ABSTRACT
Objective: The crucial aim of irrigation solutions used in root canal treatments is the elimination of biofilm which is considered an important virulence factor of Enterococcusfaecalis. Essential oils have been investigated to understand their efficacy for biofilm elimination in recent years. This study aimed to evaluate the antimicrobial and antibiofilm effects of Thymus vulgaris essential oils (TEO) and Origanum onites essential oils (OEO) on E. faecalis.
Methods
The antimicrobial effectiveness of TEO and OEO against E. faecalis (ATCC 29212) was investigated by broth microdilution method, and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The effect of TEO and OEO on preventing biofilm formation was evaluated by measuring biofilm biomass using crystal-violet method, and its effect on biofilm viability was evaluated by determining the number of living cells in the biofilm as colony-forming units. Biofilm viability was analyzed with one-sample t-test with statistical significance accepted as p<0.05.
Results
The MIC values of TEO and OEO were determined as 0.078 µL/mL, and MBC values were determined as 0.156 µL/mL for OEO and 0.078 µL/mL for TEO. The percentage inhibition of biofilm formation at MIC value for OEO and TEO was determined as 53.9% and 55.6%, respectively. Both essential oils caused a significant reduction in the number of viable cells within the biofilm.
Conclusion
It is concluded that TEO and OEO show high antimicrobial and antibiofilm activity against E. faecalis biofilm. Therefore, these essential oils can be considered an alternative irrigation solution for eliminating resistant root canal infections.
Introduction
Microorganisms, found in the oral flora, can adhere better to hard and soft tissue surfaces due to their biofilm formation ability. Enterococcus faecalis is one of the important biofilm-forming bacteria, which causes resistant periradicular dental infections. E. faecalis settles in root canals, mainly the apical part and forms biofilm layer in deeper dentin tubules (1, 2). The extracellular matrix, which forms the basic structure of the biofilm, is a protective polymer structure that bacteria constantly form. As it forms, it ensures that the bacteria are more isolated from the buried external environment (2, 3). Therefore, the biofilm layer is considered a small community with three-dimensional signal transport, nutrition, and waste channels that microorganisms create as a suitable habitat for themselves. Because of these channels, microorganisms can protect themselves from changes in the external environment and neutralize the effects of agents such as disinfectants and antiseptics used to destroy them (2).
E. faecalis forms a typical biofilm in which the physicochemical properties are modified to dominate according to the environment. In a typical biofilm, living cells are located on the surface of the biofilm, and irregular growth of adjacent cell clusters is observed in aerobic or anaerobic and nutrient deprived-medium (1, 3-5). Therefore, resistant periradicular dental infection develops because of the complex structure of the biofilm and the first step to eliminate the infection should be the destruction of the biofilm. At this point, sodium hypochlorite at a concentration of 0.5-6% is routinely applied in dental treatments as it is a cost-effective irrigation solution to eliminate E. faecalis biofilm (5). However, the search for alternative irrigation agents continues due to their toxic and caustic nature to the periradicular tissues (5-7).
When considering agents with antibacterial solid activity as well as minimal tissue irritation, herbal-based agents can be thought. Therefore, natural products with herbal ingredients are used in the development of many oral hygiene products because they have rich antimicrobial components (7, 8). Thymus vulgaris (thyme) essential oils (TEO) and Origanum onites essential oils (OEO) are herbal agents with antibacterial activities, but there is insufficient information in the literature regarding the effectiveness of these essential oils (EOs) on E. faecalis biofilms. So, this study aimed to determine the antibacterial activity of TEO and OEO on E. faecalis biofilm by evaluating minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and antibiofilm activity at different concentrations.
Methods
This microbiological research study was conducted in accordance with the principles of the Declaration of Helsinki. This study was approved by Başkent University Medical and Health Sciences Research Board Institutional Review Board (approval number: KA22/187, date: 28.04.2022). A series of microbiological tests were conducted to calculate MIC, MBC, biofilm formation inhibition percentage, and biofilm viability effect of different concentrations of TEO and OEO to evaluate their effectiveness on E. faecalis biofilm within the scope of this study. E. faecalis ATCC 29212 was used for all biofilm and antimicrobial activity tests. TEO and OEO were provided by the manufacturer (Caliskan Agriculture, Türkiye). All the EOs were obtained by the hydro-distillation method.
MIC and MBCs for TEO and EOS
For determining MIC values, broth microdilution method was used as recommended by the Clinical and Laboratory Standards Institute (9). The EOs in combination with 1% dimethyl sulfoxide, were diluted twofold in Mueller Hinton Broth (BD Difco™, France). After 24h of incubation at 37 °C, absorbance was measured at 570 nm (BioTek Instruments, ELX 800, USA). All studies were performed in triplicate. The TEO and OEO concentrations ranged from 0.039-10 µL/mL (10). The wells containing only bacterial suspension were used for bacterial growth control, and sterile Mueller Hinton Broth without bacteria or antibacterial agent was used for sterilization control. Plates were incubated at 37 °C for 24h and each experiment was repeated 3 times. MBC was determined by inoculating 10 µL of suspension from each well in Tryptic Soy Agar (Condalab, Spain) after the incubation period of MIC assay (11). MBC was considered the lowest concentration of the test substance in which no microbial growth was observed after the incubation period at 37 °C for 24h.
