No abstract

Isoflavaspidic acid PB (PB), a phloroglucinol derivative extracted from aerial parts of Dryopteris fragrans (L.) Schott, had antifungal activity against several dermatophytes. This study was aimed at exploring the antifungal mechanism of PB against Trichophyton rubrum (T. rubrum). The effectiveness of PB in inhibiting T. rubrum growth was detected by time-kill kinetics study and fungal biomass determination. Studies on the mechanism of action were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), sorbitol and ergosterol assay, nucleotide leakage measurement, and UPLC-based test and enzyme-linked immunosorbent assay. Fungicidal activity of PB was concentration- and time-dependent at 2 × MIC (MIC: 20 μg/mL) after 36 h. The total biomass of T. rubrum was reduced by 64.17%, 77.65%, and 84.71% in the presence of PB at 0.5 × MIC , 1 × MIC , and 2 × MIC , respectively. SEM analysis showed that PB changed mycelial morphology, such as shrinking, twisting, collapsing, and even flattening. TEM images of treated cells exhibited abnormal distributions of polysaccharide particles, plasmolysis, and cytoplasmic content degradation accompanied by plasmalemma disruption. There were no changes in the MIC of PB in the presence of sorbitol. However, the MIC values of PB were increased by 4-fold with exogenous ergosterol. At 4 h and 8 h, PB increased nucleotide leakage. Besides, ergosterol content in T. rubrum membrane treated with PB at 0.5 × MIC , 1 × MIC , and 2 × MIC was decreased by 9.58%, 15.31%, and 76.24%, respectively. There was a dose-dependent decrease in the squalene epoxidase (SE) activity. And the reduction in the sterol 14α-demethylase P450 (CYP51) activity was achieved after PB treatments at 1 × MIC and 2 × MIC . These results suggest that PB displays nonspecific action on the cell wall. The membrane damaging effects of PB were attributed to binding with ergosterol to increase membrane permeability and interfering ergosterol biosynthesis involved with the reduction of SE and CYP51 activities. Further study is needed to develop PB as a natural antifungal candidate for clinical use.

The adsorption of metal ions to 14 species of microorganisms including oral bacteria and samples of dental plaque was investigated. At around neutral pH, which is similar condition as the oral environment, high levels of silver adsorption to cells were demonstrated with Propionibacterium acnes and Fusobacterium nucleatum, while Porphyromonas gingivalis and Actinomyces levoris showed lower levels of silver adsorption. Adsorption of copper ions was observed at higher levels in both P. acnes and Corynebacterium matruchotii. P. gingivalis and A. levoris strains showed only low level adsorption. Samples of dental plaque from 3 different origins were collected from 2 adults respectively or collected and mixed from 124 dental students. These samples showed lower levels of nickel and silver adsorption than P. acnes, and higher levels of metal adsorption than P. gingivalis. The release of the nickel ions, which has been well known as a causative agent for mucous epithelium and skin allergies, from P. acnes cells bound the metal ions by washing was examined. The metal ions were readily released from the bacterial cells by washing with EDTA but not almost by water. This finding indicates that metal ions adsorbed to bacterial cells are not easily released by washing with water. These results suggests that several oral bacteria and dental plaque possess an increased capacity for adsorption of dental metal ions in oral environments. Yamamura, et al.: Metal adsorption to microorganisms and dental plaque 621