Kazuko Nakajo

Fluoride released from glass-ionomer cement is responsible to inhibit the acid production of caries-related oral streptococci

OBJECTIVES Glass-ionomer cements (GICs) are known to have inhibitory effects on bacterial growth, but the biochemical mechanism of this property has not been fully understood. This study aimed to evaluate inhibitory effects of GIC on the acid production of caries-related oral streptococci, and to identify the components responsible for the inhibition. METHODS An eluate was prepared by immersing set GIC in phosphate-buffered saline at 37 degrees C for 24h. Fluoride and other elements in the eluate were quantified by fluoride ion electrode and atomic absorption photometry, respectively. Streptococcus mutans NCTC 10449 and Streptococcus sanguinis NCTC 10556 were used to evaluate the pH fall and the rate of acid production after the addition of glucose in the presence or absence of the eluate. Acidic end products from glucose were also assayed by carboxylic acid analyzer. RESULTS The eluate contained silicon (1.24+/-0.26 mM), fluoride (0.49+/-0.02 mM) and aluminum (0.06+/-0.00 mM), and inhibited the pH fall and the acid production rate of both streptococci at acidic pH, with a concomitant decrease in lactic acid production. These effects were comparable to those of a potassium fluoride solution containing the same concentration of fluoride as the eluate. SIGNIFICANCE These results indicate that the GIC eluate inhibits the acid production of caries-related oral streptococci at acidic pH and that the effect is due to fluoride derived from the GIC. Thus, adjacent to GIC fillings, bacterial acid production and the subsequent bacterial growth may decrease, establishing a cariostatic environment.

Effects of the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) on bacterial viability and metabolism

The antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) is a strong bactericide when unpolymerized and has the potential to be utilized in various resinous biomaterials. To analyze the antibacterial characteristics of this monomer in detail, the ability of high concentrations of unpolymerized MDPB to kill Streptococcus mutans in planktonic or biofilm forms within a short time-period of contact, and the inhibitory effects of low concentrations of MDPB on the metabolic function of S. mutans, were examined. High concentrations of MDPB showed effective killing of planktonic and biofilm S. mutans cells within 60 s, and complete killing was obtained by contact with 1,000 μg ml(-1) of MDPB for 60 s. At a concentration of 4-8 μg ml(-1) , MDPB demonstrated growth inhibition, inducing elongation of the lag phase and of the doubling time, when the bacterial number was low. Inhibition of the production of acid from S. mutans by 8 μg ml(-1) of MDPB may have been caused by the inhibition of lactate dehydrogenase activity. At high concentrations, MDPB is lethal to both planktonic and biofilm forms of S. mutans in a short time-period, and at low concentrations, MDPB inhibits metabolic enzymatic activity.

Resistance to Acidic Environments of Caries-Associated Bacteria: Bifidobacterium dentium and Bifidobacterium longum

Oral Bifidobacteriaceae, Bifidobacterium dentium and Bifidobacterium longum, are known to be isolated together with mutans streptococci and lactobacilli from caries lesions, suggesting that these Bifidobacteriaceae are caries associated and acid resistant. This study aimed to investigate effects of acidification on B. dentium and B. longum, and to compare them with those on Streptococcus mutans, Streptococcus sanguinis and Lactobacillus paracasei. Effects of acidification, growth ability in a complex medium at a pH of 4.0–8.0, cell viability in 2-morpholinoethanesulfonic acid monohydrate (MES)-KOH buffer at pH 4.0, as well as stability of intracellular pH (pHin) at an extracellular pH of 3.5–8.0 estimated using a fluorescent dye, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester in MES-KOH, 3-(N-morpholino)propanesulfonic acid-KOH or N,N-bis(2-hydroxyethyl)glycine-KOH buffer, were investigated. B. longum grew as well as Streptococcus strains over a wide pH range, whereas B. dentium grew best in the narrow pH range around neutral. The cell viability of B. dentium decreased significantly after 2 h of acidification at a pH of 4.0, but this was significantly less than that of the Streptococcus and Lactobacillus species, whereas B. longum maintained almost 100% viability. The pHin was close to the extracellular pH at pH of 5.5–7.5 in the Bifidobacterium and Streptococcus strains, while at a pH of <5.0, the pHin was higher than the extracellular pH in all the strains, but the pHin maintenance ability of Bifidobacterium strains was higher than that of the Streptococcus strains. The high survival rate and pHin maintenance ability of bifidobacteria comparable to that of S. mutans in the acidic environment may account for why bifidobacteria exist as stable species in acidic caries lesions together with mutans streptococci.

