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ORIGINAL ARTICLE
Year : 2019  |  Volume : 22  |  Issue : 10  |  Page : 1378-1387

Antibacterial activity of Salvadora persica against oral pathogenic bacterial isolates


1 Department of Botany, Faculty of Science, Tanta University, Tanta, Egypt; Department of Biology, Faculty of Science, Taif University, Taif, KSA
2 Department of Botany, Faculty of Science, Menoufiya University, Al Minufiyah, Egypt
3 Department of Pharmacognosy, Faculty of Pharmacy, Damanhour University, Damanhour, Egypt
4 Department of Botany, Faculty of Science, Tanta University, Tanta, Egypt

Date of Acceptance18-Mar-2016
Date of Web Publication14-Oct-2019

Correspondence Address:
Prof. M A Khalil
Department of Biology, Faculty of Science, Taif University, Taif

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njcp.njcp_413_14

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   Abstract 


Objective: The objective was to determine the antibacterial activity of Salvadora persica extract against bacteria isolated from dental plaque of patients. Materials and Methods: Out of 40 different clinical specimens collected from patients suffering from plaque-induced gingivitis, 12 Staphylococcus aureus and 8 Streptococcus sp. isolates were recovered. The isolates were screened for their biofilm-forming capacity using tissue culture plate (TCP), tube method (TM), and congo red agar (CRA) method. Antibacterial activity of methanolic S. persica extract as well as of commercial antimicrobials against tested isolates was performed. High-performance liquid chromatography–mass spectrometry (HPLC-MS) and gas chromatography-MS (GC-MS) analysis were performed for S. persica crude extract and its volatile oil, respectively, to determine their constituents. Results: Out of 20 isolates, 80%, 85%, and 90% showed positive results using TM, CRA, and TCP, respectively. The highest antimicrobial activity of methanolic S. persica extract was observed at 200 mg/ml. HPLC-MS analysis shows many polyphenols in S. persica extract such as Chrysin-8-c-β-D-glucopyranoside, ferulic acid, gallic acid, and stigmasterol. Chemical composition of the essential oil of S. persica was determined by GC-MS yield; a mixture of monoterpene and hydrocarbons. The major compounds were butylated hydroxytoluene followed by benzene (isothiocyanatomethyl). Conclusion: Methanolic extract of S. persica had significant antibacterial effect against S. aureus and Streptococcus sp. isolates, and it may be gave a good alternative method for controlling oral pathogen.

Keywords: Antibacterial activity, gas chromatography-mass spectrometry, high-performance liquid chromatography–mass spectrometry, plaque-induced gingivitis, Salvadora persica, Staphylococcus aureus, Streptococcus spp


How to cite this article:
Khalil M A, El-Sabbagh M S, El Naggar E B, El-Erian R H. Antibacterial activity of Salvadora persica against oral pathogenic bacterial isolates. Niger J Clin Pract 2019;22:1378-87

How to cite this URL:
Khalil M A, El-Sabbagh M S, El Naggar E B, El-Erian R H. Antibacterial activity of Salvadora persica against oral pathogenic bacterial isolates. Niger J Clin Pract [serial online] 2019 [cited 2019 Nov 17];22:1378-87. Available from: http://www.njcponline.com/text.asp?2019/22/10/1378/269017




   Introduction Top


Dental plaque is a complex biofilm formed on saliva-coated tooth surfaces. Its development is dependent on adhesion of bacteria to salivary components adsorbed to the tooth surface.[1],[2],[3],[4] Plaque-induced gingivitis is the most common of periodontal disease, and is resulting from biofilm- forming bacteria located at the gingival margin. Gingivitis is the inflammation of gingival tissue. Clinical features include redness, swelling and bleeding of the gums. Periodontitis usually develops from untreated gingivitis and can involve loss of bone and tissue decay. The combined activities of microorganisms within the subgingival biofilms and the host responses to them, lead to the progression of the disease and tissue damage.[5],[6],[7] Periodontopathogens include Gram-negative bacteria such as Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Capnocytophaga sp., and Gram-positive bacteria such as Streptococcus faecalis and Streptococcus mutans, which are involved in development of dental caries, are causing decalcification and eventual decay. Staphylococci are considered to be transiently resident in the oral cavity.[8]

