Lactoferrin knockout mice demonstrates greater susceptibility to Aggregatibacter actinomycetemcomitans-induced periodontal disease.

BACKGROUND: Among the innate defense mechanisms in the oral cavity, lactoferrin (LF) is a vital antimicrobial that can modify the host response against periodontopathogens. Aggregatibacter actinomycetemcomitans is the main periodontopathogen of localized aggressive periodontitis. The aim of this study is to evaluate the role of LF during A. actinomycetemcomitans-induced periodontitis. METHODS: Differences in the expression levels of cytokines, chemokines, chemokine receptors, and bone loss markers between wild-type (WT) and LF knockout mice (LFKO(-/-)) were evaluated by real time-PCR. Serum IgG and LF levels were quantified by ELISA. Alveolar bone loss among the groups was estimated by measuring the distance from cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) at 20 molar sites. RESULTS: Oral infection with A. actinomycetemcomitans increased LF levels in periodontal tissue (P = 0.01) and saliva (P = 0.0004) of wild-type infected (WTI) mice compared to wild-type control mice. Pro-inflammatory cytokines such as interferon-γ, tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and IL-12 were increased in the infected LF knockout (LFKO(-/-)I) mice compared to the WTI mice, whereas the anti-inflammatory cytokines IL-4 and IL-10 were decreased. Chemokines and chemokine receptors showed different expression patterns between WTI and LFKO(-/-)I mice. The LFKO(-/-)I mice developed increased bone loss (P = 0.002), in conjunction with increased expression of receptor activator of nuclear factor-κB ligand and decrease in osteoprotegerin, compared to WTI mice. CONCLUSIONS: These results demonstrate that the infected LFKO(-/-) mice were more susceptible to A. actinomycetemcomitans-induced alveolar bone loss, with different patterns of immune responses compared to those of WTI mice.

Human salivary cystatin SA exhibits antimicrobial effect against Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Healthy subjects who do not have Aggregatibacter actinomycetemcomitans in their oral cavity may possess factors in saliva that might demonstrate antibacterial activity against the bacterium. The aim of this study was to identify and purify proteins from saliva of healthy subjects that might demonstrate antibacterial activity against A. actinomycetemcomitans and test the same against the bacteria. MATERIAL AND METHODS: Saliva from 10 healthy volunteers was tested individually for its anti-A. actinomycetemcomitans activity. Among the 10 subjects, eight demonstrated anti-A. actinomycetemcomitans activity. Saliva was collected from one healthy volunteer who demonstrated the highest antimicrobial activity against A. actinomycetemcomitans. After clarifying the saliva, it was subjected to an affinity chromatography column with A. actinomycetemcomitans. The proteins bound to A. actinomycetemcomitans were eluted from the column and identified using mass spectrometry (MALDI-TOF/TOF MS). Among other proteins that bound to A. actinomycetemcomitans, which included lactoferrin, immunoglobulin A and kallikrein, cystatin SA was observed in significantly higher concentrations, and this was purified from the eluate. The purified cystatin SA was tested at different concentrations for its ability to kill A. actinomycetemcomitans in a 2 h cell killing assay. The bacteria were also treated with a proteinase inhibitor, leupeptin, to clarify whether the antimicrobial effect of cystatin SA was related to its protease inhibitory function. Cystatin SA was also tested for its ability to prevent binding of A. actinomycetemcomitans to buccal epithelial cells (BECs) in an A. actinomycetemcomitans-BEC binding assay. RESULTS: Cystatin SA (0.1 mg/mL) demonstrated a statistically significant antimicrobial activity against A. actinomycetemcomitans. The effect of cystatin SA decreased with lower concentrations, with 0.01 mg/mL showing no effect. The addition of monoclonal cystatin SA antibodies to the purified sample completely negated the antimicrobial effect. Treatment of A. actinomycetemcomitans with leupeptin resulted in no antimicrobial effect, suggesting that the antimicrobial activity of cystatin SA is independent of its protease inhibitory function. A. actinomycetemcomitans pretreated with cystatin SA showed reduced binding to BECs, suggesting a potential role for cystatin SA in decreasing the colonization of A. actinomycetemcomitans. CONCLUSION: The present study shows that cystatin SA demonstrates antimicrobial activity against the periodontopathogen A. actinomycetemcomitans, and future studies determining the mechanism of action are necessary. The study also shows the ability of cystatin SA to reduce significantly the binding of A. actinomycetemcomitans to BECs.

