Taketo Kawarai

Inhibition of Streptococcus mutans Biofilm Formation by Streptococcus salivarius FruA

The oral microbial flora consists of many beneficial species of bacteria that are associated with the healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multi-species biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting S. mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion-exchange and gel filtration chromatography. MALDI-TOF mass spectrometry analysis was performed and the results were compared to data bases. The S. salivarius HT9R genome sequence was determined; and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-D-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose; and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. The inhibition was induced by decreasing polysaccharide production dependent on sucrose digestion rather than fructan digestion. The data indicate S. salivarius produces large quantities of FruA; and FruA alone may play an important role in multi-species microbial interactions for sucrose-dependent biofilm formation in the oral cavity.ABSTRACT The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.

Effect of high pressure gaseous carbon dioxide on the germination of bacterial spores

Effect of high pressure gaseous carbon dioxide treatment (HGCT) at 6.5 MPa, 35 degrees C on the germination of bacterial spores was investigated. Germination of bacterial spores was estimated by the decrease of heat tolerance. Approximately, 40% of Bacillus coagulans and 70% of Bacillus licheniformis were germinated by HGCT for 120 min at 35 degrees C, respectively. Germination was confirmed by phase contrast microscopy. The effect of hydrostatic pressure treatment (HPT) at 6.5 MPa, 35 degrees C on the germination of B. coagulans and B. licheniformis spores were also investigated. Spores did not germinate by HPT alone at 6.5 MPa for 120 min.

Biofilm formation by Escherichia coli in hypertonic sucrose media

High osmotic environments produced by NaCl or sucrose have been used as reliable and traditional methods of food preservation. We tested, Escherichia coli as an indicator of food-contaminating bacterium, to determine if it can form biofilm in a hyperosmotic environment. E. coli K-12 IAM1264 did not form biofilm in LB broth that contained 1 M NaCl. However, the bacterium formed biofilm in LB broth that contained 1 M sucrose, although the planktonic growth was greatly suppressed. The biofilm, formed on solid surfaces, such as titer-plate well walls and glass slides, solely around the air-liquid interface. Both biofilm forming cells and planktonic cells in the hypertonic medium adopted a characteristic, fat and filamentous morphology with no FtsZ rings, which are a prerequisite for septum formation. Biofilm forming cells were found to be alive based on propidium iodide staining. The presence of 1 M sucrose in the food environment is not sufficient to prevent biofilm formation by E. coli.

E2F-1-Deficient NOD/SCID Mice Developed Showing Decreased Saliva Production

The non-obese diabetic mouse (NOD) is the most characterized model used to study insulin-dependent type 1 diabetes mellitus (IDDM) and Sjoögren’s syndrome (SS). In a previous report, we found NOD.E2f1 −/− mice show a greater progressive development to IDDM and SS compared to NOD mice. Our previous data indicated a progressive decrease in regulatory T cells (CD4+CD25+) and a decrease in the systemic secretion systems for insulin, and saliva was associated with the progression of IDDM and SS. Therefore, to define the mechanism of early-onset IDDM SS in E2F-1 deficient NOD mice required further investigation by producing E2F-1 deficient NOD/SCID mice in which the T and B cells do not develop. The purpose here was to analyze the essential function of the E2F-1 molecule in the development of IDDM and SS; and the dysfunction of the pancreas islet and salivary gland in the NOD background using NOD/SCID mice. We produced NOD/SCID.E2f1 −/− mice using homologous recombination; determined diabetes development; measured saliva and insulin production; and performed a histological analysis. The deficient mice showed a decreasing volume of saliva; no infiltration of lymphocytes into salivary glands; no development of diabetes; and no protein localization of FGFR-2b in the ducts of the salivary gland that regulates submandibular gland proliferation and morphogenesis. Therefore, we considered a deficiency in E2F-1 induces a decrease in regulatory T cells and an increase in auto-reactive T cells; however, the E2F-1 deficiency is not associated with T and B cells-independent dysfunction of pancreatic β cell in insulin secretion. Further, the E2F-1 deficiency is associated with T and B cells-independent dysfunction of the salivary gland exhibits a decrease in saliva production volume. We suggest E2F-1 may be also associated with the differentiation of exocrine cells in the duct where FGFR-2b is expressed in the salivary gland. The E2F-1 deficient NOD/SCID mouse model is useful for showing the development of the salivary gland; and is also useful for various experiments in humanized mice.

