Scanning Electron Microscopic study of Piper betle L. leaves extract effect against Streptococcus mutans ATCC 25175

Abstract

IntroductionPrevious studies have shown that Piper betle L. leaves extract inhibits the adherence of Streptococcus mutans to glass surface, suggesting its potential role in controlling dental plaque development. Objectives: In this study, the effect of the Piper betle L. extract towards S. mutans (with/without sucrose) using scanning electron microscopy (SEM) and on partially purified cell-associated glucosyltransferase activity were determined.Material and MethodsS. mutans were allowed to adhere to glass beads suspended in 6 different Brain Heart Infusion broths [without sucrose; with sucrose; without sucrose containing the extract (2 mg mL-1 and 4 mg mL-1); with sucrose containing the extract (2 mg mL-1 and 4 mg mL-1)]. Positive control was 0.12% chlorhexidine. The glass beads were later processed for SEM viewing. Cell surface area and appearance and, cell population of S. mutans adhering to the glass beads were determined upon viewing using the SEM. The glucosyltransferase activity (with/without extract) was also determined. One- and two-way ANOVA were used accordingly.ResultsIt was found that sucrose increased adherence and cell surface area of S. mutans (p<0.001). S. mutans adhering to 100 µm2 glass surfaces (with/without sucrose) exhibited reduced cell surface area, fluffy extracellular appearance and cell population in the presence of the Piper betle L. leaves extract. It was also found that the extract inhibited glucosyltransferase activity and its inhibition at 2.5 mg mL-1 corresponded to that of 0.12% chlorhexidine. At 4 mg mL-1 of the extract, the glucosyltransferase activity was undetectable and despite that, bacterial cells still demonstrated adherence capacity.ConclusionThe SEM analysis confirmed the inhibitory effects of the Piper betle L. leaves extract towards cell adherence, cell growth and extracellular polysaccharide formation of S. mutans visually. In bacterial cell adherence, other factors besides glucosyltransferase are involved.

Comparison of SEM and VPSEM imaging techniques with respect toStreptococcus mutansbiofilm topography

“…Biofilms are composed of c. 85% EPS and 15% bacterial cells, and in S. mutans biofilm, the EPS strongly Imaging of oral in vitro oral biofilm contributes to the antibiotic resistance, host evasion, hydration, maturation, adhesion, and structural support (Fulcher et al, 2001;Rahim & Thurairajah, 2011;Xiao et al, 2012). Although previous studies (Ishii et al, 2004;Weimer et al, 2006) have demonstrated the benefits of using VPSEM to visualize other biofilms under humid conditions as SEM protocol destroys the majority of the biofilm structure (Donlan & Costerton, 2002;Priester et al, 2007), several studies analyze the matrix of S. mutans biofilm by SEM (da Cunha et al, 2013;Fu et al, 2013;Wang et al, 2013).…”

“…Streptococcus mutans is a facultatively anaerobic, Grampositive bacterium that is a major contributor to dental caries and in some cases a progenitor to endocarditis (Loesche, 1986). The bacterial cells adhere to tooth surfaces using the extracellular polymeric substance (EPS), specifically the molecule glucan in S. mutans biofilms (Rahim & Thurairajah, 2011). Production of the glucan and the EPS increases when in the presence of sucrose and glucosyltransferase activity, but with limited nitrogen, potassium, and phosphate in the medium (Fulcher et al, 2001;Donlan & Costerton, 2002;Rahim & Thurairajah, 2011).…”

“…The bacterial cells adhere to tooth surfaces using the extracellular polymeric substance (EPS), specifically the molecule glucan in S. mutans biofilms (Rahim & Thurairajah, 2011). Production of the glucan and the EPS increases when in the presence of sucrose and glucosyltransferase activity, but with limited nitrogen, potassium, and phosphate in the medium (Fulcher et al, 2001;Donlan & Costerton, 2002;Rahim & Thurairajah, 2011). The EPS is required during the initial attachment of bacterial cells to the tooth surface, as the polysaccharides form hydrophobic and ionic interactions between the host receptors and the bacterial adhesions (Marsh, 2004).…”

Peer Review #1 of "Antibacterial and anti-adherence effects of a plant extract mixture (PEM) and its individual constituent extracts (Psidium sp., Mangifera sp., and Mentha sp.) on single- and dual-species biofilms (v0.1)"

“…Manuscript to be reviewed al., 2000;Kreth et al, 2005). The positive control, 0.12% CHX appeared to have no adhering activity as the concentration used is more to the bactericidal (Rahim & Thurairajah, 2011).…”

“…The remaining beads from the experiments were processed for SEM viewing according to the method described by Rahim & Thurairajah (2011) with slight modification. Briefly, three glass beads for each of experiment were fixed with 1 ml of 4% glutaraldehyde solution (prepared in sodium cacodylate buffer, pH 7.4) and kept at 4 o C until the subsequent analysis.…”