Rapid identification of oral isolates of Aggregatibacter actinomycetemcomitans obtained from humans and primates by an ultrafast super convection based polymerase chain reaction.

Aggregatibacter actinomycetemcomitans is a Gram negative oral bacterium associated with localized aggressive periodontitis (LAP). Detection of A. actinomycetemcomitans in clinical samples is routinely done by PCR. Our aim was to develop a rapid and reliable PCR method that can be used as a chair-side tool to detect A. actinomycetemcomitans in clinical samples. Sensitivity and specificity assessment was performed on buccal and plaque samples obtained from 40 adolescents enrolled in an ongoing LAP study by comparing 20 A. actinomycetemcomitans-positive subjects and 20 who were negative. In a second study, A. actinomycetemcomitans presence was tested in oral samples from eighty-six primates that included rhesus monkeys, chimpanzees, marmosets, tamarins and baboons. All samples were processed for detection of A. actinomycetemcomitans by means of culture, conventional PCR (cPCR) and rapid PCR (rPCR) using a Super Convection based AmpXpress thermal cycler (AlphaHelix, Sweden). For human samples, culture, cPCR and rPCR showed perfect agreement. Using this method A. actinomycetemcomitans was detected in 27 of 32 rhesus monkeys, 4 of 8 chimpanzees and 1 of 34 marmosets. Rapidity of AmpXpress thermal cycler, combined with Ready-To-Go PCR beads (GE Life sciences), a quick DNA extraction kit (Epicentre Biotechnologies, Madison, Wisconsin, USA) and a bufferless fast agarose gel system, made it possible to obtain results on A. actinomycetemcomitans detection within 35 min. We conclude that AmpXpress fast PCR can be conveniently used as a chair-side tool for rapid detection of A. actinomycetemcomitans in clinical samples.

Clinical anti-microbial efficacy of a new zinc citrate dentifrice.

This crossover design clinical study compared the anti-microbial effects of a new 1% zinc citrate dentifrice with a control formulation. Thirty adults completed a washout phase and baseline samples of dental plaque, buccal mucosa, tongue, saliva, and plaque collected to enumerate anaerobes and streptococci. Subjects were randomly assigned a test dentifrice to use for the next 13 days. Oral samples similar to the baseline were collected on day 14 prior to oral hygiene for microbial analysis. The subject then placed a custom intra-oral stent with hydroxyapatite (HA) squares and brushed their teeth with their assigned dentifrice. Oral samples and HA squares were collected 5 h later for microbial analyses. This completed the study with one test dentifrice. The entire study was repeated with the alternate dentifrice after a second washout phase. Whereas baseline samples demonstrated no significant differences in microbial parameters between the two treatment groups (p > 0.05), subjects provided the zinc citrate dentifrice demonstrated 24-52% reductions in anaerobic bacteria and streptococci on day 14 versus the control paste (p < 0.05). In the 5-h post-brushing samples, subjects provided the zinc citrate toothpaste demonstrated 27-49% reductions for anaerobic bacteria and streptococci (p < 0.05). Additionally, in situ microbial biofilm formation on HA disks was significantly inhibited amongst the zinc citrate group (p < 0.05). Significant reductions in anaerobic bacteria and streptococci were observed amongst all intra-oral locations along with in situ biofilm formation after use of the zinc citrate dentifrice.

In vivo antimicrobial effectiveness of an essential oil-containing mouth rinse 12 h after a single use and 14 days' use.

