Inhibitory effects of Oenothera biennis (evening primrose) seed extract on Streptococcus mutans and S. mutans-induced dental caries in rats.

BACKGROUND: Oenothera biennis (evening primrose) seed extract (OBSE) is known to contain polyphenols, which may possess antioxidant activities. Polyphenols extracted from several plants are reported to exhibit cariostatic activities by inhibiting mutans streptococcus growth and glucosyltransferase activities. The purpose of the present study was to examine the inhibitory effects of OBSE on the development of dental caries, both in vitro and in vivo. METHODS: OBSE was investigated for its inhibitory effects on cellular aggregation, hydrophobicity, sucrose-dependent adherence and insoluble glucan synthesis. Furthermore, biofilm formation was examined in the presence of OBSE, using confocal microscopic imaging. An animal experiment was also performed to examine the in vivo effects. RESULTS: OBSE induced a strong aggregation of Streptococcus mutans MT8148 cells, while cell surface hydrophobicity was decreased by approximately 90% at a concentration of 0.25 mg/ml. The sucrose-dependent adherence of the MT8148 cells was also reduced by addition of OBSE, with a reduction rate of 73% seen at a concentration of 1.00 mg/ml. Additionally, confocal microscopic observations revealed the biofilm development phase to be remarkably changed in the presence of OBSE. Furthermore, insoluble glucan synthesis was significantly reduced when OBSE was present at concentrations greater than 0.03 mg/ml. In an animal experiment, the caries scores in rats given OBSE (0.05 mg/ml in drinking water) were significantly lower than those in rats given water without OBSE. CONCLUSION: Our results indicate that OBSE has inhibitory activity on dental caries.

Synergistic effects of streptococcal glucosyltransferases on adhesive biofilm formation.

Glucosyltransferases (GTF)-I and GTF-SI of Streptococcus mutans synthesize water-insoluble and both water-soluble and -insoluble glucans, respectively, and play essential roles in the sucrose-dependent adhesion of the organism to tooth surfaces. To examine the interactions of different GTFs on artificial biofilm formed by S. mutans and other oral streptococci, we generated GTF-I- and GTF-SI-hyperproducing isogenic mutant strains. Transformant B42-21, which hyperexpressed GTF-SI, exhibited firm adhesion in the presence of sucrose, whereas transformant B42-10, which hyperexpressed GTF-I, failed to exhibit firm adhesion. Furthermore, co-culture of transformant B42-21 with water-soluble glucan-synthesizing Streptococcus sanguinis yielded firm adhesion, while the addition of dextran T10 to B42-21 growing culture had no effect on adhesion. These findings suggest that GTF-SI has a strong effect on sucrose-dependent adhesion and is essential for biofilm formation on smooth surfaces, in cooperation with water-soluble glucans synthesized de novo by oral streptococci that inherently lack cell adhesion ability.

Legionnaires' disease associated with habitual drinking of hot spring water.

A 57-year-old man presented with pneumonia, respiratory distress, and myelodysplastic syndrome. A diagnosis of Legionnaires' disease due to Legionella pneumophila (L. pneumophila) was established. The patient had long been drinking tap water via a conduit from a hot spring resource, from which L. pneumophila was also isolated. Both the patient's strain and the water strain of L. pneumophila were identified as serogroup 1, and the genetic relatedness between the two strains as seen by pulsed-field gel electrophoresis was 87%. The patient was successfully treated with erythromycin, fluoroquinolone, and rifampicin. This case raises an important issue on public health represented by legionellosis in Japan.

Low-molecular-weight lignin-rich fraction in the extract of cultured Lentinula edodes mycelia attenuates carbon tetrachloride-induced toxicity in primary cultures of rat hepatocytes.

The extract of cultured Lentinula edodes mycelia (LEM) is a medicinal food ingredient that has hepatoprotective effects. In this study, we fractionated the LEM extract to explore novel active compounds related to hepatoprotection by using primary cultures of rat hepatocytes exposed to carbon tetrachloride (CCl(4)). The LEM extract and the fractions markedly inhibited the release of alanine aminotransferase (ALT) from hepatocytes damaged by CCl(4) into the culture medium. The strongest hepatocyte-protective activity was seen in a fraction (Fr. 2) in which a 50% ethanol extract was further eluted with 50% methanol and separated using reverse-phase HPLC. Fr. 2 had an average molecular weight of 2753, and the main components are lignin (49%) and saccharides (36%, of which xylose comprises 41%). Therefore, Fr. 2 was presumed to be a low-molecular-weight compound consisting mainly of lignin and xylan-like polysaccharides. The hepatocyte-protective activity was observed even after digestion of xylan-like polysaccharides in Fr.2 and confirmed with low-molecular-weight lignin (LM-lignin) alone. In addition, Fr. 2, the xylan-digested Fr. 2 and LM-lignin showed higher superoxide dismutase (SOD)-like activity than the LEM extract. These results suggested that the effective fraction in the LEM extract related to hepatocyte protection consisted mainly of LM-lignin, and its antioxidant activity partially contributes to the hepatocyte-protective activity of the LEM extract.

Application of colorimetric microdilution plate hybridization for rapid genetic identification of 22 Mycobacterium species.

Quantitative microdilution plate hybridization was used to identify 22 Mycobacterium species. DNAs of clinical strains were rapidly extracted and labeled with photoreactive biotin. Labeled DNAs were distributed into wells of a microdilution plate in which reference DNAs had been immobilized. After 2 h of hybridization, hybridized DNAs were quantitatively detected with peroxidase-conjugated streptavidin and the substrate, tetramethylbenzidine. This method could differentiate among 20 of the 22 Mycobacterium species tested. The type strains of Mycobacterium tuberculosis and M. bovis were genetically highly related and could not be differentiated by this method. Of 194 biochemically identified human clinical strains, 178 (90%) were genetically identified within 3 h of the small-scale DNA extraction.

Deletion and reintroduction of glucosyltransferase genes of Streptococcus mutans and role of their gene products in sucrose dependent cellular adherence.

Streptococcus mutans has three kinds of glucosyltransferases (GTases), i.e. GTase-I, GTase-SI and GTase-S. These enzymes co-operatively synthesize adhesive glucan from sucrose, which contributes to firm adherence of growing organisms to solid surfaces. In this study, the genes encoding GTase-I (gtfB) and GTase-SI (gtfC) of S. mutans MT8148 (serotype c) were inactivated by the insertion mutagenesis by allelic exchange. Three types of isogenic mutants lacking either GTase-I, GTase-SI or both, respectively were isolated. Sucrose dependent cellular adherence of these mutants were significantly lower than that of the parent. Adherence of GTase-SI deficient mutant was lower than that of GTase-I deficient mutant. We then generated Escherichia coli-Streptococcus shuttle vectors carrying the gtfB and gtfC gene. The shuttle vector containing the gtfB gene was transformed into the GTase-I deficient mutant. Western blot analysis of the transformant revealed that GTase-I protein was fully expressed. Sucrose dependent adherence of the transformant increased but did not reach that of the parent. Similarly, the shuttle vector containing the gtfC gene was transformed into the GTase-SI deficient mutant. The expression of GTase-SI and sucrose dependent adherence of the transformant was revealed to be at a level similar to those by the parent. These results indicated that GTase-SI does play an essential role in the production of adhesive glucan that can lead to firm cellular adherence to solid surfaces.