Microbiologically Induced Corrosive Properties of the Titanium Surface

Corrosion of titanium is the major concern when it is used for dental treatment. This study aimed to investigate the mechanism of the microbiologically induced corrosive properties of titanium. An experimental well was made of polymethyl methacrylate with pure titanium at the bottom. Viable or killed cells of Streptococcus mutans were packed into the well, and pH at the bacteria-titanium interface was monitored with and without glucose. Before and after 90-minute incubation, the electrochemical behavior on the titanium surface was measured by means of a potentiostat. The oxygen concentration under bacterial cells was monitored with oxygen-sensitive fluorescent film. The amount of titanium eluted was measured by inductively coupled plasma-mass spectrometry. The corrosion current and passive current under killed cells were low and stable during 90 min, while those under viable cells increased, regardless of the glucose-induced pH fall. The polarization resistance and oxygen concentration under killed cells were high and stable, while those under viable cells decreased. No elution of titanium was detected. Viable bacterial cells may form ‘oxygen concentration cells’ through metabolism-coupled oxygen consumption and subsequently induce corrosive properties of the titanium surface.

Evaluation of pH at the Bacteria–Dental Cement Interface

Physiochemical assessment of the parasite-biomaterial interface is essential in the development of new biomaterials. The purpose of this study was to develop a method to evaluate pH at the bacteria-dental cement interface and to demonstrate physiochemical interaction at the interface. The experimental apparatus with a well (4.0 mm in diameter and 2.0 mm deep) was made of polymethyl methacrylate with dental cement or polymethyl methacrylate (control) at the bottom. Three representative dental cements (glass-ionomer, zinc phosphate, and zinc oxide-eugenol cements) were used. Each specimen was immersed in 2 mM potassium phosphate buffer for 10 min, 24 hrs, 1 wk, or 4 wks. The well was packed with Streptococcus mutans NCTC 10449, and a miniature pH electrode was placed at the interface between bacterial cells and dental cement. The pH was monitored after the addition of 1% glucose, and the fluoride contained in the cells was quantified. Glass-ionomer cement inhibited the bacteria-induced pH fall significantly compared with polymethyl methacrylate (control) at the interface (10 min, 5.16 ± 0.19 vs. 4.50 ± 0.07; 24 hrs, 5.20 ± 0.07 vs. 4.59 ± 0.11; 1 wk, 5.34 ± 0.14 vs. 4.57 ± 0.11; and 4 wks, 4.95 ± 0.27 vs. 4.40 ± 0.14), probably due to the fluoride released from the cement. This method could be useful for the assessment of pH at the parasite-biomaterial interface.

Susceptibility of Enterococcus faecalis to a Combination of Antibacterial Drugs (3Mix) in vitro

Abstract It has been reported that enterococci cause significantly persistent root canal infection, especially after Ca(OH) 2 intracanal dressing, and that they often show tolerance to certain antibacterial drugs. We aimed to evaluate the susceptibility of enterococci to a combination of antibacterial drugs, i.e. ciprofloxacin, metronidazole and minocycline (3Mix), which is used for Lesion Sterilization and Tissue Repair (LSTR) therapy. The minimum inhibitory concentrations (MICs) of ciprofloxacin and minocycline on E. faecalis (6 strains) and E. faecium (1 strain) were 5–20 μg/mL, respectively, and no inhibitory effect was observed with metronidazole. However, 3Mix (100 μg each/mL), as a mixture, inhibited the growth of every strain completely. In addition, 3Mix also inhibited all bacterial growth in faeces (16 samples). The present result strongly indicates that 3Mix is sufficiently able to inhibit enterococcal growth, and may be useful for endodontic treatment, even in cases where enterococci are suspected to cause endodontic disorders.

Effects of α-Amylase and Its Inhibitors on Acid Production from Cooked Starch by Oral Streptococci

This study evaluated acid production from cooked starch by Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus mitis, and the effects of α-amylase inhibitors (maltotriitol and acarbose) and xylitol on acid production. Streptococcal cell suspensions were anaerobically incubated with various carbohydrates that included cooked potato starch in the presence or absence of α-amylase. Subsequently, the fall in pH and the acid production rate at pH 7.0 were measured. In addition, the effects of adding α-amylase inhibitors and xylitol to the reaction mixture were evaluated. In the absence of α-amylase, both the fall in pH and the acid production rate from cooked starch were small. On the other hand, in the presence of α-amylase, the pH fell to 3.9–4.4 and the acid production rate was 0.61–0.92 μmol per optical density unit per min. These values were comparable to those for maltose. When using cooked starch, the fall in pH by S. sanguinis and S. mitis was similar to that by S. mutans and S. sobrinus. For all streptococci, α-amylase inhibitors caused a decrease in acid production from cooked starch, although xylitol only decreased acid production by S. mutans and S. sobrinus. These results suggest that cooked starch is potentially acidogenic in the presence of α-amylase, which occurs in the oral cavity. In terms of the acidogenic potential of cooked starch, S. sanguinis and S. mitis were comparable to S. mutans and S. sobrinus. α-Amylase inhibitors and xylitol might moderate this activity.

