Comparing the disinfecting efficacy of pomegranate peel extract oil, Garlic oil, Tulsi leaf oil, and Clove leaf oil with standard autoclaving on dental round burs tested against Enterococcus faecalis: An in vitro study.

Background: The study was conducted to compare and evaluate the disinfecting efficacy of four different herbal oils with standard autoclaving against Enterococcus faecalis (E. faecalis). Fifty round burs were infected with E. faecalis and dipped into the chemical test solution for 10 min. The disinfecting efficacy for each test chemical was checked using culture method on blood agar plate and peptone water test to check for turbidity. Clove leaf oil and Tulsi leaf oil have very good antimicrobial efficacy against E. faecalis which are similar and comparable to that of autoclaving. Aim: This stusy aimed to compare the efficacy of pomegranate peel extract (PPE) oil, Garlic oil, Tulsi leaf oil, and Clove leaf oil with standard autoclaving on dental round burs tested against E. faecalis. Settings and Design: The disinfecting capability of four different experimental oils against autoclave was evaluated on 50 round burs exposed to E faecalis in an In-vitro study. Materials and
Methods: A total of 50 round dental burs were exposed to E. faecalis and the disinfecting capability of four different experimental oils against autoclave was evaluated by counting colony-forming units (CFUs) and turbidity test. Statistical Analysis: Statistical analysis was performed using SPSS version 20.0 (IBM), Chicago. The CFUs between the five groups (I, II, III, IV, V) were compared using the Chi-square test. The level of significance was kept at 5%.
Results: Clove leaf oil showed the best disinfection efficacy comparable to autoclaving, followed by Tulsi leaf oil with no statistically significant difference between them, whereas garlic oil and PPE oil were significantly less effective against E. faecalis.
Conclusion: Clove leaf oil and Tulsi leaf oil can be used for disinfecting dental burs, infected with E. faecalis, as alternatives to autoclaving. Copyright:

INTRODUCTION

The human microbiome is composed of a variety of organisms such as bacteria, viruses, fungi, and archaea that live in and around the human body. These are largely ubiquitous and play a major role in our human body physiology by having an important role in maintaining our surrounding environment. Only a small proportion of these bacteria induce a variety of infections which indirectly or directly lead to the spread of infectious agents.[1] The oral microbiome is a significant part of the human microbiome. The human oral cavity comprises significantly different types of microorganisms with distinct microbial communities which include bacteria, protozoa, archaea, viruses, and fungi. These microorganisms form a complex ecological community that impacts both systemic and oral health.

Infection control is an important concern in dentistry and medicine because of the rapid spread of infectious diseases in health care settings. Both the dental clinician and the patient are always at risk of communicating diseases during treatment. Sterilization is the procedure through which all the microorganisms both in vegetative or spore states are countered. The chief purpose of sterilization is to eradicate the spread of standing infectious diseases and prevent any new infections. In dentistry, it mainly relates to the reuse of instruments to prevent the spread of cross-infection and ensure optimal dental care.[2] Hence, all dental procedures should be performed using sterile instruments in a sterile environment.

Dental burs are “critical items” that are used in clinical dentistry for different procedures such as caries excavation, tooth preparation for almost all kinds of restorations, and endodontics to gain access into the root canal system. During all these procedures, burs get severely contaminated with saliva, blood, necrotic tissue, and potential pathogens and are recognized as a potential vehicle for cross-infection. Microbes transmit the disease or infection by handling them with contaminated hands, storing them in contaminated boxes, and even with improper sterilization of reused instruments. In routine dental practice, adequate disinfection and sterilization methods have to be followed to control and reduce the risk of cross-transmission of infection.[3] The burs when provided by the manufacturers comprise a high bacterial content that can be contagious and hence are required to be sterilized before they can be used on patients.[1] Many methods have been introduced to sterilize or disinfect dental burs in which autoclaving and hot air oven methods remain the gold standard and the most used method of disinfecting dental instruments. However, these methods have some drawbacks such as it increases the risk of corrosion and fracture susceptibility decreases its cutting efficiency and also decreases the life span of burs. Moreover, these are the time taken procedures that need a power source, storage facilities, and infrastructural maintenance, all of which should be weighed against its benefits.[4]

Root canal therapy is one of the most commonly carried out procedures in clinical dentistry. Endodontic instruments are “critical items” as they are introduced inside the root canal system which communicates the peri-radicular bone through apical foramen which may be one of the oro-systemic links. Hence, burs and endodontic instruments coming in contact with the root canal system should be in sterile condition before use and while reusing them.[5]

Although multispecies biofilms are the main reason for endodontic infections, the virulence factors possessed and the survival rate of Enterococcus faecalis (E. faecalis) makes it the chosen single microorganism used for laboratory research to represent persistent endodontic infections.

