Antibacterial Activity of Boswellia sacra Flueck. Oleoresin Extract against Porphyromonas gingivalis Periodontal Pathogen

Boswellia sacra Flueck. oleoresin extract (frankincense) has traditionally been used in the treatment of different diseases, but there are no sufficient studies on its potential activity against periodontal pathogens. Therefore, antibacterial and antibiofilm activity of frankincense extract against Porphyromonas gingivalis clinical isolates were studied. The phytochemical composition of the volatile components of the extract was identified by GC-MS analysis revealing 49 compounds as trans-nerolidyl formate, cycloartenol acetate, ursenoic acid 3-oxomethyl ester, bisabolene epoxide, and kaur-16-ene. It decreased the growth and increased the leakage of nucleotides in 58.3% and 33.3% of isolates, respectively. Additionally, it reduced the extracellular polysaccharide production and the cell surface hydrophobicity in 41.67% and 50% of the isolates, respectively. Crystal violet assay revealed inhibition of biofilm formation by the tested isolates. Light microscope and scanning electron microscope were used to examine the biofilms and they confirmed the reduction of biofilm formation by frankincense extract. Downregulation of the genes linked to biofilm formation (fimA, hagA, and hagB) was observed using qRT-PCR after treatment with the frankincense extract. This study suggested that the frankincense extract could exhibit antibacterial and antibiofilm activity against P. gingivalis isolates. Thus, the frankincense extract could be used as a treatment approach for periodontitis.


Introduction
Pathogenic bacteria have many virulence features, such as enzymes, toxins, fimbriae, and capsules that decrease the host response via producing tissue breakdown, impeding tissue healing and weakening the host defense [1,2]. Chronic periodontitis (CP) is an inflammatory condition involving the destruction of the gingival tissues of the oral cavity. The pathogenesis of CP encompasses a complex interaction between the periodontal pathogens with the host immunity [3]. One of the major etiologic pathogens that contributes

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
GC-MS analysis revealed 49 compounds from the frankincense oleoresin extract. The results are listed in Table 1 and presented in Figure 1. The retention times and mass spectra comparisons with the MS library were used to identify the sample. Mass fragmentation patterns of all detected compounds were displayed in supporting materials Figures S1-S49.

Bacterial Growth Curve
To evaluate the impact of the frankincense oleoresin extract on the growth process of P. gingivalis isolates, growth curves were created for the tested cells before and after the treatment of the frankincense oleoresin extract (0.5 MIC). A significant decrease (p < 0.05) in the growth of 58.3% of the tested isolates was observed and an illustrative example is shown in Figure 2. The remaining isolates showed a non-significant change (p > 0.05) after treatment.

Bacterial Growth Curve
To evaluate the impact of the frankincense oleoresin extract on the growth process of P. gingivalis isolates, growth curves were created for the tested cells before and after the treatment of the frankincense oleoresin extract (0.5 MIC). A significant decrease (p < 0.05) in the growth of 58.3% of the tested isolates was observed and an illustrative example is shown in Figure 2. The remaining isolates showed a non-significant change (p > 0.05) after treatment.

Figure 2.
A graph showing P. gingivalis growth curves: (a) a significant decrease in bacterial growth after treatment with the frankincense extract (0.5 MIC); (b) non-significant change in the growth curve after treatment with 10%DMSO.

Measurement of Nucleic Acid Leakage
We observed that the frankincense extract (0.5 MIC) increased the release of nucleotides from intracellular compartments significantly (p < 0.05) in 33.3% of the tested isolates by measuring the absorbance at 260 nm using an 1800 UV-VIS spectrophotometer. A representative example is shown in Figure 3. The remaining isolates showed a non-significant change (p > 0.05) after treatment.

Measurement of Nucleic Acid Leakage
We observed that the frankincense extract (0.5 MIC) increased the release of nucleotides from intracellular compartments significantly (p < 0.05) in 33.3% of the tested isolates by measuring the absorbance at 260 nm using an 1800 UV-VIS spectrophotometer. A representative example is shown in Figure 3. The remaining isolates showed a non-significant change (p > 0.05) after treatment.

