Materials Today: Proceedings xxx (xxxx) xxx

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Materials Today: Proceedings

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Comparison of the antibacterial efficacy of teatree oil, nisin and calcium

hydroxide against Enterococcus faecalis
Suruchi Thawre a, Rajesh Joshi a, Sonia B. Bhardwaj b,⇑, Jagat Bhushan a
a
Department of Conservative Dentistry and Endodontics, Dr.HarvanshSingh Judge Institute of Dental Sciences & Hospital, Panjab University, Chandigarh 160014, India
b
Department of Microbiology, Dr.Harvansh Singh Judge Institute of Dental Sciences & Hospital Panjab University, Chandigarh 160014, India

a r t i c l e i n f o a b s t r a c t

Article history: Enterococcus faecalis is a microorganism commonly detected in persistent endodontic infections which
Received 16 March 2020 leads to root canal failure. It can gain entry into the root canal system during treatment, between
Received in revised form 1 April 2020 appointments or even after the treatment has been completed. The use of the effective intra-canal
Accepted 28 April 2020
medicaments can significantly enhance bacterial elimination during root canal treatment. The aim of
Available online xxxx
the study was to evaluate and compare the antibacterial efficacy of Teatree oil, Nisin and Calcium hydrox-
ide against E. faecalis. Typtic Soya agar plates were prepared. The agar well diffusion method was used
Keywords:
and plates were incubated at 37 °C for 24hr. The antibacterial efficacy was then evaluated. The zone of
Root canal failure
E. faecalis
inhibition (mm) of each antimicrobial medicament was measured by standard method. Maximum
Intra-canal medicaments antibacterial efficacy was exhibited by Tea tree oil followed by Nisin and Calcium hydroxide. The results
Teatree oil showed that Teatree oil and Nisin have better antibacterial efficacy than the routinely used Calcium
Nisin hydroxide and they can be used as an alternative intra-canal medicament in endodontics.
Calcium hydroxide Ó 2020 Elsevier Ltd. All rights reserved.
Selection and peer-review under responsibility of the scientific committee of the International Confer-
ence on Aspects of Materials Science and Engineering.

1. Introduction limitations in access by instruments and irrigants. Thus the use

Enterococci are Gram positive cocci that are facultative anaer-
obes and can occur singly, in pairs or as short chains. Enterococcus
faecalis is one of the top three nosocomial bacterial pathogens
reported [1]. Around 150 microbial species have been isolated
and cultured from root canals. E. faecalis has been found in 4–
40% of primary endodontic infections as reported by Rocas et al.
[2]. It’s occurrence in root filled teeth ranges from 24 to77%as given
by Hancock et al. [3]. E. faecalis can withstand harsh environmental
conditions. It can grow at 10 °C to 45 °C at pH 9.6 into 6.5% sodium
hypochloriteand can survive even at 60°Cfor 30 min. Therefore it
can survive in root canal infections where nutrients are less, lead-
ing to root canal failures [4]. Thus the development of effective
medicament against E. faecalis is desirable, so this microorganism
was used as a test microorganism, in this study.
Chemo-mechanical preparation of the root canal reduces micro-
bial load, however it is difficult to obtain complete disinfection due
to anatomic complexities, diversity of the root canal and
⇑ Corresponding author.
E-mail address: sbbhardwaj2002@yahoo.com (S.B. Bhardwaj).
of intra-canal medicaments in between appointments can enhance
bacterial elimination before root canal filling as well as maintains
the predictable long term success of the endodontic treatment.
Traditionally, Calcium hydroxide has been the choice and is
considered a gold standard as an intra-canal medicament, because
of its wide spectrum of action against many endodontic pathogens,
which is thought to be mainly due to its alkalinity causing destruc-
tion of the bacterial cell membrane. Recent studies by Yadav et al.
have shown that E. faecalis is highly resistant to intra-canal
medicaments such as Calcium hydroxide [5]. E. faecalis passively
maintains pH homeostasis and withstands high alkalinity and so
it is mainly resistant to the calcium hydroxide dressing [6].
A new intracanal medicament investigated is Nisin, which is a
naturally occurring antimicrobial peptide and produced by Lacto-
coccus lactis, is a class I bacteriocin. It is colorless, odorless, taste-
less, and has a low toxicity. Nisin is safe to humans and is used
as a food preservative in over 40 countries and is approved to
use in unlimited concentrations in Australia, UK and France [7].
Another new intra-canal medicament investigated is Melaleuca
alternifolia (Teatree oil) which is derived from an Australian plant
and has potential as a root canal disinfectant. Its major active com-
ponent is terpinen-4-ol which has antibacterial and antifungal

https://doi.org/10.1016/j.matpr.2020.04.824
2214-7853/Ó 2020 Elsevier Ltd. All rights reserved.
Selection and peer-review under responsibility of the scientific committee of the International Conference on Aspects of Materials Science and Engineering.

