Violet I. Haraszthy

The Microbiology of Early-Onset Periodontitis: Association of Highly Toxic Actinobacillus actinomycetemcomitans Strains With Localized Juvenile Periodontitis

Recent studies of the dental plaque bacteria associated with the various forms of early-onset periodontitis confirm the importance of target periodontal pathogens such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Prevotella intermedia, and Porphyromonas gingivalis in these diseases. A. actinomycetemcomitans strains exhibit a wide range of variability in leukotoxin production. By virtue of a unique promoter for the leukotoxin (ltx) operon, highly leukotoxic A. actinomycetemcomitans strains (e.g., JP2) express 10- to 20-times greater levels of leukotoxin than minimally toxic strains (e.g., 652). In dot blot hybridization and polymerase chain reaction (PCR) assays, the distribution of leukotoxic A. actinomycetemcomitans was examined among 165 fresh isolates and strains from our culture collection obtained from 91 human patients and non-human primates. Highly leukotoxic A. actinomycetemcomitans strains were found in 22% of the subjects and represented 28% of the isolates examined. This is a much higher prevalence than reported in a similar survey of A. actinomycetemcomitans strains from Northern Europe. Patients harboring the highly leukotoxic strains were much younger (mean age 12.7 years) than those harboring minimally toxic A. actinomycetemcomitans (mean age 25.5 years). In addition, patients with localized juvenile periodontitis were shown to have a substantially higher prevalence of highly leukotoxic strains than healthy individuals or those with adult periodontitis. Fifty-seven percent of the localized juvenile periodontitis patients harbored these strains and 64% of the isolates obtained from these patients were highly toxic A. actinomycetemcomitans. No highly toxic strains were identified from healthy individuals or from patients with adult periodontitis. The polymerase chain reaction assay could readily identify and distinguish the ltx promoters from highly toxic and minimally toxic A. actinomycetemcomitans in whole plaque samples. These data point to the importance of specific A. actinomycetemcomitans strains, as characterized by their expression of high levels of leukotoxin, in the pathogenesis of certain types of early-onset periodontitis and, possibly, other forms of rapidly progressing periodontitis. J Periodontol 1996;67:282-290.

A Clinical Study of 406 Sinus Augmentations With 100% Anorganic Bovine Bone

BACKGROUNDnThe aim of the present study is to evaluate the use of anorganic bovine bone (ABB) associated with a collagen membrane (CM) for a sinus graft by means of clinical, histologic, and radiographic parameters in cases with bone availability < or =7 mm. A preliminary evaluation consisted of a clinical examination, computed tomography (CT), and a panoramic x-ray.nnnMETHODSnNinety-two patients requiring bilateral sinus grafts and 222 requiring unilateral procedures (total: 406 sinuses) participated in this study. A total of 1,025 implants were placed in the grafted sinuses. A total of 118 implants were placed simultaneously with the sinus graft (one stage), and 907 implants were placed in a subsequent surgery (two stages), 6 to 12 months after the graft was performed. In seven cases, a biopsy was harvested for histomorphometric analysis. Recall appointments were scheduled every 6 months, and panoramic and periapical x-rays were required every year for 3 years.nnnRESULTSnAmong 1,025 implants, 19 were lost (survival rate: 98.1%). The difference in survival rates for implants placed in native bone < or =3 mm (98.1%), >3 to < or =5 mm (98.6%), and >5 to < or =7 mm (97.0%) was not statistically significant (P = 0.3408). The survival rates for implants with rough and machined surfaces (98.6% and 97.0%, respectively) were not statistically significant (P = 0.0840). The histomorphometric analysis showed new bone formation (39.0% +/- 12%), marrow space (52.9% +/- 9.3%), and residual ABB (8% +/- 2.7%).nnnCONCLUSIONnOur results indicated that 1,025 implants placed in sinuses grafted exclusively with ABB combined with CM led to an excellent and predictable survival rate of 98.1%.

