Keiko Tsuruda

Antibacterial effect of silver-zeolite on oral bacteria under anaerobic conditions

OBJECTIVES The purpose of this study was to evaluate the antibacterial effect of silver-zeolite (SZ) against oral bacteria under anaerobic conditions. METHODS The antibacterial activity of SZ was evaluated by determining the minimum inhibitory concentrations (MICs) using two-fold serial dilutions of SZ in Brain Heart Infusion broth. Release of Ag+ into the broth was measured by an atomic absorption technique. RESULTS SZ inhibited the growth of the bacteria tested under anaerobic conditions. The MIC of SZ ranged between 256 and 2048 micrograms/ml, which corresponded to a range of 4.8-38.4 micrograms/ml of Ag+. All strains grew in broth containing 16,384 micrograms/ml of type-A zeolite. SIGNIFICANCE These results suggested that SZ may be a useful vehicle to provide antibacterial activity to dental materials used even under anaerobic conditions such as deep in the periodontal pocket.

Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans Induces DNA Damage, S/G2 Cell Cycle Arrest, and Caspase- Independent Death in a Saccharomyces cerevisiae Model

ABSTRACT Cytolethal distending toxin (CDT) is a bacterial toxin that induces G2/M cell cycle arrest, cell distension, and/or apoptosis in mammalian cells. It is produced by several Gram-negative species and may contribute to their pathogenicity. The catalytic subunit CdtB has homology with DNase I and may act as a genotoxin. However, the mechanism by which CdtB leads to cell death is not yet clearly understood. Here, we used Saccharomyces cerevisiae as a model to study the molecular pathways involved in the function of CdtB from Aggregatibacter actinomycetemcomitans, a cause of aggressive periodontitis. We show that A.actinomycetemcomitans CdtB (AaCdtB) expression induces S/G2 arrest and death in a DNase-catalytic residue and nuclear localization-dependent manner in haploid yeasts. Yeast strains defective in homologous recombination (HR) repair, but not other DNA repair pathways, are hypersensitive to AaCdtB, suggesting that HR is required for survival upon CdtB expression. In addition, yeast does not harbor the substrate for the other activity proposed for CdtB function, which is phosphatidylinositol-3,4,5-triphosphate phosphatase. Thus, these results suggest that direct DNA-damaging activity alone is sufficient for CdtB toxicity. To investigate how CdtB induces cell death, we examined the effect of CdtB in yeast strains with mutations in apoptotic regulators. Our results suggest that yeast death occurs independently of the yeast metacaspase gene YCA1 and the apoptosis-inducing factor AIF1 but is partially dependent on histone H2B serine 10 phosphorylation. Therefore, we report here the evidence that AaCdtB causes DNA damage that leads to nonapoptotic death in yeast and the first mutation that confers resistance to CdtB.

Topical application of Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces cell cycle arrest in the rat gingival epithelium in vivo

BACKGROUND Aggregatibacter actinomycetemcomitans is one of the etiological pathogens implicated in the onset of periodontal disease. This pathogen produces cytolethal distending toxin (CDT) that acts as a genotoxin to induce cell cycle arrest and cellular distension in cultured cell lines. Therefore, CDT is a possible virulence factor; however, the in vivo activity of CDT on periodontal tissue has not been explored. Here, CDT was topically applied into the rat molar gingival sulcus; and the periodontal tissue was histologically and immunohistochemically examined. MATERIALS AND METHODS Recombinant purified A. actinomycetemcomitans CDT was applied to gingival sulcus of male Wistar rats and tissue samples were immunohistochemmically examined. RESULTS One day after application, infiltration of neutrophils and dilation of blood vessels in the gingival connective tissue were found. At day three, desquamation and detachment of cells in the junctional epithelium was observed. This abrasion of junctional epithelium was not observed in rats treated with mutated CDT, in which a His274Ala mutation is present in the CdtB subunit. This indicates the tissue abrasion may be caused by the genotoxicity of CdtB. Expression of the proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, was significantly suppressed using CDT treatment in the junctional epithelium and gingival epithelium. CONCLUSION Using the rat model, these data suggest CDT intoxication induces cell cycle arrest and damage in periodontal epithelial cells in vivo.

Clonal distribution of enterotoxigenic Staphylococcus aureus on handles of handheld shopping baskets in supermarkets

Aims:  Shopping carts and handheld shopping baskets in supermarkets are subject to accidental bacterial contamination through contacts with a variety of food. We investigated the prevalence of Staphylococcus aureus on the handles of handheld shopping baskets in four supermarkets distantly located in Osaka district, Japan.

Effects of fluoride intake on the mineral content, acid solubility and resorption caused by experimental periodontitis of rat alveolar bone

Adult rats were given either distilled water or drinking water containing 100 parts/10(6) of fluoride. The alveolar bone of rats given fluoride for 90 days showed an increased mineral content and decreased acid solubility compared to the bone of rats given distilled water. Experimental periodontitis was initiated in both groups after 110 days of treatment to cause alveolar bone resorption. Fourteen days later, the rats were killed and it was found that the alveolar bone resorption caused by experimental periodontitis was significantly smaller in the rats given fluoride in their drinking water than in those given distilled water. The findings suggest that fluoride intake might have a protective effect on rapidly progressing alveolar bone resorption.

CdtC-induced processing of membrane-bound CdtA is a crucial step in Aggregatibacter actinomycetemcomitans CDT holotoxin formation

ABSTRACT Aggregatibacter actinomycetemcomitans is an oral pathogen causing periodontal disease and bacterial endocarditis. It produces cytolethal distending toxin (CDT) that could damage mammalian cells and tissues. CDT is a tripartite protein toxin composed of CdtA, CdtB, and CdtC. We have previously indicated that CdtA is a lipoprotein and that the proteolytic processing of CdtA is important for biogenesis and secretion of CDT holotoxin. Here, we established an in vitro processing assay of CdtA and investigated the interactions of CdtA with other Cdt subunits. This assay demonstrated that incubation of membrane-bound CdtA (MCdtA), CdtB, and CdtC immediately generated a processed form of CdtA (CdtA′), which is recovered from the soluble fraction. In contrast, incubation of soluble membrane-unbound CdtA with CdtB and CdtC did not yield any CdtA′. Furthermore, incubation of CdtC with MCdtA was enough to induce rapid processing of MCdtA, whereas CdtB alone was unable to induce the processing. Coimmunoprecipitation demonstrated that CdtA′ and CdtC formed a complex. Furthermore, subsequent addition of CdtB to this reaction mixture resulted in complete CDT holotoxin complex. The cytolethal distending activity assay demonstrated that CDT complex containing CdtA′ showed far stronger cytotoxicity than that containing CdtA. Collectively, our data suggest that CDT holotoxin formation in vivo is a sequential event: interaction of MCdtA and CdtC induces proteolytic processing of MCdtA, and the released CdtA′ forms a complex with CdtC. Subsequent binding of CdtB to the CdtA′/CdtC complex results in CDT holotoxin formation.