The pathogenesis of periodontal diseases may fluctuate from slow, chronic progressive destruction of collagen and aggressive tissue degeneration, to brief and acute with varying intensities and durations. Treatment of periodontal diseases includes biofilm control, root surface debridement or root scaling, surgery, and the use of antimicrobial agents.[5]

The prevention and treatment of plaque-induced gingivitis require control of the causative pathogens within dental plaque [9] by mechanical removing of plaque, using antimicrobial agents or oral hygiene products containing antimicrobial agents.[10] However, majority of the population may not carry out mechanical plaque removal adequately. The antimicrobial mouthrinses and toothpaste that augment daily home care may provide an efficient income of remove or controlling bacterial plaque to limit gingivitis and periodontitis.[11],[12],[13] Recently, appearance of strains with reduced susceptibility to antimicrobial agents that may be attributed to their biofilm structure and the physiological characteristic of strains within the biofilm. Indeed, biofilms can be up to 1000 times more tolerant to antibiotics than equivalent planktonic cultures.[14]

Due to dental treatment was expensive and not so easily accessible, especially in developing countries, the antimicrobial agents used were expensive and had undesirable side effects in humans, and the increasing of prevalence of multidrug-resistant bacteria, urgency search for new infection-fighting strategies to control oral microbial infections was recommended.[15]

Natural plant products are becoming increasingly popular treatments, even for oral health care. Attention is being now focused on the natural substances that possess antibacterial properties extracted from Salvadora persica. S. persica, is belongs to the Salvadoraceae family, composed of carbohydrates, alkaloid (salvadorine), steroids, terpenoids, saponins, flavonoids (quercetin and kaempferol), and glycosides (Kaempferol 3-L rhamnosyl-7-xylopyranoside).[16]

Some studies have focused on the ability of the S. persica extract to inhibit the formation of dental biofilms by reducing the adhesion of microbial pathogens to the tooth surface, which is a primary event in the formation of dental plaque and the progression to tooth decay and periodontal diseases.[17],[18],[19]

The aim of this study is to evaluate the antibacterial activity of S. persica L. extract against tested isolates obtained from patients suffering from plaque-induced gingivitis. The ability of the bacterial isolates to form biofilm was determined. Finally, constituents of the S. persica extract were determined using high-performance liquid chromatography–mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS) analysis.


   Materials and Methods Top


Patients and clinical sample collection

The clinical specimens were collected from 40 patients (24 males and 16 females), aged ranged between 25 and 35 years suffering from plaque-induced gingivitis. The patients were recruited from the Outpatient Clinic of Periodontology Department, Faculty of Dentistry, Tanta, Egypt. The study and consent form were approved by the local ethical committee (Approval code: 427/03/11). Patients with diabetes, immunocompromised, and that on systemic antimicrobial therapy, smokers, pregnant, or lactating females were excluded from the study.

The microbiology testing of the clinical samples was performed using paper point obtained from the target plaque for each patient. The patient samples were immediately placed in nutrient broth transport media and then transferred to laboratory of Bacteriology in Botany Department, Faculty of Science, Tanta University, Egypt.

Isolation and identification of bacterial isolates

Each specimen was plated to nutrient agar, blood agar, and mannitol salt agar plates. Culture plates were incubated up to 24 h at 37°C. The resultant yellow colonies of Gram-positive cocci growing on mannitol salt agar medium were preliminarily identified as Staphylococcus aureus(n = 12). Confirmation of identification of these isolates was performed using standard biochemical test including Gram stain, catalase, coagulase, and DNase tests as described by Bergey's Manual for Systematic Bacteriology.[20] Obtained buff colonies on blood agar, showed α-hemolysis, Gram-positive cocci arranged in strips and resistant to optochin disk were identified as Streptococcus spp.(n = 8).[20] The biofilm-producer S. aureus ATCC 29213 was used as reference strain.

