Effect of sampling on the composition of the human subgingival microbial flora

Eighteen adults with chronic periodontitis participated in an experiment to study the effect of sampling the subgingival microbial flora of periodontal pockets on the subsequent microbial composition of the pocket contents. Samples of bacteria were collected atraumatically with a periodontal curette from periodontal pockets. The sample was suspended in 0.85 N saline containing 1 % gelatin. A drop of the suspension suitably diluted was applied to a microscopic slide and the specimen immediately examined by darkfield microscopy. The microbial flora was classified into coccoid cells, motile cells, spirochetes and “other” microorganisms. The proportion of bacteria in each of these 4 categories was expressed as a percentage, based on the examination of the first 200 bacteria detected in a series of high power fields (mag. × 1200). The microbial composition of the contents of each of 6 pockets was examined at day 0 (baseline data) and each pocket reexamined once either 3, 7, 14, 21, 28 or 42 days later. In addition to the microbial examination, GI and P1I scores and probing depth measurements were also obtained at day 0 and at the reexamination. The results indicated a tendency toward reduced P1I and GI scores at the various reexaminations in comparison to baseline scores. Probing depth measurements did not change significantly. A slight but significant proportional increase in coccoid cells was detectable at 3 days and persisted at more or less the same level for the remainder of the experiment. Spirochetes showed a proportional decrease from baseline during the first week, while motile cells were proportionally reduced only on day 3. No significant changes occurred in the “other” microorganisms. It is not clear from the results if the alterations in the microbial flora were due to the sampling procedure per se or to a change in oral hygiene habits in a population of new patients suddenly more aware of their periodontal status. The clear-cut changes from baseline to day 3 levels and the subsequent lack of change from the 3-day through the 42-day intervals suggest that sampling per se may have contributed only to the changes from baseline noted in the first few days. The change in oral hygiene habits, however, may have contributed not only to the initial changes from baseline, but also to the maintenance of the new microbial proportions at a stable level for the remainder of the experimental period. Despite changes from baseline, all criteria remained well within the range of values that might be expected at periodontally diseased sites in patients with untreated chronic periodontitis.     

肾移植患者龈下菌群状况对牙龈增生的影响

目的了解肾移植患者牙周及龈下菌群状况，探索环胞素A（cyclosporine A，CsA）引起的牙龈增生的影响因素。方法肾移植患者20例，用CsA药前、后3个月和6个月分别记录龈下产黑菌数量和厌氧菌总量，以及菌斑指数、出血指数、探诊深度和牙龈增生指数等各项牙周指标，进行统计分析。结果用CsA药后3个月及6个月细菌数量较用药前均有明显增加，牙周炎症也明显加重，用药后6个月牙龈增生出现，以上差异均有统计学意义（P〈0．05）。细菌数量与牙龈增生指数具有相关性（P〈0．01）。结论龈下细菌数量是肾移植患者应用CsA引起牙龈增生的重要影响因素，提示肾移植患者在术前和术后应加强牙周检查及治疗。     

Relationship between clinical findings and subgingival microbial flora in periodontitis (2)

The purpose of this study was to examine the relationship between clinical findings and subgingival relationship between clinical findings and subgingival microbial flora in periodontitis at the first medical examination and after initial preparation. The results obtained were as follows: 1. Clinical findings with the exception of plaque index showed improvement after initial preparation in comparison with the first medical examination. 2. In phase contrast microscopy, both total bacteria and incidence of spirochetes and motile rods decreased after initial preparation in comparison with the first medical examination. 3. Clinical findings with the exception of plaque index were related to the total bacteria and proportional distribution of spirochetes and motile rods in periodontal pockets, observed in phase contrast microscopy. 4. Total bacteria and proportional distribution of black-pigmented Bacteroides in periodontal pockets decreased after initial preparation in comparison with the first medical examination.     

Relationship between clinical findings and subgingival microbial flora in periodontitis (1)

The purpose of this study was to examine the relationship between clinical findings and subgingival microbial flora in periodontitis. The results obtained were as follows: 1. In a phase-contrast microscopic study, no correlation was found between the clinical findings, total bacteria or proportional distribution of spirochetes or motile rods in the periodontal pocket. 2. Anaerobic incubation revealed no correlation between clinical findings, total bacteria or proportional distribution of black-pigmented Bacteroides in the periodontal pocket.     

