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

Plant collection and extraction

Fresh leaves of Psidium sp. (voucher no. 48126) and Mangifera sp. (voucher no. 48124) were obtained from Balai Ungku Aziz of the University of Malaya, Kuala Lumpur and the UPM Agriculture Park, Selangor respectively. Leaves of Mentha sp. (voucher no. 48127) grown in Cameron Highlands, Pahang, were obtained from a local market in Selangor, Malaysia. The plants were identified at the Rimba Ilmu Herbarium (University of Malaya) and deposited under the stated voucher numbers. The fresh plant leaves were washed with tap water, followed by deionised water, dried using tissue paper, weighed and cut into small pieces before boiling based on the method described by Nalina & Rahim (2006). Briefly, 1 g of fresh leaves was boiled in 1 L of deionised water for several hours until the final volume is one-third of the initial volume. The decoction was then filtered using a muslin cloth to remove any debris and the clear filtrate was further centrifuged at 1,500 × g, 4°C for 15 min to eliminate any sediment. The supernatants were filtered using Whatman No. 1 paper with a diameter of 150 cm and boiled again until the final volume of 100 ml. The supernatants were then frozen overnight at −80°C followed by freeze-drying for 2 days using freeze dryer (Eyela FDV-1200, China) in a sterile environment. The sterile dried crude aqueous extracts were stored at −20°C for further use.

Preparation of bacterial suspension

S. sanguinis ATCC BAA-1455 (strain SK36) and S. mutans ATCC 25175 used in this study were obtained from American Type Culture Collection (ATCC, USA). Prior to the experiment, the respective 20% glycerol stocks of each bacterium at −80°C were thawed at room temperature and 1% of the strains were transferred in 20 ml of sterile fresh Brain Heart Infusion (BHI) broth (Oxoid Ltd, Basingstoke, Hampshire, UK), incubated aerobically at 37°C with shaking at 150 rpm until mid-log phase of growth (6–8 h). The bacterial suspensions were centrifuged at 5,800 × g, 4°C for 10 min and the pellets were washed three times with ice-cold sterile deionised water, suspended in fresh BHI broth and incubated at 37°C for 15 min to reactivate their growth phase. The turbidity of each strain was standardised by adjusting the absorbance to 0.144 (equivalent to 1.00 × 10⁸ CFU/ml and 1.53 × 10⁷ CFU/ml for S. sanguinis and S. mutans respectively) at 550 nm using a spectrophotometer (Shimadzu UV-1700, Japan). For dual-species, the standardised suspensions of the two bacteria were mixed (1:1 vol/vol) prior to the experiment. The purity of the bacterial cultures was checked each time prior to every experiment by streaking the culture broth on BHI agar and incubated at 37°C for 24–48 h.

Determination of minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and the sum of the fractional inhibitory concentration (ΣFIC) index

A broth microdilution method in accordance with standard protocol CLSI (CLSI, 2012) and modified method of Kuete et al. (2009) were used to determine the MIC of PEM and its individual constituent plant extracts (Psidium sp., Mangifera sp. and Mentha sp.). About 1 g of sterile dried extract (Psidium sp.) was reconstituted in 7.81 ml sterile deionised water giving a final concentration of extract at 128 mg/ml. After sonication for 30 s and vortexing for 30 min, the reconstituted extract was centrifuged at 1,500 × g, 4°C for 15 min. The supernatant of the reconstituted extract was serially diluted two-fold in BHI broth to obtain a range of concentration (0.25–64 mg/ml) in respective test tubes labelled as T1–T9. The diluted extracts were transferred into the corresponding wells of a 96-well microtitre plate (NUNC™ Brand products) as follows. To the first three rows of the 96-well microtitre plate labelled as A1–A9; B1–B9; C1–C9 (carried out in triplicate to ensure reproducibility), 100 μl of the respective serially diluted plant extract in BHI broth (T1–T9) was added. To wells labelled as A11, B11, C11, 100 μl of BHI broth was added to ensure bacterial growth. To all of the wells above, 5 μl of S. sanguinis suspension (standardised to 10⁶ CFU/ml) was added. This gave the final concentration of the extract in the respective wells (A1–A9; B1–B9; C1–C9) a range of concentration from 0.24–60.95 mg/ml. In addition to these, wells labelled as A12, B12, C12 was filled with 105 μl of BHI broth just to ensure the sterility of the broth. To another row of wells, one row apart from the others (E1–E9), 105 μl of the two-fold serially diluted plant extract only was added which served as blank control. The microtitre plate was sealed and covered with aluminium foil, incubated aerobically at 37°C with shaking at 150 rpm for 24 h. Following incubation, the MIC was determined at 550 nm using ELISA reader based on turbidity determined by comparing the absorbance of the suspension in wells of test extract (A1–A9; B1–B9; C1–C9) with that of the corresponding blank control. The MIC of each compound was defined as the lowest concentration that inhibited the bacterial growth. Similar procedure was repeated with S. mutans. The similar experiment was repeated for the other plant extracts (Mangifera sp., Mentha sp. and PEM). For PEM, an equal volume of the stock solution of extracts (Psidium sp., Mangifera sp. and Mentha sp.) each with a concentration of 128 mg/ml was mixed followed by two-fold serial dilution.