Antimicrobial susceptibility test

All S. aureus (St. 1-12) isolates were screened for their susceptibility to 12 different antimicrobials using the disc agar diffusion method (modified Kirby–Bauer method) on Muller–Hinton agar media (MHA). The used antimicrobials were ampicillin (AMP, 10 μg), piperacillin (PRL, 100 μg), amoxicillin/sulbactam (SAM, 10/10 μg), imipenem (IPM, 10 μg), levofloxacin (LEV, 5 μg), ofloxacin (OFX, 5 μg), doxycycline (Do, 30 μg), gentamicin (CN, 10 μg), vancomycin (VA, 30 μg), rifamycin (RA, 15 μg), and nitrofurantoin (F, 300 μg). The entire surface of MHA agar plate was inoculated with 0.1 ml of each bacterial suspension, turbidity matching 0.5 McFarland standard, by a sterile cotton swab stick and the plate was air-dried before antibiotics discs were laid on the surface. The plates were incubated at 35°C for 24 h. The diameter of the inhibition zone (mm) was manually measured and compared according to the Clinical and Laboratory Standards Institute guidelines.[21] As the same manner, Streptococcus sp. (Strep. 13–20) isolates were screened for 11 antimicrobials, namely, ceftriaxone (CRO, 30 μg), cefotaxime (CTX, 30 μg), tetracycline (TE, 30 μg), clindamycin (DA, 2 μg), chloramphenicol (C, 30 μg), vancomycin (VA, 30 μg), erythromycin (E, 15 μg), ciprofloxacin (CIP, 5 μg), penicillin (P, 30 μg), ampicillin (AMP, 10 μg), and cotrimoxazole (sulfamethoxazole/trimethoprim) (SXT, 25 μg). For the purpose of analysis, isolates with resistant phenotypes included those that were classified as intermediate resistance.

Biofilm formation

Ability for biofilm formation by the tested isolates was determined using three different techniques as follows:

Tube method

Biofilm formation by bacterial isolates was estimated qualitatively as described previously by Christensen et al.[22] After incubations at 37°C for 18–20 h, culture was decanted. Tubes were stained with crystal violet (0.1%) and presence of a visible stained film lined the wall and bottom of the tube was considered positive for slime production. The results were visually scored as weak/nonproducers (WP/NP), moderate producers (MP), or high producers (HP) based on intensity of resultant film. A tube containing uninoculated TSB was simultaneously tested and used as a negative control. Furthermore, tubes containing TSB inoculated with S. aureus ATCC 29213 (a biofilm-positive reference strain) was used as positive control. Experiments were performed in triplicate and repeated three times.

Congo red agar method

Freeman et al.[4] had described an alternative method of screening biofilm formation by Staphylococcus isolates using brain–heart infusion broth supplemented with 5% sucrose, agar no. 1 (10 g/L) and congo red stain (0.8 g/l). Plates were inoculated with the tested isolates and incubated aerobically for 24 h at 37°C. Positive (HP) result was indicated by black colonies with a dry crystalline consistency. A darkening of the colonies with the absence of a dry crystalline colonial morphology indicated a moderate result (MP). Nonslime producers (NP) usually remained pink, though occasional darkening at the centers of colonies was observed and this gave a bull's eye appearance (WP).

Tissue culture plate method

Biofilm-forming ability was measured by determination of adhesion to polystyrene tissue culture plates (TCPs) as described by Christensen et al.[22] with slight modification. Briefly, 96-well flat-bottomed TCPs (Greiner Bio-One, Frickenhausen, Germany) were filled with 100 μl TSB, with/without supplements. A diluted overnight bacterial culture (1:100 in TSB, 100 μl TSB) was added to each well. Positive control wells contained TSB inoculated with S. aureus ATCC29213 while negative control wells contain TSB only. The plates were incubated at 37°C for 18 h followed by several washing with PBS (pH 7.3). Sodium acetate (2%) was added as fixative, decanted, and wells were stained with crystal violet (0.1% w/v). Finally, the plates were rinsed under running tap water, air-dried, and read at 570 nm by a Sunrise absorbance reader (Tecan Austria GmbH, Austria). In accordance with the original method, isolates with OD <0.120 were considered as WP/NP, those with OD values 0.120–0.240 were regarded as MP. An OD >0.240 indicates as HP. To compensate for background absorbance, OD readings from sterile medium, fixative, and dye were averaged and subtracted from all test values. The mean OD value obtained from media control well was deducted from all the test OD values. The statistical analysis was conducted using SPSS version 16.0 (SPSS Inc., Chicago, IL). P < 0.05 was considered to be statistically significant.