Please cite this article as: S. Thawre, R. Joshi, S. B. Bhardwaj et al., Comparison of the antibacterial efficacy of teatree oil, nisin and calcium hydroxide
against Enterococcus faecalis, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.04.824
2 S. Thawre et al. / Materials Today: Proceedings xxx (xxxx) xxx
properties [8]. It also holds mild solvent action, and hence could
hold potential applications in root canal treatment for dissolving
the necrotic pulp tissue. The major advantages of using herbal
alternatives are easy availability, cost-effectiveness, increased shelf
life, low toxicity, lack of microbial resistance.
Till date to our knowledge, no study in the literature has been
reported to compare the antimicrobial efficacy of Nisin, Teatree
oil and Calcium hydroxide against E. faecalis. Therefore the objec-
tive of this study was to evaluate the antibacterial efficacy of the
new medicaments Nisin and Teatree oil versus traditional medica-
ment Calcium hydroxide.
2. Materials and methods
2.1. Standard organism
A pure culture of E. faecalis (ATCC 29212) was procured from Hi
Media, Mumbai and subcultured on Tryptic Soya Agar plates incu-
bated at 37 °C overnight for 24 hrs.
2.2. Preparation of extracts
Stock solutions of the medicaments were prepared. Nisin
(Sigma Aldrich Co, USA) at a concentration of 2.5%, Tea tree oil
(Sigma Aldrich Co, USA) having miscibility in 85% (v/v) ethanol to
a concentration of 2% by volume, Calcium hydroxide (Hi Media,
Mumbai) (1.5:1) in normal saline.
2.3. Antibacterial assay
Wells of 5 mm diameter were punched using sterile cork beaker
and 100 ml of the irrigants were added to the wells with the help of
a micropipette.
1. Teatree oil (M. alternifolia).
2. Nisin.
3. Calcium hydroxide.
4. Normal Saline (Negative Control).
The plates were incubated at 37 °C for 24 hrs. After 24 hrs the
diameters of the Zones of Inhibition were recorded using vernier
caliper. The experiment was performed 3 times. The Zone of Inhi-
bition size was expressed in mm.
2.4. Antimicrobial activity
The antimicrobial activity of plant essential oils (Eos) is classi-
fied into three levels by Rota et al. [9]:
1. Weak activity (inhibition zone  12 mm).
2. Moderate activity (inhibition zone < 12 mm < 20 mm).
3. Strong activity (inhibition zone  20 mm).
3. Results and discussion
Well defined zone of Inhibition in all the groups of Teatree oil
(M. alternifolia), Nisin, Calciumhydroxide was seen but no zone of
Inhibition was seen in normal saline (negative control) against E.
faecalis [Fig. 1].
The mean zones of inhibition were recorded [Table 1]. The
results were tabulated and statistically analyzed using analysis of
variance (ANOVA). The mean zone of inhibition was maximum
with Teatree oil (27.5 mm) followed by Nisin (18.5 mm), Calcium
hydroxide (15.16 mm) and Normal Saline (0 mm).
Please cite this article as: S. Thawre, R. Joshi, S. B. Bhardwaj et al., Comparison of the antibacterial efficacy of teatree oil, nisin and calcium hydroxide
against Enterococcus faecalis, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.04.824
Fig. 1. Zones of Inhibition of Teatree oil, Nisin, Calcium hydroxideand Normal
Saline against E. faecalis.
Table 1
Readings for the Mean Zones of Inhibition in all the groups.
Groups Agent N Mean (mm) Variance ANOVA (pValue)
1. Teatree oil 3 27.5 0.25 P < 0.01 (Significant)
2. Nisin 3 18.5 0.25
3. Ca(OH)2 3 15.16 0.58
4. N. Saline 3 0 0
N: Number of plates.
Comparison of the zones of inhibition formed by Teatree oil,
Nisin, Calcium hydroxide, and Normal Saline is shown in [Fig. 2].
The agar diffusion test was used to evaluate the efficacy. This is
frequently used to test the efficacy of antimicrobials and the zone
of inhibition, which is dependent on the diffusion of the antimicro-
bials into the agar. The advantage of this method is that it allows
direct comparison of the materials against the organisms, indicat-
ing the potential of the test materials to eliminate microorganisms
in the local microenvironment of the root canal system. However,
there are certain disadvantages of using this method. The most
important is that the results may be variable depending upon the
ability of the material to diffuse across the medium and not only
on the toxicity of the material for the particular organism accord-
ing to Balakrishnan et al. [10]. In this study, the experiment was
repeated three times.
In the present study, well defined zones of inhibition in all the