OBJECTIVES: Two studies were conducted to determine the antimicrobial effect of rinsing with an essential oil-containing mouth rinse 12 h after a single rinse and 12 h after 2 weeks of twice daily rinsing, during the daytime and overnight. MATERIALS AND METHODS: These studies utilized a randomized, double-blind, controlled crossover design. Following baseline sampling of bacteria from supragingival plaque and the dorsum of the tongue, subjects began twice-daily rinsing with either an essential oil mouth rinse containing 0.09% zinc chloride (Tartar Control Listerine Antiseptic) or a negative control rinse. Bacterial sampling was repeated 12 h after the first rinse, and again 12 h after the final rinse 14 days later. The sampling schedule was adjusted according to whether the study was investigating daytime or overnight activity. Samples were plated on Schaedlers medium (total anaerobes), Schaedlers Nalidixic/Vancomycin medium (Gram-negative anaerobes), and OOPS medium (volatile sulphur compound (VSC)-producing organisms). Inter-group log10 transformed colony-forming units/ml counts from samples of supragingival plaque and tongue swabs on each of the three media were compared by analysis of covariance. RESULTS: The mean bacterial counts in subjects using the essential oil mouth rinse were significantly lower (p< or =0.005) than mean counts in subjects using the control rinse in all the comparisons, i.e., tongue and supragingival plaque samples on each of three media at two sampling periods in the daytime and overnight study, respectively. Mean bacterial count percent reductions for plaque samples ranged from 56.3 to 95.3; percent reductions for tongue samples ranged from 61.1 to 96.1. There was a trend to higher reductions after 14 days' rinsing than after the initial rinse. CONCLUSION: Rinsing with the essential oil mouth rinse can have long-lasting effects in reducing anaerobic bacteria overall as well as Gram-negative anaerobes and VSC-producing bacteria. The significant reductions in numbers of these bacteria produced by the essential oil mouth rinse, both in plaque and on the dorsum of the tongue, can play a key role in explaining the essential oil mouth rinse's effectiveness in reducing supragingival plaque and gingivitis as well as its effectiveness in controlling intrinsic oral malodor over prolonged periods.

Antimicrobial effects of a new therapeutic liquid dentifrice formulation on oral bacteria including odorigenic species.

The control of oral malodor is well-recognized in efforts to improve oral health. Antimicrobial formulations can mitigate oral malodor, however, procedures to assess effects on oral bacteria including those implicated in halitosis are unavailable. This investigation examined the antimicrobial effects of a new liquid triclosan/copolymer dentifrice (test) formulation that demonstrated significant inhibition of oral malodor in previous organoleptic clinical studies. Procedures compared antimicrobial effects of the test and control formulations on a range of oral micro-organisms including members implicated in halitosis, substantive antimicrobial effects of formulations with hydroxyapatite as a surrogate for human teeth and ex vivo effects on oral bacteria from human volunteers. With Actinomyces viscosus, as a model system, the test formulation demonstrated a dose-dependent effect. At these concentrations the test formulation provided significant antimicrobial effects on 13 strains of oral bacteria including those implicated in bad breath at selected posttreatment time points. Treatment of hydroxyapatite by the test dentifrice resulted in a significant and substantive antimicrobial effect vs. controls. Oral bacteria from subjects treated ex vivo with the test dentifrice resulted in significant reductions in cultivable oral bacteria and odorigenic bacteria producing hydrogen sulfide. In summary, microbiological methods adapted to study odorigenic bacteria demonstrate the significant antimicrobial effects of the test (triclosan/copolymer) dentifrice with laboratory and clinical strains of oral bacteria implicated in bad breath.

Lactoferrin iron levels are reduced in saliva of patients with localized aggressive periodontitis.

