INTRODUCTION
Enterococcus faecalis, a commensal bacterium of the gastrointestinal tract of some mammals, has frequently been recovered from persistent apical periodontitis and poses a challenge to eliminate during the treatment of endodontic infections. Enterococci are Gram-positive facultative anaerobes that are opportunistic pathogens. In the human body, up to 90% of enterococcal infections are caused by E. faecalis.[1] Posttreatment apical periodontitis occurs due to persistent bacteria which has survived initial chemomechanical endodontic interventions or through recontamination of the root canal system (Barbosa-Ribeiro et al.). E. faecalis has demonstrated a high-adhesion activity to root canal filling materials and can induce tissue responses by host-mediated tissue damage within the periradicular region.[23]
Endodontic infection is biofilm-mediated, which develops through surface-adherent bacterial cells and is composed of a multicellular microbial community secured within an extracellular matrix.[45] Its formation and maintenance relies upon the production of proteins and exopolysaccharides as a constituent of the extracellular polymeric substances. E. faecalis biofilms demonstrate resistance to antimicrobials.[56] The continued survival of E. faecalis in the apical aspects of root-filled teeth is attributed to its vast virulence and resistance mechanisms maintained within a biofilm. The development of an E. faecalis biofilm has been reported in association with genetic polymorphisms conferring its resistance.[2] Other virulence factors E. faecalis include lipoteichoic acid, extracellular superoxide, and hyaluronidase, which allow for secretion of toxins and resistance to lethal conditions.[7]
During environmental stresses, E. faecalis can enter a viable but noncultivable state as a survival mechanism, which is reversible when favorable conditions recommence. It grows at temperatures from 10°C to 45°C, survives at 60°C for 30 min, and grows at pH 9.6 or in 6.5% NaCl broth.[8]
Calcium hydroxide (Ca[OH]2) therapy is one of the most widely used endodontic intracanal medicaments; however, E. faecalis has demonstrated resistance to intracanal Ca(OH)2 therapy in the root canal system. E. faecalis invades dentinal tubules and possesses a proton pump allowing for its resistance against the widely used intracanal medicament, Ca[OH]2.[89] Its ability to invade dentinal tubules is attributed to its adherence to collagen in the presence of serum.[9]
A phytotherapeutic approach to medicaments has emerged in which various essential oils have been tested as a novel endodontic medicament, yielding a reduction in E. faecalis.[10] Among these, cinnamon and ginger have been reported.[1112] In a study evaluating biofilm-disruptive properties of ginger oil versus ampicillin, both agents showed similar results of inhibiting growth of a preformed biofilm.[11] Another study demonstrated among ethanol extracts of seven spices, Cinnamomum zeylanicum (cinnamon) and Zingiber officinale (ginger) achieved maximal inhibition of E. faecalis.[12] The aim of this study was to identify the antimicrobial potential of cinnamon and ginger oils against preformed biofilms of an oral strain of E. faecalis within saline and brain–heart infusion (BHI) conditions.
MATERIALS AND METHODS
The manuscript of this laboratory study has been written according to the Preferred Reporting Items for Laboratory Studies in Endodontology (PRILE) 2021 guidelines.[13] A PRILE 2021 flowchart is provided in Figure 1.
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Figure 1: Preferred Reporting Items for Laboratory Studies in Endodontology 2021 flowchart
Bacterial culture
E. faecalis, ATCC4083 (American Type Culture Collection, Virginia, USA), that originated from an oral periapical lesion associated with a previously root canal treated tooth was selected for this study. This organism was revived from −80°C freezer storage (Thermo Scientific, Waltham, MA, USA) and plated on blood agar plates supplemented with hemin and Vitamin K.[14] After streaked onto the blood agar plate, the plate was covered with parafilm and placed in a 37°C incubator with 5% CO2 for 24 h. E. faecalis cells exhibited growth following the 24 h period.
Preformed biofilm
To prepare biofilms, 5 ml of BHI broth (Becton Dickinson, Franklin Lakes, NJ, USA), was inoculated with E. faecalis and incubated at 250 rpm at 37°C for 24 h. An optical density of 600 nm (OD600) was determined and a subculture was made to achieve an OD600 0.1 in BHI. 200 μl of the subculture was added to columns 2–12 of a 96-well plate. Column 1 was seeded with 200 μl of BHI (negative control). The plates were incubated statically at 37°C for 24 h. To ensure growth, at 24 h, the OD600 was measured using a plate reader (Tecan) before the addition of the test agents.
