JADA Continuing Education
Microbiological changes associated with dental prophylaxis
J. MAX GOODSON, D.D.S., Ph.D.,
MICHAEL D. PALYS, D.M.D., M.M.Sc.,
ELIZABETH CARPINO, M.E.M.,
ELIZABETH O. REGAN, M.S.,
MICHAEL SWEENEY, B.F.A. and
SIGMUND S. SOCRANSKY, D.M.D.
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ABSTRACT
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Background. Despite the common application of dental prophylaxis as part of patient therapy, there is little reported that describes the microbiological impact of this treatment.
Methods. The authors gave 20 healthy college-aged subjects three dental prophylaxes with a fluoride-containing prophylaxis paste during a two-week period and instructed them in oral hygiene. They evaluated the microbiological composition of dental plaque samples collected before and after treatment using DNA probe analysis. They analyzed 40 representative bacterial species in seven bacterial complexes by checkerboard DNA–DNA hybridization assay techniques.
Results. After three dental prophylaxes, the patients’ mean Gingival Index score decreased from 0.82 to 0.77, the mean Plaque Index score decreased from 0.72 to zero, and the total number of bacteria per tooth decreased to approximately one-third of the original number. The authors computed two different measures of bacterial presence. The reduction in bacterial numbers was statistically significant and occurred in many species. Bacterial proportion (DNA percentage or percentage of the bacteria per tooth) did not change significantly. Greater reductions in bacterial count occurred in species that showed high numbers before treatment. The total bacterial count decreased by approximately 72 percent of its original level before prophylaxis was initiated.
Conclusions. Professional dental prophylaxis did not target any particular bacteria or bacterial groups but removed bacteria nonspecifically and in proportion to their initial numbers.
Clinical Implications. Repeated dental prophylaxes effect a reduction in bacterial amount that is commensurate with the initial amount, but they do does not alter composition. This suggests that mild gingivitis may be a bacterially nonspecific effect of plaque accumulation and emphasizes the need for regular plaque removal to maintain optimal gingival health.
There is much in the literature on the contribution of dental plaque to the development of oral disease. Dental plaque is a complex mixture of bacteria, with representatives from more than 500 species1 enmeshed in a tightly adherent biofilm.2 The plaque biofilm forms on all hard and soft tissues in the mouth. Some of the microorganisms in plaque have been identified as the principal etiologic agents in the development of caries,3 gingivitis4 and periodontal disease.5
Professional dental prophylaxis removed bacteria nonspecifically and in proportion to their initial numbers.
A central tenet of the experimental gingivitis model is scrupulous initial tooth cleaning. Before beginning the study, therefore, we provided participating subjects with repeated prophylaxes. This article details the microbiological composition of dental plaque before and after subjects were brought to the "super-clean" state, to show the effect of professional prophylaxis. After the initial two-week tooth cleaning period, we required our subjects to refrain from all home care for two weeks. The results of that subsequent part of the study will be detailed in future publications.
Professional plaque removal, or tooth cleaning, is an important component of a healthy oral hygiene regimen. In a longitudinal study, Axelsson and colleagues6 demonstrated the beneficial effects of professional mechanical tooth cleaning when combined with oral hygiene instruction. In this study, selective removal of supragingival plaque from all tooth surfaces with mechanically driven instruments and fluoride polishing paste improved gingival health (reducing scores on gingival and plaque indexes).
Löe and colleagues7 described the "super-clean" state as the first step in their experimental gingivitis model. These investigators used microscopy and, subsequently, conventional culture techniques to determine the specific bacteria involved in this process. Unfortunately, both microscopic and culturing techniques have serious limitations. Microscopy cannot identify species, and culture identification is so labor-intensive that statistical analysis of the results becomes prohibitively expensive. There also are strains that are uncultivable altogether. Microbiology by means of DNA probe analysis has the advantage of being rapid, specific and relatively inexpensive.8 We decided to conduct a study in which we would use DNA probe analysis to determine and analyze the specific bacterial changes that occurred as a result of dental prophylaxis. This article reports on changes in the bacterial composition of dental plaque that occurred during an initial prophylaxis phase before the development of experimental gingivitis.
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SUBJECTS AND METHODS
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Subject population.
We conducted this study with the approval of the institutional review board of The Forsyth Institute, Boston. Subjects came from the Boston community in response to newspaper advertisements. The subject population consisted of 20 university students who were between 18 and 28 years of age and had no periodontal disease. Nine men and 11 women participated. Subjects were qualified to participate if they were between 18 and 45 years of age, had at least 24 teeth and no periodontal sites with pocket depth greater than 4 millimeters. We excluded from the study any potential subject who was pregnant or lactating, had undergone periodontal or antibiotic therapy in the preceding three months, or had systemic conditions that might influence the course of periodontal disease or require antibiotic coverage for routine periodontal procedures.
The examining clinician explained to all subjects the purpose and nature of the study, including the types of clinical measurements and sampling methods to be used. When all questions had been answered to the subject’s satisfaction, he or she signed an institutional review board–approved consent form. Each subject received a copy of the consent form.