Antibacterial activity of Salvadora persica extract

Preparation of plant extracts

An aliquot of 50 g of grinded air-dried aerial parts of S. persica was separately exhaustively extracted with 250 ml of methanol in a Soxhlet apparatus at 60°C then filtered through 0.45 μm membrane prior to use as described by Jork et al.[23] The collected methanolic extracts were evaporated via rotavapor at 40°C–50°C under reduced pressure yielding 3 gm. The residues were suitably diluted with methanol so as to get the final concentration of extract 1000 μg/ml and then stored at 18°C to avoid decomposition.

Antimicrobial activity of the plant extract by agar-well diffusion

Different concentrations; 12.5, 25, 50, 100, and 200 mg/ml, of stored crude extract of S. persica were diluted in methanol. Their antimicrobial activities were tested against the twenty tasted isolates using agar-well diffusion method as described by Perez et al.[24] and Gislene et al.[25] Briefly, each bacterial isolate were inoculated on chocolate and mannitol salt agar media at 37°C for 24 h. Bacterial suspension of each isolates was adjusted to obtain turbidity equal to 106 CFU/ml using the turbidimeter. Appropriate volume (0.1 ml) of prepared suspensions was dropped on the center of three well-dried plates of nutrient and chocolate agar media, were then spread homogeneously using sterile glass rod and left to dry for 15 min. Wells of 5 mm in diameter were made in agar surface using sterile cork borer, 40 μl of different prepared concentrations of methanolic extract of S. persica were inserted simultaneously in hole made in plates by automatic pipette (Sovorex, Switzerland). Methanol solvent alone was used as control, for each test organism. For comparison, the standard drugs, 0.1 ml of AMP (10 μg/ml), was used as positive control. The plates were incubated at 37°C for 24 h. After incubation, inhibition zone diameter was measured in millimeters (mm) and compared with methanol and standard drug. The extracts that showed the highest inhibition zone was observed.

Identification of methanolic extracts of Salvadora persica by high-performance liquid chromatography–mass spectrometry

Sample preparation for high-performance liquid chromatography–mass spectrometry analysis

The residue extract after evaporation was dissolved in HPLC grade methanol to a concentration of 10 mg/ml then filtrated through a syringe filter membrane. Thereafter, aliquots of 5 μl were injected into the LC/MS analysis system. The HPLC–MS system consisted of electrospray ionization (ESI) interfaced BrukerDaltonik Esquire-LC ion trap mass spectrometer (Bremen, Germany) and an Agilent HP1100 HPLC system equipped with an autosampler and an ultraviolet-visible absorbance detector. The ionization parameters were negative ion mode and capillary.

When the chromatographic peak areas of the identified compound were extracted and calculated, the peak areas were found to increase with increased concentration of injected compounds. The results of quantitative profiling were further improved by normalizing the calculated peak areas.[26],[27]

Extraction of Salvadora persica volatile oil extract by steam distillation

To obtain the essential oil of S. persica aerial part root, 1.5 kg of root sticks were cut into 1–2 cm pieces in length. The pieces were ground using a stone mill and thereafter mixed with 700 ml of distilled water in a distillation flask. The mixture was heated and temperature of the steam was maintained at 80°C during the distillation. Steam from an additional container with boiling water was continuously added to the distillation flask allowing the compounds to evaporate as an oil-water mixture which was cooled to room temperature. The oil/water mixture obtained was extracted with HPLC grade hexane, and the hexane was evaporated under reduced pressure at 20°C.

