Antibacterial and Anti-biofilm Activity of Frankincense Extract Against Porphyromonas Gingivalis
Condition: Periodontal Diseases
MeSH terms: Periodontal Diseases
Study type: Interventional
Phase: Phase 1

Summary: 
      Boswellia sacra Flueck. oleoresin extract (frankincense) was traditionally used in the
      treatment of different diseases as respiratory, rheumatoid arthritis, and bacterial
      infections. Therefore, the antibacterial and antibiofilm activity of frankincense extract
      against Porphyromonas gingivalis periodontal pathogen clinical isolates were studied
    

Detailed description: 
      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 (grade III
      stage b periodontitis), and selected from the clinic of Periodontology, Faculty of Dentistry,
      Tanta University

      Gingival crevicular fluid samples (GCF) were collected from pockets having ≥ 5 mm depth and
      positive bleeding on probing (active disease) with a paper point. Samples were obtained from
      the periodontal pockets after removing the supragingival plaque from the teeth to be sampled.
      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 K1). They were then
      diluted and cultured onto blood agar supplemented with 5% sheep blood, hemin (5 μg/mL) and
      vitamin K1 (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 Gram-negative 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. The identification of P. gingivalis isolates was then confirmed using API
      20A (BioMérieux, France).

      4.8. Antibacterial screening It was performed using the agar diffusion method [40]. 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 K1, 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 was used as
      negative controls and tetracycline disc (30 µg) was used as a positive control. All Petri
      dishes were anaerobically incubated at 37°C for 48 hrs. 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.

      4.9. Determination of MIC values The MIC values of frankincense extract against P. gingivalis
      isolates were estimated using the broth microdilution method. Briefly, cultures of P.
      gingivalis were grown overnight in BHI broth containing hemin (5 µg/mL) and vitamin K1 (1
      µg/mL). Then, 100 µL of bacteria 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 hrs., 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 sub-inhibitory concentrations (0.5 MIC) of the frankincense
      extract.

      4.10. Bacterial growth curve P. gingivalis isolates were cultured in blood agar plates,
      supplemented with 5% sheep blood, hemin (5 μg/mL), and vitamin K1 (0.5 μg/mL), for 5-7 days
      under anaerobic conditions at 37°C [43]. Then, a single colony of each isolate was inoculated
      into BHI broth, containing hemin (5 µg/mL) and vitamin K1 (1 µg/mL), and grown anaerobically
      for 24 hrs. The optical density (OD) values at 600 nm were detected every 2 hrs. using UV-VIS
      spectrophotometer (Shimadzu, Japan), and the growth curves were constructed via plotting the
      log OD against the sampling time (hrs.).

      4.11. Measurement of nucleic acid leakage The release of nucleic acids from the bacterial
      cells was measured as described previously. 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 hrs. through measuring the absorbance at 260 nm using an
      1800 UV-VIS spectrophotometer (Shimadzu, Japan).

      4.12. Antibiofilm activity of frankincense extract

      It was performed as previously described . 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 hrs., the non-adherent cells were removed via washing with PBS, and
      they were left to dry for 1 hr. The biofilms were stained using a solution of 0.1% crystal
      violet and the samples were washed with distilled water. For quantitative analysis of biofilm
      production, 125 µL of 30% acetic acid was added 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 untreated-OD treated)/(OD untreated) x 100 Measurement
      of the extracellular polysaccharides The polysaccharides found in the extracellular polymeric
      substance (EPS) of the formed biofilm were estimated using the phenol-sulfuric acid method
      [43]. 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 was
      added to each well. The plates were then left at room temperature for 30 min and the number
      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.

      4.14. Bacterial cell surface hydrophobicity

      The bacterial hydrophobicity was assessed using the hydrocarbon-xylene test. 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 mixtures were left for 2 hrs 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:

      HI=(A540 control-A540 test)/(A540 control) 4.15. Examination of biofilm morphology by light
      microscope and SEM It was carried out according to Qi et al. Briefly, two groups of glass
      cover slides were flooded with P. gingivalis isolates (with and without 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 hrs 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).

      4.16. 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. 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, USA) using Rotor-Gene Q5 plex
      instrument (Qiagen, Germany). The relative gene expression was determined using the method of
      2-ΔΔCt 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
    

Intervention name(s): Frankincense Extract
Intervention type(s): Drug
Intervention description(s): The phytochemical composition of the volatile components of the extract was identified by GC-MS analysis, Crystal violet assay to test 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
Other intervention name(s): Boswellia sacra Flueck. Oleoresin Extract
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Diagnostic
Keywords: frankincense extract,; P. gingivalis ,; antibacterial activity;; susceptibility test of anaerobes; ; natural materials