Obtaining essential oils and stock solution preparation
Shelf-stable essential oils were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) for two plants: cinnamon (Ceylon type, #8015-91-6) and ginger ([6]-gingerol, Z. officinale, #23513-14-6) (Sigma-Aldrich, St. Louis, MO, USA). Oils were stored under supplier recommended storage conditions and had a supplier stated expiration period of 5 years.
Stock solutions (50%) of each oil were prepared. Dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, MO, USA) was utilized within the study to serve as a solubilizer for the test agents. 10 ml of cinnamon oil combined with 10 ml of DMSO and 5 ml of ginger oil with 5 ml of DMSO were added to separate flasks. DMSO solvent also served as a control to observe any effect of changes to biofilm susceptibility.[15]
Calcium hydroxide preparation
Calcium hydroxide (Ca[OH]2) (0.1%), used as a positive control (Calcium Hydroxide U. S. P., Pemco, Newark, NJ), was prepared by adding 0.25 g to 25 ml of BHI or normal saline (USP Normal Saline 0.9% Sodium Chloride, Nurse Assist, Inc. [Haltom City, TX, USA]).
Antibiofilm assay
The cell was incubated for 24 h as described above, and spent supernatants were removed. The biofilm cells were washed with 200 μl phosphate-buffered saline twice to remove nonadherent cells. All trials were performed in two biological replicates with a minimum of five technical replicates per plate [Figure 2 for additions]. Saline (200 μl) or BHI (200 μl) was added to the wells. Trial #1 tested cinnamon oil and (Ca[OH]2).
Figure 2: Ninety-six well plate key of additions for Trials 1, 2, and 3. Trials 1 and 3 tested cinnamon oil and calcium hydroxide. Trial 2 tested cinnamon oil, gingerol, and calcium hydroxide. DMSO: Dimethyl sulfoxide, CaOH: Calcium hydroxide, Cinn oil: Cinnamon oil, Gin: Gingerol, BHI: Brain–heart infusion
To achieve a 1% final concentration, 4 μl of the 50% cinnamon oil was added with either addition. Ca(OH)2 was added at 20 μl, and DMSO was 4 μl or 8 μl DMSO. Trial 1 included two replicate plates. Trial #2 tested cinnamon oil, ginger oil, and Ca(OH)2; all additions were the same as in Trial 1 with the addition of 4 μl of the 50% ginger oil was to achieve 1% in the well. Trial 2 included two replicate plates. In Trial 2, it was found that ginger oil did not disperse within the solution even in the presence of DMSO; therefore, it was excluded from Trial 3. Trial #3 tested cinnamon oil and Ca(OH)2 under the same conditions tested in Trial 1. Trial 3 included two replicate plates.
The plates from all trials were incubated at 37°C in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI, USA) at 20% H2, 20% CO2, and 80% N2 atmospheric conditions for 24 h. Following this, the turbidity of wells on tested plates was measured at OD600 nm using the microliter plate reader.
2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-([phenylamino] carbonyl)-2H-tetrazolium hydroxide colorimetric assay
To assess cell viability and potential metabolic output within the biofilm, the soluble tetrazolium dye 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-([phenylamino] carbonyl)-2H-tetrazolium hydroxide (XTT) was used following each trial. All plates were washed with 100 μl of saline. 50 μl of the XTT solution (10 mg of XTT) was added to 10 ml BHI (with 200 μl of 10 mg/ml menadione), and 200 μl was added to each well. The plates were incubated for 30 min at room temperature. Following the incubation, the plates were read at absorbance (Ab) 450 nm using the microplate reader. The plate was then diluted 1:2, 100 μl of the sample was diluted to 100 μl with saline, and then reread with the microplate reader.
RESULTS
Analysis was performed using Microsoft® Excel for Student's t-test and independent samples–Kruskal–Wallis test for pairwise comparisons between the different antimicrobial activity tests against reference and clinical strain in its biofilm state at a 95% level (P < 0.05).
The actual OD450 absorbance values per plate were obtained within trials. The percent of biofilm reduction, averages, and standard deviations were calculated [Figures 3-5]. Data in Figure 3 represent the averages and standard error of two plates with four replicates per plate. Data in Figures 4 and 5 represent the averages and standard error of six technical replicates per plate.
