To Evaluate and Compare the Antimicrobial Efficacy of Various Disinfecting Agents on K-file against Gram-positive and Gram-negative Bacteria of Endodontic Origin: An In Vitro Study
Corresponding Author: Kavita Dhinsa, Department of Pediatrics and Preventive Dentistry, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow, Uttar Pradesh, India, Phone: +91 9721222260, e-mail: firstname.lastname@example.org
Introduction: Microorganisms induce a variety of infections and diseases in the human body and are largely ubiquitous in the nature of the contamination, directly, or indirectly leading to transmission of infectious agents. A substantial number of bacterial species have been identified as inhabitants of the oral cavity. Infection control is a major topic of concern in medical and dental healthcare settings.
Aim: To evaluate the disinfecting ability of various herbal extracts on sterilization of endodontic files against endodontic pathogens.
Materials and methods: The study was conducted in three phases to evaluate the disinfecting ability of tea tree, neem, eucalyptus, and ajwain oil on sterilization of endodontic files against Staphylococcus aureus, Streptococcus β-hemolyticus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Peptostreptococcus species, and Bacteroides fragilis.
Results: Colony-forming units (CFU) of all tested bacteria among group I (tea tree oil) were found to be significantly lower than that among group II (ajwain oil), group III (eucalyptus oil), and group IV (neem oil), respectively.
Conclusion: The antimicrobial activity among all the essential oils was found to be maximum for tea tree oil, followed in descending order by ajwain, eucalyptus, and neem oils. Hence, the incorporation of these herbs in dental practice will prove to be a valuable adjunct in dental treatment.
Clinical significance: Herbs have also been long used in various endodontic medicaments and dressings with obtundent and soothing effects and exhibited a great microbial inhibition potential against the tested endodontic pathogens.
How to cite this article: Opi K, Dhinsa K, Tripathi AM, et al. To Evaluate and Compare the Antimicrobial Efficacy of Various Disinfecting Agents on K-file against Gram-positive and Gram-negative Bacteria of Endodontic Origin: An In Vitro Study. Int J Clin Pediatr Dent 2023;16(S-2):S161–S167.
Source of support: Nil
Conflict of interest: None
Keywords: Disinfection, Endodontic files, Herbal oils
Microorganisms cause various infections and diseases in the human body and are largely pervasive in the nature of contamination, directly or indirectly leading to the transmission of infectious agents.1 A considerable number of bacterial species have been identified as inhabitants of the oral cavity.2 Infection control is a major topic of interest in both medical and dental healthcare settings.3 However, evidence linking endodontic treatment to disease transmission is lacking. In the absence of adequate infection control procedures, there is a real potential for transmission of pathogenic microbes through endodontic instruments.4 These pathogenic organisms can originate from inside the root canal or from the periradicular tissues.5 This is especially important in endodontics because all types of debris, such as necrotic and vital tissue, bacteria, dentinal chips, blood byproducts, and other potential irritants, are encountered during root canal instrumentation.6 It is extremely important for endodontic instruments to be cleaned, disinfected, and successfully sterilized to prevent any contamination from infectious instruments.7
Although there are many procedures for sterilizing or disinfecting endodontic instruments, they still have some shortcomings. Autoclaving is considered the gold standard for sterilization but has the disadvantage of modifying the mechanical properties of files by reducing their cutting efficiency.1 A limitation of instrument sterilization with liquid chemical sterilants is that the instruments cannot be wrapped during processing; hence, keeping up sterility after processing and during storage is impossible.8
To overcome the drawbacks of conventional methods, phyto-dentistry appeared in dentistry due to its properties, such as the fact that it does not disturb the physical properties and microorganisms are not able to develop resistance against the agents used.1 Herbal products have been used in folk medicine since ancient times, spanning both Eastern and Western healing traditions.9 According to the World Health Organization, herbal medicine is considered a plant material or preparation that consists of raw or processed components from one or more plants with therapeutic values.10 The presence of phenolic compounds attributed to the antimicrobial activity of several essential oils.11 The importance of these medicinal plants for therapeutic use is that they are inexpensive, safe, effectual and easily available, making them essential tools for physicians to treat their patients.12 Azadirachta Indica comes from the family Meliaceae, which is commonly known as neem. Different parts of neem (leaf, bark, and seed oil) exhibit wide pharmacological activities including; antimalarial, antioxidant, antiinflammatory, antimutagenic, anticarcinogenic, antihyperglycemic, antiulcer, and antidiabetic properties.13Eucalyptus globulus Labill, belongs to the family Myrtaceae. The medicinal value of eucalyptus oil is largely based on the content of a specific oil component—1, 8-cineole.14Trachyspermum ammi belonging to the family Apiaceae. Ajwain seeds contain phenols which have carvacrol (1–7%), thymol (30–50%), and also monoterpenes which have 20–35% terpinene and 20–25% paracymene, pinene, and limonene.15 Tea tree oil (Melaleuca alternifolia) belongs to the Myrtaceae family. The phytocomponents characterizing the tea tree oil are monoterpenes and alcohol-monoterpene. It is best known for its antimicrobial activity against a broad spectrum of microorganisms and some viruses.16 The aim of our study is, therefore, to evaluate and compare the disinfectant ability of different herbal extracts on sterilization of endodontic files against various endodontic pathogens.