Effect of skimmed milk and its fractions on the inactivation of Escherichia coli K12 by high hydrostatic pressure treatment

We investigated the effects of skimmed milk and its protein fractions (casein, whey, globulin, and albumin) on the injury and inactivation of Escherichia coli K-12 by high hydrostatic pressure (HHP) treatment. The protective effect of skimmed milk on HHP-mediated inactivation and injury of E. coli increased with increases in the skimmed milk concentration. However, protein fractions derived from skimmed milk did not exhibit this protective effect. Microscopy analysis by DAPI/PI staining indicated that some cells were localized in the solid portion of skimmed milk, and some of these cells were alive. The coagulated fraction derived from the autoclaved whey fraction also showed a significant protective effect. We speculate that the solid portion in skimmed milk could provide the protective effect to bacterial cells.

Cytoplasmic acidification may occur in high-pressure carbon dioxide-treated Escherichia coli K12.

While studying the mechanism by which high-pressure carbon dioxide treatment (HCT) inactivates bacteria, we found that the efficiency of DNA recovery via phenol extraction was extraordinarily low from E. coli K12 cells that had been subjected to HCT. DAPI staining of the treated cells, however, revealed that nuclear DNA was present. Most DNA from the cells subjected to HCT was probably caught in the denatured protein layer during phenol extraction. The efficiency of DNA recovery from proteinase-treated crude extracts from cells subjected to HCT was high. Crude extracts of E. coli K12 cells that had not undergone HCT were intentionally acidified with acetic acid to pH 5.2 to cause acidic coagulation of cytoplasmic proteins. The efficiency of DNA recovery from the acidified extracts was low. These results suggest that in cells subjected to HCT, cytoplasmic pH is reduced to around pH 5.2, and that nuclear DNA becomes entangled in coagulated cytoplasmic proteins. Acidification of the cytoplasm might be the primary mechanism by which HCT inactivates bacteria.

Effects of short‐chain fatty acids on Actinomyces naeslundii biofilm formation

Actinomyces naeslundii is an early colonizer and has important roles in the development of the oral biofilm. Short-chain fatty acids (SCFA) are secreted extracellularly as a product of metabolism by gram-negative anaerobes, e.g. Porphyromonas gingivalis and Fusobacterium nucleatum; and the SCFA may affect biofilm development with interaction between A. naeslundii and gram-negative bacteria. Our aim was to investigate the effects of SCFA on biofilm formation by A. naeslundii and to determine the mechanism. We used the biofilm formation assay in 96-well microtiter plates in tryptic soy broth without dextrose and with 0.25% sucrose using safranin stain of the biofilm monitoring 492 nm absorbance. To determine the mechanism by SCFA, the production of chaperones and stress-response proteins (GrpE and GroEL) in biofilm formation was examined using Western blot fluorescence activity with GrpE and GroEL antibodies. Adding butyric acid (6.25 mm) 0, 6 and 10 h after beginning culture significantly increased biofilm formation by A. naeslundii, and upregulation was observed at 16 h. Upregulation was also observed using appropriate concentrations of other SCFA. In the upregulated biofilm, production of GrpE and GroEL was higher where membrane-damaged or dead cells were also observed. The upregulated biofilm was significantly reduced by addition of anti-GroEL antibody. The data suggest biofilm formation by A. naeslundii was upregulated dependent on the production of stress proteins, and addition of SCFA increased membrane-damaged or dead cells. Production of GroEL may physically play an important role in biofilm development.

Inhibition of Streptococcus mutans biofilm formation using extracts from Assam tea compared to green tea.