Figure 3: Average percent of Absorbance 450 nm for trial #1. (a) 24-hr old biofilms were treated with test agents or DMSO control with additional BHI. After a 24 hr incubation, XTT was added to the wells, allowed to incubate, and read with a plate reader at Ab450. Data represent the averages and standard error of two plates with four replicates per plate. (b) 24-hr old biofilms were treated with test agents or DMSO control with additional saline. After a 24 hr incubation, XTT was added to the wells, allowed to incubate, and read with a plate reader at Ab450. Data represent the averages and standard error of two plates with four replicates per plate. BHI versus saline (P < 0.05). 1% cinnamon oil versus negative (no test addition) and positive (calcium hydroxide) controls (P < 0.05). 1% cinnamon oil combined with calcium hydroxide (P > 0.05).
Figure 4: Average percent of Absorbance 450 nm for trial #2. (a) 24-hr old biofilms were treated with test agents or DMSO control with additional BHI. After a 24 hr incubation, XTT was added to the wells, allowed to incubate, and read with a plate reader at Ab450. Data represent the averages and standard error of six technical replicates per plate. (b) 24-hr old biofilms were treated with test agents or DMSO control with additional saline. After a 24 hr incubation, XTT was added to the wells, allowed to incubate, and read with a plate reader at Ab450. Data represent the averages and standard error of six technical replicates per plate. BHI versus saline (P < 0.05). 1% cinnamon or ginger oil versus negative (no test addition) and positive (calcium hydroxide) controls (P < 0.05). 1% cinnamon oil versus 1% cinnamon oil combined with calcium hydroxide (P > 0.05)
Figure 5: Average percent of Absorbance 450 nm for trial #3. (a) 24-hr old biofilms were treated with test agents or DMSO control with additional BHI. After a 24 hr incubation, XTT was added to the wells, allowed to incubate, and read with a plate reader at Ab450. Data represent the averages and standard error of six technical replicates per plate. (b) 24-hr old biofilms were treated with test agents or DMSO control with additional saline. After a 24 hr incubation, XTT was added to the wells, allowed to incubate, and read with a plate reader at Ab450. Data represent the averages and standard error of six technical replicates per plate. BHI versus saline (P < 0.05). 1% cinnamon oil versus negative (no test addition) and positive (calcium hydroxide) controls (P < 0.05). 1% cinnamon oil combined with calcium hydroxide (P > 0.05)
Antimicrobial potential of cinnamon
Cinnamon oil was tested upon in all three trials [Figures 3-5]. A significant difference was detected with the presence of cinnamon alone when compared to negative (no addition to BHI or saline conditions) and positive control (Ca[OH]2) groups (P < 0.05). No significant difference was found between cinnamon alone and cinnamon with Ca(OH)2 within both BHI and saline conditions (P > 0.05).
Antimicrobial potential of ginger
Ginger oil was tested in one trial [Figure 4], it was observed that its oil preparation did not disperse within the wells. Ginger showed a significant difference when compared to the “no addition” control in both BHI and saline conditions.
Antimicrobial potential of positive control: Calcium hydroxide
Among all three trials, the highest biofilm reduction values of Ca(OH)2 were 88.1% and 10.7% in saline and BHI conditions, respectively. The combination of Ca(OH)2 with cinnamon oil revealed 86.5% and 73.9% reduction in biofilm in saline and BHI conditions, respectively. These results may suggest an indifferent relationship between cinnamon oil and Ca(OH)2 in the presence of saline but a synergistic effect in BHI conditions. However, the combination of ginger oil and Ca(OH)2 revealed a higher reduction of 91.6% in saline conditions but no reduction in the presence of BHI.
Brain–heart infusion versus saline conditions
A significant difference was found between BHI and saline conditions in which greater biofilm reduction was found in saline environments for both oil types (P < 0.05).