Chemical composition of Salvadora persica volatile oil by gas chromatography-mass spectrometry

GC-MS analysis was used to determine the compounds present in S. persica volatile oil, carried out in the Biochemistry Laboratory of the Faculty of Agriculture, Cairo University. The fatty acids sample was analyzed by an HP 6890 Series Gas Chromatograph System with an HP 5973 mass selective detector. The system was equipped with a TR-FAME (Thermo 260 M142 P) (30 m, 0.25 mm ID, 0.25 μm Film (70% cyanopropyl polysilphenylenesiloxane) capillary column, 200°C temperature injector, and 250°C temperature transfer line). The oven temperature was programmed as follows: initial temperature; 80°C for 2 min, increase to 3°C min −1 up to 230°C, and then hold at 230°C for 5 min. The carrier gas was He2(1.5 ml/min). The amount of sample injected was about 1 μl (5 mg/2 ml) and the ionization energy was 70 eV. Qualitative identification of the different constituents was performed by comparison of their relative retention times and mass spectrum with those of authentic reference compounds (fatty acid methyl esters, purity 98% by GC). Furthermore, probability merge search software and the NIST MS spectra search program were used.


   Results Top


Out of 40 dental plaque specimens, 20 bacterial isolates were recovered from patients attending the Outpatient Clinics of Periodontology Department, Faculty of Dentistry, Tanta, Egypt. Production of biofilm by all tested isolates under study was assessed by three methods (tube method [TM], congo red agar [CRA], and TCP) as represented in [Table 1] and [Figure 1]. For the ease of comparison, the results of each test were classified as WP/NP, MP, or HP as aforementioned. In TM method, 16 (80%) of 20 tested isolates displayed moderate biofilm producers phenotype in TSB medium, For CRA test, a total of 85% of the isolates (n = 17) were MP of brown colonies, and 15% strains were classified as nonproducers (red colonies). The reference strain, 29213 ATCC, was found to be positive, as expected. The microtiter plate test correctly identifies the positive reference bacterial strain. Eighteen isolates (90%) were found to be biofilm producers; 2 isolates were HP and 16 isolates were MP.
Table 1: Staphylococcal and streptococcal biofilm formation as detected by three methods

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Figure 1: (a) Biofilm formation by the tested isolates using tube method. A: Positive formation, B: Negative biofilm formation. (b) Production of biofilm by the tested isolates cultured on congo red agar. A: moderate biofilm producer (MP, brown colonies), B: Nonproducer (NP, pink colonies). (c) Screening of biofilm producers by tissue culture plate method: high, moderate, weak, and nonproducers differentiated with crystal violet staining in 96-well tissue culture plate

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Antimicrobial susceptibility of the tested Streptococcus aureus and Streptococcus sp. isolates

The susceptibility of the recovered S. aureus and Streptococcus isolates to 11 different antimicrobial agents was conducted using disc diffusion method [Figure 2]. The incidence of resistance to different tested antibiotics ranged between 8.3% (gentamycin and nitrofurantoin) and 100% (AMP). For pencillins, the incidence of resistance to AMP, oxacillin, PRL, and amoxicillin/sulbactam, was 100%, 58.3%, 25%, and 16.6%, respectively, as represented in [Figure 3]a. Furthermore, no resistance against IPM, LEV, OFX, and VA was observed.
Figure 2: Antimicrobial susceptibility test of different antimicrobial agents against staphylococcal (a) and streptococcal (b) isolates

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Figure 3: Histogram showing susceptibility of Staphylococcus aureus Scientific Name Search  (a) and Streptococcus sp. (b) to tested antimicrobial agents. OX = Oxacillin; F = Nitrofurantoin; CN = Gentamicin; LEV = Levofloxacin; RA = Rifamycin; PRL = Piperacillin; VA = Vancomycin; SAM = Amoxicillin/Sulbactam; AMP = Ampicillin; DO = Doxycycline; IPM = Imipenem; OFX = Ofloxacin; CRO = Ceftriaxone; CTX = Cefotaxime; TE = Tetracycline; DA = Clindamycin; C = Chloramphenicol; E = Erythromycin; CIP = Ciprofloxacin; P = Penicillin; SXT = Cotrimoxazole (Sulfamethoxazole/Trimethoprim)

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The incidence of resistance of Streptococcus isolates to different tested antibiotics ranged between 100% to AMP, followed by 87.5% to penicillin, 37.5% to VA and clindamycin, 25% to erythromycin, and 12.5% to CTX and CRO [Figure 3]b.