Characterization of the subgingival microbial flora around endosteal sapphire dental implants in partially edentulous patients

The microbiota associated with oral endosteal sapphire ceramic implants was studied using cultural methods. Samples were taken from both implant and control (tooth) sites, and comparisons were made between healthy and diseased and between implant and control sites. Diseased sites harbored a microbiota with a large number and proportion of Gram-negative anaerobic rods, black-pigmented Bacteroides, and surface translocating bacteria. Healthy sites in the same patients had smaller amounts of bacteria dominated by facultative Gram-positive cocci and rods. The microbiota in diseased and healthy implant and control sites was very similar. It is suggested that peri-implant tissues behave very similarly to periodontal tissues and that peri-implantitis lesions should be considered as site-specific infections harboring a high number of periodontal pathogens, mainly Gram-negative anaerobic rods.     

Effect of scaling and root planing on the composition of the human subgingival microbial flora

The effect of a single session of scaling and root planing on the subgingival periodontal flora of 14 adult human subjects was investigated by darkfield microscopy. At baseline, one randomly selected periodontally diseased site in each subject was assessed for GI and P1I scores, probing depth and the percentage distribution in subgingival debris of coccoid cells, spirochetes, motile cells and other microorganisms. Following a single full mouth scaling and root planing session, these criteria were reevaluated at other initially diseased sites, one per subject and time interval. The intervals tested were days 3, 7, 14, 21, 28, 35, 42, 49, 56, 70 and 90.
The P1I and GI scores tended to decrease during the first 14 days, returning to baseline around days 21–28. After another transient decline around 35–42 days the values stabilized around baseline levels till the end of the experiment. Probing depth decreased below baseline during the first 7 days and with the exception of day 28, remained below baseline level till the end of the experiment. Coccoid cells increased from 25.1 % at baseline to 76.1 % on day 3. Return to the baseline level occurred by day 21. Spirochetes did not return to baseline until day 42. The percentage of motile cells decreased significantly from baseline on day 3 only (14.8 % to 3.8 %).
The results indicated that a single session of scaling and root planing is capable of disturbing the proportions of certain bacterial forms in the subgingival periodontal flora, and that it may require approximately 42 days for the proportions to return to baseline levels. Probing depth was significantly decreased by the debridement throughout most of the 90-day experimental period. The proportion of coccoid cells was negatively correlated with both GI and P1I scores, while the percentage of spirochetes was positively correlated with GI and P1I scores as well as probing depth measurements.     

Effect of systemic doxycycline administration on the subgingival microbial flora: a dark-field microscopy study

The effect of systemically administered doxycycline hydrochloride on subgingival microbial plaque morphotypes was evaluated using dark-field microscopy. After 1 week of drug administration, while the percentage of coccoid bacteria showed a significant increase, the percentages of spirochetes, motile rods and non-motile rods tended to decrease. Since spirochetes and/or motile rods are considered to be associated with the pathogenesis of periodontal disease, the significant decrease in the proportions of these bacteria are considered to be important. Therefore, it is suggested that doxycycline may be beneficial for the treatment of periodontal disease as an adjunct to traditional therapeutic methods.     

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.     

The efficacy of a single pocket irrigation on subgingival microbial vitality

The object of this study was to monitor the proportion of vital bacteria (microbial vitality: VF in %) present in subgingival dental plaque following one single subgingival irrigation with saline (S), chlorhexidine (CHX) or povidone iodine (I₂), but without any subgingival instrumentation. Its effect on the main composition of the microflora was also assessed. Seventeen patients with adult periodontitis took part in this investigation. In each patient four initially untreated pockets (pocket depth 5–11 mm) associated with bleeding were selected for the standardised pocket irrigation and plaque sampling at baseline (0 h) and after the following 1 h, 24 h, 7 days and 31 days. The subgingival irrigation was only performed once (0 h). One pocket per quadrant was irrigated using 0.9% prereduced S, 0.2% CHX or 0.05% I₂ (Iso-Betadine Buccale). The remaining untreated pocket without any irrigation served as an additional control (C). Using an acrylic splint as a guide, paperpoints were inserted into the pocket precisely at the same site to collect subgingival plaque. The bleeding on sampling (BOS) was thereafter noted. The proportions of bacterial morphotypes were examined by darkfield microscopy. VF was evaluated using a vital fluorescence staining. The undisturbed subgingival dental plaque was composed of 86% (median value) vital bacteria. The sampling procedure alone and the saline irrigation led to a decrease in the number of spirochetes but had no influence on the vitality of the flora. Large variations in VF could be observed in the short-term (1 h, 24 h) irrigation effect of CHX and I₂. The reduction of VF was still significant after 7 days (VF_CHX 30–80%, VF_I2 35–80%) but persisted up to 31 days only after I₂ irrigation (VF_I2 12–90%). The findings indicated that all single subgingival irrigations resulted in a temporary change of the subgingival microflora while povidone iodine produced the longest lasting antimicrobial effect. Any clinical advantage of this situation should be further investigated. Received: 16 February 1998 / Accepted: 29 May 1998     