MATERIALS AND METHODS
The study was performed to evaluate the antimicrobial activity of tea tree, neem, eucalyptus, and ajwain oil on sterilization of endodontic files against Staphylococcus aureus, Streptococcus β-hemolyticus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Peptostreptococcus species, and Bacteroides fragilis. A total of 175 endodontic K-files were set in an endodontic instrument box and autoclave at 121° C for 15 minutes at a pressure of 15 pounds. Seven endodontic pathogens were used for disinfecting the files. After the seven bacterial broths for each strain were obtained, five presterilized K-files were infected with each strain of bacteria by loading them into a sterile container with the bacterial broth separately for 30 minutes (Fig. 1). Followed which all the K-files infected with each strain were transferred to a sterile petri dish separately and were incubated for 30 minutes at 37C for drying to ensure fixation of the microbes on the files (Fig. 2). Five infected files from each of the strain were obtained and were inserted into the test chemicals (Fig. 3):
Group I—35 endodontic K-files (i.e., five files each infected with seven bacteria) were treated with tea tree oil.
Group II—35 endodontic K-files (i.e., five files each infected with seven bacteria) were treated with ajwain oil.
Group III—35 endodontic K-files (i.e., five files each infected with seven bacteria) were treated with eucalyptus oil.
Group IV—35 endodontic K-files (i.e., five files each infected with seven bacteria) were treated with neem oil.
Group V (control group)—35 endodontic K-files (i.e., five files each infected with seven bacteria) were treated with autoclaving.
The endodontic K-files in groups I, II, III, and IV were subjected to the test chemical for 1 minute, and group V was autoclaved following the standard protocol.
The disinfecting capacities of the test chemicals were checked utilizing the three strategies.
Miles and Misra’s method.
After the files were completely sterilized by the different herbal oils, all the files were again subjected to individual test tubes containing peptone water and then kept for incubation at 37 C for 18–24 hours (Fig. 4). Turbidity of the test tube indicates the growth of the contaminated bacteria and also refers that the particular file has not been sterilized completely (Fig. 5).
Miles and Misra’s Method for Colony Count
From the above test tube with disinfected files, 1 mL will be serially diluted utilizing Miles and Misra’s method of serial dilutions in 10 test tubes of 9 mL normal saline, 10 dilutions were made, which makes the stock bacterial suspension to be diluted up to 10–10 (Fig. 6). From each of the dilutions, 0.2 mL were inoculated in Mueller–Hinton agar (MHA) plate with the use of inoculation loop, then was incubated for 24 hours or longer at 37 C. The colony-forming unit (CFU) was calculated using the formula given below (Fig. 7).
Colony-forming unit (CFU) = number of colonies counted/[amount plated (in mL) × the dilutions].
Microscopic Examination (Fig. 8)
The prepared slides were examined and checked after doing a Gram stain to confirm the presence of microorganisms under a light microscope.
Data were analyzed utilizing Statistical Package for the Social Sciences version 21. Categorical variables were summarized as frequency. The frequency of samples found positive for turbidity was summarized as percentages. Colony counts were summarized as mean and standard deviation. Graphs were prepared in Microsoft Excel.
On intergroup comparison between the different herbal oils used in the study, (Fig. 9) shows that for all the seven tested bacteria, the mean log value of CFUs of all tested bacteria among group I (tea tree oil), was found to be significantly lower than that among group II (ajwain oil), group III (eucalyptus oil), and group IV (neem oil), respectively. Hence, post hoc pairwise comparison showed group I > II > III > IV.