Phytodentistry describes the use of plants and their products in the treating process of stomatological problems, directly or indirectly. It has developed with dentistry owing to its properties such as it does not interfere with the physical properties and microorganisms fail to develop resistance against the agents used.[6] Previous in vitro studies have concluded that Clove oil, Tulsi oil, and Garlic oil are effective disinfectants and can be used as a substitute for autoclaving.[7]

Pomegranates have a salient medical past and possess notable medicinal properties.[8] The pomegranate peels constitute 50% of the total weight of the fruit. Bioactive compounds such as flavonoids, phenolics, ellagitannins, and proanthocyanidin compounds are present in them. Peel of pomegranate fruits also has minerals, such as magnesium, potassium, sodium, nitrogen, phosphorus, and calcium,[9] and also contains complex polysaccharides.

A study has concluded that pomegranate peel extract (PPE) has antimicrobial properties and is highly antioxidant. It also concluded that 20% of PPE alone and also in combination with 0.2% chlorhexidine (CHX) gluconate showed efficacy against E. faecalis.[10]

Round dental burs are the most commonly used instruments to remove the decayed part of the teeth, to prepare teeth to receive restorations, and to gain entry into infected root canals during root canal treatment. Hence, they come in contact with saliva and other infected organic and inorganic tissues and encounter the highest level of infection.[1] Moreover, there is very little literature regarding the use of PPE oil to disinfect dental instruments.

A study has shown that the sterilization method of autoclaving modifies the cutting efficiency of dental burs.[4] Hence, this present study was conducted to compare the disinfecting efficacy of PPE oil, Garlic oil, Tulsi leaf oil, and Clove leaf oil with standard autoclaving on dental round burs infected with E. faecalis.

MATERIALS AND METHODS

Study setting

The study was conducted at the Department of Microbiology and Department of Conservative Dentistry and Endodontics after obtaining clearance from the ethical committee, number 20015 (February 21, 2020). A total number of fifty round burs (Manufacturer: Mani) were divided into five groups of ten burs each.

Study design: An in vitro study

For the study, all the round dental burs were presterilized twice using an autoclave (Uniclave, Confident Dental Solutions) using the standard protocol. E. faecalis, ATCC 21292 was used in the study for contaminating the burs.

Sample size

Based on the study, antimicrobial activity of garlic, CHX group, and tea tree oil (Groppo et al. 2002), a standard deviation was assumed to be 300 in each group. Using an alpha of 5% and power of 80%, the Z values for alpha and beta are 1.959964 and 0.841621, respectively. Using the formula, where S is the standard deviation and D is the clinically significant difference in proportion (assumed to be 329), we get a sample size of 7 in each group. For ease of division of groups, we have taken a sample size of 10 for each group.

Sample preparation

The E. faecalis were inoculated into brain heart infusion broth and incubated overnight at 37°C. The turbidity equivalent to 0.5 Mcfarland standard was used as inoculum for further study. Pre sterilized dental burs were put into the test tubes containing the broth sealed with cotton plugs and incubated at 370 C for 30 min. After incubation, all the burs were taken out of the test tubes and shifted to a sterile petri dish.

Samples were divided randomly and equally divided into five groups containing ten burs each:

Group I: Dental burs treated with PPE oil (concentration of 0.5% weight/volume) prepared in the Department of Rasashastra, Sri Dharmasthala Manjunateshwara College of Ayurveda, Hassan, Karnataka, India.

Group II: Dental burs treated with Garlic oil extract (concentration 0.5% weight/volume) prepared in the Department of Rasashastra, Sri Dharmasthala Manjunateshwara College of Ayurveda, Hassan, Karnataka, India.

Group III: Dental burs treated with Tulsi leaf oil (concentration of 0.5% weight/volume) (Dr. Jain”s Forest Herbals Pvt. Ltd., Andheri, Mumbai, Maharashtra, India).

Group IV: Dental burs treated with Clove leaf oil (concentration of 0.5% weight/volume) (Dr. Jain”s Forest Herbals Pvt. Ltd., Andheri, Mumbai, Maharashtra, India).