Bacterial Cell Surface Hydrophobicity
The cell surface hydrophobicity was investigated in the P. gingivalis isolates, before and after treatment with the frankincense extract via measuring of hydrophobicity index (HI). The bacterial surface hydrophobicity is an important factor contributing to bacterial adherence and biofilm formation. We found that there was a significant decrease in the HI in 50% of the tested isolates after treatment as shown in Figure 4. The remaining isolates showed a non-significant change in HI (p > 0.05) after treatment. Antibiotics 2021, 10, x FOR PEER REVIEW 6 of 18

Bacterial Cell Surface Hydrophobicity
The cell surface hydrophobicity was investigated in the P. gingivalis isolates, before and after treatment with the frankincense extract via measuring of hydrophobicity index (HI). The bacterial surface hydrophobicity is an important factor contributing to bacterial adherence and biofilm formation. We found that there was a significant decrease in the HI in 50% of the tested isolates after treatment as shown in Figure 4. The remaining isolates showed a non-significant change in HI (p > 0.05) after treatment.

Antibiofilm Activity of Frankincense Extract
Crystal violet assay was utilized to assess the production of mature biofilms by P. gingivalis isolates in 96-well microtitration plates. We noted that the frankincense extract significantly (p < 0.05) inhibited the biofilm formation in five P. gingivalis isolates (41.67% of isolates) with percentages of biofilm reduction that ranged from (62.5-80%) as shown in Figure 5. The remaining isolates showed a non-significant change (p > 0.05) in biofilm formation after treatment.

Bacterial Cell Surface Hydrophobicity
The cell surface hydrophobicity was investigated in the P. gingivalis isolates, before and after treatment with the frankincense extract via measuring of hydrophobicity index (HI). The bacterial surface hydrophobicity is an important factor contributing to bacterial adherence and biofilm formation. We found that there was a significant decrease in the HI in 50% of the tested isolates after treatment as shown in Figure 4. The remaining isolates showed a non-significant change in HI (p > 0.05) after treatment.

Antibiofilm Activity of Frankincense Extract
Crystal violet assay was utilized to assess the production of mature biofilms by P. gingivalis isolates in 96-well microtitration plates. We noted that the frankincense extract significantly (p < 0.05) inhibited the biofilm formation in five P. gingivalis isolates (41.67% of isolates) with percentages of biofilm reduction that ranged from (62.5-80%) as shown in Figure 5. The remaining isolates showed a non-significant change (p > 0.05) in biofilm formation after treatment.

Antibiofilm Activity of Frankincense Extract
Crystal violet assay was utilized to assess the production of mature biofilms by P. gingivalis isolates in 96-well microtitration plates. We noted that the frankincense extract significantly (p < 0.05) inhibited the biofilm formation in five P. gingivalis isolates (41.67% of isolates) with percentages of biofilm reduction that ranged from (62.5-80%) as shown in Figure 5. The remaining isolates showed a non-significant change (p > 0.05) in biofilm formation after treatment.

Extracellular Polysaccharide Measurement
The extracellular polysaccharides, which are the main components of the biofilms, were assayed before and after treatment with the frankincense extract using the phenolsulfuric acid method. A significant reduction (p < 0.05) of the quantity of the extracellular polysaccharides was observed in five P. gingivalis isolates (which showed inhibition of their biofilm formation by crystal violet assay) as shown in Figure 6. The remaining isolates showed a non-significant change (p > 0.05) after treatment. Figure 6. A chart showing a significant reduction of extracellular polysaccharide production by five P. gingivalis isolates after treatment with the frankincense extract.

Extracellular Polysaccharide Measurement
The extracellular polysaccharides, which are the main components of the biofilms, were assayed before and after treatment with the frankincense extract using the phenolsulfuric acid method. A significant reduction (p < 0.05) of the quantity of the extracellular polysaccharides was observed in five P. gingivalis isolates (which showed inhibition of their biofilm formation by crystal violet assay) as shown in Figure 6. The remaining isolates showed a non-significant change (p > 0.05) after treatment.

Extracellular Polysaccharide Measurement
The extracellular polysaccharides, which are the main components of the biofilms, were assayed before and after treatment with the frankincense extract using the phenolsulfuric acid method. A significant reduction (p < 0.05) of the quantity of the extracellular polysaccharides was observed in five P. gingivalis isolates (which showed inhibition of their biofilm formation by crystal violet assay) as shown in Figure 6. The remaining isolates showed a non-significant change (p > 0.05) after treatment. Figure 6. A chart showing a significant reduction of extracellular polysaccharide production by five P. gingivalis isolates after treatment with the frankincense extract. Figure 6. A chart showing a significant reduction of extracellular polysaccharide production by five P. gingivalis isolates after treatment with the frankincense extract.