Elimination of the microorganism from the root canal is the utmost objective of root canal treatment. Removal of microorganism and their metabolites present in the pulp and periapical area is important for endodontic treatment.17 Proper knowledge about the habitat of microbial location in the interior of the root canal is important for understanding the infection pathway and for successful antimicrobial procedures. To achieve successful endodontic treatment, it is important to know the specific bacterial species harbored in each root canal.18 Endodontic files are considered to be one of the “critical items” as they contact the crucial tissues of the body; thereby, sterilization before use and reuse becomes important.1 The architectural complexity of the files hinders the cleaning procedures; therefore, proper sterilization of these files are mandatory for the effective removal of microorganisms which can be achieved by using various essential oils which are a rich source of natural compounds.5 Many plant-based essential oils have great potential for antimicrobial activity. To overcome the drawbacks of the chemical irrigants and their potential to remove the microorganism, scientists have developed new sources with broad-spectrum activities.19Streptococcus ß hemolyticus, or the facultative anaerobes, have been reported to be one of the predominant bunches isolated from contaminated root canals. Apart from this, Enterococcus faecalis has been five reported to grow, and colonize more rapidly in the dentinal tubules. Therefore, it is known to be the most resistant microbial organism in the oral cavity and results in the failure of root canal treatment. Studies have reported that Pseudomonas aeruginosa has been resistant to all the antimicrobial drugs that are prescribed during endodontic diseases. In cases of periapical lesions and necrosis of pulp, the habitat inside the root canal comprises all the strict (obligate) anaerobes such as Peptostreptococcus and Bacteroides.20 Since plant-based essential oils have a great ability can affect the growth of pathogens because of the presence of certain plant-based compounds. This study has been taken to evaluate the effectiveness of the disinfecting ability of various tea tree, neem, eucalyptus, and ajwain oil on sterilization of endodontic files against Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Streptococcus β-hemolyticus, Petostreptococcus species, Pseudomonas aeruginosa, and Bacteroides fragilis. Preparation of bacterial subculture was done using the streak plate method followed by bacterial broth preparation for each strain of bacteria in which K-files were infected.21,22 After infection, files were inserted into peptone water and evaluation was done using the following method:
Turbidity assessment: The original McFarland turbidity standards were made of barium sulfate precipitates which are prepared by incorporating barium chloride into sulfuric acid. The growth of contaminated bacteria is proved by the presence of turbidity in the test tube, which indicates that it is not sterilized completely.23 Numerous studies have been done to assess the presence or absence of bacteria using the turbidity method Raju et al.,3 and Hugar et al.1
Miles and Misra colony count: The Miles and Misra method (or surface viable count) is a technique that determines the CFUs in the bacterial suspension.24 Numerous studies have been done to determine the CFU using Miles and Misra method such as Kramer and Gilbert,25 Harrison et al.,26 Tin et al.,27 and Holla et al, etc.28
Microscopic examination assessment: The Gram stain is a sensibly fast, basic method and is able to supply a good demonstration under normal circumstances of most microbes, recognizing them as either grampositive or gramnegative, as appropriate.29 Numerous studies Young,29 Adam,30 and Hiremath and Bannigidad31 have been done to determine the strains of bacteria using Gram staining procedure to view under a microscope.
In the present study, the results stated that the antimicrobial efficacy of tea tree oil was significantly higher when compared to ajwain, eucalyptus, and neem oils. As reported by Gibbons and Carson et al., the essential oil’s effectiveness is due to the presence of chemical composition in it. As mentioned by Southwell et al., terpinen-4-ol is one of the major constituents present in tea tree oil. Apart from this linalool, α and γ-terpinene, α-terpinolene, p-cymene, and together with cineole and terpinen-4-ol also plays an important role in the antimicrobial efficacy of tea tree oil.16 In addition, Brophy et al., stated that P-cymene is one of the degradation products of tea tree oil that is produced after the terpinene-4-oil is oxidized.