BACKGROUND: Actinobacillus actinomycetemcomitans (Aa) is associated with localized aggressive periodontal disease in juveniles (LAgP). Lactoferrin (LF) is an iron-binding salivary protein that has been shown to kill Aa in its iron-free form (apo) and reduce binding to host cells in its iron-saturated form (halo). However, recent in vitro studies show that LF does not kill clinical isolates of Aa, and LF with reduced levels of bound iron does not interfere with its attachment. These findings suggest that colonization of Aa may occur more readily in an environment containing LF with low iron levels. The purpose of this study was to examine the relationship of LF iron levels in saliva of LAgP patients as compared to their age-, gender-, and race-matched controls. METHODS: Whole and parotid saliva was collected from LAgP patients and matched controls. Micrograms of LF/mg of protein as well as nanograms of iron/micrograms of LF were determined. Iron binding was determined in parotid saliva by addition of nonlabeled and 59Fe labeled iron. RESULTS: LAgP patients' whole saliva had higher LF levels than controls, but their LF contained less iron (P < or =0.005). No iron was found in LF from parotid saliva in either group. When iron was added to parotid saliva, the LAgP saliva bound 20 to 30 times less iron than controls (P< or =0.001). Finally, LF was identified as the major iron-binding protein in parotid saliva by 59Fe autoradiography and Western blotting. CONCLUSIONS: This study shows that the level of bound iron in LF is significantly reduced in LAgP patients compared to controls. These data suggest that LF from LAgP patients has a reduced capacity to bind iron and that LF iron levels may play an important role in Aa-induced LAgP.

Lactoferrin iron levels affect attachment of Actinobacillus actinomycetemcomitans to buccal epithelial cells.

BACKGROUND: Prior reports have suggested that the iron-binding protein lactoferrin (LF) may either kill Actinobacillus actinomycetemcomitans (Aa) or interfere with its binding to host cells. Other studies have indicated that the degree of iron saturation of LF might play a role in these interactions. However, these studies utilized strains that had lost critical attachment characteristics found in well-preserved clinical isolates of Aa. The purpose of this work was to study the effect of LF iron levels on survival and attachment of well-preserved clinical isolates of Aa. METHODS: LF containing 0%, 30%, and 100% iron saturation was tested for its ability to kill clinical isolates of Aa and to inhibit their binding to buccal epithelial cells (BECs). RESULTS: Neither iron-free LF (apo-LF) nor iron-saturated LF killed Aa clinical isolates. Increasing the iron saturation of LF resulted in an increased inhibition of Aa binding to BECs (P < or =0.005). This effect was consistent for the 3 clinical isolates tested. Pretreatment of Aa with iron-saturated LF reduced binding to BECs by 58%, 61.8%, and 64.2%, respectively, for each of the 3 clinical strains tested (P < or =0.005). Pretreatment of Aa strains with apo-LF, iron alone, or bovine serum albumin had no effect on binding. Pretreatment of BECs with LF (either apo-LF or iron-containing LF) had no influence on Aa binding. CONCLUSIONS: These results indicate that reduction in binding of Aa to epithelial cells is maximized by pretreatment of Aa cells with iron-saturated lactoferrin. These in vitro results suggest that patients with lactoferrin containing lowered levels of iron would be more susceptible to Aa colonization.

Colonization and persistence of rough and smooth colony variants of Actinobacillus actinomycetemcomitans in the mouths of rats.

Fresh isolates of Actinobacillus actinomycetemcomitans (Aa) bind avidly to surfaces in vitro, but existing in vivo studies of the adherence of Aa are limited. This study had two goals: (1) to compare the oral colonization of two isogenic strains of Aa-CU1010, a clinical isolate that expresses the adherent phenotype, and CU1012, a minimally adherent laboratory variant-and (2) to check for phenotypic reversion of these strains in a clinical setting. Rifampicin-resistant strains, developed for tracking in Sprague-Dawley rats, were tested in vitro to determine their stability and binding. In study 1, after antibiotic suppression, six rats (group I) received CU1010 in their feed. The eight rats in group II received CU1012 in their feed and four were supplemented by oral swabbing and four by gastric gavage. Group III consisted of three sham-inoculated controls. All rats were inoculated for 4 days. Microbiological data were collected at 1, 4 and 8 weeks after inoculation. Supporting data were supplied by antibody titres and clinical measures of alveolar bone loss. Study 2 consisted of six rats in each of three groups as above, but tagged strains of Aa were delivered by food alone. At all time-points in both studies, Aa was absent before inoculation and controls had no Aa or antibody to Aa. In study 1, all six rats in group I yielded positive cultures for Aa at 8 weeks. In group II, five of eight had positive cultures for Aa at 1 week, two of eight at 4 weeks and none had Aa at 8 weeks (P < or =0.001). All six rats in group I had serum anti-Aa titres compared to group II, where titres were seen in four of eight rats (P < or =0.015). In vitro data paralleled those found in vivo. No phenotypic reversion of either strain was seen in vivo. In study 2, four of six rats in group I showed Aa and had titres to Aa, while no other animals showed Aa at any time. The model provides convincing evidence that, unlike laboratory variants, clinical isolates colonize, persist and integrate into an already established, albeit reduced, econiche.

Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f.

The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of L-rhamnose and 2-acetamido-2-deoxy-D-galactose (molar ratio, 2:1) with the structure -->2)-alpha-L-Rhap-(1-3)-2-O-(beta-D-GalpNAc)-alpha-L-Rhap-(1-->* O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903phikan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.

Comparative antimicrobial activities of antiseptic mouthrinses against isogenic planktonic and biofilm forms of Actinobacillus actinomycetemcomitans.

BACKGROUND: Bacteria contained in biofilms have been shown to have a decreased susceptibility to antimicrobial agents compared to those in planktonic form. Thus, in vitro biofilm models have been developed for screening oral antimicrobial formulations in an effort to produce findings more predictive of clinical activity. This study compared the antimicrobial activity of three mouthrinse formulations when tested against isogenic strains of Actinobacillus actinomycetemcomitans (Aa), one of which was a clinical isolate which forms tenacious biofilms in vitro and the other of which was a spontaneous variant which always grows planktonically. METHOD: Biofilm-forming Aa strains CU1000 and NJ4300, obtained as clinical isolates, and their respective spontaneous planktonic variants, CU1060 and NJ4350, were grown under standard laboratory conditions and exposed for 15 s to either a negative control (phosphate buffered saline [PBS]), an essential-oil containing mouthrinse (Listerine Antiseptic [LA]), an amine fluoride/stannous fluoride-containing mouthrinse (Meridol [M]), or a triclosan and PVM/MA copolymer-containing mouthrinse (Plax [P]). The cells were then washed, serially diluted, plated, and incubated for enumeration of viable bacteria. Colony-forming units (CFU)/ml were log10 transformed and the mouthrinse groups were compared to the PBS group using analysis of variance. RESULTS: All 3 mouthrinses produced statisically significant 99.99% reductions (p< or =0.0001) in both planktonic strains compared to the PBS control. Effects on the biofilm forms of the organisms were more variable. Exposure to LA produced statistically significant (p< or =0.0001) reductions in strains CU1000 and NJ4300 of 98.20% and 96.47%, respectively, compared to PBS. M and P produced much smaller reductions which were not statistically significant. CONCLUSIONS: The results of this study, in which antimicrobial mouthrinses were tested against biofilm-forming and planktonic strains of the same organism, provide a clear demonstration of the resistance to antimicrobial agents conferred by biofilm formation and provide additional support for employing tests using biofilms to more accurately assess the relative activities of antiplaque agents in vitro.

Proximal caries in juvenile periodontitis patients.

