Aggregatibacter actinomycetemcomitans colonization and persistence in a primate model.

Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis resulting in premature tooth loss in adolescents. Tooth adherence and biofilm persistence are prerequisites for survival in the oral domain. Here, using a rhesus monkey model, 16S rRNA sequencing, and weighted network analysis, we assessed colonization of A. actinomycetemcomitans variants and ascertained microbial interactions in biofilm communities. Variants in A. actinomycetemcomitans leukotoxin (ltx) were created, labeled, inoculated, and compared with their progenitor strain for in vivo colonization. Samples of tooth-related plaque were assessed for colonization at baseline and after debridement and inoculation of labeled strains. Null, minimal, and hyper-Ltx-producing strains were created and assessed for hydroxyapatite binding and biofilm formation in vitro. Ltx-hyperproducing strains colonized with greater prevalence and at higher levels than wild type or ltx mutants (P = 0.05). Indigenous and inoculated A. actinomycetemcomitans strains that attached were associated with lactate-producing species (i.e., Leptotrichia, Abiotrophia, and Streptoccocci). A. actinomycetemcomitans was found at 0.13% of the total flora at baseline and at 0.05% 4 wk after inoculation. In vivo data were supported by in vitro results. We conclude that hyper-Ltx production affords these strains with an attachment advantage providing a foothold for competition with members of the indigenous microbiota. Increased attachment can be linked to ltx gene expression and up-regulation of adherence-associated genes. Growth of attached A. actinomycetemcomitans in vivo was enhanced by lactate availability due to consorting species. These associations provide A. actinomycetemcomitans with the constituents required for its colonization and survival in the complex and competitive oral environment.

Ezh2 knockout in mesenchymal cells causes enamel hyper-mineralization.

Enhancer of zeste homolog 2 (EZH2) is the catalytic core of polycomb repressive complex 2 (PRC2), which primarily methylates lysine 27 on histone H3 (H2K27me3), generating transcriptionally suppressed heterochromatin. Since EZH2 suppresses expression of genes involved in dentin formation, we examined the role of EZH2 in tooth development. Intriguingly, microCT analysis of teeth from mice with conditional Ezh2 knockout in uncommitted mesenchymal cells showed hyper-mineralization of enamel, which is produced by the epithelial-lineage cells, ameloblasts. Scanning electron microscopy analysis and nano-indentation of the incisor enamel from knockout mice revealed smaller inter-rod spaces and higher hardness compared to wild type enamel, respectively. Interestingly, expression of the calcium channel subunit gene, Orai2, was decreased compared to its competitor, Orai1, both in knockout mouse incisors and the ex vivo culture of ameloblasts with the surrounding tissues under EZH2 inhibition. Moreover, histological analysis of incisor from knockout mice showed decreased ameloblastin and expedited KLK4 expression in the ameloblasts. These observations suggest that EZH2 depletion in dental mesenchymal cells reduces enamel matrix formation and increases enamel protease activity from ameloblasts, resulting in enamel hyper-mineralization. This study demonstrates the significant role of the suppressive H3K27me3 mark for heterochromatin on enamel formation.

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.

Classification and diagnosis of aggressive periodontitis.

OBJECTIVE: Since the initial description of aggressive periodontitis (AgP) in the early 1900s, classification of this disease has been in flux. The goal of this manuscript is to review the existing literature and to revisit definitions and diagnostic criteria for AgP. STUDY ANALYSIS: An extensive literature search was performed that included databases from PubMed, Medline, Cochrane, Scopus and Web of Science. Of 4930 articles reviewed, 4737 were eliminated. Criteria for elimination included; age > 30 years old, abstracts, review articles, absence of controls, fewer than; a) 200 subjects for genetic studies, and b) 20 subjects for other studies. Studies satisfying the entrance criteria were included in tables developed for AgP (localized and generalized), in areas related to epidemiology, microbial, host and genetic analyses. The highest rank was given to studies that were; a) case controlled or cohort, b) assessed at more than one time-point, c) assessed for more than one factor (microbial or host), and at multiple sites. RESULTS: Epidemiologic studies provided insight into ethnic and societal factors affecting AgP. DNA analysis of microbes showed some consistency but significant variability. Host factor analysis was less consistent. Many genetic studies were conducted but few had either sufficient power or looked at multiple genes in AgP. CONCLUSIONS: Conflicting data resulted for several reasons; 1) the classification was too broad, 2) the disease (AgP) was not studied from its inception, at differing time points (temporal), and at different locations (topographic). New technologic advances coupled with a more delimiting definition of disease will allow for genetic, host and microbial factor analyses in an unbiased manner. As such we predict that progress can be made in identifying a robust group of genetic, host, and microbial risk-markers associated with periodontal disease that can improve diagnostic capability in disease associated with juveniles, adolescents, and post-adolescent individuals.

Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions.

A new periodontitis classification scheme has been adopted, in which forms of the disease previously recognized as "chronic" or "aggressive" are now grouped under a single category ("periodontitis") and are further characterized based on a multi-dimensional staging and grading system. Staging is largely dependent upon the severity of disease at presentation as well as on the complexity of disease management, while grading provides supplemental information about biological features of the disease including a history-based analysis of the rate of periodontitis progression; assessment of the risk for further progression; analysis of possible poor outcomes of treatment; and assessment of the risk that the disease or its treatment may negatively affect the general health of the patient. Necrotizing periodontal diseases, whose characteristic clinical phenotype includes typical features (papilla necrosis, bleeding, and pain) and are associated with host immune response impairments, remain a distinct periodontitis category. Endodontic-periodontal lesions, defined by a pathological communication between the pulpal and periodontal tissues at a given tooth, occur in either an acute or a chronic form, and are classified according to signs and symptoms that have direct impact on their prognosis and treatment. Periodontal abscesses are defined as acute lesions characterized by localized accumulation of pus within the gingival wall of the periodontal pocket/sulcus, rapid tissue destruction and are associated with risk for systemic dissemination.

Subgingival microbial communities in Leukocyte Adhesion Deficiency and their relationship with local immunopathology.

Leukocyte Adhesion Deficiency I (LAD-I) is a primary immunodeficiency caused by single gene mutations in the CD18 subunit of ß2 integrins which result in defective transmigration of neutrophils into the tissues. Affected patients suffer from recurrent life threatening infections and severe oral disease (periodontitis). Microbial communities in the local environment (subgingival plaque) are thought to be the triggers for inflammatory periodontitis, yet little is known regarding the microbial communities associated with LAD-I periodontitis. Here we present the first comprehensive characterization of the subgingival communities in LAD-I, using a 16S rRNA gene-based microarray, and investigate the relationship of this tooth adherent microbiome to the local immunopathology of periodontitis. We show that the LAD subgingival microbiome is distinct from that of health and Localized Aggressive Periodontitits. Select periodontitis-associated species in the LAD microbiome included Parvimonas micra, Porphyromonas endodontalis, Eubacterium brachy and Treponema species. Pseudomonas aeruginosa, a bacterium not typically found in subgingival plaque is detected in LAD-I. We suggest that microbial products from LAD-associated communities may have a role in stimulating the local inflammatory response. We demonstrate that bacterial LPS translocates into the lesions of LAD-periodontitis potentially triggering immunopathology. We also show in in vitro assays with human macrophages and in vivo in animal models that microbial products from LAD-associated subgingival plaque trigger IL-23-related immune responses, which have been shown to dominate in patient lesions. In conclusion, our current study characterizes the subgingival microbial communities in LAD-periodontitis and supports their role as triggers of disease pathogenesis.

Reduction in bacteremia after brushing with a triclosan/copolymer dentifrice-A randomized clinical study.

AIM: The aims of this study were to; 1) test susceptibility to bacteremia in subjects with moderate gingivitis, and 2) compare the effects of brushing with a fluoride toothpaste (control) as compared to a triclosan/copolymer toothpaste (test) on those susceptible to repeated bacteremia. MATERIALS AND METHODS: One hundred and seven adult subjects were tested for repeated bacteremia after eating a hard apple. Twenty-nine bacteremia positive subjects were enrolled in a double-blind cross-over study designed to analyse the effects of a test toothpaste. After random toothpaste assignment, subjects brushed for 21 days. Following a wash-out period, subjects completed the study with the alternate toothpaste. Statistical analysis compared bacteremia between groups by analysis of covariance (ANCOVA). RESULTS: Twenty-six adult subjects completed the cross-over study. No statistically significant differences for bacteremia were seen at baseline. Mean bacterial counts at baseline and post-treatment visits were 45.5 and 10.8 counts versus 48.5 and 38.0 counts, respectively (test vs. control group; significant at p < .05). Significant reductions in blood borne bacteria were seen in the test versus control groups in both cultural and DNA data (p < .05). CONCLUSIONS: Thirty percentage of subjects showed repeated bacteremia. Brushing with a triclosan/copolymer dentifrice demonstrated significant reductions in bacteremia as compared to the control toothpaste.