Examination of the Biofilm Morphology by Light Microscope and Scanning Electron Microscope (SEM)
Light microscope analysis of the formed biofilms before and after treatment with frankincense extract confirmed the inhibition of biofilm formation in five P. gingivalis isolates which were found to be inhibited by semi-quantitative crystal violet assay. Visible Light microscope analysis of the formed biofilms before and after treatment with frankincense extract confirmed the inhibition of biofilm formation in five P. gingivalis isolates which were found to be inhibited by semi-quantitative crystal violet assay. Visible reductions of biofilms on the treated slides were noted after crystal violet staining as shown in Figure 7. It is well known that electron microscopes could produce images of the tested specimens with higher resolution than those produced by light microscopes. Therefore, we examined five P. gingivalis isolates, previously examined by light microscope, using SEM. The SEM examination demonstrated a significant reduction in the aggregation of P. gingivalis bacterial cells in the formed biofilms after treatment with frankincense extract as shown in Figure 8.

Quantitative RT-PCR
qRT-PCR was utilized to study the impact of frankincense extract on the P. gingivalis virulence factors which are related to biofilm formation. As shown in Table 3, the gene expression of fimA, hagA, and hagB was found to be significantly decreased in 50%, 41.7%, and 50% of the isolates, respectively. The remaining isolates showed a non-significant It is well known that electron microscopes could produce images of the tested specimens with higher resolution than those produced by light microscopes. Therefore, we examined five P. gingivalis isolates, previously examined by light microscope, using SEM. The SEM examination demonstrated a significant reduction in the aggregation of P. gingivalis bacterial cells in the formed biofilms after treatment with frankincense extract as shown in Figure 8. Light microscope analysis of the formed biofilms before and after treatment with frankincense extract confirmed the inhibition of biofilm formation in five P. gingivalis isolates which were found to be inhibited by semi-quantitative crystal violet assay. Visible reductions of biofilms on the treated slides were noted after crystal violet staining as shown in Figure 7. It is well known that electron microscopes could produce images of the tested specimens with higher resolution than those produced by light microscopes. Therefore, we examined five P. gingivalis isolates, previously examined by light microscope, using SEM. The SEM examination demonstrated a significant reduction in the aggregation of P. gingivalis bacterial cells in the formed biofilms after treatment with frankincense extract as shown in Figure 8.

Quantitative RT-PCR
qRT-PCR was utilized to study the impact of frankincense extract on the P. gingivalis virulence factors which are related to biofilm formation. As shown in Table 3, the gene expression of fimA, hagA, and hagB was found to be significantly decreased in 50%, 41.7%, and 50% of the isolates, respectively. The remaining isolates showed a non-significant

Quantitative RT-PCR
qRT-PCR was utilized to study the impact of frankincense extract on the P. gingivalis virulence factors which are related to biofilm formation. As shown in Table 3, the gene expression of fimA, hagA, and hagB was found to be significantly decreased in 50%, 41.7%, and 50% of the isolates, respectively. The remaining isolates showed a non-significant change in the gene expression (p > 0.05) after treatment. In addition, there was a nonsignificant change in the gene expression of rgpA and kgp in all isolates.
The bolded values point to a significant decrease in gene expression.

Cytotoxicity Assay
The cytotoxic effect of Boswellia sacra Flueck. oleoresin extract in a human skin fibroblast (HSF) cell line was determined using the SRB (sulforhodamin B) assay [31]. The results exhibited that the frankincense extract at the concentration of ≤30 µg/mL displayed no cytotoxicity, whereas that at >30-300 µg/mL exhibited mild cytotoxicity in the HSF cell line with IC 50 = 30.7 ± 4.41 µg/mL in comparison to doxorubicin as a positive control IC 50 = 4.36 ± 0.52 µg/mL.

