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Evaluation of Antimicrobial Activity of Two Endodontic Sealers
International Journal of Clinical Pediatric Dentistry, March-April 2018;11(2):79-82
79
IJCPD
Evaluation of Antimicrobial Activity of Two Endodontic
Sealers: Zinc Oxide with Thyme Oil and Zinc Oxide
Eugenol against Root Canal Microorganisms—
An in vitro Study
1Nilima R Thosar, 2Manoj Chandak, 3Manohar Bhat, 4Silpi Basak
IJCPD
ORIGINAL ARTICLE
10.5005/jp-journals-10005-1489
1,4Professor, 2,3Professor and Head
1Department of Pedodontics and Preventive Dentistry, Sharad
Pawar Dental College & Hospital, Datta Meghe Institute of
Medical Sciences, Wardha, Maharashtra, India
2Department of Conservative Dentistry and Endodontics, Sharad
Pawar Dental College & Hospital, Datta Meghe Institute of
Medical Sciences, Wardha, Maharashtra, India
3Department of Pedodontics and Preventive Dentistry, Jaipur
Dental College, Jaipur, Rajasthan, India
4Department of Microbiology, Jawaharlal Nehru Medical College
Datta Meghe Institute of Medical Sciences, Wardha, Maharashtra
India
Corresponding Author: Nilima R Thosar, Professor, Department
of Pedodontics and Preventive Dentistry, Sharad Pawar Dental
College & Hospital, Datta Meghe Institute of Medical Sciences
Wardha, Maharashtra, India, Phone: +917152287701, e-mail:
drnthosar@rediffmail.com
ABSTRACT
Aim: The present study was aimed to nd out and compare
the antimicrobial effect of the paste containing zinc oxide
cement mixed with thyme oil (ZO + Th oil) with that of the paste
containing zinc oxide and eugenol (ZO + E) against Staphylo-
coccus aureus, Escherichia coli, Enterococcus faecalis, and
Pseudomonas aeruginosa, common root canal pathogens of
deciduous teeth.
Materials and methods: An in vitro antimicrobial effect was
carried out by the agar diffusion method. The ZO + Th oil paste
was lled in the punched holes of Mueller Hinton agar at two
equidistant points. The same was followed for ZO + E paste.
For 24 hours, all the plates were incubated at a temperature of
37°C. The inhibition zones in millimeters around the wells were
calculated. There were 6 times repetitions of the test for each
microorganism. Data were tabulated and analyzed statistically
using one-way analysis of variance (ANOVA) and Tukey’s post
hoc comparison test. Level of signicance for the tests was 5%.
Results: Zones of bacterial inhibition were highest for ZO +
Th oil paste against the pathogenic microorganisms S. aureus,
followed by E. coli, E. faecalis, P. aeruginosa while for ZO + E
paste, the decreasing order against pathogenic microorgan-
isms was E. coli, S. aureus, E. faecalis, and P. aeruginosa.
Statistically signicant difference was found in both the pastes,
ZO + Th oil paste and ZO + E paste.
Conclusion: ZO + Th oil paste showed higher levels of anti-
microbial effect against the root canal pathogens.
Keywords: Antimicrobial efcacy, Zinc oxide eugenol paste,
Zinc oxide mixed with thyme oil paste.
How to cite this article: Thosar NR, Chandak M, Bhat M,
Basak S. Evaluation of Antimicrobial Activity of Two Endodontic
Sealers: Zinc Oxide with Thyme Oil and Zinc Oxide Eugenol
against Root Canal Microorganisms—An in vitro Study. Int J
Clin Pediatr Dent 2018;11(2):79-82.