BACKGROUND: Caries is recognized as the prevalent proximal dental disease in adolescents, while proximal bone loss is minimal to non-existent in this population. Adolescents demonstrating an inverse disease pattern, i.e., minimal caries and active periodontitis, could provide powerful clues with regard to both diseases. However, data are inconsistent. This study was designed to clarify this relationship by comparing proximal caries prevalence in a juvenile periodontitis (JP) group to a matched non-periodontally diseased control group. METHODS: Two groups (cases [JPs] and control patients [CPs]) were matched for age, sex, and race and evaluated for decayed, missing, filled teeth and surfaces (DMFS) by radiographic analysis. Statistical analysis was performed by ANOVA and Student t test. The study consisted of four phases. Phase I was based on data from a previous study that failed to include race in the analysis. Thus, the original 23 JP patients (mostly African-Americans from New York City) were rematched for race as well as sex and age with CPs from Newark, NJ. The effect of water fluoridation (found in NYC) was evaluated in Phase II by matching the 23 original CPs (mostly Caucasian from NYC) with 23 CPs from NJ. Since differences were seen, we rematched our original JPs from NYC with a new set of race-matched CPs from NYC (Phase III). Finally, 13 JP patients from the University of Medicine and Dentistry of New Jersey (UMDNJ) were matched with CPs from NJ (Phase IV). RESULTS: Phase I and III indicated that JP patients had significantly less proximal caries than their matched CPs (P < or =0.05). Phase II confirmed the role of fluoride in caries reduction. Phase IV (NJ sample) supported our previous data and suggested that JP patients had less proximal caries than CPs (P < or =0.05). CONCLUSIONS: JP patients had significantly less proximal caries than their matched CPs when groups were balanced and radiographic evaluations were performed. In-depth studies of JP patients could provide important clues about both caries and periodontal disease etiology and pathogenesis.

Effect of an essential oil-containing antiseptic mouthrinse on plaque and salivary Streptococcus mutans levels.

BACKGROUND: Clinical studies in which antimicrobial mouthrinses were shown to have significant antiplaque activity most frequently have used gingivitis as the clinically relevant endpoint. However, there is evidence to suggest that mouthrinses containing active agents effective against Streptococcus mutans, such as chlorhexidine, may also have a role in inhibiting dental caries. This clinical study was conducted to determine the effect of 2x-daily rinsing with an essential oil-containing antiseptic mouthrinse (Listerine Antiseptic) on levels of recoverable S. mutans and total streptococci in supragingival interproximal plaque and in saliva. Additionally, a follow-up in vitro study is reported which determined whether a differential susceptibility to the antiseptic mouthrinse exists among different strains of streptococci. METHOD: Following baseline saliva and plaque sampling for quantification of recoverable S. mutans and total streptococci, 29 qualifying subjects were randomly assigned either the essential oil mouthrinse or a sterile water control. They rinsed with 20 ml for 30s 2 x daily for 11 days and once on the 12th day, in addition to their usual oral hygiene procedures. On day 12, saliva and plaque samples were again collected and microbiological quantification performed. The procedures were repeated with the alternate rinse after a 1-week washout period. RESULTS: The essential oil mouthrinse produced respective reductions of 69.9% and 75.4% in total recoverable streptococci and in S. mutans in plaque, and corresponding reductions of 50.8% and 39.2% in saliva. The in vitro study revealed that streptococci from the mutans group were more susceptible to the bactericidal activity of the essential oil mouthrinse than streptococci from the mitis group. CONCLUSIONS: As antimicrobial mouthrinses are most frequently recommended to patients whose mechanical oral hygiene procedures are not adequate for the control of supragingival plaque and gingivitis, this study provides an additional rationale for the inclusion of the essential-oil mouthrinse as an adjunct to daily oral hygiene procedures.

A model for clinical evaluation of anti-microbial effects of agents on plaque colonization.

PURPOSE: To evaluate a new improved method of microbial analysis in a cross-over clinical study by investigating the efficacy of a single application of an essential oil-containing (EOC) dentifrice as compared to its vehicle control (VC) over a 6-hr period. MATERIALS AND METHODS: Acrylic stents with retention areas for 7, 3 mm x 3 mm hydroxyapatite (HA) squares were fabricated for 12 subjects. 30 min following stent placement, one HA square was removed and sampled for viable microflora. After stent replacement, subjects were assigned either the EOC dentifrice or its VC and brushed under supervision for 1 min. Stents remained in place for the next 6 hrs. A HA square was removed at hourly intervals for 6 hrs following brushing. The microflora was analyzed for total anaerobes on Schaedler's media, for total gram-negative anaerobes on Schaedler-NV selective media, and for total Fusobacterium species on CVE selective media. Plates were incubated anaerobically at 37 degrees C for 2-5 days. Colony forming units were calculated. For each time point, pairwise t-tests were performed using the adjusted means and the pooled error term from the analysis of variance (ANOVA). RESULTS: Treatment with the EOC dentifrice resulted in a statistically reduced plaque growth. Differences were seen as reductions in: (1) total gram-negative anaerobes seen from 1-6 hrs (P < or = 0.011), (2) total anaerobic bacteria which achieved significance at 3 hrs and continued through 6 hrs (P < or = 0.005), and (3) Fusobacterium species as seen from 4-6 hrs (P < or = 0.002).