Discussion
Considering the vast spread of bacterial resistance to different antibiotics, which is now becoming an increasing worldwide health threat, researchers are seeking alternative strategies to prevent or limit bacterial infections. Recently, there has been growing evidence demonstrating that the plant extracts could offer promising antimicrobial and antibiofilm potentials with no risk of increasing resistance to antibiotics. A great number of phytochemicals have been documented to be valuable alternatives and supplementary products that could be used in our battle against bacterial infections [32,33]. The frankincense extract reveals a wide variety of different classes of active compounds such as steroids, sesquiterpenes, hormones, vitamins, oxygenated sesquiterpenes, pentacyclic, tetracyclic triterpenes, fatty acids, and other polycyclic aromatic hydrocarbons. Among 49 compounds detected by GC/MS of the frankincense oleoresin extract, trans-Nerolidyl formate was the main compound representing 19.88% followed by cycloartenol acetate, (3á)-, urs-12-en-24-oic acid, 3-oxo-, methyl ester, (+)-7, cis-Z-à-bisabolene epoxide, and kaur-16-ene representing 13.56%, 12.42%, 9.41%, and 7.46% of total extract, respectively. Trans-nerolidyl formate belongs to sesquiterpene, kaur-16-ene belongs to diterpenes. While from triterpenes, cycloartenol acetate, (3á)-, urs-12-en-24-oic acid, 3-oxo-, methyl ester, (+)-7, and cis-Z-à-bisabolene epoxide were identified. Fragmentation patterns of these main compounds are presented in Figures S15, S43, S37, S16, and S9, while the chemical structures are represented in Figure 9. This is the first report for the identification of these compounds from the frankincense (B. Sacra) extract. activity at 30 µM against airborne microbes and inhibited the growth of P. obscurans by 49.9% in comparison with the control (99.4%) in the previous study [34]. Additionally, kaurene presented antimicrobial activity against oral pathogens [35] and bisabolene epoxide exhibited an interesting antimicrobial activity [36,37]. In addition to other sesquiterpenes, triterpenes, and hydrocarbons which were previously reported for their antimicrobial activities [38]. P. gingivalis is a Gram-negative oral anaerobe causing periodontitis, a severe gum infection, which could lead to tooth breakdown and loss in addition to many other serious complications [5].
In the present work, the frankincense extract exhibited antibacterial activity against P. gingivalis clinical isolates with MIC values ranged from 500 to 1000 µg/mL. Some researchers have reported that the frankincense extract has antibacterial activity against different bacterial pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Propionibacterium acnes, and Klebsiella pneumoniae [39,40]. Al-Hamdoni and Al-Rawi [30] reported that frankincense extract exhibited antibacterial activity against the red complex periodontal pathogens isolated from patients in Mosul, Iraq with MIC value of 2.4 mg/mL. The difference between the MIC values reported in the current study and that of Al-Hamdoni and Al-Rawi [16,30] could be explained by the finding reported by Di Stefano et al. [40]. They noticed that frankincense essential oils derived from three different Boswellia sacra cultivars had different MIC values against Staphylococcus aureus, Pseudomonas aeruginosa, and Propionibacterium acnes standard isolates.
We found that the frankincense extract inhibited cellular growth in 58.3% of the tested isolates. To explain the inhibitory effect of the frankincense extract on the growth process of P. gingivalis isolates, the leakage of nucleotides from the bacterial cells was measured before and after treatment. We observed that 33.3% of the tested isolates showed a significant increase (p < 0.05) in the release of nucleotides from the intracellular  The activity of frankincense extract could be attributed to the variety of active compounds detected in it, especially trans-Nerolidyl formate which exhibited antimicrobial activity at 30 µM against airborne microbes and inhibited the growth of P. obscurans by 49.9% in comparison with the control (99.4%) in the previous study [34]. Additionally, kaurene presented antimicrobial activity against oral pathogens [35] and bisabolene epoxide exhibited an interesting antimicrobial activity [36,37]. In addition to other sesquiterpenes, triterpenes, and hydrocarbons which were previously reported for their antimicrobial activities [38].
In the present work, the frankincense extract exhibited antibacterial activity against P. gingivalis clinical isolates with MIC values ranged from 500 to 1000 µg/mL. Some researchers have reported that the frankincense extract has antibacterial activity against different bacterial pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Propionibacterium acnes, and Klebsiella pneumoniae [39,40]. Al-Hamdoni and Al-Rawi [30] reported that frankincense extract exhibited antibacterial activity against the red complex periodontal pathogens isolated from patients in Mosul, Iraq with MIC value of 2.4 mg/mL. The difference between the MIC values reported in the current study and that of Al-Hamdoni and Al-Rawi [16,30] could be explained by the finding reported by Di Stefano et al. [40]. They noticed that frankincense essential oils derived from three different Boswellia sacra cultivars had different MIC values against Staphylococcus aureus, Pseudomonas aeruginosa, and Propionibacterium acnes standard isolates.
