|Year : 2019 | Volume
| Issue : 5 | Page : 260-267
Antibacterial activity of Algerian chewing sticks extracts on oral denture biofilm
Wafae Didi, Hafida Hassaine, Sarah Gaouar
Laboratory of Applied Microbiology in Food, Biomedical and Environment (LAMAABE), Biology Department - Faculty of Nature and Life, Earth and Universe Sciences, Abou-Bekr Belkaid University of Tlemcen, Tlemcen, Algeria
|Date of Web Publication||24-Sep-2019|
Ms. Wafae Didi
P. O. Box 119, 13000 Tlemcen.
Source of Support: None, Conflict of Interest: None
Aims and Objectives: To evaluate potent antibiofilm activity of two Algerian chewing sticks, Salvadora persica and Juglans regia, on oral species adhered to elderly dentures. Materials and Methods: The antibacterial activity of the plant extracts and the effect on the biofilm formation were evaluated in vitro by the broth micro-dilution assay. The antibiofilm effect was tested in vitro on artificial oral biofilms, adhered to resin composite, and visualized by environmental scanning electron microscope. Results: Data revealed that bacteria of artificial biofilms were less sensitive than the planktonic strains. However, all tested bacteria, planktonic or biofilms, were more susceptible in the presence of extracts, with J. regia extract showing greater activity than S. persica. Conclusion: All tested oral bacteria, planktonic or biofilm, were more susceptible to J. regia and S. persica, respectively. Considering their antibacterial activity, these extracts may be of great interest for future studies about natural antiplaque agents in daily oral-care products, especially for elderly denture wearers.
Keywords: Adhesion, Enterococcus, Juglans Regia, Resin, Salvadora Persica, Streptococcus
|How to cite this article:|
Didi W, Hassaine H, Gaouar S. Antibacterial activity of Algerian chewing sticks extracts on oral denture biofilm. J Int Oral Health 2019;11:260-7
|How to cite this URL:|
Didi W, Hassaine H, Gaouar S. Antibacterial activity of Algerian chewing sticks extracts on oral denture biofilm. J Int Oral Health [serial online] 2019 [cited 2022 Jan 18];11:260-7. Available from: https://www.jioh.org/text.asp?2019/11/5/260/267709
| Introduction|| |
Most of the microorganisms present in the oral biofilm are harmless natural inhabitants, but some have the potential to cause damage to the mineralized tissues or infections in the soft tissues. By inserting foreign bodies such as dental restorations, new niches appear for the microorganisms, promoting biofilm accumulation with a pathological potential. Wearing removable dentures induces local environmental changes, especially the dependent elderly, who do not keep their prostheses sufficiently clean, which result in denture stomatitis.,
“Nature itself is the best physician” the wise statement of Hippocrates has now been updated to include the beneficial influences from the plant kingdom against biofilm-related dental diseases.,,, Chewing sticks may play a role in the promotion of oral hygiene, and further evaluation of their effectiveness is warranted, as stated in the Consensus Report on Oral Hygiene. The traditional medicinal use of Salvadora persica as an antimicrobial stick toothbrush for oral hygiene and to treat gum inflammation is a centuries old practice and a part of the Greco-Arab system of medicine.S. persica is a member of the Salvadoraceae family, has been scientifically proven to contain more than 10 different natural chemical compounds, such as fluorides, silica, tannic acid, resins, alkaloids (salvadorine), volatile oils (simgrins), sulfur, vitamin C, sodium bicarbonate, chlorides, calcium, benzyl isothiocyanate, salicylic acids, sterols, trimethylamine, saponins, and flavonoids, all considered essential for good oral and dental hygiene.
Juglans regia or walnut species, belong to the Juglandaceae family, is a medicinal plant that has been used in traditional medicine for the treatment of a lot of diseases. Walnut bark has been claimed to own anti-inflammatory, blood purifying, anticancer, depurative, diuretic, and laxative activities. It has been reported to contain steroids, flavonoid C-glycoside, flavones, essential oils, tannins, organic acids, phenolic aldehyde, monoglyceride, sesquiterpene, and diarylheptanoid.
Despite the wide use of S. persica and J. regia, the two plants have not received much attention to be exploited rationally and suitably in Algeria. Until now there has never been any study on the antibiofilm activity of the Algerian ecotype of the two plants. In addition, information on the antimicrobial effect on denture biofilm is still scant. Thus, this study aimed at investigating the antibacterial activity of extracts from the two chewing sticks on the growth of oral bacteria, in their planktonic forms and in denture biofilms.
| Materials and Methods|| |
This study was carried out with simple stratified technique and samples of denture plaque from the internal and the external surfaces of complete dentures of outpatients undergoing treatment at University Dental Clinic, Faculty of Medicine of Tlemcen University (Algeria), which were collected and cultured to check for microorganisms during 1 year. Specimens were inoculated onto Columbia agar (Liofilchem, Roseto degli Abruzzi, Italy) supplemented with 5% (v/v) blood. The plates were incubated at 37°C for 48h under aerobic conditions. Isolates were initially identified on the basis of colony morphology, negative catalase reaction, Gram’s stain, API STREP (bioMérieux, Marcy l’Etoile, France), followed by Matrix Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) automatic identification.