Tenacious adhesion of Actinobacillus actinomycetemcomitans strain CU1000 to salivary-coated hydroxyapatite.

Adherence of Actinobacillus actinomycetemcomitans to hard-tissue surfaces was evaluated by comparing a phenotypically stable, well-maintained clinical isolate (strain CU1000) to Streptococcus gordonii G9B, an extensively studied oral-colonizing bacterium. Standard innocula of radiolabelled bacteria were added to saliva-coated hydroxyapatite (SHA) and the ratio of bound to unbound cells counted. Several other clinical isolates as well as laboratory strain Y4 were studied. In other experiments, cell detachment from SHA was compared in static and shaking vessels to calculate controlled desorption of cells over time. A sonic-displacement assay was used to measure avidity of binding to HA and SHA. To better define the attachment properties of CU1000, bacteria were treated with a variety of agents including detergents, salts and enzymes before or after incubation with SHA. Results indicated that CU1000 bound better than G9B (a minimum of 10-fold greater; p < or = 0.05) and did not desorb from SHA, while G9B desorbed to equilibrium in 4 h. Furthermore, Langmuir isotherm calculations indicated that, unlike G9B, CU1000 did not follow second-order adsorption kinetics and thus did not achieve saturation. In addition, of the agents tested only periodate reduced attachment and resulted in detachment of CU1000 from surfaces. These experiments suggest that clinical isolates of A. actinomycetemcomitans possess unique binding properties that promote adsorption to and impede desorption from SHA. The characteristics described for the actinobacillus in this study have been previously underestimated, appear to be mediated by glycoconjugates, and may resemble attachment described for several biofilm-forming, non-oral pathogens.

Efficacy of a triclosan/NaF dentifrice in the control of plaque and gingivitis and concurrent oral microflora monitoring.

PURPOSE: To compare the effect of a dentifrice containing 0.3% triclosan and 1100 ppm fluoride and a control dentifrice containing 1100 ppm fluoride on plaque, gingiva and the oral microflora in a long-term study simulating clinical usage. MATERIALS AND METHODS: 159 subjects entered the clinical study and 80 were randomly selected to participate in the microbiological evaluation. 71 subjects completed the detailed evaluation of the oral microflora after 6 months use. Plaque was collected at baseline, 3 months, and 6 months, and examined by darkfield microscopy, Gram stain, immunofluorescence, and selective and non-selective media. Changes in antimicrobial susceptibilities were determined for the first 6-month period and for 6 months post-therapy for 68 subjects who completed the entire study. Susceptibilities of whole plaque samples and MIC values for two pre-designated common plaque organisms, A. viscosus and V. parvula were performed. RESULTS: Multivariate ANOVA and non-parametric analyses revealed no statistical differences for any factor tested. No detrimental shifts were found in either; (1) the compositional make up of the normal flora, (2) the periodontopathic or cariogenic flora, or (3) the opportunistic flora in either group of dentifrice users. Both treatments resulted in decreases in Gram positive cocci over time. There was a reduction in spirochetes in the triclosan/fluoride group as compared to the control group. No overgrowth in opportunists, periodontal pathogens, or cariogenic flora was found in either group. No increase in the proportion of the whole plaque flora resistant to triclosan was found nor was an increase in the MIC values of either A. viscosus or V. parvula in either group. Overall, there appeared to be a general decrease in plaque bacteria in both groups over the course of the experiment.

Effects of sublethal exposure to an antiseptic mouthrinse on representative plaque bacteria.

