Volume 6, Issue 1, January – 2021 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

A Comparative Evaluation of Antimicrobial Efficacy

of Zinc Oxide with Thyme Oil, Zinc Oxide with
Peppermint Oil and Zinc Oxide Eugenol Sealers and
Obturating Materials Against Enterococcus Faecalis
and Candida Albicans”- An in-Vitro Study
Dr. Hennelon Joe Jesus D’Costa
Department of Pedodontics & Preventive dentistry
AJ Institute of Dental Sciences
Mangalore, India

Abstract:- The increase in failure of pulpectomies due to I. INTRODUCTION

re-infection by most common micro-organisms like
Enterococcus faecalis and Candida albicans has been Dental caries is a multifactorial microbial infectious
noticed in recent years. Thus need for an obturating disease characterized by demineralization of in-organic and
material or sealer material having a high efficacy against destruction of the organic substances of the tooth. It is also
these causative microbes is the need of the hour which one of the most commonly seen oral health problem
can eliminate these microbes even in an event of re- globally. Indian children reported to have a DMFT of
infection after the completion of the root canal treatment around 2 and caries prevalence was increasing with age
in deciduous teeth. AIM: To compare antimicrobial from 51.9% and 63.1% in 5-15 years of age group
efficacy of Zinc oxide eugenol, Zinc oxide plus thyme oil respectively in the last National Oral Health survey
and Zinc oxide with peppermint oil against Enterococcus conducted in 2002-2003 [1].Dental caries continues to pose
Faecalis and Candida Albicans. MATERIALS AND a major public health problem despite the recent scientific
METHODS:This study was conducted on 30 Muller advances. One of the main complaints of dental caries is due
Hinton agar plates which were divided into three groups to the unbearable pain caused once the carious lesion
(n=15). Three wells of 6mm diameter were made by reaches the pulp [2].
removal of agar at equidistant points and then were
filled with the test materials. Both microorganisms were When the deciduous teeth pulp tissue gets affected
grown at 37℃ for 24hrs in MH Broth. The lawn either by caries or trauma pulp therapy has to be performed
technique was used for uniform distribution of bacterial in such cases so as to maintain the primary teeth in its
dilution. The plates were maintained at room functioning and healthy state until replaced by their
temperature for 2 hours for prediffusion of material and permanent successors. Pulpectomy in primary teeth includes
then incubated at 37℃ for 24 hours. After incubation, chemo-mechanical preparation and use of intra canal
the diameters of zone of inhibition around the plates dressing with anti-bacterial properties and the success
were measured in millimeters with the help of Hi depends on proper removal of infected bacteria. Complex
Antibiotic zone scale RESULTS: The mean zones of anatomy of primary teeth root canals makes ittough for
inhibition were highest in for ZoE followed by ZoT and cleansing proper cleansing by instrumentation and
least for ZoP against Enterococcus faecalis. Similarly, irrigations[3].Presence of numerous accessary, curved, thin
ZoE had highest mean zones of inhibition, followed by tortuous, ribbon like canals which are connected to each
ZoT and the least was ZoT against Candida albicans. other with interconnected canals in primary teeth, the intra
CONCLUSION: The antimicrobial efficacy was highest canals dressing may fail to remove microorganisms from in
in ZoE against E faecalis and Candida albicans followed accessible areas[4].
by ZoT but Candida albicans were almost resistant to
ZoP and had the least efficacy against E faecalis. All the Typically poly-microbial flora consisting of gram
test materials had better antimicrobial efficacy against positive and gram negative bacteria consist of the main
Candida albicans compared to E faecalis. endodontic flora [5, 6]. Microorganisms are harboured by
the root canal dentinal tubules, the apical portion of root
Keywords:- ZoT, ZoP and ZoE. canal and extra radicular tissue may also harbour bacterial
biofilms[7,8].