For the MBC determination, 20 μl of the bacterial suspension from selected wells (A1–A9; B1–B9; C1–C9) which showed almost no turbidity was inoculated on BHI agar (Oxoid Ltd, Basingstoke, Hampshire, UK) and incubated for 48 h at 37°C. The lowest concentration of the extracts that showed no growth corresponded to the MBC value.

The sum of the fractional inhibitory concentration (ΣFIC) determines the synergy of the plant extracts, calculated using the following equation: ${\displaystyle \begin{array}{ccc}\hfill & \hfill & \mathrm{\Sigma }\mathrm{FIC}=1∕3\left({\mathrm{FIC}}_{\mathrm{I}}+{\mathrm{FIC}}_{\mathrm{II}}+{\mathrm{FIC}}_{\mathrm{III}}\right)\hfill \end{array}}$ and the FICs were calculated as follows: ${\displaystyle \begin{array}{c}\hfill \\ \hfill & \hfill & {\mathrm{FIC}}_{\text{I}}\left(\text{FIC of}Psidium\text{sp.}\right)=\frac{\text{MIC (PEM)}}{\text{MIC}\left(Psidium\text{sp.}\right)}\hfill & \hfill \\ \hfill & \hfill & {\text{FIC}}_{\text{II}}\left(\text{FIC of}Mangifera\text{sp.}\right)=\frac{\text{MIC (PEM)}}{\text{MIC}\left(Mangifera\text{sp.}\right)}\hfill & \hfill \\ \hfill & \hfill & {\text{FIC}}_{\text{III}}\left(\text{FIC of}Mentha\text{sp.}\right)=\frac{\text{MIC (PEM)}}{\text{MIC}\left(Mentha\text{sp.}\right)}.\hfill & \hfill \end{array}}$ The ΣFIC index determines the interaction between the different plant extracts in the mixture where the interaction is interpreted according to a range of values; value less than 0.5 as synergistic, value greater than 0.5–1 as additive, value greater than 1–4 as indifferent and value greater than 4 as antagonistic (Van Vuuren & Viljoen, 2011).

Preparation of sterilised saliva

Undiluted sterile saliva was prepared according to the method described by De Jong & Van der Hoeven (1987) and Nordini, Fathilah & Rahim (2013). To minimize variation, a single volunteer was used to collect 100 ml of stimulated whole saliva (SWS). The SWS collection was done by expectoration after chewing sugar-free gum (before eating or at least 2 h after eating) in ice-chilled test tubes. The aggregation of the protein in the SWS was minimised by adding 1,4-Dithio-DL-threitol (DTT) (GE Healthcare, Danderyd, Sweden) to a final concentration of 2.5 mM. The mixture was stirred slowly for 10 min followed by centrifugation at 800 × g, 4°C for 30 min. The supernatant was then filter sterilised through a disposable 0.22 μm low-protein-binding filter (Cellulose acetate syringe filters Sartorius, USA) into sterile centrifuge tubes and stored at −20 °C until further use. Prior to the experiment, the sterile SWS was thawed and centrifuged to remove any precipitate It was later used to coat glass beads (3 mm in diameter) (Merck, Damstadt, Germany) in an artificial mouth (NAM) model forming a layer that mimics the acquired pellicle on the tooth surface and thus referred as the Experimental Pellicle.

Ethical approval

The study protocol was reviewed and approved by the Ethics committee of Faculty of Dentistry University of Malaya, Kuala Lumpur, Malaysia (The Ethic committee/IRB reference number: DF OB1506/0070(P). Written informed consent was obtained from one donor for saliva collection.

Anti-adherence effect of plant extracts

Prior to use, the dried aqueous extracts were reconstituted in sterile deionised water to a final concentration of 0.5 mg/ml. The reconstituted extracts were further sterilised by filtration using 0.22 μm nylon syringe filters (Millipore, Billerica, MA, USA). For PEM, the same concentrations of the sterile reconstituted individual extracts were mixed (1:1:1 vol/vol/vol) to give a mixture of extracts of the same concentration (0.5 mg/ml).

