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. 2017 Sep 13:8:1738.
doi: 10.3389/fmicb.2017.01738. eCollection 2017.

Anti-biofilm Activities from Bergenia crassifolia Leaves against Streptococcus mutans

Yucui Liu  1 , Yanjie Xu  1 , Qiuhang Song  1 , Fei Wang  1 , Luguo Sun  2 , Lei Liu  2 , Xiaoguang Yang  3 , Jingwen Yi  1 , Yongli Bao  1 , Haifeng Ma  4 , Honglan Huang  5 , Chunlei Yu  2 , Yanxin Huang  1 , Yin Wu  1 , Yuxin Li  1
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Free PMC article

Anti-biofilm Activities from Bergenia crassifolia Leaves against Streptococcus mutans

Yucui Liu et al. Front Microbiol. .
Free PMC article

Abstract

Streptococcus mutans has been reported as a primary cariogenic pathogen associated with dental caries. The bacteria can produce glucosyltransferases (Gtfs) to synthesize extracellular polysaccharides (EPSs) that are known as virulence factors for adherence and formation of biofilms. Therefore, an ideal inhibitor for dental caries is one that can inhibit planktonic bacteria growth and prevent biofilm formation. Bergenia crassifolia (L.), widely used as a folk medicine and tea beverage, has been reported to have a variety of bioactivities. The present study aimed to explore the effect of B. crassifolia (L.) leaf extracts on the biofilm of Streptococcus mutans. The B. crassifolia (L.) leaf extracts showed inhibitory effects by decreasing viability of bacteria within the biofilm, as evidenced by the XTT assay, live/dead staining assay and LDH activity assay, and could decrease the adherence property of S. mutans through inhibiting Gtfs to synthesize EPSs. In addition, the reduced quantity of EPSs and the inhibition of Gtfs were positively correlated with concentrations of test samples. Finally, the MTT assay showed that the extracts had no cytotoxicity against normal oral cells. In conclusion, the extracts and sub-extracts of B. crassifolia leaves were found to be antimicrobial and could reduce EPS synthesis by inhibiting activities of Gtfs to prevent bacterial adhesion and biofilm formation. Therefore, B. crassifolia leaves have potential to be developed as a drug to prevent and cure dental caries.

Keywords: Bergenia crassifolia; biofilm formation; cytotoxicity; extracellular polysaccharides; glucosyltransferase activity.

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Figures

FIGURE 1
FIGURE 1
Determination of MIC for extracts/sub-extracts against S. mutans. After incubation with resazurin sodium 24 h, the lowest concentration that could protect the color from changing to pink was determined as the MIC. Other changing color pictures are displayed in Supplementary Data Sheet 2.
FIGURE 2
FIGURE 2
Percentage of bacterial viability within biofilms measured by the XTT assay. (A) Extracts of 95% ethanol, (B) extracts of water, (C) sub-extracts of EtOAc, (D) sub-extracts of CHCl3, (E) sub-extracts of BuOH. Data are presented as the mean + standard deviation based on three individual tests. ∗P < 0.05 and ∗∗P < 0.01.
FIGURE 3
FIGURE 3
Bacterial cell damage within the biofilm based on LDH activity in different test samples. The extracts of B. crassifolia leaves were serially diluted twofold in BHI broth containing 1% sucrose from 1/8 MIC to MIC respectively. The graph represents the actual concentrations of the MIC dilutions.
FIGURE 4
FIGURE 4
Percent inhibition of extracts/sub-extracts effect on EPS glucans by the phenol/H2SO4 method. (A) (%) Inhibition of water-insoluble glucans. (B) (%) Inhibition of water-soluble glucans. The extracts of B. crassifolia leaves were serially diluted twofold in BHI broth containing 1% sucrose from 1/8 MIC to MIC respectively. The graph represents the actual concentrations of the MIC dilutions. Data are presented as the mean ± standard deviation.
FIGURE 5
FIGURE 5
Effect of B. crassifolia leaf extracts on water-insoluble glucan formation by Gtfs of S. mutans from sucrose. The formation of water-insoluble glucans was expressed as a percent of the control (without sample).
FIGURE 6
FIGURE 6
Effects of B. crassifolia leaf extracts on S. mutans ATCC25175 biofilm by using a fluorescence microscope. Live cells exhibited green fluorescence (SYTO 9), whereas bacteria with damaged membranes exhibited red fluorescence (PI). (A) control; (B) HP-95% ethanol; (C) HP-Water; (D) HP-CHCl3; (E) HP-EtOAc; (F) HP-BuOH, and (0) stained with SYTO9; (1) stained with PI; (2) Merged.
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