Utilization of Variant and Fusion Proteins To Functionally Map the Aggregatibacter actinomycetemcomitans Trimeric Autotransporter Protein ApiA.

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is a causative agent of localized aggressive periodontitis. Critical to its infection process is the first and essential step of attachment, which is related to the coordinated functions of surface components comprised of proteins and extracellular polysaccharides. One such protein is the outer membrane trimeric autotransporter protein ApiA, a versatile virulence factor with numerous functions, including cell binding, invasion, serum resistance, autoaggregation, and induction of cytokine release. Here we report on the use of Escherichia coli strains expressing protein variants to define the separate functions ascribed to the N terminus and those related to the C terminus. Importantly, a hybrid protein that comprised the N terminus of trimeric ApiA and the ß-barrel domain of monomeric autotransporter Aae was constructed, which allowed the expression of a monomer surface-exposed domain of ApiA. Functional and phenotypic analyses demonstrated that the C terminus of ApiA forms an independent domain that is crucial for general stability and trimer formation, which appears to be associated with autoaggregation, biofilm formation, and surface expression. Importantly, the results show that the monomeric form of the N-terminal passenger domain of ApiA, while surface exposed, is sufficient for binding to buccal epithelial cells; however, it is not sufficient to allow aggregation and biofilm formation, strengthening the importance of the role of trimerization in these phenotypes.

Colonization and Persistence of Labeled and "Foreign" Strains of Aggregatibacter actinomycetemcomitans Inoculated into the Mouths of Rhesus Monkeys.

Aggregatibacter actinomycetemcomitans (Aa) is a pathobiont and part of a consortium of bacteria that can lead to periodontitis in humans. Our aim was to develop a model for oral inoculation of labeled Aa into a suitable host in order to study Aa traits and ecological factors that either enhance or repress its persistence. Primate species were screened for Aa to select a host for colonization studies. Macaca mulatta (Rhesus/Rh) was selected. Rh Aa strains were isolated, subjected to sequencing and functional analysis for comparison to human strains. "Best" methods for microbial decontamination prior to inoculation were assessed. Three groups were studied; Group 1 (N=5) was inoculated with Aa Spectinomycin resistant (SpecR) Rh strain 4.35, Group 2 (N=5) inoculated with Aa SpecR human strain IDH 781, and Group 3 (N=5) the un-inoculated control. Repeated feeding with pancakes spiked with SpecRAa followed high dose oral inoculation. Cheek, tongue, and plaque samples collected at baseline 1, 2, 3, and 4 weeks after inoculation were plated on agar; 1) selective for Aa, 2) enriched for total counts, and 3) containing 50 µg/ml of Spec. Aa was identified by colonial morphology and DNA analysis. Rh and human Aa had > 93-98 % genome identity. Rh Aa attached to tissues better than IDH 781 in vitro (p < 0.05). SpecR IDH 781 was not recovered from any tissue at any time; whereas, RhSpecR 4.35 was detected in plaque, but never tongue or cheek, in all monkeys at all times (> 1 × 105 colonies/ml; p < 0.001). In conclusion, the primate model provides a useful platform for studying integration of Aa strains into a reduced but established oral habitat. Primate derived SpecRAa was consistently detected in plaque at all collection periods; however, human derived Aa was never detected. The model demonstrated both microbial as well as tissue specificity.

Macrophage inflammatory protein-1α shows predictive value as a risk marker for subjects and sites vulnerable to bone loss in a longitudinal model of aggressive periodontitis.

