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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 11  |  Issue : 2  |  Page : 125-130

A comparative evaluation of the antimicrobial effect of six natural products in comparison to 2.5% sodium hypochlorite against Enterococcus faecalis: An in vitro study


1 Department of Preventive Dentistry, Umm Al Qura University Makkah, Saudi Arabia
2 Dental Intern, Umm Al Qura University Makkah, Saudi Arabia
3 Dental Student, Faculty of Dentistry, Umm Al Qura University Makkah, Saudi Arabia
4 Department of Medical Microbiology, Faculty of Medicine, Umm Al Qura University, Makkah, Saudi Arabia

Date of Submission22-Oct-2021
Date of Decision26-Apr-2022
Date of Acceptance15-May-2022
Date of Web Publication22-Aug-2022

Correspondence Address:
Jameel Abdulsalam Abuljadayel
Department of Preventive Dentistry, Umm Al Qura University, Al-Abdeyyah, Mecca 21955
Saudi Arabia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/sjhs.sjhs_149_21

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  Abstract 


Background: Enterococcus faecalis (E. faecalis) is known to play a key role in the etiology of constant endodontic infections. Recently, suggestions of natural alternatives to overcome the downsides of sodium hypochlorite (NaOCl) have been discussed in the literature. Aims: The present study investigates the antibacterial effect and compares the bacteria-killing time of six possible natural alternatives in comparison to 2.5% NaOCl, namely net Manuka Honey (MaH), crude black seed oil (BSO), 100% apple vinegar (AV), castor oil, crude sesame oil, and a novel plant extract (Bactil®). Materials and Methods: Agar well-diffusion assay was used to determine the antibacterial effect of the products against E. faecalis. Muller‒Hinton broth (MH broth) was used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC). Time-kill assay was performed to investigate the killing time required by the tested product to inhibit E. faecalis at 30, 60, 90, and 120 s. Results: Four out of the six tested products (crude BSO, 0.2% Bactil®, 100% AV, and net MH) exhibited an antibacterial activity against E. faecalis. While after MBC and MIC determination, only two of the products (0.4% BSO and 0.2% Bactil®) were able to inhibit the growth of E. faecalis within 30 s of exposure similar to the positive control 2.5% NaOCl. Conclusion: Within the limitations of this study, both 0.4% BSO and 0.2% Bactil® were as effective as 2.5% NaOCl against E. faecalis and could be suggested for further testing as promising natural alternatives to disinfect the root canal system.

Keywords: Antibacterial, disinfection, Enterococcus faecalis, root canal treatment, sodium hypochlorite


How to cite this article:
Abuljadayel JA, Shafei JS, Fairaq MA, Mirza AF, Samman AN, Abdulshakoor AA, Jalal NA, Ashgar SS, Al-Said HM. A comparative evaluation of the antimicrobial effect of six natural products in comparison to 2.5% sodium hypochlorite against Enterococcus faecalis: An in vitro study. Saudi J Health Sci 2022;11:125-30

How to cite this URL:
Abuljadayel JA, Shafei JS, Fairaq MA, Mirza AF, Samman AN, Abdulshakoor AA, Jalal NA, Ashgar SS, Al-Said HM. A comparative evaluation of the antimicrobial effect of six natural products in comparison to 2.5% sodium hypochlorite against Enterococcus faecalis: An in vitro study. Saudi J Health Sci [serial online] 2022 [cited 2022 Oct 3];11:125-30. Available from: https://www.saudijhealthsci.org/text.asp?2022/11/2/125/354159




  Introduction Top


Effective root canal disinfection and proper elimination of microorganisms are important prerequisites for long-term success of nonsurgical root canal treatment (NSRCT).[1],[2] Mechanical shaping preparation of the root canals reduces the majority of the infection-causing bacteria, along with their primary substrate from necrotic pulp debris.[3] However, the retention of microorganisms within the dentinal tubules has been accounted for the persistence of endodontic reinfection and NSRCT failure.[2],[4] Therefore, treating the entire pulp canal system chemo-mechanically with medications or washing it with agents that possess antibacterial traits is crucial to achieve a proper NSRCT.[2],[5] As aiming to decrease the intracanal microbial load and making the environment conducive to tissue repair around the apex are two of the NSRCT objectives.[2],[5]

Although several types of bacteria are found in the root canal system after pulpal infection, Enterococcus faecalis (E. faecalis) is known to play a key role in the etiology of constant endodontic and secondary infections as this anaerobic Gram-positive cocci is considered one of the most difficult bacteria to eliminate from the root canal system as it has the ability to resist the normal cleaning methods.[1],[2],[4],[6],[7],[8]