We found that the frankincense extract inhibited cellular growth in 58.3% of the tested isolates. To explain the inhibitory effect of the frankincense extract on the growth process of P. gingivalis isolates, the leakage of nucleotides from the bacterial cells was measured before and after treatment. We observed that 33.3% of the tested isolates showed a significant increase (p < 0.05) in the release of nucleotides from the intracellular compartments which could be due to a decrease in the membrane integrity induced by frankincense extract.
Besides the antibacterial activity of the frankincense extract against P. gingivalis isolates, this investigation demonstrated more possible features concerning its antibiofilm activity. In most cases, bacterial cells such as P. gingivalis are present mainly embedded in biofilms. Developing biofilms is a strategy adopted by bacteria to help their survival in the host environments [41]. Biofilms are functionally and physiologically different from planktonic bacteria [42]. An important difference is the ability of bacteria embedded in the biofilm to tolerate many antibiotics and antibacterial agents 100-1000 times higher than planktonic bacteria. Another main difference is the protection of bacteria enclosed in the biofilm from the attack by the immune system of the host which enables the bacterial cells to form a persistent infection [43]. Therefore, we studied the effect of the frankincense extract on P. gingivalis biofilms using crystal violet assay.
Crystal violet is a basic dye (i.e., produces a positive charge after ionization) that can bind to the negatively charged molecules on the bacterial surface and polysaccharides present in the extracellular matrix [44]. In the current study, the semi-quantitative measurement of the mature biofilms produced by P. gingivalis isolates revealed that frankincense extract significantly (p < 0.05) inhibited the biofilm formation in five P. gingivalis isolates. As crystal violet stains both live and dead bacterial cells, we measured the extracellular polysaccharide production by P. gingivalis isolates. Polysaccharide production by bacteria is a crucial part of biofilm formation [45]. Polysaccharides are synthesized by live bacteria only; thus, their level could be related to bacterial viability. We noticed that the polysaccharide production by the tested P. gingivalis isolates was significantly reduced after treatment with frankincense extract. The mechanism of reduction of the extracellular polysaccharide matrix of P. gingivalis after treatment with frankincense extract still necessitates further future studies.
The bacterial hydrophobic properties are responsible for adhesion and biofilm formation [46,47]. Thus, to investigate the effect of frankincense extract on the hydrophobicity of the tested isolates, we determined the HI before and after treatment. We noticed that 50% of P. gingivalis isolates displayed a decrease in their HI after treatment with frankincense extract. This outcome might be a factor contributing to the reduction of biofilm formation by P. gingivalis isolates induced by the frankincense extract.
Examination of the biofilm formation by both light microscope and SEM before and after treatment with the frankincense extract confirmed the reduction of biofilm formation by frankincense extract.
Biofilm formation by P. gingivalis bacteria is regulated by various genes. Amongst them, the long fimbriae A (FimA) encoded by the fimA gene, which plays an important role in P. gingivalis adhesion, thus affecting biofilm formation [48]. Arg-gingipain (RgpA) in addition to lysine-specific cysteine proteases (Kgp) are necessary for the maturation and processing of Fim A proteins, therefore supporting P. gingivalis adhesion to the host tissues [49]. The heme genes (hagA, hagB) facilitate the acquirement of heme from red blood cells which are necessary for the growth of P. gingivalis bacteria, and they are involved in the bacterial adhesion to the human gingival epithelial cells. Hence fimA, rgpA, kgp, hagA, and hagB genes are critical in P. gingivalis pathogenicity and biofilm formation. Consequently, we determined the relative expression of these genes in P. gingivalis isolates treated with frankincense extract by qRT-PCR. We observed a significant decrease in the gene expression of fimA, hagA, and hagB in 50%, 41.7%, and 50% of the isolates, respectively. There was a non-significant change in the gene expression of rgpA and kgp.
Therefore, many more in vitro studies should be carried out to demonstrate the activity of the frankincense extract on the different oral bacteria associated with periodontal diseases other than P. gingivalis such as Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, Campylobacter rectus, and Campylobacter gracilis [2].