Improved diagnostics remains a fundamental goal of biomedical research. This study was designed to assess cytokine biomarkers that could predict bone loss (BL) in localized aggressive periodontitis. 2,058 adolescents were screened. Two groups of 50 periodontally healthy adolescents were enrolled in the longitudinal study. One group had Aggregatibacter actinomycetemcomitans (Aa), the putative pathogen, while the matched cohort did not. Cytokine levels were assessed in saliva and gingival crevicular fluid (GCF). Participants were sampled, examined, and radiographed every 6 months for 2-3 years. Disease was defined as radiographic evidence of BL. Saliva and GCF was collected at each visit, frozen, and then tested retrospectively after detection of BL. Sixteen subjects with Aa developed BL. Saliva from Aa-positive and Aa-negative healthy subjects was compared to subjects who developed BL. GCF was collected from 16 subjects with BL and from another 38 subjects who remained healthy. GCF from BL sites in the 16 subjects was compared to healthy sites in these same subjects and to healthy sites in subjects who remained healthy. Results showed that cytokines in saliva associated with acute inflammation were elevated in subjects who developed BL (i.e., MIP-1α MIP-1ß IL-α, IL-1ß and IL-8; p<0.01). MIP-1α was elevated 13-fold, 6 months prior to BL. When MIP-1α levels were set at 40 pg/ml, 98% of healthy sites were below that level (Specificity); whereas, 93% of sites with BL were higher (Sensitivity), with comparable Predictive Values of 98%; p<0.0001; 95% C.I. = 42.5-52.7). MIP-1α consistently showed elevated levels as a biomarker for BL in both saliva and GCF, 6 months prior to BL. MIP-1α continues to demonstrate its strong candidacy as a diagnostic biomarker for both subject and site vulnerability to BL.

A consortium of Aggregatibacter actinomycetemcomitans, Streptococcus parasanguinis, and Filifactor alocis is present in sites prior to bone loss in a longitudinal study of localized aggressive periodontitis.

Aggregatibacter actinomycetemcomitans-induced localized aggressive periodontitis (LAP) in African-American adolescents has been documented but is poorly understood. Two thousand fifty-eight adolescents aged 11 to 17 years were screened for their periodontal status and the presence of A. actinomycetemcomitans in their oral cavity. Seventy-one A. actinomycetemcomitans-negative and 63 A. actinomycetemcomitans-positive periodontally healthy subjects were enrolled, sampled, examined, and radiographed yearly for 3 years. Gingival and periodontal pocket depth and attachment levels were recorded. Disease presentation was characterized by bone loss (BL). Subgingival sites were sampled every 6 months to assess (i) the role of A. actinomycetemcomitans in BL and (ii) the association of A. actinomycetemcomitans and other microbes in their relationships to BL. Sixteen of 63 subjects with A. actinomycetemcomitans developed BL (the other 47 subjects with A. actinomycetemcomitans had no BL). No A. actinomycetemcomitans-negative subjects developed BL. Human oral microbe identification microarray (HOMIM) was used for subgingival microbial assessment. On a subject level, pooled data from A. actinomycetemcomitans-positive subjects who remained healthy had higher prevalences of Streptococcus and Actinomyces species, while A. actinomycetemcomitans-positive subjects with BL had higher prevalences of Parvimonas micra, Filifactor alocis, A. actinomycetemcomitans, and Peptostreptococcus sp. human oral taxon 113 (HOT-113). At vulnerable sites, A. actinomycetemcomitans, Streptococcus parasanguinis, and F. alocis levels were elevated prior to BL. In cases where the three-organism consortium (versus A. actinomycetemcomitans alone) was detected, the specificity for detecting sites of future BL increased from 62% to 99%, with a sensitivity of 89%. We conclude that detecting the presence of A. actinomycetemcomitans, S. parasanguinis, and F. alocis together indicates sites of future BL in LAP. A synergistic interaction of this consortium in LAP causation is possible and is the subject of ongoing research.

A lactotransferrin single nucleotide polymorphism demonstrates biological activity that can reduce susceptibility to caries.