Biofilm Formation Assay
The biofilm formation assay was performed as described previously (12). Briefly, flat-bottom polystyrene microtiter plates containing 180 µL of tryptic soy broth (Condalab, Spain) were inoculated with 20 µL of bacterial culture adjusted to 1x106 CFU/mL. After 48h of incubation, biofilm formation was detected by the crystal violet staining method (CVS) (13). The absorbance was measured at 570 nm (11), and biofilm formation ability was evaluated as negative, weak, intermediate and strong for tested strain (14). Staphylococcus aureus ATCC 6538 and S. aureus ATCC 29213 were used as a positive and negative controls, respectively.
Calculation of Biofilm Formation Inhibition Percentage of EOs
The effect of TEO and OEO against E. faecalis biofilm were tested in 96-well plates at MIC and MBC concentrations and evaluated by comparing untreated wells. Briefly, 100 µL of bacterial suspension for each isolate were added to each well containing different concentration of EOs at MIC, MBC concentrations. After 24h of incubation, CVS was applied, and the OD values were quantified as described above. Wells with PBS were used as a control to calculate the percentage of biofilm inhibition (PI) as follows (11):
PI = [(Control OD570 nm-Sample OD570 nm)/Control OD570 nm] x 100
Effects of TEO and OEO on Biofilm Viability
Biofilm formation was carried out without antimicrobial agent as described above for prolonged time. In this study, the medium was refreshed every 48h and biofilm formation was provided in 2 weeks. At the end of the process, the wells containing enterococcal biofilm were washed twice with saline and 200 µL of each agent was added to each well at different concentrations. Then the sonication procedure (Daihan, Korea) for 10 seconds at 30k Hz was performed, and wells were mixed to remove excess biomass after sonication. To calculate viable microorganisms in the biofilm, serially diluted suspensions were cultured and the plates were incubated for 24h at 37 °C. The colonies were counted and the number of viable cells within the biofilm was calculated as CFU/mL. The number of viable microorganisms was compared with those in the control well with PBS (11).
Statistical Analysis
The IBM SPSS for Windows, version 22.0 was used for the statistical analysis (IBM Corp., USA). MIC and MBC were given as average values of repeated tests while calculation of biofilm formation inhibition was given as percentages. Biofilm viability data of TEO and OEO were analyzed by using one-sample t-test and statistical significance was sat at p<0.05.
Results
The MIC and MBC values of TEO and OEO were given in Table 1. MIC values of TEO and OEO were same at 0.078 µL/mL; however, TEO had higher MBC (0.156 µL/mL) than OEO (0.078 µL/mL). Biofilm formation inhibition percentages of TEO and OEO were calculated as 53.9% and 47.9% at 0.078 µL/mL concentrations while 55.6% and 44.8% at 0.156 µL/mL concentration for TEO and OEO, respectively. Antibiofilm activity of TEO and OEO on E. faecalis was given in Figure 1 indicating the OD values at 570 nm.
The effects of TEO and OEO on biofilm viability were shown in Table 2 and Figure 2. It was observed that both TEO and OEO has a significant effect on biofilm viability as both EOs caused sharp decrease in the number of viable cell within the biofilm at 0.078 µL/mL concentration. There was not statistical difference between TEO (p=0.483; p≥0.05) and OEO (p=0.169; p≥0.05) activities on biofilm viability.
Discussion
Sodium hypochlorite is considered the gold standard for root canal antisepsis and in routine clinical applications, chlorhexidine and sodium hypochlorite are the most frequently used irrigation materials in endodontic treatments to eliminate residual microorganisms (1, 8, 12). According to the literature on the elimination of endodontic pathogens, chemo-mechanical treatment did not show sufficient success due to the microbial adhesion to the dentin surface because of the specific anatomic structure of the root canal system and the adhesion ability of these endodontic pathogens.
Other than sodium hypochlorite, calcium hydroxide and triple antibiotic paste are the intracanal medicaments that can penetrate deep dentin tubules to eliminate biofilm (6, 15). Ethylenediaminetetraacetic acid (EDTA) is one of the most used agents to remove the smear layer from root canals. Since E. faecalis biofilm is embedded in deep dentin tubules, EDTA application is assumed to provide an antibacterial effect during the removal of the smear layer. However, its antimicrobial activity against biofilms is still controversial (2). According to the previous in vitro studies, irrigation with silver nanoparticles (16), photodynamic therapy with lasers (16, 17), or ozone application (18) during the irrigation might increase the antibacterial efficacy of sodium hypochlorite or EDTA.