Microbiological changes in subgingival flora after treatment with amoxicillin-clavulanic acid

The association amoxicillin-clavulanic acid can be employed as an alternative to the usual antibiotic therapy of periodontitis. The purpose of this study was to determine subgingival microbial changes in 33 patients with periodontitis after using amoxicillin (500 mgrs.-t.i.d.) and clavulanic acid (125 mgrs.-t.i.d.) for 5 days. It resulted clinically in a decrease of both gingival index and plaque index (not significant) and microbiologically in absence of the main bacterial pathogens found pretreatment, such as Bacteroides melaninogenicus, Bacteroides intermedius, Eikenella corrodens and Actinomyces sp., although it was not able to eliminate Actinobacillus asinomycetemcomitans from a juvenile periodontitis and from a prepuberal periodontitis patient. Atibiotic susceptibility testing showed that all bacteria tested were sensitive to this antibiotic. Although this short term study shows good microbial response of main periodontal pathogens, long term studies are necessary to assess the effect of this antibiotic in periodontitis therapy.     

The influence of periodontitis on the subgingival flora around implants in partially edentulous patients

The hypothesis that teeth act as reservoirs of micro‐organisms for the colonization of oral implants has recently been stated several times. The present study aimed at examining, in partially edentulous patients with severe periodontitis, whether pockets around teeth and implants harbored a comparable micro‐flora. In 6 patients (3 with refractory periodontitis and 3 with advanced chronic adult periodontitis), plaque samples were taken from a deep and shallow pocket around both teeth and implants for differential phase contrast microscopy and DNA probe analysis. The results showed important differences in the sub‐gingival flora between the 2 disease groups, as well as between deep and shallow pockets, around both implants and teeth. On the other hand, when pockets around teeth and implants with equal depths were compared a striking similarity was observed in the microbial composition. These observations confirm the hypothesis that pockets around teeth act as a reservoir and highlight the importance of periodontal health when oral implants are planned.     

The predominant cultivable subgingival flora of beagle dogs following ligature placement and metronidazole therapy

Ligature-induced periodontitis was evaluated microbiologically in 8 beagle dogs. Gingival health was established by repeated cleaning. At day 0 subgingival plaque was sampled from individual sites in two quadrants. All teeth in one quadrant were then ligated at the CEJ, and the other quadrant served as a non-ligated control which was cleaned three times each week. At day 14, four dogs were given metronidazole for seven days. Plaque was cultured anaerobically on nonselcctive media, and the predominant cultivable flora was characterized. At day 0 Gram-negative facultative rods represented 56.8 % of the flora. with motile and surface translocating organisms predominating. At day 14 Bacteroides asaccharolyticus , including catalase positive B.asaccharolyticus-like organisms, increased at ligated sites to 34.7 % of the cultivable flora. After metronidazole therapy the total bacterial count decreased, and Gram-negative anaerobic rods became non-detectable. Gram negative facultative bacteria which were motile or surface translocating represented 52.7 % of the cultivable flora after metronidazole treatment.
In the beagle dog ligature placement was associated with a shift in the flora from Gram-negative facultative rods to Gram-negative anaerobic rods. Metronidazole treatment reduced the total cultivable flora and selectively reversed the microflora shifts which followed ligature placement.     

Inhibition of Actinobacillus actinomycetemcomitans leukotoxicity by bacteria from the subgingival flora

Actinobacillus actinomycetemcomitans produces a pore-forming leukotoxin that lyses human polymorphonuclear leukocytes and monocytes. Certain proteolytic bacteria may coexist with A. actinomycetemcomitans in periodontal pockets. We aimed therefore to examine whether oral bacteria can modify the leukotoxicity of A. actinomycetemcomitans. A total of 55 strains representing 45 bacterial species of the subgingival flora were tested. Each strain was incubated with the highly toxic strain of A. actinomycetemcomitans HK 1519 and the leukotoxic activity of the suspension against human polymorphonuclear leukocytes was determined from the activity of the lactate dehydrogenase released upon lysis of the leukocytes. Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica and Prevotella loeschii inhibited the leukotoxicity of A. actinomycetemcomitans cells as well as the activity of leukotoxin purified from the same strain. The bacterial strains without the ability to block leukotoxic activity also failed to destroy pure leukotoxin even after 5 h of incubation. The proteolytic degradation of leukotoxin by P. gingivalis was mainly dependent on the activity of the enzymes R- and K-gingipains. P. intermedia and P. nigrescens also degraded the leukotoxin by enzymes. The results imply a role of the periodontal microflora in modifying the virulence of A. actinomycetemcomitans by destroying its leukotoxin.     

Clustering of subgingival microbial species in adolescents with periodontitis

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