Streptococcus mutans is prominently linked to dental caries. Saliva's influence on caries is incompletely understood. Our goal was to identify a salivary protein with anti-S. mutans activity, characterize its genotype, and determine genotypic variants associated with S. mutans activity and reduced caries. An S. mutans affinity column was used to isolate active moieties from saliva obtained from a subject with minimal caries. The bound and eluted protein was identified as lactotransferrin (LTF) by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis and confirmed by Western blotting with LTF antibody. A single nucleotide polymorphism (SNP) that produced a shift from arginine (R) to lysine (K) at amino acid position 47 in the LTF antimicrobial region (rs: 1126478) killed S. mutans in vitro. Saliva from a subject with moderate caries and with the LTF "wild-type" R form at position 47 had no such activity. A pilot genetic study (n = 30) showed that KK subjects were more likely to have anti-S. mutans activity than RR subjects (P = 0.001; relative risk = 3.6; 95% confidence interval [95% CI] = 1.5 to 11.13). Pretreatment of KK saliva with antibody to LTF reduced S. mutans killing in a dose-dependent manner (P = 0.02). KK subjects were less likely to have caries (P = 0.02). A synthetic 11-mer LTF/K peptide killed S. mutans and other caries-related bacteria, while the LTF/R peptide had no effect (P = 0.01). Our results provide functional evidence that the LTF/K variant results in both anti-S. mutans activity and reduced decay. We suggest that the LTF/K variant can influence oral microbial ecology in general and caries-provoking microbes specifically.

Can salivary activity predict periodontal breakdown in A. actinomycetemcomitans infected adolescents?

OBJECTIVE: While Aggregatibacter actinomycetemcomitans (Aa) is highly associated with localised aggressive periodontitis (LAP) many Aa-carriers do not develop LAP. This study was designed to determine whether specific salivary factors could distinguish between subjects who have Aa initially and remain healthy (H/AA) as compared to those who develop LAP (LAP/AA). DESIGN: H/AA subjects and healthy controls with no Aa (H) were enrolled in a longitudinal cohort study to investigate initiation of bone loss (LAP) over 3 years. After detection of LAP, stored saliva from 10 H, 10 H/AA, and 10 LAP/AA subjects was thawed, processed, and tested for (1) lactoferrin (Lf) concentration and iron levels; (2) agglutination of Aa; (3) killing of Gram-positive bacteria. RESULTS: LAP/AA saliva levels of Lf iron were low prior to and after bone loss (3.6+1.7ngFe/µg) (LAP/AA vs. H and H/AA p≤0.01). Saliva from H/AA subjects caused Aa to agglutinate significantly more than H or LAP/AA saliva (p≤0.01). LAP/AA saliva killed Streptococcus mutans, Streptococcus sanguis and Lactobacillus in vitro by >83%. Saliva from H individuals killed these bacteria by <3.3% (LAP/AA vs. H; p≤0.01). H/AA killing was intermediate. CONCLUSION: LAP/AA saliva showed: low levels of Lf iron, minimal Aa agglutinating activity, and high killing activity against Gram-positive bacteria. Aa-positive healthy saliva (H/AA) showed: higher levels of Lf iron, maximal Aa agglutinating activity, and moderate killing of Gram-positive bacteria. A salivary activity profile can distinguish between subjects who are Aa-positive and remain healthy from those who develop LAP.

Whole mouth antimicrobial effects after oral hygiene: comparison of three dentifrice formulations.

AIMS: This study compared the antimicrobial effects of three commercial dentifrice formulations: sodium fluoride/triclosan/copolymer (TCN/C), stannous fluoride/sodium hexametaphosphate/zinc lactate (SnF(2)/SHMP) and sodium fluoride (NaF). MATERIALS AND METHODS: Thirty-five adults (15 men and 20 women; average age 33 years and pockets <5 mm) completed this double-blind, triple-crossover study. After washout, baseline samples from four sites, plaque, saliva, tongue and buccal mucosa, were collected and evaluated for six microbial types, anaerobes, Streptococci, Actinomyces, hydrogen-sulphide (H(2) S)-producing bacteria, Fusobacteria and Veillonella. A specific dentifrice was randomly assigned for twice-daily use for 13 days. On day 14, 12 h after brushing, samples were collected for microbiological evaluations. Alternate dentifrices followed this identical protocol. RESULTS: For all four oral sites and six organisms evaluated in each site, the TCN/C demonstrated significant reductions (49-83%) as compared with the other treatments (p < 0.01). The SnF(2)/SHMP group showed significant reductions of 14-43% for 14 of 24 outcomes as compared with the NaF group (p < 0.01), with no differences in 10 outcomes. CONCLUSIONS: The TCN/C dentifrice formulation consistently demonstrated significant reductions for a range of microorganisms in diverse oral sites in comparison with the NaF, or the SnF(2)/SHMP dentifrice formulations as seen 12 h after brushing.

