Identification of HACEK clinical isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid and accurate tool for the identification of many microorganisms. We assessed this technology for the identification of 103 Haemophilus parainfluenzae, Aggregatibacter aphrophilus, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae (HACEK) clinical isolates and 20 Haemophilus influenzae clinical isolates. Ninety-three percent of HACEK organisms were identified correctly to the genus level using the Bruker database, and 100% were identified to the genus level using a custom database that included clinical isolates.

Soluble products from Eikenella corrodens stimulate oral epithelial cells to induce inflammatory mediators.

In the inflammatory response elicited by bacterial colonization in periodontal pockets, pocket epithelial cells not only serve as a barrier to isolate the pocket microenvironment from external stimuli but also regulate the functions of neighboring cells including fibroblasts and inflammatory cells. To elucidate this mechanism, we characterized the effects of periodontopathic bacterium Eikenella corrodens 1073 components on the production of some inflammatory mediators in a human oral epithelial cell line (KB). In enzyme-linked immunosorbent assay (ELISA), the E. corrodens supernatant induced interleukin-6 (IL-6), IL-8 and prostaglandin E2 but not interferon-gamma (IFN-gamma) production by KB cells. After incubation with E. corrodens supernatant, KB cells showed a marked increase in the levels of IL-6, IL-8 and PG G/H synthase (cyclooxygenase)-2, but not IFN-gamma, gene expression by reverse-transcriptase polymerase chain reaction. All these E. corrodens products responsible for production of these inflammatory mediators resisted freezing and boiling and were present in a 10-kDa filtrate. These results imply that these soluble small-molecular-mass products from E. corrodens stimulate various inflammatory mediator productions by human oral epithelial cells and may play a role in the initiation of periodontal inflammation and subsequently perpetuate the inflammatory response during chronic infection.

Purification and characterization of Eikenella corrodens type IV pilin.

Eikenella corrodens is a gram-negative human pathogen associated with periodontal diseases and soft-tissue infections. Pilin was purified by association-dissociation and fast protein liquid chromatography; it had an apparent molecular mass of about 14.8 kDa and an N-terminal amino acid sequence reflective of type IV pilins. Antibodies to the purified protein reacted with pili on whole cells. This is the first report of purification of type IV pili/pilin from this organism. Other type IV pili are important virulence factors; we are currently investigating the biological role of pili in E. corrodens.

Comparison of the osteolytic activity of surface-associated proteins of bacteria implicated in periodontal disease.

OBJECTIVES: To compare the osteolytic activity of surface-associated material (SAM) and lipid A-associated proteins (LAPs) from periodontopathogenic bacteria. MATERIALS AND METHODS: Surface-associated material was extracted from the surface and LAPs from the cell walls of a range of periodontopathic bacteria including Actinobacillus actinomycetemcomitans and Eikenella corrodens. These bacterial fractions were assayed to determine their composition and their capacity to induce bone resorption was determined by use of the neonatal murine calvarial bone resorption assay. RESULTS: The SAMs from E. corrodens and A. actinomycetemcomitans demonstrated bone-resorbing capacity at concentrations as low as 1 ng ml-1 which, given the molecular weights of the active components, is in the picomolar range of activity. In contrast, the SAMs from the other three bacteria were significantly less potent and showed a lower efficacy. The LAPs all showed significant, and similar, capacities to induce bone breakdown. CONCLUSIONS: This is the first demonstration that LAP from periodontopathic bacteria can stimulate bone degradation. The LAPs from diverse bacteria all produced similar levels of bone-resorbing activity. In contrast, the SAM showed significant differences in potency and in efficacy (maximal stimulation). This may mean that in vivo certain periodontopathic bacteria have significantly more bone-resorbing capacity than others and should be therapeutic targets.

Bone resorbing activity of surface-associated material from Actinobacillus actinomycetemcomitans and Eikenella corrodens.

