcal and actinomycotic species were common in the supragingival plaque, but their proportions, especially those of the actmomycetes, were decreased in the subgingival flora. In many respects the bacterial profile associated with disease resembled that reported in human periodontal disease.     

Clinical alterations in relation to the morphological composition of the subgingival microflora following scaling and root planning

The aim of the present study was to relate shifts in the composition of subgingival plaque in periodontal pockets to alterations of the clinical periodontal conditions following a single course of subgingival scaling and root planing during a period of professional supragingival plaque control. For this purpose, 36 pairs of contralateral periodontal pockets in 10 subjects with moderately advanced periodontitis were assessed for the degree of gingival inflammation, probing pocket depths, bleeding on probing, attachment levels and the amount of supragingival plaque. In addition, samples of subgingival plaque were analyzed morphologically by dark-field microscopy. All patients received detailed information about proper oral hygiene and every 1-2 weeks, professional removal of supragingivally located deposits. When the oral hygiene standard had been sufficiently improved, 1 course of subgingival scaling on 1 side of each jaw only (test side) was carried out. Clinical and microbiological examinations were repeated after the scaling as well as after 2 and 6 months, while patients were recalled for supragingival prophylaxis every 2nd to 4th week. Our data showed that a single course of subgingival scaling and root planing resulted in reduced probing depths, a gain in clinical attachment and a shift in the composition of the subgingival microflora to a composition found in relatively healthy periodontal conditions. In relatively shallow pockets, however, a possible influence of repeated sampling on the subgingival microflora could not be ruled out. Bleeding on gentle probing was a reliable parameter for predicting a subgingival microflora where motile bacteria hold an increased portion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relative proportions of pathogen-related oral spirochetes (PROS) and Treponema denticola in supragingival and subgingival plaque from patients with periodontitis.

The purpose of this study was to use monoclonal antibodies to enumerate spirochetes in dental plaque, including the newly recognized pathogen-related oral spirochete (PROS) and specific serovars of Treponema denticola. Plaque was collected from control subjects with no apparent periodontal disease and from sites of moderate to severe chronic periodontitis in patients with inflammatory periodontal disease. Individual monoclonal antibodies were used to determine whether spirochetes were present and then a double-staining protocol was employed to count total spirochetes and specific treponemes in individual microscopic fields. Results indicate that spirochetes are more common at diseased sites and in subgingival plaque than at healthy sites or in supragingival plaque. Together PROS and T. denticola comprised the majority of all spirochetes in all samples and PROS and T. denticola serovars "B" and D were most numerous in plaque from patients with periodontitis. PROS were the majority of all spirochetes in supragingival plaque (76.2% +/- 23.8%) and subgingival plaque (60.9% +/- 19.1%) from periodontitis patients, significantly larger than the percentage of T. denticola serovar "B" (P less than .001 for both supragingival and subgingival plaque) and serovar D (P less than .01 for supragingival and P less than .001 for subgingival plaque). These observations indicate that PROS are the predominant spirochete in plaque from sites of patients with periodontitis, but other analytical approaches are necessary to determine if PROS or T. denticola are pathogenic.     

Controlling the plaque biofilm

Aim : To examine the effect of supragingival plaque removal in conjunction with different periodontal therapies on subgingival plaque composition in different subject populations. Method : Four different studies are presented which examined the effect of repeated removal of supragingival plaque performed by professionals or by tooth brushing on subgingival plaque composition. The studies were performed in different populations including chronic periodontitis, periodontal maintenance and refractory subjects. For all studies, each subject was examined for clinical parameters at up to 168 sites and subgingival plaque samples were taken from the mesial aspect of each tooth and examined for their content of specific bacterial species using checkerboard DNA‐DNA hybridisation techniques. Results : Repeated supragingival plaque removal used in conjunction with scaling and root planing only or combined with other periodontal therapies resulted in improvements in clinical parameters as well as significant decreases in the counts of subgingival species, including those associated with periodontal disease aetiology. Meticulous tooth brushing provided similar clinical and microbial improvements. Conclusions : Meticulous removal of supragingival plaque has beneficial effects on clinical parameters of periodontal disease and on the nature of the microbiota that colonises both above and below the gingival margin and appropriately has been a major focus in the prevention and control of dental diseases, particularly periodontal disease.     