Genome sequence of Aggregatibacter actinomycetemcomitans RHAA1, isolated from a rhesus macaque, an Old World primate.

Aggregatibacter actinomycetemcomitans is implicated in localized aggressive periodontitis. We report the first genome sequence of an A. actinomycetemcomitans strain isolated from an Old World primate.

Aggregatibacter actinomycetemcomitans as an early colonizer of oral tissues: epithelium as a reservoir?

This study examined in vivo and in vitro colonization by Aggregatibacter actinomycetemcomitans, an organism highly associated with aggressive periodontitis. Thirteen volunteers (5 were A. actinomycetemcomitans positive for buccal epithelial cells [BECs] and teeth, 5 were A. actinomycetemcomitans positive for teeth only, and 3 were A. actinomycetemcomitans-negative controls) had two mandibular stents fabricated. Each stent contained 3 removable hydroxyapatite (HA) tooth surrogates. One HA square was removed from a stent at 5 time points over 7 h to assess the transfer of A. actinomycetemcomitans from teeth or BECs to HA. Streptococcus, Actinomyces, A. actinomycetemcomitans, and total anaerobic counts were evaluated on each square over time. In vitro experiments evaluated binding, desorption, transfer, and reattachment of A. actinomycetemcomitans wild-type and mutant strains to BECs and saliva-coated HA (SHA). Streptococcus and Actinomyces formed 80% of the cultivable flora on HA in all subjects. Transfer of A. actinomycetemcomitans to HA was not seen in subjects with A. actinomycetemcomitans on teeth only. All 5 subjects with A. actinomycetemcomitans on BECs showed transfer of A. actinomycetemcomitans to HA. In vitro, A. actinomycetemcomitans desorbed from BECs and transferred to SHA. A. actinomycetemcomitans binding to SHA was irreversible and did not transfer to BECs. The adhesin Aae showed specificity for BECs. Fimbrial mutants showed the greatest reduction in binding to SHA. A. actinomycetemcomitans migrated from BECs to HA in vivo and to SHA in vitro; however, A. actinomycetemcomitans movement from teeth and SHA to BECs did not occur. In vivo, A. actinomycetemcomitans colonized HA within 6 h and thus can be considered an early colonizer. BECs are a likely reservoir for A. actinomycetemcomitans tooth colonization.

Mapping the epithelial-cell-binding domain of the Aggregatibacter actinomycetemcomitans autotransporter adhesin Aae.

The Gram-negative periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) binds selectively to buccal epithelial cells (BECs) of human and Old World primates by means of the outer-membrane autotransporter protein Aae. We speculated that the exposed N-terminal portion of the passenger domain of Aae would mediate binding to BECs. By using a series of plasmids that express full-length or truncated Aae proteins in Escherichia coli, we found that the BEC-binding domain of Aae was located in the N-terminal surface-exposed region of the protein, specifically in the region spanning amino acids 201-284 just upstream of the repeat region within the passenger domain. Peptides corresponding to amino acids 201-221, 222-238 and 201-240 were synthesized and tested for their ability to reduce Aae-mediated binding to BECs based on results obtained with truncated Aae proteins expressed in E. coli. BEC-binding of E. coli expressing Aae was reduced by as much as 50 % by pre-treatment of BECs with a 40-mer peptide (201-240; P40). Aae was also shown to mediate binding to cultured human epithelial keratinocytes (TW2.6), OBA9 and TERT, and endothelial (HUVEC) cells. Pre-treatment of epithelial cells with P40 resulted in a dose-dependent reduction in binding and reduced the binding of both full-length and truncated Aae proteins expressed in E. coli, as well as Aae expressed in Aa. Fluorescently labelled P40 peptides reacted in a dose-dependent manner with BEC receptors. We propose that these proof-of-principle experiments demonstrate that peptides can be designed to interfere with Aa binding mediated by host-cell receptors specific for Aae adhesins.