Source of support: Nil
Conict of interest: None
INTRODUCTION
Microbial infections in root canals of primary teeth are
polymicrobial in nature.1 Pulpectomy of primary teeth
includes biomechanical preparation and use of intracanal
dressing with antibacterial properties. The success of
endodontic treatment depends on the removal of infected
bacteria.2 There are various methods: proper debride-
ment with instrumentation, antibacterial irrigations, and
antibacterial filling materials.3
Due to the presence of numerous accessory canals in
primary teeth, intracanal dressings may fail to remove these
microorganisms from inaccessible areas. So, it is important
to use an obturating material which has antibacterial prop-
erties to act on such microorganisms of inaccessible areas of
root canals of primary teeth. Due to antibacterial efficacy,
thyme oil shows its potential to be used in dentistry.4
Thymol inhibits the bacterial growth in oral cavity.
It shows potential to inhibit the dental infection also.5,6
Thymol and carvacrol are its important constituents in 20
to 40%. Others include B-cymene, pinene and triterpenic
acid, menthone, borneol, linalool, and cineole.7 As there
are no studies available in the literature about the use of
thyme oil as obturating material in deciduous teeth, the
present study was carried out by using ZO + Th oil paste
and compared with ZO + E paste.
Therefore, the present study was aimed to find out
the antimicrobial activity of ZO + Th oil paste with that
of ZO + E paste.
MATERIALS AND METHODS
The present study was an in vitro study. It was approved
by the institutional ethical committee of Datta Meghe
Nilima R Thosar et al
80
Institute of Medical Sciences. In this study, zinc oxide
was combined with thyme oil. It was compared with
zinc oxide and eugenol combination paste. Thyme oil
was procured from Aromatantra, Mumbai.
The powder–liquid ratio of pastes was as per specifica-
tions of Tchaou et al.3 A quantity of powder 0.2 gm was
mixed with 0.07 cc oil. Mixing of powder with liquid was
carried out with the help of spatula on a dry glass slab.8
Microorganisms from the microbiology department of
Jawaharlal Nehru Medical College, Wardha, were used
for the study.
Microbial strains studied were: S. aureus [American
Type Culture Collection (ATCC) 25923], E. faecalis (ATCC
29212), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853).
Mueller Hinton agar was used as the growth medium
for testing the susceptibility of S. aureus, E. coli, E faecalis,
and P aeruginosa.9
Stock culture of test microorganism was poured in
brain heart infusion broth (5 mL). Incubation was done
at 37°C for 24 hours. Microorganisms were then subcul-
tured on blood agar; again incubated for the same time
and same temperature. Colonies of microorganisms were
inoculated in the medium of nutrient broth for the time of
6 hours. Its density was adjusted to 0.5 as per McFarland
scale.10 Petri dishes (90 mm) with 4 mm thick Mueller
Hinton agar was used. All the work was done under
laminar air flow chamber.
The lawn technique was used for uniform distribution
of bacterial dilutions. Care was taken while punching the
holes in agar; the mean diameter of holes was 6 mm at
the level of equal distance from each other. Holes were
punched with open end of micropipette. Freshly prepared
root canal filling paste was then placed in the hole. It was
kept at room temperature for 2 hours.
The procedure was repeated for 6 times for every
microorganism. The plates were incubated for 24 hours.
Next day, the diameter of growth inhibitory zones was
calculated by using the antibiotic zone scale of HiMedia.8
Wider zones were read as having a higher antibacterial
effect against the specific microorganism.9
Data were analyzed statistically using ANOVA and
Tukey’s post hoc test at a significance level of 5% using
the GraphPad Prism 4 software.
RESULTS
For ZO + Th oil paste, the diameter in mm of the inhibition
zones for S. aureus was 36.33 ± 1.36, for E. coli, it was 35.33
± 2.73, for E. faecalis, it was 28 ± 1.78, and for P. aeruginosa,
it was 21.33 ± 7.22 (Table 1).
Table 2 is suggestive of the one-way ANOVA of bac-
terial inhibition zones for the ZO + Th oil paste group
in which the difference between and within groups was
statistically significant (p-value: 0.0001, p < 0.05).