Host-derived salivary biomarkers may be useful in diagnosing periodontal disease.

ARTICLE TITLE AND BIBLIOGRAPHIC INFORMATION: Host-derived salivary biomarkers in diagnosing periodontal disease: systematic review and meta-analysis. de Lima CL, Acevedo AC, Grisl DC, Taba M Jr, Guerra E, De Luca Canto G. J Clin Periodontol 2016; 43(6): 492-502. SOURCE OF FUNDING: Self-funded TYPE OF STUDY/DESIGN: Systematic review with meta-analysis.

Bacterial infection increases periodontal bone loss in diabetic rats through enhanced apoptosis.

Periodontal disease is the most common osteolytic disease in humans and is significantly increased by diabetes mellitus. We tested the hypothesis that bacterial infection induces bone loss in diabetic animals through a mechanism that involves enhanced apoptosis. Type II diabetic rats were inoculated with Aggregatibacter actinomycetemcomitans and treated with a caspase-3 inhibitor, ZDEVD-FMK, or vehicle alone. Apoptotic cells were measured with TUNEL; osteoblasts and bone area were measured in H&E sections. New bone formation was assessed by labeling with fluorescent dyes and by osteocalcin mRNA levels. Osteoclast number, eroded bone surface, and new bone formation were measured by tartrate-resistant acid phosphatase staining. Immunohistochemistry was performed with an antibody against tumor necrosis factor-α. Bacterial infection doubled the number of tumor necrosis factor-α-expressing cells and increased apoptotic cells adjacent to bone 10-fold (P < 0.05). Treatment with caspase inhibitor blocked apoptosis, increased the number of osteoclasts, and eroded bone surface (P < 0.05); yet, inhibition of apoptosis resulted in significantly greater net bone area because of an increase in new bone formation, osteoblast numbers, and an increase in bone coupling. Thus, bacterial infection in diabetic rats stimulates periodontitis, in part through enhanced apoptosis of osteoblastic cells that reduces osseous coupling through a caspase-3-dependent mechanism.

Nanopore film based enrichment and quantification of low abundance hepcidin from human bodily fluids.

Endogenous peptides that represent biological and pathological information of disease have attracted interest for diagnosis. However, the extraction of those low abundance peptides is still a challenge because of the complexity of human bodily fluids (HBF). Hepcidin, a peptide hormone, has been recognized as a biomarker for iron-related diseases. There is no rapid and reliable way to enrich them from HBF. Here we describe a peptide extraction approach based on nanoporous silica thin films to successfully detect hepcidin from HBF. Cooperative functions of nanopore to biomolecule, including capillary adsorption, size-exclusion and electrostatic interaction, were systematically investigated to immobilize the target peptide. To promote this new approach to clinical practices, we further applied it to successfully assay the hepcidin levels in HBF provided by healthy volunteers and patients suffering from inflammation. Our finding provides a high-throughput, rapid, label-free and cost-effective detection method for capturing and quantifying low abundance peptides from HBF. FROM THE CLINICAL EDITOR: Diagnosing diseases with low concentration peptide biomarkers remains challenging. This team of authors describes a peptide extraction approach based on nanoporous silica thin films to successfully detect low concentrations of hepcidin from human body fluids collected from 119 healthy volunteers and 19 inflammation patients.

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.

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.

The Widespread Colonization Island of Actinobacillus actinomycetemcomitans.

Genomic islands, such as pathogenicity islands, contribute to the evolution and diversification of microbial life. Here we report on the Widespread Colonization Island, which encompasses the tad (tight adherence) locus for colonization of surfaces and biofilm formation by the human pathogen Actinobacillus actinomycetemcomitans. At least 12 of the 14 genes at the tad locus are required for tenacious biofilm formation and synthesis of bundled Flp pili (fibrils) that mediate adherence. The pilin subunit, Flp1, remains inside the cell in tad-locus mutants, indicating that these genes encode a secretion system for export and assembly of fibrils. We found tad-related regions in a wide variety of Bacterial and Archaeal species, and their sequence characteristics indicate possible horizontal transfer. To test the hypothesis of horizontal transfer, we compared the phylogeny of the tad locus to a robust organismal phylogeny using statistical tests of congruence and tree reconciliation techniques. Our analysis strongly supports a complex history of gene shuffling by recombination and multiple horizontal transfers, duplications and losses. We present evidence for a specific horizontal transfer event leading to the establishment of this region as a determinant of disease.