An investigation of the effect of an essential oil mouthrinse on induced bacteraemia: a pilot study.

AIM: This pilot study was designed to assess the effect of an essential oil antiseptic mouthrinse (EOM) in reducing bloodstream bacteria after chewing an apple. MATERIAL AND METHODS: From a panel of 200, we screened 62 individuals with mild-to-moderate gingivitis. Twenty-two individuals who showed a bacteraemia after chewing an apple were enrolled. Subjects were recalled, instructed to chew an apple, had blood drawn (first baseline), and were randomly assigned EOM or a control (C) treatment for 2 weeks. Subjects were recalled, given an apple, and had blood taken for bacterial counts. Following a 1-week fluoride dentifrice wash-out, subjects were recalled, given the apple challenge, had blood drawn (second baseline), assigned the alternate treatment, and recalled for testing. Differences between baseline and 2-week post-treatment (EOM versus C) in blood-borne bacteria were assessed by analysis of covariance. RESULTS: Mean aerobic blood-borne bacteria decreased by 68.5% (17.7 viable counts from baseline; p<0.001), while anaerobic counts decreased by 70.7% (14.5 mean viable counts from baseline; p<0.001) for the EOM treatment. No reduction was seen for the C treatment. CONCLUSIONS: This double-blind, placebo-controlled, randomized, 2-week cross-over study showed that rinsing with essential oils reduced the level of bloodstream bacteria in subjects with mild-to-moderate gingivitis.

An improved cost-effective, reproducible method for evaluation of bone loss in a rodent model.

AIM: This study was designed to investigate the utility of two "new" definitions for assessment of bone loss in a rodent model of periodontitis. MATERIAL AND METHODS: Eighteen rats were divided into three groups. Group 1 was infected by Aggregatibacter actinomycetemcomitans (Aa), group 2 was infected with an Aa leukotoxin knock-out, and group 3 received no Aa (controls). Microbial sampling and antibody titres were determined. Initially, two examiners measured the distance from the cemento-enamel-junction to alveolar bone crest using the three following methods; (1) total area of bone loss by radiograph, (2) linear bone loss by radiograph, (3) a direct visual measurement (DVM) of horizontal bone loss. Two "new" definitions were adopted; (1) any site in infected animals showing bone loss >2 standard deviations above the mean seen at that site in control animals was recorded as bone loss, (2) any animal with two or more sites in any quadrant affected by bone loss was considered as diseased. RESULTS: Using the "new" definitions both evaluators independently found that infected animals had significantly more disease than controls (DVM system; p<0.05). CONCLUSIONS: The DVM method provides a simple, cost effective, and reproducible method for studying periodontal disease in rodents.

Macrophage inflammatory protein-1alpha: a salivary biomarker of bone loss in a longitudinal cohort study of children at risk for aggressive periodontal disease?

BACKGROUND: Periodontitis develops in a time-dependent manner. Cross-sectional studies document one moment in time but fail to capture the progressive nature of disease. Radiographic measures of bone loss are relatively insensitive but are reliable markers of irreversible disease. Longitudinal studies are needed to identify biomarkers that can precede radiographic evidence of bone loss and, thus, mark the period prior to clinical evidence of irreversible disease. A longitudinal study of students susceptible to localized aggressive periodontitis (LAgP) was conducted to evaluate chemokines/cytokines found in saliva derived from periodontally healthy children who subsequently developed alveolar bone loss. METHODS: Students were screened, sampled for Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans [Aa]), and divided into a cohort of Aa+ and Aa- students. Ninety-six periodontally healthy Aa+ and Aa- students were recalled every 6 to 9 months following screening. Examinations, saliva collections, and radiographs were performed. After seven students developed bone loss, the levels of 21 cytokines were assessed and matched to saliva from seven Aa+ and seven Aa- students who remained healthy for > or =1 year. Subsequently, saliva from an additional 27 students who remained healthy was analyzed. RESULTS: Nineteen cytokines were not detected or were detected at low levels. Macrophage inflammatory protein (MIP)-1alpha was elevated 50-fold in seven Aa+ students who developed disease 6 to 9 months prior to radiographic detection of bone loss compared to levels in 21 Aa+ and 20 Aa- students who remained healthy (P <0.001). Interleukin (IL)-1beta was also elevated (P = 0.01). MIP-1alpha had a specificity of 96.8% and a sensitivity of 100%, whereas IL-1beta showed 90.3% specificity and 85.7% sensitivity relative to bone loss. MIP-1alpha levels were also related to increasing probing depth and the number of pockets >6 mm. CONCLUSION: The superior sensitivity and specificity of MIP-1alpha, which correlated well with probing depths and the onset of bone loss, suggested that it could be used as an early biomarker for LAgP.