Clinical evaluation and plaque sampling.
We made clinical evaluations of Gingival Index, or GI, and Plaque Index, or PLI,9 for all sites in the mouth, and we took plaque samples from the mesial aspect of all 28 teeth, excluding third molars. We took samples separately from each tooth using individual sterile Gracey curets and placed them in individual tubes containing 0.15 milliliters each of tris ethylenediamine tetra-acetic acid buffer solution and 0.5-molar sodium hydroxide.
Prophylaxis.
After initial screening, obtaining of informed consent, clinical evaluation and sample collection, all subjects received professional plaque removal (dental prophylaxis) using a rubber cup and prophylaxis handpiece with prophylaxis paste (Prophy Paste with Fluroide, medium grit, Oral-B, Belmont, Calif.). We repeated this procedure once per week for two weeks. At each visit, we also gave subjects instruction in proper home care. After three prophylaxes, we took a second periodontal plaque sample for comparison with the pretreatment sample.
Identification and enumeration of bacteria.
We evaluated plaque samples for their content of 40 representative subgingival species using checkerboard DNA–DNA hybridization as described by Socransky and colleagues,8 a technique by which plaque samples are boiled for five minutes and neutralized in 0.8 mL of 5.0-molar ammonium acetate. Samples then were placed in a Minislot apparatus (Immunetics, Cambridge, Mass.), where they were affixed to a Nytran membrane (Boehringer Mannheim, Indianapolis) by exposure to ultraviolet light, then heated at 120 C for 20 minutes.
DNA probes.
We prepared digoxigenin-labeled, whole genomic DNA probes using a random primer technique. This method involves growing bacteria on agar or broth media, harvesting the cells and extracting, purifying and labeling the bacterial DNA from each of the test species. We labeled the DNA with digoxigenin using a random primer technique.10 We evaluated the presence of 40 bacteria shown in the box
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Standards.
We prepared whole-cell DNA standards by adjusting the optical density (600 nanometers) of cell suspensions harvested from agar plates or broth media to an optical density of 1.0 (approximately 1 billion cells). Subsequent dilutions were based on microscopic counts in a defined volume. Routine standards (100,000 and 1 million) used on each checkerboard were prepared from a DNA cocktail of all 40 bacterial species, each adjusted to provide the same signal as the whole-cell standard.
Hybridization.
We placed prehybridized Nytran membranes into a Miniblotter 45 (Immunetics) with the membrane turned 90 degrees to its original orientation. We then pipetted the probes into each lane of the Miniblotter. Each membrane was placed in a sealed plastic bag containing hybridization buffer. After membranes had been in a hybridizing solution overnight at 42 C, we washed them first at low stringency to remove loosely bound probes, then twice for 20 minutes at high stringency.
Probe detection.
We incubated the membranes with a 1:20,000 dilution of antidigoxigenin antibody conjugated with alkaline phosphatase using the modification described by Engler-Blum and colleagues.11 After washing, the membranes were incubated in AttoPhos (Clare Chemical Research, Dolores, Colo.) overnight at room temperature. Fluorescent emission was detected using the Storm FluorImager (Amersham Biosciences, Sunnyvale, Calif.). We adjusted the sensitivity of the assay for bacterial species to detect 10,000 of each species.
Data analysis.
We summarized microbiological data for analysis as two measures. We computed the numbers of bacteria as mean values (averaged across all teeth) for each subject at each visit, and we computed the proportion of bacteria as the fraction obtained by dividing the number of each species by the sum of all 40 species. We analyzed differences between species by means of the nonparametric Kruskal-Wallis method, with P values of .001 or less considered as statistically significant, to compensate for error introduced by multiple testing.
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RESULTS
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Clinical.
Mean GI scores decreased from 0.82 before the first cleaning to 0.77 after the third cleaning. PLI scores decreased from 0.72 to zero during the same period. The total number of bacteria per tooth decreased from more than 300,000 to approximately 100,000 (Figure 1).
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Figure 1. A. Tooth cleaning and microbiological sampling schedule. B. Gingival Index, or GI, and Plaque Index, or PLI, scores decreased after cleaning. C. Average total number of bacteria per tooth for 20 healthy young adult subjects before and after three dental prophylaxis visits decreased from more than 300,000 before cleaning to approximately 100,000 after the visits.
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Numbers of bacteria.
The numbers of each of the 40 bacterial strains before and after the cleaning procedures are illustrated in Figure 2. The numbers of bacteria were reduced in every case. We observed statistically significant reductions in numbers of Veillonella parvula, Fusobacterium nucleatum (subspecies vincentii and polymorphum), Fusobacterium periodonticum, Eikenella corrodens, Streptococcus mitis, Campylobacter showae and Capnocytophaga ochracea.
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Figure 2. The number (left) and percentage (right) of bacterial species, before and after the two-week regimen of professional cleaning. Note that changes (all reductions) in bacterial numbers often were significant, whereas none of the changes in percentage composition of the microbiota was statistically significant. (Complete bacterial names appear in the box on page 1560.)
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Proportions of bacteria.
