In vitro Antibacterial Activity of Zingiber officinale and Orthosiphon stamineus on Enterococcus faecalis

This study evaluates the antibacterial effects of Zingiber officinale essential oil and Orthosiphon stamineus water extract against Enterococcus faecalis. The herbs were prepared in various concentrations to determine their minimum inhibitory concentrations (MIC) and growth inhibitory effect. Anti-adhesion activities of the herbs were determined by co-incubation with E. faecalis cultures for 6 and 24 h. Biofilm disruption activities were determined by adding the studied herbs into preformed E. faecalis biofilm. The effects on the morphology of E. faecalis grown as biofilm were studied using scanning electron microscopy (SEM). The MICs of ginger oil and O. stamineus extract were 0.31 and 25 mg/mL, respectively. Between the tested herbs, ginger exhibited greater inhibitory effects on the growth of E. faecalis grown in suspension mode. Both herbs generally showed anti-adhesion activities in inverse concentration-dependent manner. No significant biofilm disruption activities by both herbs were observed. SEM analyses showed E. faecalis cell surface changes in the treated biofilm. The studied herbs may have compromised the integrity of the bacterial cell membrane. These findings suggest that the studied herbs may have better antibacterial activities against E. faecalis in suspension mode compared to biofilm mode, with ginger oil showed greater antibacterial activity compared to O. stamineus extract.


Introduction
Dental caries and periodontal disease are two of the most prevalent oral health problems worldwide (Kassebaum et al., 2017).The main cause of these conditions is bacterial infection from the dental plaque.Amongst the common pathogenic species involved in both conditions are Enterococcus faecalis, a facultative anaerobic bacteria and notoriously known to be resistant to antibacterial and clinical therapy (Stuart et al., 2006).Recent findings in drug discovery research using herbal extracts have given new hopes for supplemental or even alternative approach to managing dental infections more effectively and safely.
Although these studies have shown the beneficial effects of both plants, little is reported on the antibacterial properties of these herbs in the field of dentistry, especially on bacteria implicated in periodontal and endodontic diseases like the notoriously resistant Enterococcus faecalis.This study will help to appreciate the antibacterial properties of Z. officinale and O. stamineus toward E. faecalis in suspension culture as well as in biofilm environment.Therefore, the aim of this research was to investigate the antibacterial effects of Z. officinale and O. stamineus on E. faecalis.The findings on the structural changes of E. faecalis after exposure to these two herbs, using scanning electron microscopy (SEM) was also reported.

Preparation of Ginger and O. stamineus
Ginger essential oil was obtained from the Drug Discovery Research Laboratory of Universiti Kebangsaan Malaysia (courtesy of Dr. Shahida Mohd Said).Briefly, ground ginger was boiled for 8 h in the boiling flask with a tight-fitting adapter attached to the aspirator vacuum.The volatile components of the samples were evaporated with steam and cooled as oil at the end of the procedure.The oil was then collected, and remaining water was removed using anhydrous sodium sulphate.O. stamineus powder was obtained from Faculty of Bioprocess, Universiti Teknologi Malaysia, Skudai, Johor (courtesy of Associate Professor Dr. Adibah Abdul Majid).

Bacterial Strain and Growth Conditions
E. faecalis ATCC 29212 was used in this study.The bacteria was grown on brain heart infusion (BHI) agar (Oxoid, UK) and passaged weekly.Cultures on BHI agar or in BHI broth (Becton Dickinson, USA) were incubated for 24 h at 37 o C in the presence of 5% CO 2 with 95% relative humidity (Shel Lab).
The positive controls that were used to compare with the tested herbs were dilution series of amoxicillin or ampicillin (Sigma, USA), and 2.5% sodium hypochlorite (NaClO).Amoxicillin or ampicillin was used because they are common antibiotics clinically used for patients with any periodontal, endodontic or perio-endo infections and NaClO is a common irrigant solution used in clinics to clean the root canal system due to its antibacterial properties.
After 24 h of exposure to studied herbs, the optical density (OD) of the growth turbidity was measured with a plate reader at 590 nm wavelength (Varioskan® Flash Microplate reader, Thermo Scientific, USA).The minimum concentration of the tested herbs that inhibits the growth of the E. faecalis after 24 h of incubation was recorded as the MIC.The growth inhibition of E. faecalis grown in suspension mode by the tested herbs was expressed as the ratio relative to positive control, NaClO.

