Comparison of the virulence of exopolysaccharide-producing Prevotella intermedia to exopolysaccharide non-producing periodontopathic organisms.

BACKGROUND: Evidence in the literature suggests that exopolysaccharides (EPS) produced by bacterial cells are essential for the expression of virulence in these organisms. Secreted EPSs form the framework in which microbial biofilms are built. METHODS: This study evaluates the role of EPS in Prevotella intermedia for the expression of virulence. This evaluation was accomplished by comparing EPS-producing P. intermedia strains 17 and OD1-16 with non-producing P. intermedia ATCC 25611 and Porphyromonas gingivalis strains ATCC 33277, 381 and W83 for their ability to induce abscess formation in mice and evade phagocytosis. RESULTS: EPS-producing P. intermedia strains 17 and OD1-16 induced highly noticeable abscess lesions in mice at 107 colony-forming units (CFU). In comparison, P. intermedia ATCC 25611 and P. gingivalis ATCC 33277, 381 and W83, which all lacked the ability to produce viscous materials, required 100-fold more bacteria (109 CFU) in order to induce detectable abscess lesions in mice. Regarding antiphagocytic activity, P. intermedia strains 17 and OD1-16 were rarely internalized by human polymorphonuclear leukocytes, but other strains were readily engulfed and detected in the phagosomes of these phagocytes. CONCLUSIONS: These results demonstrate that the production of EPS by P. intermedia strains 17 and OD1-16 could contribute to the pathogenicity of this organism by conferring their ability to evade the host's innate defence response.

Reduction of two red complex bacteria by sustained-release doxycycline and correlation to improvement in mean pocket depth.

OBJECTIVE: The objective was to evaluate the effects of an 8.5% sustained-release doxycycline-containing polymer formulation (SRDF) on deep pockets (pocket depth [PD] ≥ 7 mm) in chronic periodontitis. Total bacterial counts were used to estimate the number of viable bacteria present before treatment and for up to 6 months posttreatment. METHODS: All sites had PD ≥ 5 mm and bled on probing in 23 subjects who received treatment with SRDF. There was an average of 8.7 teeth or 23 sites for each subject. One deep pocket (≥ 7 mm) in each subject was selected for monitoring. This site was sampled prior to treatment and at 7, 21, 91, and 182 days after SRDF placement. The primary endpoints were changes in the viable counts of two red complex species, Porphyromonas gingivalis and Tannerella forsythia. Secondary endpoints were changes in the number of total anaerobic bacteria recovered and changes in PD. RESULTS: Relative to baseline, SRDF reduced the proportions of P. gingivalis and T. forsythia by 88% and 99%, respectively, at day 7. At the conclusion of the monitoring period--182 days--P. gingivalis and T. forsythia were present but at 19% to 20% of the pretreatment values. Total anaerobic counts were reduced by 96% at day 7; by 87% at day 21; and by 75% and 68% at days 91 and 182, respectively. Mean PD for the sample sites (initially ≥ 7 mm) was reduced 2 mm by day 21, and this difference persisted throughout the study. CONCLUSIONS: This study demonstrates SRDF has a significant effect, not only statistically but also microbially and clinically, on deep periodontal sites in patients with chronic periodontitis. SRDF significantly reduced the number of red complex bacteria P. gingivalis and T. forsythia, as well as the number of total anaerobic bacteria. By day 21, PD was reduced by 2 mm, and this reduction was maintained for at least 6 months posttherapy.

Genetic analysis of mobile tetQ elements in oral Prevotella species.

Prevotella species are members of the bacterial oral flora and are opportunistic pathogens in polymicrobial infections of soft tissues. Antibiotic resistance to tetracyclines is common in these bacteria, and the gene encoding this resistance has been previously identified as tetQ. The tetQ gene is also found on conjugative transposons in the intestinal Bacteroides species; whether these related bacteria have transmitted tetQ to Prevotella is unknown. In this study, we describe our genetic analysis of mobile tetQ elements in oral Prevotella species. Our results indicate that the mobile elements encoding tetQ in oral species are distinct from those found in the Bacteroides. The intestinal bacteria may act as a reservoir for the tetQ gene, but Prevotella has incorporated this gene into an IS21-family transposon. This transposon is present in Prevotella species from more than one geographical location, implying that the mechanism of tetQ spread between oral Prevotella species is highly conserved.