Nordini’s Artificial Mouth (NAM) model developed by Rahim et al. (2008) was used in the in vitro study of the anti-adherence effect of PEM and its individual constituent plant extracts towards the early and late plaque colonisers in single- and dual-species biofilms. The NAM model was used in the study to represent oral cavity. The model consists of a glass chamber (1 × 6 cm length), glass beads (3 mm diameter), water bath system, saliva reservoir, bacterial reservoir and a peristaltic pump. In this study, nine glass beads were placed in the glass chamber of the NAM model. The glass beads represent the tooth surface. The glass chamber which was immersed in the water bath system, set at 37°C represents the oral cavity. The peristaltic pump (Masterflex L/S, USA) of the NAM model was used to flow the saliva/test extract/bacteria from the reservoir into the system via rubber tubing at a constant flow rate (0.3 ml/min for resting saliva). The experiment was carried out as follows. Saliva was first flowed into the NAM model for 2 min at a constant rate to form an experimental pellicle on the glass beads. Excess saliva was rinsed off by allowing sterile deionised water to flow in for 2 min. This allowed the experimental pellicle to be pretreated with sterile deionised water. Following this, S. sanguinis which was standardised to 0.144 absorbance at 550 nm wavelength was allowed to flow into the NAM model for 24 h to form a 24-h biofilm on the experimental pellicle-coated glass beads.

Of the nine glass beads, three were taken out randomly whereas the remaining six glass beads were kept for SEM viewing and reserves. The three glass beads were then individually placed in a sterile microcentrifuge tube containing 1 ml Phosphate Buffered Saline (PBS) (pH 7.4), sonicated for 10 s and vortexed for one minute to dislodge the attached bacteria. The suspension was serially diluted ten-fold (10⁻¹–10⁻⁵ dilutions) with PBS. A 100 μl of the homogeneous bacterial suspension from each tube was pipetted out and plated on three separate BHI agar plates using the Lawn method. The plates were incubated for 24 h at 37°C. The counting of all viable microorganisms formed on the plate done using colony counter. Plates from the dilution which gave a CFU number of between 30–300 colonies were selected and used in the calculation of the growth population. The bacterial suspension used in the plating was 100 μl and hence it was expressed as CFU/ml based on the following formula: ${\displaystyle \text{Total CFU/ml}=\frac{\text{Number of colonies}}{\text{Volume plated (ml)}\times \text{Dilution factor}}.}$ The deionized water treated experimental pellicle served as negative control for S. sanguinis in single-species biofilm and the bacterial population (total CFU/ml) assumed as 100% bacterial adherence. Similar procedure was repeated with S. mutans (standardised to 0.144 absorbance at 550 nm) for single-species biofilm. For dual-species biofilms, the individual strain was mixed in equal amount (1:1 vol/vol) and a similar procedure was repeated. In the dual-species biofilms, the two bacteria were differentiated according to their colony sizes. S. mutans colony is larger than S. sanguinis allowing the colonies to be counted separately.

The experiment was subsequently repeated where the experimental pellicle was pretreated with respective plant extract (Psidium sp./ Mangifera sp./ Mentha sp./PEM) at sub-MIC (0.5 mg/ml) before inoculation with bacterial suspension used for the development of single- and dual-species biofilms. For positive control, the experimental pellicle was pretreated with 0.12% chlorhexidine gluconate (CHX). The individual experiment was carried out in triplicate.

Percentage of adherence (X) was calculated using the equation below: ${\displaystyle \text{X}=\frac{\text{adhered cells (test or positive control)}\times 100}{\text{adhered cells (negative control)}}.}$ Assuming the bacterial adherence to negative control was 100%, the percentage of anti-adherence was calculated as: ${\displaystyle \text{\%Anti-adherence}=100-\text{X}.}$

Scanning Electron Microscopy (SEM) analysis of cell population on glass beads

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°C until the subsequent analysis. Prior the analysis, 4% glutaraldehyde solution was pipetted out and washed twice with sodium cacodylate buffer for 15 min, followed by postfixing with 2% (vol/vol) osmium tetroxide in 1% (wt/vol) sodium cacodylate buffer solution for 1 hour and kept at 4°C. Then, the glass beads were rinsed with deionised water twice for 15 min before dehydration process. A series of 30, 50, 70, 80, and 95% ethanol at an interval of 15 min was used for dehydration process. This was followed by dehydration carried out twice for 15 min each using 100% ethanol. Ethanol was gradually displaced with acetone in the following ratios (vol/vol); Ethanol:Acetone 3:1, 1:1, 1:3 each for 20 min and finally followed by 100% acetone three times for 20 min. The dehydrated samples were then subjected to Critical Point Drying (CPD) (Balzers CPD 030; Balzers Union, Balzers, Liechtenstein) for 1 h 40 min in liquid CO₂ under 95 bar pressure. The glass beads were then kept in a tight container in a desiccator. Prior to SEM viewing, the beads were gold-coated under low pressure with ion sputter coater (Joel JFC1100, Japan). The beads were viewed for cell population at 10,000X magnification using Scanning Electron Microscope (Quanta FEG 250; FEI, Eindhoven, The Netherlands).