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Volume 6, Issue 1, January – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
Endodontic failure corresponds with the persistence of infections.The oral cavity occurrence of Candida Albicans
peri-radicular lesion. To conserve the update in endodontic has been reported to be 30-45% in healthy humans. Their
treatment and materials is necessary, otherwise persisting incidence in the root canal ranges from 1% to 17%.In
micro-organisms or secondary infections mainly cause by infected canals, there are also strong risks that candida
insufficient coronal restorations and absence of anti- albicans may be identified. In chronic apical periodontitis
bacterial properties of sealer and obturating material can cases, as inferred from numerous studies of candida albicans
lead to loss of the tooth. In cases of tooth filled with in the range of 7-18%, it is the most commonly detected
obturating materials, 35-100% cases micro-organism will be fungus. Properties such as hyphal formation and
present in the peri-radicular tissue. Earlier studies using thrgmotropism that enable it to penetrate dentin deeply and
culture media revealed predominantly facultative anaerobes make it thrive in persistent apical periodontitis with its
and few obligate anaerobes which is distinctly different virulence[12].
microbial flora compared to primary infection, including
mostly gram-positive bacteria. Complex root morphology, Therefore because of the shortcomings of Zinc oxide
inherent physiological root resorption, close proximity of eugenol search for newer materials like zinc oxide powder
permanent successor tooth, complex diagnosis due to with thyme oil and zinc oxide powder with peppermint to
immature patient which are some of the reasons why pulp test their antimicrobial efficacy against Enterococcus
therapy of primary teeth still remains controversial despite Faecalis and Candida Albicans which are most commonly
high success rates pointed out in previous studies[9]. seen in secondary or failed root canal infections.

Therefore this makes it important to use obturating and II. MATERIALS AND METHODS
sealer materials which has high antibacterial property to act
onmicroorganism of inaccessible areas in root canals of This study was conducted on 30 Muller Hinton (MH)
deciduous teeth[10]. agar plates. The number of samples for all three groups were
15 each (n=15). Three wells of 6mm diameter were made by
Zinc oxide eugenol is widely used for root canal removal of agar at equidistant points with open end of
sealing and obturation in primary teeth3.In dental practice, micropipette and then were filled with the test materials. For
eugenol has topical uses such as relieving pain from all the test materials and ZOE paste, using a cement spatula,
different sources like pulpitis and dental hypersensitivity 1 scoop (0.2 mg) of zinc oxide powder was mixed with 7
[3,11]. Certain studies have reported that eugenol has drops of oil (0.07) on a dry, sterile glass slab.
irritating action on the tooth [11]. It may have some side
effects as its presence per-apically may deflect the path of The standard strains of bacteria and fungi, E Faecalis
eruption of permanent successors and may cause necrosis of and C albicans were used in this study.Both microorganisms
bone and cementum, periapical tissue irritation and may were cultivated in MH Broth at 37 ° C for 24 hours and then
cause tooth discoloration4. It has a disadvantage that seeded in MH agar to develop 0.5 turbidity on the
includes slower rate of resorption than the root of deciduous McFarland scale, corresponding to 108 CFU/mL
teeth. It is also believed to have limited antibacterial activity concentrations. The 2nd layer of this MH broth was
[4,12]. included. Following the injection of test materials, the
seeded agar was added directly over the plates. For uniform
Thus the search for newer materials with effective distribution of bacterial dilution the lawn technique was
antimicrobial properties has led to conduct this study with used.
use ofzinc oxide powder with thyme oil and zinc oxide
powder with peppermint oilto determine its antibacterial The plates were maintained at room temperature for 2
efficacy against most resistant microorganisms like hours for prediffusion of material and then incubated at
Enterococcus Faecalis and Candida albicans. 37℃ for 24 hours. After incubation, the diameters of zone
of inhibition around the plates were measured in millimetres
Enterococcus Faecalis is gram positive cocci with the help of Hi Antibiotic zone scale (HiMedia).
facultative anaerobes which are seen in cases with root canal
failures or persistent root canal infections,[3,6] isolated from III. STATISTICAL ANALYSIS
retreatment cases of apical periodontitis,[5,6,13,14]
enterococcus Faecalis' tendency to bind to dentinal tubule A power analysis was established by G*power, version
collagen and stay viable within tubules, which can induce 3.0.1(Franz Fauluniversitat, Kiel, Germany). A sample size
periapical disease and recurrent endodontic tooth care of 36 subjects (12 in each group which can be rounded off to
deficiencies. 5,6, it is tough to eliminate this species through 15 in each group comprising the total sample to be 45)
root canal medications when established in the dentinal would yield 95% power to detect significant differences,
tubules [5,6,13The success rate of root canal therapy at the with effect size of 0.7 and significance level at 0.05.
time of obturation is substantially decreased in the presence
of Enterococcus Faecalis[13]. Antimicrobial behavior data was statistically evaluated
at a significance level of 5 percent using one way ANOVA
The largest proportion of fungal microbiota is formed and Tukey's post-hoc test.Data was entered in the excel
by candida species. Candida albicans has been the most spread sheet. Descriptive statistics like mean, standard
commonly detected fungal species in endodontic deviation and percentages was calculated. Inferential

