Daniel H. Fine

Aggregatibacter actinomycetemcomitans and Its Relationship to Initiation of Localized Aggressive Periodontitis : Longitudinal Cohort Study of Initially Healthy Adolescents

ABSTRACT 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.

flp-1, the first representative of a new pilin gene subfamily, is required for non-specific adherence of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans, a Gram‐negative bacterium responsible for localized juvenile periodontitis and other infections such as endocarditis, produces long fibrils of bundled pili that are believed to mediate non‐specific, tenacious adherence to surfaces. Previous investigations have implicated an abundant, small (≈ 6.5 kDa), fibril‐associated protein (Flp/Fap) as the primary fibril subunit. Here, we report studies on fibril structure and on the function and evolution of Flp. High‐resolution electron microscopy of adherent clinical strain CU1000N revealed long bundles of 5‐ to 7‐nm‐diameter pili, whose subunits appear to be arranged in a helical array similar to that observed for type IV pili in other bacteria. Fibrils were found to be associated with the bacterial cell surface and smaller structures thought to be membrane vesicles. A modified version of the CU1000N Flp1 polypeptide with the T7‐TAG epitope fused to its C‐terminus was expressed in the wild‐type strain, and the presence of the modified Flp1 in fibrils was confirmed by immunogold electron microscopy with monoclonal antibody to T7‐TAG. To determine the importance of Flp1 in fibril formation and cell adherence, we used transposon IS903φkan to isolate insertion mutations in the flp‐1 gene (formerly designated flp). Mutants with insertions early in flp‐1 fail to produce fibrils and do not adhere to surfaces. Both fibril production and adherence were restored by cloned flp‐1 in trans, thus providing the first evidence that flp‐1 is required for fibril formation and tight, non‐specific adherence. One mutant was found to have an insertion near the 3′ end of flp‐1 that results in the expression of a truncated and altered C‐terminus of Flp1. This mutant produced short, unbundled pili, and its adherence to surfaces was significantly less than that of wild‐type bacteria. These findings and related observations with the Flp1‐T7‐TAG protein indicate that the C‐terminus of Flp1 is important for the bundling and adherence properties of pili. Extensive sequence comparisons and phylogenetic analysis of 61 predicted prepilin genes of bacteria revealed flp‐1 to be a member of a novel and widespread subfamily of type IV prepilin genes. Thus, Flp pili are likely to be expressed by diverse bacterial species. Furthermore, we found that it is common for bacterial genomes to contain multiple alleles of flp‐like genes, including the open reading frame (flp‐2, previously designated orfA) immediately downstream of flp‐1 in A. actinomycetemcomitans. The duplication and divergence of flp genes in bacteria may be important to the diversification of the colonization properties of these organisms.

Tight-adherence genes of Actinobacillus actinomycetemcomitans are required for virulence in a rat model

Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that has been associated with localized aggressive periodontitis and infections of the heart, brain, and urinary tract. Wild-type clinical isolates have the remarkable ability to adhere tenaciously and nonspecifically to solid surfaces such as glass, plastic, and hydroxyapatite. Adherence by A. actinomycetemcomitans is mediated by the tight-adherence (tad) gene locus, which consists of 14 genes (flp-1–flp-2–tadV–rcpCAB–tadZABCDEFG). All but 2 of the genes have been shown to be required for the secretion and assembly of long, bundled Flp1 fibrils. To test whether the tad locus is required for colonization and disease, we developed a rat model for periodontal disease. To mimic the natural route of infection, Sprague–Dawley rats were inoculated orally by adding bacteria directly to their food for 8 days. After inoculation with wild-type or mutant strains defective in adherence (flp-1 and tadA), the rats were assessed for colonization of the oral cavity and pathogenesis. Wild-type A. actinomycetemcomitans was able to colonize and persist for at least 12 weeks in the oral cavity, elicit a humoral immune response, and cause significant bone loss in rats. In contrast, rats fed flp-1 or tadA mutant strains showed no bone loss and their immune responses were indistinguishable from those of the uninoculated controls. These results demonstrate the critical importance of the tad locus in the colonization and pathogenesis of A. actinomycetemcomitans.

