Dental caries is the main public health concern ranked first for the permanent tooth decay of about 2.3 billion people. ¹ Specific bacteria are related to dental caries initiation and progressions. Streptococcus mutans is considered the principal etiological agent of dental caries due to its adherence ability to tooth surface, sucrose fermentation, extracellular polysaccharides, and acid production. ² , ³ Hence, the reduction of S. mutans or its attachment to tooth surfaces can prevent or decrease caries development. ⁴

Today, there are several mechanical, chemical, and biological preventive strategies to control the caries progression. ⁵ However, despite the progress in preventive methods, dental caries distribution in developed countries remains high. Hence, there is a great need to look for novel preventive methods. Besides, there are some well-known anti-microbial agents like chlorhexidine gluconate (CHX) which is very successful in reducing the salivary mutans Streptococci count, but it is not effective as a preventive agent due to its adverse effects like teeth and tongue discoloration and bacterial resistance. ⁶ , ⁷

However, biofilm elimination from teeth surfaces remains a challenge because the nature of structural and physiological properties of biofilm bacteria provides natural resistance to anti-bacterial agents. ⁸ Thus, further researches and development of alternative natural and safe antimicrobial agents for dental caries prevention are needed. In recent decades, much attention has been attracted to the use of traditional herbal agents due to exhibiting potent anti-bacterial, anti-cancer, anti-adhesion, or anti-cariogenic activities of plant-derived active compounds. ⁹ , ¹⁰ , ¹¹

One of the traditional plants is Pistacia atlantica (P. atlantica) which is called ”Banneh” in Iran. It is one of the most important tree species commonly distributed in different countries such as Iran, Iraq, Algeria, and turkey. ¹² The resin of P. atlantica tree has various beneficial effects and it is traditionally used in Iran as peptic ulcer treatment, mouth freshener, antiseptic, gum tissue strengthener, appetizer, astringent, diuretic, gastrointestinal disorder treatment, etc. ¹³ Other studies have reported the anti-fungal, anti-parasite, and anti-bacterial activities of P. atlantica. ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ So, this study aimed to investigate the anti-cariogenic efficacy of the essential oil (EO) and methanolic extract (ME) of P. atlantica resin through the investigation of their anti-bacterial, and anti-adhesion activities against strongly adherent (SA) S. mutans strains in vitro.

Bacterial strain and growth conditions

S. mutans ATCC 35668 and other SA strains, isolated from caries lesions, were provided from the Microbiology Laboratory of Isfahan branch, Azad University, and cultured in tryptic soy broth (TSB; Merck, Germany). The growth profile of the strains was recorded at time intervals. The antibiotic susceptibility of the strains was evaluated by the disc diffusion method (Kirby and Bauer) on Mueller Hinton agar (Merck Co. Germany). ¹⁸ The antibiotic discs (Padtan Teb Co. Iran) used were: Pencillin-G (P; 10 μg), Cefalothin (CF; 30 μg), Cefalexin (CN; 30 μg), Vancomycin (V; 30 μg), Gentamycin (GM; 10 μg) and Streptoomycin (S; 10 μg).

Biofilm formation ability

To select SA strains, the biofilm formation ability of strains was quantified by a microtiter plate method as previously described. ¹⁹ Briefly, overnight cultures of strains in TSB supplemented with and without substrates (Sucrose 1% and a mixture of 0.5% glucose and 0.5% fructose) were prepared. Their turbidity was adjusted to 0.5 on Mc Farland turbidity. Each bacterial suspension (250 μl) was transferred into wells of a microtiter plate, incubated (35 ° C, 24 h), the wells were emptied, and washed with sterile PBS solution. The remaining attached bacteria were fixed (96% methanol: 250 μl/well; 15 min), stained (2% crystal violet: 200 μl; 5 min), the excess stain was washed off, and the plates were left to dry. The formed biofilm was quantified by resolubilizing the stain in 200 μl of 33% (v/v) glacial acetic acid (Merck, Germany) per well. The optical density (OD) of the stain was measured at 492 nm by an ELISA reader (TECAN Co. Spectra SLT). Control wells only contained broth. According to the obtained ODs, strains can be classified into the different categories:

OD < ODC = nonadherent (NA); ODC < OD < 2ODC = Weakly adherent; 2ODC < OD < 4ODC = Moderatly adherent; 4ODC < OD = SA (or strong biofilm producer).