Antimicrobial efficacy of 0·05% cetylpyridinium chloride mouthrinses

This study evaluated the antimicrobial activity of two commercially available 0·05% cetylpyridinium chloride (CPC) mouthrinses with or without alcohol and examined its antimicrobial activity on oral bacterial species including fresh clinical isolates compared to a chlorhexidine mouthrinse and a control fluoride mouthrinse without CPC. Two different approaches were used to evaluate antimicrobial activity. First, the minimum inhibitory concentration (MIC) was determined for each mouthrinse against a panel of 25 micro‐organisms including species associated with dental caries, gingivitis and periodontitis. Second, supragingival dental plaque obtained from 15 adults was incubated with the four mouthrinses to evaluate antimicrobial activity on micro‐organisms in oral biofilms. Both CPC mouthrinses exhibited lower MICs, that is, greater antimicrobial activity, against oral Gram‐negative bacteria especially periodontal pathogens and species implicated in halitosis such as Aggregatibacter actinomycemcomitans, Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, Prevotella intermedia and Solobacterium moorei than the control mouthrinse. Ex‐vivo tests on supragingival plaque micro‐organisms demonstrated significantly greater antimicrobial activity by the CPC mouthrinses (>90% killing, P < 0·001) and the chlorhexidine rinse (>98% killing, P < 0·05) compared to the control fluoride mouthrinse. Whilst the chlorhexidine mouthrinse was most effective, mouthrinses containing 0·05% CPC formulated with or without alcohol demonstrated broad‐spectrum antimicrobial activity against both laboratory strains and supragingival plaque bacteria compared to a control mouthrinse without CPC.

Characterization and prevalence of Solobacterium moorei associated with oral halitosis

Previous studies suggest that Solobacterium moorei is associated with oral halitosis. In the present study, we examined the prevalence of S. moorei on the dorsal surface of the tongue in 57 adults (21 with and 36 without halitosis) by bacterial culture and direct amplification of nucleic acids. We also examined the S. moorei type strain and four clinical isolates for 16S ribosomal nucleic acid sequence, H(2)S and enzyme production, and antibiotic susceptibility. S. moorei was found on the dorsal surface of the tongue in 100% of the subjects with halitosis and 14% of subjects without halitosis. Infection with S. moorei was correlated with organoleptic measures of halitosis and with volatile sulfur compound levels. Nucleic acid probe detection of S. moorei as a test for halitosis exhibited 100% sensitivity and 86% specificity. The S. moorei type strain and all four clinical isolates showed >98% 16S rDNA sequence similarity, produced H(2)S, demonstrated acid phosphatase, beta-galactosidase, alpha-glucosidase, esterase, leucine arylamidase and naphthol phosphohydrolase enzyme activities, and were sensitive to all antibiotics tested except gentamicin, kanamycin, nalidixic acid and rifampin. This study supports the hypothesis that S. moorei is associated with halitosis.

A 6‐month study of the effects of 0.3% triclosan/copolymer dentifrice on dental implants

AIMnSupportive therapy to maintain dental implants is increasingly important. This study examined the effect of a 0.3% triclosan/2% copolymer dentifrice on oral biofilms and gingival inflammation (GI) on dental implants and peri-implant tissues.nnnMATERIALS AND METHODSnOne hundred and twenty adults with a dental implant and contra-lateral tooth were enrolled in this 6 month, double-blind, two-treatment, parallel group study. Sixty subjects were randomly assigned to a triclosan/copolymer dentifrice test group and 60 subjects to a fluoride dentifrice control group and instructed to brush twice daily for 6 months. At baseline, 3, and 6 months, a calibrated dentist assessed dental plaque, GI and collected supragingival dental plaque for microbiological analysis.nnnRESULTSnSubjects in the triclosan/copolymer group demonstrated significantly lower levels of dental plaque, gingivitis, and bleeding on probing at 3 and 6 months at both the implant and contra-lateral tooth compared with the fluoride group (p<0.05). There were significantly fewer Gram-negative anaerobes in the triclosan/copolymer group (p<0.05) including >90% reductions in Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Eubacterium saburreum, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella melaninogenica, Solobacterium moorei, and Tannerella forsythia.nnnCONCLUSIONSnTwice daily use of a triclosan/copolymer dentifrice may enhance dental implant maintenance by reducing dental plaque and GI.