Table 3 shows multiple comparisons: Tukey test was
done for the ZO + Th oil paste group to evaluate the inhib-
itory zones of bacteria. It showed that the difference was
statistically significant for S. aureus and E. faecalis with a
p-value 0.004, which is less than 0.05; however, significant
differences were also found for other bacteria also, i.e.,
Table 1: Zones of bacterial growth inhibition in mm of ZO + Th oil against four bacterial strains
Material n Mean Std. deviation Std. error
95% condence interval for mean
Minimum MaximumLower bound Upper bound
S. aureus 6 36.33 1.36 0.55 34.89 37.76 35.00 38.00
E. coli 6 35.33 2.73 1.11 32.46 38.20 32.00 38.00
E. faecalis 6 28.00 1.78 0.73 26.12 29.87 26.00 30.00
P. aeruginosa 6 21.33 7.22 2.95 13.74 28.92 14.00 30.00
Table 2: One-way ANOVA of zones of bacterial growth inhibition
of ZO + Th oil against four bacterial strains
Source of
variation
Sum of
squares Df
Mean
square f-value p-value
Between groups 2756.80 4 689.20 53.17 0.0001 S,
p < 0.05
Within groups 324.00 25 12.96
Total 3080.80 29
S: Signicant
Table 3: Multiple comparison: Tukey test of zones of bacterial growth inhibition of ZO + Th oil against four bacterial strains
Microorganisms Mean difference Std. error p-value
95% condence interval
Lower bound Upper bound
S. aureus E. coli 1.00 2.07 0.988, NS −5.10 7.10
E. faecalis 8.33 2.07 0.004, S 2.22 14.43
P. aeruginosa 15.00 2.07 0.0001, S 8.89 21.10
E. coli E. faecalis 7.33 2.07 0.013, S 1.22 13.43
P. aeruginosa 14.00 2.07 0.0001, S 7.89 20.10
E. faecalis P. aeruginosa 6.66 2.07 0.027, S 0.56 12.77
NS: Not signicant; S: Signicant
Evaluation of Antimicrobial Activity of Two Endodontic Sealers
International Journal of Clinical Pediatric Dentistry, March-April 2018;11(2):79-82
81
IJCPD
between S. aureus and P. aeruginosa with p-value 0.0001;
between E. coli and E. faecalis (0.013, p < 0.05); between
E. coli and P. aeruginosa, 0.0001; and between E. faecalis
and P. aeruginosa (p-value: 0.027, p < 0.05).
The difference for the inhibitory zones between S.
aureus and E. coli (0.988, p < 0.05) was not significant
statistically. Diameters in mm of the inhibitory zones
for other microorganisms in the ZO + E paste group in
descending order were: E. coli (19 ± 1.09), S. aureus (16 ±
0.00), E. faecalis (10.83 ± 1.47), and P. aeruginosa (10.33 ±
0.51) (Table 4) respectively.
In Table 5, one-way ANOVA for the ZO + E paste
group, differences for inhibitory zones of bacteria are
found to be statistically significant (p-value: 0.0001,
p < 0.05) between and within groups. Table 6 shows
multiple comparisons: Tukey test for inhibitory zones of
bacteria and it was observed that the difference between
S. aureus and E. coli was found to be statistically significant
(0.0001, p < 0.05). For other microorganism, the difference
was found to be statistically significant like S. aureus vs E.
faecalis (0.0001, p < 0.05); S. aureus vs P. aeruginosa (0.0001,
p < 0.05); E. coli vs E. faecalis (0.0001, p < 0.05); and E. coli
vs E. faecalis (0.002, p < 0.05).
The difference for E. faecalis vs P. aeruginosa (0.893,
p < 0.05) was not statistically significant.
DISCUSSION
Infections of root canals in primary teeth are polymicro-
bial in nature. Numerous materials have been tried in
dentistry for obturation of deciduous teeth. Zinc oxide
is widely used in dentistry.
There are disadvantages associated with this material
like slow resorption of ZOE as compared with physiologic
resorption of deciduous tooth, deflection of succedaneous
tooth, irritation to tissue at periapical area of tooth, bone
and cementum necrosis and tooth discoloration.11 Thyme
oil is found in European countries around Mediterranean
and said to belong to around 300 species of shrubs and
plants.
