Thomas E. Rams

The subgingival microbial flora associated with human dental implants

I n recent years the microbial colonization on different surfaces in the oral cavity has been studied by oral microbiologists. Resident bacterial populations have been examined from the buccal mucosa,’ the tongue,2 enamel,3 cemental root surfaces,4 epoxy resin crowns,s overhanging amalgam restorations,6 and surfaces of dentures? However, none of the studies has involved surfaces of dental implants, despite their wide use over the past 15 years for the replacement of teethin edentulous spores. Even with clear anatomic and morphologic differences, clinical and histopathologic descriptions of gingival inflammation, pocket formation, and alveolar bone loss around protruding dental implants that are remarkably similar to periodontal lesions associated with natural teeth have been reported.8s’2 With natural teeth the relationship between periodontal disease and the presence of periodontopathic microorganisms in subgingival plaque has become widely accepted. 13,14 In contrast little is known about the nature of the subgingival bacterial populations that colonize around dental implants and their relationship to peri-implant tissue conditions. Direct microscopic examinations of dental plaque enables a rapid and inexpensive study of subgingival bacterial types without encountering the many difficulties inherent in carrying out conventional bacteriologic culturing, and they have been used previously to characterize the microorganisms around natural teeth.‘5,‘6 In this investigation the subgingival microbiologic flora associated with 17 gingival tissue-protruding dental implants was studied with direct phasecontrast microscopy.

Antibiotic Resistance in Human Chronic Periodontitis Microbiota

BACKGROUND Patients with chronic periodontitis (CP) may yield multiple species of putative periodontal bacterial pathogens that vary in their antibiotic drug susceptibility. This study determines the occurrence of in vitro antibiotic resistance among selected subgingival periodontal pathogens in patients with CP. METHODS Subgingival biofilm specimens from inflamed deep periodontal pockets were removed before treatment from 400 adults with CP in the United States. The samples were cultured, and selected periodontal pathogens were tested in vitro for susceptibility to amoxicillin at 8 mg/L, clindamycin at 4 mg/L, doxycycline at 4 mg/L, and metronidazole at 16 mg/L, with a post hoc combination of data for amoxicillin and metronidazole. Gram-negative enteric rods/pseudomonads were subjected to ciprofloxacin disk-diffusion testing. RESULTS Overall, 74.2% of the patients with CP revealed subgingival periodontal pathogens resistant to at least one of the test antibiotics. One or more test species, most often Prevotella intermedia/nigrescens, Streptococcus constellatus, or Aggregatibacter actinomycetemcomitans, were resistant in vitro to doxycycline, amoxicillin, metronidazole, or clindamycin, in 55%, 43.3%, 30.3%, and 26.5% of the patients with CP, respectively. Fifteen percent of patients harbored subgingival periodontal pathogens resistant to both amoxicillin and metronidazole, which were mostly either S. constellatus (45 individuals) or ciprofloxacin-susceptible strains of Gram-negative enteric rods/pseudomonads (nine individuals). CONCLUSIONS Patients with CP in the United States frequently yielded subgingival periodontal pathogens resistant in vitro to therapeutic concentrations of antibiotics commonly used in clinical periodontal practice. The wide variability found in periodontal pathogen antibiotic-resistance patterns should concern clinicians empirically selecting antibiotic treatment regimens for patients with CP.

Clinical and Microbiologic Effects of Adjunctive Antibiotics in Treatment of Localized Juvenile Periodontitis. A Controlled Clinical Trial

The aim of this study was to assess the clinical and microbiologic effects of the combination of amoxicillin and metronidazole therapy as an adjunct to mechanical treatment in the management of localized juvenile periodontitis. Twenty-five localized juvenile periodontitis (LJP) patients from a Brazilian population were randomly allocated into an experimental group receiving mechanical treatment and antibiotics, and a control group receiving mechanical treatment and placebo. Clinical and radiographic assessments, as well as microbiologic sampling for Actinobacillus actinomycetem.comitans, were performed at baseline and one year after the end of the treatment. At the termination of the study A. actinomycetemcomitans could be isolated from the oral cavity of all patients in the control group who harbored the bacterium at baseline and in 4 out of 8 patients in the experimental group. Both treatment modalities resulted in significant benefit on an individual basis. The experimental group, however, displayed better results than did the control group regarding gingival index (GI), probing depth (PD), clinical attachment level (CAL), and radiographic analysis of crestal alveolar bone mass, but not with respect to plaque index (PI). No serious adverse effects of the antibiotic treatment were observed in the present study. J Periodontol 1998;69:1355-1363.

Prevalence of β-lactamase-producing bacteria in human periodontitis.