The results of this study demonstrate that saline extracted surface-associated material (SAM) of Actinobacillus actinomycetemcomitans and Eikenella corrodens stimulates bone resorption at picomolar concentrations. Various inhibitors of known osteolytic mediators--indomethacin, interleukin-1 receptor antagonist (IL-1ra) and a neutralising antibody to murine tumour necrosis factor (TNF) alpha--were tested to determine the mechanism of action of these SAMs. Bone resorption induced by SAM from E. corrodens was slightly inhibited by indomethacin and almost completely inhibited by blocking the action of TNF alpha; that from A. actinomycetemcomitans was not significantly affected by either of these inhibitors.

Production and characterization of monoclonal antibodies against bacterial lectin of Eikenella corrodens.

A lectin-like substance (EcLS) was purified from the Eikenella corrodens 1073 cell and monoclonal antibodies were produced against it to confirm the role of EcLS in adhesive properties of E. corrodens such as hemagglutination and coaggregation with oral bacteria. Four hybridoma clones were selected. Two of the antibodies were of the IgG1 isotype and the others were of the IgG2b isotype. These monoclonal antibodies inhibited both the hemagglutination of E. corrodens and the coaggregation with Actinomyces viscosus or Streptococcus sanguis. The reactivity of the monoclonal antibody to E. corrodens 1073 was significantly higher than that to E. corrodens 1080 of which adhesive activity was weaker than that of E. corrodens 1073. These findings suggest the difference in adhesive properties is due to the difference in the amount of EcLS expressed on the cell surface. The immunoelectron microscopic study revealed that EcLS of E. corrodens 1073 was localized in the outer space of outer membrane, not in cell surface appendages such as fimbriae where bacteria possessed adhesin. These results suggest that coaggregation of E. corrodens with A. viscosus or S. sanguis was mediated by EcLS.

The mechanisms of Eikenella corrodens aggregation by salivary glycoprotein and the effect of the glycoprotein on oral bacterial aggregation.

The mechanism of aggregation of Eikenella corrodens 1073 with E. corrodens aggregating factor (EcAF) which was purified from submandibular-sublingual (SM-SL) saliva was investigated. Heating (100 degrees C, 10 minutes) or protease treatment of E. corrodens cells abolished the aggregating activity. The aggregation was inhibited by adding N-acetyl-D-galactosamine (GalNAc) and saccharides which contain a galactose residue at the non-reducing end. The aggregating activity was sensitive to EDTA and was restored by Ca2+ but not by Mn2+ or Mg2+. Neuraminidase treatment of EcAF increased their ability to aggregate. E. corrodens, suggesting that the sialic acids on EcAF interfere with aggregation. These data suggest that the aggregation of E. corrodens 1073 with EcAF is mediated by specific interactions between a bacterial cell surface lectin-like substance and a complementary GalNAc-like receptor. EcAF also aggregated 16 strains of oral bacteria including periodontopathic bacteria such as Porphyromonas (Bacteroides) gingivalis 381 and Actinobacillus actinomycetemcomitans ATCC29522; however, those aggregations were not inhibited by GalNAc. Therefore, EcAF appears to have more than two types of bacterial binding site and plays important roles in accumulation of dental plaque by forming a complex network of plaque bacteria including periodontopathic strains.

Inhibition of bone DNA and collagen production by surface-associated material from bacteria implicated in the pathology of periodontal disease.

Gentle extraction of oral bacteria implicated in the pathogenesis of periodontal disease, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, or Eikenella corrodens, with saline removes the extracellular components while leaving the bacteria intact. This readily-solubilized surface-associated material (SAM) has been demonstrated to significantly inhibit DNA and collagen synthesis by murine calvaria at concentrations as low as 10 ng/ml. DNA and collagen synthesis in isolated calvarial osteoblasts were also inhibited by these SAM preparations with similar dose responses. The inhibitory effect of these bacterial expolymers was blocked by 1 microM indomethacin. The potent inhibitory actions on bone synthesis of the SAM from these bacteria may contribute to the alveolar bone loss found in patients with periodontal disease.