Plant Material
Dried frankincense oleoresin was obtained from a verified Boswellia sacra tree in Salalah, Oman. It was collected in December 2020. A voucher sample (No. gpfr2020-11) was deposited at the herbarium of Pharmacognosy Department, Faculty of Pharmacy, Tanta University after identification in plant ecology department, faculty of science, Tanta University. Frankincense oleoresin was ground to obtain a fine powder. The extraction was done by cold maceration method using 100 g of powder in 500 mL 100% ethanol for 48 h each at room temperature till exhaustion, then filtered using, Whatman filter paper standard size (5.0 mm diameter), evaporated under reduced pressure then lyophilized to obtain a residue. The yield of extract was 20.88 g.

Chemicals and Cell Lines
All the utilized chemicals in the present study were of the analytical grade and they were purchased from Merck, USA. HSF cell lines were obtained from Nawah Scientific Inc., (Mokatam, Cairo, Egypt).

GC-MS Data Analysis
The oleoresin extract was analyzed by a GC-MS (Perkin Elmer model: Clarus 580/560S) with a column (Elite-5MS) of 30 m length, 0.25 mm diameter, and 0.25 m film thickness. The temperature in the column oven was programmed to rise from 50 to 300 • C in 2 • C increments. Electron impact ionization was used to ionize the sample components (EI, 70 eV). The injector was set to 280 • C, while one of the detectors was set to 200 • C. At a rate of 3.0 scans/s, the mass range of 40-1000 m/z was scanned. In the split injection method, 1.0 µL of pure frankincense oleoresin extract was manually injected into the GC-MS using a Hamilton syringe for total ion chromatographic analysis. The GC-MS takes 48 min to complete. The relative percentage of extract constituents was expressed as a percentage with peak area normalization [50].

Identification of Compounds
Mass spectroscopy was used to identify the bioactive compounds by comparing their retention indices and mass spectra fragmentation patterns to those found in the National Institute of Standards and Technology (NIST) library.

Selection of Patients
The study was carried out on 30 systemically healthy patients of both sexes their ages ranged from 25 to 50 years old, diagnosed with moderate to severe chronic periodontitis (majority stage III grade b periodontitis), Table S2 contains more information about the patients, such as age, sex, stage of periodontitis of each patient. The patients were selected from the clinic of Periodontology, Faculty of Dentistry, Tanta University. All the selected patients were systemically free, non-smoker, and not under any systemic antibiotic before or at time of sampling. If female, they were not lactating or pregnant.

Ethical Statement
This clinical and microbiological in vitro study was conducted according to principles guidelines outlined in the declaration of Helsinki on experimentation involving human subjects. The study protocol and consents are approved by the Research Ethics Committee, Faculty of Pharmacy, Tanta University on 2021, and Clinical Trials.gov ID: NCT: NCT04705714.

Bacterial Isolation
Gingival crevicular fluid samples (GCF) were collected from pockets having ≥5 mm depth and positive bleeding on probing (active disease) with a paper point. The selected site was isolated with sterile cotton rolls, and the supragingival plaque was discarded with sterile cotton pellets. Two sterilized paper points (size # 40) were carefully inserted to the maximum depth of the periodontal pocket and held in position for ten seconds, and then they were placed immediately in one milliliter of transport medium in an Eppendorf tube and another sample from the same site in a test tube contains one milliliter of transport medium. Samples were obtained from the periodontal pockets after removing the supragingival plaque from the teeth to be sampled. Figure S50 shows the method of subgingival plaque sample collection using sterile paper points.
The subgingival plaque samples were then inoculated into 2 mL brain heart infusion (BHI) broth supplemented with 5 µg/mL hemin and 1 µg/mL menadione (vitamin K 1 ). They were then diluted and cultured onto blood agar supplemented with 5% sheep blood, hemin (5 µg/mL) and vitamin K 1 (0.5 µg/mL). These plates were incubated in duplicate for 7-10 days at 37 • C in an anaerobic atmosphere. The bacteria grown were finally selected based on their color, size, and shape. The black-pigmented colonies and Gramnegative rods (when examined microscopically) were examined by a fluorescence test using longwave UV light. The absence of fluorescence distinguishes between P. gingivalis and other anaerobic, black-pigmented, Gram-negative rods [51]. The identification of P. gingivalis isolates was then confirmed using API 20A (BioMérieux, France). P. gingivalis ATCC 33,277 was used as a reference strain.

Antibacterial Screening
It was performed using the agar diffusion method [52]. In brief, bacterial suspension was distributed onto blood agar plate surfaces supplemented with 5% sheep blood, 1% (v/v) hemin, and 1% (v/v) vitamin K 1 , then sterilized 6 mm blank filter paper discs were impregnated with 25 µL of frankincense oleoresin extract with different concentrations ranging from 0.5 to 1000 µg/mL. Discs loaded with 10% DMSO were used as negative controls [53,54] and tetracycline disc (30 µg) was used as a positive control. All Petri dishes were anaerobically incubated at 37 • C for 48 h. Inhibition zone diameters were measured and the concentrations of frankincense extract which showed clear zones (inhibition zone) around the discs were regarded to have an inhibitory effect on the tested bacteria.