groups of Teatree oil (M. alternifolia), Nisin, Calcium hydroxide was
seen but no zone of inhibition was seen in Normal Saline (negative
control) against E. faecalis [Fig. 1]. The results showed that the
mean zone of inhibition was formed maximum in Teatree oil
(27.5 mm) > Nisin (18.5 mm) > Calcium hydroxide Ca(OH)2
(15.16 mm) against E. faecalis [Table1, Fig. 2]. No zone of inhibition
was seen in Normal Saline (0 mm) against E. faecalis. The values
were statistically significant showing significant difference in size
of zone of inhibition formed with each group (p < 0.01).
Nisin is an antibacterial agent which is shown to be effective
against a broad spectrum of Gram positive bacteria. The mode of
action of Nisin in killing the bacteria has been shown by triggering
the activity of bacterial muscin hydrolases which results in the
lysis of the bacterial cells as seen by Severina E. et al. [11]. Nisin
based mouthwash has also been tested in beagle dogs and there
was a significant decrease in bleeding sites as compared to con-
trols, by Howell TH et al. [12]. In our study, Nisin has shown
greater efficacy against E. faecalis (Mean zone of inhibi-
tion = 18.5 mm) as compared to routinely used medicament Cal-
cium hydroxide (Mean zone of inhibition = 15.16 mm) which is
quite encouraging.
 S. Thawre et al. / Materials Today: Proceedings xxx (xxxx) xxx
Fig. 2. Comparative Mean Zones of Inhibition of Teatree oil (27.5 mm), Nisin (18.5 mm), Calcium hydroxide (15.16 mm) and Normal Saline (0 mm) against E. faecalis.
Calcium hydroxide has shown insufficient antimicrobial effi-
cacy against E. faecalis and also against other microfloracommonly
present in infected root canals such as Prevotella, Peptostreptococ-
cus, Actinomyces, Bacteroides, Fusobacterium and Eubacterium [13].
It is not much as effective in eradicating E. faecalis, as its high pH
is not maintained in the presence of E. faecalis [5]. This is also seen
in our study as both Calcium hydroxide and Nisin are showing
moderate activity against E. faecalis.
Results of our study showed that, Teatree oil having maximum
zone of inhibition i.e. 27.5 mm, exhibits very good antibacterial
efficacy against E. faecalis. This is also supported by Sinha et al.,
according to whom teatree oil exhibits antimicrobial activity due
to tarpenin-4-ol which causes lysis by leakage of ions in the
microbes [14]. Teatree oil in association with Calcium hydroxide
has also shown good results in pH and calcium release analysis
showing its effectiveness as oily vehicle in endodontic treatment
particularly during the delayed dressing stage [15]. The recom-
mended therapeutic concentration for Teatree oil is 2.5 to 5%, to
retain its antibacterial property without any toxic effect [16].
4. Conclusion
Under the limitations of this study, Tea tree oil showed strong
antimicrobial activity followed by moderate antimicrobial activity
by Nisin, Calcium hydroxide against E. faecalis. These herbal
extracts/products have high antimicrobial efficacy, are cost-
effective, show lack of microbial resistance and can be used as root
canal medicaments.
The in vitro results are promising but preclinical and clinical tri-
als are needed to evaluate dosage, allergic potential, biocompatibil-
ity and safety factors of these medicaments. Henceforth, in future
studies, these medicaments can be tested for their toxicity and
inhibition of biofilm formation by the bacteria.
CRediT authorship contribution statement
Suruchi Thawre: Conceptualization, Methodology, Software,
Writing - review & editing. Rajesh Joshi: Conceptualization, Super-
vision, Data curation, Validation. Sonia B. Bhardwaj: Writing -
Please cite this article as: S. Thawre, R. Joshi, S. B. Bhardwaj et al., Comparison of the antibacterial efficacy of teatree oil, nisin and calcium hydroxide
against Enterococcus faecalis, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.04.824
3
original draft, Visualization, Investigation, Writing - review & edit-
ing. Jagat Bhushan: Conceptualization, Supervision, Validation.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
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Please cite this article as: S. Thawre, R. Joshi, S. B. Bhardwaj et al., Comparison of the antibacterial efficacy of teatree oil, nisin and calcium hydroxide
against Enterococcus faecalis, Materials Today: Proceedings, https://doi.org/10.1016/j.matpr.2020.04.824