Two phenotypic methods were used for detection of biofilm production of all the isolates. The extracellular polysaccharide substance and slime-producing ability of the isolates were determined with the Congo Red Agar (CRA) method, and the modified tissue culture plate (TCP) test was used for the quantification of biofilm.
J. regia bark and S. persica stem as the plant materials were harvested in different area in Spring 2015, and were kept dried for 2 weeks at 30°C before extract preparation [Table 1]. Extract was prepared according to Darmani et al. In brief, ground chewing sticks samples were extracted in 80% methanol. The mixture was left for 3 days at room temperature and then filtered using Whatman No. 4 filter paper (Whatman, Maidstone, England). The ethanolic extract was then concentrated and stored at 4°C until needed for use.
Antibacterial activity was analyzed. Bacterial cells were grown in brain heart infusion broth (BHIB) (Conda Pronadisa, Madrid, Spain). The two plant extract materials were formulated in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Saint-Quentin Fallavier, France) and taken into different concentrations as per the requirements.
Minimum inhibitory concentrations (MICs) were determined by the microtiter broth method in sterile flat bottom 96-well plates. Serial dilution techniques were used to determine the MIC50 and MIC90 of extracts at concentrations of 51.2mg/mL. Optical density of each well was measured at 490nm using a microtiter reader (ELx808IU, BioTek Instruments Inc., Winooski VT, USA). The mean % inhibition of replicate tests was used to determine the final MIC values.
ODt24 = optical density of the test well at 24h postinoculation
ODt0 = optical density of the test well at 0h postinoculation
ODgc24 = optical density of the growth control well at 24h postinoculation
ODgc0 = optical density of the growth control well at 0h postinoculation
A modified microtiter plate test was used to test the effect of plant extracts on biofilm formation. Negative controls (cells +BHIB), vehicle controls (cells + BHIB + DMSO), and media controls (BHIB) were included. Biofilms were established in the 96-well plates for 24h at 37°C. At 24h after incubation, the appropriate concentration of test extract was added. The adherent 24-h biofilm layer formed was stained with 150mL of crystal violet (1%) for 15min at room temperature. Excess stain was rinsed off and the dye was re-solubilized with 150mL of 95% ethanol per well. The concentration at which the extract depleted the biofilm biomass by at least 50% was labeled as the IC50.
where, ODt = optical density of the test well at 24h postinoculation, ODgc = optical density of the growth control well at 24h postinoculation, and ODv = optical density of the vehicle control at 24h postinoculation.
To evaluate the effects of the two different plant extracts on bacterial biofilm, uniformly sized acrylic resin plates (10 ×10 × 1mm) were sterilized and immersed in bacterial suspension and incubated for 24h at 37°C under aerobic condition. After incubation, each plate was washed twice in sterile saline solution to remove loosely adherent microorganisms and then treated with the two extracts. After 8h, each plate was washed and sonicated in 10mL of sterile saline solution. For viable cell counting, each 100 uL was spread on brain heart infusion agar plate. After incubation for 48h, bacterial colonies from each plate were counted and the relative colony-forming units were calculated. All biofilm results were compared to the control, which was incubated with sterile saline solution instead of plant extracts, and were expressed as percentage of microbial survivor.
Electronic microscopy was carried out to observe the effect of extract on Streptococcus gordonii adhesion to resin, after 4h of treatment, using an environmental scanning electron microscope (XL 30 ESEM Philips, Eindhoven, Netherlands).
Growth and biofilm inhibition data were statistically analyzed by Microsoft Excel 2010. Bacterial colony counts were transformed to log values to normalize the data. Mean and standard deviation were recorded for all readings from triplicate independent experiments.
| Results|| |
Bacterial identification observed by isolations were found to be gram-positive rods in chains and catalase negative, which were presumed to be Streptococcus spp. The identity of the selected isolates was confirmed by the analytical profile index (API) of the API 20 STREP system and MALDI-TOF results.
CRA plate method and confirmation by microtiter plate assay revealed the biofilm-forming abilities of all the isolates. Furthermore, the microtiter plate adherence assay indicated that biofilm formation by S. parasanguinis, S. gordonii, Enterococcus faecalis, and E. durans recorded a higher optical density value, and were therefore selected for antibiofilm studies [Table 2].