Antibacterial activity of Salvadora persica extract

Results of the antibacterial activity of different concentrations of methanolic S. persica extract against staphylococcal (St1-12) and Streptococcus (Strep. 13-20) isolates are disseminated in [Table 2]. It is clear that the diameter of the inhibition zone depends mainly on the concentrations of the extract used and the tested antibacterial activity. The methanolic S. persica extract at concentration 200 mg/ml showed broad-spectrum activity as the highest effective zone of inhibition was recorded against tested isolates, followed by extract its concentration was 100 mg/ml. It is observed that the antibacterial activity of all the extract concentrations was significantly different (P ≤ 0.001) from each other, when tested against the selected isolates. S. aureus 1 and Streptococcus spp. 8 showed maximum zone of inhibition (35 and 36 mm) to methanolic extract (200 mg/ml) of S. persica, respectively. The negative control (solvent) showed no inhibition, whereas the positive control (antibiotic, AMP) showed varied range of inhibition zone (10–28 mm). In general, the methanolic S. persica extract (200 mg/ml) was showed antibacterial activity against tested isolates, where mostly nearest or more than that of the standard antibiotic (ampicillin).
Table 2: Antimicrobial activity of methanolic extract of Salvadora perisca against tested isolates

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High-performance liquid chromatography–mass spectrometryanalysis

Analysis of methanolic S. persica extract using HPLC-MS in negative and positive mode was represented in [Table 3], [Table 4] and [Figure 4], [Figure 5], respectively. The obtained results by negative-mode HPLC-MS show many compounds present in the tested extract such as chrysin-8-c-β-D-glucopyranoside, ferulic acid, gallic acid, isorhamnetin, chlorogenic acid, kaempferol, and isoquercitrin [Table 3] and [Figure 4].
Table 3: High-performance liquid chromatograph-mass spectrometry chromatograms of the methanolic extract of Salvadora persica (negative mode)

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Table 4: High-performance liquid chromatograph-mass spectrometry chromatograms of the methanolic extract of Salvadora persica (positive mode)

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Figure 4: High-performance liquid chromatography–mass spectrometry chromatogram of the crude methanolic extract of Salvadora persica, liquid chromatography-negative mode electrospray ionization-MSn base peak chromatogram profile allows the identification and confirmation of all peaks of interest

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Figure 5: High-performance liquid chromatography–mass spectrometry chromatogram of the crude methanolic extract of Salvadora persica, liquid chromatography-positive mode electrospray ionization-MSn base peak chromatogram profile allows the identification and confirmation of all peaks of interest

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The results of positive-mode HPLC-MS revealed that the most predominant components were stigmasta-5,22-dien-3 β-ol (stigmasterol) and stigmast-5-en-3 β-ol (β-sitosterol) as shown in [Table 4] and [Figure 5].

Gas chromatography-mass spectrometry anaylsis

S. persica volatile oil was analyzed by GC-MS as shown in [Table 5] and [Figure 6]. Analysis shows many compounds, the most predominant components were butylatedhydroxytoluene followed by benzene isothiocyanatomethyl.
Table 5: Gas chromatography-mass spectrometry analysis of Salvadora persica volatile oil

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Figure 6: Gas chromatography-mass spectrometry analysis of Salvadora persica volatile oil