Group V: Dental burs are treated according to standard autoclaving protocol.[5]

Preparation of pomegranate peel extract oil

100 g of PPE was collected made into a paste with the help of Khalva yantra (pestle). Decoction of PPE was prepared using a 1:16 ratio reduced to four parts, i.e., to 100 g of PPE, 16 l of water was added and boiled till it was reduced to four parts, i.e., to 400 ml of decoction. Later, 100 ml of decoction of PPE was boiled with 400 ml of sesame oil. The solution was boiled till the appearance of froth. After this, the oil was filtered.

Preparation of Garlic oil extract: 100 g of fresh garlic cloves was collected made into a paste with the help of Khalva yantra (pestle). Decoction of garlic extract was prepared using a 1:16 ratio reduced to four parts, i.e., to 100 g of garlic extract, 16 l of milk was added and boiled till it was reduced to four parts, i.e., to 400 ml of decoction. Later, 100 ml of decoction of garlic extract was boiled with 400 ml of sesame oil. The solution was boiled till the appearance of froth. After this, the oil was filtered.

Blood agar plate for growth of bacterial colonies and turbidity method in peptone water was used to check the disinfecting capabilities of the intended chemicals. Microscopic examination was done to confirm the presence of catalase-negative Gram-positive cocci in pairs. All the dental burs were dipped into the required test chemical for 10 min, after which five burs from each group were rolled over the entire blood agar plate using sterilized forceps and were incubated overnight at 37°C for the growth of bacterial colonies colony-forming units (CFU).

For colony-forming units, the scores were given as

Zero

0–103

103–105

>105.

For the turbidity method, five dental burs from each group were placed into the five different 1 ml peptone water and incubated overnight at 37°C and checked for turbidity, which proposed the growth of bacteria.

For turbidity, the scores were given as

No turbidity – 0

Mild turbidity – 1

Moderate turbidity – 2

Severe turbidity – 3.

After overnight incubation, a Gram stain was performed from the colonies grown on blood agar and from the overnight turbid peptone water. The presence of catalase-negative Gram-positive cocci in pairs confirmed the presence of E. faecalis and helped to rule out any contamination during the entire experiment.

Statistical analysis

Statistical product and service solutions, version 20.0 (SPSS Inc., Chicago, IL, USA).

The CFU between the five Groups (I, II, III, IV, V) were compared using the Chi-square test. The level of significance was kept at 5%.

RESULTS

The Clove leaf oil group and autoclave group completely inhibited the growth of bacterial colonies on the blood agar plate [Figure 1d and e] and no turbidity was seen in peptone water [Figure 2d and e and Tables 1 and 2]. Tulsi leaf oil group showed only 10–20 bacterial colonies on the blood agar plate [Figure 1c] with almost mild to no turbidity seen in peptone water [Figure 2c], showed no statistical difference with clove oil and autoclave, suggesting that it was efficient in reducing the CFU of E. faecalis significantly and Gram-positive cocci in pairs seen under the microscope.

Figure 1

Blood agar plate showing the CFU of Enterococcus faecalis: (a) 0.5% w/v Garlic oil; (b) 0.5% w/v pomegranate peel extract oil; (c) 0.5% w/v Tulsi leaf oil; (d) 0.5% w/v Clove leaf oil; and (e) Autoclave

Figure 2

Turbidity in test tubes with peptone water seen with: (a) Garlic oil; (b) Pomegranate peel extract oil; Mild turbidity seen with: (c) Tulsi leaf oil; No turbidity seen with: (d) Clove leaf oil; (e) Autoclave

Table 1

Association of number of colonies and type of extract

Agar plate	Garlic oil (%)	Pomegranate oil (%)	Clove leaf oil (%)	Tulsi leaf (%)	Standard autoclaving (%)	Total (%)
Zero colonies	0	0	5 (100)	2 (40)	5 (100)	12 (48)
0-103 colonies	0	0	0	3 (60)	0	3 (12)
103-105 colonies	0	2 (40)	0	0	0	2 (8)
>105 colonies	5 (100)	3 (60)	0	0	0	8 (32)
Total	5 (100)	5 (100)	5 (100)	5 (100)	5 (100)	25 (100)

χ2 value of 43.75 and P value of<0.001

Table 2

Association of turbidity in peptone water with different extracts

Peptone water	Garlic oil (%)	Pomegranate oil (%)	Clove leaf oil (%)	Tulsi leaf (%)	Standard autoclaving (%)	Total (%)
No turbidity	0	0	5 (100)	3 (60)	5 (100)	13 (52)
Mild turbid	1 (20)	1 (20)	0	2 (40)	0	4 (16)
Moderate turbid	3 (60)	4 (80)	0	0	0	7 (28)
Severe turbid	1 (20)	0	0	0	0	1 (4)
Total	5 (100)	5 (100)	5 (100)	5 (100)	5 (100)	25 (100)

χ2 value of 28.049 and P value of 0.005

Garlic oil extract and PPE oil groups showed very poor results with low disinfection ability compared to the other groups [Tables 1 and 2]. The bacterial colony count for the Garlic oil and PPE oil group was > 105 [Figure 1a and b] with maximum turbidity seen with peptone water [Figure 2a and b] and Gram-positive cocci in pairs seen under the microscope.