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Volume 6, Issue 1, January – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
statistics like ANOVA with post-hoc Bonferroni was used to were made to react against E Faecalis species, ZoE showed
compute the significant difference among the groups using the highest zone of inhibition with median score of 21mm
SPSS version 22. Any other necessary tests will be dealt at followed by ZoT 12mm and ZoP showing the least median
the time of analysis. score with 8mm forthe zone of inhibition (table 1)

IV. RESULTS ZoE showed greater zones of inhibition against

Candida albicans compared to E Faecalis and the difference
The datashows a statistically significant difference in was found to be statistically significant. The two zones of
the zones of inhibition of the different materials against the inhibition for E Faecalis & C albicans with ZoP showed no
2 test micro-organisms(Candida albicans and E.faecalis). statistically significant difference among them. ZoT showed
Zinc Oxide Eugenol (ZoE) showed the highest median of greater zones of inhibition against C albicans compared to E
zone of inhibition of 45mm against Candida albicans Faecalis and this difference was found to be statistically
followed by Zinc Oxide Thymol (ZoT) with median of significant (table 2)
38mm of zone of inhibition and ZoP showed no zones of
inhibition when made to react with Candida Albicans
species. Similar results were seen when the study materials

TABLE 1: COMPARISON OF THE ANTIMICROBIAL EFFICACY AMONG THE GROUPS USING KRUSKAL-
WALL

Kruskal-
Minimum Maximum Median IQR P value
wallis
ZoE 40 48 45 5
Candida albicans ZoP 0 35 0 8 34.86 0.00*
ZoT 0 45 38 5
ZoE 18 25 21 3
E faecalis ZoP 0 12 8 12 37.29 0.00*
ZoT 12 13 12 1
*significant

TABLE 2: COMPARISON OF THE ANTIMICROBIAL EFFICACY WITHIN THE GROUP BETWEEN CANDIDA
ALBICANS AND E FAECALIS USING MANN-WHITNEY TEST
Minimum Maximum Median IQR U value P value
C.albicans 40 48 45 5
ZoE 0.00 0.00*
E.faecalis 18 25 21 3

C.albicans 0 35 0 8
ZoP 80.00 0.126
E.faecalis 0 12 8 12

C.albicans 0 45 38 5
ZoT 15.00 0.00*
E.faecalis 12 13 12 1
*significant

V. DISCUSSION important factor when selecting it as an obturating and

sealer material [15]
Antibacterial activity is one of the most important and
desired properties for an ideal obturating material and sealer Enterococcus faecalis is a gram positive, facultative
to be used on primary teeth. Incorporation of antibacterial anaerobic microorganism found in persistent per-apical
components in an endodontic obturating material has infections and failed root canal treatments[16]. It has been
become essential to prevent the re-growth of bacteria isolated in cases of persistent root canal infections. Rocas IN
leading to secondary infection. This is usually caused by the et al concluded that the prevalence of E Faecalis is 4-40% in
residual microorganisms left behind due to the nature of the primary endodontic infections and 67-77% in secondary
root canals in deciduous teeth. It is also essential to control infections, consistently higher.E Faecalis forms a defensive
the re-entry of bacteria into the root canal system. However, biofilm capable of blocking lymphocytic function, rendering
these components should not cause any specific toxicity the bacteria 1000 times more resistant to phagocytosis,
toward the innate microorganisms found in the root canal antibodies and antimicrobials. [17]. E Faecalis can also
system because of which it may exert toxic effect on the endure prolonged periods of nutritional
host cells causing irritation and inflammation. Therefore, deprivation[14].With the involvement of E. faecalis in the
biocompatibility of the antibacterial components is very pulp canal system at the time of obturation, the effective rate