Oral microbial interactions from an ecological perspective: a narrative review.

Landscape ecology is a relatively new field of study within the sub-specialty of ecology that considers time and space in addition to structure and function. Landscape ecology contends that both the configuration (spatial pattern) and the composition (organisms both at the macro and or micro level) of an ecology can change over time. The oral cavity is an ideal place to study landscape ecology because of the variety of landscapes, the dynamic nature of plaque biofilm development, and the easy access to biofilm material. This review is intended to provide some specific clinical examples of how landscape ecology can influence the understanding of oral diseases and act as a supplement to diagnosis and treatment. The purpose of this review is two-fold; (1) to illustrate how landscape ecology can be used to clarify the two most prominent microbiologically induced infections in the oral cavity, and (2) how studies of oral microbiology can be used to enhance the understanding of landscape ecology. The review will distinguish between "habitat" and "niche" in a landscape and extend the concept that a "patch", is the demarcating unit of a habitat within a landscape. The review will describe how; (1) an individual patch, defined by its shape, edges and internal components can have an influence on species within the patch, (2) spatial dynamics over time within a patch can lead to variations or diversities of species within that patch space, and (3) an unwelcoming environment can promote species extinction or departure/dispersion into a more favorable habitat. Understanding this dynamic in relationship to caries and periodontal disease is the focus of this review.

Oral Epithelial Cells Expressing Low or Undetectable Levels of Human Angiotensin-Converting Enzyme 2 Are Susceptible to SARS-CoV-2 Virus Infection In Vitro.

The oral cavity is thought to be one of the portals for SARS-CoV-2 entry, although there is limited evidence of active oral infection by SARS-CoV-2 viruses. We assessed the capacity of SARS-CoV-2 to infect and replicate in oral epithelial cells. Oral gingival epithelial cells (hTERT TIGKs), salivary gland epithelial cells (A-253), and oral buccal epithelial cells (TR146), which occupy different regions of the oral cavity, were challenged with replication-competent SARS-CoV-2 viruses and with pseudo-typed viruses expressing SARS-CoV-2 spike proteins. All oral epithelial cells expressing undetectable or low levels of human angiotensin-converting enzyme 2 (hACE2) but high levels of the alternative receptor CD147 were susceptible to SARS-CoV-2 infection. Distinct viral dynamics were seen in hTERT TIGKs compared to A-253 and TR146 cells. For example, levels of viral transcripts were sustained in hTERT TIGKs but were significantly decreased in A-253 and TR146 cells on day 3 after infection. Analysis of oral epithelial cells infected by replication-competent SARS-CoV-2 viruses expressing GFP showed that the GFP signal and SARS-CoV-2 mRNAs were not evenly distributed. Furthermore, we found cumulative SARS-CoV-2 RNAs from released viruses in the media from oral epithelial cells on day 1 and day 2 after infection, indicating productive viral infection. Taken together, our results demonstrated that oral epithelial cells were susceptible to SARS-CoV-2 viruses despite low or undetectable levels of hACE2, suggesting that alternative receptors contribute to SARS-CoV-2 infection and may be considered for the development of future vaccines and therapeutics.

Pragmatic Return to Effective Dental Infection Control through Triage and Testing (PREDICT): A feasibility study to improve dental office safety.

Background: The COVID-19 pandemic highlights the need for practitioners to enhance workflows to increase safety and mitigate risk. As dental practice creates a highly aerosolized environment, pre-visit testing for SARS-CoV-2 has the potential to be an effective mitigation strategy to minimize disease transmission in dental offices. The Pragmatic Return to Effective Dental Infection Control through Testing (PREDICT) Feasibility Study examined the potential, logistics, and impact related to laboratory-based PCR viral testing and point-of-care (POC) antigen testing. Methods: Dental healthcare workers (DHCWs) and patients in four dental offices within the National Dental Practice-based Research Network participated in this prospective study. In addition to electronic surveys, participants in two offices completed POC testing, while participants in two offices used lab based PCR methods to detect SARS-CoV-2 infection. For this feasibility study, analysis was limited to descriptive measures. Median and interquartile ranges were reported for Likert scale responses and mean and standard deviation for continuous variables. Results: Forty-one of forty-three consented patients and twenty-eight of twenty-nine DHCWs completed the protocol. Descriptive statistics calculations including median and interquartile ranges revealed (1) saliva, tongue epithelial cells and nasal swabs were the most desirable specimens for testing for groups (2) both LAB and POC protocols took similar amounts of total time to complete the full testing protocol and (3) DHCWs and patients reported feeling more comfortable when both groups were tested. Conclusions: This feasibility study suggests that pre-visit SARS-CoV-2 testing can be effectively implemented into dental practice workflows and positively impact perception of safety for DHCWs and patients, though a larger scale, network study is necessary for generalizability of results. As new virulent infectious diseases continue to emerge, preparing dental personnel to employ an entire toolbox of risk mitigation strategies, including testing, may have the potential to decrease dental practice closure time, maintaining continuity of dental care services for patients. Trial registration: This trial was registered on ClinicalTrials.gov: NCT05123742.