Molecular Cloning of the fur Gene from Actinobacillus actinomycetemcomitans

ABSTRACT In several bacterial species, iron availability in host tissues is coordinated with the expression of virulence determinants through the fur gene product. Initial experiments showed that a cloned Escherichia coli fur gene probe hybridized to Southern blots of Actinobacillus actinomycetemcomitans strain JP2 (serotype b) chromosomal DNA. The A. actinomycetemcomitans fur gene was then cloned utilizing partial functional complementation of the fur mutant in E. coli strain H1780. Analysis of the cloned DNA sequence revealed a 438-bp open reading frame with a deduced 146-amino-acid sequence exhibiting 80% identity to Haemophilus influenzae Fur and 62% identity to E. coli Fur. The pUC Aafur gene probe (generated from JP2 serotype b) hybridized to representatives from all five A. actinomycetemcomitans serotypes as well as to two strains derived from monkeys, suggesting that fur is widely distributed in A. actinomycetemcomitans. Open reading frames having >70% identity with the E. coli and H. influenzae flavodoxin and gyrase A genes, respectively, were found. Expression of the A. actinomycetemcomitans fur gene product repressed fiu expression and siderophore production in E. coli. A gel shift assay demonstrated that the expressed A. actinomycetemcomitans Fur protein bound the bacterial fur consensus sequence. Further characterization of the fur gene product in A. actinomycetemcomitans may improve our understanding of its role in the pathogenesis of periodontal disease and may lead to specific therapeutic modalities.

Identification and Analysis of the Gap Region in the 23S Ribosomal RNA from Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus which can cause certain severe extra-oral infections as well as forms of human periodontal disease such as localized juvenile periodontitis. In contrast to many prokaryotic and eukaryotic species which exhibit an intact 23S ribosomal RNA (rRNA) molecule, examination of six A. actinomycetemcomitans strains-including three serogroup representative strains and two strains from non-human primates-revealed that this micro-organism does not produce an intact 23S ribosomal RNA (rRNA) molecule but, rather, two smaller forms of 1.8 kb and 1.2 kb designated as 23Sa and 23Sβ fragments. On the other hand, 14 other strains of Actinobacillus, Haemophilus, and Pasteurella species demonstrated intact 23S rRNA. The sequence of the region of the 23S rRNA gene in A. actinomycetemcomitans strain ATCC 43718 containing the cleavage site was determined by dideoxynucleotide sequencing, while the location of the 3 and 5 termini of the 23Sa and 23Sβ fragments was resolved by S1 nuclease mapping and cDNA primer-extension. A deletion of 112 bases was noted in comparisons of base sequences from A. actinomycetemcomitans rRNA and rDNA. The DNA intervening sequence was localized to nucleotide 1180 of the Escherichia coli 23S rRNA map. While the primary structure of the gap region showed little homology with the gap regions described in other organisms, the secondary structure was similar to that previously described in the parasitic helminth Schistosoma japonicum. Restriction enzyme and nucleotide sequence analysis of the gap region in eight other A. actinomycetemcomitans strains showed it to be highly conserved.

Detection of periodontal pathogenic microorganisms in atheromatous plaque. Preliminary results

AbstractIntroduction. Recent studies suggest that chronic infections, including those associated with periodontitis, increase the risk for coronary vascular disease. We hypothesize that oral microorganisms including periodontal bacterial pathogens enter the blood stream during transient bacteremias where they may play a role in the development and progression of atherosclerosis.n Materials and Methods. To test this hypothesis, 34 human specimens obtained during carotid endarterectomy or bypass procedures were examined by use of specific oligonucleotide primers for Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans and Bacteroides forsythus in polymerase chain reaction (PCR) assays.n Results. Twenty (59%) of the 34 specimens tested positive for bacterial 16S rDNA. Subsequent hybridization of the bacterial 16S rDNA positive specimens with species-specific oligonucleotide probes revealed that 32,4% of the 34 atheromas tested positive for at least one of the target periodontal pathogens. Further analysis of the results in the bacterial positive group (n=20) shows that 55% of the atheromas tested positive for at least one of the target periodontal pathogens.n Conclusion. These findings indicate that periodontal pathogens are present in atherosclerotic plaques, where they may play a role in the development and progression of atherosclerosis leading to coronary vascular disease and other clinical sequelae.ZusammenfassungEinleitung. In aktuellen Studien wird behauptet, dass chronische Infektionen, darunter auch Parodontitis, das Risiko für kardiovaskuläre Erkrankungen erhöhen. Wir nehmen an, dass orale Mikroorganismen während einer rezidivierend auftretenden, vorübergehenden Bakteriämie bei Pathogenese und Fortschreiten der Arteriosklerose eine entscheidende Rolle spielen können.n Material und Methode. In dieser Studie wurden von 34 Patienten Proben aus den verschiedensten Gefäßabschnitten während der Gefäßoperation (Endarteriektomie oder Bypass) entnommen und auf bakterielle 16S rDNS der mit Parodontitis assoziierten Keime Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans und Bacteroides forsythus mittels spezifischen Oligonukleotidprimern in Polymerasekettenreaktionstests (PCR) untersucht.n Ergebnisse. Zwanzig (59%) der 34 untersuchten Gefäßwandpräparate waren in einem oder mehreren der PCR-Tests auf bakterielle 16S rDNS positiv. PCR-Tests auf bakterielle 16S rDNS bestätigen ebenfalls die Anwesenheit von parodontal pathogenen Keimen in 32,4% der insgesamt 34 untersuchten Gefäßwandpräparate. Innerhalb dieser Gruppe, die auf Mikroorganismen positiv war (n=20), wurden in 55% der Proben parodontal pathogene Keime identifiziert.n Schlussfolgerung. Diese Ergebnisse zeigen, dass Mikroorganismen, die mit Parodontitis assoziiert sind, in atherosklerotischen Plaques vorhanden sind und bei der Ätiologie und Pathogenese der Atherosklerose eine wichtige Rolle spielen können.