Divalent Cations Enhance Fluoride Binding to Streptococcus mutans and Streptococcus sanguinis Cells and Subsequently Inhibit Bacterial Acid Production

One preventive effect of topical fluoride application is derived from the fact that fluoride can inhibit bacterial acid production. Furthermore, divalent cations such as Ca2+ and Mg2+ increase the binding of fluoride to bacterial cells. These findings suggest that exposure of oral bacteria to fluoride in the presence of divalent cations increases fluoride binding to bacterial cells and subsequently enhances fluoride-induced inhibition of bacterial acid production. This study investigated the effects of fluoride exposure (0–20,000 ppm F) in the presence of Ca2+ or Mg2+ prior to glucose challenge on pH fall ability by bacterial sugar fermentation, as well as fluoride binding to bacterial cells by exposure to fluoride, and fluoride release from bacterial cells during bacterial sugar fermentation, using caries-related bacteria, Streptococcus mutans and Streptococcus sanguinis. The pH fall by both streptococci was inhibited by exposure to over 250 ppm F in the presence of Ca2+ (p < 0.01), whereas in the presence of Mg2+, the pH fall by S. mutans and S. sanguinis was inhibited after exposure to over 250 and 950 ppm F, respectively (p < 0.05). The amounts of fluoride binding to and released from streptococcal cells increased with the concentration of fluoride the cells were exposed to in the presence of Mg2+, but were high enough even after 250 ppm F exposure in the presence of Ca2+. The enhanced inhibition of acid production in the presence of divalent cations is probably due to the improved efficiency of fluoride binding to bacterial cells being improved via these divalent cations.

Fluoride-sensitivity of growth and acid production of oral Actinomyces : comparison with oral Streptococcus

Actinomyces are predominant oral bacteria; however, their cariogenic potential in terms of acid production and fluoride sensitivity has not been elucidated in detail and compared with that of other caries‐associated oral bacteria, such as Streptococcus. Therefore, this study aimed to elucidate and compare the acid production and growth of Actinomyces and Streptococcus in the presence of bicarbonate and fluoride to mimic conditions in the oral cavity. Acid production from glucose was measured by pH‐stat at pH 5.5 and 7.0 under anaerobic conditions. Growth rate was assessed by optical density in anaerobic culture. Although Actinomyces produced acid at a lower rate than did Streptococcus, their acid production was more tolerant of fluoride (IDacid production 50 = 110–170 ppm at pH 7.0 and 10–13 ppm at pH 5.5) than that of Streptococcus (IDacid production 50 = 36–53 ppm at pH 7.0 and 6.3–6.5 ppm at pH 5.5). Bicarbonate increased acid production by Actinomyces with prominent succinate production and enhanced their fluoride tolerance (IDacid production 50 = 220–320 ppm at pH 7.0 and 33–52 ppm at pH 5.5). Bicarbonate had no effect on these variables in Streptococcus. In addition, although the growth rate of Actinomyces was lower than that of Streptococcus, Actinomyces growth was more tolerant of fluoride (IDgrowth 50 = 130–160 ppm) than was that of Streptococcus (IDgrowth 50 = 27–36 ppm). These results indicate that oral Actinomyces are more tolerant of fluoride than oral Streptococcus, and bicarbonate enhances the fluoride tolerance of oral Actinomyces. Because of the limited number of species tested here, further study is needed to generalize these findings to the genus level.

Experimental Ti–Ag alloys inhibit biofilm formation

We prepared experimental Ti–Ag alloys and investigated their properties. The strength and hardness of the Ti–Ag alloys increased with the concentration of Ag. Moreover, the alloys exhibited sufficient elongation, making them suitable for dental applications. The machinability of the Ti–Ag alloys was superior to that of pure titanium. By carrying out the anode polarization test and immersion test, we found that the corrosion resistance of the Ti–Ag alloys was comparable to that of pure titanium. Further, we performed a biofilm formation test and found that the amount of biofilm formed on the experimental Ti–Ag alloys was less than that on pure titanium, pure silver, and a dental alloy. It was concluded that the experimental Ti–Ag alloys are new types of biomaterials that have an inhibitory effect on biofilm formation as well as excellent mechanical properties and outstanding machinability.