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 infection enhances apoptosis in vivo through a caspase-3-dependent mechanism in experimental periodontitis.

The purpose of this study was to test the hypothesis that diabetes aggravates periodontal destruction induced by Aggregatibacter actinomycetemcomitans infection. Thirty-eight diabetic and 33 normal rats were inoculated with A. actinomycetemcomitans and euthanized at baseline and at 4, 5, and 6 weeks after inoculation. Bone loss and the infiltration of polymorphonuclear leukocytes (PMNs) in gingival epithelium were measured in hematoxylin-eosin-stained sections. The induction of tumor necrosis factor alpha (TNF-α) was evaluated by immunohistochemistry and of apoptotic cells by a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay. After A. actinomycetemcomitans infection, the bone loss in diabetic rats was 1.7-fold and the PMN infiltration 1.6-fold higher than in normoglycemic rats (P < 0.05). The induction of TNF-α was 1.5-fold higher and of apoptotic cells was up to 3-fold higher in diabetic versus normoglycemic rats (P < 0.05). Treatment with a caspase-3 inhibitor significantly blocked noninflammatory cell apoptosis induced by A. actinomycetemcomitans infection in gingival epithelium and connective tissue (P < 0.05). These results provide new insight into how diabetes aggravates A. actinomycetemcomitans-induced periodontal destruction in rats by significantly increasing the inflammatory response, leading to increased bone loss and enhancing apoptosis of gingival epithelial and connective tissue cells through a caspase-3-dependent mechanism. Antibiotics had a more pronounced effect on many of these parameters in diabetic than in normoglycemic rats, suggesting a deficiency in the capacity of diabetic animals to resist infection.

In vitro efficacy of diallyl sulfides against the periodontopathogen Aggregatibacter actinomycetemcomitans.

The in vitro antibacterial effects of diallyl sulfide (DAS) against the Gram-negative periodontopathogen Aggregatibacter actinomycetemcomitans, the key etiologic agent of the severe form of localized aggressive periodontitis and other nonoral infections, were studied. A. actinomycetemcomitans was treated with garlic extract, allicin, or DAS, and the anti-A. actinomycetemcomitans effects of the treatment were evaluated. Garlic extract, allicin, and DAS significantly inhibited the growth of A. actinomycetemcomitans (greater than 3 log; P < 0.01) compared to control cells. Heat inactivation of the garlic extracts significantly reduced the protein concentration; however, the antimicrobial effect was retained. Purified proteins from garlic extract did not exhibit antimicrobial activity. Allicin lost all its antimicrobial effect when it was subjected to heat treatment, whereas DAS demonstrated an antimicrobial effect similar to that of the garlic extract, suggesting that the antimicrobial activity of garlic extract is mainly due to DAS. An A. actinomycetemcomitans biofilm-killing assay performed with DAS showed a significant reduction in biofilm cell numbers, as evidenced by both confocal microscopy and culture. Scanning electron microscopy (SEM) analysis of DAS-treated A. actinomycetemcomitans biofilms showed alterations of colony architecture indicating severe stress. Flow cytometry analysis of OBA9 cells did not demonstrate apoptosis or cell cycle arrest at therapeutic concentrations of DAS (0.01 and 0.1 µg/ml). DAS-treated A. actinomycetemcomitans cells demonstrated complete inhibition of glutathione (GSH) S-transferase (GST) activity. However, OBA9 cells, when exposed to DAS at similar concentrations, showed no significant differences in GST activity, suggesting that DAS-induced GST inhibition might be involved in A. actinomycetemcomitans cell death. These findings demonstrate that DAS exhibits significant antibacterial activity against A. actinomycetemcomitans and that this property might be utilized for exploring its therapeutic potential in treatment of A. actinomycetemcomitans-associated oral and nonoral infections.

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.