胃病患者口腔中的幽门螺杆菌

目的 通过多聚酶链反应（ＰＣＲ）检测胃病患者的幽门螺杆菌。方法 从１３例有上消化道症状经内镜检查证实的胃病患者采集胃粘膜,口腔含漱液和６个牙位的龈上及龈下菌斑,应用ＰＣＲ检测标本中的Ｈｐ。同时用酶联免疫法（ＥＬＩＳＡ）检测静脉血,唾液、龈沟液中抗Ｈｐ的特异性ＩｇＧ抗体。结果 ＰＣＲ检测胃粘膜均Ｈｐ阳性６例患者（４６．２％）的含漱液和１１例患者（８４．６％）的至少一份菌斑样本中检测出Ｈｐ。龈下菌斑中     

OCCURRENCE OF AGGREGATIBACTER ACTINOMYCETEMCOMITANS IN BRAZILIAN INDIANS FROM UMUTINA RESERVATION,MATO GROSSO,BRAZIL

A ggregatibacter actinomycetemcomitans is associated with periodontal disease, especially localized aggressive periodontitis, produces a potent leukotoxin and its distribution is influenced by ethnic characteristics of the population.

Objective:

Using culture and polymerase chain reaction (PCR) techniques, this study evaluated the occurrence of this microorganism and the distribution of leukotoxic strains isolated from Indians belonging to the Umutima Reservation, Mato Grosso, Brazil.

Material and Methods:

Forty-eight native Brazilians with gingivitis and 38 with chronic periodontitis, belonging to Umutina, Paresi, Bororo, Bakairi, Kayabi, Irantxe, Nambikwara and Terena ethnicities, were studied. Subgingival, supragingival and saliva samples of each patient were collected and transferred to VMGA III medium and to ultra pure Milli Q water. Bacteria were grown on TSBV agar and incubated in anaerobiosis (90% N₂ + 10% CO₂) at 37°C for 72 h. The presence of the ltx promoter was determined by PCR, and a 530 bp deletion in the promoter was evaluated by using specific primers.

Results:

A. actinomycetemcomitans was isolated from 8.33% of saliva, supragingival and subgingival samples from patients with gingivitis and from 18.42% of saliva and supragingival biofilm, and 26.32% subgingival biofilm from patients with chronic periodontitis. By PCR, the bacterial DNA was detected in 8.33% of saliva, supragingival and subgingival biofilms from patients with gingivitis and from 23.68% of saliva, 28.95% supragingival biofilm and 34.21% subgingival biofilm from patients with periodontitis. All strains were grouped as non-JP2 clones based on the absence of deletion in the leukotoxin promoter. Differences among the microbial and clinical parameters in patients were analyzed by using the Mann-Whitney, Chi-square or Fisher''s exact tests.

Conclusions:

The present results suggest that A. actinomycetemcomitans can be related to the attachment loss in this population, but the presence of minimally leukotoxic strains, as well as its role in the pathogenesis of the periodontitis in these native Brazilians need to be further investigated.     

Natural distribution of oral Actinobacillus actinomycetemcomitans in young men with minimal periodontal disease

A total of 1005 subgingival and extracrevicular samples from 201 male recruits, 18–25 yr old, were selectively cultivated for Actinobacillus actinomycetemcomitans. The organism was isolated in 55 subjects (27%); 9.5% of pooled subgingival plaque samples from first molars, 14% cheek mucosa, 20% dorsum of tongue and 20% saliva samples were culture-positive. In order to divide the study population into distinct clinical categories, cluster analysis was performed, based on previous caries experience, probing pocket depth categories, bleeding scores, visible plaque and calculus. Two clusters (n=86 and n=92, respectively) were identified with no or minimal periodontal disease (mean±standard deviation % of periodontal probing depth 1–2 mm 78.7±10.4% and 57.4±12.6%, respectively; virtually no periodontal probing/depth in excess of 4 mm) and a relatively low DMF-S (22±13). A third cluster (n=22) had, in contrast, a high DMF-S (47.7±173) and a relatively high % of periodontal pockets of ≥5 mm (5.9 ±5.2%). Prevalence of A. actinomycetemcomitans in this cluster was 41%, while the organism was found in 23% and 27% in the minimally diseased populations (p<0.15). Whereas no heterogeneity of associations between subgingival and extracrevicular occurrence of the organism could be ascertained in different clusters, the organism was significantly more often identified in extracrevicular material, especially dorsum of tongue samples, compared with subgingival plaque (McNemar's X²=12.45, p<0.001). Multiple linear regression analysis revealed the number of A. actino-mycetemcomitans positive samples as well as the % of sites bleeding on probing being positively associated with the % of sites with a probing pocket depth of ≥5 mm (R2=0.345, p≤0.0001). The present large-scale investigation points to the wide distribution of this putative periodontopathogen in young individuals with minimal periodontal disease.     