Effect of rinsing with an essential oil-containing mouthrinse on subgingival periodontopathogens.

BACKGROUND: Studies have shown that the nature and amount of supragingival plaque can influence the composition of the contiguous subgingival flora. The purpose of this study was to investigate the effect of rinsing with an essential oil antimicrobial mouthrinse on levels of representative subgingival bacteria in subjects with mild to moderate periodontitis. METHODS: This controlled study used a randomized, double-masked, 2 x 2 crossover design. After baseline subgingival sampling, 37 qualifying subjects rinsed with the essential oil mouthrinse or a negative control twice daily for 14 days, with a post-treatment sample obtained on day 15. Following a washout period, the procedure was repeated with the alternative rinse. Target organisms enumerated were Porphyromonas gingivalis, Fusobacterium nucleatum, Veillonella sp., and total anaerobes. Intergroup comparisons of post-treatment log-transformed colony forming unit counts were made using analysis of covariance. RESULTS: After 14 days of twice-daily rinsing, the level of each of the target subgingival organisms was significantly lower in the essential oil group than in the control group (P < 0.001), with percent reductions ranging from 66.3% to 79.2%. CONCLUSIONS: Rinsing with the essential oil antiplaque/antigingivitis mouthrinse can have significant antimicrobial activity against subgingival periodontopathogens. Most likely, the antimicrobial effect was mediated primarily by disruption of the contiguous supragingival plaque by the mouthrinse. Because studies have shown that control of supragingival plaque can influence the onset and/or progression of periodontitis, additional studies on non-prescription antimicrobial oral care products may lead to new regimens for decreasing the burden of periodontal diseases in the population.

Aggregatibacter actinomycetemcomitans and its relationship to initiation of localized aggressive periodontitis: longitudinal cohort study of initially healthy adolescents.

Aggregatibacter actinomycetemcomitans is frequently associated with localized aggressive periodontitis (LAP); however, longitudinal cohort studies relating A. actinomycetemcomitans to initiation of LAP have not been reported. A periodontal assessment was performed on 1,075 primarily African-American and Hispanic schoolchildren, ages 11 to 17 years. Samples were taken from each child for A. actinomycetemcomitans. A cohort of 96 students was established that included a test group of 38 A. actinomycetemcomitans-positive students (36 periodontally healthy and 2 with periodontal pockets) and 58 healthy A. actinomycetemcomitans-negative controls. All clinical and microbiological procedures were repeated at 6-month intervals. Bitewing radiographs were taken annually for definitive diagnosis of LAP. At the initial examination, clinical probing attachment measurements indicated that 1.2% of students had LAP, while 13.7% carried A. actinomycetemcomitans, including 16.7% of African-American and 11% of Hispanic students (P = 0.001, chi-square test). A. actinomycetemcomitans serotypes a, b, and c were equally distributed among African-Americans; Hispanic students harbored predominantly serotype c (P = 0.05, chi-square test). In the longitudinal phase, survival analysis was performed to determine whether A. actinomycetemcomitans-positive as compared to A. actinomycetemcomitans-negative students remained healthy ("survived") or progressed to disease with attachment loss of >2 mm or bone loss (failed to "survive"). Students without A. actinomycetemcomitans at baseline had a significantly greater chance to remain healthy (survive) compared to the A. actinomycetemcomitans-positive test group (P = 0.0001). Eight of 38 A. actinomycetemcomitans-positive and none of 58 A. actinomycetemcomitans-negative students showed bone loss (P = 0.01). A. actinomycetemcomitans serotype did not appear to influence survival. These findings suggest that detection of A. actinomycetemcomitans in periodontally healthy children can serve as a risk marker for initiation of LAP.