In this study, two plant species of the Algerian traditional medicine were selected based on their popular use for antibacterial activity.
The extract showed limited activity on the effect on resulted planktonic growth, with an IC50 ranging between 0.8 and 6.4mg/mL.
E. faecalis and E. durans showed more susceptibility to S. persica extract than Streptococcus strains, with an IC50 ≥ 6.4mg/mL [Figure 1]. The activity of J. regia extract was more pronounced against S. gordonii and E. faecalis with an IC50 ≥ 0.8mg/mL [Figure 2].
|Figure 1: The percent inhibition of Salvadora persica extract for planktonic growth in tested strains. Data expressed as mean ± standard deviation (SD). Error bars represent SD|
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|Figure 2: The percent inhibition of Juglans regia extract for planktonic growth in tested strains. Data expressed as mean ± standard deviation (SD). Error bars represent SD|
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The effect of methanolic extract on the attachment and biofilm formation on microtiter plates was investigated. The J. regia and S. persica extracts showed an IC50 biofilm ≥51.2mg/mL for biofilm inhibition. None of the two extracts were able to eradicate biofilm cells completely [Figure 3] and [Figure 4].
|Figure 3: The percent inhibition of Salvadora persica extract for biofilm formation in tested strains. Data expressed as mean ± standard deviation (SD). Error bars represent SD|
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|Figure 4: The percent inhibition of Juglans regia extract for biofilm formation in tested strains. Data expressed as mean ± standard deviation (SD). Error bars represent SD|
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Bacterial attachment on acrylic resin was observed after 8h in contact with the extracts, a reduction in biofilm microbial counting was observed for the incubated systems [Figure 5]. Treated with S. persica extract, oral enterococci were the most resistant strains with a reduction of 97%, followed by S. gordonii (99%), whereas S. parasanguinis showed a complete susceptibility to the two extracts. Overall, J. regia alcoholic extract was found to be the most effective against biofilms, reducing the microbial count by approximately 99%.
|Figure 5: Antibiofilm activity of Juglans regia and Salvadora persica extracts. Data expressed as mean ± standard deviation (SD). Error bars represent SD|
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On electron microscopy, morphological examination showed the presence of elongated cells with interrupted chains after treatment with S. persica extract [Figure 6]B. Bacterial attachment and aggregation on resin had notably increased after treatment with the herbal agents, whereas the ESEM images of S. gordonii cells incubated with the J. regia extract revealed many structural changes in cell morphology [Figure 6]C. The cells were damaged and shredded and their contours became distorted. The action of the extracts led to the formation of indentations in cell membranes and subsequent leakage of cells content. However, it is worth noting that untreated control cells presented a typical streptococcal appearance as cocci [Figure 6]A, and the biofilm appeared as a compact structure with bacteria close to one another, whereas in treated biofilm, several gaps were observed.
|Figure 6: Environmental scanning electron microscope (ESEM) images showing the effect of extracts on Streptococcus gordonii biofilm formed on acrylic resin (scale bars = 10 µm). (A) S. gordonii biofilm formed on resin before antimicrobial treatment. (B) Salvadora persica extract effect. (C) Juglans regia extract effect|
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| Discussion|| |
Poor hygiene is a major factor disposing the denture wearers to development of diseases as it allows biofilm formation. Investigation with edentulous patients, in this study as in several other studies, revealed that most of the elderly subjects were not very much aware of the denture hygiene and its various complications. Removable dental prosthesis, such as full or partial dentures, or orthopedic appliance will create new surfaces for biofilm formation and this increases the total amount of biofilm dramatically. Study finding confirms the presence of Streptococcus and Enterococcus species, by biochemical techniques and MALDI-TOF, in addition to their adhesion ability by TCP and the adhesion on acrylic resin in vitro. Viridans streptococci comprise a vital part of the normal flora of the human upper respiratory tract. They play a significant role as a reservoir of antimicrobial resistance genes. Also, enterococci are regarded as a part of oral flora and form most of the primary dental root infections. For these reasons, we selected them in our study, and we included them after being evaluated for the ability of biofilm formation.
Considering the biofilm formation process, any substance acting on preventing the initial adhesion or any other development stage would be useful to control biofilm growth on denture surface and reduce the diseases caused by them. Various domestic methods are available for cleaning, such as sodium chloride solutions, effervescent tablets, and mouth rinse solutions, and also disinfection with medicinal plants such as green tea. Investigations on antibacterial plants and their active constituents have focused entirely on planktonic bacteria with little emphasis on the more resistant and difficult to control biofilm forms. For these reasons and with the purpose of evaluating another potential alternative substance with a possible application in the disinfection of complete dentures, this study was conducted for the first time to investigate the antimicrobial activities of Algerian S. persica and J. regia extracts on the biofilm on dentures of elderly wearers. The results clearly showed that alcoholic extracts of S. persica as well as J. regia could inhibit the planktonic growth of oral bacteria; however, the effectiveness varied against the different tested microorganisms.