Determination of MIC Values
The MIC values of frankincense extract against P. gingivalis isolates were estimated using the broth microdilution method [8,55,56]. Briefly, cultures of P. gingivalis were grown overnight in BHI broth containing hemin (5 µg/mL) and vitamin K 1 (1 µg/mL). Then, 100 µL of bacteria (diluted first with fresh BHI to obtain an optical density of 0.2 at 660 nm) plus 100 µL of serial dilutions (two-fold) of the frankincense extract (starting from 2000 µg/mL) in BHI were mixed in the wells of the microtitration plate. Each microtitration plate had a negative control well-containing BHI without bacteria and a positive control well containing only bacteria without the extract. After anaerobic incubation at 37 • C for 24 h, bacterial growth was inspected visually. The MIC values were recorded as the lowest concentrations that caused an inhibition of the bacterial growth. All the following experiments were performed before and after treatment of P. gingivalis isolates with subinhibitory concentrations (0.5 MIC) of the frankincense extract.

Bacterial Growth Curve
P. gingivalis isolates were cultured in blood agar plates, supplemented with 5% sheep blood, hemin (5 µg/mL), and vitamin K 1 (0.5 µg/mL), for 5-7 days under anaerobic conditions at 37 • C [57]. Then, a single colony of each isolate was inoculated into BHI broth, containing hemin (5 µg/mL) and vitamin K 1 (1 µg/mL), and grown anaerobically for 24 h. The optical density (OD) values at 600 nm were detected every 2 h using UV-VIS spectrophotometer (Shimadzu, Japan), and the growth curves were constructed via plotting the log OD against the sampling time (hrs.).

Measurement of Nucleic Acid Leakage
The release of nucleic acids from the bacterial cells was measured as described previously [58,59]. P. gingivalis isolates, at log phase, were centrifuged and the pellets were resuspended in phosphate-buffered saline (PBS), and incubated under anaerobic conditions. The release of cellular nucleic acids was measured at different times of 0, 1, 2, and 4 h. Through measuring the absorbance at 260 nm using an 1800 UV-VIS spectrophotometer (Shimadzu, Japan).

Bacterial Cell Surface Hydrophobicity
The bacterial hydrophobicity was assessed using the hydrocarbon-xylene test [55]. Briefly, P. gingivalis isolates were cultured anaerobically and the pellets collected after centrifugation were resuspended in phosphate urea magnesium sulfate buffer (PUM buffer, pH 6.9). Then, volumes of 0.3, 0.9, 1.2, and 1.8 mL of n-hexane were added to 4.8 mL of the bacterial suspension. The two phases were vortexed, the mixtures were left for 2 h, and the aqueous phase was cautiously separated. The absorbance of the tested isolates that remained in the aqueous phase was measured at 540 nm. HI was identified by the following equation:

Antibiofilm Activity of Frankincense Extract
It was performed as previously described [60]. Briefly, 100 µL of P. gingivalis suspensions were inoculated and cultured into a 96-well microtitration plate at anaerobic conditions at 37 • C. After incubation for 72 h, the non-adherent cells were removed via washing with PBS three times, and they were left to dry for 1 h. The biofilms were stained using a solution of 0.1% crystal violet and the samples were washed thrice with distilled water. For quantitative analysis of biofilm production, 125 µL of 30% acetic acid was added for 15 min and the OD at 492 nm was measured using ELISA Auto Reader (Sunrise Tecan, Austria). The percentage of biofilm reduction was calculated using the formula: % reduction of biofilm formation = OD before treatment − OD after treatment OD before treatment × 100

Measurement of the Extracellular Polysaccharides
Polysaccharide quantification assay was performed because polysaccharides are the main component in the formed biofilm and its quantity would affect biofilm formation [45,57]. The polysaccharides found in the extracellular polymeric substance (EPS) of the formed biofilm were estimated using the phenol-sulfuric acid method [57]. In brief, microtitration plates, after 3 days of biofilm formation by P. gingivalis isolates, were washed with PBS to remove the free-floating bacteria and air-dried. Then, 40 µL sterile water plus 40 µL 6% phenol solution, followed by 200 µL 97% sulfuric acid were added to each well. The plates were then left at room temperature for 30 min and the quantity of polysaccharides in the formed biofilm were determined by measuring the absorbance at an OD of 490 nm using ELISA Auto Reader (Sunrise Tecan, Austria). We used different glucose concentrations (0, 5, 10, 20, and 100 mg/L) as a standard to convert the OD values to polysaccharide concentrations.