OD and ODC are the mean OD of wells with biofilm and the control wells, respectively.

Resin collection, essential oil and methanolic extract preparation

P. atlantica resin was gathered from the Zagros Mountains of Iran. ME and EO were obtained from P. atlantica resin. The EO was extracted by hydrodistillation method (150 g; with 3 L water) and pale yellow oil (12 g) was obtained (8% v/w). ²⁰

Anti-bacterial activity assay

Anti-bacterial activity of EO and ME was evaluated by the microdilution method as described previously. ²¹ The crude EO and ME of P. atlantica were used as stock solutions with the concentration of 300 mg/ml in DMSO which was nontoxic to bacteria. The S. mutans suspension (20 μl equal to 0.5 Mc Farland suspension.), diluted extracts at different concentrations (30 μl of 60%-100%), and BHI broth (200 μl) were added to each well of 96-well microtiter plates. Wells containing CHX (0.65 mg/ml) and bacterial suspension without any inhibitory compound (non-treated) were considered as the positive and negative controls, respectively. Then, plates were incubated (37 °C, 18 h) and the optical densities of wells were read by the ELISA reader at 620 nm and compared. Each control or treatment was tested in 8 replicates.

Anti-adhesion activity assay

The anti-adhesion assay was performed according to Tahmourespour et al.(2019) with some modifications as follows: ²¹ Each column of a 96-well microplate was filled with 200 μl/well of each EO and ME concentration. The plate was shaken (1 h) and the wells were emptied. S. mutans suspension (20 μl) along with TSB containing 1% sucrose (200 μl) were added to them and incubated (4 h, 37°C). The unattached cells were rinsed then adhered S. mutans cells were fixed, stained and the optical density of every well was read as described above. This assay can estimate the microbial adhesion reduction percentage versus the control wells. Control wells contained PBS buffer instead of ME or EO.

Statistical analysis

Each experiment was done in triplicate. The obtained data were statistically analyzed using a software package SPSS-20 (SPSS Inc., Chicago, IL, USA) with one-way and two-way analysis variance. Direct comparisons between the two groups were made using the Wilcoxon W-Mann–Whitney U-test. Statistical significance was set at P < 0.05.

Figure 1shows the optical densities obtained from the growth of S. mutans strains after 24 h of incubation in the same medium and condition which did not show any significant difference using the Kruskal–Wallis test (P > 0.05). The results of in vitro antibiotic susceptibility tests of selected strains are summarized in Table 1. No significant differences (P > 0.05) in antibiotic-resistance rates between the tested strains were found. The resistance prevalence against the tested antibiotics was relatively low, ranging from 0% (Vancomycin) to 100% (Streptomycin). Figure 1

The comparison between the growth rate of Streptococcus mutansstrains after 24 h incubation in same condition. (OD: Optical density; STD: Standard strain of S. mutans ATCC35668;SM : Streptococcus mutans).

As S. mutans is the main bacterium responsible for early colonizing the oral cavity, it is necessitated in caries progression from its initiation. Dental caries is an important oral health problem that needs more investigation. In this regard, the use of different mouthwashes with anti-microbial activity is increased, but most of their major components (e.g., CHX) can cause various side effects. ⁴ Hence, to overcome and reduce such side effects, different studies have been evaluated the anti-microbial and anti-biofilm effects of various herbal extracts. ⁶ , ⁷ , ²¹ , ²² Since dental plaque formation as a biofilm initiates with the adherence of bacteria (S. mutans) to the pellicle and in situ growth of attached cells, finding herbal agents with the anti-adherence activity along with anti-microbial property can possess a beneficial role in preventing biofilm and caries development. Although P. atlantica species are considered the best known and one of the most appreciated medicinal plants in countries like Iran, Turkey, etc., still very few studies regarding its anti-cariogenic potential are available in literatures. Hence, in this study, the anti-bacterial and anti-adherence potential of EO and ME derived from P. atlantica resin was investigated, because the resin has been utilized for 5000 years ago for a wide range of purposes. ²³

The target strains of this research were selected with the aim of inhibition or suppression of growth and biofilm formation by the EO and ME of P. atlantica resin. At first, the properties of target strains such as their planktonic growth rate, antibiotic susceptibility profile, and biofilm formation ability were investigated. According to the results, no statistically significant difference in each of the examining properties was observed between the selected strains. Hence that, all 4 strains showed the same growth rate and antibiotic susceptibility profile. Also, all were found SA in the existence of sucrose (1%) while they were NA in no sugar condition.