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Volume 6, Issue 1, January – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
of root canal therapy is substantially decreased. [18]. Above Studies done by Markowitz K et al. and Saggar V et al.
all, relative to other species, E.faecalis rapidly colonizes the concluded that Zinc oxide eugenol containing sealers were
dentinal tubules and is therefore especially difficult to more superior in inhibiting the micro-organisms found in
eradicate. [19]. Due to all these reasons, it represents a primary root canal system[31,32]. A study done by Rahul et
standard against which the antimicrobial action of an intra- al in 2015, showed that ZoE based sealer Endoflas FS had
canal medicament is tested[19,20] the highest zones of inhibition against E Faecalis and C
albicans which is in agreement with the results of the present
Candida albicans is the most frequently isolated fungal study[6] . Zones of inhibition were seen highest with ZoE
species from contaminated root canals, and because of its obturating material compared to the herbal extracts in the
invasive affinity to dentin, this species is considered a study done by Shipra et al33 which was similar to the result
dentinophilic microorganism.[21].Candida albicans though seen in study done by Navit et al& Markowitz et al
are found in reduced quantities when compared to other (1992)[33,34]. A study was done to assess the antimicrobial
microorganisms which are isolated from infected dental activity of endodontic sealers on Enterococcus Faecalis by
pulp[22]. The study conducted by Baumgartner et al Anjali et al 2012 concluded that Eugenol based endodontic
assessed the presence of Candida albicans from abscess and sealers had greater zones of inhibition compared to that of
cellulite aspirates of endodontic origin and contaminated non eugenol sealers which was same as the results seen in
root canals using the PCR method and concluded the study experiments conducted by Andre Mickel et al[13,35].
by documenting the presence of Candida albicans in 5 of 24
root canal samples. [23]. Verma et al 2015 in a study concluded that Zinc oxide
eugenol (ZoE) had comparable antimicrobial effect with
Bonastre (1837) found zinc oxide eugenol, which was other obturating materials used against E Faecalis in the
later used by Chisholm (1876)[24] in dentistry. The first root primary root canal dentine [36]. Similarly, Queiroz AM et al
canal filling material to be recommended for primary teeth 2009 concluded that ZoE had the highest antibacterial effect
was Zinc oxide eugenol paste, as defined by Sweet in when compared with other materials used against E
(1930)[25] and has since been the most widely used root Faecalis[37].
canal filling material in dentistry[4].ZOE has historically
been the material of choice for filling deciduous teeth in the In a clinical study, success rates of zinc oxide eugenol
root canals[26], and was the only material specifically cement after obturation in primary teeth were reported by
prescribed in the clinical recommendations established by various authors as follows; 82.3% by Barr et al[38], 82.5%
the American Academy of Pediatric Dentistry until 2008. by Gould[39] and 86.1% by Coll et al[40]. A more recent
(AAPD) [27]. The antimicrobial effectiveness of zinc oxide retrospective evaluation by Bahrololoomi and Zamaninejad
eugenol may be due to the eugenol content. Owing to its was done in 2015 to find out the success rate of ZOE as an
capacity to induce hydrophobicity, Eugenol is bactericidal in obturating material for a mean follow‑up period of 24
nature, allowing them to separate the lipids of the bacterial months. They reported that two‑visit Pulpectomy of primary
cell membrane and mitochondria, spreading the cell molars using ZOE for root filling is one of the most reliable
structures and making them more permeable. Extensive and successful treatments for necrotic primary teeth[41].
bacterial cell leakage or the exit of essential molecules and
ions will lead to cell death[28]. Thymol is the primary ingredient of thyme oil. In
dental practice, thyme oil demonstrates antibacterial activity
Oyedemi et al 2009 investigated the bactericidal action and has proven useful[42]. Thymol tends to prevent the
mechanism of eugenol against gram-positive and gram- development of oral pathogens in the mouth and can
negative bacteria and concluded that eugenol caused damage decrease tooth decay in conjunction with other essential
to the cell wall resulting in leakage of protein from the oils[43,44]. One of the essential oils present in Listerine
bacterial cell wall. [29].Hashieh conducted a study which with antibacterial effects is Thymol.[45].
show that the level of eugenol released from a zinc oxide-
eugenol-based sealer beyond the apex is very low and In a study done by Nilima et al in 2016, the zones of
decreases over timebut still at these concentrations, eugenol inhibition were highest for ZoT, followed by ZoE and least
is said to have anti-inflammatory and analgesic properties for ZoP against E Faecalis which contradicts the results of
that are very useful after a pulpectomy procedure[30]. the present study where ZoE had highest zones of inhibition
However, it has disadvantages like slow resorption, followed by ZoT[3].
deflection of permanent tooth bud, tooth discoloration,
irritation to periapical tissues. All these disadvantages led to NilimaThosar et al 2013 conducted a study to assess
the search of numerous materials for root canal filling in the antimicrobial efficacy of 5 essential oils against oral
primary teeth [4]. pathogens which concluded that Thyme oil had the highest
antifungal effect on candida albicans followed by
In the present study eugenol has been compared to peppermint oil while the least effect was seen with eugenol
peppermint and thyme oil because of the drawbacks seen which refutes the results of the present study[11]. Studies
from the use of eugenol. These are plant essential oils which done by Gislene et al (2000), Hili et al (1997)and Nzeako et
can exhibit antimicrobial activity against the microorganism al (2006) showed identical conclusions[46,47,48] .Similar
which are commonly found in secondary infection like E results were also seen in studies done by Kovac et al
faecalis and Candida albicans. (2013)[49], Amorim et al (2006) [50]and Baumgartner et al