Treating chronic periodontitis: current status, challenges, and future directions.

Periodontitis affects roughly one-third of the US population. A timely diagnosis of chronic periodontitis at its earliest stage is essential to avoid more challenging severe stages of the disease. Most cases of slight and moderate chronic periodontitis can be successfully managed by mechanical removal and/or reduction of subgingival bacterial biofilms and calculus. However, any factor that affects either the local environment or the host response may contribute to progression of the disease and a poor treatment response. Thus, it is essential that clinicians are aware of etiologic and risk factors associated with disease development and progression in order to plan and execute a successful treatment. This paper reviews a variety of risk factors, both local and systemic, that can impact the successful treatment of chronic periodontitis.

Gene expression profile and pathogenicity of biofilm-forming Prevotella intermedia strain 17.

BACKGROUND: Prevotella intermedia (P. intermedia), a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We compared these two strains (strains 17 versus 17-2) in terms of their capacities to form biofilms and to induce abscess formation in mice as an indication of their pathogenicity. Further, gene expression profiles between these two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also compared using microarray assays. RESULTS: Strain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several genes which including those for heat shock proteins (DnaJ, DnaK, ClpB, GroEL and GroES) were up-regulated two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2. Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes which including those for levanase, extracytoplasmic function-subfamily sigma factor (sigmaE; putative) and polysialic acid transport protein (KpsD), as compared to those of strain 17 in liquid culture media. CONCLUSION: These results demonstrate that the capacity to form biofilm in P. intermedia contribute to their resistance against host innate defence responses.

Microbiologic changes following administration of locally delivered doxycycline in smokers: a 15-month follow-up.

BACKGROUND: Additional clinical benefits have been reported with the use of locally delivered antimicrobials in the treatment of smokers; however, long-term changes in the subgingival microbiota following the use of these drugs in smokers have not been documented. The aim of this study was to evaluate long-term microbiologic changes following locally delivered doxycycline in the treatment of smokers. METHODS: Sixteen smokers with chronic periodontitis presenting a minimum of four pockets (probing depth>or=5 mm) were selected. Patients were assigned randomly to receive scaling and root planing (SRP) or SRP and local doxycycline (SRP-D). Patients were treated at baseline and 12 months. Subgingival plaque samples were collected at baseline; 3, 6, and 12 months; and 45 and 90 days following retreatment. Polymerase chain reaction and DNA-DNA hybridization analyses were performed to detect the presence of selected periodontal pathogens. RESULTS: The reduction in the number of sites positive for Porphyromonas gingivalis and Tannerella forsythia (previously T. forsythensis) was statistically significant for SRP-D at 3 months (68% and 41.3%, respectively) and for SRP at 6 months (75% and 52%, respectively) following treatment. The SRP group showed a greater frequency of P. gingivalis than the SRP-D group at 3 months (58% and 25%, respectively). There also was a greater reduction in the frequency of P. gingivalis at 3 months following retreatment with SRP-D compared to SRP (47% and 8%, respectively). CONCLUSION: In smokers, adjunctive local doxycycline resulted in a greater reduction in the frequency of P. gingivalis following initial and supportive therapy compared to conventional treatment.

Detection and characterization of beta-lactamase genes in subgingival bacteria from patients with refractory periodontitis.