Anti-Adhesion Assay and Biofilm Disruption Test
For the anti-adhesion assay, both E. faecalis cultures (1 × 10 5 CFU/mL) and the studied herbs were co-incubated into sterile 96-well plates in the anti-adhesion test for 6 and 24 h as described previously.In biofilm disruption test, E. faecalis biofilm was pre-grown in 96-well plates for 48 h.The prepared herbs were added to the preformed biofilm and further incubated for 24 h.Untreated E. faecalis acted as the negative control, while ampicillin (5 mg/mL) and 2.5% NaClO-treated biofilm acted as the positive controls for both tests.The biofilm formation in both tests was quantified using crystal violet staining.The absorbance of growth turbidity was measured with a plate reader at 590 nm wavelength.

Quantification of Biofilm by Crystal Violet Staining
Biofilm formation by E. faecalis was quantified according to Yamada et al. (2005) with modifications.The media containing unattached cells was gently pipetted out.Two hundred microliters of 0.1% crystal violet solution (Sigma, USA) was added and incubated for 15 min at room temperature.The crystal violet solutions were gentl 80% ethan One-hundr The absorb

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The MICs inhibition 0.313 mg/m stamineus of the teste    3i and 3j).On higher magnification, the cell surfaces of E. faecalis showed irregular, pitted appearance (Figure 3k, arrow).