DISCUSSION
Unhealed or refractory cases of apical periodontitis lesions in previous endodontically treated teeth have been microbiologically analyzed by Sundqvist et al. with a demonstrable link to E. faecalis as the most common microorganism isolated.[16] E. faecalis possesses significant resistance to treatment, which is attributed to its ability to penetrate dentinal tubules, withstand high pH values, and form communities organized in a biofilm and its virulence factors. Its biofilm allows resistance to destruction by enabling it to be 1000 times more resistant to phagocytosis, antibodies, and antimicrobials over other nonbiofilm-producing organisms, recovered from endodontic infections.[417]
Ca(OH)2 is a standard medicament in endodontic therapy. Its success has been attributed to its ability to disrupt the cellular metabolism of bacteria by causing protein denaturation due to its high pH value of approximately 12.5.[18] However, previous literature has discussed the insufficiency of Ca(OH)2 in eliminating E. faecalis at high pH levels. Evans's in vitro study testing the effect of Ca(OH)2 pH levels on the ability of E. faecalis to survive found that E. faecalis still survived at a pH level of 11.1, but a 20-fold reduction was achieved at 11.5. It was determined that the presence of a functioning proton pump is vital to its survival.[8] In addition, in radicular dentin, the alkalinity of Ca(OH)2 may only reach a pH of 10.3 after intracanal dressing due to the dentin's buffering effect.[19]
Phytotherapeutic substances have been used in dentistry as anti-inflammatory, antibiotic, analgesic, and sedative agents as well as for other methods including endodontic irrigation, medicaments, and retreatment.[22] Cinnamon from the plant C. zeylanicum and ginger from Z. officinale are among the herbs having antimicrobial properties against oral pathogens and have shown significant activity against isolates of enterococci.[12]
The active components responsible for antimicrobial action of cinnamon and ginger are cinnamaldehydes and gingerol. Cinnamaldehydes disrupt bacterial cell membranes and ginger inhibits bacterial growth (bacteriostatic activity).[2021] This was demonstrated in an in vitro study that investigated the antibacterial activity of spices against clinical isolates from 215 strains of enterococci colonies with a result of all different species of Enterococcus maximally inhibited by cinnamon and ginger.[12] There are currently limited studies that investigate the potential of cinnamon and ginger essential oils against E. faecalis in a biofilm state. In a study by Mohd-Said et al., a dose-dependent potential of ginger oil against E. faecalis biofilm revealed an inhibitory effect of 76% at a 5.0 mg/mL concentration.[11] An 83% biofilm inhibition by ginger essential oil against multidrug-resistant bacteria, including E. faecalis, was demonstrated in another antibiofilm activity study.[22] The current study sought to compare cinnamon oil in addition to ginger oil.
The current study tested essential oils against preformed E. faecalis biofilms under two environments, in BHI and saline to simulate nutrient-rich versus nutrient-depleted conditions, respectively.[23] Sources of nutrient supply within the root canal system that bacteria can utilize include necrotic pulp tissue, proteins, and glycoproteins from tissue fluids that seep into the main canal from the apical foramen or lateral canals, saliva components that penetrate coronally, and products from metabolism of other bacteria.[24]
The colorimetric XTT assay was utilized to quantify the presence of viable cells within the biofilm, providing for the measurement of biofilm formation in a semi-quantitative manner.[25] Before placement of test solutions, optical density values were measured, correlating the cell number for biofilm analysis. Results indicate that cinnamon and ginger oils have an antimicrobial potential against a preformed, 1-day-old, E. faecalis biofilm in vitro. A greater reduction in biofilm was detected in the presence of saline than in BHI media. This could be attributed to BHI providing continued nutrition to E. faecalis, allowing it to maintain viable cells, whereas physiologic saline provides osmotic protection for microbial cells but does not provide a nutritional reservoir. Calcium hydroxide presented with a reduction of the E. faecalis biofilm, possibly due to the absence of clinical root canal conditions, such as the presence of dentin debris following canal instrumentation, smear layer, and anatomical canal complexities, which may hinder Ca(OH)2 penetration into dentinal tubules. Here, Ca(OH)2 came into direct contact with the entirety of the biofilm without hindrance by other factors.
There are limitations within this study to address for future considerations. This pilot study will require further development in which the biological trial triplicate represents consistency in its protocol measures. In addition, performing an ex vivo study on a dentin model may more closely simulate the clinical setting. E. faecalis-dentin interaction within a 6-week-old biofilm can reveal sediment formation where there is an increase in calcium phosphates and carbonates.[25] An SEM study performed by Kishen et al. concluded that this bacteria-mediated process resulted in the formation of a distinct calcified biofilm, which may be a contributing factor for increased resistance.[26] Furthermore, the presence of nutrition within the root canal environment cannot be adequately simulated.
CONCLUSION
Based on the results of this in vitro feasibility study, cinnamon oil may provide a potential adjunctive therapy in treating endodontic infections associated with E. faecalis. Additional trials are necessary to determine the efficacy of ginger oil. Further developments in research on these oils are warranted for understanding the resistance of E. faecalis biofilms as they mature and evaluating different concentrations of these reagents and their effects over time.
Financial support and sponsorship
This study was supported by Rutgers School of Dental Medicine and the American Association of Endodontists Foundation (SA).
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
This study was supported by Rutgers School of Dental Medicine and the American Association of Endodontists Foundation (SA).
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