Fifty-three beta-lactamase-producing strains of oral bacteria isolated from patients with refractory periodontitis in Norway and USA were screened for the presence of the bla(TEM), bla(SHV), bla(OXA), bla(ampC), bla(cfxA), and bla(cepA/cblA) genes by the polymerase chain reaction (PCR). The PCR products were characterized by direct sequencing of the amplified DNA. Thirty-four of the 53 enzyme-producing strains (64%) were positive in one of the PCR assays. All beta-lactamase-producing Prevotella and Capnocytophaga spp. were CfxA positive. TEM-type beta-lactamases were identified in one strain each of Escherichia coli and Neisseria sp., and one strain of Citrobacter freundii possessed an AmpC-type beta-lactamase. Screening for gene cassettes and genes known to be associated with integrons did not reveal the presence of integrons in these oral bacteria. Sequence analyses showed that most CfxA positive Prevotella and Capnocytophaga isolates from patients with refractory periodontitis harboured variants of the CfxA2 and CfxA3 enzyme. The present study also showed that many different genetic determinants of beta-lactamase production are found in bacteria isolated from refractory periodontitis, many of which remain to be characterized.

Subantimicrobial dose doxycycline enhances the efficacy of scaling and root planing in chronic periodontitis: a multicenter trial.

BACKGROUND: Previous studies have shown that subantimicrobial dose doxycycline (SDD) is of clinical benefit in the treatment of chronic periodontitis (CP). The aim of this study was to further assess the role of SDD as an adjunct to scaling and root planing (SRP) in the treatment of CP. METHODS: A double-blind, randomized, placebo-controlled, multicenter clinical study was conducted to test the efficacy of SDD (20 mg doxycycline B.I.D.) in combination with SRP in subjects with moderate to severe CP. Two-hundred ten subjects were treated with a standardized episode of SRP and randomized to receive either adjunctive SDD or placebo for 9 months. Efficacy parameters included per-subject mean changes in clinical attachment level (CAL) and probing depth (PD) from baseline, and the total number of sites with attachment gains and probing depth reductions > or = 2 mm and > or = 3 mm from baseline. RESULTS: In periodontal sites with PD 4 to 6 mm and > or = 7 mm (N = 209, intent-to-treat population), mean improvements in CAL and PD were greater following SRP with adjunctive SDD than SRP with placebo, achieving statistical significance in all baseline disease categories at month 9 (P < 0.05). At month 9, 42.3% of sites in the SDD group demonstrated CAL gain > or = 2 mm compared to 32.0% of sites in the placebo group (P < 0.01). CAL gain > or = 3 mm was seen in 15.4% of sites in the SDD group compared to 10.6% of sites in the placebo group (P < 0.05). When considering the same thresholds of change in PD, 42.9% of sites in the SDD group compared to 31.1% of sites in the placebo group demonstrated PD reduction > or = 2 mm (P < 0.01), and 15.4% of sites in the SDD group compared to 9.1% of sites in the placebo group demonstrated PD reduction > or = 3 mm (P < 0.01). CONCLUSION: Adjunctive subantimicrobial dose doxycycline enhances scaling and root planing. It results in statistically significant attachment gains and probing depth reductions over and above those achieved by scaling and root planing with placebo.

Current Status of Systemic Antibiotic Usage in Destructive Periodontal Disease.

The recognition that periodontal diseases are primarily caused by specific microorganisms has led researchers to explore the possibility that antibiotics may enhance the effect of mechanical debridement procedures such as scaling and surgery. For some selected periodontal diseases, this has proven to be true. This paper will review systemically-administered antibiotics and the clinical studies and case reports supporting their use. In periodontal therapy, the tetracyclines are the most commonly-used antibiotics in the United States. Tetracycline hydrochloride, minocycline, and doxycycline have been shown to inhibit in vitro most putative periodontal pathogens. Several studies support the use of tetracyclines in the treatment of localized juvenile periodontitis. Penicillins such as amoxicillin are effective in vitro against most periodontal pathogens but have limited efficacy due to the presence of beta-lactamases in gingival fluid. Amoxicillin/ clavulanate potassium (Au) has proven effective in treating adult refractory periodontitis characterized by a Gram-positive flora. Metronidazole is an effective adjunct in adult periodontitis associated with high numbers of "black-pigmented Bacteroides" and spirochetes. A combination of metronidazole and amoxicillin produces a synergistic effect against A. actinomycetemcomitans and has been shown to be effective at eliminating this organism. Clindamycin is an effective adjunct in the treatment of adult refractory periodontitis associated with a predominantly Gram-negative flora. The use of macrolides, quinolones, and combinations of antibiotics is discussed. Clinical studies do not support the use of systemically-administered antibiotics in routine adult periodontitis. Clinical studies do, however, support the use of antibiotics in the treatment of specific periodontal diseases. J Periodontol 1993; 64:760-771.