Data presented in [Figure 1] and [Figure 2] revealed a fluctuation in the range of MIC values varying according bacteria species showing that E. durans and E. faecalis were the most susceptible strains toward S. persica with MIC of 6.4mg/mL. The MIC values of S. persica were lower than those obtained in a recent study carried out by Chelli-Chentouf et al. They showed that the MIC values of methanolic extract varied with the test microbe from 100 to 400mg/mL. However, the study of Al-Sohaibani and Murugan showed a lower MIC (2.6mg/mL) for the Saudian S. persica. This difference in the antimicrobial activities may be attributed to differences in the timing and location of plant collection, extraction methods, variability in the tested bacterial species, and the evaluation techniques.
The antimicrobial activity observed was in agreement with classic studies in which the effects of miswak extracts on the growth of the various oral and cariogenic microorganisms were verified.,, Many researchers have studied the constituents of miswak and found it to contain more than 10 different natural chemical compounds, all considered essential for good oral and dental hygiene.,,,
Furthermore, a comparative study found that the aqueous extracts of miswak and derum (different type of chewing stick obtained from walnut tree J. regia) were both able to significantly inhibit the growth of cariogenic bacteria, with the derum extract showing greater activity than miswak, which is comparable to our study results, where Algerian J. regia extract showed significant potential against the oral strains with the highest antibacterial activity against planktonic growth with a MIC of 0.8mg/mL. Another study conducted by Sharafati-Chaleshtori et al. where the ethanolic extract of the walnut leaves had shown a significant potential and an MIC of 15.6mg/mL for S. salivarius and S. sanguinis. Results corroborate with the study carried out by Deshpande et al. where acetone extract was found to be more effective as antimicrobial against salivary microflora.
Aiming at restoring the natural antimicrobial capacity of removing adhered bacteria, in vitro, two methods were used to evaluate the antibiofilm activity, the crystal violet assay and the adhesion assay on resin. Both methods showed a good antibiofilm activity exhibited by the two plants. These observations are speculated by the fact that extracts efficiently penetrate in the matrix of extracellular polymeric substances and kill or remove the biofilm-embedded cells.
In the obtained results, a considerable variation for oral isolates was grown as sessile (biofilm) and planktonic populations. Extracts were effective in inhibiting planktonic bacterial growth at low concentrations, but biofilm bacteria appeared 8–60 times more resistant to S. persica and J. regia extracts, respectively. Crystal violet staining showed that the two extracts exhibit antibiofilm activity against Enterococcus and Streptococcus by 50%, whereas the previous investigation conducted by Al-Sohaibani and Murugan revealed a significant reduction of sessile cells, and biofilm formation was inhibited up to 87.92%, and it also indicated that S. persica contains bioactive antibiofilm agents with dual functionalities of growth inhibition and quorum sensing regulator interaction. The “battle” against oral biofilms is a very challenging task, mainly due to their tendency to persist in spite of treatment. This tendency has been attributed to numerous cell–cell communication pathways such as quorum sensing, horizontal gene transfer, and intrabiofilm metabolic transaction. Consequently, biofilm microorganisms can be up to 1000 times more resistant than planktonic bacteria to conventional antimicrobial therapies with antibacterial agents such as antibiotics or chlorhexidine.,
Treated resin showed the number of viable cells statistically smaller than that of control. This showed that the number of bacteria covered by biofilm was smaller over time with the fact of an atypical evolution of biofilm formation stages. In agreement with viable counting cells, ESEM image showed clear effects of these agents on S. gordonii growth in terms of increased attachment. Microscopic observations gave further evidence that the J. regia extract acts by damaging bacterial membranes. As a result, the cells shrink and undergo lysis. In addition, the biofilm appearance suggests that the extract may not only act on the bacterial membranes but also lead to inhibit the attachment and aggregation of the cells.
Overall, this report gives a basis for further in vivo studies and tends to reinforce the use of these extracts as antimicrobial agent in folk medicine. The two plants should be evaluated further in depth to isolate the active components and to clarify their mode of action, hence, their actual effects on denture plaque. However, additional tests, including experimental models and pharmacological applicability, are required before considering these plant extracts as alternative methods in the treatment of oral diseases or for their use in daily oral-care products, gel, rinses, or effervescent tablet preparations, especially for elderly denture wearers.
The S. persica and J. regia extracts are highly potent as antibacterial and biofilm removal agents. These findings highlighted that the bioactive components of the two plants showed useful alternatives to improve denture hygiene and oral health.
Informed consent from all participants was obtained before participating in the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]