Statistical analysis

The experiments were repeated and analysed using three triplicates and expressed as a mean ± standard deviation, where the number of determinations, n = 27. Data were analysed using IBM SPSS statistical software, version 23. The Shapiro–Wilk test was used to test assumptions of normality. The results were analysed using Kruskal–Wallis and Mann–Whitney tests for non-parametric statistical analysis to compare the difference between the two independent groups. Results were considered significant at p < 0.05.

Minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and the sum of the fractional inhibitory concentration (ΣFIC) index of plant extracts

Table 1 shows the MIC, MBC and ΣFIC index of the PEM and its individual constituent plant extracts against the early (S. sanguinis) and late (S. mutans) plaque colonisers. The MIC values of PEM (S. sanguinis 3.81 mg/ml; S. mutans 1.91 mg/ml) were lower than those of the individual extracts for the two bacteria. Among its individual constituent plant extracts, the Psidium sp. showed the lowest MIC against S. mutans (3.81 mg/ml) whereas Mentha sp. and Psidium sp. have almost similar MIC against S. sanguinis (7.62 mg/ml). It is shown that the MBC were higher than the MIC for all the extracts. Psidium sp. (MBC value of 15.24 mg/ml) showed the higher bactericidal effect compared with Mangifera sp., Mentha sp. and PEM (MBC value of 30.48 mg/ml) against S. sanguinis. The MBC value exhibited by PEM and Psidium sp. against S. mutans (30.48 mg/ml) was lower compared with Mangifera sp. and Mentha sp. (60.95 mg/ml). It was found from the calculation of the ΣFIC index, the interactions of the plant extracts are synergistic for the two bacteria where the index is lower for S. mutans (ΣFIC = 0.25) compared with S. sanguinis (ΣFIC = 0.42).

Bacterial population adhering to the experimental pellicle

The bacterial population for S. sanguinis and S. mutans adhering to experimental pellicle without treatment (negative control) either individually (single-species) or together (dual-species) is shown in Table 2. In a single-species biofilm, the population of S. mutans ((2.48 ± 0.16) × 10⁷ CFU/ml) adhering to the experimental pellicle was ten times higher compared with S. sanguinis ((2.30 ± 0.37) × 10⁶ CFU/ml) and it is statistically significant (p < 0.05). However, in dual-species biofilms, the S. mutans population ((1.54 ± 0.35) × 10⁷ CFU/ml) adhered in similar proportions with S. sanguinis ((1.49 ± 0.36) × 10⁷ CFU/ml).

Anti-adherence effect of plant extracts in single- and dual-species biofilms

Figure 1 shows the anti-adherence effects of PEM and its respective constituent plant extracts in single-species biofilm (S. sanguinis and S. mutans). The anti-adhering effect of all the extracts (except Psidium sp.) towards S. sanguinis was significantly higher when compared with S. mutans.

Figure 2 shows the anti-adherence activities of the PEM and its individual constituent plant extracts in the dual-species biofilms. It was observed that Psidium sp. also demonstrated similar and higher anti-adherence activities towards S. sanguinis (88.54 ± 4.47%) and S. mutans (88.65 ± 3.22%) compared with PEM, Mangifera sp. and Mentha sp. The Mentha sp. exhibited significantly higher anti-adherence activity towards S. sanguinis (70.12 ± 6.90%) compared with S. mutans (50.54 ± 18.64%). PEM demonstrated comparable anti-adherence effect towards S. sanguinis and S. mutans (47.50 ± 17.93% and 49.08 ± 18.90% respectively).

Bacterial adherence to plant extracts treated-experimental pellicle viewed under SEM

Figures 3 and 4 show the SEM photographs of single-species biofilm viewed at 10,000X magnification displaying the cell population adhering to the experimental pellicle (saliva-coated glass beads) treated with plant extracts as well those of the negative and positive controls. The population of S. mutans and S. sanguinis were almost zero in single-species biofilm adhering to 0.12% CHX-treated experimental pellicle (positive control) and very much reduced with plant extract-treated when compared with the negative control.

Figure 5 shows the SEM photographs of the dual-species biofilms. It was observed that the population of the two bacteria adhering to Psidium sp.-treated experimental pellicle was highly reduced (Fig. 5D). It was different for Mentha sp-treated experimental pellicle, the adhered population of S. sanguinis was less compared with S. mutans (Fig. 5F). The adherence of the two bacteria to Mangifera sp.- (Fig. 5E) and PEM-treated experimental pellicle (Fig. 5C) respectively were almost comparable.