A Role for Antibiotics in the Treatment of Refractory Periodontitis.

Refractory periodontitis is considered by many investigators to be a separate disease entity that is descriptive of a particular patient who has multiple sites, rather than a few individual sites, that do not respond to conventional periodontal treatment modalities. Such patients continue to demonstrate loss of attachment and alveolar bone despite frequent periodontal treatment which includes surgical intervention, scaling and root planing, and often systemically-administered tetracycline. Controlled clinical studies have demonstrated that both clindamycin-hydrochloride and amoxicillin/clavulanate potassium (Au) are beneficial when used in conjunction with periodontal scaling. Gordon et al. found improvements in attachment levels, inflammation, suppuration, and a decrease in pocket depths for up to 2 years following a 7-day course of Clindamycin given in conjunction with a full-mouth scaling. The incidence of disease activity decreased from an annual rate of 8% of all sites prior to antibiotic treatment to 0.5% after treatment. Magnusson, reporting on a similar group treated with a 14-day course of Au, found an average loss of attachment of 2.2 mm and an increase in pocket depth of 1.5 mm in sites demonstrating disease progression prior to antibiotic treatment. At 3 months post-antibiotic therapy, these sites had regained an average of 2 mm of attachment and pocket depths had decreased an equivalent amount. Both attachment levels and pocket depths remained relatively stable for up to 12 months post-therapy. In an ongoing study, 30 subjects with refractory Periodontitis were treated with either Clindamycin or Au in conjunction with scaling or scaling plus a placebo. Prior to antibiotic treatment, but while being scaled at 3-month intervals, sites with disease activity lost an average 2.4 mm of attachment. At 3 months post-treatment, the clindamycin-treated group showed an average gain of 2.1 mm, the Au-treated group gained 1.9 mm, and the scaling group gained 1.4 mm in attachment. The clindamycin group remained relatively stable for up to 21 months and the Au group remained stable for about 15 months without additional treatment. Five of the 6 subjects treated with scaling alone required additional treatment within 9 months. Preliminary analyses have indicated that at least two patterns or rates of attachment loss may be associated with refractory periodontitis and that each pattern may be indicative of a different microflora. The pattern associated with a relatively rapid loss of attachment was characterized by a Gram-negative flora which contained spirochetes, P. intermedia, and Fusobacterium species. A slow, continuous rate was associated with a predominantly Gram-positive flora containing a high proportion of S. intermedius and/or a S. intermedius-like organism. J Periodontol 1993; 64:772-781.

Antibiotic Susceptibilities of Periodontal Bacteria: In Vitro Susceptibilities to Eight Antimicrobial Agents.

In vitro susceptibilities of 369 to 966 bacterial isolates from periodontal lesions to eight antibiotics were determined by agar dilution technique as a means of determining which antimicrobial agents were inhibitory for bacteria frequently associated with destructive periodontal diseases. Although most bacteria were relatively susceptible to the penicillins, greater activity was generally noted with amoxicillin than with either penicillin or ampicillin with the exception of Selenomonas sputigena and Peptostreptococcus. Antibacterial activities obtained with minocycline were significantly higher than with tetracycline for Actinobacillus actinomycetemcomitans and Streptococcus but comparable for most other taxa. Clindamycin and metronidazole both demonstrated excellent activity against the anaerobic Gram-negative rods but were less effective against some of the capnophilic and facultative organisms. Eikenella corrodens was exceptionally resistant to both of these drugs; and A. actinomycetemcomitans was generally resistant to Clindamycin but relatively susceptible to metronidazole. Erythromycin was considerably less active than the other antibiotics against the majority of the periodontal bacteria. No single antibiotic, at concentrations equivalent to those achieved in body fluids, was uniformly effective in inhibiting all bacteria currently implicated or suspected as etiologic agents of periodontal diseases.