Antimicrobial efficacy of 0·05% cetylpyridinium chloride mouthrinses.

UNLABELLED: 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 MIC's, 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. SIGNIFICANCE AND IMPACT OF STUDY: These in vitro and ex-vivo studies provide a biological rationale for previous clinical studies demonstrating the efficacy of CPC mouthrinses in reducing supragingival plaque and plaque-associated gingivitis.

The effect of a microbead dentifrice on microbial load in oral microenvironments.

OBJECTIVES: The human oral cavity contains several microenvironments or ecologic niches. While mechanical plaque control is well known to reduce the number of supragingival dental plaque bacteria, there is little data on antimicrobial effects in other oral ecologic niches. The present study examined the effects of mechanical plaque control using a microbead dentifrice on bacteria colonizing oral ecologic niches. METHODS: Twenty-two adults (aged 18-70years) including nine generalized moderate chronic periodontitis subjects and 13 periodontally healthy subjects having average gingival indices ≥1 and plaque indices ≥1.5 completed a 1week washout phase and refrained from oral hygiene the morning of baseline sample collection. Microbial samples from supragingival dental plaque, buccal mucosa, dorsal surface of the tongue and whole mixed saliva were obtained. Subjects brushed with a microbead dentifrice and, after 10min, sampling was repeated. The number of anaerobic bacteria was determined by culture on non-selective media and transformed to log(10) for statistical analyses. RESULTS: Mechanical plaque control using the microbead dentifrice resulted in statistically significant reductions in bacterial numbers in each ecologic niche (P<0.001). The greatest reduction in the number of viable bacteria occurred in samples taken from the buccal mucosa (97.22%) followed by a 95.22% reduction in supragingival plaque bacteria, a 94.51% reduction in the number of bacteria on the dorsal surface of the tongue and a 91.57% reduction in the number of bacteria in whole mixed saliva. CONCLUSIONS: Mechanical plaque control using a microbead dentifrice reduces microbial load in microenvironments throughout the human oral cavity.

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.

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

AIMS: 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). METHODS AND RESULTS: Broth and agar dilution-based MIC tests were performed using 28 oral and nonoral bacterial strains representing 17 species. MICs for TCS were lowest and more than 100-fold lower than DTBBP (P < 0.0005) by both methods. MICs for TCS were lower in broth-based tests compared with TCC. The additions of defibrinated blood to agar and horse serum to broth increased MICs--in the case of TCS, 10- to 15-fold. Significantly higher proportions of nonsusceptible plaque and salivary bacteria were recovered from agar media containing DTBBP or TCC compared with TCS (P < 0.05). CONCLUSIONS: TCS is a more effective antimicrobial agent than either TCC or DTBBP as determined by in vitro testing. SIGNIFICANCE AND IMPACT OF THE STUDY: The utility of in vitro testing for antiplaque agents as a predictor of in vivo efficacy is affected by the methods used.

Prevalence of periodontal pathogens in Brazilians with aggressive or chronic periodontitis.

OBJECTIVES: Previous studies suggest differences between geographically and racially distinct populations in the prevalence of periodontopathic bacteria as well as greater periodontal destruction associated with infection by highly leucotoxic Actinobacillus actinomycetemcomitans. The present study examined these hypotheses in Brazilians with aggressive or chronic periodontitis. MATERIALS AND METHODS: Clinical, radiographical, and microbiological assessments were performed on 25 aggressive periodontitis and 178 chronic periodontitis patients including 71 males and 132 females, 15-69 years of age. RESULTS: The prevalence of Porphyromonas gingivalis was similar to that of other South American populations. The prevalence of A. actinomycetemcomitans and its highly leucotoxic subgroup was higher in Brazilians. Highly leucotoxic A. actinomycetemcomitans was more prevalent in aggressive periodontitis (chi2=27.83) and positively associated with deep pockets (>6 mm, chi2=18.26) and young age (<29 years, chi2=18.68). Greater mean attachment loss was found in subjects with highly leucotoxic A. actinomycetemcomitans than in subjects with minimally leucotoxic (p=0.0029) or subjects not infected (p=0.0001). CONCLUSION: These data support the hypothesis of differences between populations in the prevalence of periodontopathic bacteria and of greater attachment loss in sites infected with highly leucotoxic A. actinomycetemcomitans. Detection of highly leucotoxic A. actinomycetemcomitans in children and adolescents may be a useful marker for aggressive periodontitis.

[Detection of periodontal pathogenic microorganisms in atheromatous plaque. Preliminary results]. / Nachweis parodontal pathogener Mikroorganismen in atheromatösen Plaques. Vorläufige Ergebnisse.

INTRODUCTION: 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. 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. 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. 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.

Molecular cloning of the fur gene from Actinobacillus actinomycetemcomitans.

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 of periodontal pathogens in atheromatous plaques.