According to Riley and Mee, the mechanism of action of terpinen-4-ol is due to the favorable hydrophobic hydrophilic character, which in turn diffuses through the water and thereby enters into the cytoplasmic membrane of the bacteria which causes the lysis of the cell wall, thereby losing its membrane integrity leading to leakage of ions and the inhibition of respiration.32
According to Markovic et al., tea tree oil is an extremely lipophilic substance, its mechanism is explained by lipophilic terpenes (terpinen-4-ol), one of the most important active principles of this essential oil. Terpinen-4-ol enters microorganism cell membranes and acts against its structural permeability. In this way, tea tree essential oil can affect the metabolism of certain microorganisms with bactericidal or fungicidal effects.33
In the present study, the result also stated that the ajwain oil had less antimicrobial activity when compared to tea tree oil but more when compared to eucalyptus oil and neem oil. As reported by Burt and Mahmoud which showed the relationship between the chemical structure of important compounds in ajwain with antibacterial effect on plants and compounds.34 As mentioned by Hasan et al., flavor and color, also play an important role in the defense mechanism of the system, thereby protecting against parasites, pathogens, fungi, and vertebrates. As mentioned by the author it is the presence of tannin and terpenoids, which plays an important role in its antimicrobial properties. Tannin plays an important role as it acts as a free radical scavenger, and terpenoids have the ability which cause apoptosis and suppress the proliferation of the cells which in turn leads to a tumor and also causes a reduction of the cholesterol level along with the risk of cardiovascular disease.35
Apart from the above results, the result also states that eucalyptus oil has antimicrobial activity, which is greater in comparison to neem oil but less in comparison to tea tree and ajwain oil. The antimicrobial action of the essential oils is due to a large degree to the oxidized terpenoids and to some hydrocarbonates whose interaction could result in indifferent, antagonistic, complementary, or synergic effects, which have been mentioned by Pei et al.36
According to Posadzki et al., the main ingredient of eucalyptus oil contains cryptone, p-cymene, α-terpineol, 1, 8-cineole α-pinene, trans-pinocarveol, phellandral, cuminal, globulol, limonene, aromadandrene, spathulenol, and terpinen-4-ol.37 In another study by Mulyaningsih et al., Pereira et al., eucalyptus oil also contains aromadendrene, followed by globulol, 1,8-cineole, and ledene, which plays an important role in antimicrobial properties.38
The results of the present study also showed the antimicrobial efficacy of Neem oil, however, it was least effective in comparison with other essential oils, that is, tea tree, ajwain, and eucalyptus oil. This could be due to the presence of terpenoids and phytochemicals, which act on bacterial cell walls, thereby binding to the cell wall and leading to the formation of adhesions forming complex, which causes inactivation of function and proteins present in the cell wall.1 Also, Mishra et al., reported that it contains azadirachtin, nimbin, picrin, and sialin as active ingredients. Prabhat et al. stated that because of the biocompatible antioxidants present in the neem, it can be recommended as an endodontic irrigant.39 Apart from the antimicrobial efficacy of the oils, results of the present study also showed a proportion of positive turbidity with respect to all the essential oils used in the study for Bacteroides fragilis and Peptostreptococcus species.
Sakkas et al. reported that Bacteroids are considered to be the most resistant microorganisms present in the root canal. These bacteria have a tendency that they result in the formation of complexes by adhering to the cell walls, thereby not allowing the antibiotics to penetrate easily into the cell wall.40 However, a study by Riggio and Lennon stated that the maximum resistance gained by Peptostreptococcus anaerobius is because of the presence of the organic compound, which leads to bioremediation.41,42
The results of the present study also showed the proportion of positive turbidity for Streptococcus β-hemolyticus (S.pyogens) with respect to ajwain, eucalyptus, and neem oils.
It has been reported by Fischetti et al., that the Streptococcus pyogenes have a unique property of the presence of protein F, which is known as fibronectin-binding protein which acts as a virulence factor because it binds to the cells, tightly thereby preventing any kind of antimicrobial action happening in the cell wall. Apart from that, Streptococcus pyogenes also contain protein M, which resists the phagocytic activity of the cell wall. It is also mentioned by the author that Streptococcus pyogenes has an outer covering of hyaluronic acid capsule which is resistant to phagocytosis and considered vital in order for it to survive in its hosts.43
The results of the present study also showed the proportion of positive tubidity for Enterococcus faecalis with respect to eucalyptus and neem oil.
A study by Igbinosa and Beshiru et al. reported an important property of enterococci is the ability of some strains to form biofilm. Biofilm production can promote increased resistance to antibiotics and other antimicrobials.44
Based on the results of the present in vitro study, with an understanding that the results could not be completely transferable to the in vivo situation, it is concluded that the ingredients of the tested materials and the time affect the results of the microbiological studies. The potential to inhibit microbial growth and survival in the root canal has also been evaluated using in vitro tests. The limitations of the present study could be the presence of efficacy of the oils which have been studied for shorter periods of time. Apart from this, the quantitative analysis of the oils, along with their qualitative screening, would further assist in understanding the active principles responsible for the antimicrobial activities. Further studies can be taken to explore the efficacy of these oils over a longer duration of time period.
Kavita Dhinsa https://orcid.org/0000-0002-3597-787X
Sonali Saha https://orcid.org/0000-0001-5361-1698
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