BACKGROUND AND OBJECTIVE Beta-lactam antibiotics prescribed in periodontal therapy are vulnerable to degradation by bacterial β-lactamases. This study evaluated the occurrence of β-lactamase-positive subgingival bacteria in chronic periodontitis subjects of USA origin, and assessed their in vitro resistance to metronidazole at a breakpoint concentration of 4 μg/mL. MATERIAL AND METHODS Subgingival plaque specimens from deep periodontal pockets with bleeding on probing were removed from 564 adults with severe chronic periodontitis before treatment. The samples were transported in VMGA III and then plated onto: (i) nonselective enriched Brucella blood agar (EBBA) and incubated anaerobically for 7 d; and (ii) selective trypticase soy-bacitracin-vancomycin (TSBV) and incubated for 3 d in air + 5% CO2 . At the end of the incubation periods, the bacterial test species were identified and quantified. Specimen dilutions were also plated onto EBBA plates supplemented with 2 μg/mL of amoxicillin, a combination of 2 μg/mL of amoxicillin plus 2 μg/mL of the β-lactamase inhibitor clavulanic acid, or 4 μg/mL of metronidazole, followed by anaerobic incubation for 7 d. Bacterial test species presumptively positive for β-lactamase production were identified by growth on EBBA primary isolation plates supplemented with amoxicillin alone and no growth on EBBA primary isolation plates containing both amoxicillin plus clavulanic acid. A subset of such isolates was subjected to nitrocefin-based chromogenic disk testing to confirm the presence of β-lactamase activity. In vitro resistance to 4 μg/mL of metronidazole was noted when growth of test species occurred on metronidazole-supplemented EBBA culture plates. RESULTS Two-hundred and ninety-four (52.1%) of the study subjects yielded β-lactamase-producing subgingival bacterial test species, with Prevotella intermedia/nigrescens, Fusobacterium nucleatum and other Prevotella species most frequently identified as β-lactamase-producing organisms. Of the β-lactamase-producing bacterial test species strains recovered, 98.9% were susceptible in vitro to metronidazole at 4 μg/mL. CONCLUSION The occurrence of β-lactamase-positive subgingival bacterial species in more than half of the subjects with severe chronic periodontitis raises questions about the therapeutic potential of single-drug regimens with β-lactam antibiotics in periodontal therapy. The in vitro effectiveness of metronidazole against nearly all recovered β-lactamase-producing subgingival bacterial species further supports clinical periodontitis treatment strategies involving the combination of systemic amoxicillin plus metronidazole.

Aggregation of human platelets by gingipain-R from Porphyromonas gingivalis cells and membrane vesicles

The hypothesis that there is an association between periodontitis and cardiovascular disease suggests new lines of research on the mechanism whereby oral bacteria might exert systemic effects. This study was conducted to ascertain and quantitate the effect of Porphyromonas gingivalis on human platelets in vitro . A second related objective was to purify and identify the aggregating vector. Aggregation was measured by platelet turbidometry and gingipain-R was purified from P. gingivalis membrane vesicles by Sepharose 2B and hydroxyapatite chromatography. The in vitro aggregation of platelets requires that at least 1.0 2 10 4 cells be stirred with 1.35 2 10 8 platelets. The specific activity is substantially increased in the membrane vesicles that are shed by this bacterium. Aggregability was due to gingipain-R activity, a potent cysteine protease that was found to be highly concentrated in the membrane vesicle fraction. The enzyme was purified 18-fold in high yield from the membrane vesicles, and consists of two noncovalently linked proteins that migrate at 49 and 44 kDa on SDS-PAGE. Aggregation of platelets by gingipain-R was shown to be dose-dependent, and inhibited by leupeptin and arginine, but not by anti-thrombin III. This is the first report enumerating the specific number of cells and lowest concentration of membrane vesicles necessary to evoke a full human platelet response, and the first report to assign this activity to gingipain-R.

Orthodontic therapy in patients with juvenile periodontitis: Clinical and microbiologic effects

The correction of malocclusions in juvenile periodontitis (JP) patients completing periodontal therapy is a problem of increasing clinical concern to orthodontists, since many teeth with severe alveolar bone loss in these patients can now be successfully treated without extraction. In this report, fixed edgewise orthodontic therapy was carried out after the completion of periodontal therapy on four JP patients. The orthodontic therapy included extensive intrusion of teeth severely affected by JP. Phase-contrast microscopic analysis of subgingival plaque from orthodontically treated teeth was used to monitor longitudinally the effects of fixed orthodontic bands on the subgingival flora and also to monitor the efficacy of topical and systemic antimicrobial therapy aimed at suppression of suspected periodontopathic bacteria. Orthodontic movement was completed on most periodontally compromised teeth without significant evidence of additional deterioration in periodontal status. However, within the first 6 months of orthodontic band placement, all patients had significant increases in the number of spirochetes and motile rods in their subgingival flora. Three of the patients also developed high levels of crevicular polymorphonuclear leukocytes around orthodontically treated teeth, indicating significant subgingival inflammation. Intensive antimicrobial measures, including topical inorganic salt applications and systemic tetracycline, were helpful in limiting clinical inflammation and subgingival colonization by periodontopathogens during orthodontic therapy. The results demonstrate that successful orthodontic repositioning can be carried out in treated JP patients. In addition, bacteriologic monitoring and chemotherapeutic suppression of periodontal pathogens may be valuable in the prevention of further destructive periodontal disease activity in periodontitis patients undergoing orthodontic therapy.