BACKGROUND: Recent studies suggest that chronic infections including those associated with periodontitis increase the risk for coronary vascular disease (CVD) and stroke. 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 leading to CVD. METHODS: To test this hypothesis, 50 human specimens obtained during carotid endarterectomy were examined for the presence of Chlamydia pneumoniae, human cytomegalovirus, and bacterial 16S ribosomal RNA using specific oligonucleotide primers in polymerase chain reaction (PCR) assays. Approximately 100 ng of chromosomal DNA was extracted from each specimen and then amplified using standard conditions (30 cycles of 30 seconds at 95 degrees C, 30 seconds at 55 degrees C, and 30 seconds at 72 degrees C). Bacterial 16S rDNA was amplified using 2 synthetic oligonucleotide primers specific for eubacteria. The PCR product generated with the eubacterial primers was transferred to a charged nylon membrane and probed with digoxigenin-labeled synthetic oligonucleotides specific for Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, and Prevotella intermedia. RESULTS: Eighty percent of the 50 endarterectomy specimens were positive in 1 or more of the PCR assays. Thirty-eight percent were positive for HCMV and 18% percent were positive for C. pneumoniae. PCR assays for bacterial 16S rDNA also indicated the presence of bacteria in 72% of the surgical specimens. Subsequent hybridization of the bacterial 16S rDNA positive specimens with species-specific oligonucleotide probes revealed that 44% of the 50 atheromas were positive for at least one of the target periodontal pathogens. Thirty percent of the surgical specimens were positive for B. forsythus, 26% were positive for P. gingivalis, 18% were positive for A. actinomycetemcomitans, and 14% were positive for P. intermedia. In the surgical specimens positive for periodontal pathogens, more than 1 species was most often detected. Thirteen (59%) of the 22 periodontal pathogen-positive surgical specimens were positive for 2 or more of the target species. CONCLUSIONS: Periodontal pathogens are present in atherosclerotic plaques where, like other infectious microorganisms such as C. pneumoniae, they may play a role in the development and progression of atherosclerosis leading to coronary vascular disease and other clinical sequelae.

Evidence for the role of highly leukotoxic Actinobacillus actinomycetemcomitans in the pathogenesis of localized juvenile and other forms of early-onset periodontitis.

BACKGROUND: Actinobacillus actinomycetemcomitans leukotoxin is thought to be an important virulence factor in the pathogenesis of localized juvenile and other forms of early-onset periodontitis. Some highly leukotoxic A. actinomycetemcomitans strains produce 10 to 20 times more leukotoxin than other minimally leukotoxic strains. The distribution, clonality, and intrafamilial transmission of highly leukotoxic A. actinomycetemcomitans were examined in order to determine the importance of leukotoxin in the pathogenesis of periodontitis. METHODS: The polymerase chain reaction (PCR) was used to differentiate highly leukotoxic from minimally leukotoxic strains in examining 1,023 fresh A. actinomycetemcomitans isolates and strains from our culture collection. These were obtained from 146 subjects including 71 with localized juvenile periodontitis (LJP), 4 with early-onset periodontitis, 11 with post-localized juvenile periodontitis, 41 with adult periodontitis, and 19 periodontally normal subjects. The arbitrarily primed polymerase chain reaction (AP-PCR) analysis of 30 oral isolates from each of 25 subjects was used to determine the intraoral distribution of A. actinomycetemcomitans clones. AP-PCR was also used to examine the transmission of A. actinomycetemcomitans in 30 members of 6 families. The clonality of 41 highly leukotoxic A. actinomycetemcomitans strains was evaluated by both AP-PCR and ribotyping. RESULTS: Highly leukotoxic A. actinomycetemcomitans was found only in subjects with localized juvenile and early-onset periodontitis. Fifty-five percent of the LJP subjects harbored highly leukotoxic A. actinomycetemcomitans isolates. Seventy-three percent of the A. actinomycetemcomitans isolates in these subjects were highly leukotoxic. Highly leukotoxic A. actinomycetemcomitans infected younger subjects (mean age 13.95 years, range 5 to 28 years) than minimally leukotoxic (mean age 35.47 years, range 6 to 65 years). Most subjects were infected with only one A. actinomycetemcomitans genotype. However, PCR of whole dental plaques and subsequent analysis of up to 130 individual oral isolates suggested a possible shift in A. actinomycetemcomitans over time in that a few subjects harbored both highly leukotoxic and minimally leukotoxic strains. AP-PCR analysis was consistent with intrafamilial A. actinomycetemcomitans transmission. Ribotyping and AP-PCR analysis confirmed a previous report that highly leukotoxic A. actinomycetemcomitans consists of a single clonal type. CONCLUSIONS: This study suggests that localized juvenile and other forms of Actinobacillus-associated periodontitis are primarily associated with the highly leukotoxic clone of A. actinomycetemcomitans.

Differentiation of strains of Actinobacillus actinomycetemcomitans by arbitrarily primed polymerase chain reaction.

The present study describes a method for amplifying DNA in Actinobacillus actinomycetemcomitans by using short, synthetic oligonucleotides of random sequence as primers in the polymerase chain reaction. Genomic DNA from each of 20 human isolates of A. actinomycetemcomitans was successfully amplified in a thermal cycler with a single synthetic primer (GGGTAACGCC) and reproducibly produced 14 different DNA amplification profiles (amplitypes). A. actinomycetemcomitans isolates from the same subject revealed the same amplitype. The arbitrarily primed polymerase chain reaction appears to be useful in characterizing human isolates of A. actinomycetemcomitans for studies of epidemiology and bacterial transmission.

Rapid identification of Actinobacillus actinomycetemcomitans based on analysis of 23S ribosomal RNA.

Actinobacillus actinomycetemcomitans is a key microorganism in the pathogenesis of several different forms of periodontal diseases. Identification of this bacterium from clinical specimens may often be complicated by the fact that the colony morphology on TSBV selective medium closely resembles that of Haemophilus aphrophilus and a key differentiating characteristic, catalase reaction, may be variable. Recent genetic studies have shown that the 23S ribosomal RNA molecule is split into two smaller forms in A. actinomycetemcomitans, but is intact in H. aphrophilus. Based on this finding, we describe a new, rapid method for identifying A. actinomycetemcomitans in which single colonies isolated from culture on TSBV agar in 5% CO2 in air are lysed, electrophoresed on 1.5% submarine agarose gels and visualized by staining with ethidium bromide. Using this assay, A. actinomycetemcomitans can be easily distinguished from morphologically similar colonies such as H. aphrophilus strains by differences in 23S rRNA within 2 h.

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 23S alpha and 23S beta 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 23S alpha and 23S beta 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.