Comparison of plaque microflora between Chinese and Caucasian population groups

This investigation was designed to compare the predominant plaque micro-organisms from a Chinese group of patients exhibiting periodontitis with an age-, sex- and periodontal disease-matched Caucasian group of patients. In addition to race, the 2 population groups differed with respect to diet and oral hygiene habits, or effectiveness at removing plaque. Clinical measurements were determined along with an evaluation for micro-organisms in supragingival and subgingival plaque. Although the Chinese and Caucasian population groups were similar with respect to composition of micro-organisms in subgingival plaque, notable differences were observed in supragingival plaque. The Chinese group had higher mean proportions of spirochetes, motile rods. Fusobacterium spp. and dark-pigmented Bacteroides species, while the Caucasian group had higher mean proportions of cocci, total Actinomyces spp., A. viscosus and total Streptococcus spp. in supragingival plaque. The microbial differences observed in supragingival plaque may be explained at least in part, if not totally, by the higher plaque index scores of the Chinese versus Caucasian population groups.     

Supragingival and subgingival microbiota of adult patients with Down''s syndrome. Changes after periodontal treatment

In this longitudinal study, five adult Down's syndrome patients with periodontitis were placed on a frequent recall visit schedule (every 6 weeks) after treatment, in order to investigate: 1) the microbiological status, both supragingivally and subgingivally, and the changes that occurred after treatment and 2) the effect of frequent professional supragingival plaque control on the subgingival microbiota and clinical variables in these patients. The clinical variables recorded were probing pocket depth, probing attachment level, bleeding on probing and presence of plaque (full mouth, six surfaces per tooth). Microbiological examination was performed separately for supragingival and subgingival samples from the same site for 14 species, using whole genomic DNA probes and the "checkerboard" DNA-DNA hybridization technique. The findings indicate that, although a reduction of periodontal indices was noticed, plaque levels remained high (60%) even at the end of the experimental period. Periodontal pathogens including Porphyromonas gingivalis, Bacteroides forsythus and Actinobacillus actinomycetemcomitans were frequently detected both supragingivally and subgingivally (>30%). The presence of a species supragingivally and the presence at the same time points subgingivally were correlated. This finding suggested that supragingival plaque acts as a reservoir for reinfection of treated sites. A reduction of the percentages of detection of these species was noticed 1 month after an oral hygiene period as well as at 3 and 6 months after treatment. Inadequate oral hygiene as performed by these patients probably affected supragingival, and consequently subgingival, plaque composition.     

Prevalence of Helicobacter pylori detected by polymerase chain reaction in the oral cavity of periodontitis patients

Helicobacter pylori is an important gastrointestinal pathogen associated with gastritis, peptic ulcers, and an increased risk of gastric carcinoma. The oral cavity has been indicated as a possible H. pylori reservoir, and may therefore be involved in the reinfection of the stomach which sometimes follows treatment of H. pylori infection. The objective of the present study was to evaluate the prevalence of H. pylori as detected by polymerase chain reaction (PCR) in the oral cavity of periodontitis patients testing positive for this bacterium in the stomach. Thirty adult patients with alterations of the superior digestive tract, testing urease positive after endoscopy and biopsy, were selected. A full-mouth periodontal examination was performed in every patient and the subjects were allocated to two groups: gingivitis (15 patients) and chronic periodontitis (15 patients). Plaque and saliva samples collected from each patient were stored in 0.5 ml of TE buffer. DNA was extracted from the samples by the boiling method and was evaluated for the presence of H. pylori using the PCR method. JW 22/23 primers were used. The DNA of ATCC H. pylori 43629 (positive control) and water (negative control) were used for controlling the reactions. Of the 30 evaluated patients, 13 (43.3%) harbored H. pylori in the mouth. The bacterium was not found on the dorsum of the tongue of any patient, but was found in saliva in three patients (10%), in the supragingival plaque in six patients (20%), and in the subgingival plaque in eight patients (26.6%). The presence of H. pylori was similar in the gingivitis and chronic periodontitis groups. In conclusion, a high percentage of patients harbored H. pylori in their mouth. The bacterium was detected in saliva, supragingival and subgingival plaque, suggesting that these sites may be considered reservoirs for H. pylori in urease-positive patients.     