The proportion of bacteria before and after dental prophylaxis showed almost no change. The results for each species are shown in the right-hand bilateral bar chart in Figure 2.
Some bacterial numbers decreased by as much as 200,000; these bacterial strains were high in number before tooth cleaning. Other bacteria were reduced by less than 20,000 but had been lower in number before cleaning. This suggests that bacteria were removed in proportion to their initial numbers. Considering the decrease in bacteria relative to the number existing before the cleaning procedures, we found a strong linear relationship (Figure 3), which indicates that all bacteria were reduced by approximately 72 percent of their original level (with a squared correlation coefficient of .97) before tooth-cleaning procedures began.
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Figure 3. Regression analysis by species exhibits a reduction in number of bacterial cells against initial number for each of 40 bacterial species (r2 = .97). (Complete bacterial names appear in the box on page 1560.)
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Bacterial complexes.
We evaluated the distribution of bacterial complexes by computing proportions of bacteria. These values changed little as a result of dental prophylaxis. Before-cleaning and after-cleaning population percentages of microorganism groups are shown in the pie charts in Figure 4. Although the Actinomyces bacteria increased and bacteria in the orange group decreased noticeably, none of these groups’ changes was statistically significant.
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Figure 4. Changes in bacterial complexes. The change in percentage among major bacterial groups before and after three dental prophylaxes was not statistically significant.
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DISCUSSION
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Participants in this study were young and periodontally healthy. Nevertheless, repeated dental prophylaxis significantly reduced the total number of bacteria on each tooth, as well as the GI score. This indicates that even healthy people may benefit from dental prophylaxis. A similar study of supragingival professional cleaning involving subjects with periodontal disease reported that subgingival bacterial numbers were reduced by supragingival cleaning.12 In this study, however, we saw changes in proportions of bacterial strains in healthy subjects to be much less remarkable than changes in number.
Although reduction in bacterial numbers may be expected to produce a transient beneficial effect, there is no indication that the bacterial ecology was altered by the prophylaxis. The microbiological effects of frequent prophylaxis appear to be simply a reduction in the bacterial load. This finding underscores the need for regular plaque removal to obtain optimal effect.
Investigators have described the clinical condition of subjects after intensive professional cleaning as "induced normality" or "superhealthy gingiva."13 This study indicates that the "super-healthy" state is associated with a 72 percent reduction in numbers of bacteria to 100,000 or fewer per tooth without any change in species composition. We deduced this from the direct proportionality between the initial numbers of each bacterial strain and the amount reduced by dental prophylaxis (Figure 3). It also appears in the statistical significance of the changes shown in Figure 2.
Throughout the history of oral microbiology, a controversy has simmered as to whether periodontal diseases are specific or nonspecific to the presence of certain bacterial species. This divergence in opinion ultimately stems from differences in treatment approaches. Extremist supporters of the "specific" hypothesis espouse specific antibiotics or antibacterial agents for treatment, with less emphasis on other aspects of treatment. Extremist adherents to the "nonspecific" hypothesis would claim that regular mechanical cleaning is of paramount importance. Periodontal diseases are considered by many to be associated with specific bacterial species. For example, adult periodontitis has been associated with Tannerella forsythensis, Porphyromonas gingivalis and Treponema denticola, and juvenile periodontitis has been associated with Actinobacillus actinomycetemcomitans. Evidence suggests that both of these disease conditions benefit from antibiotic therapy.14 Others have proposed that gingivitis could be the result of the generic metabolic production of short-chain fatty acids.15 This study indicates that a mild level of gingivitis in healthy people may have a more nonspecific nature than does periodontitis, as simple nonspecific removal of bacteria virtually eliminates clinically visible gingivitis.
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CONCLUSION
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Dental prophylaxis, one of the most common professional periodontal treatments administered, generally can provide measurable reduction in clinical signs of gingivitis, particularly redness. This study indicates that the principal effect of this treatment on 40 periodontal bacteria is a reduction in their total numbers, but little change in the relative bacterial composition of dental plaque. These observations suggest that the earliest signs of gingivitis simply are the result of increased plaque accumulation and, consequently, the products of dental plaque accumulation. As a result, regular plaque removal appears necessary to prevent the consequences of early gingivitis.
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FOOTNOTES
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Dr. Goodson is the director, Clinical Research, The Forsyth Institute, 140 The Fenway, Boston, Mass. 02115, e-mail "mgoodson{at}forsyth.org". Address reprint requests to Dr. Goodson.
Dr. Palys is a periodontist, Harvard University Health Services, Dental Service, Cambridge, Mass.
Ms. Carpino is a research assistant, The Forsyth Institute, Boston.
Ms. Regan is a research assistant, The Forsyth Institute, Boston.
Mr. Sweeney is a research assistant, The Forsyth Institute, Boston.
Dr. Socransky is a senior member of the staff, The Forsyth Institute, Boston.
The research described in this article was supported in part by grant P30-DE11814 from the National Institute of Craniofacial and Dental Research to ABIOMED, Danvers, Mass.
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ABSTRACT
SUBJECTS AND METHODS
