Date Published: May 28, 2019
Publisher: Public Library of Science
Author(s): Chiaki Tsutsumi-Arai, Kensuke Takakusaki, Yuki Arai, Chika Terada-Ito, Yusuke Takebe, Takahiro Imamura, Shinji Ide, Seiko Tatehara, Reiko Tokuyama-Toda, Noriyuki Wakabayashi, Kazuhito Satomura, David D. Roberts.
This study aimed to investigate the cleansing effects of grapefruit seed extract (GSE) on biofilms of Candida albicans (C. albicans) formed on denture-base resin and the influence of GSE on the mechanical and surface characteristics of the resin. GSE solution diluted with distilled water to 0.1% (0.1% GSE) and 1% (1% GSE) and solutions with Polident® denture cleansing tablet dissolved in distilled water (Polident) or in 0.1% GSE solution (0.1% G+P) were prepared as cleansing solutions. Discs of acrylic resin were prepared, and the biofilm of C. albicans was formed on the discs. The discs with the biofilm were treated with each solution for 5 min at 25°C. After the treatment, the biofilm on the discs was analyzed using a colony forming unit (CFU) assay, fluorescence microscopy, and scanning electron microscopy (SEM). In order to assess the persistent cleansing effect, the discs treated with each solution for 5 min were aerobically incubated in Yeast Nitrogen Base medium for another 24 h. After incubation, the persistent effect was assessed by CFU assay. Some specimens of acrylic resin were immersed in each solution for 7 days, and changes in surface roughness (Ra), Vickers hardness (VH), flexural strength (FS), and flexural modulus (FM) were evaluated. As a result, the treatment with 1% GSE for 5 min almost completely eliminated the biofilm formed on the resin; whereas, the treatment with 0.1% GSE, Polident, and 0.1% G+P for 5 min showed a statistically significant inhibitory effect on biofilms. In addition, 0.1% GSE and 0.1% G+P exerted a persistent inhibitory effect on biofilms. Fluorescence microscopy indicated that Polident mainly induced the death of yeast, while the cleansing solutions containing at least 0.1% GSE induced the death of hyphae as well as yeast. SEM also revealed that Polident caused wrinkles, shrinkage, and some deep craters predominantly on the cell surfaces of yeast, while the solutions containing at least 0.1% GSE induced wrinkles, shrinkage, and some damage on cell surfaces of not only yeasts but also hyphae. No significant changes in Ra, VH, FS, or FM were observed after immersion in any of the solutions. Taken together, GSE solution is capable of cleansing C. albicans biofilms on denture-base resin and has a persistent inhibitory effect on biofilm development, without any deteriorations of resin surface.
Candida albicans (C. albicans), which is frequently isolated from denture plaque, is the main microorganism involved in the pathogenesis of denture stomatitis (DS) . As C. albicans has a high affinity to acrylic resin denture-base material, it adheres easily to and forms a dense biofilm on denture surfaces [2, 3]. Therefore, one of the most effective methods of treating and preventing DS is the removal of this fungus from the denture surfaces by efficient cleansing.
The CFU assays showed significantly lower quantities of biofilm on the discs from the 1% GSE, Polident, and 0.1% G+P groups than the control group. That is, the colony formation was inhibited by 57%, 98%, 80%, and 85% after treatment with 0.1% GSE, 1% GSE, Poldent and 0.1% G+P solutions, respectively. Among the cleansing solution groups, 1% GSE almost completely diminished the biofilm formed on disc surfaces compared with 0.1% GSE, Polident, and 0.1% G+P, among which no significant differences were noted (Fig 1). Another CFU assay to examine the persistent effect of each solution showed significantly lower quantities of biofilm on the discs from the 0.1% GSE, 1% GSE, and 0.1% G+P groups than the control group. That is, the colony formation was inhibited by 70%, 100%, 47%, and 76% after treatment with 0.1% GSE, 1% GSE, Poldent and 0.1% G+P solutions, respectively. Among the cleansing solution groups, the discs treated with the 1% GSE were significantly lower in biofilm content than those treated with 0.1% GSE, Polident, and 0.1% G+P. Interestingly, the addition of 0.1% GSE to Polident (0.1% G+P group) significantly inhibited biofilm formation compared with Polident alone. There were no significant differences among the 0.1% GSE, Polident, and 0.1% G+P groups (Fig 2). The comparison among the CFU assays for the discs treated with each solution followed by another 24-h incubation clearly revealed that treatment with 0.1% GSE and 1% GSE maintained the effectiveness of inhibition against biofilm formation even 24 h after a 5-min treatment. Importantly, the treatment with 1% GSE for 5 min almost eliminated the biofilm formed by C. albicans on the resins and inhibited the biofilm formation for at least 24 h after the initial treatment. Interestingly, the addition of 0.1% GSE to Polident (0.1% G+P) showed a more persistent effectiveness of inhibition of biofilm formation (Fig 3).
C. albicans biofilms recovered from discs treated with 0.1% GSE, 1% GSE, Polident, or 0.1% G+P for 5 min contained significantly much fewer vital C. albicans cells compared with those from the control group. Particularly, 1% GSE was proven to almost completely eliminate the vital cells from the biofilms. In addition, 0.1% GSE was nearly equally efficacious as Polident in the elimination of vital cells from biofilms. Microscopic images of 5-min treated C. albicans showed that this effect of GSE might be due to the cell death of C. albicans. Fluorescence microscopy observations showed that 1% GSE induced the death of C. albicans cells in both yeast and hyphal forms. Judging from the mechanism of the Live/Dead® FungaLight yeast viability kit, in which PI penetrates dead cells through the damaged cell wall/membrane and yields red fluorescence when it binds to DNA [26, 27], 1% GSE might have the potential to damage the Candida cell wall or membrane. Consistently, the SEM images of the 1% GSE-treated C. albicans showed shrinkage and surface damages in both yeasts and hyphae. The cell wall and cell membrane play important roles in cell viability, morphogenesis, response to environmental stressors, and pathogenesis . Thus, these morphological changes are thought to reflect the breakdown of cell homeostasis, resulting in cell death. In contrast, in the 0.1% GSE, Polident, and 0.1% G+P groups, only a portion of yeast and hyphal forms of C. albicans were affected by each solution.
The results of the present study strongly suggest that GSE can be very useful for the disinfection of denture surfaces. Our data also suggest that a denture treated with GSE for 5 min has the potential to have a persistent inhibitory effect on the biofilm development of C. albicans. In addition, immersion in the solution containing GSE does not cause surface deterioration of denture-base resin. Therefore, GSE has great potential as a new component of denture cleanser.