Although the mechanism responsible for the clinical antiplaque efficacy of oral antiseptics is generally considered to be primarily one of bactericidal activity, it has been suggested that oral antiseptics may have additional effects on bacteria exposed to sublethal levels. Studies reported herein, investigated the effects of sublethal levels of an essential oil-containing antiseptic mouthrinse (Listerine Antiseptic, Warner-Lambert Co., Morris Plains, NJ) on selected activities of representative plaque microorganisms using in vitro models. These studies demonstrated that sublethal exposure to the tested oral antiseptic can have significant effects in reducing intergeneric coaggregation, increasing bacterial generation time, and extracting endotoxin from Gram-negative bacteria. These in vitro activities can be correlated with features of plaque formation and pathogenicity seen in vivo; however, additional studies will be necessary to confirm that these mechanisms are, in fact, operative clinically.

Assessing pre-procedural subgingival irrigation and rinsing with an antiseptic mouthrinse to reduce bacteremia.

In this controlled clinical study, the authors examined the effect of subgingival irrigation and rinsing with an antiseptic mouthrinse before ultrasonic scaling of a quadrant containing inflamed gingivae. The results showed that pre-procedural subgingival irrigation and rinsing can significantly reduce the level of bacteremia associated with ultrasonic scaling. These results support the American Heart Association's recommendation of adjunctive subgingival irrigation prior to invasive procedures in patients at risk of developing bacterial endocarditis.

Prevalence and distribution of bacteriophage phi Aa DNA in strains of Actinobacillus actinomycetemcomitans.

phi Aa is a bacteriophage that was originally isolated by induction of a lysogenic strain of the oral bacterium Actinobacillus actinomycetemcomitans. Since the discovery of phage phi Aa, additional phages infecting several other strains of A. actinomycetemcomitans have been identified. To determine the prevalence of phi Aa or phi Aa-related temperate phages in this species, a phi Aa-specific DNA probe was prepared to screen for homologous sequences among 42 strains of A. actinomycetemcomitans. Fourteen (33%) of the 42 strains examined contained DNA sequences that hybridized with the phage phi Aa probe. A bacteriophage designated phi Aa33384 was isolated by induction from one of the strains (ATCC 33384) that contained a sequence that hybridized with the phi Aa probe. The phi Aa probe hybridized with the DNA extracted from bacteriophage phi Aa33384. The distribution of the phage phi Aa sequence among A. actinomycetemcomitans serotypes was 5/13 (38%) of the serotype a strains, 0/16 (0%) of the serotype b strains, and 9/13 (69%) of the serotype c strains. The results of this investigation suggest that the target sequence prepared from the phage phi Aa genome is fairly common in the A. actinomycetemcomitans chromosome, and that the sequence is distributed among the A. actinomycetemcomitans serotypes in a seemingly nonrandom manner.

Reduction of viable bacteria in dental aerosols by preprocedural rinsing with an antiseptic mouthrinse.

Two double-blind controlled clinical studies were conducted to determine the efficacy of preprocedural rinsing with Cool Mint Listerine Antiseptic mouthrinse on the level of viable bacteria recovered from dental aerosols when generated immediately after and 40 minutes after rinsing. Eighteen healthy subjects participated in each study. In the first study, following a 24-hour no-oral-hygiene period, subjects received a 10-minute ultrasonic scaling of a randomly chosen half mouth, rinsed with either Cool Mint Listerine or a control, and received an ultrasonic scaling of the remaining half mouth. During each scaling period, aerosolized bacteria were collected on a sterile filter using a modified air-sampling device. The filters were overlaid on culture media, incubated aerobically, and colonies were counted. The second study followed the same basic design except that ultrasonic scaling was done for 5 minutes, and the post-rinsing sampling was performed following a 40-minute simulated dental treatment period. Rinsing with Cool Mint Listerine resulted in a 92.1% reduction in viable bacteria in aerosols generated immediately after rinsing and a 91.3% reduction in aerosols generated 40 minutes after rinsing. These reductions were significantly greater than control (P = 0.0001). These studies suggest that preprocedural rinsing with Cool Mint Listerine Antiseptic may potentially reduce the risk of cross contamination in the dental operatory.