Discussion
E. faecalis are non-spore forming, fermentative, gram-positive organism (Suchitra & Kundabala, 2006).They are usually associated with endodontics infection and implicated more commonly in persistent and secondary infections (Sedgley et al., 2005;Johnson et al., 2006).The persistent infection is caused by microorganisms that can resist intracanal antimicrobial procedures and can survive under extreme conditions (Narayanan & Vaishnavi, 2010).The entry of E. faecalis into the root canal usually occurs during endodontic treatment, between appointments or even after root canal treatment (Stuart et al., 2006).E. faecalis possesses virulence factors such as lytic enzymes, cytolysin, aggregation substance, and lipoteichoic acid (Stuart et al., 2006).E. faecalis is able to form biofilm within the infected root canal system, which renders the bacteria resistant to phagocytosis, antibodies, and antimicrobials (Stuart et al., 2006;Liu 2010).The standard intracanal medicaments used in root canal treatment include calcium hydroxide and disinfectant such as sodium hypochlorite.However, E. faecalis biofilm has been reported to resist these common intracanal medicaments (Stuart et al., 2006;Liu, 2010;Sathorn et al., 2007;Haapasalo & Shen, 2012), thus contributing to the failure of root canal treatment and persistent root canal infection.The current irrigant solution, sodium hypochlorite used in cleaning the root canal system is toxic to the surrounding soft tissue (Borzini et al., 2016).Thus, there is a need to find safer, gentler irrigant.E. faecalis has been reported to be resistant towards intracanal medicament such as calcium hydroxide when the pH is not maintained at a therapeutic level.This survival capacity of E. faecalis is facilitated by a functioning proton pump with the capacity to acidify the cytoplasm whenever the pH is high (Evans et al., 2002).Apart from that, starved E. faecalis were able to form a biofilm that was more resistant to 5.25% sodium hypochlorite than those in stationary and exponential phase (Liu, 2010).
The examination of the growth inhibition effect of the studied herbs against E. faecalis was carried out using broth microdilution tests.The present study showed that both ginger oil and O. stamineus extract exhibited growth inhibition activity towards E. faecalis grown as a suspension culture.Comparatively, ginger oil showed a greater inhibitory effect to O. stamineus extract.This may be contributed by the herb preparations; ginger was used as essential oil, while O. stamineus was prepared by reconstituting the powdered plant with water, thus it can be regarded as a crude preparation of the herb.In the form of essential oil, the antibacterial components of ginger, namely gingerols and shogaol (Park, Bae, & Lee, 2008;Wang et al., 2010) may be in higher concentrations, thus showing good growth inhibition activity.The O. stamineus water extract may have lower concentrations of its antibacterial component, rosmarinic acid, which is the primary polyphenol in O. stamineus leaf (Akuwoah et al., 2004).
In most oral diseases involving microorganisms, the initiation of a disease requires the pathogens to adhere to the tooth supporting structure or tooth surface (Li et al., 2004).Substances that possess bacterial anti-adhesion activity hold a particular advantage in preventing the adhesion of bacteria to a surface, which is the required initial step for a pathogen to establish infection.In this study, both ginger oil and O. stamineus extract showed inverse dose-dependent anti-adhesion activity following both exposure time lengths of 6h and 24 h.Significant anti-adhesion activity relative to ampicillin was observed only at the two lowest ginger oil concentrations.At higher concentrations, more abundant E. faecalis adherence was observed for both herbs.It is suggested that there may be unknown compounds in ginger oil and O. stamineus extract when at higher concentrations, are able to promote E. faecalis adhesion.For O. stamineus it has no anti-adhesion activity following 6 h exposure.At 24 h, both studied herbs generally showed lower anti-adhesion activity as the adhered cell layer may have got thicker.No significant biofilm disruption activity was observed for both studied herbs compared to positive control.This may simply because the preformed E. faecalis biofilm may have matured, where a high amount of extracellular polymeric matrix that encased the E. faecalis cells protects the bacteria from external insults, such as antimicrobial substances (Rickard et al., 2003).E. faecalis in suspension mode do not possess this protective mechanism, which may suggest a greater antibacterial activity of the herbs towards this growth mode than in the biofilm mode.
Our findings suggest that ginger oil, in lower concentrations, has more potential to be developed into antibacterial agent against bacteria in suspension growth mode rather than.The O. stamineus extract used in this study was more in a crude form, which may explain its low antibacterial activity.Previous studies which reported the protective effects of O. stamineus employed methanolic (Chun-Hoong et al., 2010;Malahubban et al., 2013) and ethanolic extracts (Alshawsh et al., 2012;Mohamed et al., 2012).
The findings on the antibacterial activity of ginger oil in this study were in contrast with the study by M. N. J. Gulve and N. D. G. Gulve (2010) which showed ginger extract has strong anti-bacterial activity against E. faecalis in agar diffusion test.Our study reported that ginger oil has a significant antibacterial effect on E. faecalis in suspension culture but not in biofilm mode.This may be due to different growth inhibition assays were used, sample size difference and ginger oil concentration difference.Park et al. (2008) showed that ginger extract can inhibit the growth of oral bacteria in suspension mode, which have good agreement with our results.One of the active agents of ginger, [10]-gingerol has been reported to cause damage to the membrane of enterococcus cells (Nagoshi et al., 2006).This damage was similar to the SEM findings in this study, where changes such as pitted cell surface and disintegrated cells were seen on E. faecalis following exposure to the studied herbs.According to Lim et al. (2009), hydroethanolic ginger extract exhibits potent antibacterial activity against Gram-positive and Gram-negative bacteria.In the past, essential oils had been demonstrated to be as effective as antimicrobial agents to use in oral care (Tufekci et al., 2008).An important characteristic of essential oils and their components is their hydrophobicity, which enables them to partition into and disturb the lipid bilayer of the bacterial cell membrane, rendering them more permeable, leading to leakage of cell contents (Burt, 2004).According to Unlu et al. (2010), cells incubated in essential oil for 48 h showed obvious changes in their morphology and loss of adhesion, which was similar to our findings for E. faecalis treated with ginger.

Conclusion
Z. officinale and O. stamineus showed effective antibacterial effect towards E. faecalis, particularly in growth inhibition and anti-adhesion, but less effective in disrupting preformed biofilm.Comparatively, Z. officinale oil has more potential to be developed as antibacterial agent against E. faecalis in suspension culture compared to O. stamineus extract.
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