For many years, different concentrations (1%–5.25%) of sodium hypochlorite (NaOCl) have been widely and routinely used as a standard irrigant to disinfect the root canal system “chemically” during NSRCT due to its potent antimicrobial properties.[1],[5],[9] However, higher concentrations were described to be indistinguishably dissolving both vital and necrotic pulp remnants, toxic and irritant to the periapical tissues.[1],[5],[10],[11] Moreover, it has been mentioned in the literature that high concentrations of NaOCl could have a harmful effect on the mechanical properties of the root dentin, which may increase the probability of vertical root fractures.[12],[13],[14] The unpleasant taste, smell, hypersensitivity, allergic reactions, and the hazards of inadvertent use were also documented as concerns.[15],[16] The literature is encouraging to investigate other natural and herbal products that possess antimicrobial activity to be suggested as an option to disinfect the intracanal area during NSRCT. Therefore, the present study aims to investigate the antibacterial effect of net Manuka honey (MH), crude black seed oil (BSO), apple vinegar (AV), castor oil (CO), crude sesame oil (SO), and a novel plant extract (Bactil®) against E. faecalis using agar well diffusion assay, then compare their killing time to the most commonly used 2.5% NaOCl using the contact time-kill assay.


  Materials and methods Top


Tested natural and synthetic compounds

Five natural products were purchased and collected from the local market in Makkah city based on their published antibacterial activity (net Manuka honey, crude BSO, crude SO, AV, and CO). While the sixth product (Bactil®) was synthesized at the laboratory of the Faculty of Medicine – Umm Al Qura University (registered trademark, with the patent registration number-120410648). About 2.5% NaOCl was used as a positive control and normal saline was the negative control. Details of the products are listed in [Table 1].
Table 1: Information of the tested products

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Tested organism

E. faecalis (ATCC 29212) was used as a tested organism in this study.

Culture media

Muller‒Hinton agar (MH agar) was used for the agar well diffusion assay to determine the effect of the six natural products against E. faecalis. In addition, MH broth media was used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC).

Preparation of standard inoculum

E. faecalis strain was subcultured on MH agar medium and incubated at 37°C for overnight. A single colony was obtained using a sterile loop and inoculated in 3 ml of MH broth to form a homogeneous suspension of the organism, which was standardized to (0.5 McFarland) using calibrated VITEK 2.

Agar well-diffusion assay

Agar well-diffusion method was performed by transferring (200 μl) of E. faecalis, which was suspended in MH broth and adjusted to 0.5 McFarland turbidity to swabbing in three directions, the surface of MH agar plate using sterile cotton swabs as described in the National Committee for Clinical Laboratory Standards (manual). The plates were allowed to dry for 10 min before cutting the wells 6 mm diameter into MH agar using sterile sharp glass rods. Wells were then filled with 50 μl of the tested compounds (Bactil® −2 mg/ml), net MaH, BSO, SO, 100% AV, CO, as well as 2.5% NaOCl as a positive control and normal saline as a negative control. This experiment was repeated five times simultaneously and the results were almost identical. The plates were incubated at 37°C for 24 h. After the incubation, the plates were examined, and the diameter of the inhibition zone was measured and recorded for each product.

Determination of minimum inhibitory concentrations and minimum bactericidal concentrations for tested compounds

Using microtitration plates, 200 μl of each tested compound was added to the first column in a microtiter plate and 100 μl MH broth to other wells. About 100 μl of tested compound then transferred from the first column to the next wells to produce a dilution series of (50%, 25%, 12.5%, 6.3%, 3.2%, 1.6%, 0.8%, 0.4%, and 0.2%). Negative and positive controls were included. About 10 μl of 0.5 adjusted McFarland bacterial suspension in MH broth was then added into the wells, including the positive control as well. For validation purposes, this technique was carried out simultaneously twice in microtitration plates and gave the same results. Plates were incubated at 37°C for 24 h. MBC was recorded as the lowest concentration of the compound that prevents any growth of a tested organism after subculture on MH agar plate according to the Clinical and Laboratory Standards Institute (CLSI M26 - A, 1998).