Examination of Biofilm Morphology by Light Microscope and SEM
Examination of biofilm morphology by light microscope and SEM was carried out according to Qi et al. [57]. Briefly, two groups of glass cover slides were flooded with each P. gingivalis isolate (before and after treatment with frankincense extract) and they were left for three days under anaerobic conditions to allow the isolates to form biofilms. The first group of the formed biofilms by P. gingivalis isolates was visualized by a bright-field microscope (Labomed, America) using 100× magnification after staining with crystal violet. The second group of the formed biofilms was rinsed using PBS and submerged in 2.5% glutaraldehyde solution for 24 h at 4 • C. Then, they were sequentially dehydrated using a series of ethanol; concentrations ranging from 30% to 100%, let to dry, and sputter-coated with gold for examination with SEM (Hitachi, Japan).

qRT-PCR
Relative gene expression of fimA, hagA, hagB, rgpA and kgp genes was examined using qRT-PCR. The used primers are listed in Table S1 and the 16S rRNA gene was the housekeeping gene or endogenous control [8]. All the experiments were performed three times and the results values were expressed as mean ± standard deviation (SD). The amplification was carried out by Power SYBR ® Green master mix (Thermo SCIENTIFIC, Waltham, MA, USA) using Rotor-Gene Q5 plex instrument (Qiagen, Germany). The relative gene expression was determined using the method of 2 −∆∆Ct [61] using the untreated isolates as control samples (its expression was set to 1). Changes in the gene expression with ≥2-fold (increased or decreased) were considered to be statistically significant [62].

Cytotoxicity Assay
The SRB assay was used to determine cell viability. In 96-well plates, aliquots of 100 µL cell suspension (5 × 10 3 cells) were incubated in complete medium for 24 h. Another aliquot of 100 µL media containing the extract at various concentrations was used to treat the cells. After 72 h of the exposure to the extract, the cells were fixed by replacing the medium with 150 µL of 10% trichloroacetic acid (TCA) and incubating for one hour at 4 • C. After removing the TCA solution, the cells were washed five times with distilled water. Aliquots of 70 µL of SRB solution (0.4 percent w/v) were added and incubated at room temperature for 10 min in the dark. Plates were washed three times with 1% acetic acid and air-dried overnight. The absorbance was measured at 540 nm using a BMG LABTECH ® -FLUOstar Omega microplate reader (Ortenberg, Germany) after 150 µL of TRIS (10 mM) was added to dissolve protein-bound SRB stain [31,63].

Statistical Analysis
Data were analyzed statistically by one-way analysis of variance (ANOVA) using the SPSS software version 18.0 (IBM, New York, NY, USA). All measurements were performed three times and expressed as mean ± SD. Results with p-values lower than 0.05 (p < 0.05) were regarded to be significant.

Conclusions
In the current investigation, the phytoconstituents of the extract were identified using GC/MS technique. A total of 49 compounds were identified. In addition, the frankincense extract displayed antibacterial activity against P. gingivalis isolates and this could be attributed to the variety of active compounds identified in this extract. Moreover, it inhibited the growth of P. gingivalis bacteria which might be associated with a reduction of the bacterial membrane integrity. On the other hand, the frankincense extract inhibited the biofilm formation by P. gingivalis and reduced the expression of genes encoding for virulence factors associated with biofilm formation. This work paid attention to frankincense extract which could be applied to gums as an effective safe antimicrobial agent for periodontitis prevention and treatment. Thus, a promising future may await Boswellia sacra Flueck. oleoresin extract in antimicrobial drug discovery. In addition, in vivo future studies should be conducted on the frankincense extract as it may participate in drug discovery.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antibiotics10070859/s1, Figures from S1 to S49: Mass fragmentation pattern of identified compounds. Figure S50 shows the method of subgingival plaque sample collection using sterile paper points. Table S1: Sequences of the oligonucleotides used in qRT-PCR. Table S2: Age, sex, stage of periodontitis of the selected patient.