Efficacy of E. officinalis on the cariogenic properties of Streptococcus mutans: a novel and alternative approach to suppress quorum-sensing mechanism

Abstract

The present study was focused on evaluating the potential of Emblica officinalis against cariogenic properties of Streptococcus mutans, a causative microorganism for caries. The effect of crude extract and ethanolic fraction from Emblica officinalis fruit was analysed against S. mutans. The sub-MIC concentrations of crude and ethanolic fraction of E. officinalis were evaluated for its cariogenic properties such as acid production, biofilm formation, cell-surface hydrophobicity, glucan production, sucrose-dependent and independent adherence. Its effect on biofilm architecture was also investigated with the help of confocal and scanning electron microscopy (SEM). Moreover, expression of genes involved in biofilm formation was also studied by quantitative RT- PCR. This study showed 50% reduction in adherence at concentrations 156 µg/ and 312.5 µg/ml of crude extract and ethanolic fraction respectively. However, the biofilm was reduced to 50% in the presence of crude extract (39.04 µg/ml) and ethanolic fraction (78.08 µg/ml). Furthermore, effective reduction was observed in the glucan synthesis and cell surface hydrophobicity. The qRT-PCR revealed significant suppression of the genes involved in its virulence. Confocal and scanning electron microscopy clearly depicted the obliteration of biofilm structure with reference to control. Hence, this study reveals the potential of E. officinalis fruit extracts as an alternative and complementary medicine for dental caries by inhibiting the virulence factors of Streptococcus mutans.

Figure 1. Inhibitory effect of crude and ethanolic fractions on:(a & b) the glass-dependent adherence by S.mutans in the absence (sucrose-independent) and presence of 5% sucrose (sucrose-dependent), (c & d) Biofilm formation in the presence and absence of salivary pellicle.

Each value is an average of triplicate assays, and each bar indicates±standard deviation (n = 3).

Figure 2. Effect of crude and ethanolic fractions on: (a & b) the inhibition of water- soluble and insoluble glucan synthesis, (c & d) reduction in the hydrophobicity.

Each value is an average of triplicate assays, and each bar indicates±standard deviation (n = 3).

Figure 3. The growth curve of untreated and treated S.mutans cells over 24 h.

Figure 4. CLSM images (a) and SEM (b) of Streptococcus mutans biofilm formed in the presence and absence of the extracts after 24 h of incubation.

The assays were performed in triplicates and similar results were obtained.

Figure 5. Direct binding ELISA of total protein from untreated S. mutans (control) and S. mutans treated with crude and ethanolic fraction against polyclonal antibodies of Ag I/II rose in rabbit.

Figure 6. Binding pattern of the compounds analysed by GC-MS showing best docking score from: (a) crude extract and (b) ethanolic extracts within the active site of Ag I/ II.

Figure 7. Gene expression profile of specific genes involved in the formation of biofilm

qRT-PCR was carried out in triplicate. Data presented were generated from at least four independent sets of experiments (Data =  mean±SD).