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   Discussion Top


Dental infections are mostly caused by different oral microorganisms due to antibiotic resistance and biofilm production. These accumulations of microorganisms change the teeth and gingival tissues to high concentrations of bacterial metabolites which results in dental disease such as plaque-induced gingivitis.[28],[29],[30] S. aureus is an opportunistic pathogen and approximately 30%–50% of healthy adults are carriers of S. aureus.[31] In this study, major bacterial isolates were S. aureus 60% and other bacterial isolates were Streptococcus sp. 40%. Biofilm formation by oral bacterial isolates is important because this mode of growth is associated with the chronic nature of the subsequent infections and with their inherent resistance to antibiotic chemotherapy.[32] Consequently, investigations to understand the pathogenesis of these infections have focused upon the process of adherence of these microorganisms on plaque-induced gingivitis. The obtained results showed that, isolates displayed a biofilm positive and 20% of them were strong biofilm producers as determined by TCP method. Similar results were observed by Mathur et al.,[33] also Khalil and Sonbol [34] recommend the use of TCP technique due to its high specificity, sensitivity, and positive predictive value. However, Jain and Agarwal,[35] Grinholc et al.,[36] and Hassan et al.[37] supported the use of CRA for biofilm detection.

Herein, results of antimicrobial susceptibility tests performed on Staphylococcus isolates are entirely consistent with previously reported data by many researchers.[34],[38] The reason for the high rate of resistance of isolates to AMP (100%) and oxacillin (58.3%) may be due to the overuse of oral penicillin or oxacillin, which is often prescribed for oral treatment. However, Streptococcus isolates show resistant to 9 from 11 antibiotics. In addition, tested isolates were highly resistant to penicillin (87.5%) and AMP (100%).[39] The close contact between bacteria within a biofilm and the matrix may inhibit the penetration of antibiotics through the exopolysaccharide matrix.[40]

Instead of the good results that can be obtained by some antibiotics in treatment of oral diseases, many medical problems may be caused.[41] Hence, the need of natural active ingredients, such as S. persica is increasing. Chewing sticks may play a role in the promotion of oral hygiene. Among at least 182 plant species suitable for preparing toothbrushing sticks, Miswak harvested from S. persica, are used most extensively.[42] It has potential medicinal and research activities. S. persica is a promising product and is useful to produce antiplaque, analgesic, anticonvulsant, antibacterial, antimycotic, cytotoxic, antifertility, deobstruent, carminative, diuretic, astringent, and also used in biliousness and rheumatism.[43]

The antibacterial activity of five concentrations of methanolic S. persica extracts against twenty tested isolates from the oral cavity of patients is significantly different. The methanolic extract showed the highest effective zone of inhibition against tested was at concentrations 200 mg/ml on bacterial isolates. Similar results were also observed by Al-Bayati and Suliaman.[41] More recently, Chelli-Chentouf et al.[44] reported that 400 mg/ml of methanolic extract of S. persica showed the highest antibacterial activity against all strains. The differences may be due to the susceptibility of strains, assay methods.[45]

Discussing the results of HPLC-MS analysis of the S. persica conclude that the potential antimicrobial activity is attributed to different compounds belonging to a diverse range of chemical classes.[46] It is assumed that the higher percentage of chrysin-8-c-β-D-glucopyranoside, kaempferol quercetin in methanolic extracts of S. persica, is most probably the cause of the higher antimicrobial activity of this extract. Our results agree with those reported by many authors.[46] In addition, coumaric, caffeic, gallic, and ferulic acids were present in methanolic extract of S. persica may be effective as antibacterial agents.[47],[48]

Analysis of volatile oil by GC-MS shows the presence of caryophyllene oxide and benzene (isothiocyanatomethyl) which have antibacterial activity.[49]


   Conclusion Top


Although the S. aureus and Streptococcus sp. isolates collected from the dental plaque of patients are not resistant to some antibiotics, such as IPM and ciprofloxacin, OFX, vancomycin, chloramphenicol, and cotrimoxazole, treatment of gingivitis using antimicrobial drug may cause serious medical problems. This adds urgency to the search for new infection-fighting natural compounds to control microbial infections. We summarize in vitro data of the antibacterial activity of the methanolic extract of S. persica against tested isolates, which were collected from gingivitis patients. The highest antibacterial activity of S. persica extracts against tested isolates was observed at 200 mg/ml concentration. S. persica methanolic extract, with its active antibioticallyChrysin-8-c-β-D-glucopyranoside, stigmasterol, and butylated hydroxytoluene, presents potential and safe alternative to synthetic antibiotics for the treatment of gingivitis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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