Time-kill assay

Using a microtitration plate, 200 μl of tested compounds were added to the first column in a microtiter plate and 100 μl MH broth to other wells. Next, 10 μl of the tested bacterial strain, which was suspended in MH broth and adjusted at 0.5 McFarland was pipetted in and mixed with the tested compound in the first well of each column. At the end of 30 s from zero time, 10 μl of the suspension was transferred from the first column to the next well in the second column; this step was repeated after 60, 90, and 120 s to the third, fourth, and fifth columns, respectively. MH broth alone was used as a negative control and MH broth with tested bacterial strain was used as a positive control. This technique was carried out twice on two microtitration plates and the results were similar. Then, the plates were incubated at 37°C for 24 h. After the incubation period, subculture on MH agar plate according to CLSI M26-A, 1998 was carried out by transferring 10 μl from each well to MH agar plates. All plates were incubated for 24 h at 37°C to investigate the killing time required of the tested compound against the tested bacterial strain (E. faecalis). The absence of growth on the medium defined as effective and detected growth indicates no effect of the tested compound at the recorded time.


  Results Top


Agar well-diffusion assay, minimum bactericidal concentrations, and minimum inhibitory concentration

The results showed that four of the tested products (100% BSO, 0.2% Bactil®, 100% AV, and net MH) have exhibited antibacterial activity against E. faecalis similar to the positive control 2.5% NaOCl. In contrast, CO and SO showed no antibacterial activity against E. faecalis, even when used at undiluted concentrations [Figure 1] and [Table 2]. To provide further evidence, the minimum inhibitor concentration (MIC) and MBC of the four products that showed inhibition zones of E. faecalis were determined (BSO, Bactil®, 100% AV, and net MH). The result also confirmed that E. faecalis was sensitive to BSO and AV with 0.2% and 6.3% MIC, respectively [Figure 2] and [Table 2]. MIC and MBC for net MH were not performed due to inhomogeneity with growth media at lower dilutions. For validation purposes and to eliminate any mistakes during the procedures, the agar well diffusion assay was repeated five times, whereas MIC and MBC were performed twice and the results were almost identical.
Figure 1: Agar well diffusion plate showing the inhibition zones of each product. A: Net MH D: Bactil, B: AV E: SO, C: CO F: BSO. + ve control: 2.5% NaOCl, −ve control: Normal saline. MH: Manuka honey, AV: Apple vinegar, SO: Sesame oil, CO: Castor oil, BSO: Black seed oil, NaOCl: Sodium hypochlorite

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Table 2: Assessment of the antibacterial activity of some natural and synthetic products against Enterococcus faecalis (ATCC 29212) by agar well diffusion method and broth microdilution method (minimum inhibition concentration and minimum bactericidal concentration)

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Figure 2: Broth microdilution plates for each product that shows an antibacterial effect against E. faecalis after testing it with the agar well diffusion method. (a) CO, (b) AV, (c) Bactil, (d) BSO and (e) SO. CO: Castor oil, AV: Apple vinegar, BSO: Black seed oil, SO: Sesame oil, E. faecalis: Enterococcus faecalis

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Time-kill assay

The results showed that both 0.4% BSO and 0.2% Bactil® were able to inhibit the growth of E. faecalis within 30 s of exposure, similar to the 2.5% NaOCl [Table 3]. In contrast, net MH and 12.5% AV were unable to inhibit the growth of E. faecalis even after 120 s of exposure. The test was carried out twice to confirm the results and gave the same outcomes.
Table 3: Determination of the contact time (Time-kill assay) of the products that showed inhibition zones to Enterococcus faecalis

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  Discussion Top


A successful NSRCT has many requirements; one of them is preventing secondary infection by removing the most prominent bacteria, especially E. faecalis.[4] To eliminate this bacteria sufficiently, root canals must be properly cleaned both mechanically and chemically with a potent antimicrobial agent such as NaOCl.[5] Recently, other natural substitutes have been investigated to overcome the drawbacks of NaOCl.[17],[18] The products that have been included in the present study (Net MH, BSO, CO, and SO) have been discussed in the literature to possess an antibacterial activity.[18],[19],[20],[21],[22],[23],[24] While Bactil® has shown a bactericidal effect in a nonpublished study and this material is a novel plant extract which produced by microbiologists working at Umm Al-Qura University. This product is under copyright law with a pending patency number 120410648 at the Saudi Authority for Intellectual Property as a necessary step before launching it in the market.

Regarding the antibacterial activity of the tested products against E. faecalis, the agar well diffusion method was able to show that four of the six tested materials (BSO, 0.2% Bactil®, 100% AV, and net MH) were able to inhibit the bacterial growth with diameter zones equal to 46 mm, 25 mm, 19 mm, and 14 mm, respectively, whereas the positive control (2.5% NaOCl) showed an inhibition diameter of 26 mm [Figure 1] and [Table 2]. These numbers indicate that E. faecalis was sensitive to those four products as well as to 2.5% NaOCl. However, for these products to function as efficient and practical root canal irrigants, they must inhibit the growth of E. faecalis within a short-term exposure. To test this notion, a time-killing assay was performed [Table 3]. Interestingly, two of the products (0.4% BSO and 0.2% Bactil®) were able to inhibit E. faecalis within 30 s of exposure. These results validate that 0.4% BSO and 0.2% Bactil® would be as efficient and practical as the most commonly used agent 2.5% NaOCl on a time/antibacterial basis against E. faecalis during clinical NSRCT.