Biofilm Growth and Detachment of Actinobacillus actinomycetemcomitans

The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that A. actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony.

The Actinobacillus actinomycetemcomitans autotransporter adhesin aae exhibits specificity for buccal epithelial cells from humans and old world primates

ABSTRACT Cells of the gram-negative periodontopathogen Actinobacillus actinomycetemcomitans express a surface-exposed, outer membrane autotransporter protein, designated Aae, which has been implicated in epithelial cell binding. We constructed a mutant strain of A. actinomycetemcomitans that contained a transposon insertion in the Aae structural gene (aae) and tested the mutant to determine its ability to bind to buccal epithelial cells (BECs) isolated from healthy volunteers. Significantly fewer mutant cells than wild-type cells bound to BECs. A broad-host-range plasmid that contained an intact aae gene driven by a heterologous tac promoter restored the ability of the mutant strain to bind to BECs at wild-type levels. This plasmid also conferred upon Escherichia coli the ability to express the Aae protein on its surface and to bind to human BECs. Aae-expressing E. coli also bound to BECs isolated from six Old World primates but not to BECs isolated from four New World primates or nine other nonprimate mammals, as well as to human gingival epithelial cells but not to human pharyngeal, palatal, tongue, bronchial, or cervical epithelial cells. Our findings indicate that Aae mediates binding of A. actinomycetemcomitans to BECs from humans and Old World primates and that this process may contribute to the host range specificity and tissue tropism exhibited by this bacterium.

Adaptive immune response in osteoclastic bone resorption induced by orally administered Aggregatibacter actinomycetemcomitans in a rat model of periodontal disease

There is mounting evidence that innate and adaptive immunity are critical for periodontal disease-mediated bone resorption. These studies examined the role of B and CD4 T cells in adaptive immunity of rats infected with Aggregatibacter actinomycetemcomitans (Aa). Sprague-Dawley male rats were fed Aa-containing mash or control-mash for 2 weeks. B and CD4 T cells were obtained from draining lymph nodes at 2, 4 and 12 weeks, postinoculation. Quantitative polymerase chain reaction-based messenger RNA expression was conducted for 89 cytokine family genes. Disease-relevance of the differentially expressed genes was assessed using a biological interaction pathway analysis software. B and CD4 T cells of Aa-infected rats increased and were activated, resulting in enhanced isotype-switched serum immunoglobulin G by 2 weeks postinoculation. Bone resorption was evident 12 weeks after Aa-feeding. In B cells, interleukin-2 (IL-2), macrophage-inhibiting factor, IL-19, IL-21, tumor necrosis factor (TNF), CD40 ligand (CD40L), CD70, bone morphogenetic protein 2 (BMP2), BMP3, and BMP10 were upregulated early; while IL-7, Fas ligand (FasL), small inducible cytokine subfamily E1, and growth differentiation factor 11 (GDF11; BMP11) were upregulated late (12 weeks). BMP10 was sustained throughout. In CD4 T cells, IL-10, IL-16, TNF, lymphotoxin-beta (LTbeta), APRIL, CD40L, FasL, RANKL and osteoprotegerin were upregulated early, whereas IL-1beta, IL-1RN, IL-1F8, IL-24, interferon-alpha1, GDF11 (BMP11), and GDF15 were upregulated late (12 weeks). Adaptive immunity appears crucial for bone resorption. Several of the deregulated genes are, for the first time, shown to be associated with bone resorption, and the results indicate that activated B cells produce BMP10. The study provides a rationale for a link between periodontal disease and other systemic diseases.

Complete Genome Sequence of Aggregatibacter (Haemophilus) aphrophilus NJ8700

We report the finished and annotated genome sequence of Aggregatibacter aphrophilus strain NJ8700, a strain isolated from the oral flora of a healthy individual, and discuss characteristics that may affect its dual roles in human health and disease. This strain has a rough appearance, and its genome contains genes encoding a type VI secretion system and several factors that may participate in host colonization.

Aggregatibacter actinomycetemcomitans as an Early Colonizer of Oral Tissues: Epithelium as a Reservoir?

ABSTRACT 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.

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 β2 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.

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.