Activity of an antimicrobial peptide mimetic against planktonic and biofilm cultures of oral pathogens.

Antimicrobial peptides (AMPs) are naturally occurring, broad-spectrum antimicrobial agents that have recently been examined for their utility as therapeutic antibiotics. Unfortunately, they are expensive to produce and are often sensitive to protease digestion. To address this problem, we have examined the activity of a peptide mimetic whose design was based on the structure of magainin, exhibiting its amphiphilic structure. We demonstrate that this compound, meta-phenylene ethynylene (mPE), exhibits antimicrobial activity at nanomolar concentrations against a variety of bacterial and Candida species found in oral infections. Since Streptococcus mutans, an etiological agent of dental caries, colonizes the tooth surface and forms a biofilm, we quantified the activity of this compound against S. mutans growing under conditions that favor biofilm formation. Our results indicate that mPE can prevent the formation of a biofilm at nanomolar concentrations. Incubation with 5 nM mPE prevents further growth of the biofilm, and 100 nM mPE reduces viable bacteria in the biofilm by 3 logs. Structure-function analyses suggest that mPE inhibits the bioactivity of lipopolysaccharide and binds DNA at equimolar ratios, suggesting that it may act both as a membrane-active molecule, similar to magainin, and as an intracellular antibiotic, similar to other AMPs. We conclude that mPE and similar molecules display great potential for development as therapeutic antimicrobials.

A second Aggregatibacter actinomycetemcomitans autotransporter adhesin exhibits specificity for buccal epithelial cells in humans and Old World primates.

Previous work showed that the Aggregatibacter actinomycetemcomitans adhesin Aae demonstrated species specificity and tissue tropism to buccal epithelial cells (BECs) derived from humans and Old World primates, but a second, lower-affinity adhesin was noted. This study was designed to determine if Omp100 (also known as ApiA), a surface-expressed A. actinomycetemcomitans adhesin, is that second adhesin and if so to investigate its tissue tropism and species specificity. A targeted mutagenesis protocol was used to construct an isogenic apiA mutant and an aae apiA double mutant with wild-type A. actinomycetemcomitans. In addition, Escherichia coli strain DH5alpha was used to express apiA to further assess binding parameters. Results indicated that the apiA mutant strain showed significantly less binding to BECs than its parent strain (P < or = 0.05). Further, binding mediated by ApiA was specific to BECs from humans and Old World primates, as seen in both wild-type A. actinomycetemcomitans and E. coli expressing ApiA (P < or = 0.05). Pretreatment of wild-type A. actinomycetemcomitans cells with anti-ApiA antiserum reduced binding in a dose-dependent manner. The aae apiA double mutant completely abrogated A. actinomycetemcomitans binding to both human and Old World primate BECs. Taken together, these studies indicate that ApiA and Aae, in concert, modulate binding of A. actinomycetemcomitans to human BECs. Since the BEC is a prominent reservoir for A. actinomycetemcomitans, identification of this second adhesin could lead to important therapeutic strategies.

Effect of an essential oil-containing antimicrobial mouthrinse on specific plaque bacteria in vivo.