Antibiotic Susceptibility of Periodontal Enterococcus faecalis

BACKGROUND Enterococcus faecalis may contribute to periodontal breakdown in heavily infected subgingival sites, particularly in patients responding poorly to mechanical forms of periodontal therapy. Because only limited data are available on the antimicrobial sensitivity of enterococci of subgingival origin, this study evaluates the in vitro antibiotic susceptibility of E. faecalis isolated from periodontitis patients in the United States. METHODS Pure cultures of 47 subgingival E. faecalis clinical isolates were each inoculated onto specially prepared broth microdilution susceptibility panels containing vancomycin, teicoplanin, and six oral antibiotics of potential use in periodontal therapy. After incubation in ambient air for 18 to 20 hours, minimal inhibitory drug concentrations were determined using applicable Clinical and Laboratory Standards Institute criteria and interpretative guidelines. The organisms were additionally evaluated for in vitro resistance to metronidazole at 4 μg/mL. RESULTS Periodontal E. faecalis exhibited substantial in vitro resistance to tetracycline (53.2% resistant), erythromycin (80.8% resistant or intermediate resistant), clindamycin (100% resistant to 2 μg/mL), and metronidazole (100% resistant to 4 μg/mL). In comparison, the clinical isolates were generally sensitive to ciprofloxacin (89.4% susceptible; 10.6% intermediate resistant) and 100% susceptible in vitro to ampicillin, amoxicillin/clavulanate, vancomycin, and teicoplanin. CONCLUSIONS Tetracycline, erythromycin, clindamycin, and metronidazole revealed poor in vitro activity against human subgingival E. faecalis clinical isolates, and would likely be ineffective therapeutic agents against these species in periodontal pockets. Among orally administered antibiotics, ampicillin, amoxicillin/clavulanate, and ciprofloxacin exhibited marked in vitro inhibitory activity against periodontal E. faecalis, and may be clinically useful in treatment of periodontal infections involving enterococci.

Subgingival microbiota in adult Down syndrome periodontitis.

BACKGROUND AND OBJECTIVE The subgingival microbiota in Down syndrome and non-Down syndrome adults receiving periodic dental care was examined for 40 bacterial species using checkerboard DNA-DNA hybridization and the results were related to clinical periodontal attachment loss. MATERIAL AND METHODS A total of 44 Down syndrome, 66 non-Down syndrome mentally retarded and 83 mentally normal adults were clinically evaluated. This involved, for each subject, the removal of subgingival specimens from three interproximal sites on different teeth; all subgingival samples per subject were then pooled and assessed for the presence and levels of 40 bacterial species using species-specific whole-genomic DNA probes and checkerboard DNA-DNA hybridization. Significant group differences in species proportions averaged across subjects were evaluated using the Kruskal-Wallis test, and associations between subgingival species and mean subject attachment loss within Down syndrome and non-Down syndrome subject groups were quantified using Pearson correlation and multiple linear regression analysis. RESULTS Down syndrome subjects exhibited greater attachment loss than non-Down syndrome subjects (p=0.05). Most microbial species were present in Down syndrome subjects at levels similar to non-Down syndrome subjects, except for higher proportions of Selenomonas noxia, Propionibacterium acnes, Streptococcus gordonii, Streptococcus mitis and Streptococcus oralis in Down syndrome subjects compared with non-Down syndrome study subjects, higher proportions of Treponema socranskii in Down syndrome subjects compared with non-Down syndrome mentally retarded subjects, and higher proportions of Streptococcus constellatus in Down syndrome subjects compared with mentally normal subjects. Down syndrome adults classified with periodontitis revealed higher subgingival levels of T. socranskii than Down syndrome subjects with no periodontitis (p=0.02). Higher subgingival proportions of S. constellatus, Fusobacterium nucleatum ssp. nucleatum, S. noxia and Prevotella nigrescens showed significant positive correlations (r=0.35-0.42) and higher proportions of Actinomyces naeslundii II and Actinomyces odontolyticus showed negative correlations (r=-0.36 to -0.40), with increasing mean subject attachment loss in Down syndrome adults. CONCLUSION Individuals with Down syndrome show higher levels of some subgingival bacterial species and specific associations between certain subgingival bacterial species and loss of periodontal attachment. These findings are consistent with the notion that certain subgingival bacteria may contribute to the increased level of periodontal disease seen in Down syndrome individuals and raise the question as to the reason for increased colonization in Down syndrome.

