Effect of LongZhang Gargle on Dual-Species Biofilm of Candida albicans and Streptococcus mutans

Abstract

Bioactive natural products have become a hot spot for oral disease treatments. At the present study, LongZhang Gargle was investigated for its effects on single-species biofilms of Candida albicans and dual-species biofilms of Candida albicans and Streptococcus mutans. Two different models of single and dual-species biofilms were grown in YNBB medium under appropriate conditions. Biofilm biomass, biofilm architecture, and cell activity in biofilms were assessed using Crystal Violet Staining, MTT, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Significant reductions of biofilm biomass and fungus activity were obtained when treated with LongZhang Gargle at 2% (P < 0.05), 4% (P < 0.05), and 8% (P < 0.05) in single-species biofilms of C. albicans, and at 4% (P < 0.05) and 8% (P < 0.05) in double-species biofilms. Suppression of density, thickness, and the proportion of hyphae and fungal spores were obtained under SEM and CLSM. In conclusion, LongZhang Gargle affects single and dual-species biofilms by inhibiting biofilm biomass, cell activity, and formation of hyphae, but it does not affect the production of Extracellular polysaccharides (EPS). We speculate that LongZhang Gargle would be a promising natural drug, which can be used in treatment against C. albicans and S. mutans in oral diseases.

Figure 1

Biofilm biomass of single-species and dual-species biofilms at varying LongZhang Gargle concentrations (0, 2, 4, and 8%) at OD 595 nm. The white bars indicate C. albicans, and the black bars indicate dual-species of S. mutans and C. albicans. Asterisks indicate the statistical differences compared to the 0% LongZhang Gargle. The error bars indicate the standard deviation (SD).^∗P < 0.05.

Figure 2

Cell activity of single-species and dual-species biofilms at varying LongZhang Gargle concentrations (0, 2, 4, and 8%) at OD 570 nm. The white bars indicate C. albicans, and the black bars indicate dual-species of S. mutans and C. albicans. Asterisks indicate the statistical differences compared to the 0% LongZhang Gargle. The error bars indicate the standard deviation (SD). ^∗P < 0.05.

Figure 3

Scanning electron microscopy images of different biofilms. (a) Morphology of single-species biofilms of C. albicans treated with 0, 2, 4, and 8% of LongZhang Gargle for 24 h in YNBB broth. Magnification was 2000×, 5000×, 10000×, and 20000×, respectively, for each concentration. (b) Morphology of dual-species biofilms of C. albicans and S. mutans treated with 0, 2, 4, and 8% of LongZhang Gargle for 24 h in YNBB broth. Magnification was 2000×, 5000×, 10000×, and 20000×, respectively, for each concentration.

Figure 4

Confocal laser scanning microscopy images of different biofilms. (a) Images of single-species biofilms of C. albicans treated with 0, 2, 4, and 8% of LongZhang Gargle for 24 h. Cells were labelled green (SYTO 9), EPS was labelled red (Alexa Fluor 647), and red and green superimposed appear as yellow. Magnification was 63× for oil immersion objective. (b) Images of dual-species biofilms of C. albicans and S. mutans treated with 0, 2, 4, and 8% of LongZhang Gargle for 24 h. Cells were labelled green (SYTO 9), EPS was labelled red (Alexa Fluor 647), and red and green superimposed appear as yellow. Magnification was 63× for the oil immersion objective. (c) Mean fluorescence intensity of single-species biofilms at different concentration of LongZhang Gargle. (d) Mean fluorescence intensity of double-species biofilms at different concentrations of LongZhang Gargle. (e) Mean fluorescence intensity of EPS in double-species biofilms at different concentrations of LongZhang Gargle. All values on three duplicate samples were presented as mean with SD (^∗P ≤ 0.05; ^∗∗P ≤ 0.01).