For microscopic examination, Gram staining was done of the bacterial colonies found on the blood agar plate to confirm the presence of catalase-negative Gram-positive cocci in pairs which helped to rule out any contamination during the experiment.

DISCUSSION

Contaminated instruments are the main reasons for the spread of infection from one individual to another individual. According to recent research, it has been suggested that instruments such as burs, files, and other instruments used for endodontic purposes should be used only once and then discarded to reduce the risk of cross-contamination but because of its cost consequences, this hasn’t been implemented yet.[111]

E. faecalis is an obligate anaerobe commonly detected in asymptomatic and persistent endodontic infections.[1213] Hence, E. faecalis broth was used to disinfect the presterilized burs and to evaluate the disinfectant ability of the test chemicals.

The study was performed on round dental burs. The implication behind using these round dental burs is that they are most commonly used to prepare cavities on the decayed tooth, to remove caries from the walls and floor of the decayed tooth, for access opening for root canal treatment, and also for de-roofing the pulp chamber. Hence, they come in contact with saliva and other vital tissues and encounter the highest level of infection.[114]

In the present study, Clove leaf oil showed better antimicrobial efficacy against E. faecalis compared to all other experimental oils and showed similar results with autoclaving. These results are following the results of previous studies where clove oil was active against Staphylococcus aureus, Candida albicans, and Pseudomonas aeruginosa and completely inhibited the growth of E. faecalis.[5] Best results were seen as Clove oil contains 80%–90% of eugenol and this eugenol leads to sensitization of the phospholipid bilayer present in the microbial cytoplasmic membrane which causes increased penetrability and absence of vital intracellular elements resulting in disturbance of the membrane of the bacterial cell eventually leading to leakage of cellular contents which finally results in the bacterial cell.[1516]

PPE oil did not show good antimicrobial results against E. faecalis. This could be because of the low concentration of PPE used in the study.[17] This is not in accordance with the study conducted by Mallya et al., 2019.[10] Garlic oil extract also, in the present study, did not show good antimicrobial results against E. faecalis which is not following the results of previous studies.[51819] It could be because of the lower concentration of the Garlic oil used in this present study.[19]

In the present study, Tulsi leaf oil showed good antimicrobial properties statistically similar to Clove oil and autoclaving. The results are following the results of the previous studies to prove their efficacy against E. faecalis.[2021] This can be attributed as Tulsi has tannins (4.6%) and essential oils (up to 2%) as active constituents (up to 2%). The essential oil present in Tulsi leaf extract consists mainly of eugenol (62%) and methyl eugenol (>85%). The antimicrobial action of tannins against micro-organisms may be explained as the ability of tannins to form complexes with the substrate or the enzymes required by microorganisms to function or exhibit their action on the cell membrane of the microorganisms.[5] A higher concentration of Tulsi leaf oil may be effective in completely inhibiting the growth of E. faecalis. It was also established that Tulsi leaf extract displayed supreme antimicrobial potential at 4% and was effective against E. faecalis.[22]

An autoclave is considered to be the gold standard for disinfecting of endodontic instruments and is suggested as a supreme process for sterilization, as it results in the whole annihilation of micro-organisms and also their spores. The heat produced by the autoclave is delivered via pressurized steam that kills the microorganisms and their spores by causing loss of shape of organisms’ structural proteins and enzymes irreversibly, denaturing and coagulating the essential protein structures, resulting in cell lysis and eventually cell death.[23] The main disadvantage of using the autoclave method of sterilization is that it alters the mechanical and physical properties of the instruments.[23]

Advantages

The use of easily available and economical herbal disinfection products does not temper the mechanical and physical properties of the instruments. In addition, it does not decrease the cutting efficiency of the dental burs and has the minimum possibility of developing resistance. There is no requirement of electric supply and therefore can find its use in rural areas.

Limitation of the study

Further studies with a wide spectrum of organism need to be conducted as endodontic biofilms are multispecies. In vivo studies are required to prove the results.

CONCLUSION

Clove leaf oil and Tulsi leaf oil have very good antimicrobial efficacy against E. faecalis which are similar and comparable to that of autoclaving.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.