OBJECTIVE Streptococcus mutans, a gram-positive oral bacterium, has been identified as one of the principal etiological agents of human dental caries. To clarify the nature of the difference anti-biofilm effect against S. mutans between Assam tea from Camellia sinensis var. assamica, partially fermented, and green tea from Camellia sinensis, non-fermented, active agents from the teas were purified. METHODS Effects of Assam tea and green tea samples on biofilm were assessed by using the conventional titer plate method and the human saliva-coated hydroxyapatite discs. The purification and identification of inhibitors were performed by using ultrafiltration with centrifugal filter devices and high performance liquid chromatography. RESULTS Assam tea has stronger biofilm inhibition activity against S. mutans than green tea. A substance of <10kDa in mass in Assam tea had a high concentration of galloylated catechins and a stronger biofilm inhibiting activity than green tea. In contrast, substances >10kDa in mass from green tea included higher concentrations of polysaccharides composed of galacturonic acid, such as pectin, that enhance biofilm formation. CONCLUSIONS The higher concentrations of galloylated catechins in Assam tea may assist in prevention of dental caries, whereas in green tea, this mode of inhibition was likely offset by the presence of pectin. Purification of catechins in partially fermented Assam tea with lower-molecular-weight polysaccharide than pectin may be useful for developing oral care products such as toothpaste and oral care gel pastes.

Cessation of Cytokinesis in Schizosaccharomyces pombe during Growth after Release from High Hydrostatic Pressure Treatment

On the basis of our previous study concerning the effect of high hydrostatic pressure treatment (HPT) on Escherichia coli FtsZ ring (bacterial cytoskeleton) formation, we aimed to determine the effect of HPT on the growth properties of a representative eukaryotic microbe, Schizosaccharomyces pombe, in relation to the behavior of genuine cytoskeletons. Microtubules were visualized with GFP-linked α-tubulin. Actin-related cytoskeletons were fluorescently stained with rhodamine-phalloidin. We observed growth retardation of about 10 h in post growth after HPT (75 MPa, 30 min, 28 °C), which caused only a little loss of viable cells. In accordance with the period of growth retardation, cessation of cytokinesis and disappearance of the contractile ring (composed of actin, myosin II, and other proteins), directly participates in cytokinesis, continued for 18 h after HPT. On the other hand, the microtubules disappeared only for 6 h after HPT. Based on these observations, the contractile ring was the site most sensitive to HPT resulting in the cessation of cytokinesis.

Hinokitiol Inhibits Candida albicans Adherence to Oral Epithelial Cells

Abstract Candida spp. are an opportunistic pathogen causing serious local and systemic infections, especially in immuno-compromised hosts such as the elderly and HIV-positive patients. Hinokitiol C 10 H 12 O 2 (β-thujaplicin) is a component of the essential oils isolated from Cupressaceae and shows antibacterial activities for various bacteria. The aim of this study was to demonstrate the preventive effects of hinokitiol on the adherence of seven species of Candida to oral epithelial cells and to establish a safe and useful method for oral hygiene. A short-time treatment (30 min) with 0.25 mM hinokitiol showed 30–70% inhibition of adherence of Candida spp. to oral epithelial cells, inhibited about 11% biofilm formation, and did not inhibit the cell growth of Candida spp. Furthermore, short treatment with 0.25 mM hinokitiol was a safe method for oral hygiene against Candida infection because it did not inhibit the cell growth of commensal bacteria, such as oral streptococci existing in normal flora, or damage the epithelial cells. However, long-time treatment and a high concentration of hinokitiol demonstrated both the adherence inhibition of Candida and damage to commensal bacteria and epithelial cells. Our data suggest an appropriate procedure to apply hinokitiol that may be beneficial for the prevention of opportunistic pathogens such as Candida spp. in the oral cavity. The clinical and daily use of hinokitiol under an appropriate procedure may be a preventive and realistic therapy for Candida infection in immune-compromised hosts.