Tissue-friendly herbal products have been started to be preferred in intracanal irrigation to prevent the exposure of healthy tissues and stem cells around the root from the toxic effects of irrigation solutions or medicaments that overflow from the root apex. Therefore, natural products derived from plants are rich in antimicrobial compounds and are often used to make oral hygiene products. EOs obtained from medicinal plants have been used in the treatment of some diseases due to their antimicrobial effects (18-20). However, there are few studies on their success in root canal treatments (8). According to the improvements in dental materials, the reason for resistant root canal infections seemed to be the presence of some resistant bacteria inside the roots such as E. faecalis and its important virulence factor biofilm formation (21, 22). Therefore, the aim of this study was to evaluate MIC, MBC, and antibiofilm activity at different concentrations of TEO and OEO to evaluate their efficacy and record them for further laboratory, in vitro, and in vivo studies.
In the present study, TEO and OEO were preferred to determine antibacterial and antibiofilm efficacies in different concentrations. Thosar et al. (20) reported an in vitro study evaluating the antimicrobial activity of TEO and, according to the results zinc oxide cement mixed with TEO showed higher antimicrobial activity than zinc oxide and eugenol group against Staphylococcus aureus, Escherichia coli, E. faecalis, and Pseudomonas aeruginosa. Furthermore, Janani et al. (23) reported another in vitro study about the antimicrobial efficacy of OEO by evaluating MIC and MBC values amongst E. faecalis. According to the results, MIC was found to be 25 µg/mL while MBC was found to be 50 µg/mL. So, OEO was reported to be an effective antimicrobial agent against E. faecalis. According to the results of another study (24) comparing the antimicrobial efficacy and removal of the smear layer in dentin tubules, 1% OEO and 5.25% NaOCl had the same antimicrobial activity against E. faecalis and 2% or 5% OES had more effective antibacterial action than 5.25% NaOCl. According to these articles, the high antibacterial efficacy of OEO and TEO against E. faecalis can be seen. However, within the scope of this study, not only the antimicrobial activity of E. faecalis but also the antibiofilm activity of the biofilm it formed was investigated. According to the antibacterial results of these EOs in the present study, TEO and OEO had the same MIC values but, different MBC values. MBC values of TEO were higher than MIC values so, it can be said that TEO might be more unstable and suspicious than OEO according to higher MBC than MIC.
Biofilm formation is considered an important virulence factor of resistant root canal infections. So, in this study, antibiofilm efficacy was evaluated to determine the correct answers for resistant clinical root canal infections. According to the obtained results of biofilm formation inhibition, TEO showed higher inhibition on biofilm formation compared to OEO at both MIC and MBC concentrations. This antibiofilm feature might be a clue for the higher efficacy of TEO rather than OEO at the same MIC value.
According to the results, there were controversial results amongst MBC and biofilm formation inhibition values. TEO had bactericidal efficacy at higher MBC results than OEO. This means suspicious behavior has a toxic effect on tissues while showing higher antibiofilm activity than OEO by biofilm formation inhibition percentages at the MIC level. The results of biofilm viability showed that OEO caused a significant decrease rather than TEO and this result means that it was more effective against the formation of biofilm which is the most important virulence factor of E. faecalis. Although, these results showed that both EOs might be used due to higher antimicrobial and antibiofilm activity on E. faecalis biofilm even at low concentrations; OEO might be accepted as a more stable EO than TEO due to the same MBC than MIC values and similar antibiofilm activity results before and after the biofilm formation. However, these controversial results should be evaluated with more in vitro studies. A significant cost is spent every year for the treatment of resistant root canal infections. Despite this, patients still need to get the teeth with the high clinical and radiographic success they deserve. Therefore, biocompatible structure and lower costs are important causes besides good antibacterial and antibiofilm effects against E. faecalis biofilm to prefer TEO and OEO.
Study Limitation
The strong point of this study was the penetration ability of EOs into dentin tubules. However, to the best of our knowledge, this is the first study comparing the antibacterial and antibiofilm efficacy of TEO and OEO against the E. faecalis biofilm. Studying a single bacterial species can be considered as the limitation of the study. The results of the present study should be evaluated for future in vitro and clinical studies including E. faecalis biofilm.
Conclusion
High antimicrobial effects of different concentrations of TEO and OEO were determined as MIC, MBC, biofilm formation inhibition percentage, and biofilm viability on E. faecalis biofilm. Therefore, it can be concluded that TEO and OEO might be used in resistant and recurrent endodontic lesions to destroy and remove the biofilm layer from root canals. However, further microbiological, and clinical studies should be done to evaluate the preliminary results of TEO and OEO and compare them with other irrigation agents such as sodium hypochlorite or chlorhexidine.