Spiramycin resistance in human periodontitis microbiota

PURPOSE The occurrence of in vitro resistance to therapeutic concentrations of spiramycin, amoxicillin, and metronidazole was determined for putative periodontal pathogens isolated in the United States. MATERIALS AND METHODS Subgingival plaque specimens from 37 consecutive adults with untreated severe periodontitis were anaerobically cultured, and isolated putative periodontal pathogens were identified to a species level. In vitro resistance to spiramycin at 4 μg/ml, amoxicillin at 8 μg/ml, and/or metronidazole at 16 μg/ml was noted when putative periodontal pathogen growth was noted on the respective antibiotic-supplemented primary culture plates. RESULTS A total of 18 (48.7%) subjects yielded antibiotic-resistant putative periodontal pathogens with spiramycin at 4 μg/ml in drug-supplemented primary isolation plates, as compared to 23 (62.2%) subjects with amoxicillin at 8 μg/ml, and 10 (27.0%) subjects with metronidazole at 16 μg/ml. Spiramycin in vitro resistance occurred among species of Fusobacterium nucleatum (44.4% of organism-positive subjects), Prevotella intermedia/nigrescens (11.1%), Parvimonas micra (10.8%), Streptococcus constellatus (10%), Streptococcus intermedius (10%), Porphyromonas gingivalis (6.7%), and Tannerella forsythia (5.3%). Amoxicillin in vitro resistance was found in P. intermedia/nigrescens (55.5%), T. forsythia (15.8%), S. constellatus (10%), F. nucleatum (5.6%), and P. micra (2.7%). Only S. constellatus (70%) and S. intermedius (40%) exhibited in vitro resistance to metronidazole. When subject-based resistance data for spiramycin and metronidazole were jointly considered, all isolated putative periodontal pathogens were inhibited in vitro by one or the other of the antibiotic concentrations, except for one strain each of S. constellatus and S. intermedius from one study subject. Similarly, either amoxicillin or metronidazole at the drug concentrations tested inhibited in vitro all recovered putative periodontal pathogens, except S. constellatus in one subject. CONCLUSIONS In vitro spiramycin resistance among putative periodontal pathogens of United States origin occurred in approximately one-half of severe periodontitis patients evaluated, particularly among subgingival F. nucleatum species. In vitro resistance patterns also suggest that therapeutic concentrations of spiramycin plus metronidazole may have potential antimicrobial efficacy in non-Aggregatibacter actinomycetemcomitans-associated periodontitis similar to amoxicillin plus metronidazole, which may be beneficial, where spiramycin is clinically available, for patients hypersensitive to amoxicillin or other beta-lactam antibiotics.

Antibiotic Susceptibility of Periodontal Streptococcus Constellatus and Streptococcus Intermedius Clinical Isolates

BACKGROUND Streptococcus constellatus and Streptococcus intermedius in subgingival dental plaque biofilms may contribute to forms of periodontitis that resist treatment with conventional mechanical root debridement/surgical procedures and may additionally participate in some extraoral infections. Because systemic antibiotics are often used in these clinical situations, and little is known of the antibiotic susceptibility of subgingival isolates of these two bacterial species, this study determined the in vitro susceptibility to six antibiotics of fresh S. constellatus and S. intermedius clinical isolates from human periodontitis lesions. METHODS A total of 33 S. constellatus and 17 S. intermedius subgingival strains, each recovered from separate patients with severe chronic periodontitis (n = 50) before treatment, were subjected to antibiotic gradient strip susceptibility testing with amoxicillin, azithromycin, clindamycin, ciprofloxacin, and doxycycline on blood-supplemented Mueller-Hinton agar and to the inhibitory effects of metronidazole at 16 mg/L in an enriched Brucella blood agar dilution assay. Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing interpretative standards were used to assess the results. RESULTS Clindamycin was the most active antibiotic against S. constellatus (minimum inhibitory concentration at 90% [MIC90] 0.25 mg/L), and amoxicillin was most active against S. intermedius (MIC90 0.125 mg/L). A total of 30% of the S. constellatus and S. intermedius clinical isolates were resistant in vitro to doxycycline, 98% were only intermediate in susceptibility to ciprofloxacin, and 90% were resistant to metronidazole at 16 mg/L. CONCLUSION Subgingival S. constellatus and S. intermedius exhibited variable antibiotic susceptibility profiles, potentially complicating empirical selection of periodontitis antibiotic therapy in patients who are species positive.