Essential oil from thyme plant is prepared from its
leaves and flowers by the method of steam distillation.
Thyme oil is useful in certain conditions like relief from
gastritis and enterocolitis. It is also found to be useful
in oral thrush. It can be used for patients suffering from
asthma and respiratory infections. Other uses of it are
treatment of swelling caused by gout or rheumatic prob-
lems, for backache, joint pains, and sciatica.
Thyme oil is also used for other conditions like vaginitis,
urinary infections, etc.7 Very few review articles are avail-
able in the literature mentioning the use of thyme oil in
dentistry. Antibacterial effects of thymol have been found
in Listerine.12 The study of Sköld-Larsson et al13 had shown
the use of thymol in the form of dental varnish to reduce
the Streptococcus mutans levels in supragingival plaque near
the bracket in patients with orthodontic brackets. Thyme oil
and clove oil together had shown the antimicrobial effect
against E. coli, S. aureus, and C. albicans at various concentra-
tions of the extracts.14-16 Thosar et al17 in their study carried
out in 2013 had shown the antimicrobial susceptibility for
Table 4: Zones of bacterial growth inhibition in mm of ZOE against four bacterial strains
Microorganisms n Mean Std. deviation Std. error
95% condence interval for mean
Minimum MaximumLower bound Upper bound
S. aureus 6 16.00 0.00 0.00 16.00 16.00 16.00 16.00
E. coli 6 19.00 1.09 0.44 17.85 20.14 18.00 20.00
E. faecalis 6 10.83 1.47 0.60 9.28 12.37 9.00 12.00
P. aeruginosa 6 10.33 0.51 0.21 9.79 10.87 10.00 11.00
Table 5: One-way ANOVA of zones of bacterial growth
inhibition of ZOE against four bacterial strains
Source of variation
Sum of
squares Df
Mean
square f-value p-value
Between groups 352.20 4 88.05 95.70 0.0001 S,
p < 0.05
Within groups 23.00 25 0.92
Total 375.20 29
S: Signicant
Table 6: Multiple comparison: Tukey test of zones of bacterial growth inhibition of ZOE against four bacterial strains
Microorganisms Mean difference Std. error p-value
95% condence interval
Lower bound Upper bound
S. aureus E. coli −3.00000 0.55377 0.0001, S −4.62 −1.37
E. faecalis 5.16667 0.55377 0.0001, S 3.54 6.79
P. aeruginosa 5.66667 0.55377 0.0001, S 4.04 7.29
E. coli E. faecalis 8.16667 0.55377 0.0001, S 6.54 9.79
P. aeruginosa 8.66667 0.55377 0.0001, S 7.04 10.29
E. faecalis P. aeruginosa 0.50000 0.55377 0.893, NS −1.12 2.12
NS: Not signicant; S: Signicant
Nilima R Thosar et al
82
thyme oil for E. coli which was with minimum inhibitory
concentration (MIC): 2 µL/mL, minimum bactericidal
concentration (MBC): 8 µL/mL; for C. albicans, MIC, MBC:
16 µL/mL; for E. faecalis MIC, MBC: 32 µL/mL and for S.
aureus MIC, MBC: 32 µL/mL respectively. The present
study suggests that use of thyme oil when mixed with zinc
oxide powder in the form of paste showed wider zones of
inhibition for S. aureus, E. faecalis, E. coli, and P. aeruginosa
in comparison with zinc oxide powder which was mixed
with eugenol oil.
As the ZO + Th oil paste showed more antibacterial
activity in comparison with ZOE paste, it can be success-
fully used in pediatric dentistry as an obturating material
in primary teeth. But further elaborative animal experi-
mental studies can prove its tissue biocompatibility and
toxicity properties.
CONCLUSION
A new material, i.e., ZO + Th oil paste, used in this study
had shown strong antibacterial activity against all the root
canal microorganisms which were studied and showed
its superiority over the zinc oxide + eugenol paste group.
This material will definitely prove its success in pedi-
atric dentistry as an obturating material for primary teeth.
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