According to the results, P. atlantica resin showed moderately anti-bacterial activity against S. mutans strains. Furthermore, it is observed that its EO possesses significantly higher anti-bacterial activity than the ME. The EO could reduce the growth of S. mutans cells up to 47.1%, while the S. mutans growth reduction in the existence of ME reached up to 39.1%. Mohamed et al.(2007) also showed that the EO of the Pulicaria crispa (Forsk.) Oliv., and Pulicaria undulata had better antimicrobial activity on most of the microorganisms than the ME. ²⁴

Such anti-microbial activity is not supported by other researches as they reported complete growth inhibitory activity or higher anti-microbial activity on S. mutans; Hosseini et al.(2013) showed that the diethyl ether extracts of P. athlatica possess stronger inhibitory activity compared to aqueous extracts, although the bacterial cells in the biofilm were not affected by the extracts. ²⁵ Najafi et al. (2014) also observed the anti-microbial effect of P. atlantica (var. mutica) against Bacillus cereus, Staphylococcus aureus, and Escherichia coli (O157 H7) by the method of disc diffusion which can be attributed to the high content of α-pinene. They also indicated that in the case of Pseudomonas aeroginosa no clear zone was formed. ²⁶ Roozegar et al.(2016) showed the anti-microbial effect of the leaf extract P. atlantica on S. mutans. ²⁷ These differences are possibly due to qualitative and quantitative variations between the content of the EO which are related to several parameters such as plant species and part, cultivars sex, harvesting time, climatic conditions, geographical origin, microbial species and their resistance ability, and methods used to investigate their activity. ²⁸ , ²⁹ , ³⁰ , ³¹ In general, EO is one of the major constituents of various parts of Pistacia species which contains various types of phytochemical agents such as terpenoids, fatty acids, phenolic compounds, and sterols. ²³ Golestannejad et al.(2020) also showed that olive leaf methanolic, ethanolic, and hydroalcoholic extracts have appropriate anti-bacterial activities due to their high phenolic content but they did not study the antibacterial activity of its EO. ³²

The inhibition of S. mutans adherence onto the polystyrene microtiter plate by different concentrations of the extract and EO was also observed in this study. In all tested concentrations of EO, a significantly stronger anti-adherence activity (50%–80%) than ME (up to 22%) was seen. It is probably due to EO constituent effects on hydrophobic interactions, which were reported to be significant in bacterial adhesion onto tooth surfaces. Thus, the surface hydrophobicity modification could be used as an anti-adherence strategy. ³³ As S. mutans adherence to surfaces is mediated by glucan, the product of glucosyltransferases, the anti-adherence property of EO probably could be associated with the anti-glucosyltransferases activity of compounds such as flavonoids and tannins. Hence, the inhibition of such enzymes can be used as another anti-adherence or anti-biofilm formation strategy. The cariostatic efficacy of flavonoids and tannins (a kind of polyphenole) relates to their anti-microbial activity against S. mutans planktonic and biofilms cells; effects on acidogenic or aciduric characteristics, and down-regulating gtf gene expression. ³⁴ Zeng et al.(2019) also demonstrated the anti-biofilm effect of flavonoids, quercetin, and kaemferol against S. mutans. ³⁵ Yoo et al. (2018) showed the anti-microbial activity of β-caryophyllene, EO from clove, against S. mutans biofilm and planktonic cells. They concluded that such EO can decrease S. mutans count, inhibit biofilm formation also decrease Gtfs expression. ³⁶ As the resin of P. atlantica is composed of different substances, it is better to isolate and purify them and evaluate their anti-cariogenic potential one by one, which was the limitation of the present study.

Overall, the growth of SA S. mutans strains was inhibited moderately in presence of EO and ME of P. atlantica resin. The EO exhibited a significantly higher anti-adherence (anti-biofilm) activity than ME against the S. mutans strains. Hence, this compound with anti-bacterial and anti-adherence activities without drug resistance induction potential can serve as a key component of mouthwashes and toothpaste formulations facilitating the prevention of dental caries or other biofilm-related oral diseases. Hence, each of the different herbal extracts or their mixture can be a promising part of an ideal caries management program and still needs additional researches.

Acknowledgment

The authors would like to thank Mr. Khalkhali (Microbiology lab assistant) and Ayla Hoodaji (a student) who helped for this research progression.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.