AIM: This study was conducted to investigate the effect of rinsing with an essential oil-containing mouthrinse on levels of specific supra and subgingival bacteria in subjects with gingivitis. MATERIAL AND METHODS: Fifteen subjects meeting entry criteria completed this randomized, controlled, double-blind, crossover study. Subjects were required to have >or=1000 target organisms per millilitre in pooled samples from two subgingival sites. Following sampling of supra and subgingival plaque, subjects began twice-daily rinsing for 14 days with either an essential oil-containing mouthrinse (Cool Mint Listerine Antiseptic) or a negative control. Supra and subgingival plaque was again sampled on day 15, and the procedure repeated after a 1-week washout period with subjects using the alternate rinse. RESULTS: Compared with the negative control, the essential oil mouthrinse produced significant reductions in supragingival plaque levels of Veillonella sp., Capnocytophaga sp., Fusobacterium nucleatum, and total anaerobes ranging from 52.3 to 88.5% (p<0.001 except for Veillonella, p=0.002); respective reductions in subgingival plaque ranged from 54.1 to 69.1% (p<0.001). CONCLUSIONS: Rinsing with the essential oil mouthrinse can have an impact on the subgingival plaque flora. This study provides additional evidence indicating that reduction in supragingival plaque can reduce levels of subgingival plaque.

Saliva from subjects harboring Actinobacillus actinomycetemcomitans kills Streptococcus mutans in vitro.

BACKGROUND: Previous research indicated that patients with localized aggressive periodontitis (LAgP) had minimal proximal decay. We speculated that differences in these two proximal dental diseases (LAgP and proximal decay) in LAgP could be due to the effect of saliva on the growth of key microorganisms related to these two infections. Carbon dioxide (CO(2)) is required for growth of Actinobacillus actinomycetemcomitans (Aa), the reputed cause of LAgP. Bicarbonate, a source of CO(2), buffers acid production by Streptococcus mutans (Sm), a key organism associated with caries. The purpose of this study was to determine whether the saliva of LAgP patients and subjects with Aa had higher levels of bicarbonate, or an elevated pH, and/or reduced survival of Sm. METHODS: Eleven Aa-positive subjects (seven with LAgP) were matched with 11 Aa-negative controls. A total of 5 ml saliva obtained from each subject was tested for CO(2) levels, pH, and effects on survival of Aa and Sm. Saliva from 22 additional subjects was used for confirmatory data. RESULTS: CO(2) levels in the test group (Aa-positive subjects) and controls (Aa-negatives) were similar. No clinically relevant differences were found in salivary pH. However, saliva from the test group killed Sm by more than two logs (P <0.05). No effect was seen on Aa. The saliva from the Aa-negative group killed Aa by two logs (P <0.05). No effect was seen on Sm. CONCLUSION: Aa-positive subjects had a salivary factor that significantly reduced survival of Sm, which may help to explain the fact that this group typically has minimal proximal decay.

The antimicrobial effect of a triclosan/copolymer dentifrice on oral microorganisms in vivo.

BACKGROUND: The authors compared the in vivo antimicrobial effects on microorganisms from dental plaque, saliva and the tongue in subjects who used a triclosan/copolymer dentifrice and a fluoride dentifrice (control). METHODS: The authors assigned 15 subjects randomly to the control dentifrice or the triclosan/copolymer dentifrice for twice-daily use for one week. They collected samples of plaque, saliva and tongue scrapings six and 12 hours after the final brushing. They analyzed colony-forming units of Veillonella species, Fusobacteria species, total cultivable anaerobes and hydrogen sulfide (H(2)S)-producing bacteria. A one-week washout followed. The authors repeated the protocol with the second dentifrice. RESULTS: The results showed no differences at baseline. Significant reductions (88 to 96 percent) in oral anaerobic bacteria were observed in the triclosan/copolymer group six and 12 hours after brushing compared with the control group (P = .001). Fusobacteria decreased by 77 to 92 percent and Veillonella decreased by 84 to 89 percent six and 12 hours after brushing in the triclosan/copolymer group versus the control group. The triclosan/copolymer group also demonstrated a significant decrease in H(2)S-producing bacteria six and 12 hours after brushing (74 to 85 percent) (P = .001). CONCLUSIONS: Brushing with the triclosan/copolymer dentifrice resulted in significant reductions in microorganisms from the three sites compared with the control dentifrice. CLINICAL IMPLICATIONS: The triclosan/copolymer dentifrice produced sustained effects on oral bacteria for 12 hours.