Molecular characterization of plasminogen activator in human supragingival plaque

Extravascular participation of the serine protease plasminogen activator (PA) in tissue remodelling and cell migration may be relevant in the regulation of periodontal homeostasis and the pathogenesis of periodontal disease. The molecular nature of authentic PA in crevicular fluid has been characterized, and this study has sought to determine whether human supragingival plaque also contains PA; if so, of which molecular species and from what source. Thirty samples of supragingival plaque plaque from 10 individuals, extensively washed to remove adherent saliva, were all found by substrate analysis to have tissue-type PA (tPA) activity. Unstimulated parotid or mixed saliva also showed tPA activity, but submandibular saliva had no measurable PA activity. Freshly isolated plaque microbes cultured under aerobic or anaerobic conditions contained no PA activity (preliminary investigation). PA activity was restricted to individual epithelial cells suggesting that the origin of PA activity in human supragingival plaque is in part from plaque-adherent epithelial cells.     

Intraoral distribution of Actinobacillus actinomycetemcomitans in young adults with minimal periodontal disease

The aim of the present study was to investigate the intraoral distribution of Actinobacillus actinomycetemcomitans in young adults with minor signs of periodontal disease but harboring the organisms in the oral cavity. 17 healthy volunteers, 20 to 27 years of age, participated. Samples from mucosal surfaces of the oro-pharyngeal cavity and saliva (n = 221) as well as subgingival plaque from every tooth (n =477) were selectively cultivated for A. actinomycetemcomitans. Species identity and presence of the leukotoxin encoding gene, ltxA, were checked by multiplex polymerase chain reaction. Moreover, the leukotoxin promoter region was analyzed. No isolate harbored a 530 bp deletion in the promoter region of the leukotoxin gene, signaling minimally toxic strains. 42.1 +/- 30.4% extracrevicular and 34.4 +/- 29.5% subgingival samples were culture-positive. In extracrevicular samples, the organism could easily be recovered from cheek mucosa (62%), saliva (59%) and the palatal tonsils (41%). Mean log-transformed numbers of A. actinomycetecomitans colony forming units (CFU/ml) in culture-positive material ranged between 1.8 from the hard palate and 2.3 from 10 microl saliva. The highest prevalence in subgingival plaque was observed at maxillary 3rd molars (55%) followed by maxillary lateral incisors (50%) and mandibular 3rd molars (41%). Mean log-transformed counts of CFU/ml ranged between 2.2 at maxillary 3rd molars and 3.4 at upper central incisors. When adjusted for jaw, site and tooth type, the odds of isolating higher numbers of the organism were increased with every mm probing depth by a factor of 1.35 (p <0.05). The odds ratio for bleeding on probing was 1.38. Thus, in this young adult population with minor periodontal disease, A. actinomyetemcomitans was mainly associated with some deviation from gingival health. Of concern might be a minority of subjects (29%) with an extremely wide distribution of the organism in the oral cavity.     

Supragingival and subgingival microbiota of adult patients with Down’s syndrome. Changes after periodontal treatment

In this longitudinal study, five adult Down’s syndrome patients with periodontitis were placed on a frequent recall visit schedule (every 6 weeks) after treatment, in order to investigate: 1) the microbiological status, both supragingivally and subgingivally, and the changes that occurred after treatment and 2) the effect of frequent professional supragingival plaque control on the subgingival microbiota and clinical variables in these patients. The clinical variables recorded were probing pocket depth, probing attachment level, bleeding on probing and presence of plaque (full mouth, six surfaces per tooth). Microbiological examination was performed separately for supragingival and subgingival samples from the same site for 14 species, using whole genomic DNA probes and the “checkerboard” DNA‐DNA hybridization technique. The findings indicate that, although a reduction of periodontal indices was noticed, plaque levels remained high (60%) even at the end of the experimental period. Periodontal pathogens including Porphyromonas gingivalis, Bacteroides forsythus and Actinobacillus actinomycetemcomitans were frequently detected both supragingivally and subgingivally (>30%). The presence of a species supragingivally and the presence at the same time points subgingivally were correlated. This finding suggested that supragingival plaque acts as a reservoir for reinfection of treated sites. A reduction of the percentages of detection of these species was noticed 1 month after an oral hygiene period as well as at 3 and 6 months after treatment. Inadequate oral hygiene as performed by these patients probably affected supragingival, and consequently subgingival, plaque composition.