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Volume 6, Issue 1, January – 2021 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
(2000)[23].ZoP showed better antimicrobial efficacy [4]. Thosar N, Chandak M, Bhat M, Basak S. In vitro
compared to ZoE in study done by Nilima et al 2016 [4]. antimicrobial efficacy of zinc oxide with peppermint
oil in comparison to zinc oxide eugenol against four
The result of this study concluded that ZoE was found root canal microorganisms. J Adv Med Dent Scie
to be superior to Zinc oxide with thyme oil and Zinc oxide Res.2016;3(9):53-8.
with peppermint oil in inhibiting E faecalis and Candida [5]. Arora R, Raiwat P, Bhayya D P.A comparative
albicans but study done in 2016, Nilima et al observed that evaluation of antimicrobial efficacy of three
ZoT had a more effective anti-fungal effect on the antifungal endodontic sealers: Endoflas FS, AH Plus and
effect of zinc oxide-based pastes containing various sealapex against enterococcus Faecalis Int J Med Dent
essential oils against Candida albicans, accompanied by ZoP Sci.2014;13(3):90-3
and ZoE, this may be attributed to different culture media [6]. Bhargava R, Shashikala K, Bhargava R. Antibacterial
used in the research, which is sabourauds dextrose agar. efficacy of three root canal sealers against
[12]. In another study done by Nilima et al in 2016, the enterococcus Faecalis ans candida albicans-an in vitro
zones of inhibition were highest for ZoT, followed by ZoE study. SRM J Res Dent Sci.2018;6(3):170-4
and least for ZoP against E Faecalis which contradicts the [7]. Navit S, Jaiswal N, Khan S A, Malhotra S et al.
results of the present study where ZoE had highest zones of Antimicrobial efficacy of contemporary obturating
inhibition followed by ZoT[3] material used in primary teeth: An in vitro study. J
ClinDiagn Res.2016;10(9):9-12
The rationale for performing this in vitro study was to [8]. Hammer K A, Carson C F, Riley T V. Antimicrobial
provide information that zinc oxide eugenol has the highest activity of essential oils and other plant extracts. J
antimicrobial efficacy followed by zinc oxide with thyme oil Appl Microbiol.1999;86:985-90.
and then zinc oxide with peppermint oil against [9]. Anderson AC, Hellwig E, Vespermann R, Wittmer A,
Enterococcus faecalis and Candida albicans. However, it Schmid M, et al. Comprehensive Analysis of
should be taken into consideration that data presented here is Secondary Dental Root Canal Infections: A
related to an in-vitro condition and in-vivo conditions such Combination of Culture and Culture-Independent
as presence of saliva, presence of other micro-organisms, Approaches Reveals New Insights. PLoS ONE
presence of dentine and the serum might modify the 2012;7(11): e49576
antimicrobial efficacy of the obturating material. Therefore, [10]. Thosar N, Chandak M, Bhat M, Basak S. Evaluation of
further in vivo studies are needed to evaluate the antimicrobial activity of two endodontic sealers:- zinc
antimicrobial properties of the obturating materials used oxide with thyme oil and zinc oxide eugenol against
inthe present study. root canal microorganisms. Int J ClinPediatr Dent
2018;11(2):79-82
VI. CONCLUSION [11]. Thosar N, Basak M, Bahadure R N, Rajkumar M
.Antimicrobial efficacy of five essential oils against
All materials developed zones of microbial growth oral pathogens: an in vitro study. Eur J
inhibition against Enterococcus faecalis and candida Dent.2013;7:71-7
albicans within the limitations of the sample.On intergroup [12]. Thosar N, Chandak M, Bhat M, Basak S. Antifungal
comparisons of mean zones of inhibition in the different effect of zinc oxide based pastes containing various
group, maximum zone of inhibition was seen in Zinc oxide essential oils against candida albicans and comparison
eugenol followed by zinc oxide with thyme oil and least of its effects with zinc oxide eugenol. Indian J Med
with zinc oxide with peppermint oil which was the least Res Pharm.2016;3(11):60-5.
against Enterococcus faecalis and candida albicans. Candida [13]. Kaiwar A, Nadig G, Hegde J, Lekha S. Assessment of
albicans were almost resistant to zinc oxide with peppermint antimicrobial activity of endodontic sealers on
oil in most of the cases. It could also be concluded that ZoE, enterococcus Faecalis: an in vitro study. J
ZoT and ZoP has better antimicrobial efficacy against Dent(Shiraz).2012;3(1):26-31
Candida albicans as compared to Enterococcus faecalis. [14]. Punathil S, Bhat S S, Bhat S V, Hegde S K.
Microbiological analysis of root canal flora of failed
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