The antibacterial effect of BSO of Nigella sativa against different Gram-negative and positive bacteria including E. faecalis is documented in the literature.[19],[25],[26],[27],[28] Our results showed that 100% BSO has produced the largest diameter of inhibition among all tested compounds against E. faecalis [Table 2]. However, Rath 2014[29] and Salman 2008[30] were not able to detect a bactericidal effect of BSO against E. faecalis. The difference in the results would be attributed to the method of oil extraction, way of storage, and/or the origin of the seed plant. In the present study, the extraction procedure for BSO was not done in our laboratory and the oil product was freshly prepared and bought ready to be used from a local shop as shown in [Table 1], the oil was stored at room temperature and the tests were done within a week after purchase. Time-kill assay [Table 3] has shown that 0.4% BSO was as effective as 2.5% NaOCl against E. faecalis and was able to inhibit the bacteria within 30 s. Collectively, this study suggests that 0.4% BSO could be used as a natural alternative for 2.5% NaOCl in cleaning the infected canals in cases of retreatment.

Bactil® also has shown a promising antimicrobial activity against E. faecalis [Table 2]. Interestingly, the results showed that E. faecalis was highly sensitive to the product with 0.0008% MIC and 0.0016% MBC. In regards to the duration that was needed to kill the bacteria, Bactil® has shown a similar evidence of bacterial inhibition in comparison to the positive control (2.5% NaOCl). No data were found in the literature about the product yet. However, the patency file (unpublished data) claims that the product is a bactericidal natural plant extract, which is nontoxic and safe for use in vivo which makes it a promising substitute for 2.5% NaOCl in NSRCT after further investigations.

AV is composed primarily of malic acid, which is mentioned in the literature as a potent substance for treating different types of infections.[31] In the present study, the results show that E. faecalis was sensitive to 100% AV [Table 2], which confirms its bactericidal effect. Other researchers have also published similar data against E. faecalis.[18],[31] Although time-kill assay [Table 3] could not detect any bactericidal action of 12.5% AV against E. faecalis within 120 s. This result in combination with the previously validated antimicrobial activity of AV against E. Faecalis by agar well diffusion method indicates that 12.5% AV is able to inhibit the bacterial growth but it needs more than 120 s to show its antimicrobial activity, which compromises the practicality of 12.5% AV to disinfect the root canal system in comparison to 2.5% NaOCl.

Manuka Honey (MH) is monofloral honey obtained from the species Leptospermum scoparium which has a long-standing reputation in New Zealand for its antimicrobial effect.[32] The present study was able to show the antibacterial effect of net MH against E. faecalis [Table 2]. This result is consistent with the published literature as Kumar 2014[22] has found that MH was able to inhibit E. faecalis. However, the killing time assay [Table 3] revealed that net MH was not able to inhibit E. faecalis within 2 min. This result would suggest that replacing 2.5% NaOCl with net MH as a root canal irrigant would not be a practical option on a clinical basis, especially in a single-visit treatment. While utilizing it as intracanal disinfectant in-between visits could still be suggested, yet requires further testing and preparations as the net honey were highly viscous and its antibacterial effect was proved in the present study without dilution.

CO and SO have shown a bactericidal potential in the literature against different types of bacteria rather than E. faecalis.[20],[21],[33],[34] The present study shows that they were both ineffective against E. faecalis [Table 2]. Thus, based on the findings of the present study, they both would not be recommended to be used as disinfectants against E. faecalis in NSRCT.

The present study has not investigated the effect of the products on the different dentin structures nor their efficacy and ability to penetrate and kill the bacteria, which intrude the dentinal tubules. In addition, the products were tested against only one type of bacteria, whereas in root canal infections, many strains of bacteria are known to be involved. Therefore, further investigations with different designs are recommended before suggesting the utilization of the materials in vivo.


  Conclusion Top


Within the protocol of the present study, both 0.4% BSO and 0.2% Bactil® were as effective as 2.5% NaOCl against E. faecalis in their antibacterial outcome and killing time which makes both of them eligible to be suggested as promising natural alternatives to disinfect the root canal system. Further laboratory investigations should be done to test their applicability, efficiency, and effects on the tooth structures and against other different bacterial strains before suggesting in vivo utilization.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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