Nachweis parodontal pathogener Mikroorganismen in atheromatösen PlaquesVorläufige Ergebnisse

AbstractIntroduction. Recent studies suggest that chronic infections, including those associated with periodontitis, increase the risk for coronary vascular disease. We hypothesize that oral microorganisms including periodontal bacterial pathogens enter the blood stream during transient bacteremias where they may play a role in the development and progression of atherosclerosis.n Materials and Methods. To test this hypothesis, 34 human specimens obtained during carotid endarterectomy or bypass procedures were examined by use of specific oligonucleotide primers for Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans and Bacteroides forsythus in polymerase chain reaction (PCR) assays.n Results. Twenty (59%) of the 34 specimens tested positive for bacterial 16S rDNA. Subsequent hybridization of the bacterial 16S rDNA positive specimens with species-specific oligonucleotide probes revealed that 32,4% of the 34 atheromas tested positive for at least one of the target periodontal pathogens. Further analysis of the results in the bacterial positive group (n=20) shows that 55% of the atheromas tested positive for at least one of the target periodontal pathogens.n Conclusion. These findings indicate that periodontal pathogens are present in atherosclerotic plaques, where they may play a role in the development and progression of atherosclerosis leading to coronary vascular disease and other clinical sequelae.ZusammenfassungEinleitung. In aktuellen Studien wird behauptet, dass chronische Infektionen, darunter auch Parodontitis, das Risiko für kardiovaskuläre Erkrankungen erhöhen. Wir nehmen an, dass orale Mikroorganismen während einer rezidivierend auftretenden, vorübergehenden Bakteriämie bei Pathogenese und Fortschreiten der Arteriosklerose eine entscheidende Rolle spielen können.n Material und Methode. In dieser Studie wurden von 34 Patienten Proben aus den verschiedensten Gefäßabschnitten während der Gefäßoperation (Endarteriektomie oder Bypass) entnommen und auf bakterielle 16S rDNS der mit Parodontitis assoziierten Keime Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans und Bacteroides forsythus mittels spezifischen Oligonukleotidprimern in Polymerasekettenreaktionstests (PCR) untersucht.n Ergebnisse. Zwanzig (59%) der 34 untersuchten Gefäßwandpräparate waren in einem oder mehreren der PCR-Tests auf bakterielle 16S rDNS positiv. PCR-Tests auf bakterielle 16S rDNS bestätigen ebenfalls die Anwesenheit von parodontal pathogenen Keimen in 32,4% der insgesamt 34 untersuchten Gefäßwandpräparate. Innerhalb dieser Gruppe, die auf Mikroorganismen positiv war (n=20), wurden in 55% der Proben parodontal pathogene Keime identifiziert.n Schlussfolgerung. Diese Ergebnisse zeigen, dass Mikroorganismen, die mit Parodontitis assoziiert sind, in atherosklerotischen Plaques vorhanden sind und bei der Ätiologie und Pathogenese der Atherosklerose eine wichtige Rolle spielen können.

Media‐ and method‐dependent variations in minimal inhibitory concentrations of antiplaque agents on oral bacteria

Aims:u2002 To determine minimal inhibitory concentrations (MICs) and the percentage of nonsusceptible bacteria – those still cultivable above a threshold concentration – in human supragingival dental plaque and saliva for antiplaque/antimicrobial agents including triclosan (TCS) and trichlorocarbanilide (TCC), and a new potential antimicrobial, 2‐t‐butyl‐5‐(4‐t‐butylphenyl)‐phenol (DTBBP).